insidiouschangesinstromalmatrixfuelcancerprogression...review...

Review

InsidiousChanges inStromalMatrix FuelCancerProgression

Fayth L. Miles1,2 and Robert A. Sikes2,3

AbstractReciprocal interactions between tumor and stromal cells propel cancer progression and metastasis. A complete un-

derstanding of the complex contributions of the tumor stroma to cancer progression necessitates a careful examination ofthe extracellular matrix (ECM), which is largely synthesized and modulated by cancer-associated fibroblasts. Thisstructurally supportive meshwork serves as a signaling scaffold for a myriad of biologic processes and responses favoringtumor progression. The ECM is a repository for growth factors and cytokines that promote tumor growth, proliferation,and metastasis through diverse interactions with soluble and insoluble ECM components. Growth factors activatedby proteases are involved in the initiation of cell signaling pathways essential to invasion and survival. Varioustransmembrane proteins produced by the cancer stroma bind the collagen and fibronectin-rich matrix to induceproliferation, adhesion, andmigration of cancer cells, as well as protease activation. Integrins are critical liaisons betweentumor cells and the surrounding stroma, and with their mechano-sensing ability, induce cell signaling pathwaysassociated with contractility and migration. Proteoglycans also bind and interact with various matrix proteins in thetumor microenvironment to promote cancer progression. Together, these components function to mediate cross-talkbetween tumor cells and fibroblasts ultimately to promote tumor survival and metastasis. These stromal factors, whichmay be expressed differentially according to cancer stage, have prognostic utility and potential. This review examineschanges in the ECM of cancer-associated fibroblasts induced through carcinogenesis, and the impact of these changeson cancer progression. The implication is that cancer progression, even in epithelial cancers, may be based in largepart on changes in signaling from cancer-associated stromal cells. These changesmay provide early prognostic indicatorsto further stratify patients during treatment or alter the timing of their follow-up visits and observations.

Visual Overview: http://mcr.aacrjournals.org/content/12/3/297/F1.large.jpg.Mol Cancer Res; 12(3); 297–312. �2014 AACR.

IntroductionThe ECM has a major role in tumor development and

progression. Over the last decade, it has become muchclearer that reciprocal interactions between tumor and stro-mal cells determine the course of cancer progression.Although much remains to be understood about the chro-nology of events dictating tumor initiation and the contextfor reciprocal regulatory signals, it is clear that continuousbidirectional paracrine signaling establishes a microenviron-ment conducive for the survival of the tumor. Hence, in thestudy of the tumor stromal microenvironment, there are twosides to be examined: (i) the effects of tumor cell signaling onthe surrounding stroma and (ii) the influence of paracrine

stromal signals on tumor cells. In general, the reciprocalrelationship supporting tumor progression might be viewedas follows: tumor cells secrete cytokines, chemokines, andenzymes that activate stromal cells and fibroblasts. Among adiverse group of molecules, these stromal cells secrete pro-teases that break down the tumor cell basement membrane.Consequently, growth factors are released from the under-lying matrix, which not only initiate the signaling pathwaysin tumor cells, but also activate fibroblasts further, inducingfibroblast secretion of a host of ECM factors responsible forregulating numerous interrelated events. Tumor cells thrivein this convoluted but nourishing milieu, which ultimatelypromotes their survival, proliferation, and metastasis(Fig. 1).Cancer associated fibroblasts (CAF) have a profound role

on ECMcomposition and dynamics. The ECM is composedof fibrillar and structural proteins, proteoglycans, integrinsand proteases, all of which may be manufactured by CAFs.Hence, CAFs potentially canmodulate levels and activities ofall these factors. Indeed, various CAF-secreted soluble factorsand proteolytic enzymes modulate the tumor microenviron-ment, altering composition of the connective tissue throughremodeling. Because the ECM controls the availability andactivation of growth factors and serves as a platform forintegrin and growth factor receptors to regulate cell signaling

Authors' Affiliations: 1Department of Epidemiology, Fielding School ofPublic Health, University of California, Los Angeles, California; 2Depart-ment of Biological Sciences, Laboratory for Cancer Ontogeny and Ther-apeutics; and 3Center for Translational Cancer Research, University ofDelaware, Newark, Delaware

Corresponding Author: Robert A. Sikes, Center for Translational CancerResearch, University of Delaware, 326 Wolf Hall, Biology, Newark, DE19716. Phone: 302-831-6050; Fax: 302-831-2281; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-13-0535

�2014 American Association for Cancer Research.

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pathways, fibroblasts modulate a multiplicity of growthfactor-mediated pathways through changes in synthesis ofmatrix components.

Growth FactorsParacrine growth factors secreted by CAFs are central to

the establishment of a microenvironment conducive totumor growth and progression. Growth factors functionat various stages through tumor progression, includingproliferation, metastasis, and dissemination. Such factors,particularly heparin-binding molecules, have the ability to

induce a number of physiologic events leading to tumorprogression because of their diverse interactions with theECM. In the ECM, they may be sequestered or copresentedby other molecules, such as proteoglycans and various cellsurface receptors, accounting for their biologic diversity.The prolific contributions of stromal cell-derived factor 1

(SDF-1) to cancer progression in the tumor microenviron-ment are linked largely to its role in recruitment of mesen-chymal stem cells andmyofibroblasts to the tumor, as SDF-1is integral to CAF activation and the desmoplastic reaction.In addition to cancer cells, SDF-1 is largely secreted by CAFsand mesenchymal cells (1, 2) where it stimulates growth,proliferation, invasion, angiogenesis, metastasis, and colo-nization in a number of cancers. It is noteworthy that bonemarrow–derived CAFs secrete higher levels of SDF-1 thanwild-type myofibroblasts (3), highlighting the particularimportance of bone marrow–niche cells in cancer progres-sion. SDF-1 is an important mediator of cross-talk betweentumor cells and fibroblasts. Paracrine stromal SDF-1 signal-ing is stimulated in the presence of TGF-b1, which increasesexpression of epithelial CXCR4, resulting in SDF-1–medi-ated Akt activation and subsequent tumor progression andsurvival (4). In addition, SDF-1 promotes cell–cell and cell–matrix adhesions by regulating integrin expression (5, 6).TGF-b1 plays a major role in activation of fibroblasts by

promoting fibroblast to myofibroblast transdifferentiation.Furthermore, TGF-b1 is actively secreted by CAFs andstromal cells. TGF-b1 is notoriously a complex, pleiotropicfactor because of multiple binding partners and associatednoncanonical signaling pathways. Consequently, it plays arole in both tumor suppression and tumor promotion.Although loss of TGF-b signaling through abrogation ofTbRII in stroma has been shown to correlate with increasedproliferation and progression of cancer cells (7–9), paracrineTGF-b1 signaling frequently induces metastasis and epithe-lial to mesenchymal transformation of cancer cells. Suchdiverse actions of TGF-b seem to be dependent on contex-tual differences that include the tissue localization, cellsource, extracellular mediators, and the cell signaling con-text. We have found that although direct stimulation ofprostate cancer cells with TGF-b1 suppresses growth andinvasion (10), stimulation of bonemarrow stromal cells withTGF-b1 suppresses paracrine apoptotic signals to promoteprostate cancer cell survival (Fig. 2). Similarly, overexpres-sion of TGF-b1 in normal prostate fibroblasts inducesinvasive tumor growth (11). Stromal TGF-b is associatedwith tumor progression and metastasis in breast cancer, andhigh stromal expression is correlated with increased mortal-ity (12). In addition, orthotopic mammary carcinoma mod-els have demonstrated a role for TGF-b in regulating tumorperfusion, as abrogation of TGF-b signaling in the stromaincreases recruitment of perivascular cells, thus improvingchemotherapeutic efficacy (13).TGF-b, among other factors, is an important regulator of

insulin-like growth factor (IGF) and the IGF axis, anothervery important signaling system in prostate cancer progres-sion. In prostate stromal cells, TGF-b1 increases the expres-sion of IGF-1 and IGF-binding protein 3 (IGFBP-3; refs. 14,

Figure 1. Reciprocal tumor: stromal cell signaling in the tumormicroenvironment promotes tumor progression and stromal cellactivation. A. Paracrine signals (soluble factors) from the tumor inducealterations that prime the stroma. B. Subsequent release of stromalproteases triggers breakdown of the tumor basement membrane (BM)and release of additional growth factors from tumor cells, furtheractivating the stroma. C. Consequently, stromal cells secrete amyriad offactors such as growth factors, proteoglycans, proteases, and insolubleproteins to promote tumor growth, migration, andmetastasis. During theprocess, cancer associate fibroblasts (CAFs) may be transformed.

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15), as well as the ratio of IGF-I bound to IGFBP-3, therebydecreasing growth of prostate cancer cells in coculture (14).Such regulation of IGF and binding protein levels providesone explanation for the divergent responses of TGF-b inprostate cancer. Prospective and case–control studies havefound that elevated plasma IGF-1 predicts an increasedprostate cancer risk of several fold (16, 17), and elevatedsera levels correlate with an increased risk for breast andcolorectal cancer, although not statistically significant (18–20). However, increased IGFBP-1 seems to be significantlyassociated with decreased risk for colorectal cancer (OR ¼0.48; 95% confidence interval, 0.23–1.00, P ¼ 0.02;ref. 21). IGF-2 may also play a role in prostate and breastcancer, where it is expressed at higher levels by mammarystromal cells (22, 23), and in particularly large quantities bystromal cells in benign prostatic hyperplasia (24). Althoughthe prognostic significance of total stromal IGF-2 levels inbreast cancer is inconclusive, patients with lower serum levelsof free IGF-2 have larger malignant tumors (25). Interest-ingly, the presence of integrin-a11 on fibroblasts is associ-atedwith increased stromal IGF-2 levels in vivo in non–smallcell lung carcinoma (NSCLC), concomitant with anenhanced rate of tumor growth (26).Similar to other heparin-binding growth factors, platelet-

derived growth factor (PDGF), expressed by leukocytes andtumor cells, promotes angiogenesis, likely through upregu-lating stromal production of VEGF (27), andmay also have amore direct effect through stimulation of endothelial cells

(28). AlthoughCAFs do not commonly express PDGF, theyreadily express PDGF receptor to promote angiogenesis andproliferation, which involves upregulation of fibroblastgrowth factor (FGF)-2 and FGF-7 in cervical cancer (29,30). Inhibition of PDGF decreased stromal heparin-bindingEGF (HB-EGF)-mediated tumor cell growth in coculturesof cervical cancer cells and CAFs from patient tumor tissues,highlighting the significance of PDGF in HB-EGF signal-ing, and cancer progression (31). In addition to cervicalCAFs,HB-EGF has been found to be expressed at high levelson prostate stromal cells (32, 33), where paracrine signalsmediate prostate cancer survival and androgen-independenttumor growth as well as neuroendocrine differentiation (34).The effects of PDGF on cancer cell proliferation andprogression occur largely through EGFR/ErbB signalingleading to activation of mitogen—activated protein kinase(MAPK) and PI3K/Akt. Similarly, hepatocyte growth factor(HGF) activates the RAS/MAPK and phosphoinositide 3-kinase (PI3K) pathways in addition to many others, bybinding c-MET, another receptor tyrosine kinase with ahighly complex signaling profile (35). CAF-secreted HGFpromotes invasiveness of several cancers, including breast,colon, and oral squamous cell carcinoma (36–38). Curious-ly, all HGF signaling in cancer cells may not be mediatedthrough c-MET. Tate and colleagues clearly demonstrate aprostate cancer cell response to HGF in a c-MET-nullbackground, thereby opening up the possibility of signalingthrough cell surface nucleolin (39). Thus, heparin-binding

Figure 2. TGF-b pretreatment ofbone marrow stromal cellsreverses stromal cell conditionedmedia (CM)-induced death ofprostate cancer cells. HS-5 bonemarrow stromal cells werepretreated with or without TGF-b1for 4 days in DMEM supplementedwith 10% FBS. Following PBSwashes, fresh serum-free mediumwas replaced and conditionedmedium was collected after 48hours incubation. C4-2 prostatecancer cells were treated withDMEM, HS-5 CM with vehicle, orHS-5 CM with exogenously addedTGF-b1. A live/dead assay wasperformed using calcein AM andethidium homodimer at aconcentration of 125 nmol/L and2 mmol/L, respectively, todifferentially label live cells fromdead C4-2 cells. Representativeimages are shown from five fieldsper experiment run three timesminimum. Color images, green(calcein AM) and red (ethidiumhomodimer) were converted tograyscale and inverted inphotoshop to produce black andwhite images. Images werecaptured using a Nikon 2000TE.Final magnification �25.

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growth factor-activated tyrosine kinases engage the ECMthrough focal adhesion complexes and associated down-stream signaling cascades, ultimately inducing a variety ofresponses to promote tumorigenesis and cancer progression.

Collagen, Fibronectin, and TenascinCAFs secrete higher levels of collagens than those of

normal tissue. It is well known that deposition of a densecollagen matrix is a major feature of the desmoplasticreaction. This generates a stiff collagen matrix, which isassociated with increased activity of the matrix cross-linkingenzyme lysyl-oxidase (40). Increased collagen density hasbeen shown to promote proliferation of epithelial cells inthree-dimensional (3D) matrices, and tumor formation ofbreast cancer cells, demonstrating the role of a collagen-dense environment in tumor initiation and progression. Theconsequence of enhanced collagen deposition is increasedcell signaling pathways that promote cancer cell invasion,migration, and collagen reorganization. Isoform-specificexpression of collagen may be dependent on tumor typeand stage. Collagen I and III are upregulated commonly byCAFs or stromal cells in several cancers (41–45). In geneexpression studies, collagens X and XI-a1 were increased inthe reactive cancer stroma of invasive breast tumors, forminga prognostic signature (46, 47), although protein levels ofcollagen XI-a1 were shown to be downregulated in malig-nant breast cancer in a separate analysis (48).Upregulation ofcollagens IV, VI, XI, and XV, and downregulation ofcollagen XIV also have been observed in CAFs during theCAF–epithelial interaction in various cancers, in some casesforming a prognostic gene signature (49–52).Carcinomas may be characterized by several variants of

fibronectin or oncofetal fibronectin, which are present in thetumor stroma and expressed by myofibroblasts. Oncofetalisoforms, including ED-A andED-B fibronectin, are derivedby alternative splicing. Because of the varied specializedprotein-binding domains, fibronectin isoforms may beunique in their affinity and specificity for ECM-bindingpartners, which include fibrin, collagen, heparin, tenascin,syndecan, and integrins. Oncofetal fibronectin is associatedwith an invasive phenotype, poor prognosis, or poor differ-entiation in many cancers, as demonstrated in CAF:tumorspheroids as well as human tissues (53–56). ED-B fibro-nectin is an angiogenic marker that is involved in neovascu-larization in advanced and remodeling tumors, and thus hasa role in dissemination of cancer cells (57). Migrationstimulating factor (MSF), a truncated isoform of oncofetalfibronectin containing the gelatin-binding domain, also issecreted by both CAFs and oncofetal fibroblasts and inducesmigration (58). The migratory response of fibroblasts toMSF is based on their ability to adhere to native collagentype I. Because of this MSF-induced migratory response, amicroenvironment is established for the manifestation of aninvasive phenotype in cancer cells (58). Overexpressionof MSF is correlated with poor survival of patients withcancer (56).The dynamic role of tenascin-C in the tumor microen-

vironment stems from its ability to interact with a diverse

group of ECM molecules and surface receptors that initiatecell signaling pathways. Tenascin-C is resident in the stromaof many poorly differentiated tumors, expressed highly byfibroblasts (37, 59–61), and induced by a variety of signalingpathways. In earlier studies, it was proposed to be a stromalmarker for mammary tumors because it was not detected innormal tissue (60), and also was found to be elevated in thesera of patients with cancer (62). Now, it is quite evident thattenascin-C has a prominent role in cancer—promotingangiogenesis, tumor cell proliferation, and metastasis, withlevels increasing with tumor aggressiveness (61). Recently, itwas identified as one among 11 analytes capable of differ-entiating between benign andmalignant ovarian cancer (63).In a mouse model of breast cancer, it was shown thatsecretion of tenascin-C by stromal cells expressing s100AF,a marker associated with metastasis and poor prognosis,promotes metastatic colonization of breast cancer (64).Tenascin-C interacts with several integrins expressed on thesurface of tumor cells and fibroblasts (65, 66). It also bindsfibronectin and interferes with fibronectin-mediated adhe-sion, competing with the syndecan-4–binding site (67, 68).Its antiadhesive properties affect proliferation (68) andstimulate cancer cell migration by promoting disassemblyof focal adhesions, thus interfering with integrin-mediatedadhesions. Tenascin-C also was shown to inhibit activationof RhoA and associated stress fiber formation, and to inducefilopodial extensions (69). In particular, alternatively splicedfibronectin type III repeats are associated with increasedmotility and invasion (70). Tenascin-W, although not ascommon as tenascin-C, has been shown to be a marker foractivated tumor stroma, and is associated with increasedmigration of breast cancer cells (71). Recently, it was foundlocalized around the vasculature of tumor cells, and wasshown to stimulate angiogenic activity in vitro (72). Hence,Tenascin-W may have further utility as a marker for tumorangiogenesis.

ProteasesProteolysis regulates and induces release of latent growth

factors stored in the matrix, unveils protein domains, andgenerates bioactive fragments. Cleavage of the ECM bymatrix metalloproteinases (MMP) and other proteases leadsto activation of growth factors, as well as cytoskeletal reor-ganization and modification of downstream signaling path-ways. Accordingly, VEGF and basic fibroblast growth factormay be released and activated to promote angiogenesis (73,74). EGF receptor (EGFR) ligands, such as HB-EGF, alsocan be processed by MMPs (75), along with IGF-bindingproteins (76, 77), which regulate the activity and bioavail-ability of IGF (78), along with ligand independent effects ofIGF-BP fragments (79).Numerous MMPs are expressed and secreted by CAFs,

and enable cancer cells to evade tissue boundaries, escape theprimary site, and metastasize. The significance of fibroblastsin synthesis of MMPs has become appreciated only recently,as MMP expression was assessed previously within theconfines of the tumor. However, it is clearer now thatstromal cells are the major source of MMPs, which become

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active at the invasive front (80). Several coculture models oftumor cells and fibroblasts have demonstrated enhancedactivation of MMP-2 or expression of MT1-MMP, anactivator of proMMP-2 (gelatinase; refs. 81–83). In vivomodels also have shown the role of stromal MT1-MMP orMMP-2 in tumor cell growth (84, 85). Notably, introduc-tion of MMP-2-null fibroblasts reduced head and necksquamous cell carcinoma (HNSCC) tumor volume in severecombined immunodeficient (SCID) mice by 90% andabrogated invasion of collagen gels in coculture. MMP-2expression in CAFs has been highlighted as an indicator ofpoor prognosis forNSCLC (86); and, in the prostate,MMP-2 is associated with an invasive phenotype (87, 88). In theprostate, stromal MMP-2 levels are upregulated by TGF-b1(89). The significance of this may reside in the clinicalobservation that high-serum TGF-b levels are associatedwith poor patient survival and increased bonemetastasis (90,91). It has been suggested that proMMP2 is activated inCAFs as a consequence of collagen I binding of integrin b1.Interestingly, b1 integrin is activated by MT1-MMP–induced cleavage of the a-V integrin precursor, promotingcollagen binding and motility of cancer cells, thus providinga potential mechanism for MT1-MMP activation of MMP-2 (81, 92).The unique set of MMPs expressed in the tumor stroma

varies with tumor type and stage, and consequently, iscontext dependent. Accordingly, in a squamous cell carci-noma model of benign and malignant HaCaT clones trans-planted onto nude mice, expression of stromal-derivedMMPs and their correlation with malignancy were exam-ined, and stage-specific differences identified. mRNA ofMMP-2, -9, -13, and -14 was upregulated in the stromaof malignant and invasive tumors, and MMP-3 wasexpressed exclusively in stroma representing very late-stagedisease (93). During progression of breast cancer, MMP-9,-13, and -11 (stromelysin-3) are upregulated in reactivestroma, and have implications in invasion and osteolysis,with MMP-11 also being implicated in mammary tumordevelopment (47, 94–97). MMP-11 and -13 are associatedwith tumor progression, invasion, and poor prognosis inseveral other cancers (98–102), where they may be expressedby both stromal and tumor cells.Other stromal-relevant MMPs are ADAM proteins, of

which ADAM-17, -9, and -12 are expressed by myofibro-blasts or stromal cells in colon cancer, liver metastases, andprostate cancer, respectively (103–105). These ADAM pro-teins have a role in cancer development, progression, orinvasion although the mechanisms have not been elucidatedthoroughly. Production of ADAMTS-1 by fibroblasts ischaracteristic of many carcinomas and correlates withenhanced secretion of TGF-b1 and interleukin (IL)-1b(106). It was shown recently that fibroblasts in coculturewith breast cancer cells upregulate ADAMTS to promoteinvasion in an epigenetic mechanism, likely throughdecreased promoter-associated histone methylation (107).Matrix metalloendopeptidase (CD10), is also expressed byCAFs, elevated in tumor stroma, and has prognostic value inNSCLC and breast cancer, although its precise role in cancer

is still being examined (51, 108–111). Among the MMPinhibitors, TIMP2 was shown to interfere with fibroblast-induced growth of cancer cells in a mouse model (112).Interestingly, however, TIMP along with plasmin, may helpto activate proMMP, and is released with MMP-2(81, 113, 114). TIMP-1 and TIMP-2 activity may besuppressed by cathepsin B, which is localized to fibroblastsand correlated with invasion and shorter patient survival(115, 116).Serine-proteases and their associated inhibitors are very

significant in the continuous loop of proteolytic activitiesthat ultimately promote tumor cell invasiveness. uPA systemcomponents pro-uPA, PAI-1, and PAI-2 are produced byfibroblasts and other stromal cells, and have an importantrole in tumor growth and metastasis. Although high levels ofPAI-1 are associated with tumor progression and poorprognosis, particularly for breast cancer (117, 118),increased PAI-2 has been correlated with better patientprognosis (119). Although both PAI-1 and -2 inhibit uPA,an activator of plasmin, and hence prevent ECM degrada-tion, PAI-1 and PAI-2 interact differently with members ofthe LDL receptor family. PAI-2 binds LDL receptors withlower affinity and consequently does not induce downstreamsignaling leading to cell proliferation, potentially explainingthe observation of decreased metastasis associated with highPAI-2 levels in cancer (120).Microarray studies have shown downregulation of the

protease inhibitor and growth suppressor, WAP-type four-disulfide core (WFDC1), in CAFs (121–123). Overexpres-sion of WFDC1 inhibits proliferation of cancer cells, andWFDC1 expression was shown to decrease with progressionof prostate cancer to metastatic disease (121). The observa-tion of downregulatedWFDC1 in a diverse group of cancershighlights the potential prognostic significance of thismolecule.

ProteoglycansLike other ECM components in the convoluted tumor

milieu, proteoglycans are implicated in many cell signalingpathways. This is largely due to their associated glycosami-noglycans, which bind and interact with a myriad of ECMproteins, macromolecules, cell adhesion molecules, andgrowth factors. An important aspect of proteoglycan bioac-tivity is proteolytic cleavage or shedding. Ectodomain shed-ding affects the biologic function of proteoglycans, alteringproliferation, adhesion, and migration responses. In addi-tion, proteolytic cleavage can yield antiangiogenic peptidefragments to facilitate dissemination (124–126).Syndecan-1 is expressed in the desmoplastic stroma or

cancer fibroblasts of breast, ovarian, head and neck, andother carcinomas (127–131). Its growth-promoting abilityhas been demonstrated in two-dimensional (2D) and 3Dcocultures, as well as knockout studies, particularly in breastcancer models. This growth-promoting function may beattributed to the cleaved, soluble form of syndecan-1 in somecancers (132), whereas the soluble ectodomain may beassociated with decreased proliferation and increased inva-siveness in breast and other cancers (133). Of note,






immunohistochemical examination showed that syndecan-1, which was redistributed from the epithelium to thestroma, was more than 10-fold higher in aggressive breasttissue compared with normal tissue (134). Similarly, fibro-blast-secreted versican promotes tumor growth, migration,and metastasis in breast, ovarian, and prostate cancer (135–139), and is a product of paracrine TGF-b1 signaling in thelatter. Stromal versican is associated with relapse and pooreroutcome in patients with cancer (140–142). Glypican-1 and-3 are additional proteoglycans that may have roles in tumorpromotion, as they are overexpressed in a variety of carci-nomas or associated stromal fibroblasts (128, 143–145).On the other hand, decorin, expressed primarily by

myofibroblasts, has a tumor-suppressive role, reducingtumor growth and metastasis in murine xenograft models,downregulating EGFR (146). The decorin protein core,specifically, is tumor suppressive triple-negative breast can-cer, inducing expression of stromal genes involved in celladhesion and tumor suppression and suppressing genesinvolved in inflammation (147). Decreased expression ofdecorin is a biomarker for aggressive soft tissue tumors (148).Likewise, lumican, which is increased in the reactive stromaof primary tumors, was shown to inhibit invasion of met-astatic prostate cancer cells in vitro (149).Among the proteoglycans, perlecan is the most diverse in

its bioactivities as a consequence of the multifunctionalnature of its large protein core and heparan sulfate chains.Hence, it is not unexpected for the roles of perlecan to extendbeyond development and normal biologic processes into thetumor microenvironment (150). Perlecan is present at highlevels in the ECM and vascularized stroma of breast, colon,prostate, and other cancers (151). In metastatic melanomas,perlecan is increased up to 15-fold (152). It contributessignificantly to cancer cell proliferation, in part, throughcooperation with other growth factor pathways, such as FGF(153–155) and has additional domains that modulate angio-genic activity (156). Its ability to activate FAK highlightspotential involvement in other tumorigenic and prometa-static signaling pathways (157).The highly prevalent pericellular glycosaminoglycan hya-

luronan provides a hydration shelf to facilitate cell signalingand cancer progression. In addition to its receptor CD44, itinteracts with proteoglycans and growth factor complexes tofacilitate cell migration and metastasis. It has an importantrole in regulating inflammation, and can recruit tumor-associated macrophages, which are necessary for extravasa-tion (158). Increased stromal hyaluronan expression isassociated with poor prognosis and metastasis in breast,colon, prostate, ovarian, and lung cancer. It is clearly a vitalplayer in the stromal tumor microenvironment (159–162).

Transmembrane proteinsIntegrins, probably the most vital group of transmem-

brane proteins implicated in cancer progression, are integralcomponents of the ECM, serving as liaisons for transmittingextracellular signals. Integrins bind RGD and other speci-ficity sequences on fibronectin and tenascin, as well asproteoglycans, and growth factors. Integrin adhesions are

regulated by proteases, and integrins reciprocally activate andregulate MMPs. Because integrins form attachments withthe matrix and vasculature, they are imperative to tumor celldissemination and extravasation from the primary site (163).Integrin-a11 is localized to mesenchymal cells, and wasfound overexpressed in NSCLC, where it was shown to havea role in tumorigenicity and increased expression of IGF-2(26, 164). This was demonstrated after implantation ofintegrin-a11–deficient fibroblasts and NSCLC cells inSCID mice caused reduction of tumor volume, which wasrestored upon re-expression of a11 in knockout mouseembryonic fibroblasts. Similarly, a population of CAFs fromhepatocellular carcinoma liver tissue demonstrated signifi-cantly higher surface expression of integrin-a4 and lowerexpression of a2 when compared with nonneoplastic tissue(165). CAFs express several a-subunits, including aV anda5, which interact withmultiple b-subunits, and are capableof binding collagen, fibronectin, and soluble factors such asTGF-b1 (166–168). Integrin ligands, ICAM and VCAM,have been shown to be upregulated and form a prognosticgene signature in CAFs (51, 52). These molecules aresignificant largely because of their fundamental roles inconnecting the ECM and cytoskeleton, initiating intracel-lular signaling pathways necessary for adhesion and motility(see discussion below on mechanical modeling).Central to transmembrane proteins implicated in the

fibroblastic tumor microenvironment is fibroblast activationprotein (FAP), a serine protease with gelatinase activityexpressed by stromal cells in various cancers (169). Itincreases proliferation, adhesion, and migration of ovariancancer cells (170). When expressed in breast cancer cells, itpromoted tumor growth, increasing both the size andnumber of tumors (171, 172). However, it is downregulatedduring the transformation of melanocytes, and may have atumor-suppressive role in melanoma (173, 174). It is alsoelevated in kidney, gastric, basal cell, colon, and lungcarcinoma (175–179). Depletion of FAP-expressing cellsin the latter induced necrosis, and highlighted FAP as animmune suppressor (178). FAP interaction with integrin-a3b1 may facilitate tumor cell adhesion and invasion (180).Endosialin/TEM1 is a highly glycosylated integral mem-

brane receptor expressed predominantly by CAFs, althoughinitially characterized as a tumor endothelial marker (181,182). It has been demonstrated to bind ECM proteins suchas fibronectin and collagen, and enhances cell adhesion andmigration on these matrices. In addition, it was shown topromote activation of MMP-9 (183). In vivo experimentsshowed that deficiency of TEM1 decreased growth of lungcarcinoma and fibrosarcoma (184). Endosialin may beexpressed by a subset of pericytes, and play a role in tumorangiogenesis, but this has not been elucidated fully (182).Podoplanin is another integral transmembrane glycopro-

tein, involved in cell adhesion and the lymphatic vascularsystem. A receptor for selectins, it is recognized by theD2-40monoclonal antibody, which has allowed for detection ofthis protein in lymph nodes. Podoplanin has been shown tobe a very specific indicator for the detection of lymphaticinvasion in primary tumors (185). Podoplanin is expressed at

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higher levels in human vascular adventitial fibroblasts thanlung-derived fibroblasts. Podoplanin-positive vascular fibro-blasts demonstrated increased tumor formation, lymph nodeinfiltration, and metastasis relative to podoplanin-negativevascular fibroblasts, underscoring the significance of podo-planin inmetastasis (186). Podoplanin has prognostic utilityfor several cancers. In breast cancer, podoplanin expressionin CAFs correlated negatively with estrogen receptor status,and was associated with poor prognosis (187, 188). Podo-planin expression in CAFs also is associated with poorprognosis in lung (189) and esophageal cancer (190), butinterestingly, is a favorable prognostic indicator in colorectalcancer (191).Protease-activated receptors (PAR) have an important role

in tumor progression andmetastasis of prostate cancer. PAR-1 is expressed mainly by myofibroblasts in the reactivestroma of primary prostate cancer tissues, whereas PAR-2is expressed predominantly by prostate cancer cells. PAR-1was found to be increased, particularly, in the stroma of high-grade prostate cancers, and also was found in the endothe-lium and fibroblasts of the bone-reactive stroma (192). Inthe prostate, stromal PAR-1 may be upregulated byhuman glandular kallikrein-4 produced by prostate cancercells, triggering secretion of IL-6 from stroma, in turnpromoting cell proliferation and upregulation of kallikreinsin prostate cancer cells (193). The activation and subsequenttumor-promoting effects of PAR-1 in the prostate tumormicroenvironment provide a nice illustration of the signif-icance of reciprocal tumor:stromal signals during cancerprogression.

Secreted proteinsThe secreted ECM glycoprotein osteonectin or add

abbreviation after cysteine: (SPARC) is overexpressed instromal or tumor cells of many cancers (194–197). It wasshown to increase microvessel density in hepatocellularcarcinoma, and therefore may promote angiogenesis. It ishighly prevalent in bone, and is a chemoattractant forbone-metastasizing cancers such as breast and prostatecancer (195). Although it was antimetastatic when over-expressed in breast cancer cells (198), stromal expressionof osteonectin along with CD10 was shown to be aprognostic indicator of recurrence for ductal carcinomain situ (199). Interestingly, stromal and tumor-derivedosteonectin were shown to suppress metastasis of bladdercancer in a murine syngeneic model. Cocultures of bladdercancer and stromal cells demonstrated that osteonectinsuppressed the inflammatory phenotype of tumor-associ-ated macrophages and CAFs by inhibiting reactive oxygenspecies formation via p38-JNK-AP1 and NF-kB. Further-more, osteonectin correlated with decreased expression ofIL-6, SDF-1, VEGF, and TGF-b, and TNF-a in fibro-blasts (200). Consistent with a beneficial role for osteo-nectin, overexpression of osteonectin has been shown toincrease susceptibility of ovarian and colon cancer tochemo- and radiation therapy (201, 202).The related protein, osteopontin, is overexpressed inmany

cancers (203). It is particularly abundant in the stroma of

breast cancer, where it is associated with poor outcome, andskin fibroblasts, where it promotes preneoplastic and malig-nant tumor progression (204–206). Similar to osteonectin,osteopontin promotes prostate cancer growth and progres-sion. The finding of higher osteopontin and osteonectinexpression in bone-metastasizing human prostate cancertissue highlights the potential prognostic value of osteopon-tin and other bone-resident proteins in cancer (207, 208).Osteopontin levels are also higher in triple-negative breastcancers (209). Periostin is another secreted stromal proteinoverexpressed in many cancers, including prostate, breast,colon, ovarian, pancreatic, and melanoma stroma. It isparticularly important in the stromal reaction of cancer andfunctions at the tumor stromal interface (47, 210–213). Inthe prostate, it correlates with poor prognosis, and is report-edly elevated 9-fold compared with BPH (47, 214).Collectively, these proteins can bind collagen, fibronectin,tenascin, andmany integrins, and therefore play a potentiallysignificant role in matrix remodeling in the tumormicroenvironment.

Biophysical and mechanical modeling of the ECMThe study of tumor–stromal interactions requires appro-

priate model systems. Much progress has been made with invitromodels, as the shift from2D to 3Dmodels have allowedfor more physiologically relevant interactions—phenotypi-cally and morphologically approximating the in vivo micro-environment. Spheroids have been used to examine theseinteractions (215), as well as other 3D cocultures usinghydrogels and biologically relevant matrices (216–218),which are also useful in examining the mechanical aspectsof the ECM and related tumor responses. Cells displaydistinct responses on matrix proteins and fibers, as a reflec-tion of their physiologic environment, emphasizing theimportance ofmatrix selection in the study of the fibroblastictumor microenvironment.ECM tension and rigidity, which can be recapitulated in

vitro with mechanical loading and occur in vivo withenhanced matrix deposition and remodeling, influence stro-magenesis (219), and tumor and fibroblast responses (220,221). In vitro studies have demonstrated that fibroblasts in3D or loaded collagen gels are stressed, and this is reflected infibroblast phenotype and signaling events (222–224).Mechanical force triggers transcription of a-smooth muscleactin and collagen to promote differentiation of myofibro-blasts, with the latter requiring cooperation of TGF-b (225,226). These events are necessary for the invasion andproliferation of tumor cells (227, 228). In a mechanicalinvasion assay utilizing paramagnetic micro beads within acollagen/fibronectin matrix and a rotating rare earth mag-net, Menon and colleagues demonstrated that mechanicalforce applied to the matrix induced tumor cell invasion ina mechanism involving cofilin, and that fibronectin wasrequired for this mechanosensory response (229). Stretch-ing of fibronectin induces mechanical stress resulting ininitiation of various signaling pathways (220, 230). Theactivation state of integrins is determined by mechanicalinfluences, among other factors, and different integrin






conformations bind to different surfaces of fibronectin. Inthe case of a5b1, this generates complexes of differentialbinding strengths, which ultimately will affect integrin-mediated signaling cascades (230–232), thus affectingtumor cell responses.Mechanosensing integrins also respond to matrix rigidity

or stiffness with enhanced signaling, activating the FAK,extracellular signal—regulated kinase, and RhoGTPasepathways (40, 233–235). This is associated with increasedcontractility and migration in tumor cells, along with reor-ganization and stretching of the matrix. Examination of thisresponse using breast cancer cells in a 3D collagen geldemonstrated radial realignment of collagen fibers (236),a characteristic effect of migrating cells.Additional studies examining the mechano-signaling

pathways mediated by integrins will help elucidatemechanisms of tumor cell progression. Mechanical forcealso is associated with activity of tenascin-C, which canlead to enhanced invasiveness of tumor cells (237), as wellas TGF-b, which has mechanoregulatory ability, stimu-lates matrix contraction, and is itself activated in responseto stress (238–240). Thus, it is the interconnected anddynamic network of external (mechanical) and intracel-lular signals that promotes cancer progression in thestromal tumor microenvironment.

Targeted ApproachesExperimental studies have generated quantitative infor-

mation about ECM molecules relevant to the fibroblastictumor microenvironment that ultimately must be translatedinto therapeutic targets or clinically useful prognostic indi-cators (241). Mass screening has limitations, as it elucidatesonly transcribed genes, and may not be as sensitive as isnecessary for accurate identification of therapeutic targets.Assessment of mRNA expression and protein levels fre-quently generates different results, and therefore, proteinanalysis should parallel gene expression studies when seekingtherapeutic targets. Targeted inhibition of CAF-derivedECM proteins has proven difficult. For example, FAP wasinitially a very exciting target for reactive tumor stroma, butlater studies with inhibitory antibodies reported no signif-icant results (242). However, other transmembrane proteinsmay prove to be useful biomarkers. Podoplanin, in partic-ular, shows promise as a marker of lymph node metastasis.This has obvious implications in cancer treatment andmanagement, as there is an overwhelming need to distin-guish metastatic cancers from organ-confined or locallyinvasive tumors. PAR-1, as well as a11b1 and endosialinhave shown potential as therapeutic targets for prostate andlung cancer, respectively. Importantly, CAF-downregulatedproteins, such as WFDC1 may be equally significant in thedevelopment of therapeutic modalities, and their utilityshould not be overlooked (Table 1). Inhibition of proteaseshas been challenging most likely because of their uniqueexpression at different stages of progression, and varyingregulatory functions in tumor and stromal cells. This elu-cidates the necessity of highly specific and strategic targeting

of MMPs. Nonetheless, PAI-1 and -2 show encouragingprognostic utility particularly for breast cancer, which issupported by many studies.Matrix proteins expressed at the target site may have

prognostic significance when detected in the primarytumor tissue, as in the case of osteopontin and osteonec-tin. Because of their metastasis-promoting and chemoat-tractant properties, these proteins could be targeted spe-cifically using competitive peptides to prevent coloniza-tion of tumor cells in bone. Bone stromal cells also displayincreased expression of the ECM proteins, versican andtenascin, which promote tumor growth in 3D coculturewith advanced prostate cancer cells (243). This providesfurther rationale for targeting tumor-promoting ECMproteins at the metastatic or secondary site, as it ispresumable that cancer cells induce expression of ECMproteins in target tissue that contribute to their growth.Perlecan, with its diverse biologic activities and contribu-tions to tumor growth, is abundant in bone marrow, andmay be another desired target for bone-metastasizingcancers (150, 244, 245). Finally, given the role of pro-teases in tumor metastasis, fragments of many of theseECM molecules will likely prove to have significantbiologic effects quite varied from the native molecule. Assuch, these fragments may be either therapeutic targets oreasily detectable prognostic markers.

SummaryThe reactive stroma is characterized by a diverse milieu

of soluble and membrane-bound proteins mediating mul-tiple deregulated biologic processes. Reciprocal commu-nication between tumor and stromal cells relies on sig-naling platforms regulated by the diverse components ofthe ECM, ultimately promoting tumor growth, survival,and eventual colonization of the metastatic site. Thus, theECM deserves special attention in the study of tumordevelopment and progression and quest for therapeutictargets.Expression of ECM components and associated signal-

ing events dictating cancer cell responses are determinedby tumor stage and grade, as well as cancer type (47).Therefore, these factors must be considered whenattempting to understand the tumor stromal microenvi-ronment. In the context of the continuous and intricatestring of reciprocal signals in the tumor microenviron-ment, elucidation of relevant stromal components shouldparallel the discovery of tumor-secreted molecules inducedby paracrine signaling.Our knowledge of the functional significance of the ECM

is based predominantly on standard 2D bioassays andmurine models, but it will take more innovative modelscarefully designed to recapitulate tumor–stromal interac-tions in 3D to ascertain the clinicopathologic relevance ofspecific matrix molecules. Hence, prospective studies ofsufficient sample size, necessary first steps to uncover thetrue prognostic value of ECM proteins, must be proceededby comprehensive studies in appropriate model systems to

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analyze and confirm the roles of these prospective targets. Asthe dynamics of tumor–stromal interactions at differenttumor stages become clearer, targeted approaches to preventand predict cancer progression can be introduced.

ImplicationsTumor expansion and metastasis depend not only on the

biology and signaling from or within the epithelial com-partment of the cancer, but also to a large degree on theparacrine factors and matrix composition of the cancer-associated stroma. Future cancer therapy should targethighly relevant stromal cell-derived matrix factors, conse-

quently attenuating stromal-mediated paracrine signals asso-ciated with cancer progression.

Disclosure of Potential Conflicts of InterestR.A. Sikes is a consultant/advisory board member of Delaware Prostate Cancer

Coalition, 1st State Prostate Cancer Survivors, and Sisters on a Mission. No potentialconflicts of interest were disclosed by the other authors.

AcknowledgmentsThis work was supported by the Center for Translational Cancer Research, the

Delaware INBRE program, NIH NIGMS (8 P20 GM103446-13), and NIH P01-CA98912 on Prostate Cancer Bone Metastasis: Biology and Targeting.

Received October 7, 2013; revised December 30, 2013; accepted December 30,2013; published OnlineFirst January 22, 2014.

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Table 1. Stromal ECM factors and their clinical relevance/prognostic value in cancer

Factor Expression Clinical significance Cancer

Oncofetal fibronectin Stroma Malignancy, poor prognosis Oral (53), ovarian (54), colorectal (55),breast (56)

TGF-b stroma Mortality, tumor growth Prostate (11), breast (12)SDF-1 CAFs Tumor progression, angiogenesis Breast (1, 2)HB-EGF Stroma, CAFs Tumor growth Cervical (31), prostate (32)Tenascin-C, -W Stroma Malignancy, metastasis Breast (60, 64, 71)MMP-2 Stroma, sera Poor prognosis, metastasis,

invasionNSCLC (86), prostate (88), skin SCC (93)

MMP-13 Myofibroblasts, stroma Poor prognosis, invasion Skin (93), HNSCC (99), colorectal (100),breast (102)

MMP-11 Fibroblasts, stroma Local invasiveness Breast (47), HNSCC (101)Adam-9 Myofibroblasts Metastasis Colorectal (104)Adam-12 Stroma Tumor progression Prostate (105)PAI-1, uPA Tumor and stroma Poor prognosis Breast (117, 118)PAI-2 Tumor and stroma Favorable prognosis Breast (117, 119)WFDC-1 CAFs (decreased) Metastasis Prostate (121)Syndecan-1 Stroma Progression, poor survival Ovarian (128), endometrial (130), breast (134)Versican Stroma Malignancy, progression Ovarian (141), prostate (142)Decorin protein core Myofibroblasts Tumor suppression Triple-negative breast (147)Hyaluronan Stroma Poor survival, metastasis Ovarian (159), breast (160), prostate (161),

NSCLC (162)ICAM CAFs Progression NSCLC (51)VCAM CAFs Metastasis Colorectal (52)a11b1 Fibroblasts Tumorigenicity NSCLC (26)CD10 (MME) CAFs, stroma Progression, poor prognosis,

recurrenceNSCLC (51), melanoma (108), breast (109)

Podoplanin CAFs 1. Poor prognosis Breast (187, 188), lung (189), esoph. (190)Colorectal (191)2. Favorable prognosis

PAR-1 Stroma Progression Prostate (192)Osteonectin (SPARC) Stroma 1. Recurrence, poor prognosis Breast (199), pancreatic (194)

2. Reduced metastasis Bladder (200)Osteopontin Stroma Tumor progression Breast (204)Periostin Stroma 1. Favorable prognosis Breast (47)

2. Poor prognosis, progression Prostate (47, 214)






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Miles and Sikes





2014;12:297-312. Published OnlineFirst January 22, 2014.Mol Cancer Res Fayth L. Miles and Robert A. Sikes Insidious Changes in Stromal Matrix Fuel Cancer Progression

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