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_____________________________________________________________________________________________ Pennsylvania Department of Health 2012-2013 Annual C.U.R.E. Report Thomas Jefferson University - 2012 Formula Grant Page 1 Thomas Jefferson University Annual Progress Report: 2012 Formula Grant Reporting Period January 1, 2013 June 30, 2013 Formula Grant Overview Thomas Jefferson University received $2,776,880 in formula funds for the grant award period January 1, 2013 through December 31, 2016. Accomplishments for the reporting period are described below. Research Project 1: Project Title and Purpose The BRM (brahma) Atpase as a Gatekeeper for Prostate Cancer Development and Progression There is a critical need to discern the mechanisms that govern prostate cancer development and progression. Based on our published and preliminary data, it is evident that the Brm ATPase plays a significant role in both processes. Our findings strongly support a model wherein loss of Brm functions as a tumor suppressor by modulating cell cycle progression and checkpoints. Moreover, “weakened” BRM activity cooperates with loss of the p27 tumor suppressor to induce hyperplastic phenotypes and castrate-resistance. Studies will determine the molecular mechanisms by which p27kip1 is induced by Brm loss (Aim 1), the impact of p27kip1 as a barrier to Brm-loss induced tumorigenesis and ADT-resistance in vivo (Aim 2), and the impact of coordinate p27kip1 and Brm loss in human disease (Aim 3). Anticipated Duration of Project 1/1/2013 12/31/2016 Project Overview Prostate cancer (PCa) is the most frequently diagnosed malignancy and second leading cause of cancer death of men in the USA. While local disease can be effectively treated through radical prostatectomy or radiation therapy, no durable means of treatment has been identified for non- organ confined PCa. First line therapeutic intervention for this stage relies on chemical castration, referred to as androgen deprivation therapy (ADT), as PCa is exquisitely dependent on the action of the androgen receptor for cell survival and proliferation. However, ADT- resistant, lethal tumors form within 2-3 years for which no effective therapeutic option has been identified. Thus, there is a significant need to understand the mechanisms that lead to prostate cancer development and promote castration-resistant tumor growth.

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Page 1: Thomas Jefferson University Annual Progress … Jefferson...Thomas Jefferson University Annual Progress Report: 2012 Formula ... Research Project 1: ... Brm functions as a tumor suppressor

_____________________________________________________________________________________________

Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report

Thomas Jefferson University - 2012 Formula Grant – Page 1

Thomas Jefferson University

Annual Progress Report: 2012 Formula Grant

Reporting Period

January 1, 2013 – June 30, 2013

Formula Grant Overview

Thomas Jefferson University received $2,776,880 in formula funds for the grant award period

January 1, 2013 through December 31, 2016. Accomplishments for the reporting period are

described below.

Research Project 1: Project Title and Purpose

The BRM (brahma) Atpase as a Gatekeeper for Prostate Cancer Development and Progression –

There is a critical need to discern the mechanisms that govern prostate cancer development and

progression. Based on our published and preliminary data, it is evident that the Brm ATPase

plays a significant role in both processes. Our findings strongly support a model wherein loss of

Brm functions as a tumor suppressor by modulating cell cycle progression and checkpoints.

Moreover, “weakened” BRM activity cooperates with loss of the p27 tumor suppressor to induce

hyperplastic phenotypes and castrate-resistance. Studies will determine the molecular

mechanisms by which p27kip1 is induced by Brm loss (Aim 1), the impact of p27kip1 as a

barrier to Brm-loss induced tumorigenesis and ADT-resistance in vivo (Aim 2), and the impact

of coordinate p27kip1 and Brm loss in human disease (Aim 3).

Anticipated Duration of Project

1/1/2013 – 12/31/2016

Project Overview

Prostate cancer (PCa) is the most frequently diagnosed malignancy and second leading cause of

cancer death of men in the USA. While local disease can be effectively treated through radical

prostatectomy or radiation therapy, no durable means of treatment has been identified for non-

organ confined PCa. First line therapeutic intervention for this stage relies on chemical

castration, referred to as androgen deprivation therapy (ADT), as PCa is exquisitely dependent

on the action of the androgen receptor for cell survival and proliferation. However, ADT-

resistant, lethal tumors form within 2-3 years for which no effective therapeutic option has been

identified. Thus, there is a significant need to understand the mechanisms that lead to prostate

cancer development and promote castration-resistant tumor growth.

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The hypothesis is that Brm serves as a barrier to prostate tumorigenesis and lethal tumor

phenotype progression, but that the consequence of Brm loss could be exploited for therapeutic

gain.

Aim 1: Determine the mechanisms of Brm-loss induced p27kip1 induction. Our data indicate

that p27kip1 serves as a critical barrier to tumorigenesis associated with Brm loss. Here, the

underlying mechanisms of p27kip1 regulation will be determined.

Aim 2: Impact of p27 as a barrier to Brm-loss induced tumorigenesis and ADT-resistance. Aim 1

will identify the underlying mechanisms by which the p27kip1 checkpoint is induced as a barrier

to tumorigenesis after Brm loss. Here, the consequence of p27 induction will be assessed, by

determining the impact of this event on tumorigenesis and progression to castrate-resistant, lethal

disease.

Aim 3: Determine the impact of coordinate p27 and Brm loss in human disease.. Given the

strong correlation between these events in human disease, here we will examine clinical

correlates of dual p27kip1 and Brm loss.

Principal Investigator

Karen Knudsen, PhD

Professor

Thomas Jefferson University

125 S. 9th Street

Philadelphia, PA 19107

Other Participating Researchers

William Ostrander – employed by Thomas Jefferson University

Expected Research Outcomes and Benefits

It is expected that these studies will provide a monumental leap in the understanding of BRM

and tumor suppressor function in prostate cancer. If successful, the outlined strategies will

provide the justification for developing future pre-clinical strategies to effectively manage

tumors with loss of BRM. In summary, the studies outlined in this project will have translatable

outcomes and completion of this work will:

1. Lead to the development of new tumor markers.

2. Reveal insight for novel therapeutic design.

3. Uncover new areas of prostate cancer research.

4. Define the role of chromatin remodeling components in a therapeutic model.

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Summary of Research Completed

Aim 1

Goal: Determine the mechanisms of Brm-loss induced p27kip1 induction. Our data indicate that

p27kip1 serves as a critical barrier to tumorigenesis associated with Brm loss. Here, the

underlying mechanisms of p27kip1 regulation will be determined.

During this reporting period, we made significant progress toward understanding Brm regulation

in prostate cancer. First, we showed that Brm and its requisite cofactor in prostate cancer, the

BAF57 SWI/SNF subunit, controls genes associated with acquisition of the metastatic

phenotype. These data reveal for the first time that Brm and SWI/SNF alterations (especially

BAF57) impinge on cell migration and invasion.

Purpose:

BAF57, a component of the switching-defective and sucrose nonfermenting (SWI/SNF)

chromatin-remodeling complex conglomerate, modulates androgen receptor activity to promote

prostate cancer. However, the molecular consequences of tumor-associated BAF57 expression

have remained undefined in advanced disease such as castration-resistant prostate cancer and/or

metastasis.

Experimental design:

Clinical human specimens of primary and metastatic prostate cancer were

immunohistochemically examined for tumor-grade association of BAF57 expression. Global

gene expression analyses were conducted in models mimicking tumor-associated BAF57

expression. Aberrant BAF57-dependent gene expression changes, bypass of androgen-mediated

signaling, and chromatin-specific SWI/SNF complex alterations with respect to cytoskeletal

remodelers such as integrins were validated. Cell migration assays were used to profile the

biologic phenotypes conferred under conditions simulating tumor-derived BAF57 expression.

Results:

Immunohistochemical quantitation of primary human specimens revealed that BAF57 was

significantly and aberrantly elevated as a function of tumor grade. Critically, gene expression

analyses showed that BAF57 deregulation circumvented androgen-mediated signaling, elicited

α2 integrin upregulation, and altered other SWI/SNF complex components at the α2 integrin

locus. BAF57-dependent α2 integrin induction conferred a prometastatic migratory advantage,

which was attenuated by anti-α2 integrin antibody blockade. Furthermore, BAF57 was found to

be markedly upregulated in human prostate cancer metastases of the lung, lymph node, and dura.

Conclusion:

The findings herein, identifying tumor-associated BAF57 perturbation as a means to bypass

androgen-signaling events that facilitate novel prometastatic phenotypes, link BAF57

upregulation to tumor dissemination. These data thereby establish BAF57 as a putative marker of

metastatic potential that could be leveraged for therapeutic intervention.

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Aim 2

Goal: Impact of p27 as a barrier to Brm-loss induced tumorigenesis and ADT-resistance.

Studies for this aim have commenced which will assess the consequence of p27 and SWI/SNF

(Brm) alterations on tumorigenesis and progression to castrate-resistant, lethal disease. Major

findings to date are:

As shown below in Figure 1, we have made excellent progress towards this end by first assessing

the incidence of Brm alterations in human disease. Nuclear Brm and SWI/SNF expression is lost

as a function of tumor development and progression, and frequently co-occur with loss of p27.

These studies lend further importance to testing the impact of p27 and SWI/SNF alterations in

models of human disease.

As shown in Figure 2, analyses of a second cohort by immunohistochemistry strongly supports

the contention that nuclear Brm function is lost in advanced disease, thus showing for the first

time that not only is Brm expression reduced as a function of disease progression, but that

subcellular localization is altered. These new findings shed significant new light into Brm

alterations and the clinical ramifications of these events.

Aim 3

Goal: Determine the impact of coordinate p27 and Brm loss.

Progress has begun for this sub-aim by generating the double null p27-/- Brm-/- animals.

Multiple lines have been successfully generated and male mice are now being aged to assess

phenotypes.

Figure 1: p27 and SWI/SNF alterations frequently co-occur in advanced disease. As shown,

genomic analyses of human metastatic prostatic adenocarcinoma co-occur with high frequency.

The Odds Radio for co-occurrence is 18.75, with a 95% confidence interval of 2.756966 -

127.517905, and a p-value of p-value: 0.001994 [Fisher's Exact Test].

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Figure 2: Brm expression and subcellular localization is altered as a function of disease

progression. As shown, examination of Brm expression by immunohistochemistry strongly

demonstrates that not only is Brm expression reduced in advanced disease, but that the nuclear

function of this SWI/SNF ATPase is lost through mislocalization to the cytoplasm as a function

of disease progression.

Research Project 2: Project Title and Purpose

Modulation of Human Breast Cancer Growth In Vivo by Activated Human Breast Fibroblasts –

The work is part of a long-term objective of providing new experimental models for more

accurate drug response testing of human breast cancer in mice. Many drugs work well when

tested on human breast cancer xenotransplant lines in mice, but fail in patients. The focus of this

proposal is to characterize growth and drug-responsiveness of human breast cancer

xenotransplants in mice with or without humanized tumor stroma. We hypothesize that novel

human mammary fibroblast lines will humanize the stromal microenvironment and modulate

tumor growth and drug responsiveness more accurately.

Anticipated Duration of Project

1/1/2013 – 12/31/2013

Project Overview

Despite enormous expenditures and efforts by academic, government, and pharmaceutical

institutions, most drugs that show promise against human breast cancer in preclinical testing in

mice, fail to cure breast cancer in the clinic. The inability to predict whether candidate drugs

will work in patients is very costly, and key resources are spent on clinical trials of drugs that

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most often fail. There is a great need for more accurate predictive preclinical testing to identify

the best drug candidates for human breast cancer.

Consistent with the national initiatives to stimulate translational and interdisciplinary efforts to

accelerate medical discovery, our laboratory’s long range goal is to facilitate faster development

of effective breast cancer therapies. To this end, we have genetically engineered mice to more

accurately mimic the hormonal environment in patients. We will now extend these efforts to

employ novel strategies to humanize the stroma of human tumor xenotransplants by testing a

panel of five previously undescribed immortalized human mammary fibroblast lines.

The primary objective is to establish new experimental xenotransplant models to achieve more

accurate predictive drug response testing of human breast cancer xenotransplants. A secondary

objective is to explore the consequences of a humanized tumor stroma on breast tumor biology,

including growth, angiogenesis and progression. We hypothesize that human mammary

fibroblasts with myofibroblast characteristics will humanize the stromal microenvironment and

modulate tumor growth and drug responsiveness to better mimic conditions in patients.

Aim 1) Characterize a panel of human mammary fibroblast lines established from discarded

tissue from reduction mammoplasties for features common to activated fibroblasts.

Aim 2) Test the ability of select human mammary fibroblast lines to modulate tumor biology and

drug responsiveness of xenotransplanted human breast cancer lines in prolactin-humanized mice.

Principal Investigator

Hallgeir Rui, MD, PhD

Professor

Thomas Jefferson University

125 S. 9th

Street

Philadelphia, PA 19107

Other Participating Researchers

Alicia Yanac, MS – employed by Thomas Jefferson University

Expected Research Outcomes and Benefits

By the end of this project we expect to have accomplished the following outcomes.

Outcome 1 – Characterization of five novel human mammary breast fibroblast lines, with

emphasis on identifying lines with features of activated fibroblasts/myofibroblasts. We expect

based on preliminary data that the cell line HMF1 will be validated to display key features of

activated human fibroblasts. Such a cell line may then be used for co-culture or co-implantation

studies by our laboratory and shared with other scientists.

Outcome 2 – Validation of the ability of human mammary fibroblasts to modulate tumor growth,

biology and drug responsiveness of human breast cancer xenotransplants in mice. We expect

that the data will support the concept that a humanized stroma provides a more representative

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model system for biology and drug response testing in vivo. Human breast cancer xenotransplant

models with humanized stroma may become a standard for preclinical testing of drugs against

breast cancer.

Summary of Research Completed

We are pleased to report solid progress during the first six months of this project.

Novel Progress related to Aim 1

Purpose: Characterize a panel of human mammary fibroblast lines established from discarded

tissue from reduction mammoplasties for features common to activated fibroblasts.

Screening primary human mammary fibroblasts.(HMFs). Primary HMFs were isolated from

surgically excised non-malignant mammary tissues, which were obtained from reduction

mammoplasty or mastectomy. HMFs were then cultured in vitro. The purity of fibroblasts was

confirmed by universal expression of vimentin and lack of cytokeratin expression using

immunofluorescent microscopy (data not shown). We then examined the expression of alpha

smooth muscle actin (α-SMA), a marker for activated fibroblasts, in a panel of five isolated

primary HMFs by immunofluorescent staining (Fig1A). Most HMF-1 cells had medium to high

level of α-SMA, while almost none of HMF-2 cells expressed α-SMA at detectable levels. Low

percentage of HMF-3, 4 and 5 expressed α-SMA at detectable levels.

To immortalize HMF-1 for long-term use, we introduced a lenti-viral vector (pLoxP-hTERT-

IRED-TK, Addgene, MA, USA) in HMF-1. We then compared the immortalized line of HMF-1

(hTERT HMF-1) to the primary HMFs for α-SMA expression by western blotting (Fig1B).

hTERT HMF-1 and HMF-1 expresses similar levels of α-SMA. Consistent with the staining

results in Fig1A, HMF-2, 3 and 4 had very low levels of α-SMA, and HMF-5 expressed

intermediate levels of α-SMA. Further examination by α-SMA staining showed immortalized

line of HMF-1 maintained the high expression level of this marker as in the primary HMF-

1(Fig1C). Therefore, the line of hTERT HMF-1 was a good candidate to be used as activated

mammary fibroblasts.

Characterization of HMFs in vitro. We then characterized the hTERT HMF1 to determine

whether it shared the functional characteristics of activated fibroblasts. HMF-2 was also

included since it had much lower expression levels of α-SMA. We first examined the fibroblasts

for mRNA expression of a group of molecules that were reported to be up-regulated in activated

fibroblasts and are important mediators for their cancer-promoting function. A luminal breast

cancer cell line, MCF-7, was included in gene expression assays as baseline (Fig 2). We found

both hTERT HMF-1 and HMF2 had higher levels of SDF1α, SDF1β, VEGF, IL6, podoplanin

and HGF than MCF7. Between the two HMFs, hTERT HMF-1 had significantly higher levels

than HMF-2 for SDF-1 α (A) and β (B), VEGF (C), IL6 (D) and podoplanin (E) (Fig. 2). HMF-2

had higher HGF mRNA expression than hTERT HMF1 (Fig 2F). We also examined TGFβ gene

expression, but the levels in all three cells were not detectable by our assay.

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Next, we examined the function of HMFs by soft agar colony assays, to see whether they could

promote anchorage-independent growth of breast cancer cells in vitro. The fibroblasts were first

plated in the wells and grown to confluence. Then MCF7 cells were suspended in soft agar and

plated on top of with the fibroblasts. After 5 weeks, there were significantly more colonies with

HMFs than MCF cells alone. These result suggested both HMFs strongly promote anchorage-

independent growth of breast cancer cells in vitro (Fig. 3).

Examining function of HMFs by tumor xenotransplant in vivo. To examine whether the HMFs

promote tumor growth as activated fibroblasts would do in vivo, we tested them in a breast

cancer xenograft mouse model. hTERT HMF1 or HMF2 were admixed with MCF7 cells before

injected into the mouse mammary gland orthotopically. MCF7 cells alone were also injected as

control. The growth of xenograft tumors by each group were plotted in Fig 4A, the average

tumor sizes in both groups with HMFs were consistently bigger than the group with MCF7

alone, suggesting hTERT HMF-1 and HMF-2 both promoted tumor growth to some degree.

However, only tumors in the group of MCF7 + hTERT HMF-1 became statistically different

than MCF7 alone after day 60. In the groups of MCF7 alone or MCF7 + HMF-2 fibroblasts, the

growth of the tumors plateaued around day 40 and even started to regress thereafter. In contrast,

the group of MCF7+hTERTHMF1 tumors continued to grow throughout the experiment.

Therefore, hTERT HMF-1 indeed acted as activated fibroblasts and promoted the growth of

MCF7 tumors in vivo.

To better understand the mechanism(s), through which hTERT HMF1 promoted tumor growth,

the xenograft tumors were further studied. H&E staining of the xenograft tumors from all three

groups were shown in Fig4B. Tumors from the group of MCF7 alone comprised many big

tumor nests with granular features. In contrast, tumors from the groups with HMFs showed that

the cancer cells were more uniformly dispersed in the stroma in much smaller clusters,

resembling higher grade cancer. SMA staining demonstrated the presence of activated

fibroblasts in all the tumors. While SMA positive fibroblasts surrounded the tumor nests in the

group of MCF7 alone, the distribution of these cells intermingled with cancer cells in the groups

with HMFs. Staining of collagen in these tumors again confirmed the distinct distribution of

tumor and stromal fibroblasts in tumors arose from MCF7 alone or MCF and HMF admixture.

To examine the contribution of HMFs in the tumors, we also stained for human vimentin, a

marker for HMFs in mouse xenograft tumors. The presence of HMFs was confirmed in the

tumors arose from MCF7 and HMF admixture after 85 days. But they only accounted for a

small portion of the tumor and their number did not differ significantly between the two groups

(data not shown), suggesting they did not contribute to the size difference of the tumors.

To find the mechanism underlying the growth difference of the xenograft tumors, we examined

the xenograft tumors for their proliferation and angiogenesis. Ki67 staining revealed

significantly higher percentage of Ki67 positive cancer cells in the groups with HMFs, indicating

both HMFs promoted proliferation of MCF7 in vivo (Fig4C). The group with hTERTHMF-1 had

only slightly higher Ki67positive cancer cells than the group with HMF-2 (83% vs 79%;

p<0.05). The tumors were also stained with anti-CD31 antibody to determine the number of

micro blood vessels present, an index for angiogenesis. Fig4D shows that the group with hTERT

HMF-1 had significantly more micro vessels than the groups with HMF-2 or MCF7 alone, while

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there was no difference between MCF-7 alone and the group with HMF-2.These data suggested

hTERT HMF-1 may facilitate tumor growth mainly by enhancing angiogenesis and promoting

proliferation.

Conclusions: Once fibroblasts get in contact with tumor cells, they could become activated and

then, in turn, aid in the development, progression, and angiogenesis of breast carcinomas.

Therefore, activated fibroblasts are an important component of tumor microenvironment. When

modeling invasive breast cancer in preclinical animal models, the presence of human activated

fibroblasts would better recapitulate the cancer stroma in xenograft studies of human breast

cancer in mice. Despite of recent efforts of generating CAFs through series of procedures of in

vitro and in vivo manipulation, the availability of activated mammary fibroblasts remains limited

and may hinder the progress of research and new therapy discovery. In this study, we aimed to

establish and characterize activated HMFs for future breast cancer studies.

Aim 2. – No progress during this reporting period.

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HMF-3

Control

HMF-4 HMF-5

HMF-1 HMF-2A

Figure 1. Screening of HMFs for activated fibroblasts. A. a panel of primary HMFs isolated from

human mammary tissues was stained for α-SMA. Control was stained with only secondary

antibody. B. hTERT-immortalized HMF-1 and the panel of primary HMFs were examined for α-

SMA expression by western blotting. GAPDH, a housekeeping protein, was used to demonstrate

loading. C. hTERT HMF-1 has similar expression levels of α-SMA as HMF-1 by

immunofluorescent staining.

B

SMA

GAPDH

C

HMF-1 hTERT HMF-1

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Figure 2. Expression of secreted factors and membrane proteins by HMFs. HMFs have elevated

mRNA expression levels of proteins associated with activated fibroblast specific. MCF7 cells,

hTERT HMF-1, and HMF-2 were analyzed for relative mRNA expression levels of SDF-1 α (A)

and β (B), VEGF (C), IL-6 (D), podoplanin (E) and HGF (F). One-way ANOVA was used for

statistics, then followed by Bonferroni's Multiple Comparison Test. *, p<0.05. **, p<0.01.***,

p<0.001.

Figure 3: HMFs promote anchorage-independent growth of MCF7 cells. A. Representative

pictures of colony growth in soft agar for MCF7 alone, MCF7 + HMF-2 or MCF7 + hTERT

HMF-1are shown. B. Colonies were counted in each field and an average of all the fields (n=8)

were calculated. For comparison among the three groups, one-way ANOVA was used, then

followed by Bonferroni's Multiple Comparison Test. ***, p<0.001.

A

MCF7 MCF7 +

HMF-2

MCF7 +

hTERT

HMF-1

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Figure 4: HMFs promote tumor growth of MCF7 cells in vivo. hTERT HMF-1 promoted breast

cancer growth, angiogenesis and progression in vivo. A. The growth of MCF7 alone, or MCF7 +

HMF-2 or MCF7 + hTERT HMF-1 xenografts in vivo was followed over time (n≥8). Two-way

ANOVA was used to compare the size of xenograft tumors. B. H&E, α-SMA and collagen

staining of xenograft tumors at 100x magnification. C. The xenografts were stained for Ki67 and

percentages of positive MCF7 cells in each group were compared. D. the xenografts were stained

for CD31 to label micro vessels. The vessels counts were plotted and compared among the three

groups. Three random fields of each tumor were used for analyses. For comparison among the

three groups, one-way ANOVA was used, then followed by Bonferroni's Multiple Comparison

Test. *, p<0.05; **, p<0.01; ***, p<0.001.

B MCF7

alone

MCF7 +

HMF-2MCF7 +

hTERT HMF-1

H&E

α-SMA

collagen

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Research Project 3: Project Title and Purpose

Creating and Phenotyping New Mouse Models of Cancer Cachexia – Cachexia, or

dysmetabolism leading to muscle and fat wasting, diminishes quality of life and response to

therapy in cancer patients. Cachexia afflicts most patients with tobacco-related cancers,

including up to 80% of those with lung cancer. Cachexia itself, distinct from other tumor effects,

causes ~1/3 of cancer deaths. Despite this, few animal models of cancer cachexia have been well

characterized and ~4 are commonly used. The purpose of this project is to create and phenotype

new mouse models of cancer cachexia. Mice with tumors of mouse and human origin will be

subjected to clinical, pathological, histological, and molecular profiling in order to expand the

models available to the research community.

Anticipated Duration of Project

1/1/2013 – 6/30/2014

Project Overview

The long-term goal of my research is to define the key regulatory pathways that mediate muscle

wasting in cancer, for the purpose of developing therapeutics to combat cachexia. Cancer

cachexia is driven by the host response to the tumor, and by factors produced by the tumor which

effect changes in central and peripheral pathways modulating activity, food intake, energy

expenditure and metabolism. Experimental analysis of cachexia has centered on a handful of

mouse models, with a candidate cytokine or single gene/pathway approach to analysis. While

this approach has led to the identification of several pathways as peripheral mediators of

cachexia, many other pathways are likely involved. Identifying those is essential to find

therapeutic targets for this currently incurable condition. The application of modern molecular

techniques to existing models of cancer cachexia will enable the discovery of new cachexia

drivers. Moreover, the existing mouse models cover only colon cancer, melanoma and a form of

lung cancer. Substantially greater numbers of models across different tumor types are necessary

to determine the relevance of known and novel pathways across the spectrum of cancer types.

The objective of this project is to perform molecular phenotyping of models of cancer cachexia

to stimulate discovery of new drivers and targets. While physiological data including muscle

weights and piecemeal gene/protein expression data are available in about 12 rodent models of

cachexia, there is a paucity of large-scale data molecular data. Indeed, across all models, there

are publicly available data on multiplex serum cytokines and muscle gene expression for only a

single model, the C26 model, from my laboratory. Specific Aims are:

Aim 1: Establish models of cancer cachexia.

Aim 2: Characterize the systemic and tumor pathophysiology associated with the models.

Aim 3: Quantify cytokine levels and muscle wasting activity in these models.

Aim 4: Perform muscle microarray analysis to identify genes and pathways regulated in common

across models or uniquely within models.

Aim 5: Collect serum, cell lines, tumors and tissues to build a resource for future analysis and

sharing with the research community.

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Principal Investigator

Teresa A. Zimmers, PhD

Associate Professor

Thomas Jefferson University

125 S. 9th Street

Philadelphia, PA 19107

Other Participating Researchers

Zongxiu Zhang – employed by Thomas Jefferson University

Expected Research Outcomes and Benefits

The project objective is to perform molecular and pathophysiological profiling of existing and

new mouse models of cancer cachexia in order to stimulate discovery. There are five clear

outcomes, all of which will benefit the research community and promote understanding of the

molecular underpinnings of cachexia. First, this work will expand the number of well-

characterized cachexia models by systematically evaluating the cachexiogenic potential of

different cell lines representing a spectrum of cancer types and sub-types. This is important to

promote research beyond the 3 most-oft used models and expand upon the total of 12 models in

the literature. Our published data on these new models will permit others to use them with

confidence. Second, this work will provide large, quantitative datasets to be deposited in public

databanks, including multiplex cytokine profiling and muscle gene expression. Analysis of those

data will permit hypothesis testing (e.g., Is increased IL-6 associated with STAT3 target gene

expression across models of cachexia?), as well as discovery. Investigators in other laboratories

will be able to query their genes/pathways of interest in these well-characterized datasets. Third,

class prediction analysis on the collected datasets across all the models and time-points analyzed

will determine whether there are particular cachexia phenotypes that are similar within or across

models. Identification of such independent phenotypes would represent a paradigm shift in the

field of cachexia research, which collective assumes all cancer cachexia is alike. Fourth, the

work will result in identification of murine cachexia gene signatures. Fifth, the banking of the

serum, cell lines, tumors and tissues will permit future analysis when funds are available. In all,

the project will be a major leap forward for the world-wide cancer cachexia research effort.

Summary of Research Completed

This progress report details results obtained from January 2013 through June 2013. Overall, we

have established one new mouse model of ovarian cancer cachexia, two of colon cancer

cachexia, and six of pancreatic cancer (Aim 1). We have characterized the systemic and tumor

pathophysiology for all (Aim 2). We have begun to quantify serum cytokine levels and muscle

protein expression (Aim 3). In the murine pancreatic cancer model, we have observed that gene

expression will cluster by cell line (Aim 4). Finally, we have banked the serum, cell lines, tumors

and tissues obtained in these models for further analysis of gene expression and for sharing of

data and tissues (Aims 5).

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SPECIFIC AIMS 1-3: Establish models of cancer cachexia. Characterize the systemic and tumor

pathophysiology associated with these models. Quantify cytokine levels and muscle wasting

activity. Hypothesis/need: Cachexia is generally discussed as a single entity, while patient

characteristics and physician clinical intuition indicate that there are likely multiple cachexia

drivers and phenotypes even within a single tumor type. The paucity of data available on murine

models limits the scope of cachexia research. Approach: Cell lines of human and murine origin

were engrafted into mice, either subcutaneously or orthotopically and the cachectic phenotype

was evaluated.

Results to date:

Ovarian cancer: Ovarian cancer often presents at late stage with disseminated disease, ascites and

evidence of inflammation and muscle wasting. To model this, female athymic nu/nu mice were

injected i.p. with 107 OVCAR-3, SK-OV-3 or ES-2 human ovarian cancer cells to mimic

disseminated abdominal disease. By 60 days, the OVCAR-3 and SK-OV-3 cell lines had not

resulted in detectable tumors. In contrast, beginning 8 days after inoculation, ES-2 cells caused

severe cachexia with marked loss of body weight and massive ascites accumulation (Fig. 1). ES-

2 cells infiltrated the pancreas, diaphragm and other abdominal organs, forming multiple solid

tumors of histology consistent with clear cell carcinoma. Muscles, including tibialis,

gastrocnemius, quadriceps and heart of tumor-bearing mice appeared markedly smaller than

normal controls (~-20% vs. control; p<0.01). Fat pads and spleen were reduced in size, while the

liver was unchanged. Piximus imaging revealed decreased bone mineral density (-16% vs.

control; p<0.05). Muscle loss was due generally to fiber atrophy, as evidenced by reduced

average myofiber cross sectional area in tibialis, although some fibers were hypertrophic. In

quadriceps muscle, ES-2 cachexia was associated with high levels of phosphorylated STAT3

(pSTAT3), whose causal role in tumor-associated muscle wasting was previously reported in our

laboratory, and also high levels of pSTAT5, which might be associated with the hypertophic

fibers.

In order to understand whether tumor-derived factors were directly responsible for the wasting

effects observed in vivo, differentiated C2C12 murine myotubes were exposed to conditioned

medium from ES-2 cell cultures and evaluated for effects on myotube size. After 48 hours, the

average diameter of C2C12 myofibers was severely reduced (-28% vs. control; p<0.001). These

effects were evident at a low concentration of supernatant and were associated with elevated

pSTAT3 levels. Consistent with reports of human ovarian cancer cachexia, Interleukin (IL)-6

levels were dramatically elevated in both blood and ascites, as was IL-11 (blood not shown).

While IL-6 was detectable only in ascites and not in cultured ES-2 cells, IL-11 was also

expressed by cultured ES-2 cells, suggesting at least some of the IL-11 was of tumor origin.

Neutralizing antibodies against IL-6 and IL-11 together could block ES-2 conditioned medium

induced atrophy of C2C12 myotubes. Taken together, the data indicate that the ES-2 cachexia

model shares features of inflammatory cachexia with the established C26 colon adenocarcinoma

model, with unique features of ascites production, disseminated metastases and complex effects

on myofiber size.

Colon cancer (data not shown): Cachexia is frequent in colon cancer but only one model is

routinely available to investigators, C26 adenocarcinoma in Balb/c or CD2F1 mice. Thus male

athymic nude mice were injected with HT-29, CaCo-2, T-84, RKO, HCT-116 or Colo205 human

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colorectal adenocarcinoma cells. Initial studies using orthotopic implantation of 106 cells into the

cecal wall resulted in bowel obstruction, which caused morbidity and complicated the cachexia

analysis. Subsequent studies using subcutaneous implantation of 5x106 cells gave rise to

reproducible localized tumors. Of these, HCT-116 and Colo205 resulted in robust cachexia, as

indicated by reduced body mass, muscle mass and muscle fiber diameter. As with ES-2 ovarian

cancer and C26 colon cancer, HCT-116 and Colo205 cachexia were associated with

inflammation, high levels of IL-6 in serum, and elevated pSTAT3 in skeletal muscle. Culture of

C2C12 myotubes with HCT-116 or Colo205 conditioned medium or plasma from the

corresponding tumor-bearing mice resulted in myotube atrophy, indicating that tumor cells

secrete cachexiogenic factors, one of which is IL-6. Tissues were collected for Aims 4 and 5,

which are underway.

Pancreatic cancer: Some 80% of patients with pancreatic cancer develop cachexia and most will

die within 5 years of diagnosis, but no models of pancreatic cancer cachexia are in common use.

We modeled pancreatic cancer cachexia using lines of murine and human origin, implanted by

injection into the pancreas of syngeneic or athymic nude mice, respectively. Three lines were

given by David Tuveson, who derived the clonal cell lines from tumors arising in mice

engineered to express mutant Kras on a background of pancreas-specific deletion of p53.

Although they possess the same transgenes, in our hands the three lines (32043, 32047, 32908)

gave rise to tumors of different histological and systemic pathophysiology in C57BL/6 mice

(Fig. 2). At a dose of 5x106 cells injected, line 32908 was the most aggressive, causing near-fatal

cachexia in 8 days, while 32047 and 32043 were lethal within 14 days. All lines induced

anorexia, likely contributing to weight loss.

Histologically, tumors from line 32908 were the least differentiated and revealed a neurogenic

phenotype, while lines 32047 and 32043 had features consistent with adenocarcinoma. All three

lines led to loss of body mass, including muscle and fat mass, with variable effects on organ size.

Tumor cells metastasized outside the pancreas to infiltrate colon, mesenteric fat, and other

abdominal organs. All lines resulted in muscle atrophy as determined from reduced tibialis

myofiber diameter. Muscles from all mice showed elevated pSTAT3, while only line 32047

induced phosphorylation of SMAD2 in muscle. This result suggested that the drivers of muscle

wasting in the 3 lines might be biochemically distinct. Western blotting analysis of tumor lysates

showed that all lines expressed the cachexia inducing factors IL-6, myostatin and Activin,

however Activin levels in blood were highest for 32043 and 32908 (not shown). Plasma from

tumor bearing mice induced atrophy in cultured C2C12 myotubes, indicating that the tumors

induced a muscle wasting activity in the blood. Moreover, in vitro adipose wasting activity was

observed using plasma from tumor-bearing mice on cultured 3T3L1 adipocytes. Analysis is

ongoing, however thus far results indicate that the individual cachexia phenotypes are

reproducible and characteristic of each of these three tumor cell lines, even though they are

derived from identical genetic backgrounds.

A similar approach was used with cell lines of human origin in athymic nude mice (Figure 3). In

this comparison, MIA-PaCA-2 cells were the most cachexiogenic, followed by BxPC-3 then

PANC-1. MIA-PaCa-2 cells induced hepatosplenomegaly and cachexia, consistent with its high

levels of expression of IL-6 and myostatin, while BxPC-3 induced wasting without

organomegaly. Muscle wasting was proportionate to muscle pSTAT3. Cell lysates showed

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expression of the cachexiogenic cytokines IL-6 and myostatin/GDF-8, with MIA-PaCa-2 also

showing expression of Activin.

SPECIFIC AIMS 4, 5: Perform muscle microarray analysis to identify genes and pathways

regulated in common across models or uniquely within models. Collect serum, cell lines, tumors

and tissues to build a resource for future analysis and sharing with the research community.

Hypothesis: Phenotypic classification using molecular profiling will enable identification of

cachexia drivers. Approach: Blood and muscle from different models of cachexia will be

subjected to hypothesis-driven and unsupervised clustering to identify patterns associated with

cachexia.

Results to date:

In a proof-of-concept study, muscle from mice with Kras/p53 mutation driven orthotopic

pancreatic cancer cachexia were subjected to Affymetrix Gene 2.0 arrays. Unsupervised

hierarchical clustering separated 32908 from 32043, while 32047 was intermediate. A

framework for data analysis has been generated in R and pilot studies using Weighted Gene

Centered Cluster Analysis for clustering of clinical characteristics have been performed on data

from each mouse (not shown). We have banked cells, tumors, tissues and plasma from all of the

models above and have prepared samples for Affymetrix Gene arrays for RNA from quadriceps

and multiplex cytokine arrays for plasma.

METHODS:

Mice. C57BL/6J mice (Jackson Laboratories) or athymic nu/nu mice (Harlan) were used at 8-10

weeks. Ovarian cancer cells were injected i.p. Colon cancer cell lines were injected

subcutaneously. Pancreatic cancer cell lines were injected into the pancreas after laparotomy

under general anesthesia, followed by analgesia and frequent monitoring. All mice were

observed at least daily for body weight, condition and tumor growth. All mouse experiments

were approved by the Thomas Jefferson University Institutional Animal Care and Use

Committee. Cell culture and reagents. Ovarian, colon and pancreatic cancer cell lines from

ATCC or for pancreatic KPC lines, from David Tuveson, were cultured then trypsinized and

injected into mice. Murine C2C12 myoblasts (ATCC, Manassas, VA) were grown in DMEM

with 20% FBS then switched to DMEM with 2% horse serum for differentiation. C2C12

myotubes were treated for 48h with tumor cell conditioned medium or DMEM plus 2% murine

plasma from tumor bearing or control mice, then were fixed, stained for myosin heavy chain and

photographed. Myotube diameters were measured to quantify muscle wasting activity in the

plasma samples. Cytokine and protein assays. IL-6, IL-11 and Activin A levels were assayed by

ELISA. Tumor production of IL-6, myostatin or Activin A was determined by

immunohistochemistry of formaldehyde fixed frozen sections. As well, we performed Western

blotting of cultured tumor cell lysates and quadriceps muscle protein to determine the relative

contribution of circulating cytokines.

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FIGURE 1: ES-2 ovarian cancer cachexia. (A) Mice with ES-2 tumors showed reduced final body weight

(FBW) despite higher initial body weight (IBW), due in part to loss of carcass and muscle mass, fat mass,

bone mineral density and bone mineral content. (B) Hematoxylin and eosin stained sections of tibialis

muscle showed abnormal muscle histology, with overall reduction in cross sectional area despite a

spreading across the histogram (inset and C). (D) Western blotting of quadriceps muscle showed

increased pSTAT3, consistent with our establishment of a causal role of STAT3 in muscle wasting.

Phosphorylated STAT5 was also increased in vivo. (E-G) ES-2 conditioned medium caused atrophy of

C2C12 myotubes and increased pSTAT3 but reduced pSTAT5 in vitro. (H) Addition of neutralizing

antibodies against IL-11 and IL-6 blocked ES-2 conditioned medium-induced C2C12 myotube atrophy.

(I) Consistent with a causal role in ES-2 cachexia, IL-6 levels were high in ascites from ES-2 mice,

whereas IL-11 was present even in ES-2 cell lysates, suggesting it was tumor-derived.

Body weights

IBW

FBW

Car

cass

0

5

10

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(g)

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*

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*

CSA Tibialis ES-2 July 2012

Control

ES-20

1000

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CS

A (m

m2 )

-11% ***

200400

600800

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30003200

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DEXA scan ES-2

BMD (g

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BMC(g

)

Are

a (c

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(g)

Fat (g)

Total (

g)

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DEXA scan SOCS3 baseline

BM

D (g

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)

Are

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(g)

Fat (g)

Total (

g)

Fat (%

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0.2

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WT

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DEXA scan Colo205/HCT116

BMD (g

/cm

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BMC(g

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Area

(cm

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(g)

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g)

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DEXA scan RKO

BMD (g

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Area (c

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(g)

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Total (

g)

Fat (%

)0.00.10.20.30.4

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DEXA scan carcasses (James Carson, USC) 02-2013

Organs

Live

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CSA Tibialis ES-2 July 2012

Control

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0

1000

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200400600800

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Fiber size, C2C12 exposed to ES-2 ovarian cancer conditioned medium, 24-48h, 11/2012

24h

48h

0

5

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20

Fib

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Control medium25% ES-2 medium50% ES-2 medium100% ES-2 medium

Control medium: 100% ES-2 unconditioned medium

-8%

**

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***

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Fiber size, C2C12 exposed to ES-2 ovarian cancer unconditioned medium, 24-48h, 11/2012

24h

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FIGURE 2: Murine pancreatic cancer cachexia from three independent Kras/p53-driven tumor lines is

phenotypically separable. All three lines induced muscle wasting (shown here as QUAD for quadriceps)

and loss of adipose tissue or white fat (W-FAT) (A). Muscle wasting was associated with loss of tibialis

myofiber cross sectional area (CSA) (B). Western blotting analysis of quadriceps revealed uniformly high

pSTAT3 levels, but elevated pSMAD2 was observed only with 32047 tumor bearing mice, indicating a

biochemical difference in cachexia drivers in muscle (C). Plasma from tumor bearing mice caused C2C12

myotube wasting (D), likely in part due to the expression of pro-cachectic IL-6, myostatin/GDF-8 and

Inhibin-beta A in all cell lines as detected by Western blotting of cell lysates (E). ELISA of plasma from

tumor-bearing mice showed highest levels of Activin in 32908 tumor-bearing mice (not shown).

Microarray analysis revealed that while many genes are regulated in common across the three cachexia

types, each has a unique profile of gene expression, suggesting that these pathways are phenotypically

distinct.

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FIGURE 3: Characterization of cachexia induced by human pancreatic cancer cells implanted into

athymic nude mice. Analysis of spleen, liver, white fat and muscle (quad=quadriceps) weights indicates

that MIA-PaCa-2 cells induce the most muscle and fat loss with simultaneous organomegaly (A-D). The

next most cachectic mice were those bearing BxPC-3 tumors, which showed muscle and fat loss without

organomegaly. Western blotting analysis revealed that pSTAT3 was most elevated in muscle of MIA-

PaCa-2 tumor-bearing mice, followed by BxPC-3 mice. In contrast to murine pancreatic cancer cachexia,

pSMAD2 levels were not altered in muscle (E). Western blotting analysis of cell extracts shows that all

three human pancreatic cancer lines express IL-6 and myostatin/GDF-8, with detectable Activin A

expressed by MIA-PaCa-2 cells.

Research Project 4: Project Title and Purpose

Liganded Glucocorticoid Receptor Functionally Interacts with Transcription Factor Stat5 in

Human Prostate Cancer Cells – Glucocorticoids are the standard treatment of advanced prostate

cancer. Recent preclinical and clinical studies have challenged the benefits of glucocorticoids

because of potential induction of therapy resistance in malignant solid tumors. Stat5a/b is highly

critical for the viability of human prostate cancer cells in vitro and for prostate tumor growth in

vivo, and active Stat5a/b predicts poor clinical outcome of prostate cancer. We hypothesize here

that the glucocorticoid receptor (GR) functionally interacts with transcription factor Stat5a/b in

prostate cancer cells. The purpose of this work is to establish whether Stat5a/b interacts with GR

signaling in human prostate cancer cells, and if this functional co-action promotes prostate

cancer cell survival.

Anticipated Duration of Project

1/1/2013 – 12/31/2013

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Project Overview

Recent data from preclinical and clinical studies have challenged the benefits of glucocorticoids

for patients with solid tumors, including prostate cancer, because glucocorticoids may induce

therapy resistance in malignant solid tumors irrespective of the tumor origin. The cellular

responses to glucocorticoids are mediated through the glucocorticoid receptor (GR). There are

two major mechanisms of gene regulation by GR. The direct transcriptional regulation

(transactivation) occurs via binding of the GR to glucocorticoid-response elements. The indirect

regulation is mediated via functional interaction with other transcription factors.

Stat5a/b is highly critical for the viability of human prostate cancer cells in vitro and prostate

xenograft tumor growth in nude mice. This laboratory has shown that active Stat5a/b in primary

prostate cancer predicts early disease recurrence, and our recent work demonstrated that Stat5a/b

is in the active state in the majority of hormone-refractory prostate cancers.

We hypothesize that liganded GR increases transcriptional activity of Stat5 in prostate cancer

cells and promotes Stat5-induced prostate cancer cell survival. The objectives of this project are:

1) Establish whether liganded GR increases transcriptional activity of Stat5a/b, and Stat5a/b in

human prostate cancer cells. This will be achieved by determining whether liganded GR

increases transcriptional activity of Stat5 in reporter gene assays.

2) Determine molecular mechanisms underlying the functional co-action of GR and Stat5 in

human prostate cancer cells. This will be achieved by testing whether liganded GR increases

nuclear translocation of Stat5 by immunocytochemistry and physical interaction with Stat5 by

co-immunoprecipitation experiments.

Principal Investigator

Marja T Nevalainen, MD, PhD

Associate Professor

Thomas Jefferson University

125 S. 9th Street

Philadelphia, PA 19107

Other Participating Researchers

Lei Gu, MD – employed by Thomas Jefferson University

Expected Research Outcomes and Benefits

It is expected that liganded GR promotes Stat5 signaling in human prostate cancer cells. We will

determine if liganded GR increases transcriptional activity of Stat5a/b cells, and if Stat5a/b

physically interacts with GR in prostate cancer. In addition, we will determine if liganded GR

enhances tyrosine phosphorylation and nuclear translocation of Stat5a/b in prostate cancer cells.

Future studies should determine whether liganded GR alters the function of the PrlR-Jak2

complex, the Jak2 activity, or the rate of Stat5 dephosphorylation. We have shown previously

that Stat5a/b is critical for the viability of human prostate cancer cells in culture and for prostate

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xenograft tumor growth in nude mice. We have further shown that Stat5a/b is activated in high-

grade prostate cancer but not in normal human prostate epithelium, and that Stat5a/b is activated

in the majority of prostate cancer hormone-refractory and metastases. Importantly, activation of

Stat5a/b in primary prostate cancer predicted early recurrence. The results of the work proposed

here will determine if liganded GR promotes Stat5-induced transcription and prostate cancer cell

survival. Future work will determine whether activation of Stat5a/b combined with positivity for

nuclear GR would provide a more powerful marker of poor clinical outcome than active Stat5a/b

alone. In addition, it will be important to establish whether activation of Stat5a/b in advanced

prostate cancer predicts unfavorable response to glucocorticoid treatment. In conclusion, this

work is important since the co-action of active Stat5a/b with liganded GR may contribute to the

induction of survival signals in prostate cancer cells.

Summary of Research Completed

Glucocorticoids have been extensively used for the treatment of advanced prostate cancer, and

the combination of paclitaxel and dexamethasone is a standard treatment for prostate cancer

patients with castrate-resistant and/or disseminated disease. Although known for their anti-

inflammatory activity, the use of corticosteroids in prostate cancer patients does not aim to

destroy malignant cells, but rather to alleviate symptoms caused either by the treatment or by the

tumor, such as edema, inflammation, pain, lack of appetite, and electrolyte imbalance.

Glucocorticoids may also exert anti-tumor effects on castrate-resistant prostate cancer by

negative feedback on the hypothalamic-pituitary-adrenal axis, leading to a decrease in both

testicular and adrenal androgens. However, recent data from preclinical and clinical studies have

challenged the benefits of glucocorticoids for patients with solid tumors, including prostate

cancer, because glucocorticoids may induce therapy resistance in malignant solid tumors

irrespective of the tumor origin. Tissue-specific differences in glucocorticoid-induced apoptosis

versus promotion of tumor growth are thought to be due to cell-type-specific transcriptional

regulation. It has been suggested that the inhibition of cell cycle progression by glucocorticoids

may be crucial in shifting the balance of several interacting pathways towards survival.

The cellular responses to glucocorticoids are mediated through the glucocorticoid receptor (GR).

In the absence of glucocorticoids, GR is sequestered in an inactive complex with chaperone

proteins in the cytoplasm. Following ligand binding, the GR dissociates from the chaperones and

forms homodimers, which enter the nucleus. There are two major mechanisms of gene regulation

by GR. The direct transcriptional regulation (transactivation) occurs via binding of the GR to

glucocorticoid-response elements. The indirect regulation is mediated via functional interaction

with other transcription factors.

Stat5a/b is highly critical for the viability of human prostate cancer cells in vitro and prostate

xenograft tumor growth in nude mice (16). Stat5a and Stat5b, encoded by separate genes, belong

to the seven member family of Stat transcription factors. Stat5a/b is activated via

phosphorylation of a specific tyrosine residue in the C-terminus by a tyrosine kinase, such as

Jak2. Tyrosine phosphorylated Stat5a/b homo- or heterodimerizes, translocates to the nucleus,

and activates transcription by binding to specific Stat5a/b response elements of target gene

promoters. Stat5a/b is the principal signaling protein downstream of the prolactin/prolactin

receptor/Jak2 (Prl/PrlR/Jak2) pathway in normal and malignant prostate cells. Prl, in turn, is a

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mitogen and survival factor for prostate cells, and Prl protein and its receptor are expressed in

prostate cancer cells. In addition, both Prl protein expression and Stat5a/b activation, are

associated with high histological grade of human prostate cancer. We further showed that active

Stat5a/b in primary prostate cancer predicts early disease recurrence, and our recent work

demonstrated that Stat5a/b is in the active state in the majority of hormone-refractory prostate

cancers.

Here it is reported that GR is expressed in CWR22Rv1, CWR22Pc, DU145 and PC-3 human

prostate cancer cell lines. Liganded GR increases transcriptional activity of Stat5a and Stat5b.

Prostate cancer cell lines

DU145, PC-3, LNCaP, CWR22Rv1 and CWR22Pc prostate cancer cells were cultured in RPMI-

1640 (Mediatech, Manassas, VA) with 10% fetal bovine serum (Atlanta Biologicals, Norcross,

GA), 2 mM L-glutamine (Mediatech), 5 mM HEPES, pH 7.3 (Mediatech), 50 IU/ml penicillin

and 50 µg/ml streptomycin (Mediatech). LNCaP and CWR22Pc (26) cells were cultured with 0.5

nM and 0.8 nM dihydrotestosterone (DHT) from Sigma-Aldrich (St. Louis, MO), respectively.

Luciferase reporter gene assays

PC-3 cells and DU145 cells were transiently co-transfected using FuGENE6 transfection reagent

(Roche, Indianapolis, IN) with 0.25 µg of pStat5a or pStat5b, and/or pProlactin Receptor (pPrlR)

(0.25 µg), and/or human pGlucocorticoid Receptor (pGR) (0.25 µg), and either 0.5 µg of

pGASx4T109-Luc (pNTCP-Luc), pBeta-Casein-Luc, pCIS-Luc or pCyclin-D1-Luc, and pRL-

TK (Renilla) (0.025 µg) as an internal transfection control. The total amount of plasmid DNA

was normalized by the empty vector (pDNR). The cells were starved in serum-free medium and

stimulated with 10 nM human prolactin (hPrl) (NIDDK, Bethesda, MD) and/or 100 nM

dexamethasone (Dex) (Sigma-Aldrich) and/or vehicle for additional 16 h. The cells were lysed

and assayed for firefly and renilla luciferase activities using the Dual-Luciferase reporter assay

system (Promega, Madison, WI) with an automatic POLARstar OPTIMA (BMG LABTECH,

Germany). Three independent experiments with triplicate measurements were carried out using

at least two sets of plasmids prepared separately. Relative luciferase activity was obtained, and

from the mean values of each independent run, the overall mean and its standard error (S.E.)

were calculated.

Liganded GR increases transcriptional activity of Stat5a/b in prostate cancer cells.

Stat5a/b is constitutively active in prostate cancers of high histological grade and in the majority

of castrate-resistant prostate cancers. Given that the GR is expressed in clinical prostate cancers

and corticosteroids are a standard treatment for metastatic prostate cancer, the investigators

tested the hypothesis that GR and Stat5a/b signaling pathways interact in human prostate cancer

cells. Transcriptional activity of Stat5a/b was determined using luciferase reporter gene driven

by promoters regulated by Stat5a/b. Beta-Casein and Cyclin-D1 promoters both contain a

Stat5a/b binding site flanked by a non-consensus Stat5a/b site, and therefore constitute strong

tetrameric Stat5a/b binding promoters. The promoter of CIS (Cytokine Inducible SH2 containing

protein) contains four consensus Stat5a/b recognition sequences, whereas the NTCP

(4×GASpT109, sodium taurocholate cotransporting protein GAS-like elements)-luciferase

reporter gene construct contains an artificial Stat5 promoter of four Stat5a/b response elements

upstream of HSV thymidine kinase promoter. In the first set of experiments, PC-3 cells (Fig.

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1A) and DU145 cells (Fig.1B) were co-transfected with Beta-casein-luciferase (luc), Prl receptor

(PrlR), Stat5a, or Stat5b, and/or GR, serum-starved and stimulated with human Prl (hPrl) (10

nM) and/or dexamethasone (Dex) at indicated concentrations for 16 h. Upon co-expression of

active Stat5a/b with liganded GR, Prl-induced transcriptional activity of Stat5a and Stat5b was

increased by 2-4-fold in PC-3 cells (Fig. 1A) and by 2-15 fold in DU145 cells (Fig. 1B) (p<0.03)

over a wide concentration range of Dex (from 1 nM to 1000 nM). When assayed with CIS-luc,

Cyclin-D1-luc, or NTCP-luc, Prl-induced transcriptional activity of Stat5a or Stat5b was

increased by 2-3-fold in cells expressing liganded GR and Stat5a or Stat5b compared to cells

negative for liganded GR (Fig. 1C) (p<0.005).

To extend the findings to the protein level, the investigators employed Bcl-xL protein expression

as a quantifiable indicator of Stat5 transcriptional activity in CWR22Rv1 cells. The cells were

infected with adenoviruses expressing wild-type Stat5b (AdWTStat5b) and treated with hPrl or

Dex for 16 h (Fig. 1D). In line with the results obtained from the Stat5-regulated reporter gene

assays, the levels of Bcl-xL protein were increased if prostate cancer cells expressed both active

WTStat5b and liganded GR (Fig. 1D, lane 4) compared to cells expressing Dex-stimulated GR

only (lane 3) or cells expressing Prl-activated Stat5a/b only (lane 2). In summary, the data

presented here indicate that liganded GR increases transcriptional activity of Stat5a/b in prostate

cancer cells.

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Research Project 5: Project Title and Purpose

Increasing Patient-Centered Care for Patients with Early Prostate Cancer – More than 90% of

patients with newly diagnosed prostate cancer have localized disease, and therefore face the

difficult decision of whether to initiate active surveillance (AS) or active treatment (AT). Given

that AT may have serious side effects, and mortality rates are comparable for men who have AS

and those who have AT, it is important for men to make an informed decision about treatment.

We propose to conduct a pilot study to test the impact of a novel patient decision support

intervention, on AS versus AT uptake. The study will involve early-stage prostate cancer

patients who have a scheduled visit for consultation at the Jefferson Prostate Cancer

Multidisciplinary Clinic (JMDC) in Philadelphia.

Anticipated Duration of Project

1/1/2013 – 12/31/2014

Project Overview

The primary objective of the study is to pilot test a decision support intervention related to

treatment decisions [(active surveillance (AS) versus active treatment (AT)] among men with

low-risk prostate cancer seen in Thomas Jefferson’s Prostate Cancer Multidisciplinary Clinic

(JMDC). We propose to pilot test the intervention with 25 newly diagnosed patients. Because

the intervention is a pilot test, we propose a non-randomized design, such that all enrolled

patients are exposed to the intervention. More specifically, we plan to meet eligible patients in

the JMDC, immediately prior to their scheduled appointment with the clinical team. A nurse

educator will educate the patient about his treatment options and guide him through decision

counseling, which involves the patient identifying factors that are likely to influence him when

deciding between AS or AT. A summary sheet of the results of the decision counseling session

will be provided to the patient and to his clinical team to further guide discussions about

treatment preference. A member of the research team will follow-up with each patient to

document the patient’s decision about treatment, as well as a medical chart audit will verify

treatment status. We expect that the proportion of patients who choose and initiate AS will be

higher than that is observed historically in the JMDC.

Specific aims of the study are to:

1. Assess feasibility of implementing the decision counseling protocol in practice

2. Determine the proportion of patients who choose AS and AT

3. Among those patients who initially choose AS, determine the proportion that actually initiate

AS

4. Identify patient factors that are associated with choosing AS.

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Principal Investigator

Ronald E. Myers, PhD

Director, Division of Population Science, Department of Medical Oncology

Thomas Jefferson University

125 S. 9th Street

Philadelphia, PA 19107

Other Participating Researchers

Amy Leader, DrPH, MPH; Anett Petrich, MSN, RN; Anna Quinn, MPH; James Cocroft, MA –

employed by Thomas Jefferson University

Expected Research Outcomes and Benefits

Men diagnosed with early-stage, low risk prostate cancer face complex decisions about

treatment. This decision may provoke anxiety and is likely to affect clinical outcomes. It is

important to facilitate well-informed, shared decision making about these options in clinical care.

However, the literature suggests that men with early-stage, low-risk prostate cancer often make

decisions about treatment with a limited understanding of available options. Furthermore, little

research has been reported in the literature on the use of decision support interventions in this

patient population. It is expected that patients who participate in the pilot study will be more

knowledgeable about AS and AT, feel informed about treatment decision making, and report a

high level of satisfaction with the decisions that they make about their care. Furthermore,

clinicians will be able to engage patients in shared decision making about treatment and clinical

trials participation effectively and efficiently.

The proposed pilot will lay the foundation for a larger randomized, control trial aimed at

developing and testing the impact of decision counseling, a novel approach to facilitating

informed, shared decision making in cancer care.

Summary of Research Completed

Development of Protocol and Study Materials

Members of the research team met weekly to review potential materials, discuss study design

and flow, and prepare for study implementation. Materials, including study surveys and

educational charts and pamphlets, were pre-tested with three patients in the Jefferson

Genitourinary Multidisciplinary Clinic (JGMC), the location of patient recruitment. Pre-testing

involved assessing the amount of time each study component required for implementation (so as

not to disrupt the patient flow of the JGMC), as well as assessing readability and patient

comprehension of the study materials. From the pre-testing exercise, we altered or included

additional language in the educational materials, at the request of the patients. We also reduced

the number of items on certain surveys, to reduce the time burden to the patient.

Next, the research team finalized the study protocol. According to the study protocol, the nurse

educator receives a list of all the patients who have scheduled appointments at the JGMC,

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including the patient’s clinical information and diagnosis. If one or more patients on the list

meet the study’s eligibility criteria, the nurse educator attends the tumor board meeting, where

the patient cases are reviewed by the clinical team in preparation for the morning’s scheduled

appointments. At this meeting, the clinical team makes a final determination about the patient’s

tumor stage and study eligibility. The research assistant approaches each eligible patient as he

waits for his clinic appointment and assesses his interest in participating in the study. For

patients who are interested, the research assistant obtains informed consent.

Following consent, the research assistant administers a baseline survey. The survey assesses

knowledge about treatment options, health perceptions, and decisional conflict related to the

decision about treatment, as well as background and demographic characteristics of the

patient. After the patient completes the survey, a nurse educator reviews an informational page

about treatment decisions with the patient. Then, the nurse uses an online Decision Counseling

Program©

(DCP) protocol to identify decision factors (i.e. reasons) that are likely to influence the

patient to choose AS or AT. During the DCP session, the patient states how important each

factor is (factor weighting). From this information, a decision preference score that ranges from

0.000 to 1.000 is calculated. A higher decision preference score indicates a preference for AS,

while a lower score indicates a preference for AT. The nurse validates the score with the patient

and assesses that he understands and agrees with the results. A copy of the summary report is

given to the patient and the clinicians for use in shared decision making about treatment during

the clinical encounter.

Three to five days after the participant visit, the nurse educator calls him by phone and

administers a brief endpoint survey to assess the patient’s treatment decision, level of decisional

conflict, perception of decision counseling, and whether he needs further information from the

clinical team.

Thirty days after the initial visit, the research assistant contacts each study participant by

telephone to determine his treatment status. Additionally, the research assistant elicits reasons

for the decision and assesses patient satisfaction with their decision.

Six months after the visit, the research assistant conducts a medical chart audit to determine the

patient’s actual treatment decision.

All study data are entered into a Microsoft Excel spreadsheet and are analyzed using SAS

(v.9.1). For decisions about treatment, the proportion of patients who choose AS, along with an

exact binomial 95% confidence interval will be computed. We will test this proportion against

the JGMC’s historical rate via an exact binomial test. Furthermore, we will determine the

likelihood that patients actually undergo the type of treatment their preference report from the

DCP indicates that they preferred.

Study Recruitment and Enrollment

We approached 26 potentially eligible patients, as determined by the clinical team at the JGMC.

Of those approached, 23 consented and enrolled in the study. Only 3 patients declined

enrollment. Of the 23 who enrolled in the study, 16 have completed a 30-day survey and 5 have

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been reviewed by medical chart audit. Figure 1 outlines the flow of patients in the study.

Study Results

Of the 5 participants who have completed the study, 3 were Caucasian and 2 were African

American. Three participants were between the ages of 50 and 59, while 2 were 60 years or

older. Four participants had more than a high school diploma, and all 5 were married or living

with a partner. From the decision counseling session, we determined that participants identified

a total of 12 decision factors; 6 factors were in favor of AS (pro factors), while 6 were in favor of

AT (con factors). At the end of decision counseling, 4 participants weighed AS and AT equally,

while 1 preferred AS. From the baseline survey to the 30-day endpoint survey, treatment-based

knowledge for the 5 participants had increased by 18% from baseline, and decisional conflict

decreased by 47% from baseline. At the 90-day chart audit, 4 s participants had chosen AS and 1

participant had chosen AT.

Figure 1. Decision Counseling and Active Surveillance Study: CONSORT Flow Diagram

Approached:

26

Consented & enrolled:

23

Declined: 3

Endpoint Survey

Complete:

16

22

Endpoint Survey Pending: 5

Lost to follow-up: 2

Endpoint Chart Audit:

5

Endpoint Survey Pending: 11

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Research Project 6: Project Title and Purpose

Cell Fate Factor Protein DACH1 Modification in Signaling and Cancer – The DACH1 protein

has features of a tumor suppressor, however very little is known of its modification by

phosphorylation or acetylation, which may in turn govern signaling by this protein. DACH1 was

cloned as a Drosophila homologue (Dac) that blocks Epidermal Growth Factor Receptor (EGFR)

signaling. Hyperactive growth factor signaling is a common feature of terminal cancer.

Identifying new mechanisms to attenuate hyperactive growth signaling in cancer may provide

important new therapeutic options. Identifying the functional modifications of DACH1 may

allow us to target the DACH1 protein and its activity in tumors.

Anticipated Duration of Project

1/1/2013 – 12/31/2016

Project Overview

The aims of these studies are to define at a higher level of resolution of the mechanism by which

DACH1 governs cancer onset and progression. Hyperactive growth factor receptor signaling

remains active in many tumors that resist current therapies. The Retinal Determination Gene

Network (RDGN), consisting of the Dachshund (dac), eyes absent (eya), eyeless, and sine oculis

(so) genes, regulates cell fate determination in metazoans. The Drosophila dac gene was cloned

as a dominant inhibitor of the hyperactive EGFR (Elipse). Genetic deletion of Dach1 in the

mouse results in perinatal lethality, therefore, we will use conditional Dach1 knockout tri-

transgenic systems.

In previous studies, we showed DACH1 inhibits breast cellular proliferation and metastasis. The

current studies will determine the mechanisms by examining post-translational modification of

DACH1. We hypothesize that post-translational modification of DACH1 is a key signaling event

contributing to tumorigenesis and metastasis. We will determine the post-translational

modifications of DACH1 and assess their role in tumor growth and metastasis. Functional

analyses of DACH1 post-translational modification may lead to the identification of new cancer

targets.

Specific Aims:

Aim 1. Determine the mechanisms by which DACH1-phosphorylation determines lung and

breast tumor cellular proliferation.

Aim 2. Determine the mechanisms by which DACH1-acetylation determines lung and breast

tumor cellular proliferation.

Aim 3. Determine the role of DACH1 post-translational modification in chromatin occupancy

and gene expression.

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Principal Investigator

Richard G. Pestell, MD, PhD

Professor and Chairman

Department of Cancer Biology

Thomas Jefferson University

125 S. 9th Street

Philadelphia, PA 19107

Other Participating Researchers

Chenguang Wang, PhD; Xuanmao Jiao, PhD – employed by Thomas Jefferson University

Expected Research Outcomes and Benefits

Resistance to Non-Small Cell Lung Cancer (NSCLC) and breast cancer to current therapies kills

a substantial number of patients each year in the United States. New molecular targets governing

tumor onset and progression are needed urgently. DACH1 represents a novel tumor suppressor

pathway. Reduced DACH1 expression correlates with poor prognosis in NSCLC and human

breast cancer. Reintroduction of DACH1 into human mammary epithelial cells transformed with

distinct oncogenes (Ras, ErbB2, c-Myc) reverts the transformed phenotype in 3D culture.

Reversion of the tumor phenotype and the associated genotype led to the DACH1 gene being

referred to as a “commandeering” tumor suppressor. DACH1 is structurally distinct from the

current known tumor suppressors. We will identify key functional modifications of DACH1

required for inhibition of NSCLC and breast tumor growth. As DACH1 is a cell fate

determination factor, understanding the mechanisms by which DACH1 inhibits tumorigenesis

may have important implications for the role of cell fate in lung and breast tumorigenesis.

Summary of Research Completed

Substantial progress has been made with this project. We have shown that DACH1 is

phosphorylated, identified the individual phosphorylation sites and shown that these residues

regulate nuclear translocation. We have also shown that the phosphorylation sites determine the

ability of DACH1 to inhibit epithelial-mesenchymal transition (EMT) via translational

suppression of Snail.

DACH1 binds YB-1 in breast cancer cells. DACH1 has been implicated in the regulation of stem

cells of several tissue types, including muscle, neural and breast. Aproteomic analysis was

conducted in order to identify candidate DACH1-binding partners that may govern this function.

DACH1 protein complexes were prepared from HEK293T cells transfected with the FLAG-

DACH1 expression vector, and DACH1-associated proteins were resolved on a 6 – 10% percent

hydrochloride gel and silver stained. Analysis of the proteins recovered from the gel through in-

gel trypsin digestion, and in sequential ms/ms identified a ~50 kDa protein, identical to YB-1.

HEK293T cells expressing FLAG-tagged DACH1 and Myc-tagged YB-1, demonstrated the

association of YB-1 with DACH1 in immunoprecipitation-Western blotting. MDA-MB-231 cells

were used to examine the binding of endogenous YB-1 to DACH1. The MDA-MB-231 cells

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were stably transduced with an expression vector encoding DACH1 and immunoprecipitation,

conducted with a FLAG antibody directed to the amino-terminus of DACH1, co-precipitated

endogenous YB-1.

The cold-shock domain of YB-1 binds to the carboxyl terminus of DACH1. The YB-1 protein

consists of three key domains; a C-terminal domain (CTD), which contributes to DNA/RNA

binding, a highly conserved cold shock domain (CSD) which forms an antiparallel β barrel to

facilitate binding to nucleic acids as a chaperone protein, and an non-conserved variable N-

terminal domain characterized by four alternating clusters of basic and acidic amino acids, which

is both alanine and proline rich and is thought to have been involved in transactivation. Nuclear

cytoplasmic shuffling of YB-1 is controlled by both a distinct nuclear localization signal and

cytoplasmic retention signals in the CTD. HEK293T cells were transfected with a series of

FLAG-tagged YB-1 mutant expression vectors and HA-tagged DACH1 (Fig. 1A). The FLAG-IP

precipitated YB-1 fragment. Western blotting for DACH1 using the HA antibody demonstrated

the association of DACH1 with each YB-1 fragment but a failure to bind the CSD alone (Fig.

1B). Immunoprecipitation of DACH1 with the HA antibody demonstrated DACH1 binding to

the YB-1 C-terminus (Fig. 1C). In GST pulldown 6-His DACH1 bound GST-expressed YB-1.

The DACH1 DBD did not bind YB-1 (Fig. 1D). Thus, the C-terminus of YB-1 was sufficient for

binding DACH1 and the YB-1 CSD domain was incapable of binding DACH1 (Fig. 1A, B).

DACH1 exists in three isoforms (DACH1a, b, c), and each of the DACH1 isoforms were capable

of binding YB-1 with similar affinity (Fig. 1E,F). DACH1 mutant expression vectors were co-

expressed with YB-1 WT in order to define the domains of DACH1 governing association with

YB-1 (Fig. 1G). Deletion of the DNA-binding domain (ΔDBD) of DACH1 did not affect binding

to YB-1, however deletion of the C-terminus (DACH1 ΔC) abolished YB-1 binding and the C-

terminus was sufficient for YB-1 binding (Fig. 1H).

Phosphorylation determines DACH1 nuclear-cytoplasmic translocation. In order to determine

whether DACH1 is phosphorylated, cellular extracts from HEK293T cells were examined before

and after treatment with calf intestinal phosphatase (CIP). The treatment of cells with CIP

reduced the mobility of DACH1. Mass spectrometry was used to identify the phosphorylation

sites and point mutation of the residues with highest Mascot Scores was conducted. The

electrophoretic mobility of the DACH1 protein expressing a mutation of S439 was similar to the

CIP treated cells expressing DACH1 wt. By IHC, the DACH1 protein was located primarily in

the nucleus. The S439A S441 and S529 mutants were cytoplasmic. These findings suggest

DACH1 phosphorylation at S439 determines the cytoplasmic vs. nuclear location.

DACH1 inhibits and YB-1 induces breast cancer cellular EMT. As YB-1 located in the

cytoplasmic pool regulates translational control to induce EMT and DACH1 expression reduced

cytoplasmic YB-1, we examined the possibility that DACH1 expression may reduce YB-1-

dependent EMT. The MBA-MB-231 breast cancer cell line stably transduced with a

ponasterone-inducible DACH1 expression vector demonstrated the expression of FLAG-tagged

DACH1 upon the addition of Ponasterone A, which reduced the abundance of Ezrin, SNAIL and

ZEB-1 (Fig. 2A). Vinculin and GDI, two loading controls for protein abundance were

unchanged. Consistent with a role for YB-1 to induce EMT, shRNA to YB-1, reduced the

abundance of SNAIL and ZEB-1 (Fig. 2A). Immunohistochemical staining demonstrated a

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reduction in ZEB-1 upon expression of DACH1 (Fig. 2B). Similarly, shRNA to YB-1 reduced

ZEB-1 abundance (Fig. 2B). The abundance of SNAIL was reduced by either DACH1

expression (Fig. 2C) or shRNA to YB-1, (Fig. 2C). The reduction in SNAIL abundance by YB-1

shRNA in MDA-MB-231 cells is consistent with prior findings in MCF10A-Ras breast cancer

Cells. E-cadherin abundance is reduced upon EMT induction and DACH1 expression or YB-1

shRNA induced E-cadherin immunostaining (Fig. 2D). Together these studies suggest DACH1

inhibits YB-1 induced EMT .

DACH1 expression regulates nuclear cytoplasmic distribution of YB-1 and thereby YB-1-

mediated cap-independent translation of Snail. The finding that DACH1 expression of YB-1

shRNA induced EMT, without affecting expression of genes governing EMT, led us to consider

alternate mechanisms. Cytoplasmic YB-1 is known to activate cap-independent translation of

mRNA encoding Snail and other factors implicated in activators of mesenchymal genes. As

cytoplasmic, not nuclear YB-1, governs EMT, we considered the possibility that DACH1

expression may regulate the relative abundance of cytoplasmic vs. nuclear pools of YB-1.

To examine the effects of DACH1 expression, on YB-1 sub-cellular localization, IHC for

DACH1 and YB-1 was conducted on postasterone-inducible DACH1 expressing breast cancer

cell lines. Expression of DACH1 in SUM159 cells was readily detectable upon Ponasterone A

treatment in a nuclear compartment, detected by the FLAG antibody to the N-terminal tag of

DACH1. YB-1 was predominately cytoplasmic in location. Upon expression of DACH1, YB-1

became predominantly nuclear (Fig. 3A). Similar findings were made in MDA-MB-231 cells in

which expression of DACH1 was associated with increased nuclear abundance of YB-1, detected

either by immunohistochemistry (Fig. 3B) or by Western blot of nuclear vs. cytoplasmic

fractions (Fig. 3C). Although, relatively little is known of the factors associated with nuclear-

cytoplasmic shuttling of YB-1, the stimuli known to induce cytoplasmic to nuclear shuttling of

YB-1 include DNA damaging agents, wherein YB-1 binds to damaged DNA. HEK293T cells

were treated with DNA damaging agents (cisplatinum 2 mmol or paclitaxel, 2 nmol). The

addition of DNA damaging agents induced the nuclear localization of YB-1 which co-associated

with nuclear DACH1.

We had shown the DACH1 expression or YB-1 shRNA reduced SNAIL abundance. The role of

SNAIL1 as an inducer of EMT is well established. The Snail1 mRNA harbors specific motifs in

its 5’UTR that are conserved and are responsible for cap-independent translation by YB-1. YB-1

has been shown to induce cap-independent translation of genes enhancing EMT including Snail

in MCF10AT cells. (The MCF10AT subclone is derived by transformation of the immortalized

benign MCF10A human mammary epithelial cells with oncogenic Ha-Ras and subsequent

passaging through mouse xenotransplants). We examined the role of DACH1 in YB-1-dependent

regulation of Snail, and deployed a Snail1 5’UTR linked to the luciferase reporter gene.

Conditions of starvation, or starvation combined with rapamycin, restricts cap-dependent

translation. Consistent with previous observations that YB-1 induces cap-dependent translation

of Snail1 5’UTR-Luc activity, we also show that YB-1 induced Snail 5’UTR-Luc activity.

Ectopic expression of DACH1 repressed YB-1 activation (Fig. 3D). The induction of Snail

5’UTR-Luc activity by YB-1 requires the cytoplasmic pool. DACH1 expression reduces the

cytoplasmic YB-1. The phosphorylation point mutants of DACH1 are localized to the cytoplasm

and fail to undergo nuclear translocation. Consistent with a model in which DACH1 inhibits YB-

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1 cytoplasmic EMT function through inducing nuclear translocation, each of the DACH1

phosphorylation point mutants failed to repress YB-1-mediated induction of Snail 5’UTR-Luc

activity, indeed the mutants conveyed a dominant positive effect on Snail1 5’UTR-Luc activity .

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Figure 1. DACH1 binding requires the YB-1 Cold Shock Domain (CSD). A) Schematic representation of

DACH1, YB-1 and YB-1 mutant expression vectors. B) IP-Western blot was conducted of HEK293T cells transfected with expression vectors encoding either N-terminal 3x FLAG-tagged YB-1 or HA-tagged DACH1 expression vectors. In (B) the IP was conducted with an antibody to the FLAG-tag of YB-1 and in (C) the IP was directed to the Ha tag of DACH1. DACH1 binding to YB-1 was derived from N=3 separate experiments. (D) DACH1-YB-1 binding characterized in pulldown using 6-His-tagged DACH1 or DACH1 DBD region and GST-YB-1. (E-G) Schematic representation of YB-1 and DACH1 mutant expression vectors. (F-H) IP-Western blot of Myc-tagged YB-1 and associated DACH1 expression vectors transfected into HEK293T cells. Antibodies were directed for IP to the 3x FLAG tag of DACH1 and Western blot to the Myc epitope of YB-1. Antibodies are directed to the proteins as indicated. Data are representative of N=3 separate experiments. The DACH1 isoforms are indicated as DACH1a, b and c.

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Figure 2. DACH1 inhibits YB-1 induced EMT. A) MDA-MB-231 cells stably transduced with a Ponasterone A-inducible FLAG-tagged DACH1 expression vector were sequentially transduced with shRNA to YB-1. Pon A treatment (2 μM) was used to induce DACH1. Western blot was conducted with the antibodies as indicated. (B-D) Immunohistochemistry for markers of mesenchymal cells include, (B) ZEB1, (C) SNAIL, (D) E-cadherin.

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Figure 3. DACH1 promotes nuclear translocation of YB-1, inhibiting YB-1 dependent translation. Confocal

microscopic images of SUM159 (CA) and MDA-MB-231 cells (B) with ectopic expression of DACH1 using antibodies as indicated. (C) Western blot analysis of cytoplasmic and nuclear protein as indicated. (D) MCF10A-RAS cell transfected with YB-1, DACH1 and SNAIL-5’UTR Luc reporter were starved and treated with rapamycin (uM). Luciferase activity was measured after 24 hours. The data are mean ±SEM.

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Research Project 7: Project Title and Purpose

Molecular Aberrations in Adenocarcinoma and Neuroendocrine Prostate Cancer – We

hypothesize that Adenocarcinoma (AdCa) and Neuroendocrine (NE) Prostate Cancer carry

different molecular aberrations that may explain the aggressive phenotype of NE Prostate Cancer

(NEPC). Using two mouse models of prostate cancer, we plan to delineate the signaling

aberrations by which neuroendocrine prostate cancer (NEPC) differ from AdCa. In both in vivo

mouse models, we will profile all samples for aberrant expression and activities of signaling

molecules and identify the molecules which contribute to an aggressive cancer cell phenotype.

This study presents an unmet clinical need for NEPC patients with advanced disease.

Anticipated Duration of Project

1/1/2013 – 12/31/2016

Project Overview

We hypothesize that Adenocarcinoma (AdCa) and Neuroendocrine (NE) Prostate Cancer carry

different molecular aberrations that may explain the aggressive phenotype of NE Prostate Cancer

(NEPC). This study presents an unmet clinical need for NEPC patients with advanced disease.

The main goal of this proposal is to delineate the molecular aberrations that occur in AdCa and

NEPC. The specific aims of this proposal are as follows:

1) To delineate the signaling aberrations by which NEPC differ from AdCa. We will use

TRAMP and castrated PTEN conditional knock-out mouse models, which are known to develop

both AdCa and NEPC. Using gene expression analysis, AdCa and NEPC tumors will be isolated

by laser capture microdissection and analyzed. We will profile all samples in both in vivo mouse

models for aberrant expression of signaling molecules. We will analyze this signature in

TRAMP and castrated PTEN conditional knock-out mice; 20 mice will be used/group and

routine staining for synathophysin and chromogranin will be used to define neuroendocrine

differentiation. All techniques to be used, including qRT-PCR, are routinely performed in our

laboratories and have been previously described by our group.

2) To validate the findings in Aim 1 using primary and metastatic samples from NEPC tumors.

We will profile all samples in both in vivo mouse models for aberrant activities of signaling

molecules. Several measures of signaling aberrations will be performed using immunoblot

analysis to assess the differences in kinase activities, DNA-repair proteins, apoptotic response

and proliferation rates. Upon transfection of the identified aberrant signaling molecules,

anchorage-independent growth assay, proliferation, androgen receptor (AR) localization and

activity, mitochondrial isolation and import, DNA damage response and apoptosis will be

measured to confirm that the aberrant signaling contribute to an aggressive cancer cell

phenotype. The study will make use of a murine tissue bank available in our laboratory from

mice at various stages of AdCa and NEPC (primary tumors as well as metastatic tumors).

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Principal Investigator

Lucia R. Languino, PhD

Professor

Thomas Jefferson University

125 S. 9th Street

Philadelphia, PA 19107

Other Participating Researchers

Sheikh Aejaz Sayeed, PhD – employed by Thomas Jefferson University

Expected Research Outcomes and Benefits

This study will provide new insights for therapeutic intervention in patients with aggressive

neuroendocrine prostate cancer. Understanding signaling aberrations occurring in AdCa and in

NEPC will help to characterize the pathways that contribute to the aggressive phenotype found in

NEPC and develop strategies to successfully treat NEPC. We will make use of a murine tissue

bank available in our laboratory from mice at various stages of AdCa and NEPC (primary tumors

as well as metastatic tumors). Protein aberrations will be correlated with the degree of

neuroendocrine differentiation, other pathologic features and survival. This study presents an

unmet clinical need for NEPC patients with advanced disease. The fundamental knowledge

gained from these studies will clearly be beneficial and applicable to populations with

historically poor cancer prognoses.

Summary of Research Completed

We have started our investigation on the basis of our initial hypothesis that Adenocarcinoma

(AdCa) and Neuroendocrine (NE) prostate cancer carry different molecular aberrations that may

explain the aggressive phenotype of NE Prostate Cancer (NEPC). During the current reporting

period, we have focused predominantly on characterizing the available prostate mouse tissue

specimens isolated from TRAMP and castrated PTEN conditional knock-out mice (Aim 1). As

described in the first specific aim of this proposal, these mice are known to develop both AdCa

and NEPC. Prostate tissue specimens from ten TRAMP mice have been characterized. Indeed,

histological examination of these specimens shows expression of both types of cancer.

Additional analysis has been performed to demonstrate that the murine tissues express

synathophysin and chromogranin A, which are known to define NE differentiation. We have

optimized the conditions to detect, by immunohistochemistry, synathophysin and chromogranin

A, and show that all our specimens express both markers of NE cancer. A representative

example of this immunohistochemical staining is shown in Figure 1.

In parallel, we have optimized the conditions to analyze integrin expression and have stained all

ten specimens for av6 integrin. Using previously published protocols, we show that av6 integrin

is expressed in AdCa but not in NE cancer. This result will allow us to differentiate the two

types of cancer; however, if confirmed, this indicates that therapeutic approaches that target

integrins will inhibit only one type of cancer but not NE. We are in the process of characterizing

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additional specimens to reach the number requested to complete the first task planned in Aim 1.

We have also obtained NCI-H660 cells and N-Myc- LNCaP cells known to be NE cells, to

develop an in vitro system to characterize molecular aberrations in NE cancer. A preliminary

analysis shows that surface expression of cell adhesion receptors in these cells is different. We

are currently completing this characterization.

METHOD optimized in our laboratory to stain for Synaptophysin and Chromogranin A by

Immunohistochemistry.

DAY 1:

1. Place slides in glass holders, tissue facing out. Bake at 60o C for 2 hrs.

2. Wash with xylene to deparaffinize the slides: 3 x 10 mins each

During the 3rd

wash pour 10mM sodium citrate in the plastic coplin jars and heat to 95oC for

about 15 minutes (using DDH20 in the bottom of the steamer)

Cover steamer with lid and place a thermometer in the lid of the steamer with the tip of the

thermometer in the sodium citrate

Prepare a stock of 1M Sodium citrate (pH 6.0): 294g of sodium citrate (dehydrate granular

powder; J.T. Baker – Catalog # 3646-01) in 1 liter of ddH2O, bring to pH 6.0 using 1 M citric

acid (Make citric acid using monohydrate granular citric acid powder (J.T. Baker – Catalog #

0110-01). NOTE: Make a fresh 10mM solution of sodium citrate at pH 6.0 for each experiment.

3. Rehydrate the slides. Discard alcohol in sink.

a. 95% ethanol- 2 x 2 mins

b. 70% ethanol- 1 x 2 mins

c. 50% ethanol- 1 x 2 mins

d. DH2O- 1 x 2 mins

4. Briefly dip slides in warm water (< 30 seconds).

5. Place slides in coplin jars filled with hot sodium citrate. (Use tweezers for protection

from the heat). Make sure that the temperature of the sodium citrate does not rise above

100oC; (carefully open the lid of the steamer to decrease temp).

a. Place pipet tips between the edges of the coplin jar and the outermost slides.

b. Wait until the temperature of the sodium citrate returns to 95oC to start the timer;

boil for 23 mins.

c. Afterwards, take out the coplin jars individually, and allow the slides to cool for

about 20 minutes (until the sodium citrate is cool enough to touch).

d. Place into glass containers, filled with RT DH20 for 3-4 mins.

e. After the slides have reached RT, submerge them in TBS for (3-4 mins)

Prepare 100 ml of 3% of H202 and submerge the slides in it for 5 minutes. Wear gloves to discard

H202

6. Wash slides with tap water, place on shaker for 5 mins. Wash DDH20 for 2 mins, then

with TBS for 2 mins.

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7. Dry slides. Wipe. Block for 20 mins.

8. Prepare antibody (Ab) solutions into numbered eppendorf tubes: Rabbit Antibody to

Synaptophysin (Invitrogen, 18-0130); Rabbit Ab to Chromogranin A (Invitrogen,

180094). Store everything on ice. Dilute in TBS solution (TBS solution=TBS +

0.2%BSA + 0.02%Na3)

9. After blocking, wipe the back dry, and use a kimwipe to get around the tissue. Incubate

with the antibodies, using the humidity tray. Put on shelf in cold room overnight.

DAY 2:

1. Put slides in glass container. Wash with TBS 3 x 3 min each

2. Prepare secondary Ab in TBS. Incubate with secondary for 30 mins at room temperature

in the humidity tray.

a) If primary Ab was raised in rabbits, use 3 g/ml biotinylated Goat anti-Rabbit IgG

(Vector Laboratories; 1:500)

b) If primary Ab was raised in mice, use 3 g/ml biotinylated Horse anti-Mouse IgG

(Vector Laboratories; 1:500)

c) If primary Ab was mouse monoclonal IgM, use 3.3 g/ml biotinylated goat anti-mouse

IgM (Vector Laboratories; 1:150)

3. Wash with TBS 3 x 3 min each

4. Meanwhile, prepare strept-avidin peroxidase (SAP)-(0.5 units/ml; Boehringer) (1:1000 in

TBS)

a. wipe slides. Incubate for 30 mins at room temperature in the humidity tray

5. Wash with TBS-3x3 mins.

6. Incubate with di-aminobenzidine for 5 mins at room temperature, shaking, and wrapped

in aluminum foil. DAB—0.5mg/ml in TBS+0.012% H2O2

i.e. for 100mL DAB:

Dissolve 0.05ug DAB in 10mL TBS. (solution A)

Add 12uL H2O2 to 90mL H2O (solution B).

Mix solution A and solution B right before incubating the slides.

7. Wash under running tap water for 5 mins

8. Rinse in DH2O for 2 mins

9. Stain with hematoxylin for 1 min and then run under tap water for a few minutes

Dehydrate slides:

a. 50% ethanol-2 mins

b. 70% ethanol-2 mins

c. 95% ethanol-2 x 2 mins

d. xylene-3 x 2 mins

10. Wipe the slides, and mount using permount: 20 µl per slide

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Fig 1. Neuroendocrine markers expressed in mouse prostate tumors.

Mouse #5224, TRAMP, age 28.6 weeks. A: Rabbit Antibody to Synaptophysin (Invitrogen, 18-

0130); B: Rabbit Antibody to Chromogranin A (Invitrogen, 180094).

Research Project 8: Project Title and Purpose

Anoikis-Resistance in Metastatic Breast Cancer Cells and the Role of the Focal Adhesion Kinase

(FAK1) – Many breast cancer patients die after the removal of primary tumors because of

metastasis. A very aggressive type called inflammatory breast cancer (IBC) is responsible for a

disproportionate number of deaths due to its propensity to rapidly metastasize. IBC primarily

affects younger women under the age of 50 at diagnosis, and is difficult to detect as it does not

present as a lump, but rather occurs as tumor emboli. Anchorage-independent growth (anoikis-

resistance) is a crucial step in metastases. The focal adhesion kinase (FAK1) is an important

regulator of anoikis-resistance. In this project, we propose to study the mechanism responsible

of anoikis-resistance in IBC to determine if FAK1 inhibition suppresses the metastatic potential

of these cells.

Anticipated Duration of Project

1/1/2013 – 12/31/2015

Project Overview

The goal of this proposal is to understand the gene expression and protein changes involved in

the mechanism of epithelial-mesenchymal transition (EMT) and anoikis-resistance that drives

cancer cells to invade and metastasize. We hypothesize that the understanding of the molecular

mechanisms driving EMT and anoikis-resistance in inflammatory breast cancer (IBC) patients

will provide novel tools for blocking the metastatic process.

A B

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Specific Aims.

1) Characterize the process of EMT and anoikis-resistance in metastatic IBC cells. We propose

to study the expression of proteins related to EMT and anoikis resistance in cancer cells from

patients with IBC.

2) Determine if CEP-37440 suppresses breast tumor growth and metastasis in animal models.

We are going to determinate if FAK1 inhibition enhances anoikis of IBC cells and suppresses

their metastasis in vivo.

3) Determine if FAK1 is responsible for anoikis-resistance of circulating tumor cells (CTC).

Cancer cells in peripheral blood, known as CTCs, play an important role in tumor dissemination

and the characterization of these cells is fundamental to develop therapeutic targets against

metastatic lesions. CTCs have also be used in preclinical models to evaluate the various phases

of the metastatic process and the phenotypic properties of tumorigenic cells.

Principal Investigator

Massimo Cristofanilli, MD

Professor of Medical Oncology

Deputy Director of Translational Research, Kimmel Cancer Center

Thomas Jefferson University

125 S. 9th Street

Philadelphia, PA 19107

Other Participating Researchers

Sandra Fernandez, PhD; R. Katherine Alpaugh, PhD; Zhaomei Mu, MD – employed by Thomas

Jefferson University

Expected Research Outcomes and Benefits

Metastasis is the main cause of death of women with breast cancer. Epithelial-mesenchymal

transition (EMT) and anoikis-resistance, are key processes for metastasis and they share common

regulators, such as Twist, Snail, E-cadherin, and N-cadherin. Therefore, understanding the

pathways, as well as specific molecules, that critically regulate EMT and anoikis-resistance

could lead to the development of new therapeutic strategies that decrease metastatic potential of

breast cancer cells. Women with triple negative IBC often develop metastasis in the lungs, brain,

and, in some cases, the bones.

FC-IBC02, a new IBC triple negative cell line, and breast xenograft model recently developed

previously by the PI, will be used in this study. Moreover, we are going to generate SUM149 and

FC-IBC-02 cell lines that stably express the inducible FAK1 shRNA lentiviral vector, and to

study the effect of FAK1 downregulation on both primary tumor growth and metastasis

formation. The formation of distant metastasis in lungs, lymph nodes and other target organs will

be monitored in vivo. Our studies will determine if FAK1 inhibition could inhibit the metastatic

process of IBC. We propose to downregulate FAK1 using FAK1 shRNA although, we expect to

be able to test in the near future a commercially available small molecule FAK1 inhibitor. IBC is

a very aggressive form of breast cancer, and currently there is no adequate therapy to help these

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patients. If these studies are successful, the inhibition of FAK1 may be incorporated into future

adjuvant therapies to prevent local and distant metastasis in patients with breast cancers from

basal subtypes.

Summary of Research Completed

During this initial reporting period, we finalized the Animal Protocol necessary to conduct the

in-vivo studies and we obtained final IRB approval. Moreover, we were able to secure a contract

with Teva Pharmaceuticals that will provide additional funds (Direct plus indirect cost $150K)

for the conduct of this research project.

We also recently published a peer-reviewed original article describing the preclinical model used

in this project, FC-IBC02 cell line and xenograft model: Inflammatory breast cancer (IBC): clues

for targeted therapies. Fernandez et al., Breast Cancer Res Treat. 2013 Jul; 140 (1):23-33.

Research Project 9: Project Title and Purpose

Long Non-coding RNAs Regulate Protein-coding Transcripts in Colon Cancer with the Help of a

Specific Family of MicroRNAs – Decades of research have been invested towards understanding

genetic defects in colon cancer. Until recently, the lack of available tools and of critical insights

resulted in the community having paid only limited attention to the mechanisms of post-

transcriptional regulation of the colon cancer cell. The work we propose will explore unexpected

facets of microRNA-mediated regulation of colon cancer-related genes by long non-coding

RNAs. Our goal is two-fold: a) provide evidence for a novel paradigm of microRNA targeting

in colon cancer cells; and b) demonstrate that non-coding RNAs can regulate important protein-

coding genes through interactions that obviate direct molecular contact (“decoying”).

Anticipated Duration of Project

1/1/2013 – 12/31/2015

Project Overview

Colorectal cancer (CRC) is the third most frequently diagnosed cancer in the United States with

~150,000 new cases and ~50,000 deaths expected in 2012. The objective of this project is to

show that long non-coding RNAs (lncRNAs) use endogenous microRNAs (miRNAs) to regulate

protein-coding messenger RNAs (mRNAs) that are expressed in CRC; also, that many of the

interactions between these miRNAs and their targets transcend what has been known as the

“standard model” of targeting, and instead comprise heteroduplexes with one or more guanine-

uracil (G:U) wobbles and mismatches in the miRNA’s “seed” region (“expanded model”).

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Specific Aims:

Aim 1. Show that lncRNAs can alter the expression levels of mRNAs in colon cancer cells. For

two of the three lncRNAs that we have cloned (currently unpublished), we will show that

modulation of their expression can alter the expression of mRNAs in colon cancer cells. Using

commercial expression microarrays, we will identify those mRNAs that are affected following

the over-expression and silencing respectively of each of the two lncRNAs.

Aim 2. Show that the mRNAs of genes expressed in CRC are affected by a specific miR family,

and that many of the underlying interactions are not anticipated by the standard miRNA targeting

model. Using the results of Aim 1 and the rna22 target prediction tool, we will predict targets for

the specific miR-family, putting emphasis on heteroduplexes that are not expected by the

“standard” miRNA targeting model. These mRNAs may have known CRC relevance. Using

reporter assays with pre-miRNAs/scrambled-control, we will examine the validity of these novel

predictions.

Aim 3. Show that the expression of the two lncRNAs of interest is affected by a specific miR-

family of miRNAs. Using at least two miRNA target prediction tools that can process ncRNA

sequences, we will predict target sites in the two of three cloned lncRNAs as well as the

corresponding heteroduplexes for members of the specific miR- family of miRNAs. We will

again use reporter assays to demonstrate the validity of the predicted heteroduplexes.

Principal Investigator

Isidore Rigoutsos, PhD

Professor and Director, Computational Medicine Center

Thomas Jefferson University

125 S. 9th Street

Philadelphia, PA 19107

Other Participating Researchers

Yi Jing, MD, PhD; Phillipe Loher, MBA; Peter Clark, PhD – employed by Thomas Jefferson

University

Expected Research Outcomes and Benefits

This project will help advance our understanding of important regulatory interactions in CRC

biology. We expect to reveal facets of post-transcriptional regulation that involve currently

unsuspected players and novel interactions, and which could not be elucidated by classical

approaches. Preliminary work by us and others in different contexts has demonstrated that

lncRNAs can play important regulatory roles by modulating the expression of mRNAs through

competition for binding to endogenous miRNAs – such transcripts are known as ‘competing

endogenous RNAs’ (ceRNAs) and impact one another in the absence of direct molecular contact.

The two lncRNAs on which we focus are up-regulated in colon cancer cell lines compared to

normal samples and are predicted to be targeted extensively by multiple members of the specific

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miR-family of miRNAs. In both cancer and normal samples, the specific miR-family members

are present and abundantly expressed. Moreover, our analysis of public CLIP-seq data, i.e. next

generation sequencing data of RNAs obtained from UV-crosslinking followed by

immunoprecipitation with an Argonaute (AGO) antibody, revealed many miRNA:mRNA

heteroduplexes involving these miR-family members: importantly for this project, the majority

of these heteroduplexes can be discerned only under the “expanded” miRNA targeting model.

Upon completion, we will have demonstrated the existence of lncRNAs that can regulate

mRNAs in the CRC context. Also, we will have established the importance of this miR-family

for CRC biology. Lastly, we will have shed light on what is now a lurking regulatory landscape

in CRC biology and involves both known (miRNAs and mRNAs) and novel (lncRNAs) players

that participate in unexpected interactions with one another.

Summary of Research Completed

MiRNAs are known to regulate the expression levels of many proteins by directly targeting the

corresponding mRNAs. More recently, miRNAs were also shown to regulate the expression

levels of long non-coding RNA transcripts, generically referred to as lncRNAs. We have

initiated experiments to test our hypothesis that in the CRC context changes in the expression

levels of specific lncRNAs can modulate the expression of specific protein-coding mRNAs in the

absence of direct molecular interactions. If present, these ‘decoying-driven’ interactions would

involve lncRNAs and mRNAs that are directly and simultaneously targeted by endogenous

miRNAs and be the result of competition for binding to these endogenous miRNAs.

Aim 1. Show that lncRNAs can alter the expression levels of messenger RNAs in colon cancer

cells. This aim is focused on determining whether the two lncRNAs of interest can modulate the

expression of mRNAs in CRC.

First we investigated whether we could extend our preliminary findings from colon cell lines to

actual metastatic colorectal cancer (mCRC) samples. To this end we profiled de-identified

samples from patients undergoing operations for metastatic colorectal cancer. The samples were

a kind gift by Dr. Jen Jen Yeh from the University of North Carolina at Chapel Hill (UNC) and

released by the Tissue Procurement Core Facility under a UNC IRB-approved protocol; the

samples were harvested after engraftment into athymic (nu/nu) mice. As can be seen from

Figure 1, pyk-reg-10 was present in 13 and pyk-reg-83 was present in 16 of the 21 tested

samples.

Further, we sought to answer the question of tissue specificity for the two lncRNAs of interest. In

particular, we sought to determine whether the two lncRNAs of interest are present in cell types

other than colon. To this end, we profiled them in five pancreas cell lines: HPNE, CaPan-2,

MiaPaCa, PL-5, and PL-45 and found both to be present in all five cell lines (Figure 2).

Additionally, we profiled the two lncRNAs in five breast and two prostate cell lines: pyk-reg-83

was found present in all seven cell lines whereas pyk-reg-10 was present in six of the seven (data

not shown).

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In specific Aim 1, we proposed to design one or more siRNAs for the purpose of silencing pyk-

reg-10 and pyk-reg-83. Of these two lncRNAs, pyk-reg-83 is a) present in all of the tested cell

lines, b) present in the majority of the tested mCRC samples, and, c) consistently more abundant

than pyk-reg-10. We thus began our investigations by focusing on pyk-reg-83 and designed two

siRNAs against it. We have just completed testing the two siRNAs separately as well as in

combination using luciferase constructs and found them to be very effective (Figure 3). Next, we

will be testing the siRNA efficiency against the full length pyk-reg-83.

The specific family of miRNAs on which we focus is endogenous in both colon and pancreas

cells (normal and cancer) and so are the two lncRNAs of interest (see above). This co-

occurrence would suggest that any direct interactions between the miRNA family and pyk-reg-

10/pyk-reg-83 likely persist in both cell contexts. Given that we have just began the project, we

wanted to leverage miRNA-targeting information, which we have generated through a parallel

study on pancreatic cancer, in the context of Aims 2 and 3. The findings might help us discard

any non-promising candidates (target sites of interest, mRNAs of interest) and focus on

promising ones. To this end, we carried out these first experiments (Figure 3) with the two

pancreas cell lines HPNE and MiaPaCa. We are currently gearing up to assay the two siRNAs

we designed in colon cell lines.

Aim 2. Show that the mRNAs of genes expressed in CRC are affected by a specific miRNA

family and that many of the underlying interactions are not anticipated by the standard miRNA

targeting model. This aim is focused on demonstrating that the endogenous miRNA family on

which we have focused can regulate the abundance of mRNAs through interactions that are not

anticipated by the “standard” model of miRNA targeting.

We began with first addressing step 2 of Aim 2 as it could proceed independently. Therein, we

proposed to design and implement the next generation of the MuTaMe algorithm that we first

described in (Tay et al. Cell 2011). The original implementation that was used in this publication

was done in shell-script language, which does not render itself to speed and to the processing of

the larger datasets that will be used in this project. In light of this, during this reporting period,

we focused on re-implementing the originally described MuTaMe algorithm in the C

programming language. We have just completed the implementation and are currently in the last

stages of testing with specific test cases to ensure the accuracy of results. As intended, this

version of MuTaMe is significantly faster than the original and can handle much bigger datasets

of miRNA target predictions with great ease.

Aim 3. Show that the expression of the two lncRNAs of interest is affected by a specific miR-

family of miRNAs. This aim is focused on demonstrating that the endogenous family of

miRNAs on which we have focused can regulate the abundance of the pyk-reg-10 and pyk-reg-

83, just like they do with mRNAs. This is an interesting novel aspect of regulatory control by

miRNAs in that, among other things, the standard model does not anticipate miRNA targeting

beyond the confines of protein-coding mRNAs.

In the context of this aim, we first examined whether transfection with pre-miRNAs for several

of the miRNAs of the family of interest has any impact on the expression level of pyk-reg-83.

This would suggest the possibility of direct targeting by these miRNAs, a prerequisite for them

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to control endogenous mRNAs through decoying. A first set of transfection experiments with

five of the miRNA family members (herein denoted as miR-a, -b, -c, -d and –e) at a

concentration of 25 nM indicated that overexpression of all five of them can induce a very

notable reduction to the level of pyk-reg-83 in HPNE cells (Figure 4). We are gearing up to

repeat the experiments and establish the level of statistical significance for these findings.

In parallel, through computational analysis of the sequence of pyk-reg-83, we have identified

two sequence segments, one near pyk-reg-83’s 5´ and the other near its 3´ end. According to our

analysis, each of these two segments is likely targeted by multiple members of the miRNA

family of interest and at nearby locations. We are in the process of constructing Renilla

luciferase assays in order to test whether the two segments are indeed targeted directly by these

miRNAs.

Figure 1. Investigating the presence of pyk-reg-10 and pyk-reg-83 in 21 metastatic

colorectal cancer samples.

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Figure 2. Investigating the presence of pyk-

reg-10 and pyk-reg-83 in five pancreas cell

lines.

Figure 3. Impact of the two designed siRNAs on

the abundance of pyk-reg-83 in two different cell

lines. (* indicates a p-value that was ≤ 0.01)

Figure 4. Impact of transfection with pre-miRNAs for five

members of the miRNA family of interest on the abundance of

pyk-reg-83 (n=1).

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Research Project 10: Project Title and Purpose

Mechanisms of Presynaptic Dysfunction of Dopaminergic Neurotransmission in Human LRRK2

(R1441G) Transgenic Mouse Model – The major goal of this project is to understand the role of

synaptic mitochondria in dopamine (DA) release and DAergic axon degeneration in LRRK2

transgenic mouse. We hypothesize that mutant LRRK2 impairs mitochondrial function at the

pre-synaptic terminals, which in turn diminishes local ATP synthesis or calcium buffering

required for normal DA release. We will utilize a combination of fast cyclic voltammetry

recording and two-photon imaging in living brain slices, and mouse genetics to uncover

mechanisms underlying DAergic transmission deficits in Parkinson’s (PD). These findings will

likely provide new insights into pathogenesis of PD and open new avenues for therapeutic

intervention.

Anticipated Duration of Project

1/1/2013 – 12/31/2016

Project Overview

Emerging evidence suggests that synaptic dysfunction of dopamine (DA) neurons is an early

event in the pathogenesis of Parkinson disease (PD) occurring prior to the onset of symptoms.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent causes of

familial and sporadic PD, demonstrating an unprecedented significant role in PD pathogenesis.

Recently, a transgenic mouse model with over-expression of human LRRK2-R1441G has been

shown to recapitulate robust motor behavioral, neurochemical and pathological features of PD,

and we have found age-dependent deficits in DA release in the striatum in this model. Both

genetic and environmental causes of PD have highlighted the importance of mitochondrial

dysfunction in the pathogenesis of PD.

Essentially, synaptic mitochondria play a critical role in the function and organization of synaptic

vesicle pools and in neurotransmitter release. However, behavior of synaptic mitochondria in

mutant LRRK2 associated-PD has not been well studied. Therefore, it is important to understand

the role of synaptic mitochondria in DA release and DAergic axon degeneration in LRRK2

transgenic mice. We hypothesize that mutant LRRK2 impairs mitochondrial function at the pre-

synaptic terminals, which in turn diminishes local ATP synthesis or calcium buffering required

for normal DA release. We will utilize a combination of fast cyclic voltammetry recording and

two-photon imaging in living brain slices, and mouse genetics to uncover mechanisms

underlying DAergic transmission deficits in PD. These findings will likely provide new insights

into pathogenesis of PD and open new avenues for therapeutic intervention.

Specific Aims:

Aim 1. To study the defects in synaptic vesicle release of DAergic neurons and its association

with mitochondria.

Aim 2. To analyze synaptic mitochondrial function and its correlation with the synaptic

dysfunction in hLRRK2-R1441G transgenic mice.

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Aim 3. To evaluate basal oxidation level using mito-roGFP measurement to see whether there is

a high basal oxidation in the hLRRK2-R1441G mice and whether this is age dependent.

Aim 4. To evaluate the mitochondrial distribution and trafficking within the axons of the

nigrostriatal projecting DAergic neurons.

Principal Investigator

Hui Zhang, PhD

Assistant Professor

Thomas Jefferson University

123 S. 9th Street

Philadelphia, PA 19107

Other Participating Researchers

None

Expected Research Outcomes and Benefits

LRRK2 mutations are the most common genetic cause of Parkinson’s disease. In this study, we

plan to determine whether LRRK2 mutation will cause dysfunction of presynaptic mitochondria,

which in turn diminishes local adenosine triphosphate (ATP) synthesis or calcium buffering

required for normal dopamine release. We will utilize a combination of fast cyclic voltammetry

recording and two-photon imaging in living brain slices as well mouse genetics to address these

questions. After the completion of this project, we will know whether there are presynaptic

mitochondria defects in the LRRK2-R1441G transgenic mice and whether this is one of the

underlying mechanisms leading to synaptic transmission deficits that we have observed. These

findings will likely provide new insights into pathogenesis of PD and help to develop early

therapies for preventing and/or delaying onset of symptoms.

Summary of Research Completed

Milestone(s) for 1/1/2013-6/30/2013:

Postdoc recruitment; finish 50% of Aim 1A; mouse crossing and establish the mouse line (pTH-

GFP/LRRK2) for Aim 2.

We have successfully finished the postdoc recruitment and the postdocs will start to work on the

project on July 01, 2013.

Aim 1. To study the defects in synaptic vesicle release of DAergic neuron and its association

with mitochondria.

We have completed 50% of Aim 1A, confirming the preliminary results as planned.

We use a paired pulse stimulation to characterize evoked DA release and recycling/refilling by

cyclic voltammetry (CV) and amperometry in acute corticostriatal slices. The paired pulse ratio

reflects the recycling /refilling of synaptic vesicles from a reserve pool to the releasable pool. We

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found that mitochondria inhibitor, rotenone (1 µM, 1 hr) slows down the recovery of DA release

stimulated by paired stimuli at 5, 10 and 20 sec interval, mimicking the decreased vesicle

recycling/refilling in hLRRK2-R1441G TG mice shown in the preliminary results. Mitochondria

membrane potential depolarizing reagent carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 3

µM, 10 min) has similar effects. These results indicate that mitochondria dysfunction affects the

recycling /refilling of synaptic vesicles and the slower recovery of DA release in the mutant

could stem from mitochondria dysfunction.

We also examined the vulnerability of DA release to CCCP in 3 pairs of 5 month-old of

transgenic R1441G-LRRK2 and their WT littermates. Bath application of CCCP (3 µM, 20 min)

causes a more rapid decrease in evoked DA release and mass release of DA indicating the

“dying” of the DA terminals in the living brain slices. The onset of mass release of DA as

indicated by the shift up of the baseline is 600 ± 50 sec for WT and 386 ± 70 sec for TG

respectively (N=3, n=6, p<0.05).

Aim 2. To analyze synaptic mitochondrial function and its correlation with the synaptic

dysfunction in LRRK2-R1441G transgenic mice.

In order to use 2PLSM: TMRM mitochondrial imaging in living brain slices to examine how

mitochondrial function (membrane potential) is altered in the dopaminergic terminals in

hLRRK2-R1441G mice as specified in Aim 2A, we haves started to cross pTH-GFP mice with

LRRK2-R1441G mice to generate mice that have specific GFP labeling in the DA terminals. It is

now in the 3rd generation. We expect to have the 5th generation, which will be used for

experiments in Aim 2A, around December 2013.

We have started to measure the mitochondrial respiratory function, i.e., ATP level in the WT

and TG ahead of time. Preliminary result shows that there is a 30±1% decrease of ATP in the

striatal slices from LRRK2-R1441G mice compared to their WT littermates (2 pairs of mice at 5

month old).

General Methods

1. Slice preparation: Mice are decapitated without anesthesia. Coronal brain slices are cut on a

vibratome at a thickness of 300 µm and then separated at the midline to obtain two usable

hemislices. Slices are allowed to recover for 1.5 hr in a holding chamber in oxygenated artificial

cerebrospinal fluid (aSCF) at room temperature, and then placed in a recording chamber, and

super- fused (1.5ml/min) with aCSF (in mM: NaCl 125, KCl 2.5, NaHCO3 26, CaCl2 2.4,

MgSO4 1.3, KH2PO4 0.3, glucose 10) at 36 ºC.

2. Stimulation and recording: Striatal slices are electrically stimulated by an Iso-Flex stimulus

isolator triggered by a Master-8 pulse generator (A.M.P.I., Jerusalem, Israel) using a bipolar

stimulating electrode placed at ~100 µm distance from the recording electrode. Disk carbon fiber

electrodes of 5 µm diameter with a freshly cut surface are placed into the striatum about 50 µm

into the slice. For CV, a triangular voltage wave (-400 to +1000 mV at 300 V/s versus Ag/AgCl)

is applied to the electrode every 100 ms. Current is recorded with an Axopatch 200B amplifier

(Axon Instrument, Foster City, CA), with a low-pass Bessel Filter setting at 10 kHz, digitized at

20 kHz (ITC-18 board, Instrutech Corporation, Great Neck, New York). Triangular wave

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generation and data acquisition are controlled by a PC computer running a house-written IGOR

program. Background-subtracted cyclic voltammograms serve for electrode calibration and to

identify the released substance.

3. ATP assay was perfomed following the manual of commercil kit (K354-100, Biovision).

Briefly, one brain slice containing both stiatum and cortex (300μm, about 20mg) was

homogenized in 200μl ATP assay buffer, after centrifugation ice cold at 15,000g for 2 min, the

supernatant was deproteinized by perchloric acid method (K808-200, Biovision). Take 25μl of

the deproteinized supernatant from each sample to a 96-well plate which already contains 25μl

ATP assay buffer in the wells. Add 50μl ATP reaction mix (contains 2μl ATP probe, 2μl ATP

converter, and 2μl Developer mix in ATP assay buffer) per well and measure O.D. at 570nm

after 30 min incubation at room temperature protecting from light. Each sample was measured in

triplicate. The ATP levels were calculated based on the ATP standard curve done at the same

time, and then normalized to μg protein of the original lysates.

4. Data Analysis and Statistics: The two-tailed Student’s t-test is used for paired data and one-

way ANOVA followed by Student-Newmann-Keuls’s test is used for group’s data (GB Stat

software, Silverspring, MD). The critical level of significance is p<0.05 (*). Means will be

reported ±SEM. N: mouse number; n: experiment number.