Abstract SY23-03: Oncogenic signal transduction via the hepatocyte growth factor/Met receptor kinase pathway
Authors: Cecchi Fabiola , Pajalunga Deborah , McNeil Brian , Rabe Daniel , Fowler Andrew , Liu Yuan , Gagnon C. Robert , Kallender Howard , Shah Manish A. , Getzenberg Robert , Schoenberg Mark , Martin Anne-Marie , Srinivasan Ramaprasad , Linehan W. Marston , Byrd R Andrew , Bottaro Donald P. 
Published: May 2019

Signal Transduction via the hepatocyte growth factor (HGF)/Met receptor kinase pathway is critical for normal embryogenesis and adult homeostasis, and aberrant HGF/Met signaling contributes to tumorigenesis and metastasis in many prevalent forms of cancer. By defining the basic molecular mechanisms of pathway activation at the target cell surface, we hope to develop novel strategies to antagonize oncogenic signaling as well as methods to assess pathway status in cancer patients. These methods may help identify those most likely to benefit from HGF/Met targeted therapies and help monitor treatment response.

Distinct domains of HGF bind to the Met receptor tyrosine kinase and to cell-surface heparan sulfate (HS) proteoglycans. The latter are widely expressed, structurally diverse biopolymers that modulate many important protein-protein interactions, but their importance in eliciting HGF biological responses is controversial. We have identified basic (positively charged) amino acid residues in HGF that form the primary binding site for (negatively charged) HS proteoglycans. HS binding by these residues increases the affinity of ligand-receptor binding and, with HS polymers of sufficient length, facilitates ligand oligomerization. Ligand oligomerization, in turn, controls Met kinase activation, cell motility and proliferation under normal conditions. Opposite (negative) charge amino acid substitutions at these critical HS binding residues in HGF can mimic bound HS in promoting high affinity receptor binding, but antagonize the binding of long HS polymers on the cell surface, thereby antagonizing ligand oligomerization and its consequences. Engineering these substitutions in the context of a short HGF isoform containing only the primary HS and Met binding domains yields a potent and selective competitive inhibitor of oncogenic HGF signaling in vivo. These findings further define the role of HS in growth factor signaling and reveal a novel strategy for antagonist development that may be applicable to other HS-binding growth factors.

The Met receptor kinase is among many transmembrane proteins that are proteolytically released from the cell surface by a process known as ectodomain shedding. Consistent with the concept that Met ectodomain shedding is a normal physiological process, we found that healthy human volunteers (n &gt; 100) display soluble Met (sMet) plasma concentrations in the range of 100 - 300 ng/ml and urinary sMet concentrations that are approximately 1000-fold lower. We hypothesized that aberrantly high Met expression, a frequent occurrence in many forms of cancer, and/or dysregulated proteolytic activity, another common feature of aggressive malignancies, could lead to increased ectodomain shedding and that sMet levels could be a useful biomarker of tumorigenesis, tumor progression and overall tumor burden. In prior studies we found that increased Met shedding correlated with malignancy in several cell lines, and that urinary sMet levels were significantly elevated in metastatic prostate cancer patients relative to those with organ-confined cancer or no evidence of disease.

In an ongoing investigation of sMet as a diagnostic and/or prognostic biomarker of bladder cancer, the mean urinary sMet level among 150 patients with pathologically confirmed disease was significantly higher than that obtained from a group of 50 individuals with no evidence of disease, whether mean values were normalized to urine creatinine concentration or not (two-tailed Mann-Whitney t-test p-value &lt;0.001). Patients who underwent radical cystecomy had greater mean urinary sMet levels than patients with pTa and pT1 tumors, consistent with the likelihood that they had more advanced local disease. Our results so far confirm that sMet can be measured in human urine samples with a large dynamic range and sub-picomolar sensitivity, and reveal that urinary sMet may provide a reliable and practical indicator of malignant potential and overall tumor burden in individuals with bladder cancer. Continued sample collection and correlative analysis of sMet values and clinical data will help determine whether urinary sMet concentration will be a sensitive and reliable indicator of bladder cancer occurrence, tumor grade, stage and progression.

We have also measured plasma sMet levels in patients participating in a phase II clinical trial of Foretinib (formerly GSK1363089/XL880), a small molecule multikinase inhibitor targeting Met and vascular endothelial cell growth factor-2 (VEGFR-2) Axl and Ron, designed to evaluate the safety and efficacy of 2 dosing schedules (continuous daily dosing and intermittent 5 days on/9 days off dosing) of foretinib as a single therapeutic agent in patients with metastatic gastric carcinoma (GC). Plasma sMet concentrations, as well as plasma VEGF-A, HGF and soluble VEGFR-2 (sVEGFR-2) levels were measured before and during treatment with foretinib to measure potential pharmacodynamic (PD) effects and/or the modulation by foretinib of Met and VEGFR-2 pathways. Marker changes from baseline were analyzed at days 5, 15, 29 and 47 using analysis of variance and their relationships with plasma concentrations of foretinib (PK) and clinical outcome (sum of longest diameter [SLD] of tumor as a surrogate for tumor shrinkage, progression-free survival [PFS], and RECIST) were also examined. In GC patients on the intermittent dosing schedule, plasma sMet and VEGF-A concentrations tended to increase during the treatment periods compared with the drug holidays, suggestive of a direct short-term drug effect on sMet and VEGF-A levels. Although mean tumor burden did not change significantly from baseline to week 8, plasma levels of sMet and VEGF-A correlated positively with the change in SLD over this period, suggesting that the increases of sMet and VEGF-A may be reflective of increased GC tumor burden.

Citation Format: Fabiola Cecchi, Deborah Pajalunga, Brian McNeil, Daniel Rabe, Andrew Fowler, Yuan Liu, C. Robert Gagnon, Howard Kallender, Manish A. Shah, Robert Getzenberg, Mark Schoenberg, Anne-Marie Martin, Ramaprasad Srinivasan, W. Marston Linehan, R Andrew Byrd, Donald P. Bottaro. Oncogenic signal transduction via the hepatocyte growth factor/Met receptor kinase pathway [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY23-03