The pathophysiology of hepatocellular carcinoma at a cellular level is complex and it is very possible that there are many unknown c-Met interactions with others signaling pathways. Multiple cell pathways are aberrant in HCC, but this review just focuses on the signaling pathways related to c-Met. The activation of c-Met can take place by the canonical pathway, which involves HGF binding to c-Met resulting in c-Met homodimerization. It can also take place through non-canonical pathways, where c-Met dimerizes with different receptors.
Heterodimers of receptor proteins and c-Met are involved in overstimulation and dysregulation of c-Met signaling pathways. This occurs during hypoxia, which can cause c-Met overexpression, mutations on tyrosine kinase domain or HGF gene amplification. However, this latter event rarely occurs in HCC.
c-Met canonical downstream signaling pathways
These signaling pathways involve proteins with SH2 domains or phosphotyrosine-binding domains that are able to interact with phosphorylated tyrosine residues that, in turn, interact physically with the cytoplasmic domain of c-Met.
Growth factor receptor-bound protein 2 (Grb-2): Grb-2 interacts with Y1356 of c-Met to transduce HGF signaling to the cytoplasm. Grb-2 is considered a key protein in HGF/c-Met axis because it connects to several signaling transducers, such as Ras, SOS, and Gab1. Grb-2 is involved in cell motility, cycle progression, angiogenesis, amongst other.
GRB2-associated binding protein 1 (Gab1): Activated c-Met is phosphorylated on Y1349 and Y1356 residues which specifically interact and phosphorylate to Gab1. However, Gab1 can also be phosphorylated by Grb-2. Gab1 is involved in many signal transduction pathways by binding to effector proteins that have a role in cell motility and extracellular matrix invasion, such as Shp2, Shc, PLCγ1, p120.
Phosphoinositide 3 kinase (PI3K): PI3K is an enzyme able to phosphorylate proteins downstream of c-Met thereby linking oncogenes and many receptors essential for cellular functions. The phospho Y1356 in c-Met can phosphorylate PI3K, inducing cell mobility by activating focal adhesion kinase (FAK). However, PI3K can also be activated by Gab1 where it promotes cell survival.
Signal transducer and activator of transcription 3 (STAT3): HGF binds to c-Met inducing the phosphorylation on Y1356. This phosphorylated amino acid interacts and actives STAT3, as was shown by Boccaccio et al. When it is activated, it translocates to the nucleus where it binds to DNA and promotes gene expression (related with angiogenesis, and long-term response).
Shc-transforming protein 1 (Shc): SHC is an adaptor protein involved in the mitogenic signal transduction from tyrosine receptors. On the other hand, experiments carried out in fibroblast showed that Shc is highly stimulated by VEGF and that activation correlated with the angiogenic response.
Non-canonical c-Met signaling pathways
c-Met activation by non-canonical pathways takes place when this receptor is over-expressed and dimerizes with other receptor subunits, or may bind to ligands other than HGF. Non-canonical pathways are usually associated with c-Met gene amplification, and are common in treatment resistant cancers[17,18], tumor progression, and metastasis, as shown in in vivo experiments using mice[19,20]. It has also been reported that c-Met dimerization takes place in the absence of ligand binding when it interacts with the following proteins:
Epithelial growth factor receptor (EGFR): Physical interaction between EGFR and c-Met was found in A431 cells. In HCC, the transactivation between these two receptors takes place, inducing the common downstream signaling effectors PI3K and Ras.
Human epidermal growth factor receptor (HER): The dimerization between c-Met and HER increases activation of PI3K/AKT signaling, which is associated with resistance to EGFR inhibitors as well as cancer progression.
Integrin α6β4: Trusolino et al determined that integrin α6β4 physically interacts with c-Met on the membrane surface of carcinoma cells. This protein is necessary for cancer invasion because the cytosolic domain of β4 induces c-Met activation. In this case, the signaling transduction is performed by Shc and PI3K.
β-catenin (β-CAT): Phosphorylated β-CAT may bind to c-Met, activating its downstream signaling. Phosphorylation of Y654 in β-CAT actives FAK, which induces cyclin D1 (CKD1) expression. At the same time, β-catenin is translocated to the nucleus, and promotes c-myc gene expression.
Receptor for hyaluronic acid (CD44): The CD44v3 splice variant is the CD44 isoform with high affinity for heparin domains. This v3 may be activated by different growth factors with heparin domains, such as fibroblast growth factor (FGF) and HGF. CD44 may act as a concentrator of HGF to present it to c-Met resulting in downstream signaling transduction. On the other hand, Olaku et al reported that CD44v6 splice variant is necessary for c-Met activation. It is thought that three specific amino acid residues (RWH in human) in v6 are necessary for complete c-Met activation.
ICAM-1: This protein can substitute for the role of CD44v6 in c-Met activation, as shown in hepatocytes from Cd44 null mice.
Plexin B1: Receptor with high similarity to c-Met, which is also expressed in the same tissues as c-Met. After mutation and expression of exogenous c-Met in cells, Giordano et al shown that plexin B1 links to c-Met when it is activated by semaphorin 4D. This interaction was reported in invasive cancer cells growing in response to semaphorin 4D.
Vascular endothelial growth factor A (VEGF-A): HGF can induce VEGF expression by phosphorylation of a key transcription factor called Sp1. This characteristic of HGF increases the expression of Bcl-2, which acts as an antiapoptotic protein.
Insulin receptor (INSR) tyrosine kinase: This receptor has an extracellular α-chain and a transmembrane β-chain. This protein has a very similar structure compared to c-Met. Furthermore, it has been reported that insulin and HGF can phosphorylate INSR in its Y1146 and Y1150 or Y1151 residues. In HCC cells, Y1322 is also phosphorylated, thus activating PI3K. HGF-stimulated hepatocytes have shown to form a INSR-c-Met complex. There is evidence that c-Met can also phosphorylate insulin receptor substrates (IRS) on Y895. Likewise, INSR phosphorylates IRS on Y612. In summary, both c-Met and IRS increase downstream signaling through PI3K-AKT, promoting cell growth, cell survival and cell motility.
Fas: This protein is one of the surface death receptors and triggers apoptosis signaling when binds its ligand (FasL). Wang et al showed that Fas and c-Met associate with each other using coimmunoprecipitation experiments in Hep G2 cells. These authors proposed that c-Met promotes cell survival by two different pathways. (1) When there are low levels of FasL in the microenvironment, Fas binds to c-Met avoiding to trigger its intracellular signaling pathway. (2) However, in the presence of high levels of HGF, c-Met releases Fas activating death receptor-mediated apoptosis. Nevertheless, the c-Met/Fas signaling pathway ratio is so high that cells activate antiapoptotic signals through PI3K/AKT/Bad axis to prevent Fas-mediated apoptosis.
Mucin 1 (MUC1): MUC1 expression is increased during transformation from the normal liver to HCC, as was described by Bozkaya et al MUC1 silencing in HCC cells leads to β-catenin activation and c-Myc expression. Under this condition, high levels of HGF in the microenvironment increase cellular motility and invasiveness. However, there are also contradicting studies. For instance, Singh et al reported that MUC1-induced c-Met activation by physical interaction decreased MMP-1 transcription and cell motility.
Neuropilin-1 and -2 (Nrp-1, -2): Neuropilins are a family of transmembrane glycoproteins involved in several processes, including axonal guidance, angiogenesis, tumorigenesis, and immunologic response. Nrp-1 can bind VEGF-A165, VEGF-B, VEGF-E, and placental growth factor (PIGF). On the other hand, Nrp-2 can bind class III semaphorins and VEGF proteins (VEGF-A165, VEGF-A145, and VEGF-C). Nrp-2 binds VEGF proteins, and increases the VEGFR-2 phosphorylation threshold, promoting migration, and sprouting cells. Nrp-2 and VEGFR2 can bind each other enhancing the signaling initiated by the HGF/c-Met axis. Moreover, Nrp-1 and Nrp-2 interact with other receptor tyrosine kinase, such as VEGFRs. Neuropilins have a short cytoplasmic domain to act as catalytic domain. This evidence suggests that the intracellular domain may present a binding site involved in kinase signal transduction.
Focal adhesion kinase (FAK): Studies carried out in MEFs and HEK293 cells showed that FAK interacts directly with c-Met. FAK is a non-receptor tyrosine kinase involved in several cell signaling pathways. Notably, it is well characterized for its role in formation and disassembly of focal adhesions, as well as cell protrusions. However, FAK is also intimately involved in the regulation of cell proliferation because it is able to phosphorylate PI3K and ERK. Experiments in FAK knockout mice revealed suppressed hepatocarcinogenesis due to decreased PI3K and ERK signaling pathways.
Collectively, these pathways are responsible for promoting initiation and progression of HCC (Figure 1).
Figure 1 Representation of hepatocyte growth factor/c-Met canonical and non-canonical pathways in hepatocytes.
Canonical pathway is activated by HGF release from stromal cells, and subsequent binding to the c-Met receptor inducing c-Met dimerization. Activated c-Met binds Gab-1, Grb-2, Shc, and STAT3. These proteins are involved in signal transduction regulating cell proliferation, migration, differentiation, or invasion, depending on the activated downstream proteins. The scheme represents the proteins described in the canonical pathway, but not all of the proteins involved in executing their cell activity. Non-canonical pathways are activated when a c-Met monomer binds to a monomer of another kind of receptor, or when a c-Met homodimer binds FAK or β-catenin on its cytoplasmic domain. The arrows represent different proteins performing different cell activities. HGF: Hepatocyte growth factor.
The expression of HGF is decreased in HCC, but it is increased in the surrounding tissue. On the other hand, c-Met is expressed in HCC at higher levels than in the surrounding tissue. These observations suggest that the overexpression of c-Met, together with additional oncogenes, is responsible of HCC aggressiveness.