The Role of Hepatocyte Growth Factor Pathway Signaling in Renal Cell Carcinoma

The urgent need for effective therapies for patients with advanced renal cell carcinoma (RCC), fewer than 20% of whom will survive more than 2 years, has led to the identification of critical genetic determinants and associated molecular pathways contributing to RCC oncogenesis, progression, and spread. Among the signaling pathways dysregulated in RCC is that of hepatocyte growth factor (HGF), which through the cell surface receptor tyrosine kinase, c-Met, stimulates proliferation, motility, and morphogenesis. Germ line missense mutations in the tyrosine kinase domain c-Met are associated with hereditary papillary renal carcinoma (HPRC) type 1, while somatic mutations and frequent trisomy of chromosome 7 implicate pathway involvement in sporadic papillary RCC. In addition, loss of the VHL tumor suppressor gene results in the derepression of an embryonic HGF-driven phenotype likely to contribute to tumor invasiveness and metastasis in clear cell RCC. Our knowledge of HGF/c-Met signaling has enabled rapid progress in characterizing its contributions to RCC and in laying the framework for the development of novel anticancer therapeutics. A better understanding of how HGF/c-Met signaling is integrated with other oncogenic pathways in RCC should aid the development of combinatorial treatment strategies, and help predict potential adverse effects of long-term pathway blockade.


Introduction
Over 200,000 cases of kidney cancer are diagnosed each year worldwide, claiming more than 100,000 lives (1). Despite significant advances in the development of immunologic therapies for this disease, there is still no effective therapy for the majority of patients with advanced RCC (1;2). Four main sporadic RCC subtypes with distinct histologies are currently recognized: clear cell, papillary, chromophobe and oncocytoma.
Papillary RCC is further sub classified into types 1 and 2 based on additional clinical, histological and genetic criteria (2). Rare, inherited forms of RCC exist which characteristically present with one or two of these histological subtypes; the study of these familial diseases has facilitated the identification of their underlying genetic defects, and helped forge mechanistic links with sporadic RCC types with similar histologies (2). For two prevalent RCC subtypes, clear cell and papillary type 1, these mechanistic links strongly implicate the HGF signaling pathway in oncogenesis, tumor progression and metastasis.
HGF is a plasminogen-like protein with mitogenic, motogenic and morphogenic activities (3;4). HGF is typically produced by cells of mesenchymal origin and acts in a paracrine manner on a variety of cellular targets including epithelial and endothelial cells, hematopoietic cells, neurons, melanocytes as well as hepatocytes (3;4). HGF is essential for early embryonic development and also contributes to organogenesis in liver, lung, kidney and other tissues (5). The cell surface receptor for HGF is the transmembrane tyrosine kinase encoded by the MET protoncogene (6). The MET oncogene was isolated from a chemically mutagenized human osteogenic sarcoma cell line; its transforming activity was due to gene rearrangement where sequences from the TPR (translocated promoter region) locus on chromosome 1 fused to sequences from the MET locus on chromosome 7 (TPR-MET) (7). Subsequent isolation of the full-length MET protooncogene coding sequence revealed structural features of a membrane spanning receptor tyrosine kinase (TK) (7). The identification of HGF as the natural ligand for the Met receptor protein and the identity of scatter factor and HGF united a collection of findings demonstrating that a single receptor transduced all HGF biological activities (3).
Consistent with its relationship with HGF, MET is widely expressed early in 4 development, deletion of the gene is lethal in mice, and widespread expression persists throughout adulthood (5;8).

The HGF/Met Signaling Pathway: an Overview
Upon HGF binding, Met is autophosphorylated on two tyrosine residues (Y1234 and Y1235 per sequence for UniProt accession P08581) within the activation loop of the TK domain which significantly enhance kinase activity, while phosphorylation on two tyrosine residues near the carboxyl terminus (Y1349 and Y1356) form a multifunctional docking site that recruits a collection of intracellular adapters containing Src homology-2 (SH2) domains and other specific receptor recognition motifs that transmit signals further downstream (7;9). Among the adapter proteins and direct kinase substrates thus far implicated in Met signaling are Grb2, Gab1, phosphatidylinositol 3-kinase (PI3K), phospholipase C-gamma (PLCγ), Shc, Src, Shp2, Ship1, and STAT3 (7). Gab1 and Grb2 in particular connect larger networks of adaptor proteins involved in signaling, presumably contributing to HGF pleiotropism (3;7). The direct binding of Grb2 to Met through Y1356 links the receptor to the Ras/MAPK pathway regulating cell cycle progression (3;7;9). Gab1/Met interactions initiate branching morphogenesis in several epithelial and vascular endothelial cell types. Gab1 is also highly phosphorylated by Met, resulting in the recruitment of PI3K, contributing in turn to cell cycle progression, protection from apoptosis, as well as increased cell motility (7)(8)(9). Among the many genes upregulated by this pathway is MET itself (9), creating the potential for Met overexpression in otherwise normal target cells through persistent ligand stimulation; indeed, Met overexpression is widely observed in cancers of epithelial origin (3).
HGF/Met signaling is implicated in a wide variety of human malignancies including colon, gastric, bladder, breast, kidney, liver, lung, head and neck, thyroid and prostate cancers, sarcomas, hematological malignancies, melanoma and central nervous system tumors (3). Through paracrine signaling, overexpression of ligand and/or receptor, autocrine loop formation and/or receptor mutation and gene rearrangement, this signaling pathway can enhance tumor cell growth, proliferation, survival, motility and invasion (3;7;9). Inappropriate Met signaling in disease can resemble developmental transitions between epithelial and mesenchymal cell types normally regulated by HGF. 5 Importantly, the pathway initiates a program of cell dissociation and increased cell motility coupled with increased protease production that has been shown to promote cellular invasion through extracellular matrices, and that closely resembles tumor metastasis in vivo (9). In addition, pathway activation in vascular cells stimulates tumor angiogenesis, facilitating tumor growth for cancers that are growth limited by hypoxia and promoting tumor metastasis (9). Hypoxia alone upregulates Met expression and enhances HGF signaling in cultured cells and mouse tumor models (9).

HGF Signaling in Kidney Development
The critical roles of HGF and Met in embryonic development were first demonstrated in mice by targeted disruption of each gene; these animals displayed placental defects, defective somite migration, stunted liver and limb muscle development and death in utero (5;10). HGF promotes the development of tubular structures in organs such as mammary gland and kidney (11). Proper kidney development depends on the multicellular process of branching morphogenesis. During the metanephric phase of kidney development, nephrogenesis is initiated by ingrowth of the Wolffian duct-derived ureteric bud into the presumptive kidney mesenchyme (11;12). In response to signals from the ureter, mesenchymal cells condense, aggregate into pretubular clusters and undergo an epithelial conversion generating a simple tubule. This tubule then undergoes morphogenesis and is transformed into the excretory system of the kidney. The nephron epithelial tube gives rise to the branched collecting duct system, while the surrounding metanephric mesenchyme undergoes mesenchymal-epithelial transition to form the proximal parts of the nephron (11;12). The coordinated exchange of signals in both directions between the growing buds of epithelium and the mesenchyme that they are invading is critical. Several soluble factors act in a complementary fashion either as proor anti-tubulogenic regulators, including members of the fibroblast growth factor, transforming growth factor-beta and Wnt families as well as glial derived neurotrophic factor, epidermal growth factor and HGF (11;12).
The HGF-driven intracellular signaling events in mesenchymal/epithelial transitions during nephrogenesis presumably resemble those defined using cultured renal 6 epithelial cell models of branching morphogenesis. In that context, the recruitment of Gab1 and Grb2 to c-Met activates SOS1, contributing to Ras-MAP kinase pathway activation, adherens junction disassembly, cell motility and proliferation (13).
Reorganization of the actin cytoskeleton, which is required for observed cell shape changes, is regulated by the Rho family of small GTPases activated downstream of PI3K and Ras (13;14). Rac1 and cdc42 regulate actin polymerization at the cell periphery resulting in the extension of lamellipodia that are essential for cell migration and fillopodia that precede de novo tubulogenesis in vitro (13;14). In contrast, RhoA acting via its downstream effector Rho-associated kinase stimulates myosin light chain phosphorylation and regulates actin stress fiber formation and cell contractility (14). Thus a coordinated activation and deactivation of Rac and Rho is required for cell shape change and migration (11;13;14). HGF stimulation also results in the tyrosyl phosphorylation of β-catenin, inducing its dissociation from E-cadherin in adherens junctions, contributing to junction breakdown and freeing β-catenin for nuclear translocation and transcriptional activation (8).

HGF Signaling in Renal Homeostasis
HGF and MET expression persist in the adult kidney, but striking changes occur in the quality and magnitude of the response of renal epithelial cells to HGF stimulation upon completion of normal development. Morphogenic and proliferative responses are minimized. While the role of HGF in adult renal physiology is not yet fully understood, the kidney is an important source of circulating HGF in adults, and HGF is an endogenous renoprotective factor with potent antifibrotic activity (15;16). HGF has been shown to protect adult kidney tissue from acute toxicity and ischemic stress (15).
Endogenous HGF levels are elevated in kidneys exposed to long term stress, and HGF counteracts TGF-b signaling associated with renal fibrosis, a major cause of chronic renal failure (15)(16)(17). At the cellular level, these tissue protective effects are most likely to be mediated through HGF-driven cell survival pathways and pathways that control extracellular matrix composition and turnover (15-17).

Dysregulated HGF Signaling in RCC
Most of the intracellular mediators and pathways activated by Met persist through development into adulthood, and it is unclear which signals are modified or silenced to provide a homeostatic, as opposed to developmental or pathological, HGF response.
Given the functional similarities between tumorigenesis and epithelial/mesenchymal transitions at the cellular level, the loss of such signal attenuation mechanisms are likely to contribute to tumorigenesis, invasiveness and metastasis. Among the four main RCC subtypes, an oncogenic role of HGF/Met signaling has been firmly established for hereditary papillary renal carcinoma (HPRC), where inherited missense mutations in the MET gene were first found; similar somatic mutations were also found in a small subset (13%) of sporadic papillary renal carcinoma (PRC) tumor samples (18)(19)(20)(21). The biochemical and biological impact of these MET mutants have been investigated in several model systems, confirming their suspected oncogenic potential, as described in greater detail below (22)(23)(24)(25)(26)(27)(28). A growing body of evidence also supports HGF/Met pathway involvement in clear cell RCC, where loss of von Hippel-Lindau (VHL) tumor suppressor gene function occurs in familial and most sporadic cases (2). VHL loss results in the aberrant expression of genes that control cell proliferation, invasion and angiogenesis (2).  (27). Overall, these findings predicted that mutant Met forms are more easily activated than WT Met, and more likely to remain active, but did not that clearly eliminate the need for an initiator of kinase activation such as ligand binding or other environmental cue.

HGF/Met Pathway Activation in HPRC and
In a study complementary to that of Miller, et al., Chiara and colleagues later compared the autophosphorylation events in WT and mutant Met receptors expressed in cultured cells using phosphorylation-site specific antibodies, and proposed that mutant receptors possessed a lower threshold for kinase activation (30). HGF binding to WT Met triggers autophosphorylation of Y1235 and Y1234 in the TK activation loop; substitution of F for Y at either position severely impairs kinase function, suggesting that phosphorylation at both sites is required for kinase activation (30,31) A more recent study further showed that mutation in Y1235D reduced k cat compared with the activated, autophosphorylated wild-type enzyme (32). Unlike WT Met, the D1246H/N and M1268T Met mutants did not undergo Y1234 phosphorylation, and were not catalytically impaired by F substitutions at that site (30). Thus these mutants were not constitutively active, but mutation overcame the normal requirement for a second phosphorylation step leading to kinase activation (30). Importantly, the apparent need for ligand activation of HPRC and PRC associated Met mutant forms suggests that therapeutic strategies aimed at ligand blockade remain viable possibilities for these patient populations. and E-cadherin are initially expressed during renal development, specifically upon transition of the mesenchyme surrounding the branching ureteric buds to the epithelium that will form the tubules of the nephron (45). As described above, this mesenchymal to epithelial transition and ensuing tubule formation involves several Wnt family members acting in an autocrine manner (12;46), as well as HGF acting in a paracrine mode (47).

HGF/Met Signaling in Clear Cell RCC
Dysregulated b-catenin signaling in the adult is potently oncogenic: mutations in the genes encoding APC or b-catenin are frequent in colorectal cancer (48). Both types of mutation allow b-catenin to bypass APC-mediated ubiquitination and proteosomal degradation and it is now known that cytoplasmic b-catenin can be stabilized by a variety of genetic defects (48). showed similar preclinical anti-oncogenic potential and revealed that HPRC-associated Met mutations could impact drug sensitivity (77)(78)(79). More selective and potent synthetic inhibitors of Met ATP binding have been developed and tested in various model systems (78)(79). Most Met TKIs competitively antagonize occupancy of the intracellular ATP binding site, preventing TK activation and downstream signaling. Among these, foretinib targets Met, VEGFR2, Axl, Ron and Tie-2 with high affinity. In the largest clinical trial devoted to papillary renal cell carcinoma, foretinib demonstrated anti-tumor activity, modulation of several target indicator plasma proteins, and a manageable toxicity profile (80). Unlike previous trials of Met pathway antagonists, this trial was restricted to patients with papillary histology (both type 1 and 2 histologies were included). In addition, patients enrolled on this trial were stratified based on the presence of indications of Met pathway activation to determine if Met status impacted response to the agent (80).
The overall response rate in the trial was 13.5%, and the median duration of response was 18.5 months. The median progression-free survival (PFS) was 9.6 months for the whole study population. When analyzed by dosing cohort, the intermediate dosing group treated with 240 mg/day on days 1 to 5 of a 14-day cycle had a slightly longer progression-free survival (PFS) at 11.6 months than patients treated with continuous dosing of 80 mg/day at 9.1 months. Fifty out of the 68 evaluable patients had some degree of tumor shrinkage, although most did not meet the criteria for partial response by RECIST. Remarkably, 5 out of 10 patients with germline MET mutations had a partial response. The other five had stable disease for at least six weeks, and four of them had more than 10% tumor shrinkage but less than the 20% necessary for a partial response (81).
Tivantinib is the only Met-directed TK inhibitor currently in human clinical trials that is not ATP-competitive; it reportedly binds to the Met TK domain near the ATP binding site and acts allosterically (82). A phase II, multicenter, single-arm study assessing the safety and efficacy of tivantinib monotherapy in adolescent and adult patients with metastatic or surgically unresectable microphthalmia transcription factor (MITF)-associated (MiT) tumors, including translocation-associated RCC (tRCC), was recently completed. Median progression-free survival was 1.9 months in tRCC and tivantinib was safe and tolerable in patients with MiT tumors, but antitumor activity was modest (83). A randomized phase II clinical trial is recruiting patients with metastatic or locally advanced kidney cancer that cannot be removed by surgery. The primary objective is to assess the response rate (confirmed complete and partial response) of patients with locally advanced or metastatic pRCC treated with either tivantinib or tivantinib combined with erlotinib hydrochloride (NCT01688973).
A cross-tumoral phase II clinical study is recruiting patients to study the antitumor activity of crizotinib across predefined tumor types harboring specific alterations in ALK and/or Met. One arm of the study will test crizotinib in PRC type 1 at doses of 500, 400 or 250 mg/day, depending on toxicity. A phase I/II multiple ascending dose study of BMS-777607 in subjects with advanced or metastatic gastroesophageal cancer, hormone refractory prostate cancer, head and neck squamous cell carcinoma, and PRC type 1 has been completed and results are awaited (NCT00605618). Preliminary analysis of an ongoing phase I clinical trial testing cabozantinib in 25 patients with metastatic clear cell renal cancer showed a median progression-free survival (PFS) of 14.7 months. Of 21 patients evaluable for response, seven had a partial response by modified RECIST criteria, 13 had stable disease and one had progressive disease. Interestingly, the investigators saw responses in patients who had prior anti-VEGF therapy, suggesting that the combination of Met-VEGF inhibition is therapeutically valuable. Further trials in this disease setting are planned (NCT01100619) (84,85) The requirement of the carboxyl-terminal docking site for WT or mutant Met transforming activity in cultured cells (24;25), and the known roles of intracellular effectors including Gab1, PI3K, Grb2, Shc and STAT3 in cell transformation (4;7), suggest that targeting one or more of these interactions could effectively disrupt Met driven oncogenesis. Knowledge of the unique structure of the Grb2 SH2 domain provided the basis for the development of small synthetic Grb2 selective binding antagonists (86). Further refinement of these early structures has yielded compounds that block HGF-stimulated cell motility, matrix invasion and morphogenesis in normal and tumor derived cultured cells, as well as vascular endothelial cells, at low nanomolar concentrations (87). Beyond effector targeting, compounds that block HSP90/client interactions, such as geldanomycin (88), also potently block Met oncogenic signaling (89,90). Human clinical trials of geldanomycin related compounds are underway for a variety of cancers where the Met pathway is active, including RCC.
While the potential efficacy of HGF/Met targeted drugs for treating subtypes of RCC as single agents is promising, combining agents such as geldanomycin that attenuate receptor supply with inhibitors of other critical receptor functions could lower the effective dose of each, reducing drug toxicity as well as the emergence of drug resistant mutations. Improving our understanding of the molecular basis of oncogenic HGF/Met signaling in RCC should facilitate the development of other combinatorial treatment strategies, and help overcome other challenges facing drug development, such as identifying patients most likely to benefit from HGF/Met targeted therapeutics, assessing drug activities in tumor tissues, and predicting the potential toxicity of longterm pathway blockade.