Extracellular Matrix Influencing HGF/c-MET Signaling Pathway: Impact on Cancer Progression

The extracellular matrix (ECM) is a crucial component of the tumor microenvironment involved in numerous cellular processes that contribute to cancer progression. It is acknowledged that tumor–stromal cell communication is driven by a complex and dynamic network of cytokines, growth factors and proteases. Thus, the ECM works as a reservoir for bioactive molecules that modulate tumor cell behavior. The hepatocyte growth factor (HGF) produced by tumor and stromal cells acts as a multifunctional cytokine and activates the c-MET receptor, which is expressed in different tumor cell types. The HGF/c-MET signaling pathway is associated with several cellular processes, such as proliferation, survival, motility, angiogenesis, invasion and metastasis. Moreover, c-MET activation can be promoted by several ECM components, including proteoglycans and glycoproteins that act as bridging molecules and/or signal co-receptors. In contrast, c-MET activation can be inhibited by proteoglycans, matricellular proteins and/or proteases that bind and sequester HGF away from the cell surface. Therefore, understanding the effects of ECM components on HGF and c-MET may provide opportunities for novel therapeutic strategies. Here, we give a short overview of how certain ECM components regulate the distribution and activation of HGF and c-MET.


Introduction
Cancer progression is not only defined by the numerous characteristics acquired by tumor cells but also includes the contributions of the tumor microenvironment [1,2]. The elaborate tumor microenvironment composed by tumor and stromal cells within the extracellular matrix (ECM) promotes neoplastic transformation, protects the tumor from the immune system and supports tumor growth, invasion and metastasis [3,4]. ECM consists of numerous proteins and macromolecules, including proteoglycans (PGs) and glycosaminoglycans (GAGs), acting as a biological barrier [5]. However, since intercellular communication in the tumor microenvironment is driven by a complex and dynamic network of cytokines, chemokines, growth factors and proteolytic enzymes, the ECM works as a reservoir for these bioactive molecules that influence tumor cell behavior [6][7][8][9].
Among the many bioactive molecules released by tumor and stromal cells, hepatocyte growth factor (HGF) is a crucial cytokine linked to promoting cancer progression [10,11]. The binding of HGF to its receptor tyrosine kinase c-MET triggers the activation of multiple downstream signaling pathways, which contribute to proliferation, growth, migration, survival, angiogenesis, invasion and metastasis [12][13][14][15]. Indeed, the HGF/c-MET system plays an essential role in tumor-stromal crosstalk [16].
The ECM s role in cancer progression is still unclear. Several ECM components, particularly proteoglycans and glycoproteins, act as signal co-receptors and/or bridging molecules, promoting and Y1235 residues, which is involved in signal transduction; and a docking site for adaptor proteins containing Y1349 and Y1356 residues [42,46,47].
Physiologically, HGF and its receptor, c-MET, play an essential role in embryonic development, organ morphogenesis, wound healing and tissue repair through activation of different signaling pathways that are involved in cell proliferation, motility, survival, differentiation, scattering and morphogenesis [15,38,39,48].

HGF/c-MET Signaling Pathway Mediates Cancer Progression
The c-MET receptor interacts with HGF in a paracrine, endocrine or autocrine manner [49-51]. As soon as the Sema and IPT domains recognize HGF, two c-MET subunits dimerize, leading to the autophosphorylation of Y1234 and Y1235 residues present in the tyrosine kinase catalytic domain. This activation induces subsequent autophosphorylation of Y1349 and Y1356 residues, thus providing a docking site for the recruitment of adapter molecules (e.g., GAB1, GRB2, SHC, CRK, PI3K, PLCγ1, SHP2 and STAT3) responsible for downstream signaling. In this way, the HGF/c-MET pathway mediates Erk/MAPK, JNK, FAK, Akt/PKB and STAT3/5 activation [42,52,53]. Nonetheless, it is important to mention that the autophosphorylation of Y1003 residue located in the juxtamembrane domain leads to internalization and degradation of the c-MET receptor. Therefore, Y1003 residue negatively regulates c-MET signaling [54].
Therefore, tumor and stromal cells communicate with each other through HGF, creating a microenvironment that contributes to cancer progression. For example, the HGF secreted by CAFs acts on tumor cells stimulating them not only to proliferate, invade and metastasize but also to produce a variety of HGF-inducers, such as bFGF, IL-1β, TGF-α, PDGF and prostaglandin E2 (PGE2), that act on stromal fibroblasts. Thus, the mutual interaction between tumor and stromal cells mediated by tumor-derived HGF-inducers and stroma-derived HGF, stimulates tumor cell invasion and metastasis [2,16,[70][71][72][73]. Similarly, the HGF produced by adipose-derived stem cells (ASCs), together with the c-MET expressed in primary breast carcinoma cells, increases tumor cell migration, metastasis and self-renewal through PI3K-mediated GS3K inactivation and β-catenin stabilization and nuclear accumulation [74].
On the other hand, it was shown that c-MET aberrant activation can promote glioma cell survival via PI3-kinase/Akt signaling [75], squamous cell carcinoma invasion via STAT3 signaling [76], lymphoma cell adhesion via PI3K signaling [77], head and neck squamous cell carcinoma (HNSCC) proliferation via MAPK signaling [78], gastric cancer growth via Akt and Erk signaling [79], prostate cancer EMT and invasion via Erk/MAPK signaling [80], and breast carcinoma cell motility and lung cancer invasion via FAK signaling [81,82]. Importantly, HGF-induced tumor cell motility and invasion are accompanied by an increase in cell dissociation and protease production [e.g., MMP-2 and urokinase-type plasminogen activator (uPA)] [38,47]. Moreover, HGF/c-MET axis can stimulate the metastatic spread of colorectal tumor cells via WNT signaling [83], prostate tumor cells through induction of c-Src activity [84] and HNSCC via Erk and Akt signaling [85]. It was observed that in esophageal squamous cell carcinoma (ESCC), HGF-induced angiogenesis is driven by either pro-angiogenic cytokines (e.g., VEGF and IL-8) [86,87] or independently of VEGF, through Akt and Erk activation in endothelial cells [88]. Thus, the HGF/c-MET signaling pathway induces different phenotypes and enhances the aggressive nature of tumor cells during cancer progression.
Some ECM components such as cell surface heparan sulfate proteoglycans (HSPGs) can bind to different growth factors and function as signal co-receptors or presenters. Indeed, CD44 and syndecan-1 can interact and recruit HGF on the cell membrane, thereby facilitating the presentation of this ligand to c-MET [17,[98][99][100]. CD44 also interacts with c-MET and improves tumor cell migration, invasion and metastasis [47]. Moreover, α5β1 and α6β4 integrins directly bind to c-MET and enhance its signaling. In addition, glycoproteins including laminin and fibronectin, have the ability to promote the association between c-MET and integrin α5β1 or α6β4, leading not only to ligand-free activation of c-MET but also to amplification of signaling by both receptors [101][102][103]. It was shown that c-MET displaces α5 integrin from β1 integrin, forming a powerful structural c-Met/β1 complex that exhibits a greater affinity for fibronectin during the invasive and metastatic process [104].
On the other hand, some proteoglycans can interact with a large number of macromolecules (e.g., growth factors and water molecules) [105]. In general, proteoglycans consist of one or more sulfated GAG chains covalently attached to a core protein [106,107]. All GAGs (except hyaluronic acid) contain sulfate groups that render the proteoglycans negatively charged. In this way, the proteoglycan through its sulfated GAGs chains can attract positively charged growth factors [105,108]. It was shown that HGF binds with high affinity to heparin, specifically with N-sulfate, 2-O-sulfate, and 6-O-sulfate groups [109,110]. Also, heparin has the ability to displace HGF from the HSPGs, suggesting that cell surface HSPGs present low-affinity binding sites to HGF. Specifically, the minimal heparin/heparan sulfate size for binding to HGF is an octasaccharide [111,112].
Cell-surface HSPGs act as signal co-receptors or presenters for c-MET, thus playing an important role in normal biological functions. The proteoglycans not only regulate the delivery of HGF to the target cells but also control the activity of HGF [112]. The heparin on the ECM acts as a reservoir and impairs the interaction of HGF with c-MET, leading to reduced mitogenic and motogenic responses of cells to HGF [111,[113][114][115]. However, recent studies show that heparin can induce c-MET activation in the absence of HGF. Indeed, heparin promotes tumor-cell motility and invasion through activation of c-MET signaling pathway [116][117][118].
Another ECM component that is capable of sequestering HGF is the thrombospondin-1 (TSP-1), a matricellular protein involved in angiogenesis, inflammation and cancer [119]. TSP-1 binds to HGF likely through its heparin-binding domains and mobilizes HGF away from the cell surface HSPGs, thus preventing c-MET activation [120,121]. Recently, we have shown that HGF/c-MET signaling pathway can also be affected by ADAMTS-1 through a mechanism speculated to involve degradation of HGF by ADAMTS-1 or its sequestration by ADAMTS-1 TSP-1 motifs [122]. However, growth factors attached to ECM components can be released by MMPs and ADAMTSs and thus activate different signaling pathways involved in cancer progression [94].

Strategies to Target HGF/cMET-ECM Interactions
An understanding of the effects of ECM components on HGF and c-MET pathway has provided ideas to therapeutic strategies. Initially, heparin sulfate-binding peptides were designed to block interactions with its ligands, hence disturbing involved signaling pathways [123,124]. Inhibiting heparan sulfate proteoglycan synthesis or altering heparin posttranslational modifications were also considered. The sulfatase HSulf-1 is responsible for modulating heparin-binding growth factors, including HGF. It is involved in the sulfation of specific sites of heparan sulfate glycosaminoglycans (HSGAGs), which is critical for HGF/HS interaction. Also, HSulf-1 was described as a negative regulator of HGF interaction with c-MET [125]. Another idea is based on the fact that HGF basic residues comprise the primary heparan sulfate (HS) binding sites. The substitution of residues on HGF sequence transformes it into a selective competitive antagonist, with the ability to block the c-MET pathway [126]. Concerning HGF/c-MET specific drugs, two major classes are described: the small molecules tyrosine kinase inhibitors (TKIs), whose mechanism of action is mainly intracellular and the human monoclonal antibodies that recognize extracellular components. A comprehensive review of these drugs was performed by De Silva et al. (2017) [127]. Monoclonal antibodies targeting HGF/c-MET pathways have been evaluated in clinical trials and are able to recognize the HGF or the c-MET extracellular domains, ultimately disturbing HGF/c-MET interaction and signaling. It has been described that the tumor-associated extracellular matrix (TAECM) masks epitopes and becomes an impediment for monoclonal antibody therapy, since accumulation of hyaluronan (HA) hampers tumor infiltration by natural killer (NK) cells and consequent tumor cell-mediated cytolysis. In addition, the use of PH20 hyaluronidase depletes tumoral HA and results in an increase of tumor cell death in the presence of trastuzumab [128]. In this way, hyaluronidase could likely be used in combination with monoclonal antibodies that target HGF/c-MET components to increase success in disturbing this signaling pathway.

Conclusions
The ECM works as a reservoir for bioactive molecules, including cytokines, growth factors and proteases that modulate tumor cell behavior. HGF produced by tumor and stromal cells acts as a multifunctional cytokine that plays an important role in cancer progression by promoting proliferation, survival, motility, angiogenesis, invasion and metastasis. However, this signaling pathway can be influenced directly or indirectly by ECM. Figure 1 summarizes the roles of several ECM components in the regulation of c-MET signaling. Among these, proteases play an essential role not only in HGF activation but also in the shedding of HB-EGF and TGF-α, which induce the ligand-free activation of c-MET through its interaction with the activated EGFR. In addition, proteoglycans and glycoproteins can act as signal co-receptors or bridging molecules and activate c-MET. However, this activation can be disturbed by some ECM components, including matricellular proteins, proteoglycans and proteases, which bind and sequester HGF away from c-MET. Consequently, ECM components that interact with HGF and c-MET can regulate the distribution and activation of these two molecules, leading to either progression or inhibition of tumorigenesis.  We also acknowledge Iuri Cordeiro Valadão for critically reading the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.

ADAM
A disintegrin and metalloproteinase ADAMTS A disintegrin and metalloproteinase with thrombospondin motifs ADSCs Adipose-derived stem cells bFGF Basic fibroblast growth factors CAFs Cancer-associated fibroblasts Funding: This study was supported by The State of São Paulo Research Foundation (FAPESP grants 2018/05566-6,). We also acknowledge Iuri Cordeiro Valadão for critically reading the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.