Levels Of Total MET Research Articles

ADOLESAN SPORCULARIN ERGOJENİK DESTEK KULLANIMINDA, FİZİKSEL VE MENTAL YORGUNLUK DÜZEYİNİN YORDAYICILIĞI

Being involved in the developmental period of adolescent athletes may cause more energy and fatigue levels. At this point, the correct and appropriate use of ergogenic supports can contribute to athletes. The study aims to reveal the predictors of fatigue and physical activity levels in adolescent athletes' use of ergogenic support. 171 (female: 98, male: 73) licensed athletes from the Provincial Directorate of Youth and Sports participated in the research voluntarily. Demographic information questionnaire, International Physical Activity Questionnaire-short form (IPAQ-SF), and Chalder Fatigue Scale were used in the research. The data collected in the study were analyzed in the Jamovi (2.0.0) statistical program at a 95% confidence interval and 0.05 significance level. In the analysis of the data, frequency (N), mean (x̄), standard deviation (ss), percentage (%), minimum (Min.), and maximum (Max.) values, Pearson's correlation and binomial logistic regression analysis were used. According to the findings of the study, most of athletes do not prefer to use ergogenic support. Those who use ergogenic support mostly use sports drinks, fish oil, and protein powder. Fatigue and total MET levels do not predict the use of ergogenic support by athletes. As a result, it can be said that adolescent athletes do not prefer to use ergogenic support and although their total MET scores are high, their fatigue levels are at a normal level.

Measuring phospho-MET by multiplex immunofluorescence to aid in selection of patients with MET activation in tumors.

3131 Background: Currently, patient selection criteria for clinical testing of MET inhibitors are limited. Robust studies selecting patients based on MET protein expression, MET gene amplification, or mutations have not met their efficacy goals. Development of microscopy-based assays to quantify levels of phospho-MET (pMET) in tumors has been hampered by poor antibody specificity. Here, we present the development and validation of a robust, highly specific multiplex immunofluorescence assay (IFA) that measures pY1235-MET and total MET in tumor tissue. Methods: This assay utilizes antibodies to pY1235-MET (NCI-23111), total MET (D1C2), and plasma membrane (PM) marker Na+/K+-ATPase, each conjugated to a different Alexa Fluor dye. We used tumor tissue from crizotinib-treated SNU5 xenograft models to demonstrate pY1235-MET assay fitness-for-purpose and cross-platform assay concordance with our validated pMET ELISA. In addition, this IFA was validated by phospho-peptide competition using custom tissue microarrays (TMA) derived from patients with colorectal carcinoma (CRC). Finally, we developed quantitative algorithms to assess pY1235 MET levels in the plasma membrane and nucleus using PM and DAPI masks, respectively. Patient-derived xenograft models (PDX) were obtained from NCI’s Patient-Derived Models Repository (www.pdmr.cancer.gov). Results: The prevalence of high pY1235-MET expression in CRC patient specimens was greater than expected; of the 64 TMA cores evaluated, 29 (45%) and 19 (29%) had high pY1235-MET and total MET levels, respectively, as defined by mean marker area of ≥ 30 μm2/cell. To address the potential utility of pY1235-MET as a diagnostic biomarker, we examined 15 CRC PDX models by pMET ELISA and IFA. Two CRC tumor models were positive for pY1235-MET expression in both assays. The pY1235-MET IFA results and gene expression data were used to select PDX models for ongoing preclinical trials of potent MET inhibitors. Conclusions: This novel pY1235-MET IFA will enable clinicians to address the utility of activated MET as a biomarker for patient selection and/or prediction of response in clinical trials of MET inhibitors. Funded by NCI Contract No. HHSN261200800001E.

Abstract 3063: Predicting efficacy of c-Met targeting therapy in autocrine and paracrine tumor cell lines

Abstract Tumor cells frequently harbor abnormalities in signaling pathways, leading to increased migration, invasion, survival, angiogenesis, and proliferation. C-Met is one such commonly aberrant pathway. Because of its effects on tumor cells, c-Met has been a target of interest for anti-metastatic therapies. Clinical trials of c-Met inhibition select patients based on total Met expression, or amplification of the Met gene. However, c-Met inhibitors target the activated pathway. We therefore predict that phospho-Met will be a better marker for patient sensitivity to, and hence selection for, c-Met inhibitor trials. C-Met is activated by the endogenous ligand Hepatocyte Growth Factor (HGF). In order to mimic the paracrine signaling during in vitro experiments, many studies add exogenous HGF at concentrations of 25-50 ng/mL. However, these concentrations may not be representative of in vivo conditions. HGF serum levels of healthy humans is 0.4 ng/mL. While HGF levels do rise in cancer patients, it is unusual to see serum levels higher than 1 ng/mL. Conditioned media from several cell lines (MDA-MB-231, PC-3, DU145, LNCaP, OS156, OS521, U87, U118, RKO, KHT, SCC7, and RIF) was collected to analyze for secretion of HGF in conditioned media. These cells were then examined for basal phosphorylation of Met. The cell lines that secreted HGF also had higher levels of phospho-Met in comparison to non-HGF-secreting cell lines. Importantly, levels of total Met did not predict for levels of phospho-Met. In migration and invasion assays, the HGF-secreting cell line KHT was found to be sensitive to the c-Met inhibitor BMS-777607.The non-HGF-secreting cell line DU145 only became sensitive to the inhibitor when high, non-physiologic exogenous levels of HGF (25-50 ng/mL) were added.These results suggest that autocrine activation of the c-Met pathway may be a factor in predicting sensitivity to c-Met inhibition. This may indicate the existence of a subset of patients most likely respond to c-Met inhibition therapy. Furthermore, consideration of exogenous HGF concentration should be considered when conducting preclinical trials of c-Met inhibitors, as addition of exogenous HGF may change the apparent efficacy of the inhibitor. Citation Format: Veronica S. Hughes, Dietmar W. Siemann. Predicting efficacy of c-Met targeting therapy in autocrine and paracrine tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3063. doi:10.1158/1538-7445.AM2017-3063

Abstract 5391: Preclinical evaluation of AMG 337, a highly selective small molecule MET inhibitor, in hepatocellular carcinoma

Abstract Purpose: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Aberrant hepatocyte growth factor / MET signaling has been implicated in hepatocarcinogenesis, suggesting that MET may serve as an attractive therapeutic target in HCC. The purpose of this study was to investigate the anti-tumor activity in vitro and in vivo of AMG 337, a potent and highly selective small molecule MET kinase inhibitor, in preclinical models of HCC. Experimental Design: The anti-proliferative activity of AMG 337 was evaluated across a panel of 40 HCC cell lines in a 72-hour viability assay. Daily oral administration of AMG 337 at doses of 3, 10 and 30 mg/kg was used to evaluate the in vivo anti-tumor activity of AMG 337 in two patient-derived mouse xenograft (PDX) models of HCC, LI0612 and LI1078. Effects on MET signaling were assessed by western blot analysis of downstream effector proteins including p-GAB1, p-AKT and p-ERK. MET protein levels and gene copy number for both PDX models and a subset of HCC cell lines were assessed by immunohistochemistry (IHC; Dako) and fluorescence in situ hybridization (FISH; Dako), respectively. High MET expression was defined as the presence of any tumor cells with membrane staining at 3+ intensity. MET amplification was defined as MET:CEN-7 ratio >2.0 or MET SNP array log2 copy number >2.5. Results: AMG 337 displayed potent anti-proliferative activity in two of 40 HCC cell lines (MHCC97H and HCCLM3) (IC50 0.015 μM and 0.025 μM, respectively); both cell lines were amplified for MET (MET/CEN-7 >2.0) and showed high MET expression (3+ IHC). AMG 337 potently inhibited p-MET in all cell lines with detectable levels of total MET. However, dose-dependent inhibition of p-GAB1, p-AKT and p-ERK was limited to those cell lines that were sensitive to AMG 337 in viability assays. AMG 337 significantly inhibited tumor growth at all doses tested (p < 0.001) in the MET-amplified and MET-high expressing HCC PDX model LI0612. However, AMG 337 had no effect on tumor growth in the non-MET-amplified and MET-low expressing HCC PDX model LI1078. Analysis of The Cancer Genome Atlas (TCGA) data identified four of 164 (2.4%) HCC tumor samples with MET amplification (https://tcga-data.nci.nih.gov/tcga), representing a patient population with potential to derive clinical benefit from AMG 337. Conclusions: These preclinical studies show that the efficacy of AMG 337 in HCC was highly associated with MET amplification. AMG 337 represents a promising, novel clinical therapeutic strategy for targeting HCC with a dependence on the MET pathway. Citation Format: Zhiqiang Du, Sean Caenepeel, Yuqing Shen, Karen Rex, Yanni Zhang, En-Tzu Tang, Ouhong Wang, Wenge Zhong, Hui Zhou, Jacqueline Huang, Eric Huang, Liaoyuan Hu, Angela Coxon, Mingqiang Zhang. Preclinical evaluation of AMG 337, a highly selective small molecule MET inhibitor, in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5391. doi:10.1158/1538-7445.AM2015-5391

Abstract 2162: Inhibition of pancreatic cancer cell and tumor growth using MET inhibitors in combination with inhibitors of downstream signaling mediators, MEK and PI3K.

Abstract Oncogenic signaling through the receptor tyrosine kinase (RTK) MET has been implicated in driving tumor cell invasive growth. High expression of MET or its cognate ligand, the hepatocyte growth factor (HGF) have been associated with poor prognosis in multiple cancer types, including pancreatic cancer. MET has also been implicated in co-operative signaling with other RTKs, including those in the HER family. We examined the expression of MET and HGF in a panel of pancreatic cancer cell lines and examined the impact of MET activation on cell signaling. While most lines were uniformly positive for MET protein, 6/12 lines not co-expressing HGF and MET, and all sub-lines of the autocrine KP4 line, demonstrated constitutive phosphorylation of MET (p-MET) in normal growth conditions. All lines were positive for epidermal growth factor receptor (EGFR) protein and had basal levels of phosphorylated-EGFR (p-EGFR) with evidence of downstream activity in the MAPK and PI3K signaling pathways. Treatment of some lines expressing constitutive p-MET with the small molecule MET inhibitor GDC-0712 resulted in suppression of both p-MET and p-EGFR. However, treatment of these same lines with erlotinib resulted in no inhibition of p-EGFR or p-MET, strongly suggesting that MET activates EGFR phosphorylation. Co-suppression of MET with GDC-0712 or onartuzumab (MetMAb) and erlotinib resulted in greater inhibition than either agent alone. We assessed whether anti-tumor activity could be further improved by targeting downstream molecules of MET or EGFR signaling, including modulators of the Ras or PI3K pathways, in combination with MET inhibitors. Improved cell inhibition was seen with combinations of MET inhibitors and the MEK inhibitor GDC-0973 compared with either agent alone, with greater activity than combinations of MET and EGFR inhibitors. Analysis of the combination of MET inhibitors with GDC-0973 revealed that Ras signaling contributed to the stability of total MET levels, resulting in lower total p-MET burden when MEK was inhibited. The combination of MET and MEK inhibitors produced greater suppression of downstream signaling through ERK and the ribosomal S6 kinase, which resulted in better induction of the pro-apoptotic mediator Bim and induction of cleaved caspase 3. These effects were not seen with combinations of MET and PI3K inhibitors in vitro. Lastly, MET inhibitors were evaluated in combination with MEK, PI3K or EGFR inhibitors in pancreatic xenograft tumor models. The combination of MET and MEK inhibitors resulted in additive anti-tumor activity whereas there was little to no apparent increase in activity with combinations of MET and PI3K or EGFR inhibitors. These data provide evidence for an interplay between MET and mutant Ras signal transduction and provide a rationale for combination of MET and MEK inhibitors in the treatment of pancreatic cancer. Citation Format: Nai-Ying Yang, Jing Peng, Janet Tien, Mark Merchant. Inhibition of pancreatic cancer cell and tumor growth using MET inhibitors in combination with inhibitors of downstream signaling mediators, MEK and PI3K. [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 2162. doi:10.1158/1538-7445.AM2013-2162

Abstract 1088: γ-Tocotrienol inhibits HGF-dependent mitogenesis and Met activation in highly malignant mammary tumor cells

Abstract Met is a receptor tyrosine kinase that plays a role in regulating numerous biological functions including mitogenesis, motogenesis, migration, invasion, and eventual metastasis. Met is activated by its natural ligand the hepatocyte growth factor (HGF). It is now well-established that aberrant Met signaling is associated with aggressive cancer phenotypes characterized by highly invasive and metastatic growth. γ-Tocotrienol, a member of the vitamin E family of compounds, displays potent antiproliferative and apoptotic activity against tumor cells. Previous studies have shown that γ-tocotrienol inhibits EGF-dependent growth of +SA murine mammary epithelial cells by suppressing ErbB3 receptor activity and subsequent reduction in the PI3K/Akt signaling. Initial studies have shown that combined treatment with relatively low doses of γ-tocotrienol and the tyrosine kinase inhibitors erlotinib or gefitinib significantly inhibited +SA mammary cell growth. Therefore, the goal of the present study was to investigate the effects of γ-tocotrienol treatment on the Met expression and activation in +SA mammary epithelial tumor cells and to evaluate the effect of combination treatment of γ-tocotrienol and the novel Met inhibitor SU11274. In these experiments, neoplastic +SA mammary epithelial cells were maintained in serum-free defined media containing 10 ng/mL of HGF as the mitogen. Cell culturing studies measuring cell viability were determined by the colorimetric MTT assay, whereas protein expression was determined by Western blotting. Immunofluorescent staining was used to determine treatment effects on Met receptors level and activation. Results showed that treatment with γ-tocotrienol or SU11274, significantly inhibited HGF-dependent proliferation of neoplastic +SA cells in a dose-responsive manner. Western blot analysis showed that treatment with a growth inhibitory dose of γ-tocotrienol (4 μM) caused a large relative reduction in total Met receptor levels, and a corresponding reduction in HGF-induced Met autophosphorylation in +SA mammary tumor cells. In contrast, similar treatment with growth inhibiting doses of SU11274 (5.5 μM) inhibited HGF-induced Met autophosphorylation, but had no effect on total Met levels. Combined treatment with subeffective doses of γ-tocotrienol (2 μM) and SU11274 (3 μM) resulted in a significant inhibition of +SA cell growth as compared to treatment of individual agents alone, and this effect was found to be cytostatic, not cytotoxic. These findings show for the first time the inhibitory effects of γ-tocotrienol on Met expression and activation, and strongly suggest that γ-tocotrienol treatment may provide significant health benefit in the prevention and/or treatment of breast cancer in women with deregulated HGF/Met signaling. This study was supported by a grant from First Tech International Ltd., and the Malaysian Palm Oil Council. 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 1088. doi:1538-7445.AM2012-1088

Abstract 3672: Preclinical assessment of MET modulation by a VEGFR inhibitor/MET inhibitor combination that shows additive antitumor efficacy

Abstract Background: Suppression of VEGFR signaling promotes activation of the MET signaling pathway, which may compensate for the VEGFR blockade, potentially leading to cellular invasion and metastasis. These findings have led to the hypothesis that VEGFR inhibitors will be more effective when combined with MET inhibitors in the clinic. This hypothesis is currently being tested in a clinical trial of Pazopanib with Tivantinib (ARQ197) at the NCI. We have replicated this trial in a xenograft model to evaluate the pharmacodynamic (PD) response of MET and HIF-1α using newly developed and validated quantitative assays and relate the biomarker response to anti-tumor efficacy. Methods: Ectopic SNU5 (human gastric cancer cell line) xenografts were staged to 200 mm3 in athymic nude mice and treated for 8 days with vehicle, Pazopanib (100 mg/kg/day, QD), Tivantinib (200 mg/kg/day, QD), and combinations of Pazopanib (QD) with two dose levels of Tivantinib (200 mg/kg/day QD and 400 mg/kg/day BID). Trucut needle biopsies (18Ga) were collected at 4 hours and 24 hours after Tivantinib administration on Day 8 and processed according to procedures developed to minimize analyte degradation. Tumor lysates were evaluated for MET, pY1235MET, pY1356MET, and HIF-1α using ELISA assays. Tumor volumes were measured at baseline and on Day 8. Results: PD-biomarker analysis in biopsy specimens revealed that treatment with Tivantinib alone did not significantly change MET, pY1235MET, and pY1356MET levels. Pazopanib treatment did not significantly increase total MET levels, but significantly increased pY1235MET and pY1356 MET at 4 hours after the Day 8 dose. The Pazopanib-induced activation of pY1235MET and pY1356MET was prevented by Tivantinib (p<0.05) in combination (BID schedule). Analysis of intratumoral levels of HIF-1α is currently underway. Compared to the vehicle treated group, single agent Tivantinib and Pazopanib inhibited tumor growth measured on Day 8 by 19% (p<0.05) and 20% (p<0.05), respectively, whereas the combination of Pazopanib and Tivantinib achieved 38-46% inhibition of tumor growth (p<0.05). The combination regimen was more effective when Tivantinib was administered BID than QD. Conclusions: The combination of Pazopanib with Tivantinib exhibited additive anti-tumor activity that was greater than either agent alone. Enhanced efficacy of combination therapy was associated with Tivantinib block of Pazopanib-induced activation of MET. These results confirm the hypothesis underlying clinical trials of combinations of VEGFR with MET inhibitors. The study is being expanded with additional xenograft models and treatment schedules to confirm and further examine the generality of these findings. Funded by NCI Contract #HHSN261200800001E. 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 3672. doi:1538-7445.AM2012-3672

448 CHARACTERIZATION OF THE AKT-MTOR PATHWAY IN TFE3-FUSION RENAL CELL CANCERS AND IMPLICATIONS FOR TARGETED THERAPY

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2012448 CHARACTERIZATION OF THE AKT-MTOR PATHWAY IN TFE3-FUSION RENAL CELL CANCERS AND IMPLICATIONS FOR TARGETED THERAPY Eric Kauffman, Gopal Gupta, Fabiola Cecchi, Kristen Raffensperger, W. Marston Linehan, Donald P. Bottaro, and Ramaprasad Srinivasan Eric KauffmanEric Kauffman Bethesda, MD More articles by this author , Gopal GuptaGopal Gupta Bethesda, MD More articles by this author , Fabiola CecchiFabiola Cecchi Bethesda, MD More articles by this author , Kristen RaffenspergerKristen Raffensperger Bethesda, MD More articles by this author , W. Marston LinehanW. Marston Linehan Bethesda, MD More articles by this author , Donald P. BottaroDonald P. Bottaro Bethesda, MD More articles by this author , and Ramaprasad SrinivasanRamaprasad Srinivasan Bethesda, MD More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.515AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Xp11 chromosomal translocations causing gene fusions of the TFE3 transcription factor gene may be responsible for up to 5% of all renal cell carcinomas (RCC). TFE3-fusion cancers (TfRCC) represent a high risk RCC subtype, with common extrarenal involvement, yet key signaling pathways for targeted therapy in the metastatic or adjuvant/neoadjuvant setting are yet to be identified. The PI3K-Akt-mTOR pathway is frequently upregulated in clear cell RCC (CcRCC) by various mechanisms including upstream MET activation or PTEN inactivation. Here we investigate the role of MET and PI3K-Akt-MTOR targeting in TfRCC. METHODS Five TfRCC cell lines established at the National Cancer Institute were studied in comparison to a large cell line panel derived from CcRCC patients. Immunoblot and Mesoscale Discovery (MSD) electrochemiluminescence assays were performed to assess protein levels and activation within the PI3K-Akt-mTOR and MET pathways. TfRCC cell growth was measured in vitro by MTT assay in response to the PI3K inhibitor LY294002, mTOR inhibitor sirolimus and MET inhibitors PHA665752 and PF02341066. RESULTS Levels of phosphorylated Akt-Thr308 or -Ser473 and the phosphorylated Akt kinase target, Glycogen Synthase Kinase 3beta, were equal or higher in all 5 TfRCC cell lines compared to CcRCC cell lines, with the exception of PTEN-null CcRCC lines which had the highest levels. In 4 of 5 TfRCC lines, increased levels of phosphorylated mTOR-Ser2448 and/or -Ser2481 were seen compared to CcRCC lines. Activation of the mTORC1 complex, as measured by phospho-S6 levels, was comparable between TfRCC and CcRCC lines, as were levels of total MET. In contrast, phosphorylated MET and endogenous MET ligand (HGF) were undetectable in TfRCC lines, despite variable expression in CcRCC lines. LY294002 inhibition of Akt activation dramatically reduced proliferation of all TfRCC cell lines, with growth-IC50s of 10-20 μM, and mTOR inhibition with sirolimus yielded a >30% growth reduction in 4 of 5 TfRCC cell lines at concentrations of </= 100 nM. MET inhibition with either PHA665752 or PF02341066 failed to suppress growth at concentrations (up to 1000 nM) well beyond the IC50 for these drugs reported in sensitive cell lines. CONCLUSIONS The Akt-mTOR pathway is activated in TfRCC cell lines. This activation appears independent of MET signaling as TfRCC lines revealed no evidence of MET activation. Pharmacologic inhibition of Akt and mTOR but not MET effectively suppresses TfRCC growth in vitro, supporting a role for therapeutic targeting of the Akt-mTOR pathway in TfRCC patients. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e183-e184 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Eric Kauffman Bethesda, MD More articles by this author Gopal Gupta Bethesda, MD More articles by this author Fabiola Cecchi Bethesda, MD More articles by this author Kristen Raffensperger Bethesda, MD More articles by this author W. Marston Linehan Bethesda, MD More articles by this author Donald P. Bottaro Bethesda, MD More articles by this author Ramaprasad Srinivasan Bethesda, MD More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

γ-Tocotrienol inhibits HGF-dependent mitogenesis and Met activation in highly malignant mammary tumour cells

Aberrant Met signalling is associated with aggressive cancer cell phenotypes. γ-tocotrienol displays potent anti-cancer activity that is associated with suppression of HER/ErbB receptor signalling. Experiments were conducted to investigate the effects of γ-tocotrienol treatment on HGF-dependent +SA mammary tumour cell proliferation, upon Met activation. The +SA cells were maintained in serum-free defined media containing 10 ng/ml HGF as the mitogen. Cell viability was determined using the MTT assay, western blot analysis was used to measure protein expression, and Met expression and activation were determined using immunofluorescent staining. Treatment with γ-tocotrienol or Met inhibitor, SU11274, significantly inhibited HGF-dependent +SA cell replication in a dose-responsive manner. Treatment with 4 μmγ-tocotrienol reduced both total Met levels and HGF-induced Met autophosphorylation. In contrast, similar treatment with 5.5 μm SU11274 inhibited HGF-induced Met autophosphorylation, but had no effect on total Met levels. Combined treatment with subeffective doses of γ-tocotrienol (2 μm) and SU11274 (3 μm) resulted in significant inhibition of +SA cell expansion compared to treatment with individual agents alone. These findings show, for the first time, the inhibitory effects of γ-tocotrienol on Met expression and activation, and strongly suggest that γ-tocotrienol treatment may provide significant health benefits in prevention and/or treatment of breast cancer, in women with deregulated HGF/Met signalling.

Abstract 3401: Genetic down-regulation of MET alters the metastatic phenotype of osteosarcoma cells

Abstract The MET tyrosine kinase receptor is important during human development for normal cell growth and migration. Activating alterations of MET have also been identified in several carcinomas. Previous immunohistochemistry studies in our laboratory found high levels of MET in 85% of osteosarcoma tumor samples. In addition, pharmacologic modulation of MET resulted in a migratory decrease in some osteosarcoma cell lines. This led us to hypothesize that MET is necessary for osteosarcoma metastasis, and genetic down-regulation of MET expression would reduce the metastatic phenotype of osteosarcoma cells. We used lentiviral shRNA constructs to knockdown MET expression in several osteosarcoma cell lines. These included MNNG-HOS, a metastatic chemically transformed line that expresses constitutively activated MET, MG63.2, a highly metastatic variant of poorly metastatic MG63, AI and LR, which are two newly derived cell lines that have extremely high MET levels. We quantified levels of MET expression using a sensitive and quantitative electrochemiluminescence (ECL) assay and assessed in vitro parameters of metastasis including motility, migration, and invasion. MET expression was assessed by ECL in protein lysates from frozen osteosarcoma tumor samples and cell lines, and 15-20% of samples showed extremely high levels of total MET. AI, LR and MG63.2 had the highest levels of MET expression. MNNG-HOS has activation of MET due to translocation of the 3′end of MET with the 5′end of TPR. We utilized these lines for shRNA knock down of MET. ECL quantification of total MET levels revealed greater than 70% decrease in MET expression with shRNA knockdown in all four cell lines, compared with cells infected with scramble shRNA controls. As predicted for MNNG-HOS, shRNA constructs targeting the 3’ tyrosine kinase domain of MET resulted in marked decreased motility, compared to intermediate decreases using shRNA constructs targeting the 5’ region. Knock down of MET in MG63.2 resulted in inhibition of both motility and migration. AI and LR assays are in progress. Taken together, these data are the first evidence that genetic down-regulation of MET results in a decreased metastatic phenotype in osteosarcoma. This is a promising finding, as several small molecule tyrosine kinase inhibitors targeting MET are in development. We are currently testing our hypothesis in a mouse model to determine the ability of the above genetically knocked-down MET cell lines to decrease metastasis in vivo. Concurrently, in preparation for translational studies, we are also assessing MET specific tyrosine kinase inhibitors utilizing the parental cell lines described above. 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 3401.