The Matrix Revolution: Matricellular Proteins and Restructuring of The

Author Manuscript Published OnlineFirst on March 19, 2020; DOI: 10.1158/0008-5472.CAN-18-2098 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 The Matrix Revolution: Matricellular Proteins and Restructuring of the

2 Cancer Microenvironment

4 Casimiro Gerarduzzi1,2,*,#, Ursula Hartmann3, Andrew Leask4 and Elliot Drobetsky1,2

7 1Centre de Recherche de l'Hôpital Maisonneuve-Rosemont, Montréal, Québec H1T 2M4, Canada

8 2Département de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada

9 3Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany

10 4College of Dentistry, University of Saskatchewan, 105 Wiggins Rd, Saskatoon SK S7N 5E4, Canada

13 *To whom correspondence should be addressed: Casimiro Gerarduzzi, PhD, Maisonneuve-Rosemont

14 Hospital Research Center, Division of Nephrology, 5415, boul. de l'Assomption, Montreal, Quebec,

15 Canada, H1T 2M4. Phone: (514) 252-3400 ext.2813, email: [email protected]

16 #Senior Author

19 Running title: The Role of Matricellular Proteins in Cancer

20 Keywords: Matricellular Proteins, Extracellular Matrix, Microenvironment, Cancer Progression,

21 Biomarkers/Therapeutics

22 Number of Figures: 1

23 Conflict of interest: The authors declare no potential conflicts of interest.

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 19, 2020; DOI: 10.1158/0008-5472.CAN-18-2098 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

25 Abstract

26 The extracellular matrix (ECM) surrounding cells is indispensable for regulating their

27 behavior. The dynamics of ECM signaling are tightly controlled throughout growth and

28 development. During tissue remodeling, matricellular proteins (MCPs) are secreted into the

29 ECM. These factors do not serve classical structural roles, but rather regulate matrix proteins

30 and cell-matrix interactions to influence normal cellular functions. In the tumor

31 microenvironment, it is becoming increasingly clear that aberrantly expressed MCPs can

32 support multiple hallmarks of carcinogenesis by interacting with various cellular components

33 that are coupled to an array of downstream signals. Moreover, MCPs also reorganize the

34 biomechanical properties of the ECM to accommodate metastasis and tumor colonization. This

35 realization is stimulating new research on MCPs as reliable and accessible biomarkers in cancer,

36 as well as effective and selective therapeutic targets.

38 Introduction

39 The behavior of individual cells is influenced by a plethora of signals originating from the

40 surrounding microenvironment, which includes the extracellular matrix (ECM). Previously

41 regarded as merely a static scaffold for cell/tissue organization, the ECM is now viewed as a

42 critical niche contributing to the regulation of cellular survival, proliferation and migration. This

43 realization has positioned the ECM at the center stage of normal physiological processes such

44 as development, tissue homeostasis and tissue remodeling.

45 The dynamic nature of ECM signaling is determined by a secreted subset of non-

46 structural matricellular proteins (MCPs) (1), in contrast to the structural roles of “classical” ECM

47 proteins such as collagen and fibronectin (2). MCP functional versatility is achieved by its

48 multiple domains that either (i) bind ECM proteins and/or cell surface receptors, (ii) bind and

49 regulate the activity or accessibility of extracellular signaling molecules such as growth factors,

50 proteases, chemokines, and cytokines, or (iii) mediate intrinsic enzymatic activities to precisely

51 orchestrate the assembly, degradation, and organization of the ECM. MCPs are tightly

52 controlled, with expression promptly occurring in context-specific scenarios. Typically, they are

53 highly expressed during early development, ultimately subsiding in adult tissues under

54 physiological conditions. However, transient re-expression is observed during injury repair, and

55 can also be sustained in chronic pathologies such as cancer (2-7). Indeed, chronic unscheduled

56 expression of various MCPs, either by tumor cells or the surrounding stroma (8), leads to

57 abnormal ECM remodeling and stimulation of mitogenic pathways essential for cancer

58 progression. This may underlie the correlation between the upregulation of many MCPs and

59 poor prognosis in cancer patients (9) and, moreover, provide rationale for exploring the utility

60 of MCPs as cancer biomarkers and therapeutic targets.

61 This review will focus on the burgeoning roles of the MCP families SPARC, CCN, SIBLING,

62 Tenascin and Gla-containing proteins in both cancer development, detection and treatment.

63 Certainly, members of these particular families are aberrantly expressed in various tumor types,

64 and moreover exhibit biochemical, biomechanical and metastatic properties influencing cancer

65 progression.

67 Normal physiological roles of MCPs

68 The ever-growing number of newly-discovered MCPs has necessitated their

69 classification into families. Members are grouped based on shared domains, which in turn

70 reflect the functional diversity between families.

71 The SPARC protein (Secreted Protein Acidic and Rich in Cysteine; hereafter alternative

72 protein names are included in parentheses; BM40, osteonectin), one of the original MCPs to be

73 characterized, is considered prototypical due to its simple structure and rich functionality. The

74 subsequent discovery of other MCPs with structural similarity revealed a broader family of

75 SPARC-related proteins (10). Such SPARC family members share follistatin-like and extracellular

76 calcium-binding (EC) domains, and are classified into five distinct groups based on sequence

77 homology of their EC domains (10): SPARCs, SPARCL1, SMOCs, SPOCKs and Follistatin-like

78 protein-1 (FSTL1). SPARC family members were shown to regulate ECM assembly and

79 deposition, influence cytokine activity, inhibit cell adhesion and cell cycle progression, regulate

80 cell differentiation and activate matrix metalloproteinases (10). While most SPARC members

81 exhibit ubiquitous expression throughout early development, in adults, expression is largely

82 limited to tissues that are diseased or undergoing wound repair/remodeling.

83 The vertebrate CCN (Centralized Coordination Network) family is composed of six

84 homologous cysteine-rich members (11): CCN1 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4

85 (WISP-1), CCN5 (WISP-2) and CCN6 (WISP-3). Each is comprised of an N-terminal secretory

86 peptide and four functional domains: insulin-like growth factor-binding protein domain (IGFBP),

87 Von Willebrand factor domain (VWR), thrombospondin type-1 repeat module (TSR) and

88 carboxy-terminal cysteine-knot (CT) motif (11). In response to tissue remodeling, CCN proteins

89 are expressed, principally in mesenchymal cells, during development and in connective tissue

90 pathologies (12). The postnatal role of CCN proteins is known for promoting collagen stability or

91 organization (13).

92 Tenascins (TNs) comprise a family of four large extracellular matrix glycoproteins, i.e.,

93 TN-C, -R, -W and -X, which exist as either trimers or hexamers (14). TNs share a characteristic

94 modular structure composed of tandem epidermal growth factor (EGF)-like domains,

95 fibronectin-type III domains and a C-terminal fibrinogen related domain (FReD). Consequently,

96 TNs share functions in modulating cellular responses to the ECM and growth factors, specifically

97 regulating growth, differentiation, adhesion and migration during tissue remodeling events

98 (15). However, each member has distinct spatial and temporal expression. TN-C expression is

99 typically present in all organs during fetal development and mechanical stress, whereas TN-W

100 expression is restricted to developing/remodeling bone and certain stem cell niches (14). TN-R

101 is expressed exclusively in the developing and adult nervous system, while TN-X represents a

102 constitutive ECM component of most connective tissues, being hardly influenced by external

103 factors (14).

104 The SIBLING (Small Integrin-Binding Ligand N-Linked Glycoprotein) family includes Bone

105 Sialoprotein (BSP), Osteopontin (SPP1, aka OPN), Dentin Sialophosphoprotein (DSPP), Matrix

106 Extracellular Phosphoglycoprotein (MEPE) and Dentin Matrix Protein-1 (DMP1). These proteins

107 are primarily implicated in bone morphogenesis and biomineralization, and were thus thought

108 to be exclusively localized to mineralized tissue such as bone and teeth (16). However, apart

109 from these traditional functions, SIBLING members were also shown to influence cellular

110 proliferation/survival pathways, collagen fibrillogenesis, MMP activities and response to injury

111 (17-20).

112 The Gla-protein family members contain vitamin K-dependent γ-carboxyglutamic acid

113 residues (21), which have high affinity for calcium ions, thus conferring important roles in

114 coagulation and bone homeostasis (22). Among the 17 Gla-protein members, Periostin (POSTN)

115 and Matrix Gla-Protein (MGP) are known to affect ECM crosslinking and various cellular

116 behaviors, such as migration, adhesion and proliferation in epithelial, endothelial, fibroblast,

117 osteoblast and myocyte cells (23-27). POSTN is expressed in osteoblast, mesangial, fibroblast,

118 mesenchymal and vascular smooth muscle cells (22), while MGP is typically secreted and

119 localized in the surrounding ECM of chondrocytes or endothelial cells (28).

120 Considering that MCP expression is context dependent, MCP knockout mouse models

121 generally lack any postnatal phenotype unless challenged by injury or disease, in which case

122 they exhibit an impaired yet subtle response (see references for further details: SPARC family

123 (29-34); CCN family (11,35); TN family (36-38); SIBLING family (39-42); POSTN (43-45)).

124 However, some MCP mouse knockouts are characterized by severe complications. For example,

125 FSTL-1 and CCN2 null mice die shortly after birth, while CCN1 and CCN5 whole body knockouts

126 are embryonic lethal, showing that these proteins are essential for development (11,46,47). As

127 for MGP knockout mice, they show severe vascular calcification, arteriovenous malformation

128 and craniofacial anomalies, and die within 8 weeks after birth (48-50).

129

130 Expression of MCPs in Cancer

131 MCP overexpression is characteristic of tissue remodeling processes, including those

132 occurring during carcinogenesis, as opposed to low/undetectable levels in normal tissue. Tumor

133 cells and the surrounding activated stromal cells are the major cell types that aberrantly secrete

134 MCPs into the tumor microenvironment, in turn promoting cancer development (5,51).

135 Nonetheless, we note there are certain cases where MCP expression has been shown to oppose

136 cancer development (51,52).

137 SPARC protein is highly expressed in cancer cells and the stroma of certain cancers,

138 including glioma, breast and cervical melanoma (53-56), where it exhibits oncogenic roles in cell

139 growth, invasion and apoptosis. Interestingly, SPARC has also been associated with tumor

140 suppression by influencing these same processes (57). This discrepancy might be explained by

141 cancer type and stage, and/or the concentration of SPARC in the tumor microenvironment (57).

142 Like SPARC, the role of FSTL1 in carcinogenesis has generated significant controversy.

143 Endometrial and ovarian cancers exhibit low FSTL1 levels; moreover, ectopic FSTL1 expression

144 exerts anti-neoplastic activity by inducing apoptosis (58). Among SPOCK isoforms (SPOCK1-3),

145 SPOCK1 is upregulated in different tumor types, and its expression positively correlates with

146 invasive/metastatic potential and hence poor prognosis (59-61). However, in brain tumors,

147 expression of all SPOCK family members decreases with increasing tumor grade (62). SMOC2

148 was shown to be upregulated in hepatocellular-, endometrial- and colorectal- cancers where it

149 modulated proliferation, chemoresistance and metastasis, respectively (63-65). Very little is

150 known regarding any role for SMOC1 in carcinogenesis, although its expression is increased in

151 brain tumors, where it interacts with TN-C to counteract the chemo-attractive effect of the

152 latter on glioma cells in vitro (66).

153 Among the CCN family, CCN1 and CCN2 are the most studied in cancer (11). Specifically,

154 CCN1 expression is elevated in many tumor types including brain, breast, prostate and pancreas

155 (67-70); similarly, CCN2 upregulation is implicated in proliferation, apoptosis and migration for

156 numerous cancers (71), including gastric (72), pancreatic (73), melanoma (74,75) and breast

157 (76). In addition to cancer cells, a potential origin of these MCPs may be cancer-associated

158 fibroblasts (CAFs) (77), and indeed this cell type was shown to be the source of CCN1/CCN2

159 expression in murine models of skin cancer (78,79). Although unscheduled expression of CCN1

160 and CCN2 are generally associated with tumor promotion, in some cases these proteins were

161 reported to inhibit cancer development (80,81). Like CCN1/2, CCN3 and CCN4 exhibit a mixture

162 of pro- vs anti- tumorigenic effects, whereas CCN5 and CCN6 are predominantly regarded as

163 tumor suppressors (11,82).

164 Each TN family member differs substantially in spatial- (tissue specificity) and temporal-

165 expression patterns (14). In the case of TN-C and TN-W, de novo expression is prominent in

166 tumors versus healthy tissue, where they promote tumor progression on multiple levels, i.e.

167 proliferation, invasion, metastasis and angiogenesis. TN-C is recovered in the stroma of most

168 solid cancers, while TN-W is primarily restricted to brain, colon, kidney and lung cancers (14). In

169 contrast, TN-R and TN-X are constitutively expressed and largely unaffected by tumorigenic

170 signals, i.e. to date have not been reported to play a substantial role in carcinogenesis (14).

171 Among SIBLING proteins, SPP1 and BSP have been the most extensively studied in the

172 context of cancer (16). Consistent with their roles in osteogenesis, SPP1 and BSP have been

173 implicated in bone malignancy (16). However, while these proteins were initially thought to be

174 expressed only during bone morphogenesis, both were subsequently shown to be broadly

175 expressed in human epithelial carcinomas, including but not limited to breast (83,84), lung

176 (85,86), prostate (87), liver (88), pancreas (89) and colon (87,90), where their

177 pathophysiological roles have recently been thoroughly reviewed (16,91). Furthermore, CAFs

178 have been shown to produce and secrete SPP1, which contributes to melanoma tumor growth

179 (92).

180 POSTN, the most well-characterized Gla-protein family member, was shown to be a

181 major determinant in proliferation for a number of aggressive, advanced solid tumors with poor

182 prognosis (22). MGP is much less known than POSTN, but is gradually emerging as a

183 determinant in cancer progression, exhibiting increased expression in colorectal, glioblastoma,

184 breast, cervical, osteosarcoma and skin cancers with unfavorable prognosis (93-97).

185 In general, MCPs are capable of regulating a variety of mechanisms necessary for

186 tumorigenesis, such as survival, proliferation, migration, matrix stiffness and development of a

187 signal reservoir and metastatic microenvironment. These versatile functions depend on the

188 diverse biochemical, biomechanical and metastatic niche effects induced by MCPs (Fig. 1), as

189 discussed in more detail immediately below.

190

191 MCP Biochemical Effects

192 Much evidence has shown that MCPs possess biochemical properties essential for

193 regulating various cellular behaviors, including ones implicated in tumor development. These

194 properties mainly pertain to the ability of MCPs to activate a number of cell surface receptors

195 and elicit their downstream signaling (Fig. 1A). Most MCPs are well-known to directly bind

196 integrins, which are αβ heterodimers composed from 18 α subunits and 8 β subunits (98).

197 Integrins are commonly bound by members of the SPARC, CCN, SIBLING, TN and Gla-protein

198 families (99-101), each bound to varying heterodimer combinations. Other than integrins,

199 members of CCN and TN families can also bind syndecans, while SPP1 is reported to also bind

200 CD44 receptors (99,100). In addition, MCPs can act indirectly by binding a variety of ligands (i.e.

201 growth factors and cytokines), thereby limiting ligand distribution and accessibility, or either co-

202 activate or inhibit their function (101).

203 SPARC has been reported to mediate a variety of signaling pathways. For example,

204 SPARC can bind directly to integrin receptors (αvβ1, αvβ3 and αvβ5) resulting in the activation

205 of the proximal intracellular kinases Akt, focal adhesion kinase (FAK), and integrin-linked kinase

206 (ILK) (102-105). These kinases were associated with SPARC-mediated invasion and survival of

207 glioma cells (105). SPARC may also directly interact with the TGFβ1 receptor to mediate Smad

208 signaling, as shown in lung cancer cells (106). Recently, SPARC was reported to bind TGFβ1 to

209 regulate its deposition in the ECM (107). In addition, SPARC may bind other growth factors but

210 with unknown effects (108,109). Interestingly, SPOCK1 was identified as a downstream target

211 of TGFβ1 and a key player in lung cancer metastasis and proliferation (110), as well as in anti-

212 apoptosis via activation of the PI3K/Akt pathway (111,112). SMOC2 acts to maintain ILK activity

213 during G1 which in turn influences cell cycle progression by modulating cyclin D1 expression

214 and DNA synthesis (113). This possibly involves ILK interaction with integrin β1 and β3

215 cytoplasmic domains, which also leads to inhibition of anoikis and apoptosis through activation

216 of PI3K/Akt signaling (114). Studies by Maier et al suggested that SMOC2 can bind directly to

217 integrins αVβ1 and αVβ6 (115), consistent with recent data showing that SMOC2 binds integrin

218 β1 to activate FAK in kidney fibroblasts (29).

219 CCNs act through multiple mechanisms to regulate a plethora of dynamic cellular

220 processes (11,101,116). In particular, these proteins activate ILK/Akt, MAPK, and associated

221 growth promoting pathways in cancer, with each CCN member exerting distinct effects and

222 temporal expression profiles. For example, CCN1 signals through integrin αVβ3/Sonic hedgehog

223 to promote motility in vitro and tumorigenic growth in vivo (117) as well as integrin α6β1-

224 mediated invasion (118), in pancreatic cancer. In glioma, CCN1 overexpression enhances

225 tumorigenicity through integrin αVβ3- and αVβ1-linked ILK-mediated activation of Akt, β-

226 catenin-TCF/Lef and associated survival and proliferation pathways (119). In breast cancer cells,

227 CCN1 can promote (i) resistance to anoikis, partly via integrin β1 (120), as well as (ii)

228 proliferation, survival and apoptosis resistance through the αvβ3-activated ERK1/2 pathway

229 (121). Similar to CCN1, ectopic expression of CCN2 (i) promotes migration and angiogenesis

230 (122), and (ii) confers apoptosis resistance through integrin αvβ3/ERK1/2 upregulation of anti-

231 apoptotic Bcl-xL and cIAP (76) in breast cancer cells. Although most CCNs act primarily through

232 binding various integrin heterodimer combinations, they also bind several other receptors

233 (11,101,123,124), for example syndecan-4 and Notch in the case of CCN1/2 and CCN3,

234 respectively. Interestingly, CCN proteins may be activated by proteolytic cleavage (125,126).

235 The opposing effects of CCN3 and CCN4 in different cancers raise the question of which

236 biochemical pathways are responsible for their signaling diversity. In colorectal cancer cells,

237 CCN3 inhibits survival by regulating caspase-3/-8 while inhibiting JNK-mediated migration (127).

238 On the contrary, CCN3 promotes osteoclastogenesis through the FAK/Akt/p38/NF-κB pathway

239 (128). CCN4 also promotes FAK and p38 signaling through αvβ1 integrin in prostate cancer cells;

240 however, this pathway specifically induces migration and vascular cell adhesion molecule-1

241 (VCAM-1) expression by down-regulating miR-126 (129). Furthermore, osteoblast-derived CCN4

242 plays a key role in prostate cancer cell adhesion to bone through VCAM-1/integrin α4β1 (130).

243 CCN4 also promotes lymphangiogenesis in oral squamous cell carcinoma (SCC) via integrin

244 αvβ3/Akt signaling and upregulation of VEGF-C expression, as well as promotes integrin

245 αvβ3/FAK/JNK signaling to induce VEGF-A-activation of angiogenesis in osteosarcoma cells

246 (131,132). Conversely, CCN4 inhibits migration in melanoma and lung cancer cells by

247 inactivating the family of Rho-like GTPases (133,134).

248 TN-C has been shown to stimulate proliferation and survival in a variety of cancers by

249 activating several pathways downstream of integrins and syndecans (135), including integrin

250 α9β1 activation of Akt and MAPK (136) and αvβ3 activation of FAK and paxillin (137). However,

251 a recent study showed that TN-C signaling through integrin α2β1, but not α9β1 or αvβ3,

252 induced autocrine growth in brain tumor cells (138). Through an indirect mechanism of

253 tumorigenesis, TN-C is able to compete with syndecan-4 binding to fibronectin, thereby

254 interfere with fibronectin inhibition of proliferation (139). Instead, the FBG domain of TN-X was

255 reported to convert latent TGFβ1 into its biologically active form to indirectly control

256 mesenchymal differentiation (140).

257 POSTN is primarily known for binding integrins αvβ3 and αvβ5 to elicit activation of

258 FAK/JNK and PI3-K/Akt signaling pathways controlling cell proliferation, survival, or migration in

259 various cancers (141-143). POSTN may also signal through epidermal growth factor receptor

260 (EGFR) to influence migration in esopahageal SCC (144), potentially through cross-talk with

261 integrin αvβ5. Unlike POSTN, little is known regarding the mechanism of MGP in cancer

262 development, although the latter can influence the TGFβ superfamily, including activation of

263 TGFβ1 receptor and inhibition of the bone morphogenetic proteins BMP-2 and BMP-4 (27,145).

264 The SIBLING family members BSP and SPP1 exhibit similar activities in cancer

265 development. BSP supports adhesion, proliferation and migration through αvβ3 and αvβ5, and

266 the pro-metastatic activity of TGFβ1 in breast cancer cells (146,147). SPP1 can interact with

267 several integrin receptors (αvβ1,3,5, α8β1, α9β1,4 and α4β1) to regulate cell proliferation,

268 angiogenesis, adhesion and migration (116,148). SPP1 can also signal through CD44 (149) to

269 activate HIF-2α-induced stemness in hepatocellular carcinoma and glioblastoma cells (150,151),

270 and Akt-mediated cell survival in mesothelioma and colorectal cancer cells (152,153).

271

272 MCP Biomechanical Effects

273 Remodeling of the ECM is an integral process in cancer development that

274 accommodates the structural architecture of the tumor and provides necessary physical

275 changes such as increased matrix and tissue stiffness to promote and sustain neoplastic

276 transformation (154). Mechanotransduction is a process in which perturbations in ECM

277 mechanical stiffness are transduced into biochemical signals. ECM stiffness can communicate

278 with cells through mechano-responsive integrins (98). In a normal setting, the ECM forms a

279 structural microenvironment of relaxed non-oriented fibrils that exerts homeostatic stiffness on

280 embedded cells. In cancer, disruption of this local ECM structure can occur through MCP-

281 mediated remodeling (5,8,155-157), which result in structures that are often stiffer, more

282 highly linearized and have a different orientation, relative to normal stroma (7). In response to

283 this, matrix bound integrin structures convert these physical mechanical signals into

284 conventional integrin biochemical signals to influence survival, proliferation and growth (158).

285 Moreover, integrin and their associated intracellular cytoskeleton mature into reinforced focal

286 adhesions and stress fibers, respectively, to compensate for changes in ECM stiffness (Fig. 1B).

287 Stress fibers are formed from the bundling of actin, and generate a counter-force, both of

288 which are regulated by phosphoactivating myosin through the stiffness-induced integrin-RhoA-

289 ROCK axis (159). Some of the biomechanical processes regulated by MCPs that affect ECM

290 stiffness include increased matrix organization, collagen crosslinking and deregulation of

291 enzymatic activity.

292 SPARC is well-known to be implicated in rearranging the matrix through collagen

293 crosslinking. SPARC binds to several fibrillary collagens (I, II, III and V) as well as to collagen IV, a

294 prominent constituent of basement membranes (160,161), and is critical for organization of

295 collagenous extracellular matrices. SPARC knockout mice manifest significant changes in

296 collagen fibril morphology, as well as a substantial decrease in adult tissue concentrations of

297 collagen (for review see Bradshaw and references therein). SPARC also influences the response

298 of host tissue to implanted tumor cells and a lack of endogenous SPARC engenders decreased

299 capacity to encapsulate the tumor as well as a reduction in the deposition of collagen (162).

300 SPARC exerts at least two roles in collagen fibril assembly, i.e., by modulating interactions of

301 collagen with cell surface receptors and directly regulating collagen incorporation into fibrils

302 (163). Loss of SPARC also disrupts the homeostasis of basement membranes and alters tissue

303 biomechanics and physiological function (164). Finally, SPARC can act as an extracellular

304 chaperone for collagens that enhance the tumorigenic environment (164-166).

305 In a recent study, TN-C significantly co-localized with aligned collagen fibers in breast

306 cancer patients, compared to the wavy and randomly organized layout of collagen (167)

307 typically observed in normal tissue (168). TN-C contains multiple ECM binding partners,

308 including collagen; however, its involvement in collagen alignment may be mediated through

309 binding to fibronectin, which serves to direct collagen organization (169-171). Similarly, POSTN

310 plays a mechanistic role in inter-matrix interactions through formation of a POSTN-BMP-1-LOX

311 complex, where BMP-1 promotes LOX activity for collagen crosslinking (172,173). In fact, POSTN

312 knockout animal models exhibit aberrant collagen fibrillogenesis (174). Furthermore, the

313 mechanotransduction pathways of both ROCK in SCC and the transcription factor TWIST from

314 various mechanical stress models are known to increase POSTN deposition (24,175). The POSTN

315 family member MGP was recently shown to be incorporated into crosslinked multimers of

316 fibronectin, which enhanced cancer cell attachment to fibronectin (23). As for the CCN family,

317 recent studies have shown CCN1, CCN2 and CCN4 to promote alignment and stability of

318 collagen fibers (13,157,176).

319

320 MCP Influence on the Metastatic Niche

321 The matrix environment needs to achieve a level of plasticity for cellular displacement

322 during metastasis. In order to disseminate, cancer cells require a local ECM niche to support

323 cellular differentiation and intravasation, and an ECM at the secondary metastatic site to

324 permit invasion and colonization (Fig. 1C). There are various ways in which MCPs are able to

325 establish a metastatic niche by influencing the ECM and its embedded cells. First, MCPs induce

326 cancer cells to undergo an epithelial-to-mesenchymal transition (EMT), a genetic program that

327 promotes metastatic dissemination of cancer cells from primary epithelial tumors (177).

328 Second, MCPs reorganize the ECM architecture and integrity to promote cancer cell

329 accessibility into intact structures, i.e. basement membrane (178). MCPs can also affect physical

330 properties of the ECM, including spatial arrangement, orientation, rigidity, permeability and

331 solubility, in such a way as to alter anchorage sites and create motility tracks suitable for

332 metastasis (178).

333 Normally, epithelial cells maintain their polarity, intercellular tight-junctions and

334 adherence to the basement membrane necessary for a proper tissue architecture and function

335 (179). During EMT, epithelial cells undergo reorganization of adhesion and cytoskeletal

336 structures to acquire a mesenchymal morphology. This allows cells to detach which, in

337 conjunction with enhanced migratory capacity associated with the mesenchymal phenotype,

338 stimulates metastasis (179).

339 SPARC family members promote EMT in a variety of cancers (Fig. 1C). Recently, SMOC2

340 was shown to participate in a pro-metastatic secretome mediated by the ARNTL2 transcription

341 factor in lung adenocarcinoma (180), and SMOC2 induction is required for colon cancer

342 invasion by stimulating EMT (65). Several studies also show that SPOCK1 promotes EMT

343 (110,181). Among SPARC family members, SPARC is the most characterized for influencing EMT

344 (106,182,183): (i) In lung cancer cell lines, TGFβ1 activation of migration and EMT is in part

345 through SPARC (106), (ii) In head and neck cancer cells, SPARC enhances EMT signaling via

346 activation of Akt (182), and (iii) Overexpression of SPARC in melanoma cells increases

347 invasiveness mediated by phosphorylation of FAK and Snail repression of E-cadherin promoter

348 activity (184).

349 The CCN family exerts varying effects on EMT. An early study using pancreatic cancer

350 cells reported that CCN1 promotes EMT and stemness, and that silencing this MCP forestalled

351 aggressive tumor cell behavior by reversing the EMT phenotype (67). Recent studies have

352 continued to dissect CCN1 signaling leading to EMT. In osteosarcoma, pharmacological or gene

353 knockdown of integrin αvβ5/Raf-1/MEK/ERK signaling components inhibited CCN1-induced

354 EMT (185), as well as CCN1-mediated expression of EMT markers and cell spreading through an

355 IGF1Rβ-JNK-dependent pathway (186). In contrast, CCN5 and CCN6 exert opposing effects on

356 EMT. In triple negative breast cancer cells, CCN5 activates the Bcl-2/Bax apoptotic pathway and

357 inhibits both EMT and migration (187), while activation of the JAK/Akt/STAT pathway reverses

358 such CCN5-mediated events (188). Similarly, CCN6 reversed the EMT features and inhibited

359 metastasis of breast cancer cells in vivo, but through a Slug signaling axis that regulates Notch1

360 activation (189). Another mechanism involves CCN6-BMP-4 binding in breast cancer cells, which

361 reduces BMP-4 signaling through p38/TAK1 and subsequent downstream activation of invasion

362 and migration (190).

363 TN-C and POSTN have also been associated with metastasis (191-195). While the

364 influence of TN-C (196,197) and POSTN (198,199) can be exerted through the EMT process,

365 interestingly, these MCPs are also capable of remodeling the ECM to form migratory tracks that

366 support rapid dissemination of cancer cells (Fig. 1C). TN-C is frequently observed to be

367 expressed along the border of matrix tracks in skin (200), pulmonary (201), colorectal (202) and

368 breast (203) cancers. In fact, TN-C assembles into matrix tracks with ECM molecules such as

369 fibronectin, laminins and several collagens (200,204), which are also linked to metastatic

370 potential (200,205,206). Evidence reveals that these TN-C matrix tracks have a functional

371 purpose in metastasis. In co-culture experiments, leading fibroblasts were able to create matrix

372 tracks composed of TN-C and fibronectin, which were left behind for the movement of SCC cells

373 (207). For fibronectin and TN-C to co-assemble into such tracks, POSTN is responsible for

374 incorporating TN-C into the meshwork architecture (208). While integrating TN-C, it is possible

375 that POSTN could also serve as a scaffold for BMP-1, LOX-1 and collagen to accelerate collagen

376 crosslinking into migratory tracks during metastasis (172). Mechanistically, track mobility

377 involves TN-C competing for syndecan-4 binding to fibronectin, which blocks integrin α5β1–

378 mediated cell adhesion for detachment (139), followed by TN-C promotion of migration

379 through integrin α9β1 following YAP inactivation (209). As previously discussed, TN-C and

380 POSTN can bind multiple ECM proteins (i.e. fibronectin and various collagens) and enzymes (i.e.

381 LOX) to serve as scaffolds of collagen crosslinking needed for cancer cell proliferation and

382 survival. This is a similar process that comes into play when TN-C and POSTN interact to build

383 ECM scaffolds for migration tracks (24,167,204,208,210).

384 Finally, for metastatic cells to exit the embedded state for intravasation, and then

385 return to the ECM for colonization after invasion at a distant site, a degree of matrix plasticity is

386 required. Such ECM remodeling is achieved by the degradative activity of extracellular

387 proteases (Fig. 1C), in particular metalloproteinases (MMPs). Like several other MCPs, SIBLINGs

388 bind and activate MMPs to promote metastasis. SPP1 binds CD44 to activate MMP-3 while BSP

389 binds to integrin αvβ3 to activate MMP-2 to increase invasiveness in various cancer cell types

390 (20,211). Furthermore, SPP1 and BSP bind and activate MMP-3 and MMP-2, respectively (20).

391 Early studies reported that SPARC upregulates the expression and activity of MMP-2 and MMP-

392 14 in glioma cells and MMP-2 in breast cancer cells (212,213). On the other hand, the SPARC

393 family member SPOCK2 was recently shown to inhibit the expression of MMP-2 and MMP14,

394 and activation of MMP-2 in endometrial cancer cells (214,215). TN-C may also influence the

395 invasion of chondrosarcoma, colon cancer and glioma cells by interacting with and upregulating

396 MMP-1, -2/9 and -12, respectively (216,217).

397 MCPs can also serve as a substrate to MMPs (218), i.e., SPARC and SPP1 in the case of

398 MMP-2, -3, -7, -9, -12 and -14, and MMP-3, -7, -9 and -12, respectively. From the SPARC family,

399 SPARCL1 and SPARC are cleaved by MMP-3 in gliomas (219) and cathepsin K in bone cancer

400 (220), respectively, whose fragments could affect SPARC activity. Recently, MMP-9 cleavage of

401 SPARC was reported to enhance SPARC-collagen binding, preventing collagen degradation by

402 MMPs in lung cancer (166). As for SPP1, thrombin and plasmin can cleave its C-terminal, which

403 increases adhesion of melanoma cells (221) and migration of breast cancer cells (222), while

404 cleavage of SPP1 by MMP-9 is essential for hepatocellular carcinoma invasion, which correlates

405 with metastatic potential (223).

406 Apart from targeting MMPs, the role of TN-C in influencing the ECM to promote invasion

407 is multifaceted. In Ewing sarcoma, TN-C expression and Src activation cooperate to promote

408 invadopodia formation, an actin-rich protrusion of the plasma membrane involved in

409 degradation of the ECM during cancer cell extravasation (224). In order for distant sites to

410 accommodate disseminated tumor cells, MCPs are also required at the secondary target tissue

411 to prime the metastatic niche for colonization. TN-C has been shown to be involved in

412 metastatic colonization since loss of this MCP in breast cancer, melanoma or metastatic niche

413 stromal cells inhibited colonization in the lungs (225-227). Gla-containing proteins have also

414 been implicated in establishing a metastatic niche. Tumor-derived POSTN was reported to form

415 a microenvironmental niche supportive of breast cancer stem cells via the integrin αvβ3/ERK

416 pathway (228). In various mouse models, POSTN was responsible for metastatic colonization of

417 the lung by breast and melanoma cells as evidenced by POSTN neutralizing antibodies,

418 antisense oligonucleotides and KO mice, all independently inhibiting metastasis (229-231).

419 Given their significance for breast cancer cell dissemination to the lungs, it remains possible

420 that both POSTN and TN-C are interdependent in promoting colonization of the metastatic

421 niche, since POSTN anchors TN-C to the ECM (208). MGP was also recently shown to influence

422 the metastatic niche by promoting osteosarcoma adhesion, extravasation and MMP activities in

423 murine lung endothelium in vitro (94).

424

425 Future Clinical Applications

426 MCPs are generally expressed at low levels in adult tissues but highly upregulated in

427 various pathologies or injuries (4-6). This has prompted researchers to elucidate the potential

428 functions of different MCPs in diseases such as cancer. As discussed throughout this review,

429 numerous studies have shown that MCPs play critical roles in cancer development. In addition,

430 the presence of certain MCPs in circulation as well as diseased tissue indicate their utility as

431 non-invasive diagnostic and prognostic cancer biomarkers. Furthermore, their extracellular

432 location and involvement in cancer pathology indicate that MCPs represent accessible and

433 potentially effective therapeutic targets. In the following sections, we discuss various pre-

434 clinical studies and clinical trials exploring the above possibilities.

435 MCPs as Cancer Biomarkers

436 SPARC has been suggested as a prognostic biomarker for certain cancers such as soft

437 tissue sarcoma, esophageal SCC and glioblastoma since its expression correlates with poor

438 survival (232-234). In addition, SPP1 may be prognostic for breast, lung, gastric, liver and colon

439 cancers since it associated with tumor progression and decreased patient survival (235-238).

440 Subsequently, a number of ongoing clinical trials have been established to validate their

441 application. Recently, SPARC has been the subject of a clinical study probing its utility as a

442 diagnostic marker for brain cancer (registered number clinical trial (NCT) 01012609), given prior

443 investigations correlating increased tumor vascular SPARC expression with decreased brain

444 cancer patient survival (239). Several groups have also reported that high plasma SPP1

445 concentrations might be predictive of poor outcome for several cancers, including breast

446 cancer (240). Consequently, one clinical study is currently probing the relevance of SPP1 serum

447 levels for diagnosis of breast cancer (NCT 02895178). Other MCP families await successful

448 clinical trials since the expression of several CCN family members in pancreatic, breast, oral,

449 esophageal and brain cancers (241-245), TN-C in colorectal, glioma, pancreatic and bladder

450 cancers (196,246-248), and POSTN in various solid cancers (249) have all been touted as

451 potential diagnostic and prognostic biomarkers.

452 MCPs as Therapeutic Targets

453 Targeting MCPs for therapeutic purposes has received relatively little attention,

454 primarily due to limited data concerning mechanisms of action. The fact that MCPs are located

455 in the extracellular space during cancer development renders them attractive as accessible

456 targets for drug delivery; moreover, their context-specific expression implies that targeting

457 these proteins would result in minimum pleiotropic side-effects. Neutralizing antibodies against

458 MCPs have shown success in various preclinical settings; however, translation to the clinic has

459 been difficult. One group showed that an SPP1 monoclonal antibody (AOM1) significantly

460 inhibited tumor growth and metastasis in a mouse model of non-small lung cancer (250). In

461 addition, a commonly-used monoclonal antibody for antagonizing CCN2 (FG-3019) has

462 reportedly been used in preclinical models with success in both monotherapy and combination

463 therapy for different tumor types, including pancreatic and melanoma (251-256). With such

464 progress, neutralizing-antibodies targeting MCPs have advanced to registered clinical trials. For

465 example, FG-3019 is currently in phase III for CCN2-targeted treatment of pancreatic cancer

466 (LAPIS, NCT03941093).

467 Alternatively, MCPs could be targeted by inhibiting gene expression in patients. In fact,

468 one of the first studies using RNA interference (RNAi) to treat cancer with promising results

469 involved targeting TN-C in 11 glioma patients (257). This was followed up with an investigation

470 of a larger cohort of 46 patients, which reported significant improvement in overall survival

471 (258). Other promising MCP targets for posttranscriptional gene silencing include SPP1, POSTN,

472 CCN1 and CCN2, where inhibition of RNA expression was shown to reduce cancer progression in

473 various animal models (68,73,229,235). Another therapeutic approach involves exploiting the

474 high expression of MCP within the tumor environment as a strategy to deliver therapeutic

475 molecules. Using a high affinity antibody to deliver radiotherapy (monoclonal antibody 81C6),

476 TN-C was targeted for treatment of glioma and lymphoma (259,260), showing safe and

477 promising anti-tumor benefit.

478

479 Conclusion and Future Perspective

480 Upon perturbation of tissue homeostasis during multistage carcinogenesis, MCPs are

481 upregulated in the tumor microenvironment to become key mediators of cell-ECM

482 communication that in turn promotes cellular proliferation, survival and metastasis. The

483 functional diversity of MCPs stems from their ability to interact with a variety of extracellular

484 signaling molecules such as ECM components and growth factors. Moreover, as emphasized in

485 this review, many MCPs have been implicated in cancer development, and thus may certainly

486 exert additive and/or antagonistic effects in this process. Our current understanding of MCP

487 pathways gives an impression of redundancy, and so a primary aim in the ECM field is to

488 elucidate the precise manner in which MCPs mechanistically converge, both functionally and

489 temporally, to remodel the tumor microenvironment and orchestrate critical neoplastic

490 processes.

491 This overarching goal highlights a major challenge, that of developing experimental

492 systems which better model the physical state of the native interstitial ECM. The usefulness of

493 various existing models, including 2D monolayers (261), 3D matrigels (262) and tissue-extracted

494 ECM (263), is limited since these models fall well short of fully recapitulating the complexities of

495 tissue ECM in vivo. This inadequacy may underlie some of the discrepancies in the literature

496 regarding MCP functionality in cancer. Furthermore, the identification of naturally occurring

497 protein-protein interactions and post-translational modifications among MCPs in the ECM have

498 been difficult to characterize. Overall, as concisely reviewed elsewhere (264), new approaches

499 are clearly needed to dissect the daunting complexity of the ECM environment and its role in

500 carcinogenesis.

501 While confronting the above challenges it remains important to concomitantly work

502 towards characterizing particular MCPs, alone or in combination, as impactful

503 diagnostic/prognostic cancer biomarkers and therapeutic targets. In fact, given their

504 burgeoning roles in cancer development and extracellular accessibility, MCPs have long been

505 regarded as potentially useful for diagnosing and treating various pathologies such as fibrosis

506 and cancer; nonetheless clinical data supporting this notion have been relatively scant. Towards

507 addressing this knowledge gap, over the past decade, progress has been made in defining

508 better the fundamental mechanisms of MCPs, opening new questions that entice the

509 generation of the next needed tools to understand sufficient detail for optimal therapeutic

510 design.

511 Herein we have summarized some important ways in which misregulation of MCP

512 expression promotes cancer development, including perturbation of intracellular signaling and

513 aberrant coordination of ECM remodeling. Although we focused on MCP families with the most

514 well-characterized roles, others are emerging as potentially important players, such as the

515 EMILIN and R-Spondin families. Recently, R-Spondin-1 and 2 were shown to promote liver,

516 glioblastoma and ovarian cancer through their well-defined influence on Wnt/β-catenin

517 signaling (265-267). In addition, EMILIN2 promotes the formation of tumor-associated vessels

518 in melanoma (268), and EMILIN1 exerts an oncosuppressive role in colon and skin (269,270).

519 Clearly, there is still much to be discovered regarding the exquisite spatiotemporal regulation of

520 MCP expression patterns and functions in the extracellular space during cancer tissue

521 remodeling, similar to the approach taken in fibrosis (17). In this respect, as more and more

522 knowledge accumulates, it should be possible to design appropriate clinical studies that could

523 firmly establish MCPs as useful biomarkers and therapeutic targets in cancer.

524

525 Acknowledgements

526 This work was supported by the Operating Grant Funding Program 24347 (co-funded by Cancer

527 Research Society and the Kidney Cancer Research Network of Canada to C. Gerarduzzi), and

528 start-up funds from Hôpital Maisonneuve-Rosemont Foundation (to C. Gerarduzzi). C.

529 Gerarduzzi is a recipient of the Kidney Research Scientist Core Education and National Training

530 (KRESCENT) Program New Investigator Award KRES180003 (co-funded by the Kidney

531 Foundation of Canada, Canadian Society of Nephrology, and Canadian Institutes of Health

532 Research) and the Cole Foundation Early Career Transition Award.

533

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1270

1271 Figure 1. Activation of the biochemical, biomechanical and metastatic effects by Matricellular

1272 Proteins. Tumor cells and the surrounding activated stromal cells are the major cell types that

1273 abnormally secrete MCPs into the microenvironment to affect cellular behavior and

1274 extracellular matrix (ECM) remodeling. A) Biochemical pathways - Matricellular Proteins (MCPs)

1275 can activate an array of cell surface receptors. Most MCPs can bind and signal through

1276 integrins, with a specific heterodimer signature accounting for signaling diversity (See text for

1277 details). In addition, it has been shown that CCN and tenascin-C (TN-C) can bind and signal

1278 through syndecans, while osteopontin (SPP1) mediates its effects through CD44. B)

1279 Biomechanical pathways – MCPs are able to increase the stiffness of the normal ECM tension

1280 by influencing matrix organization and collagen crosslinking, as well as deregulating enzymatic

1281 activity. Stiffness is converted by integrins into biochemical signals that can influence pathways

1282 in A). In addition, matrix stiffness can lead to the maturation of integrin and the actin

1283 cytoskeleton into focal adhesions and stress fibers, respectively. This occurs by activating the

1284 integrin-RhoA-ROCK-myosin axis, which is reviewed in detail elsewhere(158),(159). C)

1285 Metastatic niche – Various MCPs prepare cancer cells and the local and secondary tumor sites

1286 for metastasis through numerous steps. MCPs stimulate cancer cells into a motile phenotype

1287 through the epithelial-to-mesenchymal transformation (EMT) but also to promote invadopodia

1288 formation at the invasion site. For metastatic cells to exit the embedded state for intravasation,

1289 MCPs can break down the ECM basement membrane through MMPs and guide cells out of

1290 their embedded state by crosslinking collagens into migration tracks. At the secondary site,

1291 MCPs once again activate MMPs to remodel the ECM for colonization after invasion. At the

1292 distant site, MCPs also prime the ECM for colonization in order to accommodate disseminated

1293 tumor cells in the new environment. Figure was produced using Servier Medical Art

1294 (http://smart.servier.com/).

A) BIOCHEMICAL B) BIOMECHANICAL SMOC2 ECM CCN1 SPARC BSP BSP MGP CCN2 CCN6 MGP INCREASE IN ECM STIFFNESS ECM SPARC TN-X SPARC POSTN Increased matrix organization CCN4 TN-C Collagen crosslinking SPP1 Deregulation of Enzymes (i.e. LOX)

TN-C POSTN TGFβ1 BMPs CCN3 TN-C SPP1 CCN TGFβ1 Receptor ECM Syndecan-4 Integrin Family CD44 Matrix β α Assembly β α β α & Signaling Paxillin ILK FAK Focal FAK Src ILK ILK P FAK FAK ILK ILK Adhesion P FAK FAK Force Maturation Filamentous Recruited RhoA Crk Ras Crk ILK Actin Fibers RhoA Proteins

ROCK Rac1 ERK Jnk Akt ROCK P Myosin Contraction Force NFκB HIF-2α P P Myosin MMP2 AP1 P

MIGRATION INVASION SURVIVAL and PROLIFERATION STEMNESS P Formation MMP9 Myosin Actin of Stress bundling Fibers

SPARC Migration Tracks MGP C) METASTATIC NICHE POSTN Collagen POSTN Crosslinking TN-C MGP TN-C POSTN MGP SPARC TN-C Colonization

MMP MMP POSTN MGP TN-C

SPARC POSTN POSTN SPARC TN-C MGP Epithelial-to- Enzymatic MMP MMP Invasion BSP Activity MMP MMP Mesenchymal TN-C SPP1 TN-C Transition CCN1

SMOC2 Intravasation SPOCK1 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 19, 2020; DOI: 10.1158/0008-5472.CAN-18-2098 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

The Matrix Revolution: Matricellular Proteins and Restructuring of the Cancer Microenvironment

Casimiro Gerarduzzi, Ursula Hartmann, Andrew Leask, et al.

Cancer Res Published OnlineFirst March 19, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-2098

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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