Matricryptins Network with Matricellular Receptors at the Surface of Endothelial and Tumor Cells

The extracellular matrix (ECM) is a source of bioactive fragments called matricryptins or matrikines resulting from the proteolytic cleavage of extracellular proteins (e.g., collagens, elastin, and laminins) and proteoglycans (e.g., perlecan). Matrix metalloproteinases (MMPs), cathepsins, and bone-morphogenetic protein-1 release fragments, which regulate physiopathological processes including tumor growth, metastasis, and angiogenesis, a pre-requisite for tumor growth. A number of matricryptins, and/or synthetic peptides derived from them, are currently investigated as potential anti-cancer drugs both in vitro and in animal models. Modifications aiming at improving their efficiency and their delivery to their target cells are studied. However, their use as drugs is not straightforward. The biological activities of these fragments are mediated by several receptor families. Several matricryptins may bind to the same matricellular receptor, and a single matricryptin may bind to two different receptors belonging or not to the same family such as integrins and growth factor receptors. Furthermore, some matricryptins interact with each other, integrins and growth factor receptors crosstalk and a signaling pathway may be regulated by several matricryptins. This forms an intricate 3D interaction network at the surface of tumor and endothelial cells, which is tightly associated with other cell-surface associated molecules such as heparan sulfate, caveolin, and nucleolin. Deciphering the molecular mechanisms underlying the behavior of this network is required in order to optimize the development of matricryptins as anti-cancer agents.


INTRODUCTION
Matricryptins are biologically active fragments released from extracellular matrix (ECM) proteins and glycosaminoglycans by proteases (Davis et al., 2000). We have extended the definition of matricryptins to the ectodomains of membrane collagens and membrane proteoglycans, which are released in the ECM by sheddases, and to fragments of ECM-associated enzymes such as Abbreviations: ECM, extracellular matrix; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; HA, hyaluronan; MAPK, mitogen-associated protein kinase; MMP, matrix metalloproteinase; mTOR, mammalian target of rapamycin; RHAMM, receptor for hyaluronic acid-mediated motility; TLR, toll-like receptor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. lysyl oxidase, which initiates the covalent cross-linking of collagens and elastin, and matrix metalloproteinases (MMPs), which contribute to ECM remodeling (Ricard-Blum and Ricard-Blum and Vallet, 2015). The molecular functions of matricryptins and the biological processes they regulate have been reviewed with a focus on collagen and proteoglycan matricryptins (Ricard-Blum and Ballut, 2011), on matricryptins regulating tissue repair (Ricard-Blum and , angiogenesis (Sund et al., 2010;Boosani and Sudhakar, 2011;Gunda and Sudhakar, 2013), cancer (Monboisse et al., 2014), and on the proteases releasing matricryptins (Ricard-Blum and Vallet, 2015).
Synthetic peptides and/or domains derived from matricryptin sequences and recapitulating their biological roles are also able to regulate angiogenesis and/or cancer in various tumor cells and cancer models (Rosca et al., 2011). They include sequences of tumstatin (He et al., 2010;Han et al., 2012;Wang et al., 2015a), laminins (Kikkawa et al., 2013), endostatin (Morbidelli et al., 2003), endorepellin (Willis et al., 2013), and the hemopexin domain of MMP-9 (Ugarte-Berzal et al., 2012. Neither these peptides nor the ectodomains of membrane collagens and syndecans are described here due to space limitation. We focus on the major matricryptins, which control cancer, metastasis, and angiogenesis, a pre-requisite for tumor growth and a therapeutic target (Folkman, 1971;Welti et al., 2013;Huang et al., 2015), and on their receptors.
Matricryptins derived from collagens IV and XVIII target tumoral cells. Arresten inhibits migration and invasion of squamous cell carcinoma and induces their death (Aikio et al., 2012). Endostatin inhibits the proliferation of some cancer cells (e.g., the HT29 human colorectal adenocarcinoma cell line) but not of others (e.g., the MDA-MB-231 human mammary adenocarcinoma cell line) (Ricard-Blum et al., 2004). Matricryptins enhance the sensitivity of tumor cells to a cytotoxic drug and even reverse in part their resistance to this drug. A tumstatin peptide increases the sensitivity of non-small cell lung carcinoma cells to cisplatin (Wang et al., 2015c) and Endostar enhances the sensitivity to radiation of nasopharyngeal carcinoma and lung adenocarcinoma xenografts in mice .
Matricryptins regulate angiogenesis, tumor growth, and metastasis by various molecular mechanisms. The antiangiogenic activities of tumstatin and endostatin contribute to tumor suppression by p53 via the upregulation of the α(II) collagen prolylyl hydroxylase (Folkman, 2006;Teodoro et al., 2006). Endostatin inhibits proliferation and migration of glioblastoma cells by inhibiting T-type Ca 2+ channels (Zhang et al., 2012), and its ATPase activity contributes to its antiangiogenic and antitumor properties (Wang et al., 2015b). This matricryptin inhibits hemangioendothelioma by downregulating chemokine (C-X-C motif) ligand 1 via the inactivation of NF-κB (Guo et al., 2015).

RECEPTORS AND CO-RECEPTORS OF MATRICRYPTINS
Matricryptins regulating angiogenesis, tumor growth and metastasis bind to several receptors, and co-receptors (Figure 1, Faye et al., 2009a) to modulate signaling pathways and fulfill their biological functions ( Table 1). The other ligands of the receptors (e.g., ECM proteins, proteoglycans, growth factors, and chemokines) are not represented in Figure 1 for the sake of clarity. Pathways regulated by matricryptins in endothelial or tumor cells via unidentified receptors and/or in other cell types are mentioned below but are not listed in Table 1.

Integrins
There are 24 integrins comprised of an α subunit and a β subunit (Barczyk et al., 2010). They lack intrinsic kinase activity and are the major adhesion receptors of the ECM, controlling ECM assembly, cell-matrix interactions, cell migration, and tumor growth (Missan and DiPersio, 2012;Xiong et al., 2013). A number of matricryptins bind to integrins at the surface of tumor and/or endothelial cells (Table 1). Matricryptins also interact with purified integrins (e.g., αvβ5 integrin for endostatin; Rehn et al., 2001;Faye et al., 2009b), or on other cell types. The αvβ3 integrin is the main receptor targeted by matricryptins (Figure 1). Anastellin decreases the activation state of α5β1 integrin on endothelial cells (Ambesi and McKeown-Longo, 2014). Arresten interacts with α3β1/αvβ3 and α1β1/α2β1 integrins at the surface of HPV-16-immortalized proximal tubular epithelial cells and mesangial cells respectively, whereas tumstatin binds to immortalized glomerular epithelial cells through α3β1 and α2β1 integrins (Aggeli et al., 2009). The above integrins are also involved in the effects of matricryptins on other cell types. Endostatin, generated by cerebellar Purkinje cells, contributes to the organization of climbing fiber terminals in these neurons by binding and signaling through α3β1 integrin (Su et al., 2012). The adhesion of smooth muscle cells to anastellin is mediated by both β1 integrins and cell surface heparan sulfate proteoglycans, which triggers ERK1/2 activation in these cells (Mercurius and Morla, 2001) and the induction of osteoclast apoptosis by the N-propeptide of procollagen II is mediated by αv or β3 integrin subunits (Hayashi et al., 2011).
Anastellin inhibits lysophospholipid (Ambesi and McKeown-Longo, 2009) and VEGF165-dependent signaling in endothelial cells by preventing the formation of the complex containing VEGFR2 and neuropilin-1 at the surface of endothelial cells (Ambesi and McKeown-Longo, 2014). One matricryptin of collagen I interacts with a member of the chemokine receptor family, the CXC chemokine receptor 2 (Stadtmann and Zarbock, 2012;Veenstra and Ransohoff, 2012).

Elastin Receptors
The Elastin Receptor Complex (ERC) is composed of two membrane associated proteins (the protective protein/cathepsin A and neuraminidase-1) and of the elastin-binding protein, an inactive spliced variant of lysosomal β-galactosidase (Blanchevoye et al., 2013). Elastin peptides activate extracellular signal-regulated kinase 1/2 via a Ras-independent mechanism in fibroblasts (Duca et al., 2005), the enzymatic activity of neuraminidase-1 being responsible for signal transduction (Duca et al., 2007). Another, still unidentified, receptor of elastin peptides exists at the surface of macrophages (Maeda et al., 2007). Furthermore, elastin peptides regulate tumor cell migration and invasion through an Hsp90-dependent mechanism (Donet et al., 2014).

CD44, Receptor for HA-Mediated Motility (RHAMM) and Toll-Like Receptors (TLRs)
Hyaluronan, a non-sulfated glycosaminoglycan, has two major receptors, CD44 and RHAMM, which mediate its roles in inflammation and cancer (Toole, 2009;Misra et al., 2015;Nikitovic et al., 2015). The binding to, and activation of, receptors depend on the size of HA, its oligosaccharides stimulating angiogenesis (Gao et al., 2008). CD44, which has several isoforms regulates cell proliferation, adhesion and migration, and is involved in tumorigenesis (Jordan et al., 2015). A sequence in the hemopexin domain of MMP-9 (PEX9) impairs tumor cell adhesion to PEX9/MMP9 through interaction with CD44 (Ugarte-Berzal et al., 2014). RHAMM has splicing variants and is located inside the cell and on the cell surface, where it is anchored via a glycosylphosphatidylinositol (Tolg et al., 2014;Misra et al., 2015). Toll-like receptors are pattern recognition receptors involved in innate immunity (Rakoff-Nahoum and Medzhitov, 2009). Low-molecular weight hyaluronan induces the formation of a complex containing CD44, TLR2/TLR4, the actin filamentassociated protein AFAP-110, and a myeloid differentiation factor MyD88, which triggers cytoskeleton activation and results in tumor invasion (Bourguignon et al., 2011).

Other Membrane and Cell Surface-Associated Proteins
Matricryptins form complexes with membrane or membraneassociated proteins. Caveolin-participates in the formation of membrane caveolae, which are platforms for signal transduction (Fridolfsson et al., 2014) and forms a complex with α5β1 integrin and endostatin in lipid rafts at the endothelial cell surface (Wickström et al., 2002) (Table 1). Nucleolin, a multifunctional protein, localized inside the cell and at the cell surface (Berger et al., 2015), binds to endostatin and triggers its internalization in endothelial cells in association with the urokinase plasminogen activator receptor and the α5β1integrin (Shi et al., 2007;Song et al., 2012).

MATRICRYPTINS AS POTENTIAL DRUGS
Matricryptins are potential anti-cancer drugs, either alone, or in combination with other treatments, but their use in pre-clinical and clinical studies remains challenging. Indeed matricryptins may display opposite activities depending on the context. The anti-tumoral effect of endostatin is enhanced by silencing of the proteoglycan versican, which decreases the inflammatory and immunosuppressive changes triggered by anti-angiogenic therapy (Wang et al., 2015d). However, endostatin may induce the proliferation of carcinoma cells, whereas its effect on cancer invasion is modulated by the tumor microenvironment (Alahuhta et al., 2015). Endorepellin stimulates angiogenesis in a stroke model by increasing VEGF levels, and exerts a neuroprotective effect in this model via α5β1 integrin and VEGFR2 . In addition, endostatin exhibits a biphasic response curve both for its antiangiogenic and anti-tumoral properties (Celik et al., 2005;Javaherian et al., 2011), which requires to test a large range of concentrations to determine the optimal dose for treatment. Another challenge is that matricryptins may themselves contain cryptic sequences displaying opposite activities as reported for the anti-angiogenic matricryptin endostatin, which contains an embedded pro-angiogenic sequence (Morbidelli et al., 2003). Different matricryptins regulate the same biological process in an opposite way as reported for the regulation of the angiogenic signaling in choroidal endothelial cells by hexastatin and elastokines , or distinct steps of a biological process as described for anastellin and endostatin (Neskey et al., 2008).
Matricryptins can be modified to extend the half-life, and their efficacy (Xu et al., 2007;Zheng, 2009;Ricard-Blum and Ballut, 2011;Ricard-Blum and Salza, 2014). Most of the examples detailed below concern endostatin, which is extensively studied and has been approved for the treatment of non-small-cell lung cancer in China (Biaoxue et al., 2012) under a recombinant form, Endostar, which contains an extra metal chelating sequence (MGGSHHHHH) at the N-terminus enhancing its solubility and stability (Jiang et al., 2009). PEGylation (Nie et al., 2006;Tong et al., 2010;Tan et al., 2012;Guo et al., 2014), and the fusion of endostatin to low molecular weight heparin or to the Fc region of an IgG enhance its half-life and its anti-angiogenic, or antitumoral activities (Lee et al., 2008;Jing et al., 2011;Ning et al., 2012;Tan et al., 2012;Li et al., 2015b).
Tumors may escape from anti-tumoral and anti-angiogenic matricryptins by upregulating factors, which stimulate angiogenesis (Fernando et al., 2008). The combination of matricryptins with inhibitors of pro-angiogenic pathways, chemotherapy, or radiotherapy enhance their therapeutic efficacy. Tumstatin has been fused to another endogenous inhibitor of angiogenesis, vasostatin (Gu et al., 2014) and to tumor necrosis factor α, which has anti-tumoral and antiangiogenic properties, which results in a more effective fusion protein than tumstatin alone (Luo et al., 2006). Endostatin has been fused to the proapoptotic domain (BH3) of the BAX protein (Chura-Chambi et al., 2014), to tumor necrosis factor-related apoptosis-inducing ligand (Zheng et al., 2013) and one of its anti-angiogenic sequences to an heptapeptide inhibitor of MMPs (Qiu et al., 2013). Endostatin has also been fused to protein sequences targeting it to tumors and/or tumor vasculature such as humanized antibodies against tyrosine kinase-type receptor HER2 (Shin et al., 2011) or against tumor-associated glycoprotein 72 highly expressed in human tumor tissues , the RGD integrin-binding sequence , and a liver-targeting peptide (circumsporozoite protein CSP I-plus (Ma et al., 2014;Bao et al., 2015).
Several approaches improve the delivery of matricryptins to tumors and endothelial cells (Xu et al., 2007;Ricard-Blum and Ballut, 2011) such as conditionally replicating oncolytic adenoviral vector for arresten (Li et al., 2015a), naked plasmid electrotransfer in muscle for tumstatin overexpression (Thevenard et al., 2013), and the nonpathogenic and anaerobic bacterium, Bifidobacterium longum, which proliferates in the hypoxic zones within tumors for tumstatin expression (Wei et al., 2015). Endostatin has been delivered in polylactic acid nanoparticles (Hu and Zhang, 2010), in gold nanoshells, which are very efficient on lung cancer cells when associated with nearinfrared thermal therapy  and into microbubbles in combination with ultrasonic radiation in a cancer model . Dendrimers mimicking the surface structure of endostatin and loaded with anticancer drug, result in both angiogenesis inhibition by endostatin and death of cancer cells by the anticancer drug (Jain and Jain, 2014).
Clinical trials of endostatin mostly focus on solid tumors in association with cytotoxic drugs (https://clinicaltrials.gov/). These trials include phase I (Lin et al., 2007;Chen et al., 2014), II , and III trials (Wang et al., 2005). Although endostatin did not improve overall survival, progression-free survival, and objective response rate when combined with etoposide and carboplatin in patients with extensive-stage small-cell lung cancer phase II trial , it significantly improves the response rate and median time to tumor progression when combined with vinorelbine and cisplatin in advanced non-small cell lung cancer patients compared to chemotherapy alone (Wang et al., 2005). Promising results have been obtained with endostatin associated with paclitaxel and 5-fluorouracile in patients with refractory malignant ascites secondary to ovarian cancer (Zhao et al., 2014).

CONCLUDING REMARKS
Several matricryptins such as the propeptide of lysyl oxidase, which is a tumor suppressor (Min et al., 2007;Ozdener et al., 2015) and the netrin-like domain of procollagen Cproteinase enhancer-1, a new anti-angiogenic matricryptin , warrant further investigation in angiogenesis, and tumor models to decipher their mechanisms of action at the molecular and cellular levels. Matricryptins are useful for treating fibroproliferative disorders (Yamaguchi et al., 2012;Wan et al., 2013) and fundus oculi angiogenesis diseases . The finding that a peptide derived from endostatin can be delivered orally in vivo and exerts anti-fibrotic activity (Nishimoto et al., 2015) paves the way for the development of new matricryptin drugs with oral bioavailability, which is a preferred administration route for long-term treatment. Matricryptins are also used as biomarkers in serum and in cerebrospinal fluid (Ricard-Blum and Vallet, 2015;Salza et al., 2015) and may serve as imaging agents when labeled with (99m)Tc as shown for endostatin (Leung, 2004) and tumstatin  and for tumstatin conjugated to iron oxide nanoparticles (Ho et al., 2012).

AUTHOR CONTRIBUTIONS
SV drafted the Section Receptors and Co-receptors of Matricryptins and Table 1 and made the figure. SB made bibliographical searches for all the sections and wrote the manuscript.