C-met mRNA Research Articles

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.

Role of miR-128 in hypertension-induced myocardial injury.

The present study aimed to investigate the role and mechanism of micro RNA (miR)-128 in hypertension-induced myocardial injury. The peripheral blood of patients with hypertension was collected and the expression of miR-128 was detected using fluorescence reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Primary myocardial cells isolated from rat in vitro were cultured under conditions of hypoxia and glucose deprivation, and miR-128 expression was measured by RT-qPCR. The expression of c-Met protein was measured using western blot analysis and the apoptosis of transfected cells was measured by flow cytometry in rat myocardial cells following transfection with miR-128 mimics or c-Met siRNA. A luciferase assay was applied to assess the binding of miR-128 to c-Met mRNA. miR-128 expression was significantly higher in hypertension patients compared with controls (P<0.05). miR-128 expression was higher in patients with stage III/IV hypertension compared with patients with stage II hypertension. Similarly, miR-128 expression in primary cardiomyocytes cultured under deprivation of oxygen and glucose increased with the culture time and reached a peak at 12 h. c-Met expression decreased significantly (P<0.05) and the ratio of apoptotic cells increased significantly (P<0.05), following transfection of miR-128 mimics. The number of apoptotic cells also increased when c-Met expression was knocked down by siRNA. The dual luciferase assay indicated that fluorescence intensity decreased significantly in miR-128 mimics and wild type c-Met group (P<0.05), indicating that miR-128 can directly target c-Met. Therefore, the results of the current study suggest that miR-128 may promote myocardial cell injury by regulating c-Met expression.

Long non-coding RNA HOTAIR acts as a competing endogenous RNA to promote malignant melanoma progression by sponging miR-152-3p.

HOX transcript antisense RNA (HOTAIR) is associated with the growth and metastasis of many human tumors, but its biological roles in malignant melanoma remain unclear. In this study, we show that HOTAIR is overexpressed in melanoma tissues and cells, especially in metastatic melanoma. High HOTAIR levels correlate with poor prognosis in melanoma patients. We also determined that HOTAIR functions as a competing endogenous RNA (ceRNA) for miR-152-3p. miR-152-3p was decreased and acted as a tumor suppressor in melanoma, and c-MET was the functional target of miR-152-3p. Furthermore, HOTAIR promotes the growth and metastasis of melanoma cells by competitively binding miR-152-3p, which functionally liberates c-MET mRNA and results in the activation of the downstream PI3k/Akt/mTOR signaling pathway. We determined that HOTAIR acts as a ceRNA to promote malignant melanoma progression by sponging miR-152-3p. This finding elucidates a new mechanism for HOTAIR in melanoma development and provides a potential therapeutic target for melanoma patients.

Abstract 4095: c-Met activation mediates resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer

Abstract Background: Inhibiting polo-like kinase 1 PLK1 may be an effective treatment for non-small cell lung cancer (NSCLC). PLK1 is a key regulator of mitosis and DNA damage checkpoints. PLK1 inhibitors are well tolerated, but only a few unselected patients with NSCLC respond to single-agent therapy. However, predictive biomarkers have not been used to select patients who are likely to experience a response to PLK1 inhibitors, and the mechanisms of resistance to PLK1 inhibitors have not been elucidated, making these unknowns a major gap in knowledge. To address this gap, we compared basal gene and protein expression in 63 NSCLC cell lines and discovered that mesenchymal NSCLC cell lines were more sensitive to PLK1 inhibitors than epithelial cell lines in vitro and in vivo. The induction of apoptosis in some NSCLC cell lines at very low drug concentrations and the need to find better therapy for mesenchymal NSCLC motivated us to further study PLK1 inhibition. Methods: To identify the pathways involved in PLK1 inhibitor-induced apoptosis, we used 3 pairs of isogenic NSCLC cell lines in which we had induced a mesenchymal phenotype using TGF-β. These isogenic lines were treated with the PLK1 inhibitor (volasertib) for 24 hours and levels of 301 proteins and phosphoproteins were simultaneously measured before and after treatment with volasertib using reverse phase protein array (RPPA). Results: The induction of a mesenchymal phenotype using TGF-β increased PLK1 inhibition-induced DNA damage and apoptosis in 3 NSCLC cell lines. To further elucidate mechanisms of resistance to PLK1 inhibition, we compared gene and protein expression in these isogenic cell lines, before and after PLK1 inhibition. There were 35, 12 and 43 proteins differentially regulated following PLK1 inhibition in epithelial vs. mesenchymal lines in HCC366, H1975, and HCC4006 cell lines, respectively at False Discovery rate 0.1. Phosphorylated FAK (Y397) and c-Met (Y1234/1235) were consistently inhibited following PLK1 inhibition in the mesenchymal lines but activated in the epithelial lines. These changes were confirmed by Western blotting. Total FAK and c-Met protein and mRNA levels were not affected, demonstrating post-translational changes. The inhibition of c-Met using EMD 1214063 led to FAK inhibition but FAK inhibition did not affect c-Met activation. The combination of c-Met inhibitor and volasertib increases sensitivity in NSCLC cell lines tested. The combinations led to more apoptosis than the single-agent inhibitors. Conclusions: NSCLC cell lines have diverse sensitivities to PLK1 inhibition, which is consistent with the results of clinical trials of PLK1 inhibitors in solid tumors, but no studies to date explain these diverse responses to PLK1 inhibition. We have identified c-Met activation as a previously unknown pathway of resistance to PLK1 inhibition in epithelial NSCLC. Citation Format: Ratnakar Singh, Li Shen, Pan Tong, Jing Wang, Faye M. Johnson. c-Met activation mediates resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer [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 4095. doi:10.1158/1538-7445.AM2017-4095

Hepatocyte growth factor/c-Met signaling pathway decreases doxorubicin sensitivity in hepatocellular carcinoma in vitro

Objective To explore the effect of hepatocyte growth factor (HGF)/c-Met signaling in doxorubicin (DOX) treatment of hepatocellular carcinoma (HCC). Methods Different biologic and genetic characteristics human HCC cell lines, Huh7, HepG2, MHCC97-L and MHCC97H were used in this experiment. Variation in c-Met mRNA expression level among different HCC cell lines was analyzed by RT-PCR. Western blot analysis was performed to detect c-Met and p-Met expression levels in these cell lines. CCK-8 experiment was carried to analyze the DOX sensitivity in various cell lines. t test and repeated measure analysis of variance were used for statistical analysis. Results Both c-Met and p-Met were overexpressed in MHCC97-L and MHCC97-H cell lines and these cell lines were resistant to DOX compared to Huh7 and HepG2. However, treatment of HGF in Huh7 and HepG2 cells activated c-Met signaling pathway and decreased the sensitivity of these two cell lines to DOX [inhibition rate: Huh7 (34.848±5.370) vs. (66.409±5.792) %, HepG2 (34.351±3.305) % vs. (62.308±5.453) %, both P = 0.002]. Whereas administration of c-Met inhibitor in MHCC97-L and MHCC97-H cell lines significantly increased the sensitivity to DOX [inhibition rate: MHCC97-L (73.106±3.472) % vs. (13.636±4.097) %; MHCC97-H (64.444±4.006) % vs. (6.296±2.796) %, both P < 0.001]. Conclusion HGF/c-Met signaling pathway is related the treatment efficacy of DOX in HCC. Key words: Carcinoma, hepatocellular; Hepatocyte growth factor; Doxorubicin; Signal transduction

Effects of small interfering RNA silencing metastasis-associated in colon cancer-1 gene on the proliferation, migration and invasion of liver cancer cells

Objective To investigate the effects of small interfering RNA (siRNA) silencing metastasis-associated in colon cancer-1 (MACC1) gene on the proliferation, migration and invasion of liver cancer cells, and the possible mechanism. Methods HepG2 cells were cultured. According to the different transfectants, the cells were divided into siRNA-MACC1 group, siRNA-NC group and blank control group. The cell proliferations in the different transfected groups were measured by methyl thiazol tetrazolium (MTT) assay. The cell apoptosis in the different transfected groups was examined by flow cytometry. The cell migration and invasion abilities in the different transfected groups were tested by Transwell methods. The expression levels of MACC1, hepatocyte growth factor (HGF) and c-Met genes in the different transfected groups were detected by real-time fluorescent quantitative polymerase chain reaction(FQ-PCR). The expression levels of MACC1, HGF, c-Met, MEK, phosphorylated MEK (p-MEK), extracellular signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK) proteins in the different transfected groups were detected by Western blotting. Results (1) MTT results showed that absorptivity (A) values in the siRNA-MACC1 group at 24, 48, 72 and 96 h (0.27±0.05, 0.36±0.06, 0.46±0.05, 0.58±0.07) were lower than the siRNA-NC group (0.39±0.06, 0.47±0.05, 0.64±0.07, 0.75±0.08) and blank control group (0.41±0.05, 0.49±0.07, 0.62±0.06, 0.77±0.09), the differences were statistically significant (F=25.945, 32.286, 40.264, 38.165, P=0.000, 0.000, 0.000, 0.000). (2) Flow cytometry results showed that the apoptosis rate in the siRNA-MACC1 group was (13.52±2.61)%, which was significantly higher than the siRNA-NC group and blank control group [(4.94±1.84)%, (5.10±2.13)%, F=73.918, P=0.000]. (3) The number of migration cells and the number of invasive cells in the siRNA-MACC1 group (73.62±12.71, 59.23±11.53) were significantly lower than those in the siRNA-NC group and blank control group (98.53±14.22, 85.44±13.12 and 102.51±15.13, 87.62±14.24, F=11.149, 9.500, P=0.000, 0.000). (4) Compared with siRNA-NC group and blank control group, the relative expression levels of MACC1, HGF and c-Met mRNA in the siRNA-MACC1 group were significantly lower than the siRNA-NC group and blank control group (F=51.235, 137.507, 139.708, P=0.000, 0.000, 0.000). (5) Compared with the siRNA-NC group and blank control group, the relative expression levels of MACC1, HGF, c-Met, p-MEK and p-ERK proteins in the siRNA-MACC1 group were decreased (F=49.803, 33.564, 24.391, 228.400, 137.410, P=0.000, 0.000, 0.000, 0.000, 0.000), while the the relative expression levels of MEK and ERK proteins were increased (F=12.751, 23.829, P=0.000, 0.000). Conclusion Specific inhibition of MACC1 gene expression in HepG2 cells could effectively inhibit cell proliferation, decrease cell migration and invasion, and induce cell apoptosis. The mechanism might be related to HGF/Met and MEK/ERK signaling pathways. Key words: Liver cancer; Metastasis-associated in colon cancer-1; Small interfering; Biological characteristics

Human papillomavirus oncoprotein E6 upregulates c-Met through p53 downregulation

PurposeHuman papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) carries a distinct clinical behaviour. c-Met oncogene is an important driver for tumour progression and its relationship with HPV in OPSCC was explored in the present study. Experimental designKnockdown of HPV oncogene E6 or p53 alone and in combination was performed to examine their effects on c-Met expression by Western blot and quantitative real-time polymerase chain reaction. The effects of c-Met inhibition on cell proliferation, migration, and colony formation were examined in HPV-positive head and neck squamous cell carcinoma (HNSCC) cells. Retrospectively collected OPSCC patient specimens (N = 78) were stained for c-Met by immunohistochemistry and the staining levels were correlated with HPV status and patient outcomes. ResultsE6 knockdown decreased c-Met protein and mRNA expression in HPV-positive HNSCC cells, which was partially abolished by the elimination of p53. Reducing c-Met decreased cell proliferation, migration, and colony formation in HPV-positive HNSCC cells. In OPSCC patient samples, high c-Met expression was associated with HPV-positive status (OR = 4.11, 95%CI: 1.16–14.55, P = 0.028) and tumour stage (OR = 0.27, 95%CI: 0.08–0.93, P = 0.039) by multivariable analysis. In T3/T4 stage patients, high c-Met expression was associated with HPV positivity and low p53 levels, supporting an axis of E6-p53-c-Met regulation. Furthermore, high c-Met expression was marginally associated with poor disease-free survival in HPV-positive patients. ConclusionsOur results suggest that c-Met may serve as a novel target for treating HPV-associated OPSCC. The data also demonstrate that HPV E6 upregulates c-Met expression partially through p53 downregulation.

Effect of metastasis suppressor 1 gene expression on migration and apoptosis of human gastric cancer cell BIU87 by regulating the hepatocyte growth factor/c-Met signaling pathway

Objective To investigate the effect of metastasis suppressor 1 (MTSS1) gene on the biological function of bladder cancer BIU87 cells by regulating the hepatocyte growth factor (HGF)/c-Met signaling pathway. Methods Cultured gastric cancer BIU87 cells and passaged cells were divided into three groups: group A (PEF MTSS1 and PSV2neo cotransfection group); group B: (shNC and PSV2neo co transfection group); group C (non transfection group). Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR) were used to detect MTSS1, c-Met, E-cadherin and N-cadherin protein and mRNA expression levels. Flow cytometry and scratch test were performed to examine the migration, proliferation and apoptosis of BIU87 cells transfected with PEF MTSS1. Results (1) Western blotting and Real-time PCR results showed that as compared with groups B and C, MTSS1 and c-Met protein and mRNA expression levels were significantly down-regulated in group A (P 0.05). (4) Flow cytometry showed that cell apoptosis rate in groups A, B and C was respectively (26.73±3.72)%, (4.28±0.68)% and (2.73±0.41)%. The apoptosis rate in group A was significantly higher than in group C (P<0.01), and there was no significant difference between groups B and C. Conclusion The transfection of MTSS1 gene can promote the proliferation and inhibit the apoptosis of gastric cancer cell line BIU87 cells probably by regulating the HGF/c-Met signal transduction pathway. Key words: Bladder cancer; Metastasis suppressor 1 gene; Hepatocyte growth factor/c-Met signaling pathway

Effect of colon cancer metastasis associated gene-1 on biological function of gastric cancer cell line MGC-803 by regulating hepatocyte growth factor/c-Met signaling pathway

Objective To study the effect of colon cancer metastasis associated gene-1 (MACC-1) expression on the proliferation and apoptosis of gastric cancer cell line MGC-803 in vitro. Methods Cultured gastric cancer MGC-803 cells and passaged cells were divided into three groups: group A [MACC-1-short hairpin RNA (shRNA) and PSV2neo3 co transfection group]; group B (shNC and PSV2neo co transfection group); group C (non transfection group). By Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR), the expression of MACC-1 protein and mRNA was detected in the three groups, and that of of c-Met, E-cadherin and N-cadherin in 3 groups of MGC-803 cells were detected. Flow cytometry and scratch test were used to analyze migration, proliferation and apotosis of MGC-803 cells transfected with MACC-1-shRNA. Results (1) Western blotting and Real-time PCR test results showed that as compared with groups B and C, MACC-1 and c-Met protein and mRNA expression in group A were significantly down-regulated (P<0.01). (2) Western blotting and Real-time PCR detection results showed as compared with groups B and C, E-cadherin protein and mRNA expression levels were significantly increased (P<0.01), c-Met and N-cadherin mRNA and protein levels were significantly decreased (P<0.01) in group A. There was no significant difference between groups B and C. (3) Scratch assay showed the scratch space was wide, and healing was inhibited in group A. the migration ability of MGC-803 cells in group A was reduced as compared with groups B and C (P<0.01), but there was no statistically significant difference in the scratch space between groups B and C. (4) Flow cytometry revealed that the apoptosis rate in groups A, B and C was (21.06±1.12)%, (2.18±0.14)% and (2.04±0.11%) respectively. The apoptosis rate in group A was significantly higher than in group C (P<0.01), and there was no significant difference between groups B and C. Conclusion The transfection of MACC-1 gene can promote the proliferation and inhibit the apoptosis of gastric cancer cell line MGC-803 cells, which may play a role in the regulation of hepatocyte growth factor (HGF)/c-Met signal transduction pathway. Key words: Gastric cancer; Colon cancer metastasis associated gene-1; Apoptosis; Biological function

Inhibition of long non-coding RNA TUG1 on gastric cancer cell transference and invasion through regulating and controlling the expression of miR-144/c-Met axis.

To discuss the expression of long noncoding RNA TUG1 (lncRNA-TUG1) in gastric carcinoma (GC) and its effects on the transferring and invading capacity of gastric carcinoma cells. Forty cases of carcinoma tissue and para-carcinoma tissue were selected from GC patients who underwent surgical removal in Zhejiang Provincial Hospital of Chinese Traditional Medicine and Wenzhou Central Hospital from January, 2013 to December, 2014; the expressing level of lncRNA-TUG1 in GC and para-C tissues was detected by applying the qRT-PCR technique. The correlation between lncRNA-TUG1 expression and patients' clinical data was classified and analyzed. SGC-7901 cells were transfected using lncRNA-TUG1 specific siRNA. Changes of the transferring and invading capacity of siRNA-transfected SGC-7901 cells were scratch-tested and transwell-detected. qRT-PCR was applied to detect the expression level of microRNA-144 after lncRNA-TUG1 was silenced. Changes of c-Met mRNA and protein expressions was detected by qRT-PCR and western-blot test. The expression level of lncRNA-TUG1 in GC tissue was significant higher than that in para-C tissue (P<0.05) and the high expression level of lncRNA-TUG1 in GC tissue was significantly correlated with tumor lymph nodes metastasis and advance TNM phasing (P<0.05). The transferring and invading capacity of SGC-7901 cells was highly inhibited after being transfected by lncRNA-TUG1 specific siRNA (P<0.05). The results of qRT-PCR and western-blot proved that the expression of microRNA-144 was significantly boosted and the expression level of c-Met mRNA and protein was inhibited after lncRNA-TUG1 was silenced (P<0.05). lncRNA-TUG1 shows an up-regulated expression in GC tissue and that bears a correlation with clinicopathological features of malignant tumor. lncRNA-TUG1 can promote the transferring and invading capacity of GC by inhibiting the pathway of microRNA-144/c-Met.

The role of miR-139-5p and miR-206 in non-small cell lung cancer (NSCLC) by targeting c-Met

Increasing evidences have confirmed that ectopic miRNAs are key regulatory factors in various types of cancers. Both hsa-miRNA-206 (miR-206) and hsa-miRNA-139-5p (miR-139-5p) have been demonstrated that possess anticancer properties in a variety of tissues and organs. In our recent investigations, we discovered miR-139-5p and miR-206 played a crucial role in lung cancer progression. There were extremely low levels of miR-206 and miR-139-5p in NSCLC (non-small cell lung cancer) cell lines as well as expression of them were inhibited in lung cancer tissues, respectively. Moreover, we found that miR-206 through targeting 3′-UTR（3'-untranslated region ）of Bcl-2 and c-Met mRNA that promoted cell apoptosis as well as suppressed non-small cell lung cancer SK-MES-1 and A549 cells colony formation, growth, invasion and metastasis. The analogical phenomenon was also discovered in miR-139-5p that targeted tumorigenic c-Met, which contributed to promotion of apoptosis and inhibition of cell proliferation and migration in non-small cell lung cancer.

Low c-Met expression levels are prognostic for and predict the benefits of temozolomide chemotherapy in malignant gliomas.

Aberrant c-Met has been implicated in the development of many cancers. The objective of this study was to identify an unfavorable prognostic marker that might guide decisions regarding clinical treatment strategies for high-grade gliomas. C-Met expression was measured using immunohistochemistry in 783 gliomas, and we further analyzed c-Met mRNA levels using the Agilent Whole Genome mRNA Microarray in 286 frozen samples. In vitro, we performed cell migration and invasion assays. Cell sensitivity to temozolomide (TMZ) chemotherapy was determined using MTT assays. Both mRNA and protein levels of c-Met were significantly associated with tumor grade progression and inversely correlated with overall and progression-free survival in high-grade gliomas (all P < 0.0001). These findings were nearly consistent at the mRNA level across 3 independent cohorts. Multivariable analysis indicated that c-Met was an independent prognostic marker after adjusting for age, preoperative Karnofsky Performance Status (KPS) score, the extent of resection, radiotherapy, TMZ chemotherapy, and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status. Further analysis in vitro revealed that downregulating the expression of c-Met dramatically inhibited cell migration and invasion capacities, enhanced sensitivity to TMZ chemotherapy in H4 and U87 glioma cells. Our results suggest that c-Met may serve as a potential predictive maker for clinical decision making.

Partial cloning of c-MET cDNA and its tissue distribution in Arabian camel (Camelus dromedarius)

The present study was, aimed to clone Hepatic growth factors receptor (c-Met). Arabian camels RT-PCR was conducted using liver tissue RNA and primers designed based on data from different animal species published in gene bank. The PCR product was sequenced and analysed using different bioinformatics programs. The results confirmed that the obtained sequence is related to c-Met gene family. The nucleotides sequence was deposited in the DDJB with accession number KC794957. Furthermore, the data showed base frequencies of A = 27.5%, C = 23.9%, G = 20.6% and T = 28.00%. Of the 600 nucleotides used for tree analyses, 454 were constant and 146 were variables. The neighbour-joining tree showed clustering of the species of family Camelidae with each other with strong bootstrapping (100 BP for MP and NJ methods). The deduced amino acids showed two non-synonymous substitutions discriminating C. dormedarius from other camelids; aspartic acid (D94) into histidine (H94) in other camelids at G281→C281 and glutamine (Q144) into histidine (H144) at A430 →C430. The results exhibited ubiquitous expression of c-Met mRNA in the tested tissues; kidney, liver, skeletal muscle, spleen, testis and heart. The obtained results are expected to be important for addressing the genetic diversity of the Afro-Arabian camel and clarifying the relationships among its available breeds.

Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells Derived from Human Arteries: A Novel Strategy to Accelerate Vascular Ulcer Wound Healing.

Vascular ulcers are a serious complication of peripheral vascular disease, especially in diabetics. Several approaches to treat the wounds are proposed but they show poor outcomes and require long healing times. Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is a pleiotropic cytokine exerting many biological activities through the c-Met receptor. This study was aimed at verifying whether HGF/SF influences proliferation, migration, and angiogenesis on mesenchymal stem cells isolated from human arteries (hVW-MSCs). hVW-MSCs were exposed to NIBSC HGF/SF (2.5, 5, 10, and 70 ng/mL) from 6 hrs to 7 days. HGF and c-MET mRNA and protein expression, cell proliferation (Alamar Blue and Ki–67 assay), migration (scratch and transwell assays), and angiogenesis (Matrigel) were investigated. hVW-MSCs displayed stemness features and expressed HGF and c-MET. HGF/SF did not increase hVW-MSC proliferation, whereas it enhanced the cell migration, the formation of capillary-like structures, and the expression of angiogenic markers (vWF, CD31, and KDR). The HGF/SF effects on hVW-MSC migration and angiogenic potential are of great interest to accelerate wound healing process. Local delivery of HGF/SF could therefore improve the healing of unresponsive vascular ulcers.

Effect of bevacizumab on proliferation and invasion of human lung cancer A549 cells

To study the effect and mechanism of bevacizumab on proliferation and invasion of human lung cancer A549 cells. A549 cells were treated with bevacizumab. Proliferation and invasion of the bevacizumab-treated A549 cells were detected using cell counting kit CCK-8 and Transwell assay, respectively. The expression of the mRNA and protein of MMP-2, MMP-9 and c-Met were detected by real-time PCR and Western blotting, respectively. Proliferation activity was inhibited at the concentration of 10 µg/ml and promoted at the concentration of 100 µg/ml bevacizumab. Bevacizumab in the concentration of 50 µg/ml had a stronger inhibitory effect on the invasion of A549 cells (16 406.19 ± 5 674.23 penetrated cells) than that of control group (36 108.68 6 263.83, P<0.05). The real-time PCR showed that bevacizumab had a stronger inhibitory effect on the expression of MMP-2 and MMP-9 mRNA at the concentration of 50 µg/ml and on the expression c-Met mRNA at the concentration of 10 µg/ml bevacizumabin the A549 cells. However bevacizumab at the concentration of 100 µg/ml showed a promoting effect on the expression of MMP-2, MMP-9 and c-Met mRNA (1.82 ± 0.31, 1.60 ± 0.25, 2.63 ± 0.48), significantly higher than that of the control group (1.00 ± 0.19, 1.00 ± 0.23, 1.00 ± 0.22, P<0.05). The expression of MMP-2, MMP-9 and c-Met mRNA and protein was inhibited by 10 µg/ml bevacizumab in a time-dependent manner. The Western blot assay showed that bevacizumab had a bi-directional effect on the expression of MMP-2 and c-Met proteins in the A549 cells: a promoting effect at 100 µg/ml and inhibitory effect on the expression of MMP-2 at 50 µg/ml bevacizumab, and inhibitory effect on the expression of c-Met protein at 10 µg/ml bevacizumab. Our findings indicate that in a certain range of concentrations, bevacizumab has prominent inhibitory effect on the proliferation and invasion of A549 cells. However,over the concentration of 100 µg/ml, bevacizumab shows a weakening anti-invasion effect, even has a promoting effect on cell proliferation. This phenomenon may be related to the inhibiting effect on the expression of MMP-2 and c-Met proteins in a non-concentration-dependent manner by bevacizumab.

Abstract 813: Human papillomavirus 16 oncoprotein E6 upregulates c-Met partially through p53 in squamous cell carcinoma of the head and neck

Abstract Background: Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) carries a distinct clinical behavior. The c-Met oncogene is an important driver for tumorigenesis, however, its relationship with HPV oncoproteins remains unclear. The present study aims to uncover the role of HPV in regulation of c-Met in OPSCC. Methods: We first knocked down the HPV oncogene E6 by siRNA to check alteration of c-Met expression through western blot and qRT-PCR in several HPV-positive HNSCC cell lines and the cervical cancer cell line CaSki. Next, we tested if E6 regulated c-Met expression is p53 dependent. Simultaneous knockdown of p53 and E6 was compared to E6 knockdown alone for the impact of p53 on c-Met expression using western blot analysis. In addition, the effects of c-Met inhibition by both siRNA and a c-Met specific inhibitor SU11274 on cell proliferation, migration, and colony formation were tested by sulforhodamine B colorimetric assay (SRB), wound healing assay, and colony formation assay, respectively, in HPV-positive cancer cell lines. Using retrospectively collected tissue specimens from 78 patients with OPSCC (N = 58 for HPV-positive vs. N = 20 for HPV-negative), we stained for c-Met and p53 by immunohistochemistry and correlated their expression with HPV status determined by HPV DNA in situ hybridization as well as other clinical characteristics. Results: E6 knockdown significantly decreased c-Met protein and mRNA expression in HPV-associated HNSCC and CaSki cell lines. E6 regulated c-Met expression was partially blocked by the elimination of p53. c-Met inhibition significantly decreased cell proliferation, migration, and colony formation. In patient samples, multivariate analysis revealed a significant association of c-Met expression with HPV status (positive vs. negative, OR = 4.11, 95%CI: 1.16-14.55, P = 0.028) as well as tumor stage (T1/2 vs. T3/4, OR = 0.27, 95%CI: 0.08-0.93, P = 0.039). In T3/T4 stage patients (N = 21), c-Met expression was significantly higher in HPV-positive patients and inversely related with p53 levels, supporting an axis of E6-p53-c-Met regulation. Conclusion: Our results show that c-Met expression is upregulated by HPV E6, which is partially mediated by p53. The data suggest that targeting c-Met may serve as a novel approach for treating HPV-associated OPSCC. (This study was supported by grants from Small Business Innovation Research (SBIR) Program (HHSN261201200097C), National Institutes of Health (R33 CA161873), and National Cancer Institute (NCI P50 CA 128613, Head and Neck SPORE). Citation Format: Guoqing Qian, Dongsheng Wang, Kelly R. Magliocca, Praveen Duggal, Sreenivas Nannapaneni, Sungjin Kim, Zhengjia Chen, Dong M. Shin, Nabil F. Saba, Zhuo G. Chen. Human papillomavirus 16 oncoprotein E6 upregulates c-Met partially through p53 in squamous cell carcinoma of the head and neck. [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 813. doi:10.1158/1538-7445.AM2015-813

Abstract 794: Prognostic value of high c-Met expression in patients with poor prognosis pancreatic adenocarcinoma following surgical resection: comparison of three c-Met scoring methods and exploration of underlying mechanisms of c-Met overexpression

Abstract Context: The HGF/c-Met pathway is an hypoxia-inducible pathway involved in tumor-stroma interactions and invasion in pancreatic ductal adenocarcinoma (PDAC). Assessment of c-Met expression is a critical issue as c-Met inhibitors are under clinical development and are suggested to display antitumor activity only in high c-Met tumors. We aimed to assess the prognostic value of c-Met overexpression in PDAC and explore underlying mechanisms (hypoxia, gene amplification, post transcriptional deregulation). Patients and methods: Patients (Pts) with resected PDAC who had received no perioperative chemo/radiotherapy were retrospectively selected. c-Met immunostaining was graded using a simplified score (high c-Met: ≥20% of cancer cells with 3+ staining) and compared to a standard scale combining staining surface and intensity (SI score) and to the MetMab score. Concordance between entire section and tissue microarray (TMA) was assessed and a computer-assisted quantification algorithm was developed (Aperio® software). Hypoxia was assessed by visual grading of HIF-1α/CA9 immunostaining, necrosis, and automated microvascular density (Aperio®). c-Met gene copy number was assessed by fluorescent in situ hybridization (FISH) and Taqman® based copy number variation (CNV) assay. c-Met mRNA levels were quantified using reverse transcription PCR (RT-PCR). c-Met pathway activation was assessed by immunostaining of phospho-c-Met, phospho-GAB1 and downstream signaling (pAKT, pERK, and pSTAT3) and graded visually. Clinical, pathological, and molecular biomarkers were correlated with disease-free (DFS) and overall (OS) survivals. Results: Thirty-seven Pts were analyzed. The simplified c-Met score displayed the best prognostic value and reproducibility vs both the SI score and the MetMab score; using this score: (a) high c-Met (7/37) was associated with shorter DFS (6.3 vs 33.0 months, HR: 3.456, p = 0.0035) and OS (10.8 vs 39.0 months, HR: 4.257, p = 0.0006); and (b) the kappa index was 0.773±0.122. In multivariate analysis, high c-Met was independently associated with both DFS and OS. c-Met expression was concordant on entire section and TMA in 87.9% of cases, and quantifiable using a specific computer-assisted algorithm. There was no correlation between hypoxia-related markers and c-Met expression. FISH and CNV analysis did not show c-Met gene amplification. mRNA quantification and downstream pathway activation data will be presented. Conclusion: c-Met is an independent prognostic marker in resected PDAC that may help identifying Pts at high risk of recurrence and poor survival. High c-Met expression was not associated with hypoxia or gene amplification in this study. The simplified c-Met scale is a robust and reproducible scoring method that could be used for ongoing larger studies on TMA. Citation Format: Cindy Neuzillet, Annemilaï Tijeras-Raballand, Jérôme Raffenne, Armand de Gramont, Pierre Bedossa, Valérie Paradis, Alain Sauvanet, Jean-Baptiste Bachet, Eric Raymond, Pascal Hammel, Anne Couvelard, Jérôme Cros. Prognostic value of high c-Met expression in patients with poor prognosis pancreatic adenocarcinoma following surgical resection: comparison of three c-Met scoring methods and exploration of underlying mechanisms of c-Met overexpression. [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 794. doi:10.1158/1538-7445.AM2015-794

Abstract 3131: Prognostic significance of miR-34a and its inverse correlation with c-MET and CDK6 in lung adenocarcinoma

Abstract Background: The microRNA(miR)s affect all aspects of cellular physiology including proliferation, apoptosis, development, aging, and metabolism. miR-34a has tumor suppressive role in various human cancers including non-small cell lung cancer (NSCLC). Recently, various targets of miR-34a including c-MET and G1-phase regulators have been reported in NSCLC cell lines. The aim of this study is to determine whether miR-34a is correlated with c-MET and G1 phase regulators such as cyclin dependent kinase (CDK) 4, CDK6 and cyclin D (CCND)1 in human NSCLC and assess the correlation between miR-34a and clinicopathologic characteristics and recurrence free survival (RFS). Material and Methods: In fresh frozen, paired tumor/normal adjacent tissue samples from 58 NSCLC patients underwent curative resection, expressions of miR-34a and c-MET, CDK4/6, and CCND1 were investigated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Results: There was inverse correlation between miR-34a and c-MET (R = -0.316; P = 0.003) and CDK6 (R = -0.4582; P = 0.004), however, neither CDK4 (P = 0.556) nor CCND1 (P = 0.143). Of 14 patients with low miR-34a, the patients showing high expression of c-MET protein were 12 (85.7%), whereas, in 5 patients with high miR-34a, high expression of c-MET protein was observed in 3 (60.0%). The expression level of miR-34a was significantly lower in the patients with squamous cell histology (P &amp;lt; 0.001) and lymphatic invasion (P = 0.001). The patients expressing high level of miR-34a were significantly associated with a longer RFS (55.6 months; 95% CI, 47.83-63.36 vs 21.6 months; 95% CI, 17.42-25.82, P = 0.020, respectively) for adenocarcinoma subtype. We could not detect any correlation between the c-MET and RFS (P = 0.666), however, adenocarcinoma patients exhibiting low c-MET mRNA and high miR-34a expression had a significantly longer RFS (54.4 months; 95% CI, 44.59-64.20 vs 18.7 months; 95% CI, 8.93-28.42, P = 0.042). Likewise, although CDK6 expression was not associated with RFS (P = 0.601), the adenocarcinomas having low CDK6 mRNA and high miR-34a expression tended to be longer RFS (55.1 months; 95% CI, 46.40-67.73 vs 27.8 months; 95% CI, 23.04-32.56, P = 0.059). In univariate Cox regression analysis, the relative risk (RR) for recurrence for adenocarcinoma patients with low miR-34a expression was 8.1 (95% CI, 1.01-65.58; P = 0.049) as well as with TNM stage, LN involvement, and the presence of lymphatic invasion of 13.6 (95% CI, 2.93-62.64; P = 0.001), 4.2 (95% CI, 1.05-16.76; P = 0.043) and 7.1 (95% CI, 1.46-34.00; P = 0.015), respectively. In multivariate analysis, miR-34a retained its prognostic independency for RFS (RR, 11.5; 95% CI, 1.31-100.68; P = 0.027). Conclusions: We demonstrated the inverse correlation between miR-34a and c-MET and CDK6 in human NSCLC and their prognostic significance for RFS especially in lung adenocarcinoma. Citation Format: Ji Hyung Hong, Kang San Roh, Sung-Suk Suh, Suk Chan Lee, Jae Ho Byun, Myung ah Lee, Jin Hyoung Kang. Prognostic significance of miR-34a and its inverse correlation with c-MET and CDK6 in lung adenocarcinoma. [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 3131. doi:10.1158/1538-7445.AM2015-3131

Deregulation of ARID1A, CDH1, cMET and PIK3CA and target-related microRNA expression in gastric cancer.

Genetic and epigenetic alterations play an important role in gastric cancer (GC) pathogenesis. Aberrations of the phosphatidylinositol-3-kinase signaling pathway are well described. However, emerging genes have been described such as, the chromatin remodeling gene ARID1A. Our aim was to determine the expression levels of four GC-related genes, ARID1A, CDH1, cMET and PIK3CA, and 14 target-related microRNAs (miRNAs). We compared mRNA and miRNA expression levels among 66 gastric tumor and normal adjacent mucosa samples using quantitative real-time reverse transcription PCR. Moreover, ARID1A, cMET and PIK3CA protein levels were assessed by immunohistochemistry (IHC). Finally, gene and miRNAs associations with clinical characteristics and outcome were also evaluated. An increased cMET and PIK3CA mRNA expression was found in 78.0% (P = 2.20 × 10-5) and 73.8% (P = 1.00 × 10-3) of the tumors, respectively. Moreover, IHC revealed that cMET and PIK3CA expression was positive in 63.6% and 87.8% of the tumors, respectively. Six miRNAs had significantly different expression between paired-samples, finding five up-regulated [miR-223-3p (P = 1.65 × 10-6), miR-19a-3p (P = 1.23 × 10-4), miR-128-3p (P = 3.49 × 10-4), miR-130b-3p (P = 1.00 × 10-3) and miR-34a-5p (P = 4.00 × 10-3)] and one down-regulated [miR-124-3p (P = 0.03)]. Our data suggest that cMET, PIK3CA and target-related miRNAs play an important role in GC and may serve as potential targets for therapy.

High basal Wnt signaling is further induced by PI3K/mTor inhibition but sensitive to cSRC inhibition in mammary carcinoma cell lines with HER2/3 overexpression.

BackgroundElevated basal, ligand-independent, Wnt signaling in some canine breast cancer cells is not caused by classical mutations in APC, β-Catenin or GSK3β but, at least partially, by enhanced LEF1 expression. We examined the expression and function of EGFR/HER-regulated pathways on the ligand-independent Wnt signaling.MethodsTwelve canine mammary tumor cell lines with previously reported differential basal Wnt activity were used. The expression levels of genes related to EGF-signaling were analyzed by cluster analysis. Cell lines with a combined overexpression of EGF-related genes and enhanced basal Wnt activity were treated with PI3K/mTor or cSRC inhibitors or transfected with a construct expressing wild-type PTEN. Subsequently, effects were measured on Wnt activity, cell proliferation, gene expression and protein level.ResultsHigh basal Wnt/LEF1 activity was associated with overexpression of HER2/3, ID1, ID2, RAC1 and HSP90 together with low to absent cMET and PTEN mRNA expression, suggesting a connection between Wnt- and HER-signaling pathways. Inhibition of the HER-regulated PI3K/mTor pathway using the dual PI3K/mTor inhibitor BEZ235 or the mTor inhibitor Everolimus® resulted in reduced cell proliferation. In the cell line with high basal Wnt activity, however, an unexpected further increased Wnt activity was found that could be greatly reduced after inhibition of the HER-regulated cSRC activity. Inhibition of the PI3K/mTor pathway was associated with enhanced expression of β-Catenin, Axin2, MUC1, cMET, EGFR and HER2 and a somewhat increased β-Catenin protein content, whereas cSRC inhibition was associated with slightly enhanced HER3 and SLUG mRNA expression. A high protein expression of HER3 was found only in a cell line with high basal Wnt activity.ConclusionsHigh basal Wnt activity in some mammary cancer cell lines is associated with overexpression of HER-receptor related genes and HER3 protein, and the absence of PTEN. Inhibition of the PI3K/mTor pathway further stimulated, however, canonical Wnt signaling, whereas the inhibitory effect with the cSRC inhibitor Src-I1 on the Wnt activity further suggested a connection between Wnt and HER2/3-signaling.