c-Met Pathway Research Articles

Crizotinib: An Anaplastic Lymphoma Kinase Inhibitor

Crizotinib is an oral small-molecule inhibitor of ALK and c-Met tyrosine kinases that is being developed by Pfizer. ALK mutations or gene rearrangements result in growth factor-independent ALK activation. Most of the available clinical data with crizotinib are in patients with tumors that have an activated ALK, and the drug has shown very promising clinical benefit in these patients. ALK gene rearrangement occurs in approximately 3-5% of non-small-cell lung cancer patients, most of whom are never- or light smokers and have adenocarcinoma histology. In these patients, crizotinib at a dose of 250 mg twice daily demonstrated a response rate of 56% and a disease control rate of 87% in previously treated patients. The drug is generally well tolerated, with the most common adverse events being nausea, vomiting and some minor visual disturbances that are fleeting. Grade 3/4 elevations in hepatic transaminases occurred in 6% of the patients, which in most patients resolved with dose reduction. Ongoing studies will define the true utility of this drug in ALK-rearranged non-small-cell lung cancer patients and in patients with other tumors that also have ALK activation. Preliminary data suggest that the drug may also be active in tumors with an activated c-Met pathway.

Signatures of Drug Sensitivity in Nonsmall Cell Lung Cancer

We profiled receptor tyrosine kinase pathway activation and key gene mutations in eight human lung tumor cell lines and 50 human lung tumor tissue samples to define molecular pathways. A panel of eight kinase inhibitors was used to determine whether blocking pathway activation affected the tumor cell growth. The HER1 pathway in HER1 mutant cell lines HCC827 and H1975 were found to be highly activated and sensitive to HER1 inhibition. H1993 is a c-MET amplified cell line showing c-MET and HER1 pathway activation and responsiveness to c-MET inhibitor treatment. IGF-1R pathway activated H358 and A549 cells are sensitive to IGF-1R inhibition. The downstream PI3K inhibitor, BEZ-235, effectively inhibited tumor cell growth in most of the cell lines tested, except the H1993 and H1650 cells, while the MEK inhibitor PD-325901 was effective in blocking the growth of KRAS mutated cell line H1734 but not H358, A549 and H460. Hierarchical clustering of primary tumor samples with the corresponding tumor cell lines based on their pathway signatures revealed similar profiles for HER1, c-MET and IGF-1R pathway activation and predict potential treatment options for the primary tumors based on the tumor cell lines response to the panel of kinase inhibitors.

Development of c-MET pathway inhibitors

Introduction:The aberrantly upregulated c-mesenchymal-epithelia transition factor (c-MET) signaling pathway has been considered to be an attractive target for cancer intervention owing to the important roles it plays in tumor formation, progression, metastasis, angiogenesis and drug resistance. Based on the historical preclinical evidence, a number of c-MET pathway targeted agents are being developed in the clinic, and recent clinical data have begun to provide some insight into which tumor types and patient populations a c-MET pathway inhibitor may be beneficial for.Areas covered:Through reviewing recent publications in the literature and information disclosed in other public forums, we describe the current understanding of c-MET biology in human malignancies and discuss the latest progress in the development of c-MET pathway inhibitors for cancer treatment.Expert opinion:The c-MET pathway inhibitors currently being evaluated in the clinic have demonstrated compelling evidence of clinical activity in different cancer types and may provide significant therapeutic opportunities. The challenges, however, are to identify the tumor types and patient populations that benefit most, and find the most effective combinations of therapies while minimizing potential toxicity.

Dual Blockade of EGFR and c-Met Abrogates Redundant Signaling and Proliferation in Head and Neck Carcinoma Cells

Head and neck squamous cell carcinoma (HNSCC) is usually fatal, and innovative approaches targeting growth pathways are necessary to effectively treat this disease. Both the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF)/c-Met pathways are overexpressed in HNSCC and initiate similar downstream signaling pathways. c-Met may act in consort with EGFR and/or be activated as a compensatory pathway in the presence of EGFR blockade. Expression levels of EGFR and c-Met were determined by Western analysis in HNSCC cell lines and correlated with antitumor responses to inhibitors of these pathways. Combining the c-Met inhibitor PF2341066 with the EGFR inhibitor gefitinib abrogated HNSCC cell proliferation, invasion, and wound healing significantly more than inhibition of each pathway alone in HNSCC cell lines. When both HGF and the EGFR ligand, TGF-α, were present in vitro, P-AKT and P-MAPK expression were maximally inhibited by targeting both EGFR and c-Met pathways, suggesting that c-Met or EGFR can compensate when phosphorylation of the other receptor is inhibited. We also showed that TGF-α can induce phosphorylation of c-Met over sixfold by 8 hours in the absence of HGF, supporting a ligand-independent mechanism. Combined targeting of c-Met and EGFR resulted in an enhanced inhibition of tumor volumes accompanied by a decreased number of proliferating cells and increased apoptosis compared with single agent treatment in vivo. Together, these results suggest that dual blockade of c-Met and EGFR may be a promising clinical therapeutic strategy for treating HNSCC.

The role of the c-Met pathway in lung cancer and the potential for targeted therapy

Hepatocyte growth factor receptor (HGFR), the product of the MET gene, plays an important role in normal cellular function and oncogenesis. In cancer, HGFR has been implicated in cellular proliferation, cell survival, invasion, cell motility, metastasis and angiogenesis. Activation of HGFR can occur through binding to its ligand, hepatocyte growth factor (HGF), overexpression/amplification, mutation, and/or decreased degradation. Amplification of HGFR can occur de novo or in resistance to therapy. Mutations of HGFR have been described in the tyrosine kinase domain, juxtamembrane domain, or semaphorin domain in a number of tumors. These mutations appear to have gain of function, and also reflect differential sensitivity to therapeutic inhibition. There have been various drugs developed to target HGFR, including antibodies to HGFR/HGF, small-molecule inhibitors against the tyrosine kinase domain of HGFR and downstream targets. Different HGFR inhibitors are currently in clinical trials in lung cancer and a number of solid tumors. Several phase I trials have already been completed, and two specific trials have been reported combining HGFR with epidermal growth factor receptor (EGFR) inhibition in non-small cell lung cancer. In particular, trials involving MetMAb and ARQ197 (tivantinib) have gained interest. Ultimately, as individualized therapies become a reality for cancers, HGFR will be an important molecular target.

Comparative analysis of pathway proteins in cancer cells in primary and metastatic tumors and circulating tumor cells.

e11132 Background: Changes in the tumor biomarker status between primary site and metastatic lesions are known to occur at a significant frequency. Correlating biomarker status in primary and metastatic tumors as well as circulating tumor cells (CTCs) may provide critical information for overall diseases pathogenesis. Here we report correlative profiling of tumor cells obtained from metastatic regions and CTCs to primary disease. Methods: The Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) is a multiplexed protein microarray platform requiring co-localization of two detector enzyme-conjugated-antibodies. In this study, fine needle aspirate (FNA) samples were collected from various metastatic sites (lymph nodes, lung, liver, chest well, etc.) from 58 breast cancer (BCA) patients and matching CTCs for correlative analysis of HER1, HER2, p95HER2, HER3, cMET, cKIT, IGF-1R, PI3K, AKT, ERK and other pathway proteins for their expression/activation. Results: Using the CEER assay, a comprehensive profile of pathway proteins can be determined from limited amounts of cancer cells in FNAs taken from metastatic sites. HER2 expression and activation was shown to be the primary pathway in some patients; however, a significant subset of HER2 positive patients also showed expression/activation of other redundant pathway proteins such as HER3, cMET, and IGF1R as well as truncated HER2 (p95HER2). Expression/activation of other targetable pathway proteins were found in BCA-FNAs with no HER2 activity. Varying levels of HER2 expression/activation were found in CTCs. The concordance of HER2 status between primary tumor, metastatic-FNAs and CTCs will be presented when samples IDs are un-blinded. Conclusions: The sensitivity and specificity of CEER assay is sufficient to provide a single-cell-level target phosphorylation. This enables profiling of pathway proteins in samples with limited amount of tumor cells, allowing frequent monitoring of tumor profile during the course of the treatments. Determination of comprehensive pathway analysis is critical in selecting the most effective therapy options and overcoming potential drug resistance through treatment combination or sequencing.

Molecular pathway profiling as a tool for personalized medicine.

e21118 Background: Patients selected for targeted therapy based on single biomarker status often show inherent resistance or develop de novo resistance. Hence, comprehensive pathway profiling is critical for rational selection/combination of suitable targeted agents at base line and during the course of treatments. Here we report a comparative profiling of receptor tyrosine kinases (RTKs) and downstream signal proteins (HER1, HER2, HER3, cMET, IGF1R, PI3K, AKT, ERK and other pathway proteins) in 10 diverse tumor cell lines treated with a panel of therapeutic agents and show the correlation between pathway inhibition and proliferation block as well as insights for potential resistance mechanisms. Methods: The Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) is a multiplexed protein microarray platform requiring co-localization of two detector enzyme-conjugated-antibodies. Channeling events between two detector enzymes in proximity provides extremely high sensitivity. In this study, cell lysates were prepared from 10 cell lines treated with 6 compounds at various concentrations, and inhibition of target proteins as well as modulations of other signaling proteins were measured on the multiplexed CEER platform. Results: Profiling of pathway proteins determined the efficacy of inhibitors for target kinase and downstream signal protein modulations. The most dramatic suppression in AKT activity was observed with PI3K and AKT inhibitors and correlated with proliferation suppression. Inhibitors of RAF and MEK strongly reduced ERK activity, also blocking cell growth. On the other hand, inhibition of single RTKs often showed ineffective suppression of AKT and ERK activity. Treatment with single agents often showed adverse profile shifts through feedback loops or pathway cross-talk. Conclusions: As CEER is a suitable technology to evaluate modulations in multiple targeted pathways and associated signalling proteins from very small samples (such as FNA or CTCs), it is now possible to test pathway activity to select the most effective targeted agents. Clinical samples should be evaluated during the course of the therapy to monitor the efficacy and treatment regimens should be adjusted according to profile shifts.

Profiling signal transduction pathways in ascites tumor cells of gastric cancer.

e14583 Background: Malignant ascites is a manifestation of end stage events in a variety of cancers and associated with a poor prognosis with limited options for therapies. We evaluated the potential use of tumor cells isolated in ascites collected from gastric cancer (GCA) patients. Here we demonstrate successful profiling of signal transduction pathway proteins in isolated ascites-tumor cells (ATCs) and their modulations in response to drug treatments. Methods: The collaborative enzyme enhanced reactive-immunoassay (CEER) is a multiplexed protein microarray platform requiring co-localization of two detector enzyme-conjugated-antibodies. Once target proteins are captured on the microarray-surface, channeling events between two detector enzymes in proximity enable the profiling of the target proteins with high sensitivity. In this study, ATCs were isolated from ascites fluid obtained from advanced GCA patients and analyzed for HER1, HER2, HER3, cMET, cKIT, IGF-1R, PI3K, and other pathway proteins for their expression/activation and the degree of pathway modulation in response to targeted drug treatments. Results: Here, we report successful profiling of activation and expression of proteins in RTK pathway in ATCs of GCA patients. Varying levels of HER2 expression was detected in some patients and with significant levels of activation in subset of these patients. A complete in vitro ablation of phosphorylation of HER2 was observed when EpCAM positive ATCs were treated with HER2 targeted inhibitor in one GCA patient. Differential patterns of other RTKs profile and downstream pathway proteins were also observed. Conclusions: The ATCs from GCA are suitable resource for real-time profiling for the status of proteins in RTK and downstream proteins in signal transduction pathways. Monitoring the profile shift of pathway proteins over time, either treatment-related or due to the natural history of disease in GCA patients, may allow more effective selection and adjustment of targeted therapy for an individual patient. As CEER requires limited amounts of sample, real-time biopsy can be performed on tumor cells obtained from ascites, FNA or CTCs for therapy optimization (combination/sequencing) during the course of treatments.

Signatures of drug sensitivity in non-small cell lung cancer.

10602 Background: We profiled receptor tyrosine kinase pathway activation and key gene mutations in eight human lung tumor cell lines and 50 human lung tumor tissue samples to define molecular pathways which may help to evaluate the efficacy of targeted therapies. Methods: A panel of eight kinase inhibitors was used to determine whether blocking pathway activation affected the growth of the tumor cell lines. Clustering the primary tumor samples with the corresponding tumor cell lines based on their signatures was used to predict treatment options for the patients based on the response of the selected tumor cell lines to the panel of kinase inhibitors. Results: Subsets of patients were identified as candidates for treatment with inhibitors of EGFR, c-Met, IGF-1R, MEK, and PI3K. The EGFR pathway in EGFR mutant cell lines HCC827, H1975 and H1650 were found to be highly activated and sensitive to inhibitors blocking EGFR signaling. Primary tumors were identified with similar profiles, and we predict that those patients would be candidates for anti-EGFR therapies. H1993 is a cell line containing c-Met amplification and with c-Met and EGFR pathway activation and patients sharing this genotype may respond to c-Met inhibitor treatment. IGF-1R pathway activated H358 and A549 cells are sensitive to IGF-1R inhibition and patients sharing a similar profile with these cells may be candidates for this type of therapy. The downstream PI3K inhibitor, BEZ-235, effectively inhibited tumor cell growth in most of the cell lines tested, except the H1993 and H358 cells, while the MEK inhibitor PD-325901 was effective in blocking the growth of KRAS mutated cell line H1734 but not in the other KRAS mutated cell lines, H358, A549 and H460. Patients with a similar signature may benefit from treatment with a combination of two kinase inhibitors. Conclusions: Thus, by combining pathway biomarker information, mutational status and clinical features, we were able to cluster the patient tumor samples to the corresponding lung tumor cell lines and propose treatment options for the patients based on their signature overlap with the tumor cell lines.

A comprehensive profiling of colorectal cancer patients through multiplexed functional pathway signature and somatic mutation analysis for effective therapy selection.

e14103 Background: Colorectal cancer (CRC) is a leading cause of death in the developed nations. The development of effective forms of cytotoxic chemotherapy for colorectal cancer has been paralleled by the emergence of monoclonal antibodies against proteins that are thought to be important in the proliferation of malignant cells. Recent clinical studies showed that EGFR-targeting agent improved response rate when added to chemotherapy, however patients with KRAS mutations did not benefit from such treatment. Here we report a comprehensive profiling of pathwy signal proteins for their level of expression/activation and presence of somatic mutations in tumor tissues obtained from 136 CRC patients. Methods: The collaborative enzyme enhanced reactive immunoassay (CEER) is a multiplexed protein microarray platform requiring co-localization of two detector enzyme-conjugated-antibodies. Once target proteins are captured on the microarray-surface, channeling events between two detector enzymes in proximity enable the profiling of the target proteins with high sensitivity. In this study, tumor lysates were prepared from frozen tissues obtained from 136 CRC patients. In addition to a multiplexed analysis for panel of mutations in KRAS and BRAF genes, the level of expression and activation of HER1, HER2, HER3, cMET, cKIT, IGF-1R, PI3K, AKT, ERK and other pathway proteins were determined using CEER technology. Results: KRAS and BRAF mutations were found in ~40% (54/136) and ~5% (6/136) respectively in this cohort. The most frequent mutant alleles were G12D (14%) and G13D (13%). Varying levels of RTKs and associated activation patterns were observed. Levels of phosphorylated ERK and AKT correlated to expression/activation patterns of HER1, cMET, HER3 and prevalence of each biomarker along with pathway redundancy will be presented. Conclusions: A comprehensive profiling of multiplexed pathway/ somatic mutation analysis provides critical information needed to select the most effective targeted agents to overcome pathway redundancy /activating mutation related drug resistance through treatment combination or sequencing.

Selection of targeted agent(s) based on matching differential protein expression and mutational profile in NSCLC patients.

e21085 Background: Treatment of nonsmall-cell lung cancer (NSCLC) with tyrosine kinase inhibitors (TKIs) shows initial response, but most tumors develop acquired resistance to TKIs due to secondary resistance mutations (T790M) and amplification of other RTKs. Patients stratified for mono therapy treatment based on genotype alone is not sufficient as other kinases contribute to survival and necessitate inhibition of multiple kinases. Methods: The Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) is a multiplexed protein microarray platform requiring co-localization of two detector enzyme-conjugated-antibodies. CEER is capable of high detection sensitivity from FNA/CTC viable samples (23-35 gage needle). Lysates were prepared from frozen tissues obtained surgically from 71 Caucasian and 29 Asian patients with NSCLC. Expression and activation of EGFR, ErbB2, ErbB3, cMET, IGF1R, cKIT, Shc, PI3K, AKT, ERK, VEGFR2, Src, FAK, Stat5, JAK2, CrKL, and other pathway proteins were profiled. Genotyping on panel of mutations in KRAS, EGFR, and BRAF were performed on all samples. Results: We have observed differential expression patterns between Asian and Caucasian patients. KRAS is mutated in 30% of all the patients. In Asians, EGFR over expression is higher (27%) compared to the Caucasians (3%). Caucasian patients expressed high Her3 (58%), cMET (25%), and co expression of both cMET and HER3 (27%). VEGFR2 is highly expressed in 17% of all patients. Patients over expressing cMET, HER3, and HER2 will benefit from combination of pan-HER and cMET inhibitors. Those over expressing cMET and EGFR will benefit from cMET and EGFR inhibitors. Lastly, patients over expressing VEGFR2 and cMET will benefit from cMET and VEGFR inhibitors. A comprehensive differential biomarker prevalence for each NSCLC sub-populations for all tested RTKs and signaling proteins will be presented. Conclusions: Mono therapy and patient selection based on genotype is not an effective criteria for treatment options. Instead, a comprehensive pathway profile using FNA/CTC should guide selection of appropriate therapy (combined or sequenced), and shifts in pathway profile should be monitored for appropriate response.

Is There a Role for IGF1R and c-MET Pathways in Resistance to Cetuximab in Metastatic Colorectal Cancer?

The KRAS mutation is not responsible for all cases of resistance to anti-epidermal growth factor receptors (EGFRs) in metastatic colorectal cancer (mCRC), and new predictive and prognostic factors are actively being sought. We retrospectively evaluated the efficacy of a cetuximab-containing treatment in 73 patients with mCRC according to KRAS and BRAF mutational status as well as PTEN, c-MET, and insulin-like growth factor receptor (IGF1R) expression. Overall response rate (ORR), median progression-free survival (mPFS), and median overall survival (mOS) were significantly lower in patients with KRAS mutation than in patients with KRAS wild-type; among the population with KRAS wild-type, only 2 patients with BRAF mutations were found and neither of them achieved a response. No significant association was found between PTEN and clinical outcome. Compared with low/normal expression, c-MET overexpression significantly correlated with shorter mPFS and mOS: 3 vs. 5 months (P = .018) and 11 vs. 10 months (P = .037), respectively. In patients with high IGF1R expression, mOS was significantly longer than in those with low/normal expression (14 vs. 8 months; P = .015). KRAS mutation significantly correlates with a worse outcome in patients treated with cetuximab, whereas no definitive inference can be drawn about the role of BRAF mutation and PTEN loss of expression. Instead, c-MET overexpression might represent a negative prognostic factor in mCRC and may have a role in resistance to anti-EGFR therapy. Interestingly, IGF1R overexpression seems a favorable prognostic factor in mCRC.

Diosgenin Suppresses Hepatocyte Growth Factor (HGF)-Induced Epithelial–Mesenchymal Transition by Down-regulation of Mdm2 and Vimentin

Substantial activation of the hepatocyte growth factor (HGF)/c-Met pathway leads to cancer cell scattering and invasion and has been observed in several types of cancers, including prostate and colorectal cancers. The phosphorylation cascade downstream of HGF, particularly PI3K/Akt signaling, regulates epithelial-to-mesenchymal transition (EMT). How this signaling governs EMT and whether specific kinases respond to particular EMT effectors remain unclear. This study found specific increases in Mdm2 and vimentin rather than the coregulation of an array of EMT marker proteins in response to HGF-induced EMT in DU145 prostate cancer cells. Importantly, it was further found that diosgenin abrogated HGF-induced DU145 cell scattering and invasion. Moreover, diosgenin effectively inhibited the HGF-induced increases in Mdm2 and vimentin by down-regulating phosphorylated Akt and mTOR. In summary, the results suggest that diosgenin may be a potential compound for use in prostate cancer therapy to target the major HGF-induced EMT pathway.

Abstract 4126: Optimization of clinical sample collection for detection of c-Met phosphorylation

Abstract The receptor tyrosine kinase c-Met is being investigated as a target for cancer chemotherapy. c-Met and its ligand HGF (hepatocyte growth factor) play important roles in cell growth, survival and migration. Dysregulation of the HGF-c-Met pathway (overexpression, constitutive activation, gene amplification, ligand-dependent activation, mutation or epigenetic mechanisms) can lead to oncogenic changes, and has been described in multiple types of cancer including lung, gastric, and colorectal cancer. For cancer therapies targeting the HGF-c-Met signaling pathway, elevated levels of c-Met expression and increased phosphorylation of c-Met have been explored as both the predictive and PD biomarkers. However, detection of c-Met phosphorylation by IHC in human clinical cancer samples has proven to be extremely challenging. We have conducted a study aimed at finding a way to detect phosphorylation of c-Met in clinical samples. In the first attempt, we have acquired a set of 30 gastric cancer samples which were fixed in formalin in the presence of phosphatase inhibitors (PhosphoGuardTM fixation kit). While this approach has helped us to improve detection of several other phospho-proteins in tissues, it did not help with the detection of phospho-c-Met. We then hypothesized that this is a very labile phosphorylation which is rapidly lost during the cold ischemia time occurring between the surgical excision of the tissue and its placement in formalin for fixation (which could last 30-60 minutes or longer in the operating room). Studies with xenograft tissues showed that a 15 minute delay in fixation resulted in approximately 50% decrease in c-Met phosphorylation, while a 30 minute delay resulted in almost complete disappearance of phospho-c-Met. We therefore acquired a set of colorectal cancer samples which were placed into formalin within 5 minutes of surgical excision. Using these rapidly fixed samples, we were able to detect strong phosphorylation of c-Met in a small number of samples. The staining pattern for phospho-c-Met correlated with that of phospho-ERK (a downstream signaling molecule in the c-Met pathway) suggesting that the relatively low occurrence of c-Met phosphorylation may reflect the status of activation of this pathway in vivo at a given time point. This work suggests that rapid fixation of human tissues facilitates detection of phospho-c-met and its use as a PD and predictive biomarker in clinical trials. 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 4126. doi:10.1158/1538-7445.AM2011-4126

Abstract 1446: Dissecting the role of PTEN/Akts in metastatic melanoma

Abstract Melanoma is an aggressive human cancer whose incidence is increasing more rapidly than any other cancer in the United States. It is characterized by an extremely high potential to develop metastases and poor response to current therapy. Although the genetic studies indicated that melanoma genesis related with molecular lesions in c-MET, INK4a, Ras/Raf, p53 and PTEN pathways, the molecular mechanism inciting metastatic melanoma are poorly understood. PTEN is a major tumor suppressor in humans. To study the role of PTEN in progression of human melanoma, we first used tissue microarrays to examine PTEN expression in 30 lymph node melanoma metastases, 30 primary melanomas, and 9 normal skin tissues. Our results showed that PTEN loss was significantly correlated with progression to the metastatic state. Moreover, analysis of various human melanoma cell lines demonstrated that PTEN expression was related to metastatic behavior, supporting the notion that PTEN is involved in the metastatic regulation of melanoma. PTEN has phosphatase activity that can act upon both polypeptide and phosphoinositide substrates. The PTEN lipid phosphatase is a ubiquitous regulator of PI3K signaling pathway. The loss of expression or mutational inactivation of PTEN protein enhances the phosphorylation of PKB/AKT and thus influences cell survival signaling. Interestingly, a PTEN mutant, which loses lipid but not protein phosphatase activity, lacks the ability to induce either G1 arrest or apoptosis, but nonetheless retains the ability to inhibit cell spreading and motility, suggesting that PTEN protein phosphatase activity is important for these behaviors. To further dissect the role of this PTEN activity in metastasis, we have established a model system containing exploiting mutant PTEN proteins with various phosphatase activities. We found that wild type PTEN can inhibit cell motility and invasiveness, while PTEN mutants cannot. Notably, wild type PTEN was able to reduce the metastatic potential of melanoma cells, whereas phosphatase deficient PTEN could promote the melanoma cell metastatic behavior. We found that PTEN mediates metastatic activity through the AKT pathway. We further determined that Akt 3 was key mediators of metastatic potential in melanoma cells in vivo. Akt3 could also rescue PTEN-mediated inhibition of metastasis. PTEN loss and Akt3 overexpression functioned synergistically to promote metastasis. Our data demonstrate that metastatic behavior in melanoma cells is dependent on the status of PTEN protein phosphatase activity and downstream Akt3, and suggest that AKT/PTEN signaling should be considered as a therapeutic target in metastatic melanoma 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 1446. doi:10.1158/1538-7445.AM2011-1446

Abstract 4045: c-Met inhibition synergistically enhances death receptor-induced apoptosis via upregulation of DR5 in papillary thyroid cancer

Abstract The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are overexpressed and/or activated in variety of human malignancies. Previously we have shown that c-Met was overexpressed in Middle Eastern Papillary Thyroid Cancer (PTC) and significantly associated with poor prognoses. In this study, we investigated the functional link between c-Met/AKT signaling pathway and Dearth Receptor 5 (DR5) in a large cohort of PTC patient samples, a panel of PTC cell lines, and xenografts in a NUDE mouse model by using tissue microarray, MTT, flow cytometry and DNA fragmentation assays and western blotting. Immunohistochemistry staining showed a significant association of p-Met with DR5 in PTC patient samples. The PHA665752, an inhibitor of c-met tyrosine kinase, inhibited cell proliferation and induced apoptosis in a dose dependent manner in all cell lines studied. PHA665752 treatment resulted in dephosphorylation of c-Met, AKT and its downstream effector molecules FOXO1, GSK-3 and pBad. Additionally, treatment of PTC cell lines with PHA665752 resulted in activation of caspases-9 and 3, cleavage PARP and apoptosis. Moreover, HGF, a ligand of c-Met, stimulated the growth of all PTC cell lines via activation of c-MET and AKT. In addition, pre-treatment of PTC cell line with PHA665752 abrogated the HGF stimulated growth and activation of c-met and AKT further suggesting the critical role of c-met and AKT pathway in PTC pathogenesis. Furthermore, c-Met inhibitor PHA665752 up-regulated DR5 expression in PTC cell line cells, and synergistically potentiated death receptor-induced apoptosis with TRAIL. Finally, co-treatment with PHA665752 and TRAIL causes more pronounced effect on PTC xenograft tumor growth in NUDE mice. Our data suggest that c-Met/HGF pathway may be a potential target for therapeutic intervention for treatment of PTC with c-Met inhibitor alone or in combination of conventionally therapeutic agents. 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 4045. doi:10.1158/1538-7445.AM2011-4045

Abstract LB-197: Hepatocyte Growth Factor Activator (HGFA) Inhibitors of c-MET/RON Kinase Signaling

Abstract Continuous advances have been made in the treatment of cancer however 70% of the patients still die of cancer every year strongly correlated to tumor metastasis. The treatment options available at present are rarely able to effectively treat metastatic cancer. Targeting hepatocyte growth factor (HGF) signaling with receptor tyrosine kinase (RTK) inhibitors is a well-developed strategy for metastatic cancer therapy however no studies have explored targets upstream of kinase activation. One such target is hepatocyte growth factor activator (HGFA) which is upregulated in a large number of cancer cell lines and patient samples. HGFA is a trypsin-like serine protease which stimulates both the c-MET and RON signaling pathways by proteolysis of the inactive single-chain zymogen growth factor ligand precursors to activated two-chain HGF and macrophage stimulating protein (MSP) respectively. Increased activation of these pathways has been shown to trigger a number of downstream signaling events necessary for metastasis including cell migration, invasion, proliferation, and angiogenesis. The present study was undertaken to discover inhibitors of HGFA based on the naturally occurring bicyclic peptide, sunflower trypsin inhibitor (SFTI). Using a combination of solution and solid-phase peptide methods, we have synthesized the natural product SFTI and analogs thereof. The compounds were tested for HGFA activity in a competitive fluorescence resonance energy transfer (FRET) assay of proteolytic cleavage by using a fluorogenic peptide substrate. We have identified several inhibitors of HGFA with low μM activity in the FRET assay. We have also demonstrated one inhibitor effectively prevented the conversion of native pro-HGF to HGF as determined by western blotting in a biochemical assay of HGFA activity. This ground breaking discovery of novel HGFA inhibitors will enable future studies on the role of HGFA and other serine proteases in the regulation of growth factors, cell signaling, and cancer metastasis. Inhibitors of HGFA offer an innovative therapeutic strategy upstream of kinase activation and signaling for the treatment of metastatic cancer. 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-197. doi:10.1158/1538-7445.AM2011-LB-197

Abstract 3309: Inhibiting c-Met activation depletes glioblastomas of stem-like tumor-initiating cells

Abstract Hepatocyte Growth Factor (HGF) and its tyrosine kinase receptor c-Met are overexpressed in glioblastomas (GBM) and expression levels correlate with poor prognosis. Previously, we showed that anti-HGF therapy using the monoclonal antibody L2G7 inhibits the progression of orthotopic xenografts derived from HGF+/c-Met+ GBM cell lines by inducing apoptosis, and inhibiting tumor cell proliferation and angiogenesis. Increasingly, there is an interest in cancer therapies targeting the stem-like tumor-initiating pool of cells within GBMs (GBM-TICs), as this subset of cells may be particularly resistant to conventional therapies and associated with tumor recurrence. This study examined the effects of c-Met pathway inhibition on the pool of stem-like cells in GBM xenografts. We used the HGF/c-Met dependant U87 cell line as a GBM model system. Wild-type U87 cells were passaged subcutaneously (s.c.) in nude mice for 2-3 weeks. Tumors (>200mm3) were removed and primary tumor-derived cells were cultured in vitro in serum-free stem cell medium containing EGF and FGF to study neurosphere formation, a marker of stem-like cells. We confirmed the expression of stem/progenitor cell markers Sox2, CD133, Nestin, and Musashi in neurosphere-forming cells (U87-NS). Under conditions of forced differentiation, U87-NS cells revealed multipotent capacities and expressed lineage specific differentiation markers such as Neurofilament, BIII-tubulin, O1, and GFAP. Animals bearing intracranial or s.c. xenografts were treated with +/- L2G7 prior to tumor removal and isolation of tumor cells. Overall, L2G7 treated tumors were substantially smaller than control in both intracranial (p<0.0001) and s.c. (p<0.0001) sites. Equal numbers of primary cells derived from control or L2G7 treated s.c. xenografts were either re-transplanted intracranially (10,000 and 1,000 cells per brain), or cultured in stem cell medium containing EGF and FGF to determine the effect of L2G7 on the pool of tumor-initiating and neurosphere-forming stem-like cells, respectively. Cells derived from L2G7 treated xenografts had smaller tumors overall, and demonstrated decreased tumor initiation (50% fewer intracranial tumors at 1000 cells/animal) as compared to the control cells. Similarly, intracranial xenografts were derived from U87-NS cells and treated with +/- L2G7. Cells isolated from L2G7 treated intracranial xenografts were less able to form neurospheres (50% inhibition, p<0.05) than cells derived from control xenografts. Orthotopic and s.c. derived tumors are currently being tested to determine the effects of c-Met inhibition on the expression of stem cell markers in tumor xenografts. In conclusion, we show that treating HGF/c-Met dependant U87 xenografts with the ligand-sequestering antibody L2G7 appears to preferentially deplete tumors of their neurosphere-forming tumor-initiating cells. 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 3309. doi:10.1158/1538-7445.AM2011-3309

C-met as a therapeutic target using ARQ 197 in renal cell carcinoma.

360 Background: c-Met is a receptor tyrosine kinase involved in normal cellular growth and vascular development. It has also been identified as a proto-oncogene. Activating c-Met mutations and indirect c-Met activation via von Hippel-Lindau gene silencing have been demonstrated in renal cell carcinoma (RCC) for papillary and clear cell histologic subtypes, respectively. We studied c-Met expression in a large cohort of RCC tumors and investigated in vitro activity of the new selective c-Met inhibitor, ARQ 197. Methods: Tissue microarrays were performed to analyze c-Met protein expression in situ in 317 RCC tumor specimens using Automated Quantitative Analysis (AQUA). Western blots were performed for c-Met, phospho-c-Met and downstream pathway markers in 6 RCC cell lines (A498, ACHN, Caki-1, Caki-2, 769P, and 786-O). In vitro activity of ARQ 197 was studied by cell viability and clonogenic assays, and by changes in c-Met and down-stream mediators. Results: c-Met expression is higher in RCC than adjacent normal renal tissue (p < 0.0001) and is expressed in all subtypes of RCC, including clear cell carcinoma; expression is highest in papillary and sarcomatoid subtypes. Greater c-Met expression correlates with higher tumor grade (p = 0.0019) and clinical stage (p = 0.0208), and is an independent predictor of poor overall survival (Multivariate Cox LR 5.7, p = 0.017). c-Met expression correlates to decreased survival in the clear cell subset as well. c-Met protein is activated (phosphorylated) in all 6 RCC cells. ARQ 197 inhibits cell growth in RCC cell lines at nanomolar concentrations (IC50 350-680 nM) and inhibits colony formation at 500 nM. ARQ 197 decreases phosphorylation of c-Met in time- and dose-dependent patterns with dose-dependent decreases in phospho-ERK. Conclusions: c-Met expression is an independent predictor of survival in RCC, including clear cell subtype, suggesting that it is a potential therapeutic target worthy of further investigation. ARQ 197 effectively inhibits proliferation of clear cell carcinoma cell lines. Further preclinical studies are planned to determine mechanism of cellular arrest and apoptosis and markers of drug sensitivity and resistance. [Table: see text]

Involvement of c-Met- and Phosphatidylinositol 3-Kinase Dependent Pathways in Arsenite-Induced Downregulation of Catalase in Hepatoma Cells

Catalase protects cells from reactive oxygen species-induced damage by catalyzing the breakdown of hydrogen peroxide to oxygen and water. Arsenite decreases catalase activity; it activates phosphatidylinositol 3-kinase (PI3K) and its key downstream effector Akt in a variety of cells. The PI3K pathway is known to inhibit catalase expression. c-Met, an upstream regulator of PI3K and Akt, is also involved in the regulation of catalase expression. To examine the involvement of c-Met and PI3K pathways in the arsenite-induced downregulation of catalase, catalase mRNA and protein expression were analyzed in the human hepatoma cell line HepG2 treated with arsenite and either an inhibitor of c-Met (PHA665752 (PHA)) or of PI3K (LY294002 (LY)). Arsenite treatment markedly activated Akt and decreased the levels of both catalase mRNA and protein. Both PHA and LY attenuated arsenite-induced activation of Akt. PHA and LY treatment also prevented the inhibitory effect of arsenite on catalase protein expression but did not affect the level of catalase mRNA. These findings suggest that arsenite-induced inhibition of catalase expression is regulated at the mRNA and post-transcriptional levels in HepG2 cells, and that the post-transcriptional regulation is mediated via c-Met- and PI3K-dependent mechanisms.