(CANCER RESEARCH54, 5241—5245,October 1, 19941

Meeting Report

Molecular Mechanisms of Progression and Metastasis of Human Tumors:

A Pathology B Study Section Workshop'

Working Report from the Division of Research Grants, NIH

An understanding of the molecular origins of cancer and the determinants of its progression has been largely accomplished for humancolon cancer. Progress is being made toward understanding the molecular origins and determinants of progression of other human cancers. The key to this progress has been the recognition that multiple,sequential genetic changes occur in the development of clinical cancers. Each of these changes is being analyzed, and its particular rolein dictating the malignant phenotype delineated. Much of this knowledge is at the point that it can be used in routine diagnosis, as well asin identifying clinically useful prognostic or therapeutically responsive groups. Comparatively speaking, the progress in understandingthe molecular bases of metastasis and how to intervene in the metastatic process is in its infancy. Furthering research in this area that canbridge the chasm from basic science to clinical application, whetherdiagnostic, prognostic, or therapeutic, is of utmost importance.

There is the potential for the realization of major advances in theprevention, diagnosis, and therapy of metastatic disease. However,detection and analysis of the genomic changes that underlay progression and metastasis of human tumors is far outstripping the number ofinsightful and innovative studies applying this knowledge to clinicallyimportant problems.

To consider the spectrum from the identification of genomicchanges associated with tumor progression and metastasis to theirexploitation in the clinical management of metastasis, the NIH Pathology B Study Section sponsored a workshop entitled “MolecularMechanisms of Progression and Metastasis of Human Tumors.― Amajor goal of the workshop was to bring together investigators involved in identifying the molecular mechanisms underlying progression and metastasis of human tumors (breast, colon, prostate, ovarian,and melanoma) and those designing new therapies designed to arrestprogression and suppress metastasis. Through the interactions of theseinvestigators and the discussions generated by their presentations, itwas hoped that potential targets for sensitive detection, discriminatingdiagnosis, and effective therapies for cancer would be identified,targets that would exploit the most recent findings on the molecularmechanisms for tumor progression and metastasis.

J. McCarthy (University of Minnesota, Minneapolis, MN) discussed the use of synthetic peptides to study the molecular mechanisms of tumor cell adhesion, migration, and metastasis. Tumor cellrecognition of the extracellular matrix is important in invasion andmetastasis. Therefore, understanding the structural/functional properties of extracellular matrix components may lead to new insightsregarding the mechanism(s) by which the extracellular matrix modifies tumor cell adhesion, motility, and invasion. Such research mightresult in the design of conformationally constrained peptides and/orpeptidomimetics for the management of metastasis formation. Melanoma, and other malignant tumor types, adhere and migrate onfibronectin and on proteolytic fragments of fibronectin that consist ofthe carboxyl terminal heparin binding domain and the a4@3,integrin

Received 4/5/94; accepted 8/2/94.1ThisworkshopwasheldJanuary30,1994,inBigSky,Montana.Itwassponsoredby

the PathologyB StudySectionandwassupportedby the Divisionof ResearchGnats,NIH.

binding domain of the molecule. Molecular dissection of the carboxylterminal heparin-binding domain of fibronectin has resulted in theidentification of several cell adhesion-promoting synthetic peptides.Some of these peptides promote cell adhesion by interacting with cellsurface proteoglycans, whereas others exert their effects by interactingwith a4fJ, integrin. Several peptides can promote melanoma celladhesion in vitro, yet none of these peptides can completely duplicatebiological activities of the larger fragment. Collectively, the resultsindicate that the functional properties of a4(3, integrin are influenceddirectly or indirectly by the molecular action of cell surfaceproteoglycans.

Analyzing melanoma cell adhesion on artificial, chimeric substratasupports the notion that melanoma cell surface proteoglycans canmodulate the activity of a43, integrin. As is observed with fibronectin-derived, proteoglycan-binding synthetic peptides, melanoma cellsadhere to, but do not spread on, monoclonal antibodies against thelarge melanoma-associated proteoglycan core protein. Importantly,chimeric substrata, consisting of surfaces coated with the a4(31 integrin-binding synthetic peptide CS1 and anti-core protein monoclonalantibodies can support melanoma cell adhesion, spreading, and focalcontact formation. Furthermore, this effect can be observed whetheror not the proteoglycan is engaged by antibodies coated onto thesubstratum, or by the addition in suspension of antibody-coated agarose beads to cells already adherent on peptide CS1. This suggests thatthe influence of the proteoglycan on integrin function is at least, inpart, mediated by intracellular signaling pathways. Thus, cell surfaceproteoglycans could influence tumor cell metastasis at steps that arelikely to involve the functional activity of a431 integrin, such as tumorcell arrest to activated vascular endothelial cells, or other processesrelated to arrest and extravasation. Understanding the complex molecular and functional interactions between cell surface proteoglycansand integrmns may provide important new insights into developingcompounds that modify intracellular signaling pathways thatmodulate tumor cell adhesion and metastasis formation.

Data using conformationally constrained novel mini-helix collagenpeptides suggest a possible role for such peptides as novel antimetastatic agents. A synthetic peptide, IV-H1, from the helical region ofthe a, chain of type IV collagen can directly support the adhesion andmigration of melanoma cells in vitro, in part through a CD44-dependent mechanism. It also can specifically inhibit melanoma cell adhesionto basement membrane type IV collagen, and it is effective at inhibiting melanoma cell invasion through reconstituted basement membranes in vitro or experimental metastasis in vivo. This linear 15 â€m̃erwas synthesized as a mini-triple helix using a novel solid-phasebranching methodology. Triple helicity of the three carboxyl terminally coupled N-Hi nascent peptides was induced by the addition ofeight repeats of Gly-Pro-Hyp at their amino termini. The resultingmini-triple helix was two orders of magnitude more effective atsupporting melanoma cell adhesion than was the linear form, with a50% effective dose of approximately 1 @.LM.Constructs such as thisshould be more resistant to protease attack and may, therefore, haveenhanced half-lives in vivo. This, and other triple helical collagen

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MEETING REPORT

based peptides are currently being compared with linear counterpartsin vivo for their ability to inhibit metastasis formation.

w. Birchmeier(Max-DelbrUck-CenterforMolecularMedicine,Berlin, Germany) described recent studies by himself and colleaguesJ. Hülskenand J. Behrens suggesting that E-cadherin is an invasionsuppressor. Recent experimental evidence suggests that the properformation of intercellular junctions is critical for the maintenance ofepithelial differentiation and that destabilization of junctions allowsinvasiveness of epithelial cells and thus the progression of carcinomas. Accordingly, various structural components of intercellular junctions have been found to be related to products of tumor suppressorgenes. For instance, the cell adhesion molecule E-cadherin, which isconcentrated in adherent junctions of epithelia, has been shown tosuppress invasiveness of carcinoma cells in vitro. E-cadherin cxpression is down-regulated in various human carcinomas, and theE-cadherin gene has been found to be mutated in diffuse-type gastriccarcinomas. The tumor suppressor gene product APC binds to f3-catenm, which is cytoplasmically associated with E-cadherin. Furthermore, the tight junction-associated protein ZO-1 is related to the dlgtumor suppressor gene product of Drosophila, and the neurofibromatosis-2 tumor suppressor gene product (merlin) is related to theezrin/radixin/moesin family of junctional proteins. Conversely, products of oncogenes have been found to destabilize junctions, such assrc, ras, los, and the scatter factor/hepatocyte growth factor receptor

met. As decreased expression of E-cadherin as well as mutations in

E-cadherin are associated with increases in cell motility and invasionand with progression of human cancers, e.g., squamous cell carcinomaof the head and neck, prostate carcinoma, and gastric carcinoma,E-cadherin may prove to be a diagnostic and prognostic marker forhuman epithelial cancers.

Birchmeier and colleagues have established a novel model for theinteractions of f3-catenin within adherent junctions. f3-Catenin is adirect linker between the membrane and the actin cytoskeleton; itbinds to E-cadherin via its internal repeat region and associates witha-catenin through its NH2-terminal domain. The desmosomal proteinplakoglobin can mediate a similar linkage. Whether there are therapeutic strategies that can exploit the interactions among E-cadherin,@3-catenin, APC, and cytoskeletal proteins is not yet known.

M. J. C. Hendrix (Saint Louis University and Cardinal GlennonChildren's Hospital, St. Louis, MO) discussed the role of dual intermediate filaments, specifically vimentin and keratins, with respect totumor cell invasion and metastasis. There is compelling evidence tosuggest a strong correlation between the expression of these dualintermediate filaments and metastatic aggressiveness observed inmany cancers, including melanoma and breast carcinoma. Althoughintermediate filaments are considered principal components of thecytoskeleton of mammalian cells, their function heretofore has remained relatively unknown. Commonly, vimentin is found in mesenchymal cells, and keratins are present in epithelial cells. Indeed,earlier studies have emphasized the use of intermediate filaments ascell type-specific markers in differentiation and pathology; however,recent reports have confounded the literature with numerous demonstrations of the coexpression of vimentin and keratins in epithelial aswell as nonepithelial neoplasms.

Questions related to the characterization of the role(s) of vimentinand keratin coexpression were examined in three models: a highlymalignant human melanoma model, a human melanoma model demonstrating low invasive potential, and a mouse L cell model. In thefirst of these, the highly metastatic human melanoma cell line, C8i6i,which endogenously expresses both vimentin and keratins 8 and 18,was genetically manipulated to produce disrupted keratin intermediatefilaments. Specifically, C8161 cells were transfected with the plasmid

LK442-K18—1070, which contains a truncated human K18 cDNA2under the control of a human @-actinpromoter. This plasmid alsocarries the gene that codes for Escherichia coli xanthine-guaninephosphoribosyltransferase as a drug-selection marker. The plasmidpH@3Apr-1-neo harbors a gene for resistance to the antibiotic G418and was used for a control transfection (C-NEO). Subsequently, 14stably transfected clones were produced. Two of these were selectedfor further study, and they are designated Ci070—iO and C1070—14.Indirect immunofluorescence microscopy of these clones demonstrated an intracellular staining pattern of short, discontinuous striatedbundles of keratin filaments, compared with the normal filamentousmorphology seen in the C-NEO controls and in the C8161 wild type.Also, double immunofluorescence labeling indicated that the vimentinintermediate filament network was intact under all parameters. Cellswere tested for their ability to invade Matrigel-coated filters over72 h or gelatin-coated filters over 6 h in vitro. In both assays, therewas a 3- to 4-fold decrease in the invasive and migratory ability,respectively, of the C-1070—iOand C1070—i4 clones compared withthe C-NEO controls. In addition, when Ci070—iO and C1070—14cells were tested for metastatic potential via s.c. administration, theywere unable to form primary tumors or spontaneous metastases.Hence, these findings show a correlation between the coexpression ofvimentin and keratin intermediate filaments and the invasive andmetastatic behavior of human melanoma. If one of these intermediatefilament networks is disrupted, such as keratin(s) in this study, thereis a dramatic reduction in invasive, migratory, and metastatic ability.

In the second model, the dual intermediate filament hypothesis wasfurther tested in a more direct manner using a human melanoma cellline of low invasive potential A375P. In this study, vimentin-positiveA375P cells were transfected with DNAS for both keratins 8 and 18.The resultant stable transfectants express vimentin- and keratin-positive intermediate filaments and show a 2- to 3-fold increase in theirability to invade Matrigel in vitro, commensurate with a 2.5-foldincrease in cell motility through gelatin and Matrigel, as determinedby video-cinematography. In addition, these transfectants maintain amore rounded morphology, compared with the P-NEO controls, during the process of attachment and spreading, which is more conduciveto a migratory phenotype. Collectively, these date provide directevidence for a role for vimentin and keratin coexpression in humanmelanoma cell invasion. Potential changes in metastatic ability arecurrently under investigation.

Lastly, in a nontumor model, a similar experimental protocol wasexecuted using vimentin-positive mouse L cell fibroblasts transfectedwith keratins 8 and 18. The results indicate that the cells expressingkeratin filaments, in addition to vimentin, demonstrate a higher migratory and invasive ability (through Matrigel and gelatin in vitro)

compared with the parental and control-transfected clones. Furthermore, there is an enrichment of keratin-positive cells from a heterogeneous population of L clones selected over serial migrations. Thismigratory activity was directly correlated with the spreading ability ofthe cells on Matrigel matrix, in which the keratin-positive transfectants maintain a round morphology for a longer duration, comparedwith controls. In summary, the results would appear to corroborateobservations made in the A375P human melanoma model and provideconcrete evidence for an important role ascribed to vimentin andkeratin coexpression with respect to migration, invasion, and alteredinteractions with the extracellular environment.

The obvious significance of these studies rests in the translation of

2 The abbreviations used are: cDNA, complementary DNA AMF, autocrine motility

factor; CIEBP, CCAAT/enhancer binding proteins; PCD, programmed cell death; Cs,,intracellular free Ca HSVtk, herpes simplex virus-thymidine kinase; TG, thapsigargin;ER, endoplasmicreticulum;CAT, chloramphenicolaminotransferase.

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MEETING REPORT

this basic science research into future use as a diagnostic marker ofdisease progression. In this respect, patients presenting with melanomas coexpressing keratin and vimentin will more than likely experience a more aggressive disease course than patients with only vimentin-positive tumors. Hence, a more biologically based clinicalmanagement could be adopted based on using these intermediatefilament markers to determine disease progression.

A. Raz (Michigan Cancer Foundation, Detroit, MI) discussed therole of the autocrine motility factor receptor in metastasis. The introduction and regulation of active locomotion in eukaryotic cells iscentral to the development and well-being of higher organisms. Thedirected migration of cells is crucial to diverse biological processesincluding embryogenesis, morphogenesis, wound healing, and immunity as well as enabling the most devastating aspect of neoplasticdisease, metastasis. However, despite many studies, the mechanismswhich regulate and direct cell migration are largely unknown.

To understand the role that active cell locomotion plays in variousbiological processes, efforts have focused on chemotactic and motilityfactor induction of cell locomotion. A tumor-secreted cytokine wasimplicated in the induction of both random and directed cell migrationof producing cells and has, therefore, been denoted AMF. Thesefactors and others may represent a family of cytokines whose regulated expression induces cell motility in noncancerous situations suchas wound-healing (scatter factor) and embryogenesis (motility-simulating factor) and whose autocrine expression (AMF) may confermetastatic capabilities on neoplastic cells. The receptor for AMF wasidentified by Raz and colleagues as a cell surface glycoprotein with amolecular weight of 78,000 (gp 78); it shows sequence homology toP53, the expression of which may be associated with metastasis ofseveral experimental tumor systems.

The temporary loss of cell-cell adhesion and the ability to migrateare apparently fundamental to the acquisition of invasive properties oftumor cells. As summarized by Birchmeier, it is known that reducedexpression of the epithelium-specific cell-cell adhesion moleculeE-cadherin might be correlated with progression. Due to the apparentopposite roles of E-cadherin and gp78 in tumor cell invasion andmetastasis, studies were initiated to evaluate the expression of thesetwo cell surface antigens in human bladder carcinoma. Concomitantreduced expression of E-cadherin and up-regulation of gp78 expression was found. Dr. Raz proposed that the relative expression of thosetwo tumor antigens may be of prognostic value in epithelial tumors.

S. 0. Freytag (Henry Ford Hospital) discussed the role of CIEBPs

in the regulation of cell growth and differentiation. The CIEBPs aresequence-specific DNA-binding proteins that regulate gene expression at the level of transcription initiation. C/EBPa, the paradigm ofleucine-zipper proteins, binds to and activates genes that are abundantly expressed in adipocytes and hepatocytes. CIEBPa is dramatically induced during 3T3-L1 adipogenesis and is highly expressed inthe mature, post-mitotic adipocyte. Based on this observation and thefact that transcription factors lie at the heart of developmental controlmechanisms, Freytag and others examined whether ectopic expressionof C/EBPa could promote adipogenesis. Infection of a variety ofmouse fibroblastic cell lines with retroviruses containing the C/EBPagene induced adipogenesis in a significant fraction (30—100%) ofinfected cells. This effect required the potent transcriptional activationdomain of C/EBPa. The results indicate that C/EBPa is a pivotalcomponent of the adipogenic differentiation control mechanism. Because C/EBPa is highly expressed in mature hepatocytes and it actiyates liver-specific genes, it is possible that CIEBPa may regulateliver development and function.

Mature adipocytes and hepatocytes express two forms of C/EBPa(Mr 42,000 and 34,000) that are translated from a single mRNA.Translation of the smaller CIEBPa protein initiates at an internal

AUG. The smaller Mr 34,000 protein contains the DNA-bindingdomain of C/EBPa but lacks the potent transcriptional activationdomain. Interestingly, expression of the Mr 34,@)0 protein inBALB/c 3T3 cells is restricted to the post-mitotic state. Transfection studies showed that expression of the Mr 34,000 protein infibroblastic cells suppressed G418-resistant colony formation to thesame extent as authentic (Mr 42,000) C/EBPa however, expression ofthe Mr 34,000 protein could not induce adipogenesis. The Mr 34,000protein could antagonize the ability of authentic C/EBPa (and otherCIEBPs) to activate genes that promote proliferation. These resultsand others suggest that the Mr 34,000 form of C/EBPa may regulateentry into the post-mitotic state during terminal differentiation.

Expression of the CIEBPs is highly regulated during differentiationof myelomonocytic cells. CIEBPa is abundantly expressed in prolifcrating, immature myelomonocytic cells, and its expression is extinguished upon terminal differentiation. By contrast, expression ofC/EBPI3 is dramatically induced during myelomonocytic cell differentiation. These observations raise the possibility that the C/EBPsmay regulate development of the myelomonocytic lineage in vivo.Dr. Freytag's laboratory has discovered that expression of CIEBPa infibroblastic cells induces specific markers for the myelomonocyticlineage. The various C/EBP genes have been transduced into hematopoietic stem cells using retroviruses and infected cells engrafted intolethally irradiated mice. Studies are in progress that will directly testthe hypothesis that the CIEBPs regulate development of the myelomonocytic lineage in vivo. If forced expression of the C/EBPspromotes myelomonopoiesis in vivo, it may be possible to apply thistechnology to treat human conditions in which production of maturemyelomonocytic cells is impaired (e.g., myeloid leukemias andcongenital neutropenia).

J. Isaacs (Johns Hopkins University, Baltimore, MD) described his

studies with colleagues Y. Furuya and L. J. Cisek on PCD as a therapyfor prostate cancer. Previously, they demonstrated that androgendependent prostatic cancer cells like normal prostatic glandular cellsare induced to undergo an active process of cellular suicide termedPCD or apoptosis by androgen ablation induced by either surgical orchemical means. Androgen ablation-induced programmed death ofnormal and malignant prostatic cells does not require the cells to enterinto or progress through the proliferation cell cycle. Activation ofPCD in the nonproliferative androgen-dependent prostatic cancer cellis thus the mechanism for the positive response of men with metastaticprostatic cancer to androgen ablation. Unfortunately, androgen-independent prostatic cancer cells also present in men with metastaticprostatic cancer do not activate PCD following androgen ablation;thus, androgen ablation is not curative. Attempts to eliminate theseandrogen-independent prostatic cancers with chemotherapy has beenof limited success. Current anticancer drugs are toxic for rapidlyproliferating cells and, although lethal in metastatic patients, <5% ofandrogen-independent prostatic cancer cells proliferate per day, making antiproliferating cytotoxic therapy of limited value. Methods forinducing the death of nonproliferating androgen-independent prostaticcancer cells are urgently needed.

One approach is to activate PCD within these cells. Previously,Isaacs and colleagues have demonstrated that androgen-independentprostatic cancer cells can be induced to undergo PCD without entrance into or progression through the cell cycle by treatments whichsustain a 3—4-foldincrease in the intracellular free Ca2@ (Ca1), demonstrating that the PCD pathway is intact in these cells. Extendingthese studies, they demonstrated that TG, a sesquiterpene-lactone, is acell-permeable, potent inhibitor (50% inhibitory concentration, 30 nM)of ER Ca2@ pumps within androgen-independent prostatic cancercells of both rodent (AT-3) and human (TSU, PC-3, and DU-145)origin. TG treatment thus depletes the ER pool of Ce2@ resulting

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within mm in a 3—4-foldelevation in Ca1. This in turn generates adiffusible messenger which increases permeability of the plasmamembrane, thereby allowing the influx of extracellular Ca2@ andsustaining the elevation in Ca1 for h. This leads to morphologicalchanges and new protein synthesis within 6—12h. Within 24 h, cellsarrest in G0 and irreversibly lose proliferative ability. During the next24—48h, the cells fragment their DNA and undergo fragmentationinto apoptotic bodies. Since these prostatic cancer cell lines in vitrohave an exceptionally high rate of proliferation, this highlights theissue of whether activation of PCD by TG requires cells to beinhibited during progression through the cell cycle or whether cellsneither in nor attempting to enter the cycle (i.e., G0 cells) can beinduced to undergo PCD by TG treatment.

To resolve this question, prostatic cancer tissue obtained fromprostatectomy specimens was collagenase dissociated to produceorganoids which were then plated in scram-free defined media(i.e., BRFF-HPCL1) in type I collagen-coated dishes. Following plat

ing, cells in organoids attach, spread out, and undergo a limitednumber of cell divisions. During a 1—2-weekperiod, cells becomeconfluent and stop proliferating as determined by [‘4C]thymidineincorporation, flow cytometric analysis, and Ki67 immunostaining.The cells in the confluent culture remain viable by trypan blueexclusion and video microscopic analysis for more than 1 month. if ascrape-wound is made in such long-term confluent cultures, no proliferation is detectable, demonstrating that contact inhibition is notrequired to keep the cells in G0. Treatment with 500 n@ TG results inmorphological changes within 24 h and cytotoxic effects within 2—4days. In contrast, treatment of such nonproliferating cultures with 10@LMof the proliferation-dependent cytotoxic agent, 5-fluorodeoxyru

ridine, has no effect. These results demonstrate that TO can induce thePCD of G0 human prostatic cancer cells without requiring them toattempt to enter the cell cycle.

To determine whether induction of PCD by TG is due to depletionof ER pools of Ca2@ or elevation in Ca1, the calbindin-D gene,encoding a Mr 28,000 calcium-binding protein, was transfected intoPC cells. Calbindin-D expressing clones, confirmed by iminunohistochemical and Western analyses, were isolated. Exposure of parentaland control transfectants to 500 nM TG for 36 h results in a 3—4-foldelevation in Ca1 and in 50% of cells undergoing PCD, even whensubsequently maintained in TG-free media. In contrast, 36 h TOtreatment of calbindin-D expressing transfectants produces less than a2-fold increase in Ca1 and 60% inhibition of the number of cellsundergoing PCD. if calbindin-D transfectants are exposed to TO for

48 h, however, this protection is lost, consistent with influx ofextracellular Ca2@, eventually exceeding the binding capacity ofintracellular calbindin-D and thus sufficiently elevating Ca in thesecalbindin transfectants to activate PCD. These results demonstrate thatTO can sustain an elevation in Ca1derived from the extracellular Ca2@activating PCD in nonproliferating androgen-independent prostaticcancer cells. In conclusion, these studies have identified both a newtarget (i.e., ER Ca2@ pumps) and a new agent (i.e., TO), which offera completely novel approach to the elimination of androgen-independent cancer cells in advanced prostatic cancer without requiring thesecells to enter or progress through the proliferation cell cycle.

P. Furmanski (New York University) discussed the search for novel

approaches to the treatment of advanced cancers and metastatic disease. Initial studies were aimed at determining the efficacy of radioconjugated monoclonal antibodies in imaging and therapy of breasttumors. Using a series of antibodies directed against human milk fatglobule and related antigens, radioconjugates with good localizationand therapeutic activity in experimental systems were identified.Phase I trials in patients established that potentially therapeutic dosesof antibody could be delivered to tumor sites with acceptable dose

limiting toxicity to normal organs such as the marrow. Phase II trialsare currently in progress to assess therapeutic activity. However, themajor limitation to the use of monoclonal antibodies in cancer treatment remains the very low accumulation of the antibodies in targettissues. One approach to resolving this limitation involved a search fornew targets for development of improved monoclonal antibodies. Thissearch serendipidously focused attention on the lactoferrins. Firstidentified as an avid iron-binding protein present in human milk andthought to function mainly in iron metabolism and transport, lactoferrin is now recognized to be involved in the primary defense againstmicrobial infection through sequestration of iron required for microbial growth. An examination of the functions of lactoferrin revealedthat lactoferrin reduced growth of several solid tumor models in miceand was a potent inhibitor of experimental metastasis by Bi6-FiOcells. These antitumor effects appeared to be mediated through naturalkiller cells; elimination of natural killer cells by pretreatment withanti-asialo OM, antibody eliminated the lactofemin effect, whereasreduction of macrophage function by pretreatment with silica did not.The mechanisms involved in cell activation by lactofemin were cxplored by analysis of the interaction of lactoferrin with DNA. Although long-recognized, the significance of the ability oflactoferrin toavidly bind DNA was not known. Using multiple cycles of binding oflactoferrin to a pool of random DNA sequences under stringentconditions followed by polymerase chain reaction amplification ofbound sequences demonstrated that binding of lactoferrin to DNAexhibited sequence specificity. Three robust consensus sequences forDNA binding were identified and confirmed by gel-shift analysis,inhibition studies, supershifting using monoclonal anti-lactoferrin antibodies, Southwestern blotting, and DNase I footprinting. The consequences of binding of lactoferrin to these sequences was examinedby transfection of K562 human leukemia cells with CAT reporterconstructs containing one of the consensus sequences. Incubation ofthe transfected cells in medium containing lactoferrin resulted inpotent activation of expression of the CAT reporter. Iron-saturatedlactoferrmn was a much stronger inducer of CAT reporter activity thanapo-lactoferrin. The results are consistent with an active role oflactoferrin in primary defense against infection and neoplasia, boththrough high-avidity binding of iron and by activation of immuneeffector cells through gene induction. The results also suggest theexistence of a novel class of cell communication molecules secretedfrom one cell and taken up by another, in which it causes transcriptional activation.

I. Hart (Ouy's and St. Thomas's Hospitals, University of London,

London, England) pointed out that while a knowledge of the mechanisms which underlay the processes of progression and metastasis wasan important aim, the clinical utility of such information was likely tobe limited. Using malignant melanoma as a model tumor to analyzethe metastatic process has been a popular approach both in the UnitedStates and United Kingdom. However, it has to be borne in mind thatonly one in three afflicted with cutaneous melanoma will succumb tothe disease; a total of only 1,000 people in the United Kingdom. Whileincidence rates for this cancer are rising, the mortality rates remainroughly constant so that the popularity of work on this system is nota reflection of its clinical importance. While melanoma undoubtedlyis a good model for investigating certain aspects of the metastaticprocess, care ought to be taken in extrapolating from findings in thissystem to other tumor types. Of course, what killed one in threemelanoma patients was secondary disease. Since this often was afaitaccompli in many of these patients, the challenge in practical termswas not to elucidate how tumors get to and grow in distant sites, buthow to deal with them once they are in these locations. The fact thatmelanoma is refractory to chemotherapy and radiotherapy means thatit is an excellent candidate tumor for developing novel therapeutic

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approaches. Hart's own laboratory has initiated experiments in genetherapy attempting to insert foreign DNA into melanoma cells in situ.Their approaches are based on the use of cDNAs encoding forimmune-stimulating products (such as interleukin 2) or for prodnigactivating enzymes (such as HSVtk, whose expression renders cellssusceptible to ganciclovir). In order to limit expression to the tumorcells, they relied on “transcriptionaltargeting―by placing the therapeutic cDNAs downstream of promoter sequences which were activeonly in melanocytic cells. A 760-base pair sequence of the 5'-flankingregion of the murine tyrosinase gene was shown to restrict expressionof heterologous genes to cells of melanocytic origin while not permitting expression in other cell types. Using a Tyr-HSVtk constructinjected into established tumors growing at a s.c. site, followed byganciclovir administration, significant and dramatic reductions intumor burden were obtained. Retroviral constructs were made whichretained specificity of transcription of HSVtk. Repeated i.v. inoculations of supernatants containing ectopic virus into C57 mice preseeded with B16 lung tumors resulted, upon subsequent administration of ganciclovir, in very dramatic reductions in tumor nodulesrelative to water-treated controls. Despite the systemic administrationof retroviruses, no general toxicity was observed upon ganciclovirtreatment. These experiments demonstrate that gene therapy approaches can be devised which are capable of limiting disseminatedcancer, whereas transcriptional targeting provides a means of avoidingpotential bystander toxicity.

The novel approaches presented in the workshop and the intensity

of the discussions generated by these presentations indicate thatsubstantial effort is being dedicated toward translating the molecularmechanisms of tumor progression and metastasis into diagnostic andtherapeutic approaches. The translation of the basic studies of molecular mechanisms into the clinic is already substantially improving ourability to detect and diagnose human cancers and hopefully will soonalso impact on our ability to treat human cancers.

Bonnie F. SloaneDepartment of PharmacologyWayne State UniversityDetroit, Michigan 4820i

Chester J. HermanDepartment ofAnatomic PathologyGrady Memorial HospitalAtlanta, Georgia 30335

Martin Padarathsingh3Division of Research

Grants, NIHBethesda, Maryland 20892

3 To whom requests for reprints should be addressed, at Division of Research Grants,NIH, Pathology B Study Section, Westwood Biology Room A-26, Bethesda MD 20892.

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1994;54:5241-5245. Cancer Res   Bonnie F. Sloane, Chester J. Herman and Martin Padarathsingh  Report from the Division of Research Grants, NIHTumors: A Pathology B Study Section Workshop: Working Molecular Mechanisms of Progression and Metastasis of Human

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