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Poster # Title Author(s)

1 Is the Mammary Stroma of Copenhagen Rats a Tumor Suppressor? Drexler, Schaeberle, Maffini

2 Stromal Influences on the MCF7 Cells Phenotype in 3-Dimensional CulturesKrause, Maffini, Soto, Sonnenschein

3 In Utero Exposure to Bisphenol A: Links with Mammary Gland Cancer DevelopmentMurray, Maffini, Sonnenschein, Soto

4 Parity Alters Responses to Ionizing Radiation in the Mammary GlandCarter, Troester, Johnson, Jerry, Smith Schneider

5 Defining the Cellular Origins of Human Breast Cancer HeterogeneityKeller, Klebba, Gupta, Gilmore, Schnitt, Kuperwasser

6Morphology and Proliferation Control of Normal and Malignant Breast Epithelial Cells in a Collagen-Based 3-Dimensional Model of Tissue Microenvironment

Dhimolea, Maffini, Soto, Sonnenschein

7Knockdown of Spry2 by RNA Interference in Human Mammary Epithelial Cells Results in Increased Cell Cycle Progression and Migration

Friesel, Toher, Liaw

8The Wnt/β-Catenin Signaling Antagonist, SFRP1, Directly Affects the Tumorigenic Properties of Non-Malignant and Malignant Mammary Epithelial Cells

Gauger, Hugh, Ostrander, Smith Schneider

9 Elucidating the Role of Wnt Signaling in Breast CancerDiMeo, Anderson, Naber, Kuperwasser

10 Inhibitory Effects of Rhodiola Crenulata on an Invasive Immortal Human Cell LineRodríguez-Cortés, Gauger, Smith Schneider

11Tumor-Targeted Delivery of TRAIL Using Salmonella Typhimurium Enhances Breast Cancer Survival

Ganai, Arenas, Forbes

12 Disease Modeling and Tissue Engineering: Breast Cancer Metastasis to Bone Reagan, Goldstein, Rosenblatt, Kaplan

14A Humanized Model of Breast Cancer Metastasis Revealing a Human-Specific Metastasis Gene Signature

Goldstein, Anderson, Rosenblatt

15Role of Bone Sialoprotein (BSP) Overexpression in Osteolytic Metastasis in CMV-BSP Transgenic Mice

Tu, Chen, Jang, Kim

16 Targeted Overexpression of BSP in Osteoclasts Promotes Bone Metastasis of Breast Cancer Tu, Fix, Tang, Zhang, Chen

17Treating Ductal Carcinoma in Situ (DCIS) of the Breast with Tamoxifen: A Decision Analysis of the Risks and Benefits of Warfarin Anticoagulation in a Patient at Increased Risk for Thromboembolism

Evans, Wong, Pauker

18Determining the Role of Immune System Function in Breast Cancer Using an Imagable Syngeneic Model of Breast Cancer

Fang, Tao, Sahagian

19Molecular Basis for Intravasation and Metastasis to Lung Using an Orthotopic Mouse Model of Breast Cancer

Fang, Tao, Sahagian

20Use of a Standardized Web-Based Tool for Evaluation of Bone Health in Breast Cancer Patients

Arnaoutakis, Wong, Parameswaran

21 Progenitor-Progeny Relationships of the Marker Defined Cell Subsets in the Human ProstateMakarovskiy, Geck, Parmelee, Tai, Carpinito

22New Target Genes in Prostate and Breast Cancer: Methylation and MicroRNA Silencing of a Stem Cell Differentiation Gene, APRIN, in Clinical Studies

Pilichowska, Denes, Kim, Makarovskiy, Carpinito, Geck

23 β1 Integrin Participates in Endoglin-Dependent Inhibition of Prostate Cancer Cell Migration Romero, Roth, Brooks, Vary

24PVR/Necl-5 Promotes Glioblastoma Invasion by Activation of the Akt Pathway and MMP-2 Production

Enloe and Jay

25Role of Histone H3K27 Demethylases in Maintenance of Multipotency in Glioblastoma Stem Cells

Sherry, Reeves, Wu, Cochran

26 A Conditional RNAi Strategy to Investigate Signaling Networks in Glioblastoma MultiformeAcquaviva, Ramachandran, Woolfenden, Charest

27 Somatostatin Therapy for Recurrent Meningiomas Kandil, Zhu, Heilman, Wu

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28Clinical Impact of Adjuvant Gamma Knife Radiosurgery to the Surgical Cavity of Resected Metastases: A Retrospective Review of the Tufts Medical Center Experience

Hwang, Eisenberg, Hale, Dvorak, Boskovitz, Yao, Pfannl, Mignano, Zhu, Strauss, Wu

28AImprovement of Neurological Function after Concurrent Treatment IVIG and Chemotherapy in a Patient with Cerebellar Degeneration Paraneoplastic Syndrome

Zhu, Li, Hedges, Wakeley

29 Larger Lentigo Maligna Lesions and Invasive Melanoma: Size Matters Zeikus, Kakullavarrupu, Olbricht

30Mapping Genes Associated with Spontaneous Canine Hemangiosarcoma and Expression Profiling, Potential Animal Model for Human Angiosarcoma

Azuma, Lindblad-Toh, Karlsson, Tonomura, Barber, Burgess, Shaw, Keating, Meola, Xu

31 YKL-40 Demonstrates a Protective Role in Rhabdomyosarcoma by Decreasing MMP-2 Scully, Yan, Shao

32 MicroRNA Expression Profiling: Connecting Lung Development and Lung Cancer Mujahid, Volpe, Nielsen

33Fibroblasts Affect the Phenotype of Normal Human Bronchial Epithelial Cells when Co-Cultured in Three-Dimensional (3D) Organotypic Cultures

Pageau, Maffini, Soto, Sonnenschein

34Laser Capture Microdissection to Isolate Primary and Metastatic Thyroid Tumors from Formalin Fixed Paraffin Embedded Tissue

Kim and Cheng

35 Mrc1 and Tof1 Participate in the Maintenance of CAG•CTG Repeat Tract IntegrityGellon, Lahiri, La Porte, Freudenreich

36The Importance of Spatial Distribution of Stemness and Proliferation State in Determining Tumor Radio-Response

Enderling, Park, Hlatky, Hahnfeldt

37 The Yin and Yang of ERK Regulation by Polyomavirus Middle T (MT) Zhu and Schaffhausen

38Solution Structure of the Hdlg/SAP97 PDZ2 Domain and its Mechanism for Interaction with HPV-18 E6 Protein

Liu, Henry, Hegde, Baleja

39 Scaffold Proteins Regulate Localized Rac Activation by Tiam1Rajagopal, Li, Wicks, Wong, Isberg, Buchsbaum

40 Sequence-Specific Chromatin Remodeling of the C-Myc Promoter by hSWI/SNFSims, Baughman, Lane, Schnitzler

41Inhibition of CBF-1-Mediated Notch Signaling Induces FGF-Dependent Transformed Cell Phenotype

Kacer, McIntire, Kirov, Prudovsky

42 Investigating Cancer Progression of Cells in 3D Matrix with Non-Invasive Fluorescent ImagingXylas, Alt-Holland, Garlick, Georgakoudi

43 Cancer Cells Modulate DNA DSB/Repair in Nontransformed Cells Beheshti, Enderling, Perkins, Burg, Hahnfeldt, Hlatky

44 Antiangiogenic Effects of Proton Irradiation

Girdhani, Hahnfeldt, Beheshti, Anaya, Peluso, Lamont, Raychowdhury, Schwager, Huber, Enderling, Abdollahi, Hlatky

45 Impaired Angiogenesis in Aged Tumor Microenvironment Attenuates Tumor GrowthKalinga, Beheshti, Hahnfeldt, Abdollahi, Hlatky

46Neuregulin-1 Alleviated Doxorubicin-Induced Down-Regulation of Cardiac Troponin Proteins in the Heart

Bian, Sun, Silver, Ho, Marchionni, Caggiano, Morgan, Yan

47 Evaluating the Efficacy of Stereotactic Spinal Radiosurgery Yao and Bakes

48Selective Inhibition of Growth of Tuberous Sclerosis Complex 2-Null Cells by Atorvastatin is Associated with Impaired Rheb and Rho Gtpase Function and Reduced Mtor/S6 Kinase Activity

Finlay, Malhowski, Liu, Fanburg, Kwiatkowski, Toksoz

49Genetic Variants in Uracil Processing Enzymes are Associated with Abnormal DNA Uracil Content

Chanson, Parnell, Crott, Choi, Han, Mason

50Dipeptidyl Peptidase 2 is a Novel Prognostic Factor and Therapeutic Target in Chronic Lymphocytic Leukemia

Danilov, Danilova, Klein, Brown, Rabinowitz, Miller, Huber

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51Distinct Gab2-Mediated Signaling Pathways are Essential for Myeloid or Lymphoid Transformation and Leukemogenesis by BCR-ABL

Chan, Mohi, Li, Neel, Van Etten

52Environmental Risk Factors and Risk of Canine Malignant Lymphoma: A Model for Human Non-Hodgkin’s Lymphoma

Zagarins, Barber, Procter-Gray, Gollenberg, Bertone-Johnson

53 Comparative Oncology Trials Consortium Aids in Development of Human Cancer Therapies Barber, Burgess, Marshall

54GS-9219 – A Novel Acyclic Nucleotide Analogue with Potent Antineoplastic Activity in Dogs with Spontaneous Non-Hodgkin’s Lymphoma

Burgess, Vail, Thamm, Hawkins, Tumas

55Tissue Banking at the Cummings School as Part of the Canine Comparative Oncology Genetics Consortium

Burgess, Barber, Azuma, Marshall, Meola, Berg

56Novel, 3D Human Tissue Platforms for Cancer Discovery: The Core for Experimentation in 3D Tissues

Garlick and Egles

57 Tufts Medical Center Transgenic Core FacilityRwayitare, Riggs, Mendelsohn, Lem

58 Tufts Animal Pathology and Histology Core Laboratory Services Richey, Lagace, Papalegis

59 Proteomic Research and Services of the Tufts Proteomic Core Facility DeGnore

60 Getting Your Research Done with University Information Technology (UIT) Services Zupan

61 Light Scattering Spectroscopic Characterization of Healthy and Cancerous White Blood Cells Hsiao

62 Hyperspectral Image Reconstruction for Diffuse Optical Tomography Larusson

63 Avenanthramides Inhibit Proliferation of Human Colon Cancer Cell Lines in vitroGuo, Nie, Wu, Wise, Collins, S. Meydani, M. Meydani

64 A Novel Mouse Model for Sporadic Colorectal CancerHung, Maricevich, Georgeon Richard, Kunin, Kho, Mahmood, Kucherlapati

65Nonalcoholic Steatohepatitis Induced by a High-Fat Diet Promotes Diethylnitrosamine Initiated Early Hepatocarcinogenesis in Rats

Wang, Ausman, Greenberg, Russell, Wang

66Nasopharyngeal Carcinoma at Tufts Medical Center: Evaluation of the Chinatown and Greater Boston Incidence and Response to Treatment

Wein and Merdad

67 Dose De-Escaltion with Gamma Knife Radiosurgery in the Treatment of Choroidal MelanomaMignano, Chan, Schirmer, Melhus, Williams, Do-Dai, Duker, Yao

68 Molecular Characterization of EBV Associated Nasopharygeal Carcinoma Merritt and Thorley-Lawson

69Frequency of Presentation and Risk Profile for Human Papillomavirus in Oropharyngeal and Hypopharyngeal Squamous Cell Carcinoma at Tufts Medical Center

Kraus, Oreadi, Wein, Laver, Papageorge

70RalA Suppresses Invasion by Ras-Transformed Keratinocytes in a Bioengineered Human Tissue Model of Squamous Cell Carcinoma

Sowalsky, Alt-Holland, Shamis, Garlick, Feig

71The Tumor Microenvironment Meets Epigenetics: Regulation of E-Cadherin Expression in Squamous Cell Carcinoma Progression through DNA Methylation

DesRochers, Kudo, Takata, Garlick

72Loss of E-Cadherin-Mediated Cell-Cell Adhesion Induces the Transition from Precancer to Squamous Cell Carcinoma through Activation of FAK and Src Kinases

Alt-Holland, Szwec-Levine, Green, Garlick

73 Quality of Life in Patients with Resected and Reconstructed MandiblesOreadi, Shastri, Chapman, Papageorge

iii

Jean Mayer USDA Human Nutrition Research Center on Aging Ground Floor

iv

Jean Mayer USDA Human Nutrition Research Center on Aging Mezzanine

v

Poster No. 1 Title:

Is the Mammary Stroma of Copenhagen Rats a Tumor Suppressor?

Authors:

Amorette Drexler, Cheryl Schaeberle, Maricel Maffini

Presented by:

Maricel Maffini

Department:

Department of Anatomy and Cellular Biology, Tufts University School of Medicine

Abstract:

Copenhagen (COP) is a rat strain that does not develop tumors when exposed to chemical carcinogens such as

N-nitrosomethylurea (NMU) during the window of vulnerability of 50-60 days of age commonly used in cancer

research. However, comparative studies showed that COP rats develop a similar number of preneoplastic lesions

as the tumor-susceptible Wistar-Furth (WF) rats. While in the COP rats, these lesions regress those in the WF

rats progress and develop into carcinomas. The resistance phenotype was attributed to the presence of mammary

carcinoma susceptibility genes in COP mammary epithelium and their absence in WF rats, although

contradictory results have been published. Previously, we showed that, on one hand, NMU-exposed WF

mammary stroma is able to induce normal mammary epithelial cells (MEC) to form carcinomas. On the other

hand, normal WF mammary stroma has also the ability to reverse the tumor phenotype of malignant epithelial

cells and induce them to form normal mammary ducts. In light of these results, we postulated that the stroma is

also responsible for the tumor-resistant phenotype observed in the COP rats.

The experimental design was 2-fold:

1) Tissue Recombination: We recombined stromal and epithelial cells from WF and COP rats; mammary

fibroblasts (MF) were collected from cleared-fat pads of animals exposed to NMU or vehicle (VEH). MECs

were collected from virgin 55 day-old rats. The experimental groups were:

Group 1: MF from NMU-exposed COP rats were recombined with COP MEC (positive control for tumor

resistance)

Group 2: MF from VEH-exposed COP rats with COP MEC

Group 3: MF from NMU-exposed COP rats with WF MEC

Group 4: MF from VEH-exposed COP rats with WF MEC

Group 5: MF from NMU-exposed WF rats with WF MEC (positive control for tumor susceptibility)

Group 6: MF from VEH-exposed WF rats with WF MEC

Group 7: MF from NMU-exposed WF rats with COP MEC

Group 8: MF from VEH-exposed WF rats with COP MEC

1

Poster No. 1

Both cell types were mixed with collagen I and grafted under the kidney capsule of host SCID mice. All tissue

recombinants developed normal ducts; however, no recombinants developed macroscopic tumors. Preneoplastic

and neoplastic lesions were only observed in Groups 5 and 7 (NMU-exposed WF fibroblasts). Groups 1 and 3

(NMU-exposed COP fibroblasts) did not show any neoplastic or preneoplastic lesions. Instead, most structures

were normal ducts. These findings suggest that the tumor resistant phenotype observed in COP rats resides in

the mammary stroma.

2) Global Gene Expression Analysis: COP and WF mammary stroma were collected from NMU- or VEH-

exposed animals after 15 (COP and WF have equal number of preneoplastic lesions) or 60 (lesions regressed in

COP and progressed in WF) days after treatment (DAT). Our data indicate that the amount of HOXA1 and

STAT3 mRNAs is lower in the COP-NMU rats compared to the WF-NMU. STAT3 mRNA is lower at 5 and

15DAT while HOXA1 is lower at 15 and 60DAT. COP-NMU animals have an increase in TGF beta2 and

procollagen type X mRNAs at 15DAT compared to WF-NMU. These and other genes expressed at 15DAT,

when the animals showed the highest number of preneoplastic lesions, seem to be setting the stage for their

disappearance by remodeling the stroma to reduce (or shut off) epithelial cell proliferation and to induce

normalization of the lesions.

2

Poster No. 2 Title:

Stromal Influences on the MCF7 Cells Phenotype in 3-Dimensional Cultures

Authors:

Silva Krause, Maricel Maffini, Ana Soto, Carlos Sonnenschein

Presented by:

Maricel Maffini

Departments:

Department of Cell, Molecular and Developmental Biology and of Anatomy and Cellular Biology,

Tufts University School of Medicine

Abstract:

Stromal-epithelial interactions mediate mammary gland development and the formation and progression of

breast cancer. In order to study these interactions in more detail, the development of defined 3-dimensional

in vitro models is essential. In the present study, we have successfully developed novel 3-dimensional in vitro

breast cancer models, which allow the study of both tumor reversion and tumor formation. Co-cultures of a

human breast cancer cell line MCF7 and human mammary fibroblasts obtained from reduction mammoplasties

embedded in either a type I collagen or a mixed Matrigel™-collagen matrix were carried out for up to 6 weeks.

Histological and ultrastructural analysis confirmed the formation of epithelial structures. The importance of the

stromal cells was apparent in both matrices; in the collagen gels the presence of reduction mammoplasty

fibroblasts reduced epithelial cell apoptosis and allowed them to become polarized before obtaining tumor-like

structures and in the mixed Matrigel™-collagen gels the presence of those fibroblasts initially resulted in the

reversion of the neoplastic phenotype and later on, these epithelial structures formed tumor-like structures.

These models provide an excellent system to study tissue organization, epithelial morphogenesis and breast

carcinogenesis.

3

Poster No. 3 Title:

In Utero Exposure to Bisphenol A: Links with Mammary Gland Cancer Development

Authors:

Tessa Murray, Maricel Maffini, Carlos Sonnenschein, Ana Soto

Presented by:

Tessa Murray

Department:


Abstract:

Epidemiological studies suggest that fluctuating estrogen levels in the fetal environment have long-term

consequences regarding the risk of developing breast cancer during adult life. In addition, experimental studies

showed that perinatal exposure to pharmacological doses of diethylstilbestrol (DES) increased the incidence and

decreased the latency period of mammary cancer. We observed that fetal exposure to environmentally relevant

doses of the xenoestrogen bisphenol A (BPA), a compound used in the manufacture of polycarbonate plastics,

alters the normal development of the mouse mammary gland resulting in long-lasting effects manifested mainly

during adult life. Of particular interest were: (i) an increase in the number of terminal end buds and terminal

ends, (ii) an increase in ductal density (breast density is considered a risk factor), and (iii) an increased

sensitivity to estradiol, suggesting enhanced susceptibility for mammary cancer development. Hence, we

hypothesize that in utero exposure to low doses of BPA increases the risk of developing mammary cancer.

To explore this hypothesis, we used a Wistar-Furth rat model. Animals were exposed to BPA (2.5, 25, 250 or

1000μg/kg/day) or vehicle (50% DMSO) from gestational day 9 to birth using an osmotic pump. In utero, BPA

exposure caused an increase in the incidence of preneoplastic lesions (ductal hyperplasias) at postnatal day 50,

95 and 140 regardless of the BPA dose. Interestingly, animals exposed to the lowest dose tested

(2.5µg BPA/kg/day) showed a consistent and statistically significant increase in ductal hyperplasias relative to

unexposed animals at all ages. More importantly, carcinomas in situ (CIS) were also observed in these animals.

We postulate that the persistent ductal hyperplasias may develop into CIS later in life. To test this hypothesis

we are currently analyzing tissue collected from animals at postnatal day 140 and 200. Also, we are conducting

morphometric analyses at all four developmental stages to determine whether BPA exposure has caused

additional long-lasting morphological changes in the mammary gland, especially paying attention to the terminal

end buds as carcinomas are thought to originate in these structures.

In summary, in utero BPA exposure induces the development of preneoplastic and neoplastic lesions in the

mammary gland in the absence of any additional treatment aimed at increasing tumor development. Our results

support the hypothesis that environmental exposure to xenoestrogens during fetal life may contribute to the

increased incidence of human breast cancer observed over the past 5 decades.

4

Poster No. 4 Title:

Parity Alters Responses to Ionizing Radiation in the Mammary Gland

Authors:

Matthew Carter, Melissa Troester, Melissa Johnson, Joseph Jerry, Sallie Smith Schneider

Presented by:

Matthew Carter

Department:

Pioneer Valley Life Sciences Institute, Baystate Medical Center

Abstract:

Parity is associated with a significant reduction in breast cancer risk. Identification of the key pathways altered

by parity will provide novel molecular targets for the development of chemo-protective agents. Radiation is a

potent carcinogen in the mammary gland and causes mutations through its ability to induce DNA damage, as

well as through its ability to induce “bystander effects.” These bystander effects have been shown to be tumor

promoting in immortalized cells and they involve the modulation of various growth factors required for stem

cell maintenance. We hypothesized that we could identify specific genes involved in parity-induced protection

by examining gene expression responses of normal breast tissue from parous and nulliparous women.

Methods: Women undergoing elective reduction mammoplasties were asked to enroll in our study and donate

their excised tissue. Small portions of this tissue were put into tissue culture for 24 hours and then exposed to

5Gy of radiation. Subsequently, the tissue was left in culture for an additional six hours to allow for

uninterrupted changes in gene expression. Finally, the tissue was fixed for immunohistochemical analysis or

RNA was harvested for microarray analysis.

Results: A SAM analysis revealed a loose 200 gene signature which showed differential transcriptional

responses between parous and nulliparous women in response to radiation. Breast tissue harvested from

nulliparous women showed the most striking changes with an increased expression of 80% of genes in response

to radiation whereas tissue from parous women showed no change or decreased expression of these same genes.

The other 20% of signature genes were strongly down-regulated in tissue from nulliparous women in response

to radiation, while they were up-regulated or remained unchanged in parous tissue. Many of the genes which

seem to be inversely affected by radiation indicate that permanent changes caused by parity involve pathways

that regulate tissue homeostasis, wound repair, as well as DNA replication and repair. For instance, transforming

growth factor-beta (TGF-β) is a multifunctional cytokine which regulates both tissue development as well as

repair processes. The data obtained from the microarray indicated that in nulliparous women, the expression of

the type III TGF-β receptor (TβRIII) is reduced in response to radiation. Reductions in the levels of TβRIII are

interesting as it is down-regulated in a wide range of human cancers, including breast cancer, and re-expression

can inhibit cancer progression. Our current work is aimed at verifying these microarray data utilizing

quantitative real-time PCR analysis to measure mRNA expression levels and IHC to assess cellular protein

levels. Taken together, these findings suggest a putative mechanism by which a full-term pregnancy conveys its

protective effect against breast cancer. 5

Poster No. 5 Title:

Defining the Cellular Origins of Human Breast Cancer Heterogeneity

Authors:

Patricia Keller, Ina Klebba, Piyush Gupta, Hannah Gilmore, Stuart Schnitt, Charlotte Kuperwasser

Presented by:

Patricia Keller

Departments:

Department of Anatomy and Cellular Biology, Tufts University School of Medicine; Molecular Oncology

Research Institute, Tufts Medical Center; Broad Institute of Massachusetts Institute of Technology and

Harvard University; Department of Pathology, Beth Israel Deaconess Medical Center

Abstract:

Human breast cancers can be broadly classified based on their molecular and gene expression profiles into

luminal and basal tumors. These tumor subtypes express markers corresponding to the two major differentiation

states of epithelial cells in the breast; luminal cells that line the breast ducts and the outer myoepithelial/basal

cells that provide contractile functions. Although there is likely a complex interplay of factors that contribute to

tumor phenotype, the persistence of characteristics of normal breast cell types in tumors suggest that the major

tumor subclasses could arise from different cells of origin. We have recently described an experimental model in

which both the epithelium and stromal compartments are derived from human tissues. This model has enabled

the creation of normal and neoplastic breast tissues in vivo. Breast cancers were created from single cell

suspensions of human breast epithelial cells that were transformed and injected into humanized mammary fat

pads. The epithelial cells were maintained in vitro for no more than 24 hours. Histological analysis revealed that

the tumors were heterogeneous invasive carcinomas with features of both basal and luminal subtypes. To

determine if different cells of origin influence the cancer phenotype, we enriched cells of the basal/myoepithelial

lineage and cells of the luminal lineage by sorting freshly isolated human mammary epithelial cells for the

markers CD10 and ESA, respectively. These enriched populations were infected and injected in the same

fashion as unsorted cells. CD10+ basal cells formed well-circumscribed tumor nodules that exhibited increased

basal differentiation. Tumors from the ESA+ luminal enriched fraction formed expansive ER+ invasive

carcinomas containing reduced squamous differentiation, consistent with luminal differentiation. These results

suggest that the cellular differentiation state of the cell of origin can persist in the resultant tumors. To determine

if the genetic background of the breast tissue also influences the tumor phenotype, we transformed breast

epithelial cells derived from patients who have had prophylactic mastectomies due to inherited BRCA1

mutations. Tumors that formed from unsorted BRCA1 cells exhibited increased basal/myoepithelial phenotype

than those from non-BRCA1 patient samples. These results suggest that the association of basal-type tumors

with BRCA1 breast cancers may result from an altered differentiation state in breast tissues of BRCA1 patients.

6

Poster No. 6 Title:

Morphology and Proliferation Control of Normal and Malignant Breast Epithelial Cells in a Collagen-Based

3-Dimensional Model of Tissue Microenvironment

Authors:

Eugen Dhimolea, Maricel Maffini, Ana Soto, Carlos Sonnenschein

Presented by:

Eugen Dhimolea

Department:


Abstract:

In order to understand carcinogenesis and predict the outcome of pharmacological treatments, in vitro models

that recapitulate the three-dimensional (3D) structural and functional context of normal and malignant tissues

will be very informative. Surrogate models have provided significant insight on the morphology of normal and

tumorigenic breast cells in 3D matrices. However, the cell-mediated re-organization of the Extracellular Matrix

(ECM) has not been thoroughly investigated at the tissue level because the exclusive use of the epithelia cell

type and estrogenic contamination in the tissue culture plastic labware prevent addressing important questions

regarding the significance of interactions between epithelial and stromal cells in breast cancer and tumor

response to hormone therapy, respectively.

We developed an estrogen-free 3D culture of normal and breast cancer cell lines and primary breast fibroblasts

in collagen type I gels. A normal human breast epithelial cell line (MCF10A) contracts the collagen gels and

increases their stiffness while acini- and ductal-like structures that resemble the morphology of normal

mammary gland are formed. This happens independently of the presence of breast fibroblasts. Analysis of

collagen fiber configuration by picrosirius red staining demonstrates a differential rearrangement of collagen

around the cellular structures, similar to the fibers surrounding the normal gland epithelium in vivo. Impeding of

gel contraction prevents the formation of organized structures by the MCF10A cells and results in random

distribution of cellular aggregates. In contrast, the estrogen-sensitive breast cancer MCF-7 cells do not cause

contraction of the collagen gel and form loose disorganized colonies. Interestingly, the combination of

fluorescent protein-labeled MCF-7 and MCF10A induces contraction of collagen and formation of compact

spherical MCF-7 structures that are different from the colonies in MCF-7 cells alone in culture. Given that circa

70% of breast tumors express the Estrogen Receptor, we tested the sensitivity of our model to estrogens. The

MCF-7 cells retain the responsiveness to proliferative and anti-proliferative stimuli of estradiol and

antiestrogens (OHT and fulvestrant) respectively, in 3D conditions. Thus, collagen type I gels are useful 3D

in vitro models for studying the role that cell-cell and cell-ECM interactions and biophysical forces play on the

morphology of normal and epithelial breast cancer cells. This model provides the opportunity for experimental

hormonal manipulations aiming at elucidating the development of drug resistance mechanisms at the tissue

organizational level in estrogen-dependent breast cancer. 7

Poster No. 7 Title:

Knockdown of Spry2 by RNA Interference in Human Mammary Epithelial Cells Results in Increased Cell

Cycle Progression and Migration

Authors:

Robert Friesel, Jessica Toher, Lucy Liaw

Presented by:

Robert Friesel

Department:

Center for Molecular Medicine, Maine Medical Center Research Institute

Abstract:

Dysregulation of receptor tyrosine kinase (RTK) signaling is a major contributor to human carcinogenesis.

Spry2 is a feedback antagonist of RTK signaling targeting components of the Ras-Raf-ERK pathway, a pathway

known to be elevated in many types of cancer. Spry2 is down-regulated in a variety of human tumors, which

may contribute to tumor progression and metastasis. Using an immortalized human breast cell line, MCF10A,

as a model, we investigated the impact of the loss of Spry2 by RNA interference on the oncogenic

transformation of MCF10A cells in vitro. Lentiviral vectors encoding Spry2-specific shRNAs were stably

transduced into MCF10A cells. MCF10A cells, in which Spry2 was stably knocked down, exhibited loss of

contact inhibition, increased cell growth, increased cell migration and a dramatic increase in the number of cells

in S phase. In addition, there was an increase in β-catenin protein levels and redistribution away from the cell

membrane. Overexpression of Spry2 in the MDA-MB-231 breast cancer cell line results in decreased cell

growth and migration. Taken together, these data indicate that Spry2 plays an important role in the regulation of

breast epithelial cell growth and supports the clinical observation that loss of Spry2 in human breast cancers

contributes to the malignant phenotype.

8

Poster No. 8 Title:

The Wnt/β-Catenin Signaling Antagonist, SFRP1, Directly Affects the Tumorigenic Properties of

Non-Malignant and Malignant Mammary Epithelial Cells

Authors:

Kelly Gauger, Jeremy Hugh, Jennifer Ostrander, Sallie Smith Schneider

Presented by:

Kelly Gauger

Departments:

Pioneer Valley Life Sciences Institute, Baystate Medical Center; Departments of Biology and of Veterinary and

Animal Science, University of Massachusetts Amherst

Abstract:

Breast cancer is the most frequently occurring cancer in women and represents the second leading cause of

cancer death among women. It is essential to understand the mechanistic actions by which breast cancer occurs

and how it can be prevented. For example, aberrant activation of the Wnt/β-catenin signaling pathway

contributes to the genesis of a wide range of human cancers, including breast cancer. Secreted frizzled-related

proteins (SFRPs) are a family of proteins that antagonize the Wnt/β-catenin signaling pathway. Considering that

the SFRP1 isoform is down-regulated in breast tumors, our hypothesis is that loss of SFRP1 expression will

render mammary epithelial cells more susceptible to tumorigeneis in vitro and in vivo, and re-expression of

SFRP1 into breast carcinoma cells will subsequently impede their cancerous characteristics. We have created a

non-malignant immortalized mammary epithelial cell line (76N Tert) that stably expresses a siRNA construct

that reduces the expression of SFRP1 by 80%. The phenotypic changes observed when SFRP1 alone is

knocked-down in these cells, which include a loss of cell polarity causing a spindle-cell morphology and an

increase in the formation of pseudopodia. Not only do these cells show signs of a more mesenchymal

phenotype, but they also exhibit mesenchymal properities. Scratch-wound assays and matrigel assays revealed

that SFRP1 loss in 76N Tert cells remarkably increases their ability to migrate and invade. Additionally, Wnt3a

stimulation significantly increases proliferation as well as β-catenin-mediated luciferase activity when compared

to empty vector transfected control cells. We have also generated a malignant breast cancer cell line

(MDA-MB-231) that overexpresses SFRP1 and when these cells are stimulated with Wnt3a, β-catenin-mediated

luciferase activity is significantly reduced compared with empty vector transfected control cells. Moreover,

FACS scan analysis demonstrates that SFRP1 expressing MDA-MB-231 cells are more susceptible to death. We

are presently utilizing a SFRP1 knockout mouse model to elucidate the involvement of SFRP1 in mammary

gland development and breast cancer susceptibility. Taken together, these data could lay the foundation for the

development of preventative treatments aimed at antagonizing the Wnt/β-catenin signaling pathway in patients

with reduced levels of SFRP1.

9

Poster No. 9 Title:

Elucidating the Role of Wnt Signaling in Breast Cancer

Authors:

Theresa DiMeo, Kristen Anderson, Stephen Naber, Charlotte Kuperwasser

Presented by:

Theresa DiMeo

Department:


Abstract:

The Wnt family of secreted proteins is essential for normal embryonic development, as well as self renewal and

differentiation of adult tissues. Mutations in the Wnt signaling pathway (for example, APC) are well

documented in promoting the initiation of colon cancer. Interestingly, mutations in the Wnt pathway have not

been linked to the progression of breast cancer; however, many breast cancers exhibit aberrant stabilization of

β-catenin and overexpression of Wnt ligands. Additionally, many negative regulators of the Wnt pathway are

silenced in breast cancer.

Recently, we have shown that human breast cancer cell lines maintain tumor cell heterogeneity in culture

(Fillmore et al 2008). A small subpopulation of cells in the cell line displays self-renewal properties to give rise

to phenotypically diverse progeny and can initiate tumor growth in vivo. One such able cell line, the SUM1315

line, does not express endogenous sFRP1, a negative regulator of the Wnt pathway, and displays active Wnt

signaling in culture. Given the importance of the Wnt signaling pathway in maintenance of self-renewal in

various systems, we set out to elucidate the impact of inhibiting this pathway in breast cancer using a xenograft

mouse model system. The secreted Wnt inhibitors sFRP1 and Dkk1 were transduced by retroviruses into the

SUM1315 cell line and characterized for their phenotypes in vitro and in vivo. While both sFRP1 and Dkk1 act

similarly to inhibit Wnt1-induced TOPFlash activity, serial CFU formation, and tumorsphere formation in vitro,

only Dkk1 is able to inhibit primary tumor formation in vivo. Knockdown of Lrp6 in SUM1315 cells

phenocopied the Dkk1-SUM1315 overexpressing cells, indicating that Dkk1 is acting through the canonical Wnt

pathway to mediate its inhibitory effects. We found that both Dkk1 overexpression and shRNA mediated

knockdown of Lrp6 promote differentiation of the SUM1315 cells to a myoepithelial phenotype. We are

currently trying to elucidate the gene targets that are responsible for the reduction in tumor growth and are

altered in response to Dkk1 expression.

10

Poster No. 10 Title:

Inhibitory Effects of Rhodiola Crenulata on an Invasive Immortal Human Cell Line Authors:

Adaris Rodríguez-Cortés, Kelly Gauger, Sallie Smith Schneider

Presented by:

Adaris Rodríguez-Cortés

Department:


Abstract:

The root of the Tibetan plant Rhodiola crenulata is part of eastern traditional medicine. Studies have suggested

that members of the Rhodiola genus display anticancer properties. In this study we examine the effect of

R. crenulata in a cellular model of invasive breast cancer, this disease being the second cause of cancer death

among women in the United States.

Deregulation of the Wnt/β-catenin pathway has been frequently observed in breast cancers and appears to have a

key role in the transformation of benign cells to a malignant form. Although mutations of the Wnt growth factor

are rarely observed in cancer, the Wnt signaling pathway is often up-regulated by either mutations that result in

stabilization of β-catenin or by hypermethylation and subsequent loss of expression of Wnt signaling antagonists

like secreted Frizzled-Related Protein 1 (SFRP1). We used an engineered cell line in which SFRP1 expression

has been knocked down. These cells were derived from 76NTert cell line, an immortalized human mammary

epithelium cell line. The resulting 76NTert-siSFRP1 cells display a mesenchymal-like phenotype, invasive

behavior and are more resistant to apoptosis triggered by anchorage independent conditions, or anoikis.

Treatment of 76NTert-siSFRP1 cells with an extract of R. crenulata inhibited migration and invasion of the

76NTert-siSFRP1 cells, as compared to untreated cells. Furthermore, R. crenulata sensitizes cells to anoikis but

does not increase γ-irradiation induced cell death. We provide evidence that death induced by R. crenulata does

not occur through the inhibition of an epithelial-to-mesenchymal transition (EMT). Taken together, our initial

results suggest R. crenulata as a potential therapeutic agent for breast cancer patients with mutations in the

Wnt/β-catenin signaling pathway.

11


Tumor-Targeted Delivery of TRAIL Using Salmonella Typhimurium Enhances Breast Cancer Survival

Authors:

Sabha Ganai, Richard Arenas, Neil Forbes

Presented by:

Sabha Ganai

Department:

Surgical Oncology Service, Baystate Medical Center

Abstract:

Background: Attenuated Salmonella typhimurium has been demonstrated experimentally as a novel anticancer

agent because of its favored growth within tumors, limited toxicity, and antibiotic susceptibility. In order to

study the ability of S. typhimurium to provide spatiotemporal control of cytotoxic protein delivery, a radiation-

inducible expression system for secretion of TNF-related apoptosis-inducing ligand (TRAIL) was developed.

Methods: Prokaryotic-expression plasmids for TRAIL or green-fluorescent protein using the RecA promoter

were electroporated into the msbB- purI- strain, VNP20009. In a syngeneic model of mammary carcinoma using

BALB/c mice, the effect of systemic infection of bacterial vectors with or without induction by 2Gy gamma-

irradiation at two days after colonization was assessed, examining outcomes of tumor growth and thirty-day

survival.

Results: In vitro confirmation of extracellular TRAIL secretion and caspase-3 and caspase-8 activity were

verified, with increased apoptosis measured by annexin-V/propidium iodide flow cytometry (p<0.05). The

expression vector for TRAIL induced by radiation led to a significant delay in tumor growth and improved

thirty-day survival in vivo, with a hazard ratio of 0.24 (95% confidence interval, 0.08–0.75; p<0.05) in

comparison with irradiated controls. Repeated dosing and irradiation after one week limited tumor growth from

baseline, with a significant survival benefit from 0% to 100% at one month after initial treatment (p<0.05).

Conclusions: By capitalizing on the intrinsic motility of bacteria and their preferential accumulation within

tumors, the pre-clinical utility of targeted therapy using Salmonella typhimurium as a TRAIL expression vector

has been demonstrated as an effective method to reduce tumor growth and improve host survival.

12


Disease Modeling and Tissue Engineering: Breast Cancer Metastasis to Bone

Authors:

Michaela Reagan, Robert Goldstein, Michael Rosenblatt, David Kaplan

Presented by:

Michaela Reagan

Departments:

Department of Biomedical Engineering, Tufts University School of Engineering; Department of Physiology,


Abstract:

Osteotropism is a complex disease involving multiple causes and courses, with invasion and metastasis

comprising 90% of cancer deaths. According to US statistics, breast cancer, one of the most common cancers to

metastasize to bone, is the most frequently diagnosed cancer in women and the second most fatal, mainly due to

metastasis. Understanding the underlying biological reasons for skeletal breast cancer metastasis is imperative

for treatment and prevention. Our lab utilizes human tissue engineered (TE) bone, specifically designed with

cellular, mineral, and growth factor components, in a disease model with NOD/SCID mice. Our TE bone is

formed using porous, biocompatible, 3D silk fibroin scaffolds and human mesenchymal stem cells differentiated

into osteoblasts. This TE bone has proven to be valuable in modeling metastasis to bone in vivo using the breast

cancer cell line SUM1315. The model established species-specific metastasis from an orthotopic location to

human TE bone, exclusively, and not to the mouse skeleton. TE bone is well defined physically, chemically, and

biologically, and can also be analyzed with immunohistochemistry and RNA-extraction without decalcification.

Building on previous findings, current studies are focused on expanding the in vivo disease model from bone

tissue to bone marrow by using undifferentiated bone marrow-derived stem cells to establish a humanized

in vivo model of metastasis to human bone marrow. We also hypothesize that different maturational stages of

bone development cause different metastatic potentials. Hence, further studies are focused on characterizing TE

bone development in vitro and in vivo over time to determine effects of bone maturation and bone components

such as BMP2 on metastasis.

13


A Humanized Model of Breast Cancer Metastasis Revealing a Human-Specific Metastasis Gene Signature

Authors:

Robert Goldstein, Kristen Anderson, Michael Rosenblatt

Presented by:

Ellen Scepansky

Department:

Department of Physiology, Tufts University School of Medicine

Abstract:

The skeletal complications of malignancy represent some of the most serious complications associated with

cancer, signaling the entry of the disease into an incurable phase. Despite many recent advances in cancer

research and therapy, there remains a need for a better understanding of the metastatic spread of cancer from its

primary location to distant “soils” within the body. Animal models are critical to the understanding of the

complexities of tumor metastasis. Through creation of an animal model that is more representative of human

pathophysiology, it should be possible to elucidate the genetic alterations that are required for cancer migration

from orthotopic locations to distant sites. Further, clinical observations indicate that organ and stromal

environments greatly influence the response of tumors to chemotherapy, suggesting that orthotopic implantation

of tumor cells in animal models can recapitulate the process of cancer metastasis to bone. Currently, orthotopic

implantation of cancer cells is not widely used in animal modeling. Work in our lab has developed a humanized

orthotopic injection model of breast cancer metastasis. We use SUM1315 breast cancer cells injected into the

murine mammary fat pad. Subsequently, metastases to subcutaneously implanted human bone cores develop.

When compared to results obtained from an intracardiac injection model of cancer metastasis to murine bone,

results from our gene array studies and genetic profiling have illuminated a novel metastatic gene signature.

While different in specific gene identities (intracardiac signature=MMP1, IL-11, CTGF, and CXCR4; orthotopic

signature=MMP13, IL-17BR, and HUNK), the two signatures contain genes of overlapping function (i.e.,

homing, invasion, angiogenesis and osteolysis). Comparison of the expression levels of the specific gene

signatures across different breast cancer cell lines used in the intracardiac or the orthotopic injection models of

breast cancer metastasis using qRT-PCR, provides the basis for our hypothesis, suggesting that the humanized

animal model of breast cancer metastasis provides a novel, human-species specific, metastatic gene signature.

14


Role of Bone Sialoprotein (BSP) Overexpression in Osteolytic Metastasis in CMV-BSP Transgenic Mice

Authors:

Qisheng Tu, Jake (Jinkun) Chen, Jin Jang, Min Kim

Presented by:

Min Kim

Department:

Department of General Dentistry, Tufts University School of Dental Medicine

Abstract:

Objective: BSP is a protein normally found only in mineralizing tissues and is a major non-collagenous protein

in bone. More recent studies indicate a positive correlation between the expression level of BSP and metastasis

of tumor cells to bone. BSP is also a marker for tumor size, lymph-node status, and a poor prognosis for breast

cancer patients. The objective of this study was to determine the role of bone sialoprotein (BSP) overexpression

in osteolytic metastasis in our CMV-BSP transgenic mice.

Methods: We first generated transgenic mice, in which a mouse BSP is linked to a CMV promoter

(CMV-BSP). 4T1 breast cancer cells were cultured and were transplanted into CMV-BSP transgenic mice as

well as wild type littermates by intracardiac injection. At week 1, 2, 3 and 4 after the first set of injections, the

tumor metastasis level and the bone resorption level were analyzed. As the transplanted 4T1 breast cancer cells

express the luciferase gene, the transplanted cancer cells were easily detected and localized in the metastatic

sites by an optical (IVIS) imaging system which tracks the luciferase expressing cells. X-ray analysis was used

to measure the level of bone resorption. Four weeks after cancer cells transplantation, recipient animals were

sacrificed. Half of the bone tissues with cancer metastases were used for histological analysis and the remaining

half for RNA extraction. RT-PCR was preformed to determine the BSP and Luciferase expression levels in both

experimental and control mice.

Results: The IVIS Imaging System demonstrated that among the mice receiving intracardiac inoculation, all the

ten CMV-BSP mice developed metastases one week after injection while only four out of seven wild type mice

showed metastatic lesions. The results demonstrated that BSP overexpression in the whole body and a high

serum BSP level dramatically increase skeletal as well as systemic metastases of 4T1 murine breast cancer cells

which originally show a primary characteristic of bone-seeking metastases. Autopsy, gross examination and

histological analyses demonstrated that the CMV-BSP mice receiving 4T1 cell inoculation died soon due to

multiple metastatic lesions involving vital organs such as liver, lung, and kidney before more osteolytic lesions

were developed and could be detected.

Conclusions: 4T1 murine breast cancer cells caused more osteolytic metastasis in CMV-BSP transgenic mice

than in wild type mice.

15


Targeted Overexpression of BSP in Osteoclasts Promotes Bone Metastasis of Breast Cancer

Authors:

Qisheng Tu, Amanda Fix, Jean Tang, Jin Zhang, Jake (Jinkun) Chen

Presented by:

Qisheng Tu

Department:

Department of General Dentistry, Tufts Univeristy School of Dental Medicine

Abstract:

Bone is one of the most common sites of breast cancer metastasis. Recent studies have shown that almost 90%

of human breast tumors produce bone sialoprotein (BSP), a protein normally found only in mineralized tissues.

We have recently found that BSP and receptor activator of nuclear factor kB ligand (RANKL) synergistically

induce osteoclastogenesis and bone resorption and decrease apoptosis of osteoclasts. While BSP is thought to

play an important role in bone metastasis of malignant tumors, there is no evidence confirming that BSP is

directly responsible for osteolytic metastases.

The objective of this study is to determine the role of BSP overexpression in osteolytic metastasis in vivo by

using a homozygous transgenic mouse line that constitutively overexpressed mouse BSP cDNA driven by an

osteoclast specific cathepsin K (CtpsK) promoter.

We first generated the BSP overexpressing transgenic mice in which the BSP gene is driven by a promoter of

osteoclast specific gene cathepsin K. Southern hybridization and PCR confirmed the integration of CtpsK/BSP

chimeric gene into the mouse genome. An elevated level of expression of BSP in bone tissue and osteoclast

cells was detected. RANKL induced osteoclast differentiation from bone marrow-derived monocytes/

macrophages and bone resorption were significantly greater in CtpsK/BSP transgenic mice than wild-type mice

(P<0.05). Compared to wild-type mice, 6-week-old CtpsK/BSP mice had reduced trabecular bone volume

(BV/TV%), cortical thickness (Ct.Th), and bone mineral density (BMD). Using an in vivo bone metastasis

model and the real-time IVIS Imaging System, we found that 9 of 10 CtpsK/BSP mice that were injected with

4T1 murine breast cancer cells (1x105, labeled by luciferase reporter) developed large osteolytic bone lesions

3 weeks after injection. In contrast, only 4 of 9 wild type mice had lesions in their hindlimbs. The lesion area

was significantly larger in CtpsK/BSP mice than in the controls as determined by X-ray, gross and histological

analyses. It was found that targeted BSP overexpression in osteoclasts could activate the master regulator of

osteoclastogenesis nuclear factor of activated T cells (NFAT)-2 and increased the mRNA expression of other

differentiation markers such as cathepsin K or TRACP. We conclude that BSP is a mediator of osteolytic

metastasis of malignant tumors through the induction of osteoclast activity, which provides the molecular basis

of and treatment for bone destruction associated with osteolytic metastasis.

16


Treating Ductal Carcinoma in Situ (DCIS) of the Breast with Tamoxifen: A Decision Analysis of the Risks and

Benefits of Warfarin Anticoagulation in a Patient at Increased Risk for Thromboembolism

Authors:

Stewart Evans, John Wong, Stephen Pauker

Presented by:

Stewart Evans

Department:

Division of Clinical Decision Making, Informatics and Telemedicine, Tufts Medical Center

Abstract:

Aim: We performed a decision analysis to determine if a 57-year old female with a history of atrial fibrillation,

but without prior systemic emboli and DCIS, should receive lifelong anticoagulation and/or tamoxifen.

Methods: Using published data, we built a Markov model comparing four treatment strategies: combination

therapy (warfarin plus tamoxifen); warfarin alone; tamoxifen alone; and neither warfarin nor tamoxifen. We

considered risks of morbidity and mortality from major thromboembolism, major bleeding, and recurrent breast

cancer that might occur over a lifetime (lifetime time horizon). To account for morbidity, we applied published

quality of life measures.

Results: In the baseline analysis, when life expectancy alone was considered, treatment with warfarin alone

yielded 21.7 years, compared with 21.5 years with neither warfarin nor tamoxifen, 21.3 years with combination

therapy, and 19.6 years with tamoxifen alone. When quality of life was also considered, combination therapy

yielded 19.7 quality-adjusted life years (QALYs), compared with 19.6 QALYs with warfarin alone, 19.5

QALYs with no medication, and 18.3 QALYs with tamoxifen alone. All parameters were varied over plausible

ranges in sensitivity analyses. Combination therapy remained the preferred strategy over warfarin alone (other

strategies were inferior) unless: the annual probability of thromboembolism without tamoxifen was higher than

1.1% (baseline 1.0%), the annual probability of breast cancer recurrence was below 2.6% (baseline 3.0%), the

efficacy of warfarin was below 0.61 (baseline 0.65), the quality of life taking tamoxifen was below 0.99

(baseline 1.00), or the efficacy of tamoxifen was below 0.29 (baseline 0.32). Neither warfarin nor tamoxifen

became preferred over all other strategies if quality of life while taking warfarin fell below 0.98 (baseline 0.99).

Conclusions: This analysis was consistent with current guidelines for breast cancer risk reduction with

tamoxifen and reflects the complexity of the decision, particularly for this postmenopausal patient with an

increased risk of thromboembolism secondary to atrial fibrillation. Tamoxifen alone was clearly the least

preferred strategy; the choice among the other strategies was a relatively close call. Our analysis demonstrates

how patient preferences and carefully individualized calculation of risks and benefits might inform the decision-

making process.

17


Determining the Role of Immune System Function in Breast Cancer Using an Imagable Syngeneic Model

of Breast Cancer

Authors:

Min Fang, Kai Tao, G. Gary Sahagian

Presented by:

Min Fang

Departments:

Department of Physiology, Tufts University School of Medicine; Molecular Oncology Research Institute,

Tufts Medical Center

Abstract:

The 4T1 mouse mammary tumor model is one of only a handful of models that metastasize effectively when

introduced orthotopically in immuno-competent mice. Because of its propensity to metastasize to bone and other

sites in immuno-competent animals, the model is being used extensively for identifying drug targets and testing

therapeutics aimed at inhibiting processes related to tumor metastasis. For this study, the 4T1 cell line was

modified for stable expression of firefly luciferase in the absence of selective pressure and for targeted

integration of siRNAs and cDNAs with FLP recombinase. Luciferase expression allows quantitative analysis of

primary tumor growth and facilitates the detection and quantitation of metastases to lungs, liver and bone, as

well as to previously unreported organs including brain, kidney and adrenals. A longitudinal experiment

following mammary tumor growth and metastasis weekly after introduction of tumor cells into syngeneic

BALB/c mice, revealed biphasic growth at the primary site with rapid growth during the first two weeks,

regression in weeks 3 and 4, and renewed growth in weeks 5 and 6. A progressive increase in extramedullary

hematopoiesis in spleen and liver occurred during the 6-week period. Regression in weeks 3 and 4 was

associated with extensive inflammation and necrosis at the primary site. Increasing levels of neutrophils,

monocytes and lymphocytes were observed in the circulation during the experiment and many of these cell types

were recruited to the primary site. Growth at the primary site was substantially affected by the immune system

function, in that regression of growth observed in normal mice was not seen in immuno-compromised BALB/c

nude or SCID mice.

Metastases appeared during the second growth phase suggesting the involvement of innate and acquired immune

responses in metastasis for this model. Microarray analysis comparing the gene expression in the 4T1 cell line to

67NR and 168FARN, two non-metastatic sibling cell lines, was carried out to investigate the molecular basis for

the 4T1 metastatic phenotype. Pathway analysis of genes with significant expression differences indicated

activation of the p38 MAPK signaling resulting in production of hematopoietic factors Csf2 and Csf3, acute

18

Poster No. 18

phase proteins Ssa3 and C3, metalloproteinases Mmp3, Mmp9 and Mmp13 and cytokines Ccl5, Cxcl16, Cxcl1

and Tslp. Factors involved in angiogenesis, including Vegfc and Agpt2, were also significantly over-expressed

in 4T1 cells. Further analysis of the interactions of these genes, and the pathways and processes in which they

function, suggest that factors produced by this breast cancer model orchestrate the production and recruitment of

cells of the innate and acquired immune responses, which in turn regulate tumor growth and modify the

intratumoral environment to allow dissemination of tumor cells to distant sites. The results highlight the

importance of innate and acquired immune system function in tumor growth and metastasis, and emphasize the

need for using immuno-competent animals and species matched models for the study of tumor metastasis.

19


Molecular Basis for Intravasation and Metastasis to Lung Using an Orthotopic Mouse Model of Breast Cancer

Authors:

Min Fang, Kai Tao, G. Gary Sahagian

Presented by:

Min Fang

Departments:

Department of Physiology, Tufts University School of Medicine; Molecular Oncology Research Institute,


Abstract:

As an approach for identifying genes involved in intravasation and metastasis to lungs, variant luciferase-

expressing 4T1 tumor cells were isolated from lung metastases after orthotopic injection of the parental line into

the mammary gland of BALB/c mice. Pools of tumor cells isolated after one and two rounds of injection and

metastasis (4T1-L1 and 4T1-L1L1, respectively) and two cell lines cloned from the 4T1-L1 pool (4T1-L1-c7b

and 4T1-L1-c12b) were shown to metastasize more effectively to lungs after injection into the mammary gland.

Analysis of cell doubling times in normal and immuno-compromised BALB/c mice, intravasation in vivo, and

migration in vitro, indicated that the selected cells had increased ability to escape from the primary tumor and

evade immune system clearance mechanisms. Comparison of gene expression profiles for selected cell pools

and lines to the parental line revealed loss of tight junction components, increased expression of extracellular

proteases, and other alterations associated with EMT (epithelial-mesenchymal transition). Expression of

Sip1/ZEB2, a member of the δEF-1 family of two-handed zinc finger nuclear factors and a known effector of

EMT, was elevated in the selected cells suggesting a role for this transcription factor in the metastatic

phenotypes that were observed. Silencing and overexpression experiments and analysis of expression of

Sip1/ZEB2 in human breast tumors indicated Sip1/ZEB2 plays an important role in the induction of EMT and

the establishment of lung metastases in breast cancer.

20


Use of a Standardized Web-Based Tool for Evaluation of Bone Health in Breast Cancer Patients

Authors:

Konstantinos Arnaoutakis, Glenn Wong, Rekha Parameswaran

Presented by:

Konstantinos Arnaoutakis

Department:

Division of Hematology-Oncology, Caritas St. Elizabeth's Medical Center

Abstract:

Objectives: ASCO guidelines recommend aromatase inhibitors (AI) adjuvant therapy in post-menopausal

hormone receptor positive breast cancer patients. Aromatase inhibitors can cause significant bone loss over the

long-term and increase treatment related morbidity. This study measures a web-based tool use for osteoporosis

evaluation, using the American Board of Internal Medicine Practice Improvement Module (ABIM PIM) for

osteoporosis. Methods: We performed a retrospective chart review (2004-06) of Breast Cancer Patients (BCP) on adjuvant AI

without prior tamoxifen use; documentation of osteoporosis risk factors and processes of care were reviewed

using the ABOIM PIM for osteoporosis.

Results: Of 228 BCP identified from the hospital tumor registry, 28 (93% Caucasian, 7% race not documented)

were eligible for analysis; mean age was 64 years (range 42-90). Post-menopausal status was documented by

history in 26 (92%); by LH, FSH and estradiol levels in 2 (8%) BCP. All tumors expressed estrogen receptors;

26 (92%) expressed progesterone receptors. Chart review revealed documentation of tobacco use in 19 (71%);

exercise level in 12 (43%); fall screen in 1 (4%); vitamin D level in 0 (0%); appropriate vitamin D intake in

10 (36%) and calcium intake in 6 (21%) charts respectively. Bone density scan was done in 21 (75%) BCP. Of

17 (60%) BCP eligible for antiresorptive therapy; only 1 (6%) had documentation of prescribed therapy.

Conclusions: The ABIM PIM osteoporosis survey is a useful web-based tool in AI treated BCP. It identifies

patients at high risk of osteoporosis and reveals deficiencies in chart documentation. By identifying high-risk

patients in a timely fashion, management modifications can be made, potentially reducing osteoporosis related

complications. We conclude that use of this web-based tool in select patient populations enhances quality of

patient care and could potentially decrease treatment related morbidity.

21


Progenitor-Progeny Relationships of the Marker Defined Cell Subsets in the Human Prostate

Authors:

Andrew Makarovskiy, Peter Geck, Allen Parmelee, Albert Tai, Gennaro Carpinito

Presented by:

Andrew Makarovskiy

Departments:

Departments of Anatomy and Cellular Biology and of Pathology, Tufts University School of Medicine;

Department of Urology, Tufts Medical Center

Abstract:

There is a gap in understanding stem cell lineages in the prostate and our knowledge is also incomplete about

how stem cells become involved into malignant prostate tissue growth. Consequently, this limits our ability to

develop new therapy alternatives to target and eliminate prostate tumor initiating cells. A recent landmark study

showed that CD133+ prostate tumor cells can be clonogenic when injected into male mice. We used a panel of

new monoclonal antibodies (MAb) against differentially displayed rare surface antigens to further characterize

the stem cell populations of the prostate. The new MAbs define subsets of prostate epithelial cells both in fetal

and adult normal prostate, which persist in prostate carcinomas. MAb specificity to the cell subsets was also

retained in vitro. One of the new antibodies (SCS7) consistently labeled a rare population of cells residing in the

stroma of the developing and adult prostate. Analysis showed their immature morphology and phenotype and

indicated lack of proliferative activity. Sporadic intermediate phenotypes detected by multicolor fluorescent

microscopy using the new and conventional cell type specific markers provided antigenic links to the prostate

parenchyma and suggested SCS7+ cells are not a stromal cell subset. Importantly, quiescent in normal tissues,

SCS7+ cells, demonstrated mitotic activity following androgen deprivation, suggesting androgens might be

acting as a negative regulator of SCS7 proliferative activity and withdrawal of androgens removes the

proliferative blockade. A similar phenomenon was observed in vitro, following purification of the normal adult

SCS7+ cells which were able to produce a progeny with phenotype of the prostate basal cells, suggesting a

higher lineage position of SCS7+ cells. To establish a germane link between the CD133+ prostatic stem cells

and the SCS7+ population, we analyzed these cell subsets, using both normal and PC-3 carcinoma cells, by

FACS. Analysis indicated that either population was present at a <1% rate with a minor overlap suggestive of

transitional phenotype. To determine their progenitor-progeny relationship, we purified these subsets from

prostate carcinoma cell line PC-3 first. While the morphology and growth characteristics of the fractionated

subsets were similar, the progeny differed in phenotype. SCS7+/CD133- cells produced SCS7+/CD133-,

SCS7+/CD133+ and SCS7-/CD133+ subsets as in parental cultures. On the contrary, the CD133+/SCS7- subset

was unable to generate SCS7+ progeny suggesting restricted differentiation potential of the CD133+ stem cells.

We hypothesize that a network of resident stem cells that includes phenotypically and functionally different

stem cell types maintains the human prostate lineage. Our future studies will compare the differentiation

potential and androgen responsiveness of these cell subsets in vivo.

22


New Target Genes in Prostate and Breast Cancer: Methylation and MicroRNA Silencing of a Stem Cell

Differentiation Gene, APRIN, in Clinical Studies

Authors:

Monika Pilichowska, Viktoria Denes, Byung Kyu Kim, Andrew Makarovskiy, Gennaro Carpinito, Peter Geck

Presented by:

Peter Geck

Departments:

Departments of Pathology and of Urology, Tufts Medical Center; Department of Anatomy and Cellular Biology,


Abstract:

Focus Area: New targets

Introduction: Differentiation programs are disrupted early in prostate cancer. Disruptions of differentiation

genes, therefore, may be the earliest diagnostic markers of cancer. In our search for new target genes in cancer,

we searched for novel genes in differentiation that also play a role in cancer. We isolated a nuclear protein,

APRIN, and we found that APRIN was involved in stem cell differentiation. APRIN mutations in the germ line

were shown to generate extensive birth defects (Cornelia de Lange Syndrome), suggesting a critical regulatory

function for APRIN. We found that the APRIN gene was silenced in a variety of cancers, e.g., it was

downregulated or silenced in 70-80% of breast and ovarian carcinomas.

Objectives: The goals of this project were to investigate:

(i) if APRIN was also silenced in prostate cancer;

(ii) to identify the mechanisms involved; and

(iii) to investigate the correlation between APRIN silencing and cancer, as an early marker for prostate cancer

diagnosis or prognosis.

Methods: Clinical cancer samples were investigated for APRIN silencing at three levels:

(i) Protein expression: We used polyclonal anti-APRIN antibodies on formalin fixed, paraffin embedded cancer

samples (immunohistochemistry).

(ii) Promoter methylation analyses: Microdissected DNA samples were bisulfite converted. We used

methylation sequencing and methylation-specific quantitative PCR.

(iii) MicroRNA-based mechanisms: Total RNAs were prepared from microdissected paraffin sections (Trizol).

MicroRNA levels were detected by quantitative miRNA PCR.

Results:

(i) Immunohistochemistry showed high APRIN levels in normal prostatic epithelial cells. In prostate cancer, we

found APRIN silencing in 30-50% of the samples.

23

Poster No. 22

(ii) Promoter methylation studies detected methylation hot-spots in the APRIN promoter. Methylation assays by

Q-PCR indicated 20-80% methylation levels in the prostate carcinoma samples we studied.

(iii) APRIN-specific microRNAs were predicted by computer analysis (PicTar, MiRNA-Viewer, TargetScan,

MiRBase etc.). We identified hsa-miR-17-3p, 27a, 200 and 223 that may potentially target APRIN. In cell lines

where APRIN was silenced by miRNA-based mechanisms, miR-27a and 200 were highly increased. In the

control lines where APRIN was highly expressed, microRNA levels were low. In prostate cancer samples, our

pilot studies with miRNA Q-PCR showed that in cancers where APRIN was silenced, miR-17-3p, 27a and 200

were highly upregulated.

Conclusions: APRIN regulates differentiation programs in reproductive and other tissues. We showed that

oncogenesis in the prostate involves APRIN silencing through promoter methylation and microRNA

mechanisms. Our ultimate goal is to establish novel APRIN epigenetic markers for early diagnosis and

prognosis.

24


β1 Integrin Participates in Endoglin-Dependent Inhibition of Prostate Cancer Cell Migration

Authors:

Diana Romero, Jennifer Roth, Peter Brooks, Calvin Vary

Presented by:

Diana Romero and Calvin Vary

Department:


Abstract:

Endoglin is a transmembrane glycoprotein involved in the regulation of TGF-β signaling. PC3-M metastatic

prostate cancer cells have undetectable levels of endoglin, whereas their non-metastatic counterpart expresses

endoglin. When the expression of endoglin was restored in PC3-M cells, we observed a (cytoplasmic domain)

CD-dependent inhibition of cell migration and a reduction of the tumorigenicity of PC3-M cells in scid mice.

Using these cells, we determined that endoglin phosphorylation by the TGF-β receptors ALK2 and ALK5 is a

mechanism involved in TGF-β1-dependent regulation of cell migration.

Our current objective is to analyze the signaling pathways downstream of endoglin that lead to the inhibition of

prostate cancer cell migration and invasion. We have observed that endoglin expression has a dramatic effect in

the organization of the actin cytoskeleton in PC3-M cells. Interestingly, endoglin co-localized with the actin

fibers in focal adhesion sites. In addition, endoglin expression affected PC3-M cell adhesion on the extracellular

matrix proteins collagen types I and IV, suggesting that there is a functional proadhesive interaction between β1

integrin and endoglin. Further, elucidation of these interactions will provide a mechanistic explanation for

endoglin regulation of cell migration in a highly metastatic prostate cancer cell line.

25


PVR/Necl-5 Promotes Glioblastoma Invasion by Activation of the Akt Pathway and MMP-2 Production

Authors:

Brian Enloe and Daniel Jay

Presented by:

Brian Enloe

Department:

Department of Cellular and Molecular Physiology, Tufts University School of Medicine

Abstract:

Glioblastoma is a severe brain cancer, with medial survival of approximately one year after diagnosis, even

when treated aggressively with surgery, chemotherapy, and radiation. Treatment failure is due to tumor

recurrence, which is the result of the cancer cells ability to invade from the main tumor into the surrounding

areas of the brain. PVR/Necl-5 was identified by our lab as a protein involved in glioblastoma cell invasion

using a functional proteomic screen. PVR/Necl-5 is a transmembrane protein that is localized at the leading edge

of invading cells. Depletion of PVR/Necl-5 using RNAi leads to decreased invasion, and reduced secretion of

matrix metalloproteinase 2 (MMP-2). Glioblastoma severity in vivo correlates with MMP-2 expression, and this

protease is responsible for degrading the extracellular matrix. PVR/Necl-5 depletion also results in reduction in

activity of integrin-linked kinase (ILK) as measured by reduced Akt phosphorylation at serine 473. Depletion of

ILK leads to a nearly complete loss of MMP-2 production. Thus, we propose that PVR/Necl-5 links ILK and

Akt activation, resulting in increased expression of MMP-2.

26


Role of Histone H3K27 Demethylases in Maintenance of Multipotency in Glioblastoma Stem Cells

Authors:

Maureen Sherry, Andrew Reeves, Julian Wu, Brent Cochran

Presented by:

Maureen Sherry

Departments:

Department of Physiology, Tufts University School of Medicine; Department of Neurosurgery,


Abstract:

Signal transducer and activator of transcription-3 (STAT3) is an important mediator of growth factor and

cytokine responses. It is constitutively activated in a variety of human cancers. In addition, STAT3 is

indispensable for murine embryonic stem cell self-renewal, neural stem cell self-renewal, and astrocyte

differentiation. In order to determine whether STAT3 has a similar role in glioblastoma stem cells, we have

isolated several stem cell lines from primary human GBM tumor samples. We have found that inhibition of

STAT3 with small molecules as well as with RNAi causes growth inhibition in the GBM stem cells. Markers of

multipotency such as Olig2 and nestin are also decreased upon STAT3 inhibition. Strikingly, STAT3 inhibition

is irreversible. Cells exposed to the STAT3 small molecule inhibitor STA-21 for as little as 4 hours do not

regain the ability to form neurospheres while still remaining viable. This observation suggests that STAT3 may

regulate epigenetic changes in glioblastoma. It is clear that the multipotency of stem cells is maintained in part

by the polycomb repressor complex, which inactivates expression of differentiation-specific genes by

methylation of histone H3 on lysine 27. Histone modifying enzymes have recently been found that demethylate

this residue and lead to derepression of the polycomb regulated genes. One of these demethylases, Jmjd3,

appears to mediate neuronal differentiation in mice. We have found that inhibition of STAT3 leads to a 100 fold

increase in expression of the Jmjd3 demethylase mRNA. This induction is rapid (within 1 hour), suggesting that

Jmjd3 is directly regulated by STAT3. This induction is accompanied by an increase in the expression of

known Jmjd3 target genes, providing further evidence that Jmjd3 may be mediating epigenetic changes in

glioblastoma stem cells. The ability of STAT3 to modulate epigenetic changes via its regulation of Jmjd3 may

explain the irreversibility of STAT3 inhibition in GBM-SC. Thus, targeting STAT3 may provide an effective

and potentially irreversible means of depleting the cancer stem cell population in glioblastoma.

27


A Conditional RNAi Strategy to Investigate Signaling Networks in Glioblastoma Multiforme

Authors:

Jaime Acquaviva, Pranatartiharan Ramachandran, Steve Woolfenden, Al Charest

Presented by:

Jaime Acquaviva

Departments:

Department of Neurosurgery and Molecular Oncology Research Institute, Tufts Medical Center

Abstract:

Glioblastoma multiforme (GBM) is the most common and aggressive form of malignant astrocytic glioma.

These rapidly fatal intracranial tumors are characterized by a high rate of cell proliferation, diffuse infiltration,

and resistance to traditional and targeted therapy. Increased receptor tyrosine kinase (RTK) activity and

subsequent activation of various downstream signaling pathways is often associated with GBM and thought to

contribute to tumor initiation and maintenance. Here, we investigate the potential of an RNAi based system to

study important GBM signaling events. Amplification and/or mutation of the epidermal growth factor receptor

(EGFR) gene are common genetic alterations in GBM. An EGFR mutant allele with deletion of exons 2-7

(known as EGFRvIII) results in expression of a constitutively active variant of EGFR and is the most frequent

EGFR mutation found in GBM. The tumorigenic properties of EGFRvIII are well established making it an

attractive therapeutic target. We show conditional, lentiviral driven, shRNA mediated knockdown of EGFRvIII

in the human GBM cell line, GBM6. To apply this strategy in vivo, we modified a population of lentiviral-

shRNA infected GBM6 cells to express luciferase and injected them into the striatum of nude mice. Our results

show that intracranial tumors can be established using human GBM cells modified for conditional RNAi and

tumor progression can be monitored by in vivo bioluminescence imaging (BLI). In addition, conditional

knockdown of EGFRvIII is retained in primary tumor derived cultures. We are currently using this orthotopic

xenograft model to optimize conditions for effective shRNA mediated knockdown of EGFRvIII in vivo.

Achieving sustained EGFRvIII knockdown in vivo will lead to valuable insight regarding the role of EGFRvIII

in GBM and, importantly, validate the use of this technique to study other signaling components relevant to

GBM. This GBM model system will provide a means to assess the potential of RNAi based therapeutics in vivo

and enable us to establish functional links between signaling pathways and various aspects of GBM tumor

biology.

28


Somatostatin Therapy for Recurrent Meningiomas

Authors:

Abdullah Kandil, Jay Zhu, Carl Heilman, Julian Wu

Presented by:

Abdullah Kandil

Department:

Boston Institute of Neurosurgery, Tufts Medical Center

Abstract:

Introduction: Most meningiomas have been shown to overexpress somatostatin receptor 2, subtype 2A (sst2A)

and somatostatin has been found to inhibit meningioma growth in vitro. Recently, Chamberlain et al. have

demonstrated a response to Sandostatin, an extended release somatostatin analog, in patients with recurrent

meningiomas.

Methods: By using the same protocol as reported by Chamberlain et al., we enrolled patients with recurrent,

atypical or malignant meningiomas who had measurable tumors by neuro-imaging as well as by 111In-octreotide

SPECT scanning after previous surgeries and radiation therapies. Sandostatin was administered subcutaneously

every month. Most patients were followed with monthly blood counts and neurological examinations.

Responses were determined by contrast-enhancing neuro-imaging every 3 months. Toxicity is monitored and

graded according to the Common Toxicity Criteria (CTC) version 2.0.

Results: Over a 10 month period, 3 males and 3 females with a median age of 70.5 years (range from

34-87 years) were enrolled. Median time of follow-up was 9.5 months (range from 5-10 months). One patient

died 9 months later due to progressive disease. Radiographic evaluation showed no reduction of tumor sizes

among all 6 patients. Three patients had grade I reaction with fatigue and injection site skin erythema. No grade

2 to 4 toxicity was observed.

Conclusion: In our small series of 6 patients with recurrent meningiomas with a median follow-up of

9.5 months while on somatostatin analog therapy, there was no radiographic response by the tumor that was

detected in any patient. Although it is well tolerated, we conclude that somatostatin alone is not likely to halt

meningioma progression.

29


Clinical Impact of Adjuvant Gamma Knife Radiosurgery to the Surgical Cavity of Resected Metastases:

A Retrospective Review of the Tufts Medical Center Experience

Authors:

Steven Hwang, Rebecca Eisenberg, Andrew Hale, Tomas Dvorak, Abraham Boskovitz, Kevin Yao,

Rolf Pfannl, John Mignano, Jay Zhu, Gary Strauss, Julian Wu

Presented by:

Julian Wu

Departments:

Departments of Medicine, of Neurosurgery, of Neurology, of Pathology, and of Radiation Oncology,


Abstract:

Purpose: To help define the role of stereotactic radiosurgery (SRS) in treating brain metastases, we analyzed

the impact of Gamma Knife (GK) stereotactic radiosurgery to the tumor cavity following surgical resection of

brain metastases.

Patients and Methods: In this retrospective cohort study, 48 patients underwent surgical resection of at least

one brain metastasis between December 1999 and December 2005. Thirty-two received post-operative GK

treatment while the remaining did not. Both univariate and multivariate Cox proportional hazards regression

were utilized to model the influence of various prognostic factors on survival.

Results: The median survival of the entire group was 18.3 months. Patients treated with GK achieved a median

survival of 19.3 months as compared to 6.6 months for the untreated group (p=0.43). As expected, younger

patients (<50 years) had longer mean survival than older patients (32.3 versus 9.2 months). However, GK did

not appear to influence survival in the younger subset, but did have an impact on the older group (9.2 vs 18.6

months, p=0.019).

Conclusions: Our study suggests that the addition of GK to surgical resection of cerebral metastases may

contribute to improved survival in patients. This report suggests that adjuvant SRS to the resection cavity may

improve outcome in some of these patients. No decline in survival was seen in any subset, and the addition of

GK to surgical resection appears to be particularly beneficial among patients >50 years of age. We conclude

from our study that Gamma Knife radiosurgery to the surgical resection cavity may contribute to improved

survival without significant additional morbidity in patients with brain metastases.

30

Poster No. 28A Title:

Improvement of Neurological Function after Concurrent Treatment IVIG and Chemotherapy in a Patient with

Cerebellar Degeneration Paraneoplastic Syndrome

Authors:

Jay-Jiguang Zhu, XueMei Li, Thomas Hedges, Katie Wakeley

Presented by:

Jay-Jiguang Zhu

Departments: Departments of Neurology, of Ophthalmology, and Divisions of Hematology and Oncology,

and Gynecologic Oncology, Tufts Medical Center

Abstract:

Paraneoplastic syndrome is a rare manifestation of systemic cancers, often associated with lung or ovarian

malignancy. Paraneoplastic cerebellar degeneration (PCD) is one of the common presentations of paraneoplastic

syndrome. Clinically, patients present with cerebellar symptoms including dysarthria, gaze evoked nystagmus,

as well as, gait ataxia. Frequently, anti-Purkinje cell antibodies, (anti-Yo) are detected in the serum and

cerebrospinal fluid. There is no effective treatment for such conditions. Intravenous immunoglobulin (IVIG) is

one of the commonly used therapies, but with limited success.

We reported a 55 year old woman with ovarian cancer, who was diagnosed with paraneoplastic cerebellar

degeneration, with clinical manifestation of cerebellar syndrome and positive anti-Yo antibody (1:1500 dilution)

in CSF only and mild elevation of tumor markers for ovarian cancer, CA-125. She presented with one and a half

months history of sub-acute onset of dysarthria, gait ataxia and intermittent blurry vision with upright position.

Exam showed downbeat nystagmus and alternating skew deviation, dysmetria, as well as gait ataxia.

Incidentally, her symptoms significantly improved 1 week after a routine influenza vaccine injection. However,

it did not last long and the same symptoms recurred 2-3 weeks later. Magnetic resonance imaging (MRI) head

with and without contrast, at the time of diagnosis, did not reveal cerebellar atrophy. She received IVIG at 0.4

gm/kg/day x 5 days every 4 weeks with concurrent chemotherapy with intravenous doxorubicin (Doxil) and

cyclophosphamide (Cytoxan) monthly. Three months after such treatment, her speech and dysmetria have

markedly improved. She continued to receive the treatment monthly for a total of 12 cycles. Updated outcome

of this patient and review of current treatment options of paraneoplastic syndromes will be reported at the

meeting.

31


Larger Lentigo Maligna Lesions and Invasive Melanoma: Size Matters

Authors:

Priya Zeikus, Jyotsna Kakullavarrupu, Suzanne Olbricht

Presented by:

Suzanne Olbricht

Departments:

Departments of Dermatology and of Research, Lahey Clinic; Department of Dermatology, University of Texas

Southwestern Medical Center

Abstract:

Background: Lentigo maligna is traditionally recognized as a slow growing lesion that undergoes malignant

transformation to invasive melanoma. Treatment regimens for lentigo maligna vary widely from watchful

waiting to surgical excision via Mohs technique as it can not be predicted which lentigo maligna lesions develop

invasive melanoma.

Objective: 1) To examine the cases of lentigo maligna that were found to be invasive after surgery was

performed, and 2) To determine whether specific clinical features exist that can predict invasive potential.

Methods and Materials: Eighty-nine cases of lentigo maligna that underwent staged excision were

retrospectively reviewed. Clinical features and treatment details were recorded. Pathological reports were

reviewed for the presence of invasion. Statistical analysis was performed with both Fisher T and Pearson Chi

square tests.

Conclusion: There were 13 (14.6%) lesions that were found to have invasive melanoma only after subsequent

slow Mohs excision. A strong correlation was observed between large lesion size and invasive potential. Nine of

18 (50%) of lesions greater than 4 cm2 area were invasive compared to 4 of 71 (5.6%) less than 4 cm2

(p<0.0001). There was no correlation of invasion based on gender, age, number of Mohs stages, length of time

between biopsy and excision, or previous treatment rendered. Superficial therapies for larger lentigo maligna

lesions are absolutely contraindicated and must be used with extreme care for smaller lesions which still have a

significant albeit smaller risk for invasive melanoma.

32


Mapping Genes Associated with Spontaneous Canine Hemangiosarcoma and Expression Profiling, Potential

Animal Model for Human Angiosarcoma

Authors:

Chieko Azuma, Kerstin Lindblad-Toh, Elinor Karlsson, Noriko Tonomura, Lisa Barber, Kristine Burgess,

Scott Shaw, John Keating, Dawn Meola, Jun Xu

Presented by:

Chieko Azuma

Departments:

Departments of Clinical Sciences and of Biomedical Sciences, Cummings School of Veterinary Medicine

Abstract:

Spontaneous canine hemangiosarcoma (HSA), a malignant tumor of vascular endothelial cells, is a significant

health concern in dogs. A particularly high disease incidence in Golden Retriever (15%) and certain other breeds

suggests a genetic susceptibility and adds considerably to the power of mapping the genetic risk factor. HSA

metastasizes rapidly through a hematogenous route or local seeding after tumor rupture. The median survival

time with surgical removal of a splenic HSA with or without chemotherapy is 2-5 months. We have proposed to

map genes associated with an increased risk of HSA in Golden Retrievers, as well as other, breeds in

collaboration with the Broad Institute of Massachusetts Institute of Technology and Harvard University.

Artificial forces for the breed creation in dogs provide a unique opportunity to study genetics for complex

diseases and the application for use in human diseases. Characteristics of canine genetic structures include

longer Linkage Disequilibrium (LD) and smaller variation of haplotypes within the breeds compared to general

human population resulting in a higher chance to identify genes associated with complex diseases in dogs with

smaller number of subjects.

Genome-wide association mapping with ~50,000 SNPs (Affymetrix canine SNP array) in 100 Golden Retrievers

with HSA and 102 normal Golden Retrievers has identified 7 preliminary loci. Six loci have been validated by

fine mapped in 5 breeds including Golden Retrievers, Labrador Retrievers, German Shepherds, Leonbergers and

Boxers. All loci are found in at least one or more breeds and are discrete in size (<600kb). So far no coding

mutations have been identified. We are also investigating their functional consequences by an expression profile

of the canine HSA and to test if the mutations alter the expression of any of possible candidate genes and related

pathways. Once the mutations have been identified and their presence in different breeds assessed, this will

allow for rapid development of genetic tests for carriers of HSA. Furthermore, canine HSA resembles human

angiosarcoma and may serve as an excellent model for this and other cancers with a high metastatic potential.

Ultimately, understanding of the disease biology will lead to identify target genes for prevention, early detection

and novel therapeutics in dogs and humans with cancer.

33


YKL-40 Demonstrates a Protective Role in Rhabdomyosarcoma by Decreasing MMP-2

Authors:

Steve Scully, Wie Yan, Rong Shao

Presented by:

Steve Scully

Department:


Abstract:

YKL-40 is a secreted glycoprotein, which has gained much attention as a sensitive and specific biomarker for

several inflammatory diseases and as a prognosticator in thirteen different carcinomas. It is a chitinase like

protein without chitinase activity and is phylogenetically conserved among animals, especially mammals. It is

present and secreted in numerous cell types and tissues during development and is present in the most

metabolically active cells in adult tissues under normal physiological conditions. Despite all of its recognition,

little is known about the functions of YKL-40 in human development, normal physiology and disease. Some

data have shown that YKL-40 plays a role in tissue remodeling. Since the case for YKL-40 as a biomarker has

been made in multiple carcinomas, we decided to explore a similar role in sarcomas. We chose to look at

Rhabdomyosarcoma since it is the most common soft tissue sarcoma in children and adolescents and results in

high morbidity and mortality.

Our pilot data showed that YKL-40 was not expressed among various types of Rhabdomyosarcomas in vitro.

Therefore, we decided to examine what role, if any, YKL-40 could play if it was overexpressed in these cancers

in vitro. These studies demonstrated that YKL-40 decreased invasion, survival and proliferation. YKL-40

overexpression also reduced the levels of Matrix Metalloproteinase-2 and N-Cadherin in the RD cell line.

Previous evidence has shown that Rhabdomyosarcomas are dependent on Matrix Metalloproteinases (MMPs)

for their invasive and metastatic capabilities. In particular, MMP-2, MMP-9 and collagenase MMP-1

overexpression seem to contribute to the more aggressive phenotype of Rhabdomyosarcoma. Thus, further

understanding of YKL-40 and its role as an inhibitor of MMPs in Rhabdomyosarcoma might lead to the

development of novel anticancer therapies.

34


MicroRNA Expression Profiling: Connecting Lung Development and Lung Cancer

Authors:

Sana Mujahid, MaryAnn Volpe, Heber Nielsen

Presented by:

Sana Mujahid

Departments:

Department of Cell, Molecular and Developmental Biology, Tufts University School of Medicine;

Department of Pediatrics, Tufts Medical Center

Abstract:

MicroRNAs are a class of small RNAs that regulate gene expression by imperfectly pairing with the 3’UTR

sequence of their target mRNAs, resulting in translational repression. Previous studies report a correlation

between those genes up regulated during lung cancer and those involved in controlling normal fetal lung

development. Our lab has been studying mechanisms necessary for normal fetal lung development. We recently

have begun to study how miRNA mechanisms contribute to the regulated expression of genes involved in lung

development. We have begun by using a miRNA qRT-PCR based screen of fetal mouse lungs during the critical

period of development from a pseudoglandular to a saccular lung in preparation for survival at birth. One goal of

this study is to find specific miRNA species which actively regulate gene expression for normal development

and which also become reactivated during the progression of lung cancer.

35


Fibroblasts Affect the Phenotype of Normal Human Bronchial Epithelial Cells when Co-Cultured in

Three-Dimensional (3D) Organotypic Cultures

Authors:

Steven Pageau, Maricel Maffini, Ana Soto, Carlos Sonnenschein

Presented by:

Steven Pageau

Department:

Department of Anatomy and Cell Biology, Tufts University School of Medicine

Abstract:

The stromal microenvironment plays an important role in the development and progression of adult respiratory

diseases. Pulmonary diseases such as asthma, fibrosis, and cancer are thought to be the result of altered

communications between epithelial and mesenchymal cells. In order to study epithelial and mesenchymal

interactions in vitro, we have developed a three dimensional (3D) organotypic model of the human peripheral

lung. This model consists of a type I collagen gel, normal human fetal lung fibroblasts (IMR-90), or primary

human adult lung cancer-associated fibroblasts (LuCAFs), derived from lung cancer resective surgery and a

surface epithelium of normal human bronchial epithelial cells (NHBECs). Our studies revealed that collagen

gels lacking fibroblasts failed to promote the differentiation of a typical peripheral respiratory epithelium.

Collagen gels containing NHBECs and IMR-90 fibroblasts at a ratio of 10:1 supported the development of an

extensive surface respiratory epithelium containing goblet, basal, and ciliated epithelial cells. Gels containing

LuCAFs supported the invasion of NHBECs into the 3D constructs and contracted the collagen gels 2-fold more

than gels containing IMR-90 fibroblasts. Masson’s trichrome staining indicated collagen-rich fibrotic lesions in

the lung tissue from which the primary LuCAFs were derived. Indirect immunofluorescence staining revealed

that LuCAFs express alpha-smooth muscle actin (α-SMA), whereas IMR-90 fibroblasts did not express this

marker. From these results and observations, we conclude that: 1) Normal fetal fibroblasts support the formation

of a well differentiated surface respiratory epithelium in 3D organotypic cultures; and 2) LuCAFs inhibit the

differentiation of respiratory epithelial cells and promote their invasion into the 3D collagen gels. A plausible

explanation for this invasive behavior is that when compared to IMR-90 fibroblasts, LuCAFs differentially

remodel their extracellular matrix and generate an altered stromal microenvironment that provides unique

biophysical cues to the overlying epithelial cells, thereby, altering their phenotype.

36


Laser Capture Microdissection to Isolate Primary and Metastatic Thyroid Tumors from Formalin Fixed

Paraffin Embedded Tissue

Authors:

Caroline Kim and Sheue-yann Cheng

Presented by:

Caroline Kim

Departments:

Division of Endocrinology and Molecular Oncology Research Institute, Tufts Medical Center

Abstract:

Identifying the determinants of thyroid cancer metastasis is often limited by tissue availability. Studies utilizing

a mouse model of follicular thyroid cancer (TRßPV mouse) which spontaneously develops thyroid cancer that

recapitulates human follicular thyroid cancer behavior, including lung metastases, have identified candidate

genes central for thyroid cancer tumorigenesis. Previous microarray analysis on TRßPV mice has not included

lung metastatic tumors1. Using archived formalin fixed paraffin embedded tissue blocks, we are isolating wild-

type, primary thyroid tumors, and metastatic thyroid cancer to lung for eventual microarray analysis.

Laser capture microdissection (LCM) of wild-type thyroids, primary thyroid tumors at sites of capsular or

vascular invasion, and lung metastases was performed. RNA integrity for each tissue block was assessed by

comparing the 3’/5’ ratio of the housekeeping gene, ß-actin prior to LCM. As proof-of-principle, quantitative

real-time PCR was performed examining the expression level of a known upregulated gene in the thyroids of

TRßPV mice, PTTG1. Similar to previous reports, PTTG mRNA isolated from laser captured thyroid cancer of

TRßPV mice was expressed ~5-fold higher compared to the mRNA from age-matched wild-type thyroids.

These findings suggest that LCM of these archived tissue blocks is a viable approach to obtain RNA for

microarray analysis. Future studies will compare and contrast the gene expression profiles of primary versus

metastatic thyroid tumors that may identify novel contributors to thyroid cancer metastasis.

(1) Ying H., Suzuki H., Furumoto H., Walker R., Meltzer P., Willingham MC., Cheng SY. 2003 Alterations in genomic profiles during tumor progression in a mouse model of follicular thyroid carcinoma. Carcinogenesis 24 (9):1467-79.

37


Mrc1 and Tof1 Participate in the Maintenance of CAG•CTG Repeat Tract Integrity

Authors:

Lionel Gellon, Mayurika Lahiri, AnnaLena La Porte, Catherine Freudenreich

Presented by:

Lionel Gellon

Department:

Department of Biology, Tufts University School of Arts and Sciences

Abstract:

Expansion of CAG•CTG repeats is the cause of a number of neurodegenerative diseases. In addition, expanded

CAG•CTG repeats have been shown to be fragile sites on a yeast chromosome. Previously, we showed that

proteins involved in the S-phase replication checkpoint, which senses stalled forks and induces a checkpoint

response, were important for preventing fragility of expanded CAG•CTG repeats (Lahiri et. al, 2004;

Freudenreich and Lahiri, 2004). In this study, we have tested the effect of the S. cerevisiae Tof1 and Mrc1

proteins, which are known to play a direct role in stabilizing stalled replication forks, on repeat stability. Using a

yeast artificial chromosome containing either a CAG•CTG-85 or a CAG•CTG-140 repeat tract or a no repeat

control, we show that deletion of either the MRC1 or TOF1 gene resulted in increased repeat tract fragility. In

addition, the repeat tract is destabilized in the absence of either protein, resulting in a high level of contractions

as well as expansions. Interestingly, complete deletion of MRC1 resulted in a greater rate of fragility as well as

frequency of repeat instability compared to checkpoint deficient alleles of MRC1, indicating that the role of

Mrc1 in replication fork progression is as important in maintaining CAG•CTG repeats as its checkpoint

signaling role. The role of Tof1p appears to be especially crucial at the CAG•CTG-140 tract as repeat fragility

and instability was much more pronounced at this longer repeat compared to CAG•CTG-85. Cell growth

monitoring showed a significant increase in growth retardation for the ∆tof1 strain containing the CAG•CTG-

140 tract, and an increase in arrests/swelling for the strains deleted for MRC1. This result corroborates the

phenotypes observed in the fragility and instability assays. Thus, these data show that expanded CAG•CTG

repeats slow replication fork progression in a eukaryotic system, and that proteins involved in fork restart are

very important in maintenance of chromosomal stability at these structure-forming sequences.

38


The Importance of Spatial Distribution of Stemness and Proliferation State in Determining

Tumor Radio-Response

Authors:

Heiko Enderling, Derek Park, Lynn Hlatky, Philip Hahnfeldt

Presented by:

Heiko Enderling

Department:

Center of Cancer Systems Biology, Caritas St. Elizabeth’s Medical Center

Abstract:

Tumor growth and progression is a complex phenomenon dependent on the interaction of multiple intrinsic and

extrinsic factors. Necessary for tumor development is a small sub-population of potent cells, so-called cancer

stem cells, that live forever, can undergo an unlimited number of cell divisions, and with a small probability

divide symmetrically to produce more such cancer stem cells. The majority of a tumor is made up of progeny

cancer cells that have a limited life span and a limited replicative potential. Tumor development is dependent on

the cancer cell's proliferative behavior, their migratory ability and cell death occurrences. With increasing

number of cells in the tumor, competition for space limits tumor progression and the majority of cancer cells

becomes quiescent, with proliferation primarily occurring on the outer rim where space is available. We present

an agent-based model early tumor development that captures the spatial heterogeneity of proliferating and

quiescent cells. We combine the model with the established linear-quadratic model of radiotherapy to predict

treatment outcomes for different quiescence radiosensitivities. We show that considering homogeneous

radiosensitivity throughout the tumor populations will lead to different results compared to a probable reduced

sensitivity of quiescent cells.

Beyond just considering mass effects of stem to non-stem cell ratios and proliferating to quiescent cell ratios, we

show that the spatiotemporal evolution of the developing heterogeneous population plays a pivotal role in

determining radioresponse and treatment optimization.

39


The Yin and Yang of ERK Regulation by Polyomavirus Middle T (MT)

Authors:

Yanni Zhu and Brian Schaffhausen

Presented by:

Yanni Zhu

Department:

Department of Biochemistry, Tufts University School of Medicine

Abstract:

Polyomavirus is noted for its ability to cause tumors in a wide range of tissues. Middle T (MT) is the principal

transforming protein of polyomavirus. It is required for viral transformation, and is sufficient to cause tumors

when expressed as a transgene. Over the years, MT has provided important insights into mammalian growth

control. The importance of tyrosine phosphorylation and phosphoinositide 3-kinase (PI3K) activity were first

recognized there.

Our work emphasizes that the effect of MT on cellular signaling of the Ras pathway is conditional.

Phosphorylation of MT of Y250 is known to activate the adaptor SHC, that leads to the activation of Ras. This is

expected to lead to the activation of Ras effectors including Raf. In turn, this is expected to lead to the activation

of ERK1 and ERK2. As expected, MT can activate ERK in MT-transformed cells.

ERK is also strongly activated by stress conditions such as serum starvation, UV radiation, or drugs, e.g.,

thapsigargin, which triggers the unfolded protein response (also called ER-stress). This can be readily shown

with phosphospecific antibodies. In sharp contrast to normal conditions, MT strongly suppresses ERK activation

resulting from stress. Examination of other aspects of the UV and unfolded protein response stresses that the MT

targets ERK without affecting other aspects of the cellular response.

To better understand how MT suppresses the activation of ERK induced by stress. The canonical ERK

activation pathway [MT (Growth Factor)→Adaptor(Shc/Grb2)→SOS→Ras→Raf→MEK→ERK] has been

examined and indicated that Ras is activated by MT, whether stress is occurring or not, while MEK activation in

response to stress is suppressed. The lost connection between Ras and MEK suggests the regulation of Raf could

be the switch to turn off ERK activation by MT. Interestingly, MT315 (PI3K-) is defective on blocking ERK

phosphorylation. Inhibition of PI3 kinase by LY294002 partially mimics MT315. Together, this suggests that

PI3K or its downstream targets such as AKT is involved in negative regulation of ERK activation by MT under

stress. This is an indication of cross-talk between the Ras and PI3-kinase pathways.

Why would a viral protein wish to suppress a stress response? Different kinds of stress (viral infection

generating dsRNA, starvation, or as here ER-stress) induce eIF2α phosphorylation to shut down cell protein

synthesis. MT also appears to reduce eIF2α phosphorylation under stresses, which would stimulate translation.

40


Solution Structure of the Hdlg/SAP97 PDZ2 Domain and its Mechanism for Interaction with

HPV-18 E6 Protein

Authors:

Yuqi Liu, Gillian Henry, Rashmi Hegde, James Baleja

Presented by:

Yuqi Liu

Departments:

Department of Biochemistry, Tufts University School of Medicine; Division of Developmental Biology,

Cincinnati Children's Hospital Medical Center

Abstract:

High-risk Human Papillomavirus (HPV) E6 proteins, in association with E6AP, can target cellular proteins for

degradation, and contribute to the development of cancer. They can bind PDZ domain-containing proteins,

including hDlg for proteasome-mediated degradation through their C-terminal PDZ-binding motifs. Our

hypothesis is that the complex of E6 and hDlg shows a unique molecular surface that can be used for the

assembly with other proteins, and provides a basis for understanding the mechanism of E6-promoted

degradation.

The second PDZ domain (PDZ2) from hDlg and a series of short synthesized peptides from C-terminal high-risk

HPV E6 were used to mimic the interaction of hDlg and E6. Isothermal titration calorimetry was used to

determine the binding affinities. The solution structure of PDZ2 in complex with HPV-18 E6 peptide was

determined by NMR with backbone root-mean-square deviations of less than 0.5Å. The structure shows a

typical PDZ architecture containing six β-strands and two α-helices. The C-terminal six residues of E6 form a

short β-strand that binds in a groove formed by αB and βB and anti-parallel to βB strand. However, a novel

mode of interaction was found in which six residues of the HPV-18 E6 peptide are contacted by the PDZ2

domain, in contrast to the typical four residues used by PDZ domains.

Molecular dynamics simulations support a model in which the C- and N-terminal ends of the peptide have

different mobilities within the complex. Comparison of the NMR complex structure to previously determined

X-ray structures of PDZ2 by itself and bound to different peptides allows a description of conformational

changes required for PDZ2 to bind to HPV-18 E6. The binding of E6 peptide with loop 2 of PDZ2 induces

additional contacts between loop 2 and the αB helix, pulls the C-terminus of αB inside and makes the PDZ2

domain more compact.

41


Scaffold Proteins Regulate Localized Rac Activation by Tiam1

Authors:

Soumitra Rajagopal, Yuhuan Li, Kathleen Wicks, Ka-Wing Wong, Ralph Isberg, Rachel Buchsbaum

Presented by:

Soumitra Rajagopal

Departments:

Molecular Oncology Research Institute and Department of Medicine, Tufts Medical Center; Department of

Molecular Biology and Microbiology, Tufts University School of Medicine; Department of Microbiology and

Immunology, Albert Einstein College of Medicine; Boston Medical Center; Oregon State University

Abstract:

Tiam1 is a widely expressed exchange factor for the Rac GTPase with effects on multiple cell functions

including growth, apoptosis, polarity, and motility. We have previously found that Tiam1 interaction with

different scaffold proteins leads to activation of different specific pathways downstream of Rac due to

recruitment of specific Rac effector proteins to Tiam1-scaffold complexes. Tiam1 is known to interact with a

number of proteins and many of these interactions are mediated through the same N-terminal region of the

protein containing a pleckstrin homology (PH) domain immediately upstream of a coiled-coil (CC) domain.

This raises the question of how signaling specificity is achieved. We reasoned that there must be regulatory

mechanisms governing the specific conditions under which each interaction occurs. We hypothesized that

Tiam1-interaction with one scaffold complex might lead to Tiam1-mediated Rac activation in response to one

upstream signal, while interaction with another scaffold complex would lead to Tiam1-mediated activation in

response to another upstream signal. Fibroblasts expressed at least two Tiam1-interacting scaffold proteins,

IRSp53 and spinophilin. We used fluorescent resonance energy transfer (FRET) to measure localized Rac

activation in association with IRSp53 and spinophilin complexes in individual fibroblasts as a way to test this

hypothesis. We found that treatment with pervanadate or PDGF induced localized Rac activation was dependent

on both Tiam1 and IRSp53, but not spinophilin. Treatment with forskolin or epinephrine induced localized Rac

activation was dependent on both Tiam1 and spinophilin, but not IRSp53. In contrast to the localized Rac

activation seen using the FRET assay, overall levels of activated Rac in cell lysates did not change with any

treatment. In addition, downstream effects of Rac activation were stimulus and scaffold-specific. Pervanadate-

induced ruffling and membrane translocation of Tiam1 were dependent on IRSp53, but not spinophilin.

IRSp53, but not spinophilin, was required to maintain Tiam1-dependent cell adhesion. Epinephrine stimulation

led to decreased IRSp53-dependent adhesion in a Rac and spinophilin-dependent fashion. Taken together, these

results support the idea that interactions of Tiam1 with multiple different scaffold proteins couple the

transmission of distinct upstream signals to localized Rac activation, with subsequent activation of specific

downstream pathways. In addition, our findings suggest that manipulating the interactions of exchange factors

such as Tiam1 with scaffold protein complexes can modulate cellular behaviors involving Rac activation.

42


Sequence-Specific Chromatin Remodeling of the C-Myc Promoter by hSWI/SNF

Authors:

Hillel Sims, Cassandra Baughman, Jacqueline Lane, Gavin Schnitzler

Presented by:

Gavin Schnitzler

Departments:

Departments of Biochemistry and of Anatomy and Cellular Biology, Tufts University School of Medicine

Abstract:

Increased transcription of the protooncogene c-myc is associated with more than half of all human tumors. One

factor that is important for c-myc transcription is the ATP-dependent chromatin remodeling complex, human

SWI/SNF (hSWI/SNF). This suggests that chromatin changes introduced by hSWI/SNF will activate

transcription by changing the accessibility of the c-myc promoter to transcription factors. The precise nature of

these chromatin changes and the role of c-myc promoter sequence in directing them, however, is unknown.

Here, we examine the effects of hSWI/SNF on c-myc promoter chromatin templates. Our results show that,

before remodeling, two favored nucleosome positions exist on c-myc. These positions, which are also observed

on the inactive promoter in vivo, block access to many important transcription factor binding sites. Strikingly, in

a single nucleosome system, hSWI/SNF responds to c-myc promoter sequence to quantitatively move

nucleosomes away from these default repressive positions. In a multinucleosome system, hSWI/SNF moves

these nucleosomes in a somewhat different way, pushing them together to form normal or structurally-altered

dinucleosomes. Overall, our results indicate that hSWI/SNF alters chromatin in a promoter-specific manner, and

provide new insights into the mechanism of c-myc transcriptional regulation.

43


Inhibition of CBF-1-Mediated Notch Signaling Induces FGF-Dependent Transformed Cell Phenotype Authors:

Doreen Kacer, Christian McIntire, Alexander Kirov, Igor Prudovsky

Presented by:

Igor Prudovsky

Department:


Abstract:

Notch signaling is an evolutionarily conserved regulatory pathway, which is involved in cell proliferation,

differentiation and cell survival decisions. Once the Notch receptor is bound by the ligand, the intracellular

domain is detached by γ-secretase and is translocated to the nucleus where it acts as a transcriptional regulator of

the CBF-1-dependent Notch signaling cascade. Our laboratory previously demonstrated that soluble forms of

Notch ligands, such as soluble Jagged 1, induce the expression and stress-independent non-classical release of

FGF1 (D. Small et al, J. Biol. Chem, 2003, v. 278, pp. 16405-16413). This effect correlated with the inhibition

of Notch signaling. We suggested that downregulation of Notch signaling in damaged tissues could stimulate the

expression and release of FGF1, which is required for tissue repair. However, we did not have direct proof of the

hypothesis that the inhibition of Notch signaling induces FGF1 transcription and export. The aims of the present

work are: 1) To verify the hypothesis about the role of Notch signaling in FGF1 release and expression using

the dominant negative (dn) form of CBF-1, a key transcription factor of the Notch signaling cascade; and 2) To

explore the cell phenotype induced by the inhibition of the CBF-1-dependent Notch signaling.

NIH 3T3 cells were stably transfected with dnCBF-1 and examined for the following characteristics: (i) FGF1

expression (using RT-PCR); (ii) FGF1 release (using adenoviral transduction of FGF1 followed by heparin

chromatography of conditioned media, SDS-PAGE, and Western blotting); (iii) growth rate; (iv) ability to grow

in soft agar; and (v) tumor formation in nude mice. DnCBF-1 expression resulted in the appearance of a

detectable level of the FGF1 transcript. Unlike control cells, dnCBF-1 transfectants released FGF1 at non-stress

conditions. dnCBF-1 transfectants exhibited a loss of growth contact inhibition, anchorage independent growth

in soft agar, and rapid formation of highly angiogenic tumors in nude mice. Significantly, the inhibition of

FGFR signaling and of the function of S100A13, a key component of FGF1 export pathway, resulted in a drastic

inhibition of anchorage independent growth of dnCBF-1 cells and restored growth contact inhibition.

Interestingly, DAPT, inhibitor of g-secretase and Notch signaling considered for the treatment of Alzheimer

disease, strongly enhanced the growth of NIH 3T3 cells, and this effect was FGFR-dependent. We suggest that

the export of FGF1 induced as a result of Notch signaling inhibition contributes to the development of a

transformed cell phenotype and to enhanced angiogenesis in tumors.

44


Investigating Cancer Progression of Cells in 3D Matrix with Non-Invasive Fluorescent Imaging

Authors:

Joanna Xylas, Addy Alt-Holland, Jonathan Garlick, Irene Georgakoudi

Presented by:

Joanna Xylas

Departments:

Department of Biomedical Engineering, Tufts University School of Engineering; Departments of Endodontics

and of Oral and Maxillofacial Pathology, Tufts University School of Dental Medicine

Abstract:

Epithelial cell adhesion to adjacent cells and matrix is crucial to invasive potential and influences cell

organization and behavior. It is unclear what triggers cancer cells to lose these abilities and the relation to

metastasis onset. Since most cancers can be cured if detected before they attain strong invasive capacity,

understanding and detecting the changes that occur in the premalignant-to-malignant transformation would

significantly advance the medical field.

Our study aims to investigate how genetic makeup and cell local environment influence metastatic behavior

with two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). By exploiting natural

non-linear fluorescence and scattering properties of cell and matrix, these techniques have potential to

non-invasively characterize tissues as well as develop a method to directly assess these optical biomarkers of

cancer in real time.

We created a model consisting of collagen type-I, human fibroblasts, and ras-transformed human epidermal

keratinocytes (HEKs) or ras-transformed, E-cadherin-deficient HEKs as benign or malignant cell equivalents,

respectively. Our data indicate that upon E-cadherin suppression and loss of cell-cell contact, E-cadherin-

deficient tumor cells migrate into surrounding matrix as single cells. E-cadherin-competent cells are organized

in rounded, compact clusters that expand evenly in all directions. However, upon incorporation of fibroblasts

into the collagen gels, we observe significant enhancements in the degree E-cadherin-competent cell migration,

suggesting that environmental factors also play a significant role in cell migration and potentially metastasis.

Fourier-based image analysis approaches indicate that an inverse power law dependence characterizes the power

spectral density (PSD) of the intensity fluctuations of the TPEF images over a range of spatial frequencies,

indicating differences in the benign and malignant cell features on the micrometer scale.

We expect these optical biomarkers to provide a foundation for non-invasive clinical evaluation of the malignant

potential of precancerous cells.

45


Cancer Cells Modulate DNA DSB/Repair in Nontransformed Cells

Authors:

Afshin Beheshti, Heiko Enderling, Matthew Perkins, Aaron Burg, Philip Hahnfeldt, Lynn Hlatky

Presented by:

Afshin Beheshti

Department:


Abstract:

Gamma-H2AX (phosphorylated H2AX from the core histone H2AX) and 53BP1 (p53 binding protein 1)

co-localize at sites of DNA damage with a number of proteins involved in DNA damage repair and signaling,

thus facilitating DSB repair and rejoining. Gamma-H2AX and 53BP1 play a critical role in suppressing

oncogenic translocations. Although induction of Gamma-H2AX foci and 53BP1 foci are frequently utilized as

assays to investigate DSB/repair following irradiation, minimal attention has been given to the generation of

spontaneous DSB/repair foci in cancer cells and cells of the tumor microenvironment. We investigated the

constitutive level of DNA damage/repair, as indicated by standard Gamma-H2AX and 53BP1 foci assays, in

cells of the tumor and adjoining stroma in vivo. In vitro studies were also done on a panel of murine tumor cells

(lung carcinoma) and human tumor cells (lung carcinoma), in both direct and indirect co-cultures with primary

nontransformed cells (murine lung fibroblast, human dermal fibroblast). In vivo studies on a panel of tumor

models, including human tumor xenografts (liposarcoma), murine tumors (Lewis lung carcinoma), and the

spontaneous K-ras LA2 lung tumor model were also performed. DSB repair foci in both in vitro and in vivo

tumor stromal populations were quantified. In vivo spatial distributions were compared to other measured tumor

features (e.g. vascularization, oxygenation, etc). An increase in DSBs occurred in nontransformed cells in the

presence of tumor cells in vitro. Conversely, preliminary data suggest that stromal fibroblasts may induce a

reduction of DSB foci in the tumor cells. In the in vivo tumor models, gamma-H2AX and coupled 53BP1

expressions were detected in several cell types (e.g., endothelial, fibroblasts, adipocytes, etc.) in the adjacent

tumor microenvironment. At distances away from the tumor, these same cell types, showed null or minimal

levels of gamma-H2AX and 53BP1. Our studies demonstrate, both in vivo and in vitro, that tumor cells can

induce DSB/repair foci in adjacent nontransformed cells. This suggests that tumor cells may transmit signals

into the microenvironment that result in DNA damage to neighboring nontransformed cells. We termed this

effect the “constitutive-break bystander effect.”

46


Antiangiogenic Effects of Proton Irradiation

Authors:

Swati Girdhani, Philip Hahnfeldt, Afshin Beheshti, Zachary Anaya, Michael Peluso, Clare Lamont,

Raktima Raychowdhury, Christian Schwager, Peter Huber, Heiko Enderling, Amir Abdollahi, Lynn Hlatky

Presented by:

Swati Girdhani

Departments:

Center of Cancer Systems Biology, Caritas St. Elizabeth’s Medical Center; Department of Radiation Oncology,

German Cancer Research Center (DKFZ), Germany

Abstract:

Tumor angiogenesis, i.e., the recruitment of microvascular endothelial cells to the tumor site, has emerged as an

important target in cancer therapy. How radiation therapy modulates tumor angiogenesis, and conversely, how

angiogenesis factors modulate cell radio-response, is a central question for the optimization of radiation

treatment. The superior physical characteristics of dose delivery by proton- vs. conventional photon-

radiotherapy have attracted considerable attention in clinical oncology. This is evidenced by increasing numbers

of proton facilities being built worldwide. In contrast to the physical aspects of proton radiation, which are

heavily exploited, the radiobiological, molecular and cell-level responses triggered by proton (vs. photon)

radiations are understudied and thus underexploited.

We demonstrate that proton radiation (at least at higher energies) is antiangiogenic and this advantageous

biological aspect of protons should be further explored to optimize treatment efficacy. We report that proton

irradiation inhibits major pro-angiogenic factors in a dose-dependent manner in both the tumor and tumor-

microenvironment (tumor-stroma and microvasculature). Differentially regulated genes 6h after 0, 0.5, 1 and

2 Gy proton irradiation were detected in human dermal fibroblasts and human lung microvascular endothelial

cells (HMVEC-L) using pan-genomic human microarrays. Of note, critical pathways in pro-angiogenic

signaling such as vascular endothelial growth factor (VEGF), interleukin 6 and 8 (IL6, IL8) and the hypoxia

inducible factor 1α (HIF-1α) were significantly downregulated after proton irradiation in both cell types. The

proton induced dose and time dependent downregulation of these pro-angiogenic genes were confirmed by real

time quantitative RT-PCR and ELISA. To investigate the regulation of these genes in tumor cells, their

expression was tested in human non-small cell lung cancer cell line A549 and mouse Lewis lung carcinoma

cells (LLC). Both tumor cell lines showed similar patterns of downregulation of angiogenic genes on RNA and

protein levels. Additionally, we investigated the effects of proton irradiation on cell invasion/migration using the

matrigel invasion assay. It was found that proton irradiation decreased cell invasion in all cell lines tested. To

functionally validate the role of VEGF and IL8 paracrine signaling in proton induced anti-angiogenic and anti-

invasive effects, in vitro, co-culture models were used. Importantly, addition of recombinant IL8 or VEGF into

47

Poster No. 44

the media partially rescued the cells from the proton radiation induced anti-invasion effects, suggesting a

functional role for these factors. Furthermore, proton-irradiated A549 cancer cells, exhibited delayed growth

in vivo, demonstrating that radiation-induced interactions with host tissues can slow the transition through

critical carcinogenesis bottlenecks. These findings suggest that proton irradiation suppresses the regulation of

key pro-angiogenic and pro-invasive proteins such as VEGF and IL8 at clinically relevant doses. The

antiangiogenic and anti-invasive effects of proton irradiation demonstrated here provide novel preclinical

evidence for beneficial radiobiological effects of proton vs. photon radiations that may be exploited clinically.

48


Impaired Angiogenesis in Aged Tumor Microenvironment Attenuates Tumor Growth

Authors:

Shiva Kalinga, Afshin Beheshti, Philip Hahnfeldt, Amir Abdollahi, Lynn Hlatky

Presented by:

Shiva Kalinga

Department:


Abstract:

The incidence of cancer increases progressively with age in both animals and humans, however, there are no

uniformity patterns of age-related distribution of tumors. To study age-dependent induction of tumor in mice,

we injected carcinomic human alveolar basal epithelial cells (A549) in nude mice in the age group of 3-12

months. Preliminary results suggest that in aged animals the tumor growth is repressed to certain extent and the

development of tumor in young mice is more prominent compared to older mice. In addition, we observed

similar tumor growth kinetics after exposing nude mice to 0.2 Gy iron radiation. Two of the several hypotheses

will be discussed: 1) levels of angiogenesis expression in both young and old mice, and 2) age-induced

expression of tumor suppressor genes such as P53 and p16INK4a.

49


Neuregulin-1 Alleviated Doxorubicin-Induced Down-Regulation of Cardiac Troponin Proteins in the Heart

Authors:

Yun Bian, Maoyun Sun, Marcy Silver, Kalon Ho, Mark Marchionni, Anthony Caggiano, James Morgan,

Xinhua Yan

Presented by:

Xinhua Yan

Departments:

Division of Cardiovascular Research, Caritas St. Elizabeth’s Medical Center; Cardiovascular Division, Beth

Israel Deaconess Medical Center; NRG Biotech; Acorda Therapeutics Inc.

Abstract:

Chemotherapy-induced cardiotoxicity is a major hurdle in cancer therapy. Recently developed new cancer

therapy targeting the erbB2 receptor by Trastuzumab significantly reduced the recurrence and early mortality in

breast cancer patients. Yet, when Trastuzumab was used in combination with the chemotherapy drug,

doxorubicin, the incidence of class III and IV heart failure increased nearly 20%.

Previous studies form our lab and others showed that Neuregulin-1 (NRG1) was effective in protecting the heart

from doxorubicin. Since NRG1 activates the erbB2 receptor on cardiomyocytes, understanding how NRG1

provides cardioprotective effects from doxorubicin is crucial for developing a safe and successful therapy in the

clinical setting.

In this study, by using an in vivo doxorubicin cardiotoxicity mouse model, we demonstrated that: 1) NRG1

improved both survival and cardiac function in doxorubicin-injured mice, and 2) doxorubicin caused down-

regulation of multiple thin filament proteins in the heart; NRG1 selectively maintained troponin proteins but not

others. Further, we used neonatal rat ventricular myocytes (NRVM) to study how NRG1 preserved cardiac

troponin proteins. We demonstrated that NRG1 inhibited doxorubicin-induced down-regulation of the mRNA

levels of cardiac troponin I (cTnI) and cardiac troponin T (cTnT), as well as doxorubicin-induced deactivation

of the mTOR pathway. Doxorubicin activated multiple caspases that were responsible for the decreasing of cTnI

and cTnT. Doxorubicin also increased proteasome degradation of cTnI. NRG1 inhibited these effects of

doxorubicin. These effects of NRG1 depended on the erbB2 receptor, as well as the PI3K, Akt and mTOR

pathways, but not by the erbB4 receptor, PKC or p38. These results demonstrated that NRG1 restored the levels

of cTnI and cTnT by increasing the transcription and translation, as well as by decreasing caspase activation and

proteasome degradation of these proteins.

This study provided new experimental evidence on how NRG1, via the erbB2 receptor, protects the heart from

chemotherapy-induced heart failure. A clearer understanding of how the NRG-erbB pathway regulates survival

signaling in diseased hearts will contribute to the development of more efficacious and safer therapies for

chemotherapy-induced heart failure, and also, to the improvement of overall health of cancer patients.

50


Evaluating the Efficacy of Stereotactic Spinal Radiosurgery

Authors:

Kevin Yao and Debbie Bakes

Presented by:

Kevin Yao

Department:

Boston Institute of Neurosurgery, Tufts Medical Center

Abstract:

Hypothesis: Spinal radiosurgery offers an important new therapeutic modality for the treatment of spinal

metastases and represents an extension of the current state-of-the-art radiation therapy. The purpose of this

study was to evaluate the accuracy and efficacy of spine metastasis radiosurgery. Over the past several years,

literature reviews have anecdotally suggested that radiosurgery for spine metastases can be effective for pain

management, tumor control, and maintaining neurological function. However, it is unclear who are the most

appropriate patients to be treated, and what are the optimal radiation planning and dosing regimens. By creating

and periodically reviewing a prospective clinical database of spine radiosurgery treatments, we hope to critically

address these issues.

Materials and Methods: The initial portion of this study involved the creation of a prospective clinical

database as to collect and organize the records of patients treated using single-fraction or hypofractionated

radiosurgery for spinal metastases. Data include patient demographics, tumor histology, description of patient

immobilization, treatment planning, and treatment delivery. The second portion of this study involves periodic

evaluation of the database to critically assess clinical efficacy of radiosurgery treatment for spinal metastases.

Outcome parameters include pain relief, local and systemic tumor control, and neurological function.

Results and Discussion: To date the database includes five patients with spinal metastases who were treated

with image guided radiosurgery at Tufts Medical Center. Four of 5 (80%) patients have shown pain

improvement while all patients are either stable or improved neurologically. All patients are alive with stable

disease. Five out of 5 (100%) treated tumors have demonstrated radiographic tumor control at last follow-up.

The mean follow-up period was 2.5 months (range, 1.5-4 months), with a median follow-up of 2.25 months.

This preliminary data suggest not only that radiosurgery is feasible and safe in the treatment of spinal

metastases, but also that excellent tumor control, pain relief, and preservation of neurological function can be

achieved.

51


Selective Inhibition of Growth of Tuberous Sclerosis Complex 2-Null Cells by Atorvastatin is Associated

with Impaired Rheb and Rho Gtpase Function and Reduced Mtor/S6 Kinase Activity

Authors:

Geraldine Finlay, Amy Malhowski, Yinglin Liu, Barry Fanburg, David Kwiatkowski, Deniz Toksoz

Presented by:

Deniz Toksoz

Departments:

Department of Medicine, Tufts Medical Center; Department of Medicine, Brigham and Women’s Hospital

Abstract:

Inactivating mutations in the tuberous sclerosis complex 2 (TSC2) gene, which encodes tuberin, result in the

tumorigenic conditions of TSC and lymphangioleiomyomatosis (LAM), which are ultimately fatal. The tumor

suppressor effect of tuberin lies in its GTPase-activating protein (GAP) activity toward Rheb small GTPase, a

Ras superfamily member. The statins, HMG CoA reductase inhibitors, have pleiotropic effects which may

involve interference with Ras and Rho GTPase prenylation and function. We show that atorvastatin selectively

inhibits serum- and estrogen-induced proliferation of Tsc2-/- fibroblasts and smooth muscle cells. The

isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) significantly reverse

atorvastatin-induced Tsc2-/- cell growth inhibition, suggesting that atorvastatin dually targets a farnesylated

protein, such as Rheb, and a geranylgeranylated protein, such as Rho, both of which are activated in Tsc2-/-

cells. Atorvastatin reduced Rheb isoprenylation, GTP loading, and membrane localization. Atorvastatin also

inhibited the constitutive phosphorylation of mTOR, S6 kinase, and S6 found in Tsc2-/- cells in an FPP-

reversible manner, and attenuated the high levels of phosphorylated S6 in Tsc2-heterozygous mice. Atorvastatin,

but not rapamycin, attenuated the increased levels of activated RhoA levels in Tsc2-/- cells, and this was

reversed by GGPP. These results suggest that atorvastatin may inhibit both rapamycin-sensitive and rapamycin-

insensitive mechanisms of tuberin-null cell growth, at least in part via Rheb and Rho inhibition, respectively.

Atorvastatin may have potential therapeutic benefit in TSC syndromes, including LAM.

52


Genetic Variants in Uracil Processing Enzymes are Associated with Abnormal DNA Uracil Content

Authors:

Aurelie Chanson, Larry Parnell, Jimmy Walter Crott, Sang Woon Choi, Haewook Han, Joel Mason

Presented by:

Aurelie Chanson

Departments:

Vitamins and Carcinogenesis Laboratory and Genomics Laboratory, Jean Mayer USDA Human Nutrition

Research Center on Aging at Tufts University; General Clinical Research Center, Tufts Medical Center

Abstract:

Introduction: Maintenance of DNA integrity through DNA repair is critical in cancer prevention. Among the

enzymes involved in DNA repair are those that prevent or correct uracil misincorporation in DNA. Repair of

misincorporated uracil is important since it can cause double-stranded DNA breaks and other mutagenic events

that can initiate or promote carcinogenesis. Also relevant is the B-vitamin folate as it is a co-factor in the

enzymatic conversion of uracil to thymidine and its deficiency promotes uracil misincorporation in DNA.

Moreover, folate and other B vitamins are implicated as factors modulating cancer risk. Five genes encode the

enzymes involved in uracil-repair in humans, for which many single nucleotide polymorphisms (SNPs) have

been identified. However, their phenotypes have not yet been explored. Our goal is to identify SNPs in uracil

processing genes that are determinants of DNA uracil levels, and to study how B vitamin status affects these

relationships.

Methods: Twenty-two SNPs were selected with a bioinformatics approach that identified variants with probable

functional consequences and from distinct linkage groups. Association between SNP genotypes and uracil levels

in blood DNA was tested in a cohort of 91 unrelated South Koreans.

Results: Multiple linear regression analyses showed that five SNPs in two of the uracil-repair genes were

significantly associated with uracil levels. Four of them were associated with an increase in uracil in the

homozygous variant individuals compared to the homozygous wild-type subjects and one was associated with a

decrease (see table below). Four SNPs were located in non-coding regions whereas one was a non-synonymous

SNP within an exon that is predicted to change the protein sequence. No interactions between B vitamin levels,

SNP genotypes and uracil levels were found in this study.

Conclusion: We identified five SNPs with a biochemical phenotype that may impact on cancer risk. We are

presently confirming our observations in a larger study.

53

Poster No. 49

Details of the five SNPs in the two uracil-repair genes found to be associated with DNA uracil levels:

Gene/SNP Designation SNP Type/Location ∆Uracil1 Adjusted P Value2

MBD4/rs140693 exon, non-synonymous 1.84-fold increase 0.011

MBD4/rs2005618 intron 6, Tag SNP 2.07-fold increase 0.033

MBD4/rs4128193 3’-UTR (downstream) 2.42-fold increase 0.006

MBD4/rs9821282 promoter 2.38-fold increase 0.007

UNG/rs1018783 promoter 1.55-fold increase* 0.038

* No aa individuals, so the reported value is for Aa vs AA individuals1 aa vs AA individuals (a: variant allele, A: wild-type allele) 2 adjusted for homocysteine, gender, serum creatinine, B vitamin status

54


Dipeptidyl Peptidase 2 is a Novel Prognostic Factor and Therapeutic Target in Chronic Lymphocytic Leukemia

Authors:

Alexey Danilov, Olga Danilova, Andreas Klein, Jennifer Brown, Arthur Rabinowitz, Kenneth Miller,

Brigitte Huber

Presented by:

Alexey Danilov

Departments:

Division of Hematology/Oncology, Tufts Medical Center; Center for Hematologic Oncology,

Dana Farber Cancer Institute; Department of Hematology and Oncology, Lahey Clinic

Abstract:

Chronic lymphocytic leukemia (CLL) is a malignancy with heterogeneous outcome. Expression of CD38 and

ZAP-70 and low rate of IgVH mutations correlate with adverse outcome in CLL. In our lab, we have cloned a

serine protease, Dipeptidyl peptidase 2 (DPP2), which is implicated in maintenance of quiescence in normal

lymphocytes. Inhibition of DPP2 leads to apoptosis of normal resting cells. We previously reported resistance of

CLL cells to DPP2 inhibition-induced apoptosis in ~40% of CLL cases, which correlated with an adverse

clinical outcome. Here, we confirm our initial observation on a large cohort of patients and demonstrate strong

correlation with IgVH mutation status. We also demonstrate that inhibition of hsp90 may restore DPP2

sensitivity to resistant CLL.

The patient cohort included 140 subjects with B-CLL from the Hematology clinics at Tufts Medical Center, the

Dana-Farber Cancer Institute (both in Boston, MA), and the Lahey Clinic (Burlington, MA). Median follow up

from diagnosis in the study was 6 years. Forty seven patients (33.6%) received at least one treatment during the

course of their disease. CLL B-cells were isolated from peripheral blood with standard Ficoll-Hypaque

technique, treated with ValboroPro (VbP, Point Therapeutics), a non-specific inhibitor of DPPs, and AX8819

(ActivX), a DPP2-specific inhibitor, incubated for 16 hours and stained with anti-CD19 antibodies, propidium

iodide and Annexin V. Cells were treated with 17-Allylaminodemethoxygeldanamycin (17-AAG, Calbiochem),

an inhibitor of hsp90 (a protein involved in stabilization of ZAP-70), or with R-406, a Syk kinase inhibitor.

IgVH mutational status was available for 44 samples. ZAP-70 expression by flow cytometry was available for

55 samples.

Inhibition of DPP2 with either VbP or AX8819 resulted in CLL cell apoptosis in 93 cases (66.4%). The

remaining CLL cases were resistant to both agents (R-CLL). In the R-CLL subgroup, 32 patients required

treatment for their disease (68.1%), contrary to S-CLL, where 22 patients initiated treatment (23.7%). Patients

with R-CLL required treatment earlier than S-CLL (p=0.03). Among S-CLLs, 91% had a mutated IgVH, 76%

55

Poster No. 50

expressed low ZAP-70 and 82% low CD38. Among R-CLL, 91% of samples had an unmutated IgVH, 100%

expressed high ZAP-70 but only 45% high CD38.

Concomitant treatment of B-cells with AAG-17 and AX8819 increased apoptosis by an average of 8.1% in

R-CLL, but not in S-CLL. R-406 alone caused 14% death in S-CLL and 6% in R-CLL. R406 displayed a

synergistic effect with AX8819, causing 7% death in S-CLL and 5% in R-CLL samples, in addition to that

caused by AX8819.

DPP2 inhibition discriminates two subsets of CLL based on their ability to undergo apoptosis upon disruption of

the quiescent program. Patients with R-CLL have worse disease outcome, with unmutated IgVH and expresses

high ZAP-70 levels, in contrast to S-CLL, which have mutated IgVH and low ZAP-70 expression. Inhibition of

SYK kinase enhances apoptosis in both subgroups. Whereas, decreasing the amount of stable ZAP-70 with

17-AAG, an hsp90 inhibitor does not affect apoptosis in S-CLL, it partially reverses the phenotype in some

R-CLL samples, implicating a role for ZAP-70 or other stabilization-dependent factor in resistance to apoptosis

in CLL.

56


Distinct Gab2-Mediated Signaling Pathways are Essential for Myeloid or Lymphoid Transformation

and Leukemogenesis by BCR-ABL

Authors:

Wayne Chan, Golam Mohi, Shaoguang Li, Benjamin Neel, Richard Van Etten

Presented by:

Wayne Chan

Departments:

Departments of Physiology and of Medicine, Tufts University School of Medicine; Molecular Oncology

Research Institute, Tufts Medical Center; Department of Pharmacology, SUNY Upstate Medical University;

Department of Medicine, Harvard Medical School; The Jackson Laboratory

Abstract:

The BCR-ABL oncogene encodes an activated fusion tyrosine kinase that causes chronic myelogenous leukemia

(CML) and B-lymphoid acute lymphoblastic leukemia (B-ALL) in humans. An autophosphorylation site at

Tyr177 of BCR-ABL recruits Grb2 via its SH2 domain and is required for efficient induction of CML-like

myeloproliferative disease by BCR-ABL in a mouse BM retroviral transduction/transplantation model. We

previously showed (Sattler et al., Cancer Cell 2002; 1:479) the scaffolding/adapter protein Gab2 is recruited to

Y177 of BCR-ABL via a Grb2/Gab2 complex and in vitro transformation of primary myeloid and lymphoid

progenitors by BCR-ABL was impaired in bone marrow from mice with homozygous null mutations in the

Gab2 gene (Gab2–/– mice), coincident with decreased activation of the ERK and Akt signaling pathways. Here,

we demonstrate an essential requirement for Gab2 in myeloid and lymphoid leukemogenesis by BCR-ABL.

Whereas, recipients of BCR-ABL-transduced Gab2+/+ BM develop fatal CML-like myeloproliferative disease

within 4 week of transplantation, recipients of BCR-ABL-transduced Gab2–/– BM fail to develop CML but

succumb after a long latent period to T-cell acute lymphoblastic leukemia, phenocopying the disease induced by

the BCR-ABL Y177F mutant. These results suggest that the Y177F and Gab2 mutations have an epistatic

relationship and that the critical transforming signals from Tyr177 of BCR-ABL are transmitted through Gab2.

Co-expression of Gab2 with BCR-ABL in Gab2–/– BM restored efficient induction of CML-like leukemia, but

mutants of Gab2 that lacked either the pleckstrin homology domain or Tyr binding sites for the SH2 domains of

the downstream Gab2 effector molecules SHP2 or p85 PI3K failed to rescue myeloid leukemogenesis by

BCR-ABL, although the mutant Gab2 proteins were expressed in circulating myeloid cells. Gab2 deficiency

attenuated B-lymphoid transformation by BCR-ABL in vitro, and significantly prolonged the latency of B-ALL

induced by BCR-ABL in mice. In contrast to CML, induction of B-ALL in Gab2–/– BM was rescued by either

WT Gab2 or the Gab2 p85 binding site mutant. These results demonstrate that BCR-ABL absolutely requires

signaling via Gab2 to both SHP2 and PI3K to cause CML, while a Gab2–SHP2 signaling pathway contributes to

the pathogenesis of BCR-ABL+ B-ALL.

57


Environmental Risk Factors and Risk of Canine Malignant Lymphoma: A Model for Human

Non-Hodgkin’s Lymphoma

Authors:

Sofija Zagarins, Lisa Barber, Elizabeth Procter-Gray, Audra Gollenberg, Elizabeth Bertone-Johnson

Presented by:

Lisa Barber

Departments:

Department of Clinical Sciences, Cummings School of Veterinary Medicine; Department of Public Health,

School of Public Health and Health Sciences, University of Massachusetts Amherst

Abstract:

Epidemiologic studies of companion animals offer an important opportunity to identify risk factors for cancers

of relevance to both animals and humans. Canine malignant lymphoma (CML) has been established as a model

for non-Hodgkin’s lymphoma (NHL) and previous studies have suggested that exposure to environmental

chemicals such as 2,4-dichlorophenoxyacetic acid (2,4-D) may relate to the development of CML. We

conducted a case-control study of dogs presented to the Foster Hospital at Tufts University between 2000 and

2006. Cases were 263 dogs with biopsy-confirmed CML. Controls included 240 dogs with benign tumors and

230 dogs undergoing surgeries unrelated to cancer. Dog owners completed a 10-page questionnaire measuring

demographic, environmental, and medical factors. After adjusting for age, weight, and other factors, we did not

find either household smoking or use of flea/tick control products to be associated with risk of CML. Use of

professionally-applied lawn care products was positively associated with CML risk. In particular, herbicide use

was associated with a significant 50% higher risk of CML [odds ratio (OR): 1.5; 95% confidence interval (CI):

1.1-2.3], while pesticide use was associated with an 80% higher risk (OR: 1.8; 95% CI: 1.2-2.9). Home owner

application of these chemicals was unrelated to risk, though the OR for use of insect growth regulators was 2.7

(95% CI: 1.1-6.9). These findings suggest that some lawn care chemicals may increase the risk of CML.

Additional analyses are needed to evaluate the specific chemical components of these products, such as 2,4-D,

that may be related to risk of CML and perhaps to human NHL.

58


Comparative Oncology Trials Consortium Aids in Development of Human Cancer Therapies

Authors:

Lisa Barber, Kristine Burgess, Kristin Marshall

Presented by:

Lisa Barber

Department:

Department of Clinical Sciences, Cummings School of Veterinary Medicine

Abstract:

Spontaneous tumors in dogs share many characteristics with human malignancies. Nonclinical studies in dogs

have provided information into cancer biology that would be infeasible to obtain in humans or rodents. The

commonalities in oncogenic pathways between species and the lack of gold-standard treatments for many canine

cancers provide opportunities for early ethical and humane investigation of novel therapies in dogs with

naturally occurring cancers.

To this end, the Comparative Oncology Trials Consortium (COTC) was founded in 2004 as an active network of

academic comparative oncology centers (primarily colleges of veterinary medicine) that is centrally coordinated

by the NIH-National Cancer Institute’s Comparative Oncology Program. The COTC functions to design and

conduct clinical trials of novel cancer therapies. The goal of this endeavor is to answer biological questions that

can be integrated into the development path of new cancer therapies for people. Trials conducted by the COTC

are focused on providing pharmacokinetic and pharmacodynamic information that can be applied to human

Phase I and II clinical trials.

Cummings School of Veterinary Medicine was one of the COTC charter member institutions, which has

expanded to include 18 veterinary schools. Recent studies have included evaluations of RGD targeted delivery

of phase expressing TNF-alpha, mTOR inhibitor rapamycin in metastatic osteosarcoma, immunocytokine fusion

protein, and cryobiopsy instrumentation in dogs with lymphoma.

59


GS-9219 – A Novel Acyclic Nucleotide Analogue with Potent Antineoplastic Activity in Dogs with

Spontaneous Non-Hodgkin’s Lymphoma

Authors:

Kristine Burgess, David Vail, Doug Thamm, Michael Hawkins, Daniel Tumas

Presented by:

Kristine Burgess

Departments:

Departments of Clinical Sciences and of Medical Sciences, Cummings School of Veterinary Medicine;

Gilead Sciences, Inc.

Abstract:

Canine lymphoma (NHL) accounts for 24% of all canine neoplasia and 83% of all canine hematopoietic

malignancies. Likewise, in humans, NHL is the second fastest growing form of cancer and the fifth leading

cause of cancer deaths in the United States. Multi-agent chemotherapy protocols will induce a remission period

for 60% to 90% of dogs with a median survival time of 6 to 12 months with approximately 20% of dogs alive at

2 years.

Despite the plethora of published chemotherapy protocols, no dramatic improvement has been made in

extending the 12 month median survival or the 20% 2-year survival in dogs. Advances in remission and

survival durations await the development of novel methods of delivering or targeting old chemotherapeutic

drugs and the development of newer generation chemotherapeutics or novel non-chemotherapeutic treatment

modalities. These are current areas of active research for both human and veterinary medicine.

GS-9219 is a double prodrug of acyclic nucleotide phosphonate 9-(2-phosphonylmethoxyethyl) guanine

(PMEG) and is only being developed for the treatment of hematological malignancies in humans. GS-9219 was

designed to preferentially deliver and accumulate PMEG and its active phosphorylated metabolite, PMEG

disphosphate (PMEGpp) in lymphoid cells while avoiding systemic exposure of PMEG. GS-9219 has

substantial antiproliferative activity against proliferating lymphoblasts and tumor cell lines of hematopoietic

origin due to potent inhibition of DNA synthesis. As an animal model of the human disease, a phase I/II trial

(dose-finding/efficacy) was performed in 38 dogs with naive or refractory NHL. These dogs received GS-9219

as a monotherapy in variable dosing schedules. This study reported an overall response rate of 79%.

Importantly, responses were documented in both previously untreated dogs and dogs with refractory NHL. This

implies a role both in induction therapy and in the rescue of drug resistant disease.

To obtain further evidence of efficacy in a relevant animal model, the comparative oncology group at the

Cummings School of Veterinary Medicine, in conjunction with other veterinary institutions, is contributing to a

60

Poster No. 54

large prospective clinical trial to evaluate the safety, efficacy, pharmacokinetc and pharmacodynamic response

of a novel cytotoxic drug, GS-9219, in a large number of dogs with relapsed spontaneous non-Hodgkin’s

lymphoma (NHL). GS-9219 is a promising development candidate and Phase I investigations of GS-9219 in

human patients with lymphoid malignancies are in progress.

61


Tissue Banking at the Cummings School as Part of the Canine Comparative Oncology Genetics Consortium

Authors:

Kristine Burgess, Lisa Barber, Chieko Azuma, Kristin Marshall, Dawn Meola, John Berg

Presented by:

Kristine Burgess

Department:

Department of Clinical Sciences, Cummings School of Veterinary Medicine

Abstract:

The recent publication of the canine genome sequence has unleashed the opportunity for comparative genomic

analysis to explore fundamental questions regarding diseases that affect humans, dogs and other species.

Success in these endeavors is dependant upon the availability of high-quality biologic specimens for future

investigations. In an effort to address this need, the National Cancer Institute Comparative Oncology Program

(NCI-COP) has created an innovative multi-institutional tissue repository of spontaneous canine cancers. The

Pfizer Canine Comparative Oncology Genetics Consortium (CCOGC) Biospecimen Repository provides a

unique opportunity for a large scale tissue banking program which will benefit all facets of comparative cancer

research.

The CCOGC consists of a collaboration of veterinary and medical oncologists, pathologists, surgeons,

geneticists, and molecular and cellular biologists. This organization consists of 7 contributing veterinary

institutions throughout the country and was developed to understand the genetics and biology of certain cancers

in companion animals. The ultimate goal of the initiative is to facilitate the comparative study of canine and

human genomics and cancer.

Spontaneous tumors in companion animals can serve as an ideal model for human cancer due to the similarities

within gene families, in particular those associated with cancer. Neoplasms of comparative interest include

melanoma, non-Hodgkin’s (NHL), leukemia, osteosarcoma, soft tissue sarcomas, and prostate, mammary, lung,

head, and neck and bladder carcinomas.

62


Novel, 3D Human Tissue Platforms for Cancer Discovery: The Core for Experimentation in 3D Tissues

Authors:

Jonathan Garlick and Christophe Egles

Presented by:

Christophe Egles

Department:

Department of Oral and Maxillofacial Pathology, Tufts University School of Dental Medicine

Abstract:

Our understanding of the interplay between cancer cells and their immediate tissue microenvironment is

undergoing a revolution that requires integration of multiple scientific disciplines to provide novel, biologically-

relevant, in vitro tissues for cancer research. The Core for Experimentation in 3D Human Tissues provides

novel cancer research tools for discovery in in vitro and in vivo three-dimensional (3D), human tissue models

that recapitulate the complex tissue architecture and signaling networks present in human cancer. Through the

fabrication and analysis of 3D tissues that mimic the human cancer microenvironment, we can now generate

novel experimental paradigms that enable investigation into the complex interplay between multiple cell and

tissue types as they occur during cancer development in vivo, in a 3D tissue context. This provides a more

global picture of human cancer-associated pathways and their local microenvironment and serves as human,

“pre-clinical” or “surrogate” tissues to translate discoveries to the clinic. The resources in our Core can now

leverage its expertise in 3D tissue biology as a portal to discovery of pathways linked to human cancer that serve

as a paradigm for future clinical translation. Our Core can fabricate 3D human tissues that incorporate a variety

of different cancer cell types to mimic the complex microenvironment of human cancers using in vitro tissue

models. These tissues also incorporate mesenchymal cells and a broad variety of stromal matrices and scaffolds

to enable cross-talk between the tumor cells and the mesenchymal cells. Tissues are now being fabricated as a

platform technology for the pharmaceutical design and screening of drugs and small molecules designed to treat

cancer. In addition, the Core has the capability to genetically-modify 3D tissues to either overexpress or knock-

out specific gene targets that are important during cancer progression. Fabrication of human tissues that mimic

human cancers provide for more reliable correlations between in vitro studies and in vivo outcomes during

human clinical studies. In this light, tissues generated through this Core will provide a “pre-clinical,”

experimental setting that will accelerate development of translational medicines for novel cancer treatments.

63


Tufts Medical Center Transgenic Core Facility

Authors:

Kibibi Rwayitare, Danielle Riggs, Michael Mendelsohn, Janis Lem

Presented by:

Kibibi Rwayitare

Department:

Molecular Cardiology Research Institute, Tufts Medical Center

Abstract:

The Tufts Medical Center Transgenic Core Facility (TCF) is a fee-for-service facility providing the following

services:

Standard Transgenic Production: The transgenic core facility guarantees at least one founder carrying all or

part of the transgene.

Knockout/Knock-in Production: Embryonic stem cells clones will be microinjected into a minimum of 40

C57BL/6 blastocysts per session.

Pathogen-free Rederivation: Embryos harvested from mice known to harbor pathogenic viruses are washed

and transferred into clean foster mothers to provide the investigator with Specific Pathogen Free (SPF) pups.

Embryonic Cryopreservation: A substantial amount of time and money is invested in making a transgenic

mouse line, and upon project completion, the need for large numbers of animals drops. Cryopreservation will

save you on time and money spent to maintain your mouse colony for future use. Cryopreserved embryos from

other institutions can also be reconstituted by the TCF.

These services are available to all Tufts Medical Center/Tufts University investigators, as well as any

investigators at non-profit institutions.

The MCRI offers cost subsidies to its members.

Please e-mail your questions to the TCF administrator at [email protected]

64


Tufts Animal Pathology and Histology Core Laboratory Services

Authors:

Lauren Richey, Brian Lagace, Derek Papalegis

Presented by:

Lauren Richey

Department:

Division of Laboratory Animal Medicine, Tufts University

Abstract:

The Tufts Research Animal Health and Pathology Support (RAHPS) laboratory provides pathology and

histology support for investigators working with research animals. A board certified veterinary pathologist

trained in the comparative pathology, physiology, health, strain variation, and phenotypic analysis of animals is

able to assist researchers at every step of their projects. This ranges from model selection, to sample collection,

lesion documentation, and interpretation of findings. Services include gross necropsy examination,

photodocumentation of lesions, histology by a certified histotechnician experienced in research animal tissues,

review of slides by the veterinary pathologist, consultation for pathology study design, review of pathology data

and reports, training in pathology techniques, and assistance with clinical pathology needs. The RAHPS

laboratory supports the research of Tufts University and Tufts Medical Center investigators by working with

each person to meet individual research pathology and histology needs.

65


Proteomic Research and Services of the Tufts Proteomic Core Facility

Author:

Jon DeGnore

Presented by:

Jon DeGnore

Department:

Department of Physiology, Tufts University School of Medicine

Abstract:

Tufts’ research community has access to state-of-the-art protein identification and characterization. The Tufts

University Core Facility (TUCF) in the School of Medicine’s department of physiology now includes high-tech

mass spectrometry equipment that allows for highly sensitive proteomic analysis. A relatively new field,

proteomics is the study of proteins and their functions. Proteomics pioneer, Marc Wilkins, coined the term less

than a decade ago, but it quickly has become one of the keys to identifying proteins and mapping their pathways

and interactions in the cellular context. By revealing new disease markers and drug targets, proteomic analysis

promises to help researchers develop new means of preventing, diagnosing and treating illnesses. Information

about core research, services, sample preparation, and research projects is also available on the web at

www.tucf.org or proteomics.med.tufts.edu.

66


Getting Your Research Done with University Information Technology (UIT) Services

Author:

Lionel Zupan

Presented by:

Lionel Zupan

Department:

UIT Academic Technology Services, Tufts University

Abstract:

Dr. Zupan will give an overview of the research technology services provided by University Information

Technology (UIT) and also discuss how Tufts faculty are using research technology in their work. UIT services

currently include: research high performance computing; research network storage; software network licensing;

tools for visualization; statistical consulting; technology consultation and planning; and monitoring, design,

implementation and application of emerging technologies for research applications.

67


Light Scattering Spectroscopic Characterization of Healthy and Cancerous White Blood Cells

Author:

Austin Hsiao

Presented by:

Austin Hsiao

Department:

Department of Biomedical Engineering, Tufts University School of Engineering

Abstract:

Leukemia is uncontrolled proliferation of immature white blood cells, significantly detrimental to the functions

of blood and the immune system. The conventional method of diagnosis requires invasive and medically

extensive biopsies and blood samplings at great discomfort of the patients. Leukemia is the most prevalent type

of cancer in children, where blood drawing is particularly painful and difficult. Therefore, a non-invasive

screening modality could significantly improve the detection and monitoring of these patients. For this reason,

we performed an initial set of studies to assess the potential of light scattering spectroscopy to determine

whether unique light scattering signatures can differentiate leukemic from healthy white blood cells. We

acquired angle-dependent and wavelength-dependent light scattering maps of the samples in the backscattering

geometry. Specifically, we acquired polarized light scattering maps from isolated cell populations along the

parallel and perpendicular polarizations and computed the differential light scattering maps, representing mostly

singly backscattered light. From these LSS maps, the wavelength-dependence of the biological samples,

characterized by a power law exponent value, was used to quantitatively differentiate between the healthy

lymphocytes, granulocytes and the leukemia cells. Therefore, these initial findings provide the basis for

detection of leukemia in in vivo flow cytometry and demonstrate the potential of a non-invasive leukemia

screening test.

68


Hyperspectral Image Reconstruction for Diffuse Optical Tomography

Author:

Fridrik Larusson

Presented by:

Fridrik Larusson

Department:

Department of Electrical and Computer Engineering, Tufts University School of Engineering

Abstract:

Diffuse optical tomography (DOT) has emerged in the last decade as a new and exciting tool for functional

medical imaging with applications in a range of areas including breast cancer detection and diagnosis. DOT

employs observations of near infrared (NIR) light that has propagated through tissue to reconstruct the spatial

distribution of various chromophores present in the region of interest. In the case of breast cancer, oxygenated

and de-oxygenated hemoglobin are of particular interest in identifying and characterizing tumors. It is well

known that the DOT reconstruction process can be quite sensitive to noise and other un-modeled effects due to

the diffusive nature of the underlying physics as well as the limited aperture over which data can be acquired in

many practical systems. While there exists a wide array of mathematical techniques for stabilizing the

reconstruction, ideally one would like a richer data set. Most DOT instruments employ no more than 5 NIR

wavelengths to probe the tissue; however, recent work in Professor Fantini’s lab has led to the development of a

hyperspectral system in which hundreds of wavelengths can be acquired. With the increase in data, however,

comes an associated rise in the complexity of the image formation process. In this poster, we explore the

development and performance of algorithms for hyperspectral DOT. We detail an efficient method for forming

the images based on the use of iterative algorithms applied to a linearized measurement model. Statistical

analysis techniques are then employed to quantify the information content of the enlarged data set and

automatically remove un-informative data from the reconstruction process in order to reduce complexity with

limited impact on accuracy. Simulation results will be provided with details for future work involving phantom

studies.

69


Avenanthramides Inhibit Proliferation of Human Colon Cancer Cell Lines in vitro

Authors:

Weimin Guo, Lin Nie, Dayong Wu, Mitchell Wise, F. William Collins, Simin Nikbin Meydani,

Mohsen Meydani

Presented by:

Weimin Guo

Departments:

Vascular Biology Laboratory and Nutritional Immunology Laboratory, Jean Mayer USDA Human Nutrition

Research Center on Aging at Tufts University; United States Department of Agriculture, ARS Cereal Crops

Research; Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada

Abstract:

High intake of whole grain food is associated with reduced risk of colon cancer, but the mechanism underlying

this protection has yet to be elucidated. Chronic inflammation and associated cyclooxygenase-2 (COX-2)

expression in the colon epithelium are causally related to epithelial carcinogenesis, proliferation, and tumor

growth. We examined the effect of avenanthramides (Avns), unique polyphenols from oats with anti-

inflammatory properties, on COX-2 expression in macrophages, colon cancer cell lines, and on proliferation of

human colon cancer cell lines. We found that Avns enriched extract of oats (AvExO) had no effect on COX-2

expression, but inhibited the COX enzyme activity and prostaglandin E2 (PGE2) production in LPS-stimulated

mouse peritoneal macrophages. Avns (AvExO, Avn-C, and the methylated form of Avn-C (CH3-Avn-C))

significantly inhibited cell proliferation of both COX-2-positive HT29, Caco-2, and LS174T, and COX-2-

negative HCT116 human colon cancer cell lines, with CH3-Avn-C being the most potent. However, Avns had

no effect on COX-2 expression and PGE2 production in Caco-2 and HT29 colon cancer cells. These results

indicate that the inhibitory effect of Avns on colon cancer cell proliferation may be independent of COX-2

expression and PGE2 production. Thus, Avns might reduce colon cancer risk through inhibition of macrophage

PGE2 production and non-COX-related antiproliferative effects in colon cancer cells. Interestingly, Avns had no

effect on cell viability and proliferation of confluence-induced differentiated Caco-2 cells, which display the

characteristics of normal colonic epithelial cells. Our results suggest that the consumption of oats and oat bran

may reduce the risk of colon cancer not only through high fiber content but also through Avns, which attenuate

proliferation of colonic cancer cells.

70


A Novel Mouse Model for Sporadic Colorectal Cancer

Authors:

Kenneth Hung, Marco Maricevich, Larissa Georgeon Richard, Alexandra Kunin, Alvin Kho, Umar Mahmood,

Raju Kucherlapati

Presented by:

Kenneth Hung

Departments:

Department of Medicine, Tufts Medical Center; Center for Molecular Imaging Research, Harvard University,

Department of Medicine, Brigham and Women’s Hospital; Harvard Partners Center for Genetics and Genomics,

Harvard Medical School

Abstract:

Traditional genetically engineered mouse colorectal cancer models are based on germ line manipulations,

resulting in mutation in both intestinal epithelium and surrounding stroma. Whereas these are appropriate for

cancer predisposition syndromes, such as familial adenomatous polyposis, they are poor surrogates for the

majority of human cases, i.e., Sporadic Colorectal Cancer (SCC). In addition, unlike human disease, these

models present with predominantly small intestinal rather than colonic lesions. To generate a model for sporadic

cancer that is truly localized to the colon, we have administered adenovirus expressing cre recombinase to

focally target genes of interest. Using this approach, we have shifted the tumor compartment from the small to

the large intestine, allowing endoscopic surveillance to monitor tumor growth. When this technique was applied

to mice bearing a conditional mutation in the Apc gene, tumors were noted as early as 6 weeks of age. When

mice bearing both the Apc mutation and a latent oncogenic K-ras allele were infected, multiple prominent

tumors were noted by 6 weeks. The results from the transcriptome and signaling pathway analysis will be

discussed. In summary, we have devised a model for SCC that allows the study of tumor formation by mutated

epithelial cells in the setting of normal stroma and permits longitudinal analysis by endoscopic modalities.

71


Nonalcoholic Steatohepatitis Induced by a High-Fat Diet Promotes Diethylnitrosamine Initiated Early

Hepatocarcinogenesis in Rats

Authors:

Yan Wang, Lynne Ausman, Andrew Greenberg, Robert Russell, Xiang-Dong Wang

Presented by:

Yan Wang

Departments:

Nutrition and Cancer Biology Laboratory and Obesity and Metabolism Laboratory, Jean Mayer USDA

Human Nutrition Research Center on Aging at Tufts University

Abstract:

It has been suggested that patients with nonalcoholic steatohepatitis (NASH) have a high risk for liver cancer.

However, it is unknown whether high-fat diet induced NASH promotes hepatocarcinogenesis. In the present

study, Sprague-Dawley rats were injected with a low dose of hepatic carcinogen diethylnitrosamine (DEN) and

then fed either Lieber-DeCarli control diet (CD) or high-fat diet (HFD) for six weeks. Liver histology and the

hepatic placental form of glutathione S-transferase (P-GST) positive foci were examined. Expression levels of

proliferating cell nuclear antigen (PCNA), cyclinD1, phosphorylated MAPK including extracellular signal-

regulated kinase (ERK) and p38, as well as TNF-α and NF-κB were measured in the liver. Induction of lipid

peroxidation end products (malondialdehyde plus 4-hydroxynonenal) and apoptotic hepatocytes in the liver were

also assessed. Results showed that HFD-fed rats developed significantly higher incidence and multiplicity of

P-GST positive foci along with the significant fat accumulation, infiltration of inflammatory cells and higher

lipid peroxidation in the liver, as compared with rats fed the CD. This high prevalence of hepatic lesions in the

liver was accompanied by greater PCNA expression and cyclinD1 protein concentration but little change in

hepatocyte apoptosis. HFD feeding elevated hepatic phosphorylated ERK but reduced phosphorylated p38 as

compared with the CD feeding. In addition, a significantly higher expression of TNF-α mRNA and nuclear

NF-κB p65 protein were observed in HFD group than those in CD group. These data clearly demonstrate that

NASH, induced by high-fat diet, promoted DEN-initiated early hepatocarcinogenesis, which was associated

with elevated TNFα/NF-κB signaling and MAPK related hepatocyte proliferation.

72


Nasopharyngeal Carcinoma at Tufts Medical Center: Evaluation of the Chinatown and

Greater Boston Incidence and Response to Treatment

Authors:

Richard Wein and Mazin Merdad

Presented by:

Richard Wein

Department:

Otolaryngology-Head and Neck Surgery, Tufts Medical Center

Abstract:

Nasopharyngeal carcinoma is a neoplasm with an incidence of approximately 1:100,000 in the North American

population. This incidence is 30-50 times greater in southern China (Guangdong province), Hong Kong, Macua,

Singapore, Vietnam, Malaysia and Taiwan. Nasopharyngeal carcinoma, when compared to other sites within the

upper aerodigestive tract treated at tertiary care medical centers, typically represents only 3% of all presenting

cancers. At Tufts Medical Center, because of the high number of Southeast Asian patients seen for evaluation,

nasopharyngeal carcinoma represents 15% of all head and neck cancers diagnosed. This presentation will

discuss the associated risk factors and the typical presentation for patients with nasopharyngeal carcinoma, the

survival of treated patients treated at Tufts Medical Center, in addition to reviewing the multiple research efforts

currently underway at Tufts Medical Center.

73


Dose De-Escaltion with Gamma Knife Radiosurgery in the Treatment of Choroidal Melanoma

Authors:

John Mignano, Michael Chan, Clemens Schirmer, Christopher Melhus, Lloyd Williams, Daniel Do-Dai,

Jay Duker, Kevin Yao

Presented by:

John Mignano

Departments:

Department of Neuroscience, Tufts University School of Medicine; Departments of Radiation Oncology, of

Neurosurgery, and of Ophthalmology, Tufts Medical Center

Abstract:

Stereotactic radiosurgery (SRS) provides an alternative to plaque brachytherapy in the treatment of choroidal

melanoma when brachytherapy is contraindicated. The potential late toxicity to the optic apparatus has limited

the number of patients treated with this approach in the past, however, prior reports on the use of SRS have

utilized margin doses in excess of 30Gy. We present our single institutional experience of a dose de-escalation

approach with the intent of visual sparing in the treatment of choroidal melanoma in cases where plaque

brachytherapy is contraindicated.

74


Molecular Characterization of EBV Associated Nasopharygeal Carcinoma

Authors:

David Merritt and David Thorley-Lawson

Presented by:

David Merritt

Department:

Department of Pathology, Tufts University School of Medicine

Abstract:

The Epstein-Barr Virus (EBV) is a ubiquitous gamma herpes virus that is associated with several human

epithelial and lymphoid malignancies, including nasopharyngeal carcinoma (NPC). The nonkeratinizing forms

of nasopharyngeal carcinoma are 100% associated with EBV infection, and are divided into two subtypes based

on histology (“differentiated” WHO type II and “undifferentiated” WHO type III). Our analysis of gene

expression data from an Affymetrix study of type II and type III NPC using the Connectivity Map (developed at

the Broad Institute) suggests that the PI3K/Akt pathway may be more highly activated in NPC type III.

Alternatively, the array data may reflect differential sensitivity to PI3K/Akt pathway inhibitors and that the

underlying genetic lesions that drive Akt activity in NPC type II and NPC type III are different. In addition,

analysis using an algorithm that analyzes sets of related genes, Gene Set Enrichment Analysis (GSEA), revealed

that in NPC type III tumors, there is an increased expression of genes associated with epithelial-mesenchymal

transition (EMT), a developmental program characterized by loss of cell adhesion, repression of E-cadherin

expression and increased cell mobility. EMT has been reported to be a feature of several metastatic cancers.

Taken together, this suggests that the activation of the PI3K/Akt pathway and EMT in NPC type III tumors may

provide a molecular basis for the distinction between NPC type II and NPC type III. It may also provide

prognostic and therapeutic implications for more efficacious treatment specifically targeting NPC type II or

NPC type III.

75


Frequency of Presentation and Risk Profile for Human Papillomavirus in Oropharyngeal and Hypopharyngeal

Squamous Cell Carcinoma at Tufts Medical Center

Authors:

James Kraus, Daniel Oreadi, Richard Wein, Nora Laver, Maria Papageorge

Presented by:

James Kraus

Departments:

Department of Oral and Maxillofacial Surgery, Tufts University School of Dental Medicine; Departments of

Otolaryngology-Head and Neck Surgery and of Pathology, Tufts Medical Center

Abstract:

Background: Recently, there has been significant molecular evidence implicating human papillomavirus (HPV)

as the causative agent in the development of squamous cell carcinoma of the head and neck, specifically, in the

oro-hypopharynx. High-risk HPV-16 has been identified in the greater part of these HPV-positive tumors.

Methods: A review of all the oropharyngeal and hypopharyngeal squamous cell carcinomas treated at Tufts

Medical Center between the years 2000-2008 was performed. History of initial presentation, staging, smoking

history and response to treatment were noted from the patients’ medical records. Pathologic specimens were

processed to determine presence or absence of HPV. If HPV was present in the tumor, it was identified as a

high-risk (HPV-HR) or low-risk (HPV-LR) strain.

Discussion: Correlation between staining properties of specimens and the initial stage at presentation, response

to therapy, need for surgical salvage (or sequential neck dissection), and survival of patients will be made. Given

the unique cultural population of patients seen at Tufts Medical Center, additional analyses will attempt to

determine if the rate of presentation of HPV-, HPV+ LR, HPV+ HR squamous carcinomas in the oropharynx

and hypopharynx parallels reported frequencies at other medical centers.

76


RalA Suppresses Invasion by Ras-Transformed Keratinocytes in a Bioengineered Human Tissue Model of

Squamous Cell Carcinoma

Authors:

Adam Sowalsky, Addy Alt-Holland, Yulia Shamis, Jonathan Garlick, Larry Feig

Presented by:

Adam Sowalsky

Departments:

Departments of Endodontics and of Oral and Maxillofacial Pathology, Tufts University School of Dental

Medicine; Departments of Biochemistry and of Anatomy and Cellular Biology,


Abstract:

Squamous cell carcinomas represent 90% of head and neck cancers and ~25% of skin cancers. A significant

proportion of these cancers contain either mutations in Ras genes that lead to constitutively-activated Ras

GTPases or amplified levels of Ras proteins. Ras proteins function by binding to and activating a set of

downstream effector proteins, including Raf and PI-3 kinases, as well as exchange factors that activate RalA and

RalB GTPases. Most previous studies have suggested that Ral GTPases play a positive role in Ras-induced

cancer progression, but these studies did not necessarily mimic the natural setting of epithelial cancer

development. In order to study the contribution of the tumor microenvironment, consisting of intercellular and

stromal interactions, to the transition from a precancerous to cancerous state, we utilized an in vitro 3D tissue

model of Ras-mediated squamous cell carcinoma. In this system, skin-derived preneoplastic keratinocytes

expressing activated Ras are used to populate the engineered epithelium. These cells form a dysplastic

epithelium that mimics premalignant disease, as they do not invade across the basement membrane into the

fibroblast-laden dermal layer. To our surprise, we found that shRNA-mediated depletion (~65%) of RalA, but

not RalB, in these keratinocytes, generated an invasive phenotype that mimicked the earliest stages of cancer

progression. This invasion was associated with focal degradation of the basement membrane, and was similar to

that observed when a known tumor suppressor, E-cadherin, was inactivated in these cells. Interestingly, loss of

E-cadherin led to decreased levels of RalA that were comparable to those generated in shRNA expressing

keratinocytes. Taken together, these results suggest that RalA activity suppresses the induction of early stages of

squamous carcinoma by Ras, and that a modest suppression of RalA expression may be a required step in the

transition from premalignant to malignant disease.

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The Tumor Microenvironment Meets Epigenetics: Regulation of E-cadherin Expression in Squamous Cell

Carcinoma Progression through DNA Methylation

Authors:

Teresa DesRochers, Yasusei Kudo, Takashi Takata, Jonathan Garlick

Presented by:

Teresa DesRochers

Departments:

Department of Anatomy and Cellular Biology, Tufts University School of Medicine; Department of Oral and

Maxillofacial Pathology, Tufts University School of Dental Medicine; Department of Oral Maxillofacial

Pathobiology, Hiroshima University

Abstract:

Epigenetic silencing of E-cadherin through DNA hypermethylation of the promoter has been well documented

in many cancer types. The observed pattern of gene silencing is heterogeneous throughout the tumor and

changes during cancer progression indicating a possible role for the tumor microenvironment in the regulation

of epigenetic-mediated silencing. As it is well established that the tumor microenvironment has a significant

impact on the phenotypic properties of a broad spectrum of cancers, we propose that the microenvironment

modifies epigenetic control of gene expression, thereby regulating transient changes in the expression of key

proteins during tumor progression. We have studied whether the tumor microenvironment plays a role in the

epigenetic control of E-cadherin expression during the progression of oral squamous cell carcinoma (OSCC).

We have used bio-engineered, 3D human tissue models that mimic various stages of OSCC progression to

elucidate if methylation-mediated gene silencing of E-cadherin is regulated by the tissue microenvironment.

Tumor cells were derived from a lymph node metastasis that originated as an OSCC and a clonal isolate

(C1-Inv-1) was characterized in 2D, monolayer culture and in 3D, engineered tissues. E-cadherin expression

was analyzed by Western blot, immunofluorescence, and RT-PCR while the methylation status of the

E-cadherin promoter was examined by both methylation-specific PCR (MSP) and bisulfite sequencing (BSP). In

2D culture, C1-Inv-1 cells demonstrated complete loss of E-cadherin expression due to promoter

hypermethylation.

We observed that methylation was limited to only a few distinct CpGs rather than being widespread throughout

the CpG island of the E-cadherin promoter, suggesting regulated DNA methylation in these cells. When

C1-Inv-1 cells were grown as a 3D tissue at an air-liquid interface in order to induce homotypic cell-cell

interactions in a tissue that mimicked the premalignant stage of tumor progression, E-cadherin expression and

localization were restored in cells in the suprabasal layers of the tissue. In addition, tumor cells manifested their

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Poster No. 71

in vivo behavior and spread as single cells into the underlying stroma. These invasive cells maintained loss of

E-cadherin expression, suggesting that continued suppression of E-cadherin and the acquisition of invasive

properties were dependent upon contact with the stromal microenvironment that sustained promoter

hypermethylation.

Currently, we are examining the methylation state of the E-cadherin promoter in those cells that maintained

E-cadherin loss as they invaded into the underlying stroma in comparison to those cells that re-expressed

E-cadherin as they stratified above the epithelial-stromal interface. Initial work has revealed an increase in

methylation in those cells grown in 3D tissues compared to 2D monolayer culture, and a significant change in

the methylation of specific CpGs in the E-cadherin promoter between those cells that maintained loss of

E-cadherin and invaded into the stroma and those that re-expressed E-cadherin and stratified above the

epithelial-stromal interface. We propose a model in which transient and heterogeneous patterns of E-cadherin

expression during cancer progression are due to epigenetic control that is dynamically sensitive to regulation by

the microenvironment and results in the unstable and reversible tumor cell phenotypes seen in invasive and

metastatic human cancers.

79


Loss of E-Cadherin-Mediated Cell-Cell Adhesion Induces the Transition from Precancer to Squamous Cell

Carcinoma through Activation of FAK and Src Kinases

Authors:

Addy Alt-Holland, Yonit Szwec-Levine, Daniel Green, Jonathan Garlick

Presented by:

Addy Alt-Holland

Departments:

Division of Cancer Biology and Tissue Engineering, and Departments of Endodontics and of Oral and

Maxillofacial Pathology, Tufts University School of Dental Medicine

Abstract:

The association between loss of cell-cell adhesion and acquisition of enhanced cell migration during early stages

of cancer progression is not well understood. We showed that human skin equivalents (HSEs) harboring

E-cadherin-deficient, early-stage tumor cells mimic premalignancy and are linked to a switch to highly invasive

carcinoma. As FAK (Focal Adhesion Kinase) and Src kinases play key roles in cell adhesion, migration and

invasion, we studied their contribution to the migratory properties of E-cadherin-deficient cells in 2D cultures

and in 3D, bioengineered HSEs. Upon scrape-wounding, E-cadherin-deficient cultures demonstrated single cell

scattering and accelerated wound closure, while E-cadherin-competent cells migrated slowly as polarized cell

sheets. Immunostaining of E-cadherin-competent cells revealed focal localization of FAK at the cell periphery

and of Src at cell-cell borders. In contrast, E-cadherin-deficient cells demonstrated a perinuclear distribution of

FAK and Src, and immunoblotting revealed elevated FAK expression and increased FAK and Src tyrosine

phosphorylation. A pharmacological approach to inhibit cell migration demonstrated that FAK and Src

inhibitors, Tyrphostin-AG1007 and PP2, reduced the phosphorylation of these kinases and reversibly inhibited

E-cadherin-deficient cell migration. Finally, in a novel transepithelial tumor cell migration model, we showed

that loss of cell-cell adhesion was associated with increased FAK expression and allowed E-cadherin-deficient

tumor cells to individually migrate between normal keratinocytes and transverse the epithethlial-stromal

interface. Taken together, E-cadherin suppression is associated with redistribution and upregulation of FAK and

Src kinases directing a highly motile tumor cell phenotype. These kinases may contribute to the transition from

precancer to malignancy during squamous cell carcinoma development. Therapeutic approaches designed to

target these kinases and block transepithelial tumor cell migration may pave the way towards elimination of

such lesions and cancer prevention.

80


Quality of Life in Patients with Resected and Reconstructed Mandibles

Authors:

Daniel Oreadi, Kalpakam Shastri, Robert Chapman, Maria Papageorge

Presented by:

Daniel Oreadi

Departments:

Departments of Oral and Maxillofacial Surgery and of Prosthodontics and Operative Dentistry, Tufts University

School of Dental Medicine

Abstract:

Objective: The aim of this study was to assess the quality of life and oral health impact in patients who have

undergone extensive mandibular resections for the treatment of both malignant and benign pathologies.

Methods: Twenty patients; 10 males and 10 females, ages 20 to 67, with a mean age of 46.2, different ethnic

backgrounds, and diagnosed with a variety of head, neck and oral pathologies, including Squamous Cell

Carcinoma (SCC) (15%), Ameloblastoma (65%), Glandular Odontogenic Cyst (GOC) (5%), Odontogenic

Keratocyst (OKC) (5%), Fibrous Dysplasia (5%) and Fibromyxoma (5%), underwent resection as primary

treatment of their respective condition with either immediate (80%) or delayed reconstruction (20%).

Results: Seventy percent of the patients to date have been restored to function and the remaining 30% are in

progress. A quality of life questionnaire (QOLQ) was administered to each patient along with an Oral Health

Impact Profile (OHIP). Evaluation of neurosensory function was also assessed and data were collected based on

severity of complaints and problems reported by patients on a 1 to 5 scale that ranged from mild (2) to moderate

(3) to severe (4-5).

Conclusion: The surgical treatment of patients with oral malignant and benign pathology is considered one of

the most difficult tasks oral and maxillofacial surgeons face while attempting to preserve function and esthetics

as much as possible with the ultimate goal of a disease free patient. Currently, different modalities allow us to

provide adequate treatment to successfully treat these patients while obtaining good results in terms of function

and esthetics. However, quality of life is affected to some degree, which varies from decreased sensory function

to inability to perform daily routine activities.

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