Co-expression Of Met Research Articles

Programmed death ligand-1 and MET co-expression is a poor prognostic factor in gastric cancers after resection.

Programmed death-ligand 1 (PD-L1) plays an essential protein for immune evasion, contributing to tumor development and progression. Recent studies have reported MET as an upregulator for PD-L1 overexpression through an oncogenic pathway. However, an association between PD-L1 expression with MET has not been reported in gastric cancer.The prognostic significance of PD-L1 and its association with Epstein-Barr virus (EBV), microsatellite instability (MSI), and mucin phenotype remain controversial.We performed in situ hybridization for EBV-encoded RNA and immunohistochemistry in tissue microarrays for 394 gastric cancers. A multiplex polymerase chain reaction with five quasimonomorphic markers was performed for MSI.PD-L1 expression was observed in 123 cases (31.2%), and clinicopathological features such as MET overexpression, high pT stage, and a lack of lymphatic invasion represent significant risk factors associated with PD-L1 overexpression in gastric cancers. No associations of EBV, MSI, or mucin phenotype with PD-L1 expression were statistically significant. PD-L1 expression was a strong indicator for worse overall survival (OS) but borderline significant in disease-free survival (DFS). A combined analysis of PD-L1 and MET expression indicated that the PD-L1+/MET+ subgroup showed the worst prognosis when compared to the PD-L1-/MET- subgroup, which had the best clinical outcome. Furthermore, PD-L1 overexpression exhibited poor prognosis in terms of both OS and DFS in EBV-negative, microsatellite stable, and intestinal mucin phenotype tumors. In conclusion, this is the first study to evaluate the overexpression of MET as a risk factor for PD-L1 positivity in gastric cancer tissue as well as the reliability and prognostic relevance of PD-L1/MET co-expression after surgery.

Co-expression of MET and CD47 is a novel prognosticator for survival of luminal-type breast cancer patients

Although luminal-type primary breast cancer can be efficiently treated, development of metastatic disease remains a significant clinical problem. We have previously shown that luminal-type circulating tumor cells (CTCs) co-expressing the tyrosine-kinase MET and CD47, a ligand involved in cancer cell evasion from macrophage scavenging, are able to initiate metastasis in xenografts. Here, we investigated the clinical relevance of MET-CD47 co-expression in 255 hormone receptor positive breast tumors by immunohistochemistry and found a 10.3- year mean overall-survival difference between MET-CD47 double-positive and double-negative patients (p<0.001) MET-CD47 co-expression defined a novel independent prognosticator for overall-survival by multivariate analysis (Cox proportional hazards model: HR: 4.1, p<0.002) and CD47 expression alone or in combination with MET was strongly associated with lymph node metastasis. Furthermore, flow cytometric analysis of metastatic patient blood revealed consistent presence of MET+CD47+ CTCs (range 0.8 - 33.3% of CTCs) and their frequency was associated with increased metastatic spread. Finally, primary uncultured CTCs with high MET+CD47+ content showed an enhanced capacity to initiate metastasis in mice. Detection and targeting of MET and CD47 may thus provide a rational basis for risk stratification and treatment of patients with luminal-type breast cancer.

Abstract 4391: MET is a potential therapeutic target and a candidate for combination therapy with EGFR inhibition in triple-negative/basal-like breast cancer.

Abstract Triple-negative breast cancer (TNBC)/basal-like breast cancer (BLBC) are known as poor outcome subtypes with deficiency of targeted molecular therapy. Previous studies showed that MET may play a critical role in the development of TNBC and may be a rational therapeutic target. In order to evaluate MET as a potential therapeutic target for TNBC, we investigated genomic aberration and expression status of MET and evaluated efficacy of MET inhibitor, PHA-665752 in TNBC cell lines. MET protein expression was relatively high in TNBC cell lines. The depletion of MET using small interfering RNA (siRNA) led decreased cell proliferation and migration. PHA-665752 treatment resulted in decreased cell viability, and most of these cell lines were found to be moderately sensitive in their response to PHA-665752. The most resistant cell line, MDA-MB-468, showed exclusively high expression of EGFR, and then EGFR depletion using siRNA induced increased sensitivity to PHA-665752. In our cohort, BLBC was the subtype showing the highest relationship with MET expression among 5 subtypes (77/134, 57.5%, P&amp;lt;0.001). MET expression had a poorer OS (P=0.001) and DFS (P=0.010). Interestingly, TNBC patients with co-expression of MET and EGFR worsen the DFS significantly compared with EGFR expression only. Our findings highly suggest that MET can be a potential therapeutic target in TNBC, and the combined inhibition of MET and EGFR may benefit for the treatment of BLBC patients. Citation Format: Yu Jin Kim, Jong-Sun Choi, Ji-Young Song, Kyungsoo Jung, Ensel Oh, Young Kee Shin, Yoon-La Choi. MET is a potential therapeutic target and a candidate for combination therapy with EGFR inhibition in triple-negative/basal-like breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4391. doi:10.1158/1538-7445.AM2013-4391

Abstract 1087: TGF-β activated EGFr as a bypass mechanism for the acquired resistance of MET inhibitors in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) patients with an active HGF/c-Met signaling pathway have a significantly worse prognosis. Although targeting strategies using MET tyrosine kinase inhibitors (TKI) are in Phase I/II testing for HCC, monotherapy using TKIs in other cancers has not demonstrated durable responses. In non-small cell lung cancer, bypass mechanisms for resistance to EGFr TKIs include MET amplification and activation. We utilized human HCC MET+ cell lines to explore a mechanism of EGFr activation during MET inhibition, and we examined the effect of combination MET and EGFr TKI therapy on suppressing this bypass mechanism in HCC in vivo. Analysis of 149 HCC specimens using transcriptome profiling and immuno-histochemical staining demonstrates a strong negative correlation between EGFr and MET expression. HCC is predominantly either MET positive [44%] or EGFr positive [45%], and co-expression of MET and EGFr is extremely rare in the same tumor. We have previously demonstrated that treatment of xenograft tumors of human HCC MET+ MHCC97-H cells with a MET TKI resulted in stasis of tumor growth in vivo (You … Rountree. Hepatology. 2011). To understand the mechanism of survival in the MHCC97-H cells after inhibition of MET, we developed an MHCC97-H cell line with stable expression of MET shRNA. This MHCC97-H METneg line demonstrated the slower growth kinetics and the inhibition of both PI3K and MAPK signaling comparable to MET TKI treated xenografts. Screening against 850 kinases and phosphatases identified EGFr activation as a mechanism of survival in this MHCC97-H METneg line. Gene expression analysis of this MHCC97-H METneg cell line demonstrated a 6 fold increase in TGF-β, a ligand of EGFr. MHCC97-H xenograft tumor analysis demonstrates a 20 fold increase in TGF-β expression after MET TKI therapy. In vivo treatment of MHCC97-H tumors with combination MET and EGFr TKI therapy resulted in complete regression of all small tumors (15 mm3 at start of TKI) compared to partial regression when MET TKI was used as monotherapy. Treatment of large tumors (200-300 mm3 at start of TKI) with combination TKI therapy resulted in a significant reduction in tumor volume compared to MET TKI monotherapy. EGFr TKI monotherapy had no effect on tumor growth kinetics. Within MET positive HCC models, MET TKI therapy results in a bypass mechanism through up-regulation of TGF-β and activation of EGFr. This bypass mechanism can be partially overcome with combination EGFr and MET TKI therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1087. doi:1538-7445.AM2012-1087

Abstract LB-124: RON (MST1R) is a novel prognostic marker and therapeutic target for gastroesophageal adenocarcinoma

Abstract Background: RON (MST1R) is a member of the MET receptor tyrosine kinase family with a putative role in cancer, but its expression and function is poorly characterized in gastroesophageal cancer (GEC). Methods: We used immunohistochemistry (IHC), immunoblotting (IB) and immunofluorescence (IF) to determine RON and MET expression in human GEC tissues obtained from curative intent resection (n=94) and cell lines (n=20), and correlated tissue expression levels to overall survival using REMARK standards for prognostic markers. We assessed MST1R and MET gene copy number using quantitative polymerase chain reaction (qPCR) and confirmed findings using fluorescence in situ hybridization (FISH) and array comparative genomic hybridization in select samples. We assessed tumor tissue samples and GEC cell lines for MST1R mutation and single nucleotide polymorphisms (SNPs). We assessed the function of RON in GEC cell lines using viability, apoptosis, and invasion assays, with further evaluation using pooled small interfering RNA (siRNA) protein knockdown. The role of RON and MET coexpression and costimulation was evaluated along with downstream pathway activation of STAT3, using global and specific phophoantibodies, in order to evaluate potential synergistic signaling effects. GEC growth inhibition was evaluated using RON specific novel monoclonal blocking antibodies, small molecule tyrosine kinase inhibitors and RON siRNA/shRNA, alone or in combination with MET and STAT3 specific inhibition to evaluate for inhibitory synergism as a result of RON and MET functional reciprocity and signaling redundancy. Results: By IHC, RON was highly over-expressed in 74% of GEC samples and over-expression was prognostic of poor survival (p=0.008); MET and RON co-expression occurred in 43% of samples and was prognostic of worst survival (p=0.03). High MST1R gene copy number (GCN) was seen in 35.5% (16/45) of cases, correlated with poor survival (p=0.01), and was associated with high MET and ERBB2 GCN. A novel somatic MST1R juxtamembrane mutation R1018G was found in 11% of samples. RON was functional in cell lines, activating downstream effector STAT3, and resulting in increased viability over controls. RON and MET co-stimulation led to enhanced malignant phenotypes over either alone. Growth inhibition and apotosis was optimal using novel blocking monoclonal antibodies to both RON and MET, versus either alone. Small molecule tyrosine kinase inhibitor, SU11274, inhibited both receptors, and proved synergistic when combined with STAT3 inhibition (combination index &amp;lt;1). Conclusions: These preclinical studies define RON as a potential important novel prognostic marker and therapeutic target for GEC, and supports continued investigation of its role in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-124. doi:10.1158/1538-7445.AM2011-LB-124

Mechanism of Action of a Novel c-Met Inhibitor MK2461 In Multiple Myeloma

AbstractAbstract 4078 Background:Cytokine stimulated signaling pathways contribute to multiple myeloma (MM) disease progression and in acquired resistance to current treatment options making MM an incurable malignancy. It is very well documented that HGF is a cytokine that is secreted by bone marrow stromal cells which has an autocrine and paracrine role in the disease progression in a myeloma setting. HGF binds to its receptor tyrosine kinase on MM cells, MET and this binding at the extracellular domain results in activation of MET which interacts with several of its target proteins resulting in increased survival, increased proliferation, cell cycle progression, motility, migration and invasion. In normal bone marrows, co-expression of MET and its ligand HGF is a rarity whereas co-expression is a common feature of MM. Elevated levels of HGF have been observed in serum and bone marrows of MM patients with a negative correlation to disease progression. In addition, increased HGF levels cause abnormal and reduced bone formation in patients. HGF gene levels in MM samples have been observed to be significantly up regulated in myeloma cells when compared to normal cells. Recently, studies have identified that HGF facilitates the MM cells to adhere to fibronectin, a bone marrow matrix protein, thereby positively impacting MM cell invasion and proliferation. Overall, HGF and its receptor mediated pathway influences tumor progression in myeloma by targeting several different aspects of the disease biology and hence is a very attractive and potentially very important target for improving treatment regimens in a myeloma setting. Methods:MK2461 was synthesized by Merck Inc. (Whitehouse Station, NJ, USA). Stock solutions were made using DMSO and working stock solutions were made using RPMI 1640 media containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In order to study the mechanism of action of the drug, immunoblotting studies were performed on lysates made from cell lines incubated with the drug for various durations. Results:MK2461 treatment led to dose and time dependent cytotoxicity in a few myeloma cell lines (OPM2, DOX40, RPMI8226 and LR5) but not in others (MM1S, MM1R, H929 and U266). The IC50 values for the sensitive lines varied from 1μ M (OPM2) to 10μ M (DOX40, RPMI8226 and LR5). However, MK2461 significantly inhibited the proliferation of MM cells at sub IC50 concentrations in all cell lines tested except MM1S. This inhibition of proliferation was observed when cells were co-cultured with stromal cells or cytokines, namely VEGF, IL6 or HGF. Culturing MM cells with increasing doses of HGF was still unable to protect them from drug induced inhibition of proliferation. MK2461 was able to induce time dependent increase in apoptosis (as measured by annexin/PI), decrease in proliferation (as measured by BrdU assay) and induction of cell cycle arrest in the drug sensitive cell lines. This effect was not observed in MM1S cells. Exploring the mechanism of action of the drug indicated that MK2461 treatment led to down regulation of pc-Met, pGab1, pAkt and pErk in both the drug sensitive (OPM2) and drug resistant (MM1S) cell lines. However, proteins down stream of Akt in the PI3K/Akt pathway, namely pGSK3β, p70S6K, Bcl2, cyclin E and cyclin D3 were down regulated only in OPM2 cells. On the contrary, we observed up-regulation of these proteins in the drug resistant cell line offering a possible explanation for the drug resistant phenotype. We have also examined combinations of MK2461 with inhibitors of PI3K/Akt pathway. Conclusion:These studies demonstrate significant in-vitro activity of MK2461 in MM. Our results suggest the presence of two populations one very sensitive to MK2461 and one insensitive. Differential effects on the signaling pathways provide important clues to the mechanisms of action of c-met inhibitors in myeloma. The results form the basis for clinical evaluation of MK2461 in MM. Disclosures:Kumar:Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Abstract 3609: MGCD265, an orally active Met/VEGFR multitargeted kinase inhibitor in Phase II clinical development, potently inhibits clinically relevant Met mutants

Abstract MGCD265 is a multitargeted receptor tyrosine kinase inhibitor in Phase II clinical development, which inhibits the activation of regulators of cancer development and progression by targeting Met, the three members of the VEGFR family, Tie-2 and Ron. Met is a key player in the epithelial-mesenchymal transition (EMT) and Met deregulation is described in several types of cancer. The mechanisms involved in Met activation include co-expression of Met with its ligand HGF, overexpression of the Met protein, Met and/or HGF gene amplification or activating mutations. Originally found in hereditary papillary renal carcinoma (HPRCC), activating missense mutations in the kinase domain provided the first evidence that Met activation was causative of this disease. Mutations outside of the tyrosine kinase domain, such as those in the juxtamembrane and extracellular sema domains have also been analyzed, and found in NSCLC, SCLC, melanoma, head and neck cancer, mesothelioma and pancreatic cancer. For the clinical development of MGCD265, we demonstrated the ability of MGCD265 to inhibit clinically relevant Met mutants described in a variety of cancers, including HPRCC and NSCLC. In cell-based assays, MGCD265 inhibits the activation of Met kinase domain mutants (e.g. Y1230C) as well as mutants within the juxtamembrane region (e.g. R988C), and the extracellular domain (e.g. N375S) with nanomolar potency. This is of particular interest, as several kinase domain mutations confer resistance of Met to Met-selective agents such as PF-4217903. MGCD265 also inhibits signaling pathways activated downstream from Met mutants. The phosphorylation of Gab1 multisubstrate docking protein following the activation of Met mutants is inhibited by MGCD265 as is the ensuing activation of the Erk pathway. Furthermore, MGCD265 inhibits the motility of renal carcinoma cells expressing an extracellular domain Met mutant, and blocks tumor growth in a NSCLC cell line expressing a juxtamembrane domain Met mutant. These results, together with our previous findings, demonstrate that MGCD265 is particularly efficient at inhibiting tumor growth in animal models in which wild-type Met or Met mutants are active, suggesting that MGCD265 may particularly benefit patients with activated Met. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3609.

β4 Integrin Is a Transforming Molecule that Unleashes Met Tyrosine Kinase Tumorigenesis

Cell multiplication in the absence of integrin-derived adhesive signals (anchorage-independent growth) is the phenotypic hallmark of neoplastic transformation. Therefore, the frequently observed up-regulation of some integrins in tumors has been interpreted as an epiphenomenon and not as a causative factor of oncogenic conversion. beta4 integrin stimulates proliferation and survival of epithelial cells and is overexpressed in human carcinomas, often in concomitance with up-regulation of the Met tyrosine kinase receptor for hepatocyte growth factor. Met is not endowed with transforming ability but can exploit the beta4 cytoplasmic tail as a substrate/adaptor for amplification of mitogenic and antiapoptotic responses, independently of cell adhesion. Here, we show that overexpression of beta4 is sufficient to transform rodent fibroblasts, enhances anchorage-independent growth of breast carcinoma cells, and induces tumorigenesis in nude mice; conversely, RNA interference-mediated depletion abrogates the transformed phenotype of neoplastic cells. These autonomous oncogenic properties are dramatically exacerbated upon Met coexpression, suggesting that the integrin can instigate the latent tumorigenic potential of the kinase. A beta4 nonadhesive variant still cooperates with Met for cellular transformation, confirming the adhesion-independent function of beta4 in magnification of Met biological effects. Conversely, a beta4 signaling-incompetent mutant that cannot be efficiently tyrosine phosphorylated by Met and displays reduced ability to activate phosphatidylinositol 3-kinase-dependent and Ras-dependent pathways aborts transformation. Our findings define beta4 as a signaling accomplice (a "servo-oncogene") of tyrosine kinase proto-oncogenes in primary carcinogenesis, evoke an unorthodox function for a prototypic adhesion molecule in the positive regulation of anchorage-independent growth, and suggest the use of beta4 as a target for anticancer therapy.

RON and MET co-expression is a prognostic indicator in bladder cancer patients

RON and MET co-expression is a prognostic indicator in bladder cancer patients

Regulation of P311 Expression by Met-Hepatocyte Growth Factor/Scatter Factor and the Ubiquitin/Proteasome System

P311 is a mouse cDNA originally identified for its high expression in late-stage embryonic brain and adult cerebellum, hippocampus, and olfactory bulb. The protein product of P311, however, had not been identified previously, and its function remains unknown. We report here that P311 expression is regulated at multiple levels by pathways that control cellular transformation. P311 mRNA expression was decreased sharply in both neural and smooth muscle cells when the cells were transformed by coexpression of the oncogenic tyrosine kinase receptor Met and its ligand hepatocyte growth factor/scatter factor. The P311 mRNA was found to encode an 8-kDa polypeptide that was subject to rapid degradation by the lactacystin-sensitive ubiquitin/proteasome system and an unidentified metalloprotease, resulting in a protein half-life of about 5 min. These data suggest that P311 expression is dramatically decreased by several pathways that regulate cellular growth.

The tyrosine kinase receptor met and its ligand HGF are co-expressed and functionally active in HHV-8 positive primary effusion lymphoma.

Primary effusion lymphoma (PEL) harbors consistent infection by human herpesvirus-8, preferentially develops in immunodeficient patients and selectively localizes to the serous body cavities. Histogenetic analysis has suggested that PEL originates from post-germinal center, pre-terminally differentiated B cells sharing phenotypic features with plasma cells. Here we have investigated the expression status and functional integrity of the Met tyrosine kinase receptor and of its ligand hepatocyte growth factor (HGF). Thirteen PEL (nine cell lines and four primary specimens) were analyzed for Met and HGF expression and function by multiple assays. For comparison, a panel of 34 high grade B cell non-Hodgkin lymphomas (NHL) other than PEL was also investigated. Co-expression of Met and HGF was found in all PEL analyzed, whereas it was restricted to 1/34 B cell NHL other than PEL (P < 0.001; chi2 test). The Met protein expressed by PEL displays biochemical characteristics typical of Met expressed by other cell types and is capable of tyrosine autophosphorylation. By using a combination of immunological and biological assays, production and secretion of a functional HGF species was identified in all PEL cell lines analyzed. HGF stimulation of PEL cells rapidly induces Met tyrosine phosphorylation, demonstrating the functional integrity of the Met/HGF loop. Because of the well known mitogenic and motogenic properties of Met/HGF interactions, these data may bear implications for PEL growth and dissemination. Among B cell neoplasms, Met/HGF co-expression selectively clusters with PEL and, as demonstrated by previous studies, with multiple myeloma plasma cells, thus reinforcing the notion that PEL displays biologic similarities with tumors derived from late stages of B cell differentiation.

Decreased fibronectin expression in Met/HGF-mediated tumorigenesis.

The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor (HGF)/scatter factor are involved in the etiology and progression of a number of human cancers. Coexpression of Met and HGF in mesenchymal cells increases the tumorigenic and metastatic potential of the cells. In the studies described here, we used differential display screening to identify changes in gene expression that are initiated by Met/HGF, and that may lead to these phenotypes. We learned that Met/HGF signaling resulted in greatly decreased fibronectin mRNA production in three different human and mouse tumor cell lines; these decreases in fibronectin mRNA were paralleled by decreases in fibronectin protein. We also found a progressive decrease in fibronectin in tumor explants and metastases derived from the Met/HGF transformed cells. The absence of fibronectin expression is a frequent cancer phenotype; our results indicate that decreases in fibronectin correlate with, but are not essential for, MetHGF/SF-mediated tumorigenesis.