C-met mRNA Research Articles

Abstract 844: Targeting the pro-survival protein c-MET with ARQ 197 inhibits growth of multiple myeloma cells

Abstract Multiple myeloma (MM) is a B-cell disorder characterized by the accumulation of mature plasma cells in the bone marrow. Earlier studies have established that patients with MM have high plasma concentrations of hepatocyte growth factor (HGF) which is correlated with poor prognosis and advanced disease stages. HGF is the ligand for the c-MET receptor tyrosine kinase. Our group has previously shown that inactivation of the c-MET receptor by siRNA and ribozyme approaches inhibited proliferation and induced apoptosis in MM cell lines. Hence, we hypothesized that the HGF/c-MET axis plays a critical role in myeloma cell survival and targeting this pathway would be an effective strategy to treat MM. To further test our hypothesis, we used MM1.S, U266 and OPM-2 myeloma cell lines and ARQ 197 (tivantinib), a small molecule non-ATP-competitive and selective c-MET inhibitor (Ki=355 nM). This drug is orally bioavailable, achieving steady-state levels of median 6-7 µM in plasma of patients with solid tumors. Cell growth was inhibited by at least 50% and annexin V/propidium iodide positive cells increased by at least 60% within 48 hours of treatment with 1 µM (U266 and OPM-2) and 3 µM (MM.1S) ARQ 197. Consistent with this biological response, downstream effectors of c-MET signaling including phospho-GAB1 and phospho-ERK1/2 were decreased. MM patients develop resistance to the current drugs such as lenalidomide and bortezomib. We tested ARQ 197 in paired MM cell lines - RPMI 8226, ANBL-6 and KAS-6/1 - that are sensitive and resistant to lenalidomide or bortezomib. In general, all the tested cell lines expressed readily detectable levels of proHGF as well as detectable albeit low levels of c-MET. Interestingly, the resistant cell lines expressed significantly higher c-MET protein levels compared to drug-naïve controls, but were equally sensitive to ARQ 197 indicating that this c-MET kinase inhibitor overcomes inherent drug resistance. The bone marrow microenvironment is also a source for acquired drug resistance, yet ARQ 197 was effective in inhibiting growth of MM cell lines grown on human stromal cells (NKtert) which mimic the bone marrow microenvironment. Consistent with these data, exogenous addition of the cytokines interleukin-6 (0.5 and 1.0 ng/ml) or HGF (50 ng/ml) did not abrogate ARQ 197-mediated cell death or growth inhibition. Importantly, plasma cells from healthy donors (n = 15) showed no difference in c-MET mRNA expression compared to plasma cells from MM patients (n = 147) but showed relatively lower levels of HGF expression compared to MM patients. These results suggest that the HGF/c-MET pathway is upregulated in MM plasma cells. In conclusion, inhibition of the c-MET receptor tyrosine kinase activity with ARQ 197 is a novel-target based strategy to impact on the pathobiology of MM. Based on these data, we are initiating a clinical trial of ARQ 197 in patients with relapsed/refractory MM. 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 844. doi:1538-7445.AM2012-844

MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo

BackgroundMicroRNAs (miRNAs) are a class of endogenously expressed, small noncoding RNAs, which suppress its target mRNAs at the post-transcriptional level. Studies have demonstrated that miR-34a, which is a direct target of the p53 tumor suppressor gene, functions as a tumor suppressor and is associated with the tumor growth and metastasis of various human malignances. However, the role of miR-34a in osteosarcoma has not been totally elucidated. In the present study, the effects of miR-34a on osteosarcoma and the possible mechanism by which miR-34a affected the tumor growth and metastasis of osteosarcoma were investigated.Methodology/Principal FindingOver-expression of miR-34a partially inhibited proliferation, migration and invasion of osteosarcoma cells in vitro, as well as the tumor growth and pulmonary metastasis of osteosarcoma cells in vivo. c-Met is a target of miR-34a, and regulates the migration and invasion of osteosarcoma cells. Osteosarcoma cells over-expressing miR-34a exhibited a significant decrease in the expression levels of c-Met mRNA and protein simultaneously. Finally, the results from bioinformatics analysis demonstrated that there were multiple putative targets of miR-34a that may be associated with the proliferation and metastasis of osteosarcoma, including factors in Wnt and Notch signaling pathways.Conclusion/SignificanceThe results presented in this study demonstrated that over-expression of miR-34a could inhibit the tumor growth and metastasis of osteosarcoma probably through down regulating c-Met. And there are other putative miR-34a target genes beside c-Met which could potentially be key players in the development of osteosarcoma. Since pulmonary metastases are responsible for mortality of patient carrying osteosarcoma, miR-34a may prove to be a promising gene therapeutic agent. It will be interesting to further investigate the mechanism by which miR-34a functions as a tumor suppressor gene in osteosarcoma.

Lentivirus-mediated RNA silencing of c-Met markedly suppresses peritoneal dissemination of gastric cancer in vitro and in vivo

To investigate the expression of c-Met in peritoneal free cancer cells isolated from human gastric cancer ascites, and its relationship to peritoneal dissemination of gastric cancer. Peritoneal free cancer cells (PFCCs) were isolated from ascites specimens of gastric cancer patients. c-Met expression in PFCCs was detected with immunocytochemistry. In human gastric cancer cell line SGC7901, c-Met expression was detected using RT-PCR and Western blot, and was suppressed with lentivirus-mediated RNAi. The proliferation of SGC7901 cells was measured using MTT assay, and the invasion ability was detected with invasion assay. The adhesion of SGC7901 cells to peritoneum was observed in human peritoneal mesothelial cells (HPMCs) monolayer in vitro and in mice in vivo. PFCCs were isolated from ascites of 6 out of 10 gastric cancer patients. c-Met expression in PFCCs was detected in 5 of the 6 gastric cancer patients. In SGC7901 cells, Lentivirus-mediated RNAi significantly reduced both c-Met mRNA and protein expression, which resulted in suppressing the cell proliferation, invasion and adhesion to peritoneum. The expression of α3β1 integrin and E-cadherin was significantly inhibited in SGC7901 cells transfected with Lenti-miRNAc-Met. In the peritoneal dissemination model of gastric cancer, intraperitoneal injection of Lenti-miRNAc-Met markedly suppressed the tumor Progression of SGC7901 cells. c-Met is expressed in PFCCs from the ascites of gastric cancer patients. Down-regulation of c-Met expression markedly suppresses the multistep process of peritoneal dissemination, thus may be a potential target for the treatment of gastric cancer.

Expression of the Hepatocyte Growth Factor and C-Met in Colon Cancer: Correlation with Clinicopathological Features and Overall Survival

The hepatocyte growth factor (HGF)/c-Met signaling system has been implicated in the development and progression of colon cancer, but the relationship between the expression of HGF or c-MET and clinicopathologic features remains controversial. In the study, we analyzed the expression of HGF and c-Met in colon cancer and assessed the influence of the expression of this growth factor and its receptor on clinical and histological parameters and patient survival. We investigated the mRNA expression of HGF and c-Met with real-time PCR in 90 unselected colon carcinomas and the corresponding normal mucosa. Furthermore, HGF and c-Met protein expression was investigated with immunohistochemistry in all the samples. The mRNA and protein expression levels of HGF and c-Met were significantly higher in colon cancer than in matched normal mucosa. The protein level in most of the cases investigated was correlated with the mRNA level. Overexpression of HGF and c-Met, at both protein and mRNA levels, was correlated with depth of invasion, lymph node metastases and overall AJCC stage. According to univariate analysis, the mean survival time was shorter in the HGF-positive and c-Met-positive groups. Multivariate Cox analysis showed that high M stage and the expression of c-Met independently had a negative impact on overall survival. The HGF/c-Met signaling pathway may be involved in the pathogenesis and progression of colon cancer. C-Met overexpression can be used as a useful parameter to evaluate the prognosis of colon cancer.

Identification of MACC1 as a novel prognostic marker in hepatocellular carcinoma

BackgroundMetastasis-associated in colon cancer-1 (MACC1) is a newly identified gene that plays a role in colon cancer metastasis through upregulation of c-MET proto-oncogene (c-MET). However, the value of MACC1 as a potential biomarker for hepatocellular carcinoma (HCC) remains unknown.MethodsMACC1 mRNA expression in 128 HCC tissues was examined by quantitative polymerase chain reaction. To show the potential correlation of MACC1 and c-MET, c-MET was also analysed.ResultsMACC1 was more highly expressed in HCC than in non-HCC tissues (P = 0.009). High MACC1 expression was significantly increased in cases with high alpha fetoprotein (AFP) (P = 0.025). A positive correlation was found between MACC1 and c-MET mRNAs (r = 0.235, P = 0.009). Both univariate and multivariate analyses revealed that MACC1 expression was associated with overall survival (OS) and disease-free survival (DFS). Moreover, stratified analysis showed that tumour-node-metastasis (TNM) stage I patients with high MACC1 levels had shorter OS and DFS than those with low MACC1.ConclusionsMACC1 may identify low- and high-risk individuals with HCC and be a valuable indicator for stratifying the prognosis of TNM stage I patients. MACC1 may serve as a novel biomarker for HCC.

Adenovirus-mediated siRNA targeting c-Met to inhibit proliferation and invasion of small cell lung cancer (SCLC) cells.

7080 Background: The hepatocyte growth factor receptor c-Met and its ligand hepatocyte growth factor (HGF) have been reported to be involved in cellular motility, growth, and invasion by activating mitogenic signaling pathways. The overexpression of c-Met gene has been found in many malignant cancers, but the roles of c-Met overexpression in SCLC tumors still remain unclear. The aim of the present study was to explore its roles and potential as a therapeutic target for SCLC. Methods: Quantitative real-time RT-PCR and immunohistochemistry assays were performed to detect the expression of c-Met mRNA and protein in SCLC tissue or corresponding non-tumor lung tissue samples. Adenovirus-mediated small interfering RNA (siRNA) was employed to down-regulate the expression of c-Met gene in SCLC cell line (NCI-H446). MTT and colony formation assays were performed to detect in vitro proliferation of NCI-H446 cells. In vitro wound-healing and transwell invasion assays were performed to detect in vitro invasion and metastasis of NCI-H446 cells. Finally, in vivo tumorigenicity and metastasis assays were done to analyze in vivo proliferation and metastasis of NCI-H446 cells in a xenograft model. Results: We showed that the levels of c-Met mRNA expression were significantly higher in SCLC tissue samples (0.97 ± 0.08) than those in corresponding nontumor lung tissue samples (0.21 ± 0.02; P<0.05). Additionally, the immunostaining of c-Met protein in SCLC tissues was stronger than that in corresponding non-tumor tissues. Adenovirus-mediated siRNA targeting c-Met could significantly down-regulate c-Met expression, and the specific down-regulation of c-Met expression in SCLC cells could strongly inhibit proliferation of SCLC cells both in vitro and in vivo. Moreover, c-Met down-regulation could also reduce invasion capacity in vitro and metastasis capacity in vivo of SCLC cells. Conclusions: Taken together, our results indicated that the overexpression of c-Met gene played an important role in the progression and development of SCLC, and adenovirus-mediated siRNA targeting c-Met could potentially be an experimental approach for SCLC gene therapy.

Effects of urokinase type plasminogen activator gene transfected bone marrow-derived liver stem cells transplantation on hepatocyte regeneration in liver fibrosis rats

Objective To explore the effects of urokinase type plasminogen activator (uPA) gene-modified bone marrow-derived liver stem cells ( BDLSC) transplantation on hepatocyte regeneration in CCl4-induced liver fibrosis rats. Methods Ten male Fisher 344 rats were donor rats of BDLSC. The BDLSC of male rat was transfected with AduPA. Thirty-six female Fisher 344 rats were equally divided into normal group (injected subcutaneously with olive oil) , model group (CCl4 induced the model, injected through tail vein with 0. 9% sodium chloride), BDLSC group (CCl4 induced the model, injected through tail vein with BDLSC) and gene transfected group (CCl4 induced the model,injected through tail vein with gene transfected BDLSC). Liver function and area of collagen were observed. The expression of hepatic growth factor ( HGF) and its receptor c-met mRNA in rats' liver tissues were tested by semiquantitative RT-PCR. The expressions of proliferating cell nuclear antigen (PCNA) in rats' liver tissues were determined by immunohistochemistry staining. Results The areas of collagen in normal group, model group, BDLSC group and gene transfected group was 0. 12% ± 0.03%, 14. 49%±1.40%, 8. 25%±0. 82% and 5. 12%±0. 40% accordingly, there were significant differences between groups (P<0. 05). Compared with model group and BDLSC group, the liver function of gene transfected group significantly improved, the serum levels of hyaluronic acid (HA),procollagen Ⅲ (PCⅢ) and the content of hydroxyproline in liver tissues decreased dramatically. The expression of HGF and c-met at mRNA levels were up-regulated significantly, and the expression of PCNA protein in liver tissues increased obviously. Conclusion uPA gene-modified BDLSC transplantation may induce proliferation of hepatocytes, and then improve the liver functions of fibrotic rats induced by CCl4. Key words: Uninary plasminogen activator; Stem cell transplantation; Hepatocytes; Liver cirrhosis; Hepatocyte growth factor; Proliferating cell nuclear antigen; Carbon tetrachloride; Disease models, animal

Abstract 3577: TAS-115: a highly potent c-Met + VEGFRs dual inhibitor with prominently safer profile

Abstract c-Met is a multi-functional proto-oncogenic receptor tyrosine kinase (RTK) involving various malignant phenotypes and aberrant expressions of HGF/c-Met axis, which are associated with poor prognosis of diverse cancers. On the other hand, VEGF/VEGFR inhibition has achieved a notable status of standard therapy for several cancers. Moreover, recent studies suggest that both RTKs work in a complementary way on cancer malignant phenotypes including angiogenesis, metastasis, and hypoxia reaction. Therefore, their dual inhibition seemed to be an attractive strategy for cancer therapy, and indeed several compounds inhibiting both targets have been subjected to clinical evaluation. However, clinical efficacy appeared to be limited so far, probably due to their poor selective of mode of action and toxicity resulting from VEGFR inhibition which result in poor tolerability restricting the dosage in the clinical setting. The optimized potent c-Met + VEGFRs dual inhibitor with better safety profile may become a successful treatment modality meeting the medical needs. To address this issue, we have developed TAS-115, a novel small molecule c-Met + VEGFRs dual inhibitor. In biochemical assay, it was identified to be a potent ATP competitive inhibitor of both c-Met and VEGFR2 at IC50 of around 10 nM that is equal or more potent than those of other known inhibitors such as crizotinib, foretinib, or sunitinib. In cellular assays, it also potently inhibited the phosophorylation of both kinases with the same potency as HGF/c-Met or VEGF dependent cellular growth at IC50 range of 10 nM. Importantly, the IC50 values under no HGF/c-Met or VEGF dependent condition were over 20,000 nM. The difference between both conditions were more than 2000-fold, while the other inhibitors showed only 10∼300 hold difference when compared to control, not stimulated cells. This selectivity was also confirmed in a panel of 40 cell lines; the IC50 values were highly correlated with HGF, c-Met mRNA expression level as revealed by coefficient value of R2&amp;gt;0.65.In vivo studies, orally administered TAS-115 exhibited highly potent anti-tumor activity against c-Met positive and negative xenografts including complete tumor eradications accompanying by abolishing of c-Met and VEGFR2 signal cascade. The effective doses (ED50) determined in about 12 xenograft models ranged from 4 to 30 mg/kg. The tolerability study of consecutive dosing for 4 wks revealed no animal death incidence, even at more than 28 times higher exposure over the ED50 values of TAS-115, which was surprising when compared with safety of other VEGFR inhibitors including foretinib, sunitinib and sorafenib, whose therapeutic indices (MTD/ED50) were only 2∼4. Taken together, TAS-115 is a highly potent c-Met + VEGFRs dual inhibitor with prominent selectivity and safer profile indicating its therapeutic potentials in human cancers. 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 3577. doi:10.1158/1538-7445.AM2011-3577

Hyperbaric oxygenation promotes regeneration of biliary cells and improves cholestasis in rats

To investigate the effects of hyperbaric oxygenation (HBO) on regeneration of the biliary ductal system and postoperative cholestasis in hepatectomized rats. HBO was performed in Wistar rats daily starting 12 h after a 70% partial hepatectomy. Regenerated liver weight, serum parameters and the proliferating cell nuclear antigen labeling index of hepatocytes and biliary ductal cells were measured. Hepatocyte growth factor (HGF), c-Met and transforming growth factor (TGF) β-1 mRNA expression levels were analyzed by quantitative reverse transcription polymerase chain reaction. HBO improved the postoperative serum levels of total bile acid but not transaminase levels. HBO promoted hepatocyte and biliary ductal cell proliferation. The hematoxylin and eosin-stained specimens revealed fewer ballooned hepatocytes and higher cell densities in the HBO group compared to the control group. HBO suppressed c-Met mRNA levels at 15 h but did not modulate HGF or TGF β-1 mRNA expression levels. HBO promoted regeneration of biliary ductal cells and improved postoperative cholestasis after a partial hepatectomy.

The effect of hepatocyte growth factor on the initial stages of mouse follicle development

Interactions between theca and granulosa cells of the follicle are critical for the coordination of ovarian follicle development. The cell-cell interactions are mediated through the local production and actions of a variety of factors. The current study is designed to investigate the expression of Hgf and its receptor, c-Met, in the mouse ovary during in vivo folliculogenesis. We found that Hgf and c-Met mRNAs were already expressed in 2-day-old ovaries, and that, while c-Met levels remained constant until 22-day-old, Hgf levels slightly but not significantly increased with age. The expression of Hgf mRNA in theca/interstitial cells was higher than in granulosa cells in 22-day-old ovaries. Immunohistochemistry analysis confirmed the expression pattern demonstrated by RT-PCR. We investigated the role of hepatocyte growth factor (HGF) at the beginning of mouse folliculogenesis and its possible interaction with kit ligand (KL). Interestingly, both KL and HGF were able to increase the expression of each other, creating a positive feedback loop. In the presence of HGF, we observed an increase of granulosa cell proliferation and an increase in the number of pre-antral and early antral follicles in ovary organ cultures. We also observed a significant increase in the diameters of follicles in individual follicle cultures. Moreover, HGF stimulated the expression of the FSH receptors, both in the whole ovary and in isolated pre-antral follicle cultures. Based on the data presented, we concluded that HGF exerts multiple levels of control over follicular cell functions, which collectively enable the progression of follicular development.

Identification of Basophils as Source of Hepatocyte Growth Factor (HGF) In CML: a Potential Trigger of Disease Acceleration.

AbstractAbstract 1202Chronic myeloid leukemia (CML) is a myeloproliferative disorder in which BCR/ABL leads to enhanced survival of leukemic cells. Several different angiogenic molecules, including vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), have been implicated in the pathogenesis of CML. Enhanced production of these pro-angiogenic molecules in CML may be associated with disease acceleration. However, little is known so far about the exact origin of these growth factors and about mechanisms underlying their production and secretion in CML cells. In the current study, we analyzed the cellular distribution of HGF and its receptor c-MET in CML cells, and explored the mechanism of expression of HGF in BCR/ABL-transformed cells. As assessed by immunostaining of bone marrow sections and isolated blood and bone marrow samples, the HGF protein was found to be expressed in a subset of leukemic cells in all patients tested. In addition, CML cells were found to express HGF mRNA and c-MET mRNA. In consecutive experiments, we were able to show that basophils are the primary source of HGF in CML. In particular, highly enriched sorted CD203c+ CML basophils were found to express substantial amounts of HGF mRNA as well as the HGF protein. Correspondingly, leukemic cell samples obtained from patients with accelerated phase CML were found to contain higher HGF mRNA levels compared to cells obtained from patients in chronic phase or blast phase CML. Finally, HGF mRNA and the HGF protein were detectable in the basophil-committed CML cell line KU812, but not in the more immature Ph+ cell line K562. We next asked whether expression of HGF in CML cells depends on BCR/ABL. To address this question, Ba/F3 cells with doxycycline-inducible expression of BCR/ABL were employed. However, BCR/ABL failed to induce expression of HGF mRNA or the HGF protein in Ba/F3 cells. Correspondingly, the BCR/ABL-blocker imatinib was found to inhibit expression of VEGF mRNA, but did not inhibit HGF mRNA expression in KU812 cells. Next, we examined the expression of c-MET in CML cells. c-MET mRNA was found to be expressed in KU812 and K562 cells, in highly enriched CD34+/CD38- CML stem cells, and less abundantly in more mature CD34+/CD38+ CML cells and CML basophils. The c-MET inhibitor MSC-2156119J-15 (Merck-Serono Darmstadt, Germany) was found to counteract growth of primary CML cells, K562 cells, and KU812 cells with comparable IC50 values (0.5-1.0 μM). In summary, our data suggest that HGF is a BCR/ABL-independent basophil-derived mediator in CML. Basophils and basophil-derived mediators may play a more active role in CML-acceleration as has been considered previously. Whether targeting of HGF or/and c-MET is an effective approach to block acceleration in CML remains to be elucidated. Disclosures:Valent:Novartis: Honoraria, Research Funding; Merck-Serono: Research Funding.

Sprouty-2 controls c-Met expression and metastatic potential of colon cancer cells: sprouty/c-Met upregulation in human colonic adenocarcinomas

Sprouty negatively regulates receptor tyrosine kinase signals by inhibiting Ras/ERK pathways. Sprouty is down-regulated in breast, prostate and liver cancers and appears to function as a tumor suppressor. The role of Sprouty in colonic neoplasia, however, has not been investigated. Sprouty-2 protein and mRNA transcripts were significantly up-regulated in human colonic adenocarcinomas. Strikingly, the c-Met receptor was also upregulated in tumors with increased sprouty-2. To delineate a potential causal relationship between sprouty-2 and c-Met, K-ras mutant HCT-116 colon cancer cells were transduced with purified TAT-sprouty-2 protein or stably transfected with full-length human sprouty-2 gene. Sprouty-2 up-regulation significantly increased cell proliferation by accelerating cell cycle transition. Sprouty-2 transfectants demonstrated strong up-regulation of c-Met protein and mRNA transcripts and hepatocyte growth factor stimulated ERK and Akt phosphorylation and enhanced cell migration and invasion. In contrast, knockdown of c-Met by siRNA significantly decreased cell proliferation, migration and invasion in sprouty-2 transfectants. Further, knockdown of sprouty-2 by siRNA in parental HT-29 and LS-174T colon cancer cells also decreased cell invasion. Sprouty-2 transfectants formed significantly larger tumor xenografts and demonstrated increased proliferation and angiogenesis and suppressed apoptosis. Sprouty-2 tumors metastasized to liver from cecal orthotopic implants suggesting sprouty-2 might also enhance metastatic signals. Thus in colon cancer sprouty functions as an oncogene and its effects are mediated in part by c-Met up-regulation.

Abstract 1639: The novel c-MET inhibitor, ARQ 197, shows additive growrh-inhibitory effect with erlotinib through enhanced degradation of c-MET protein via ubiquitin/proteasome pathway

Abstract Background c-MET, a receptor of hepatocyte growth factor (HGF), is known to be overexpressed in a variety of human cancers. Recently several c-MET inhibitors are being developed as cancer chemotherapy against this compelling molecular target. ARQ 197 is a new small molecule c-MET inhibitor. Since ARQ 197 shows efficient anti-cancer activity without many severe adverse events in Phase I clinical trials, it is expected to be a useful anticancer agent for clinical treatment. This drug is known to bind to c-MET at neighbor area of its ATP binding site and it does not work as an ATP mimic. In this study, we investigated the molecular mechanism of inhibitory effect on c-MET by ARQ 197 in a non-small cell lung cancer cell (NSCLC) line. Materials and Method We established a c-MET-overexpressed NSCLC line, PC-9/MET, by long term exposure of PC-9 cells to EGFR-tyrosine kinase inhibitor. The cytotoxicity of ARQ 197 was measured by MTT assay. The expression of c-MET protein and mRNA were detected by Western blotting analysis and real time RT-PCR method, respectively. In a c-MET degradation analysis, bortezomib was used for inhibition of proteasome. Results ARQ 197 is equally cytotoxic to parental PC-9 and EGFRTKI-resistant PC-9/MET cell line with GI50 values around 200 nM (?). ARQ 197 showed an additive growth- inhibitory effect with erlotinib in PC-9/MET cells as assessed by an Isobologram analysis. In ARQ 197-treated PC-9/MET cells, a clear decrease of phospho c-MET protein as well as phospho AKT and ERK proteins was observed, however, ARQ 197 also induced a down-regulation of c-MET protein in the cells without any effect on c-MET mRNA level. This ARQ 197 induced degradation was reversed by a concomitant treatment with a proteasome inhibitor, bortezomib in the cells. However, bortezomib did not affect cytotoxicity of ARQ 197 in the same cells. Conclusion These data suggest that ARQ 197 may inhibit c-MET through downregulation of this protein in addition to inhibition of its kinase activity. Furthermore, it may enhance c-MET degradation by the ubiquitin/proteasome pathway. These c-MET inhibitory activities by ARQ 197 should be important for the additive combined effect with erlotinib in PC-9/MET cells. 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 1639.

Adenovirus-Mediated SiRNA Targeting c-Met Inhibits Proliferation and Invasion of Small-Cell Lung Cancer (SCLC) Cells

The hepatocyte growth factor receptor c-Met and its ligand hepatocyte growth factor (HGF) have been reported to be involved in cellular motility, growth, and invasion by activating mitogenic signaling pathways. The overexpression of c-Met gene has been found in many malignant cancers, but the roles of c-Met overexpression in SCLC tumors still remain unclear. The aim of the present study was to explore its roles and potential as a therapeutic target for SCLC. Quantitative real-time RT-PCR and immunohistochemistry assays were performed to detect the expression of c-Met mRNA and protein in SCLC tissue or corresponding non-tumor lung tissue samples. Adenovirus-mediated small interfering RNA (siRNA) was employed to down-regulate the expression of c-Met gene in SCLC cell line (NCI-H446). MTT and colony formation assays were performed to detect in vitro proliferation of NCI-H446 cells. In vitro wound-healing and transwell invasion assays were performed to detect in vitro invasion and metastasis of NCI-H446 cells. Finally, in vivo tumorigenicity and metastasis assays were done to analyze in vivo proliferation and metastasis of NCI-H446 cells in a xenograft model. We showed that the levels of c-Met mRNA expression were significantly higher in SCLC tissue samples (0.97±0.08) than those in corresponding non-tumor lung tissue samples (0.21±0.02; P<0.05). Additionally, the immunostaining of c-Met protein in SCLC tissues was stronger than that in corresponding non-tumor tissues. Adenovirus-mediated siRNA targeting c-Met could significantly down-regulate c-Met expression, and the specific down-regulation of c-Met expression in SCLC cells could strongly inhibit proliferation of SCLC cells both in vitro and in vivo. Moreover, c-Met down-regulation could also reduce invasion capacity in vitro and metastasis capacity in vivo of SCLC cells. Taken together, our results indicated that the overexpression of c-Met gene played an important role in the progression and development of SCLC, and adenovirus-mediated siRNA targeting c-Met could potentially be an experimental approach for SCLC gene therapy.

RNA interference targeting c-Met inhibits proliferation of human laryngeal carcinoma Hep-2 cell line in vitro and in its xenografts in nude mice

The proto-oncogene c-Met was found to express on human laryngeal carcinoma Hep-2 cell line in previous research. In the present study, the author further examined whether inhibition of c-Met by RNA interference (RNAi) might inhibit biologic activity of Hep-2 cell line in vitro and proliferation using a murine laryngeal carcinoma model. RNAi plasmid that can express small interfering RNA targeting c-Met or siRNA that did not match any known human coding mRNA(control siRNA plasmid)was designed, constructed, and transfected into Hep-2 cell line by using cationic liposome Lipofectamine2000 as transfecting agent. In vitro, the transfection efficacy was tested by RT-PCR and Western Blot method, then elected the most inhibitive c-Met-siRNA sequence. Cell proliferation, movement and invasion were studied using MTT, cell migration assay and cell invasion assay, respectively. The Hep-2 cells were transplanted into nude mice, then the time of tumor formation and growth were observed. After tumor formation, c-Met-siRNA was given as the anti-tumor therapy. Expression of c-Met, MMP-9 and VEGF were detected by Western Blot method. After the pSilencer2.0/c-Met-shRNA recombinant plasmid transfection into laryngeal carcinoma Hep-2 cells, the expression of mRNA and protein of c-Met decreased significantly in Hep-2 cells. On the 35th day after tumor vaccination, the tumor volume was (138 ± 27) mm³ in c-Met-siRNA transfection group, Which was diminished significantly in contrast with control group (P < 0.01). The expression of c-Met, MMP-9 and VEGF in the tumor of experiment group was decreased significantly, respectively (P < 0.05). The results indicated that c-Met-siRNA can down-regulate the expression of c-Met and markedly inhibit laryngeal carcinoma Hep-2 cell proliferation, movement and invasion and the growth of transplantation tumor of nude mice. The siRNA expressing plasmid mediated gene therapy might be a new strategy in targeting molecular therapy of cancer of larynx.

3,3'-Diindolylmethane Inhibits Activation of Akt through Inhibition of Hepatocyte Growth Factor Receptor c-Met Signaling.

Abstract Background: 3,3'-Diindolylmethane (DIM), the major acid condensation product of indole-3-carbinol, is a promising anticancer compound that is bioavailable, non-toxic, and easily obtainable. Although DIM has strong anticancer activity against breast cancer cells in vitro and in vivo, the exact mechanism for this activity is not known. We and others have found that DIM inhibits activation of the Akt pathway in breast cancer cells. Akt is a serine threonine protein kinase involved in many important cellular functions including proliferation and cell survival. Akt is active when phosphorylated at serine-473 and threonine-308. In breast cancer cells, Akt can be activated by various stimuli, including signaling from growth factor receptors like the hepatocyte growth factor (HGF) receptor c-Met, which is often overexpressed in breast cancer. In this project, we show that c-Met is an upstream point of inhibition of the Akt pathway by DIM.Materials and Methods: When MDA-MB-231 breast cancer cells reached 70-80% confluency, we serum-starved them by reducing the fetal bovine serum concentration in the media from 10% to 1% for 20 hours and then treated with DIM or the vehicle control DMSO for 4 hours or the indicated time. When using recombinant growth factors to activate Akt or c-Met, we incubated cells in medium containing no fetal bovine serum and added the growth factors for 10 minutes after incubation with DIM. To analyze protein expression and protein phosphorylation we performed Western blotting. To analyze c-Met kinase activity we used a commercially available kit.Results: DIM does not inhibit activation of Akt downstream of either epidermal growth factor or insulin-like growth factor-1. However, DIM does inhibit Akt activation downstream of HGF and of fetal bovine serum. DIM inhibits tyrosine phosphorylation of c-Met at Y1349 and Y1234/1235. DIM does not affect total c-Met protein or mRNA levels and does not directly inhibit c-Met kinase activity.Discussion: While we have determined that DIM inhibits c-Met phosphorlyation, and therefore HGF signaling, the mechanism of this inhibition requires further study. We hypothesize that DIM induces serine phosphorylation of c-Met, since c-Met can be inhibited by reactive oxygen species (ROS)-induced phosphorylation of serine-985 by protein kinase C (PKC), and DIM increases ROS levels in cells.Components of the Akt pathway are frequent targets for mutations in breast cancer and c-Met and its ligand HGF are associated with lower survival and increased risk of distant metastases in breast cancer. This project represents an important step in determining the mechanism of action for DIM, a promising phytochemical for the treatment of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6117.

MicroRNA-1/206 Targets c-Met and Inhibits Rhabdomyosarcoma Development

MicroRNAs (miRNAs) are endogenous short (approximately 22) nucleotide RNAs that regulate gene function by modification of target mRNAs. miRNA-1 (miR-1) and miRNA-206 (miR-206) are highly expressed in skeletal muscle. Due to the tissue-specific nature of miR-1/206 for skeletal muscles, we investigated the role of miR-1/206 in the development of rhabdomyosarcoma. Initially, we demonstrated that miR-1/206 expression was suppressed in rhabdomyosarcomas and found at very low levels in a rhabdomyosarcoma RD cell line. Transient transfection of miR-1/206 into cultured RD cells led to a significant decrease in cell growth and migration. Using bioinformatics, we identified two putative miR-1/206 binding sites within the 3'-untranslated region of the human c-Met mRNA. miR-1/206 was then shown to have activity on mRNA expression by targeting the c-Met 3'-untranslated region. The expression of c-Met protein was shown to be down-regulated by subsequent Western blot analysis. Conversely, up-regulation of c-Met was confirmed in tissue samples of human rhabdomyosarcoma, with its level inversely correlated with miR-1/206 expression. In vivo, miR-1/206-expressing tumor cells showed growth delay in comparison with negative control. Our results demonstrated that miR-1/206 suppressed c-Met expression in rhabdomyosarcoma and could function as a potent tumor suppressor in c-Met-overexpressing tumors. Inhibition of miR-1/206 function could contribute to aberrant cell proliferation and migration, leading to rhabdomyosarcoma development.

Down-Regulation of c-Met Expression Inhibits Human HCC Cells Growth and Invasion by RNA Interference

Cell migration is a basis for invasion and metastasis of malignant tumors. Receptor tyrosine kinases are recognized as important therapeutic targets in antineoplastic strategies. C-met is a receptor tyrosine kinase highly expressed in human hepatocellular carcinoma (HCC) cell line MHCC97-H. Higher expression of c-met in tumor tissue can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventually, metastasis. To explore the roles of c-met in modulating the motility of cell, we silenced c-met expression in the HCC line MHCC97-H by RNA interference (RNAi). For transient expression, c-met-siRNA 1,2 recombinant plasmids were transfected into phoenix A cells. The MHCC97-H cells were cultured in Dulbecco's modified Eagles's medium (DMEM) with 10% fetal bovine serum (FBS) to establish MHCC97-H HCC cells stably expressing c-met-siRNA. MHCC97-H cells were treated with the recombinant virus for assay of c-met mRNA and protein, evaluation of growth and invasion of MHCC97-H cells, and identification of hepatitis B virus X (HBX) protein correlation with c-met. After transfection of c-met-siRNA for 48 h, the expression of c-met decreased markedly in MHCC97-H cells; the most effective site of the siRNA target sequence is at the 537 upstream, far from the transcription start. In addition, the proliferation, motility, and invasive ability of MHCC97-H cells were significantly inhibited. Furthermore, we showed that hepatitis B virus (HBV) X protein (HBX) potentiated the activities of the extracellular signal-regulated kinase 1/2 (ERK1/2) in MHCC97-H cells. Treatment with extracellular signal-regulated kinase (ERK) inhibitor (U0126), but not P38 MAPK inhibitor (SB203580) or phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), markedly suppressed the expression of c-met protein in MHCC97-H cells. These results indicate that the over-expression of c-met protein plays an important role in the cell invasion of MHCC97-H, and HBX protein may promote the expression of c-met by ERKs pathway.

Expression of Hepatocyte Growth Factor and the Proto-oncogenic Receptor c-Met in Canine Osteosarcoma

Hepatocyte growth factor (HGF) and the proto-oncogenic receptor c-Met are implicated in growth, invasion, and metastasis in human cancer. Little information is available on the expression and role of both gene products in canine osteosarcoma. We hypothesized that the expression of c-Met is associated with malignant histologic characteristics, a short survival time, and a reduced disease-free interval in canine osteosarcoma. Quantitative real-time polymerase chain reaction was used to analyze the messenger RNA (mRNA) expression of both HGF and c-Met in 59 canine osteosarcoma samples. The relationship between HGF and c-Met expression, patient outcome, and histologic characteristics of the tumor were studied. Western blot analysis was performed to investigate the presence of active HGF protein. The expression pattern of c-Met in 16 slides of canine osteosarcoma was identified by immunohistochemistry. Coexpression of HGF and c-Met mRNA in all canine osteosarcoma samples suggested autocrine or paracrine receptor activation. A significant, moderately positive correlation was found between c-Met and HGF mRNA expression. c-Met mRNA expression was not associated with survival time or disease-free interval. Expression of c-Met was significantly associated with metastasis via the lymphogenic route. Immunolabeling with c-Met revealed a cytoplasmic staining pattern in all osteosarcoma cell types. In this study, c-Met mRNA expression in canine osteosarcoma was found to be of no influence on survival time and disease-free interval. Further studies are necessary to confirm the involvement of the c-Met pathway in the lymphogenic route of metastasis.

MicroRNA‐23b mediates urokinase and c‐met downmodulation and a decreased migration of human hepatocellular carcinoma cells

Urokinase-type plasminogen activator (uPA) and c-met play a major role in cancer invasion and metastasis. Evidence has suggested that uPA and c-met overexpression may be coordinated in human hepatocellular carcinoma (HCC). In the present study, to understand whether the expression of these genes might be coregulated by specific microRNAs (miRs) in human cells, we predicted that Homo sapiens microRNA-23b could recognize two sites in the 3'-UTR of uPA and four sites in the c-met 3'-UTR by the algorithm pictar. The miR-23b expression analysis in human tumor and normal cells revealed an inverse trend with uPA and c-met expression, indicating that uPA and c-met negative regulation might depend on miR-23b expression. Transfection of miR-23b molecules in HCC cells (SKHep1C3) led to inhibition of protein expression of the target genes and caused a decrease in cell migration and proliferation capabilities. Furthermore, anti-miR-23b transfection in human normal AB2 dermal fibroblasts upregulated the expression of endogenous uPA and c-met. Cotransfection experiments in HCC cells of the miR-23b with pGL4.71 Renilla luciferase reporter gene constructs, containing the putative uPA and c-met 3'-UTR target sites, and with the pGL3 firefly luciferase-expressing vector showed a decrease in the relative luciferase activity. This would indicate that miR-23b can recognize target sites in the 3'-UTR of uPA and of c-met mRNAs and translationally repress the expression of uPA and c-met in HCC cells. The evidence obtained shows that overexpression of miR-23b leads to uPA and c-met downregulation and to decreased migration and proliferation abilities of HCC cells.