MET mRNA Research Articles

Down‐regulation of MET in hippocampal neurons of Alzheimer's disease brains

We found that mRNA of MET, the receptor of hepatocyte growth factor (HGF), is significantly decreased in the hippocampus of Alzheimer's disease (AD) patients. Therefore, we tried to determine the cellular component-dependent changes of MET expressions. In this study, we examined cellular distribution of MET in the cerebral neocortices and hippocampi of 12 AD and 11 normal controls without brain diseases. In normal brains, MET immunoreactivity was observed in the neuronal perikarya and a subpopulation of astrocytes mainly in the subpial layer and white matter. In AD brains, we found marked decline of MET in hippocampal pyramidal neurons and granule cells of dentate gyrus. The decline was more obvious in the pyramidal neurons of the hippocampi than that in the neocortical neurons. In addition, we found strong MET immunostaining in reactive astrocytes, including those near senile plaques. Given the neurotrophic effects of the HGF/MET pathway, this decline may adversely affect neuronal survival in AD cases. Because it has been reported that HGF is also up-regulated around senile plaques, β-amyloid deposition might be associated with astrocytosis through the HGF signaling pathway.

Abstract A241: Differential roles of STAT3 depending on the mechanism of STAT3 activation in gastric cancer cells.

Abstract Background: Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated in response to growth factors and cytokines, and which contributes to the regulation of cell proliferation, apoptosis, and motility in many human tumor types. Methods: We investigated the mechanisms of STAT3 activation and the function of STAT3 depending on its mechanism of activation in gastric cancer cells. Results: All cell lines positive for constitutive MET activation manifested STAT3 phosphorylation. The MET-tyrosine kinase inhibitor (TKI) and cell transfection with a small interfering RNA (siRNA) specific for MET mRNA inhibited STAT3 phosphorylation in MET-activated cells, indicating that STAT3 activation is linked to MET signalling. Forced expression of a constitutively active form of STAT3 also attenuated MET-TKI-induced apoptosis, suggesting that inhibition of STAT3 activity contributes to MET-TKI-induced apoptosis in MET-activated gastric cancer cells. On the other hand, MKN1 and MKN7 cells, both of which are negative for MET activation, produced interleukin-6 (IL-6) that activated STAT3 through the Janus kinase (JAK) pathway but which did not appear to be required for cell survival. Depletion of STAT3 by transfection with a specific siRNA inhibited both the migration and invasion activities of MKN1 and MKN7 cells, suggesting that STAT3 activated by IL-6 contributes to regulation of cell motility. Conclusion: Our data thus show that activated STAT3 contributes to either cell survival or cell motility in gastric cancer cells, and that these actions are related to different mechanisms of STAT3 activation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A241. Citation Format: Wataru Okamoto, Isamu Okamoto, Katsuya Tuchihara, Kazuyoshi Yanagihara, Kazuto Nishio, Kazuhiko Nakagawa. Differential roles of STAT3 depending on the mechanism of STAT3 activation in gastric cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A241.

Abstract C25: The use of a patient derived tumor xenograft collection to assess different Met and HGF detection methods and their predictive values for therapy response.

Abstract Aims: Met inhibitors are targeted drugs which hold promise for the treatment of tumors with HGF/Met pathway activation. To accurately identify such tumors, relevant and well validated predictive biomarkers are needed. In the present study, we used our large collection of tumor models to investigate the value of Met and HGF as determined by multiple methods for predicting sensitivity to Met-inhibitors. Materials and Methods: HGF and Met were assessed at DNA, RNA and protein levels in a panel of 292 tumor models covering 15 different histotypes. The tumors were analyzed for Met amplification and protein expression in a tissue microarray analysis using standard FISH and IHC assays. Met and HGF were also assessed for gene copy number variations, mutations, mRNA and protein expression levels and phosphorylation. For relevant tumors, sensitivity to the Met inhibitors PF-04217903 and/or JNJ-38877605 was evaluated by Tumor Clonogeneic Assays (TCA) and/or in vivo testing. Results: Regarding Met and HGF expression, results obtained at the DNA, RNA and protein level were highly consistent. Affymetrix analyses showed expression of Met mRNA in a large proportion of the models, with a good correlation to protein levels detected by IHC or Western blot. Western blot analyses revealed that most tumors which overexpressed Met showed a high phosphorylation level of the receptor. The FISH and SNP6 analyses allowed identification of a subset of tumors with Met gene amplification, associated with overexpression of Met mRNA, protein and receptor phosphorylation. These models were sensitive to Met-inhibitors in both TCA and in vivo assays. In addition, a subset of tumor xenografts displayed very high Met expression levels and sensitivity to Met-inhibitors, although they did not carry an amplified Met gene. IHC analyses revealed that Met was diversely expressed in some tumors, indicative of intra tumoral heterogeneity that could impact on response to Met inhibitors. HGF, both at mRNA and protein level, was detectable in a subset of tumors, including tumors which did not overexpress Met. Some of these tumors were strongly sensitive to Met inhibitors, suggesting a predictive value of HGF expression. Of interest, these tumors had different topography, such as the liver, the kidney and the lung as well as sarcomas. Conclusion: This study reveals Met overexpression as one of the major determinants of tumor sensitivity to Met-inhibitors. Accurate assessment of Met levels by mRNA array, western blot and IHC analyses showed that Met overexpression was not only due to gene amplification, demonstrating the importance of gene expression assessment complementary to gene copy number evaluation. The in situ analysis also demonstrated that intra-tumoral heterogeneity in Met expression levels can be of importance. Furthermore, HGF levels may be a good predictor for response to Met inhibitors in tumors. Taken together, out data show that in addition to the well-established ex vivo 3D assay with PDX, Met and HGF expression emerges as a powerful parameter to predict tumor response to cMet-directed therapies in vivo. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C25. Citation Format: Vincent Vuaroqueaux, Torsten Giesemann, Luigi Tornillo, Armin Maier, Rebekka Krumbach, Anne-Lise Peille, Tim Kees, Jianing Guo, Frederic Foucault, Zakia Amalou, Serenella Eppenberger, Luigi Terracciano, Heinz-Herbert Fiebig. The use of a patient derived tumor xenograft collection to assess different Met and HGF detection methods and their predictive values for therapy response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C25.

Induction of MET by Ionizing Radiation and Its Role in Radioresistance and Invasive Growth of Cancer

Ionizing radiation (IR) is effectively used in cancer therapy. However, in subsets of patients, a few radioresistant cancer cells survive and cause disease relapse with metastatic progression. The MET oncogene encodes the hepatocyte growth factor (HGF) receptor and is known to drive "invasive growth", a regenerative and prosurvival program unduly activated in metastasis. Human tumor cell lines (MDA-MB-231, MDA-MB-435S, U251) were subjected to therapeutic doses of IR. MET mRNA, and protein expression and signal transduction were compared in treated and untreated cells, and the involvement of the DNA-damage sensor ataxia telangiectasia mutated (ATM) and the transcription factor nuclear factor kappa B (NF-κB) in activating MET transcription were analyzed by immunoblotting, chromatin immunoprecipitation, and use of NF-κB silencing RNA (siRNA). Cell invasiveness was measured in wound healing and transwell assays, and cell survival was measured in viability and clonogenic assays. MET was inhibited by siRNA or small-molecule kinase inhibitors (PHA665752 or JNJ-38877605). Combinations of MET-targeted therapy and radiotherapy were assessed in MDA-MB-231 and U251 xenografts (n = 5-6 mice per group). All P values were from two-sided tests. After irradiation, MET expression in cell lines was increased up to fivefold via activation of ATM and NF-κB. MET overexpression increased ligand-independent MET phosphorylation and signal transduction, and rendered cells more sensitive to HGF. Irradiated cells became more invasive via a MET-dependent mechanism that was further enhanced in the presence of HGF. MET silencing by siRNA or inhibition of its kinase activity by treatment with PHA665752 or JNJ-38877605 counteracted radiation-induced invasiveness, promoted apoptosis, and prevented cells from resuming proliferation after irradiation in vitro. Treatment with MET inhibitors enhanced the efficacy of IR to stop the growth of or to induce the regression of xenografts (eg, at day 13, U251 xenografts, mean volume increase relative to mean tumor volume at day 0: vehicle = 438%, 5 Gy IR = 151%, 5 Gy IR + JNJ-38877605 = 76%; difference, IR vs JNJ-38877604 + IR = 75%, 95% CI = 59% to 91%, P = .01). IR induces overexpression and activity of the MET oncogene through the ATM-NF-κB signaling pathway; MET, in turn, promotes cell invasion and protects cells from apoptosis, thus supporting radioresistance. Drugs targeting MET increase tumor cell radiosensitivity and prevent radiation-induced invasiveness.

Prognostic implication of MET overexpression in myxofibrosarcomas: an integrative array comparative genomic hybridization, real-time quantitative PCR, immunoblotting, and immunohistochemical analysis

It remains obscure in myxofibrosarcoma about the basis of tumorigenesis, progression, and metastasis. Chromosome 7 gains are common in some sarcomas, including myxofibrosarcoma, whereas the specific oncogenes are yet to be characterized. We performed an integrative study of MET gene at 7q31.2 to elucidate its implication in myxofibrosarcoma. Focused on candidate oncogenes on chromosome 7, 385K array comparative genomic hybridization was used to profile DNA copy number alterations of 12 samples. MET transcript was successfully quantified by real-time RT–PCR for 16 laser-microdissected tumors and two myxofibrosarcoma cell lines (NMFH-1, OH931). MET immunoexpression was assessable in 86 primary localized tumors with follow-up. To analyze endogenous MET expression and activation, NMFH-1 and OH931 cells, both with wild-type MET gene, were subjected to Western blotting and hepatocyte growth factor-treated NMFH-1 cells were evaluated for the kinetics of MET tyrosine phosphorylation. Non-random large-scale gains on 7q were detected in five cases, delineating three recurrent amplicons, 7q21.11–7q21.3, 7q22.1–22.3, and 7q31.1–7q32.3, in which the locus of MET displayed increased copy number, among others. MET mRNA was upregulated in OH931, NMFH-1, and nine tumors (56%), whereas neither gene dosage nor mRNA expression of MET was associated with clinicopathological factors. In contrast, MET protein overexpression, present in 67% of cases, was highly related to deep location (P=0.004), higher grades (P=0.001), and more advanced stages (P<0.001). Importantly, MET overexpression independently portended inferior metastasis-free survival (P=0.004) and overall survival (P=0.0221). Expressing activating phospho-MET at Tyr1234/Tyr1235, OH931 cells had more abundant total MET than NMFH-1 cells, whereas the latter became promptly phosphorylated on stimulation of hepatocyte growth factor. In primary myxofibrosarcomas, MET overexpression, as a frequent event, is likely driven by 7q gains with mRNA upregulation, associated with important prognosticators, and independently predictive of worse outcomes, highlighting its possible causative function in tumor aggressiveness and potentiality as a therapeutic target.

The Met protooncogene is a transcriptional target of NFkappaB: Implications for cell survival

NF kappaB transcription factor regulates gene expression in response to extracellular stimuli such as TNF alpha. The genes regulated by NF kappaB encode for proteins which control cell growth and survival. Met is the tyrosine kinase receptor for hepatocyte growth factor, and it too promotes cell mitogenesis and survival. Previously, we showed that Met gene expression is regulated by TNF alpha. In this report, we identify and characterize a TNF alpha response element in the Met promoter. This element contains tandem C/EBP sites adjacent to an NF kappaB site. Binding of the NF kappaB p65 subunit and C/EBP beta to this element is induced by TNF alpha. To examine the interplay of NF kappaB and Met in vivo, we determined that Met mRNA and protein levels are reduced in the livers of p65-/- mice as compared to controls. In p65-/- mouse embryonic fibroblasts (MEFs), Met induction by TNF alpha is abrogated while Met's basal gene expression is reduced by half as compared to controls. When overexpressed in p65-/- MEFs, Met confers resistance to TNF-alpha-mediated cell death. Conversely, expression of dominant negative Met in wild-type MEFs renders them sensitive to cell death induced by TNF alpha. A similar response following TNF alpha challenge was observed in hepatocytic cells treated with siRNA to knockdown endogenous Met. Together, these results indicate that the Met gene is a direct target of NF kappaB and that Met participates in NF kappaB-mediated cell survival.

MetMAb, the One-Armed 5D5 Anti-c-Met Antibody, Inhibits Orthotopic Pancreatic Tumor Growth and Improves Survival

The hepatocyte growth factor (HGF) and its receptor, c-Met, have been implicated in driving proliferation, invasion, and poor prognosis in pancreatic cancer. Here, we investigated the expression of HGF and c-Met in primary pancreatic cancers and described in vitro and in vivo models in which MetMAb, a monovalent antibody against c-Met, was evaluated. First, expression of HGF and MET mRNA was analyzed in 59 primary pancreatic cancers and 51 normal samples, showing that both factors are highly expressed in pancreatic cancer. We next examined HGF responsiveness in pancreatic cancer lines to select lines that proliferate in response to HGF. Based on these studies, two lines were selected for further in vivo model development: BxPC-3 (c-Met(+), HGF(-)) and KP4 (c-Met(+), HGF(+)) cells. As BxPC-3 cells are responsive to exogenous HGF, s.c. tumor xenografts were grown in a paracrine manner with purified human HGF provided by osmotic pumps, wherein MetMAb treatment significantly inhibited tumor growth. KP4 cells are autocrine for HGF and c-Met, and MetMAb strongly inhibited s.c. tumor growth. To better model pancreatic cancer and to enable long-term survival studies, an orthotopic model of KP4 was established. MetMAb significantly inhibited orthotopic KP4 tumor growth in 4-week studies monitored by ultrasound and also improved survival in 90-day studies. MetMAb significantly reduced c-Met phosphorylation in orthotopic KP4 tumors with a concomitant decrease in Ki-67 staining. These data suggest that the HGF/c-Met axis plays an important role in the progression of pancreatic cancer and that targeting c-Met therein may have therapeutic value.

Targeting MET transcription as a therapeutic strategy in multiple myeloma

Multiple myeloma (MM) is an incurable indolent malignancy with an average lifespan of 3 years, underscoring the need for new therapies. Studies have shown that the receptor MET and its ligand hepatocyte growth factor play an important role in proliferation, migration, adhesion, and survival of MM cells. Hence, an effective way to decrease MET receptor may act as a viable therapeutic option. Since MET mRNA and protein have short half-lives, we hypothesized that transcription inhibitor will reduce MET transcript and protein levels and this will lead to cell death. Pharmacological (flavopiridol) and molecular (shRNA) transcription inhibitor were used to impede formation of MET transcripts. The diminution of global RNA synthesis with flavopiridol was related to phosphorylation status of Ser residues (r (2) = 0.90 and 0.92 for Ser2 and Ser5) on the C-terminal-domain of RNA polymerase II. This was accompanied with a time-dependent decrease in MET transcript, which reached to less than 30% (1 microM) and 10% (3 microM) by 24 h. This decline in transcript level was directly associated with a reduction in MET protein level (r (2) = 0.82) and resulted in cell death. Assessment of MET in MM survival was done by using shRNA targeted towards MET. When cells were infected with shRNA viral construct, there was increased cell death with a decline in MET transcript and protein. Taken together, our study demonstrates that MET plays a critical role in the survival and removal or lowering of MET by flavopiridol or shRNA results in the demise of MM cells.

MET overexpression in colorectal cancer (CRC) liver metastases predicts poor survival

METHODS: In 63 patients with CRC liver metastases, we quantified MET mRNA from microdissected tumor and adjacent normal liver by Real-Time RT-PCR. Using random primed cDNA and genespecific PCR, specimen threshold cycle was determined and standard curves generated. Samples were normalized using 18S RNA. Tumor MET gene overexpression was defined as mRNA levels above the median of normal adjacent liver. Overall survival (OS) was compared by Kaplan-Meier and Log-rank. Median follow-up for survivors was 44 months.

The HGF Receptor c-Met Is Overexpressed in Esophageal Adenocarcinoma

The hepatocyte growth factor (HGF) receptor, Met, has established oncogenic properties; however, its expression and function in esophageal adenocarcinoma (EA) remain poorly understood. We aimed to determine the expression and potential alterations in Met expression in EA. Met expression was investigated in surgical specimens of EA, Barrett's esophagus (BE), and normal esophagus (NE) using immunohistochemistry (IHC) and quantitative reverse transcriptase polymerase chain reaction. Met expression, phosphorylation, and the effect of COX-2 inhibition on expression were examined in EA cell lines. IHC demonstrated intense Met immunoreactivity in all (100%) EA and dysplastic BE specimens. In contrast, minimal immunostaining was observed in BE without dysplasia or NE specimens. Met mRNA and protein levels were increased in three EA cell lines, and Met protein was phosphorylated in the absence of serum. Sequence analysis found the kinase domain of c-met to be wild type in all three EA cell lines. HGF mRNA expression was identified in two EA cell lines. In COX-2-overexpressing cells, COX-2 inhibition decreased Met expression. Met is consistently overexpressed in EA surgical specimens and in three EA cell lines. Met dysregulation occurs early in Barrett's dysplasia to adenocarcinoma sequence. Future study of Met inhibition as a potential biologic therapy for EA is warranted.

MET and HGF predict outcome in colorectal cancer

Abstract Introduction: MET, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor (HGF), are critical in cellular proliferation, motility, and invasion. We hypothesize that elevated MET and HGF gene expression predicts metastatic spread and poor survival in colorectal (CR) cancer patients. Methods: In 60 primary CR adenocarcinomas, MET and HGF mRNA was quantified with Real-Time RT-PCR. Using random primed cDNA and gene-specific PCR, specimen threshold cycle was measured and standard curves generated. Tumor mRNA levels were extrapolated, normalized to 18S RNA and compared to pooled matched normal mucosa. Median follow-up for survival analysis was 6 years. Results: When compared with normal tissue, a > 2 fold elevation in MET mRNA was seen in 69% of tumors with 48% having > 10 fold increases. Elevated HGF mRNA was noted in 47% of tumors with 19% having a > 10 fold increase. MET and HGF mRNA levels were correlated (p = 0.01). 33 patients had low expression of both genes (LOW MET/HGF) while 27 patients had high expression of both (HIGH MET/HGF). HIGH MET/HGF was associated with nodal and distant metastases (Table). Further, HIGH MET/HGF gene expression predicted poor survival (RR = 2.3, p Factor High MET/HGF n (%) Low MET/HGF n (%) p Value Relative Risk All patients 27 (100) 33 (100) — — Grade 1 1 (4) 1 (3) 0.57 — 2 23 (85) 24 (72) 3 3 (11) 8 (24) T stage 1 1 (4) 1 (3) 0.96 — 2 5 (19) 8 (24) 3 18 (67) 21 (64) 4 3 (11) 3 (9) N stage 0 9 (33) 21 (64) 0.02 1.9 1/2 18 (67) 12 (36) M stage 0 15 (56) 26 (79) 0.05 2.1 1 12 (44) 7 (21) AJCC stage 1/2 5 (19) 18 (55) 0.004 1.8 3/4 22 (81) 15 (45) Conclusions: Over-expression of MET and HGF identifies a metastatic phenotype. These molecular markers may be exploited for identifying tumors with aggressive biology and are potential therapeutic targets.

Osteopontin-induced, integrin-dependent migration of human mammary epithelial cells involves activation of the hepatocyte growth factor receptor (Met).

Osteopontin (OPN) is a secreted glycophosphoprotein which induces migration of mammary carcinoma cells, and has been implicated in the malignancy of breast carcinoma. Hepatocyte growth factor (HGF) induces cell migration of several mammary epithelial cell (MEC) lines, via activation of its cognate receptor (Met). This study examines the mechanism of OPN-induced MEC migration, in terms of the cell surface integrins involved and induction of the HGF/Met pathway. Three different MEC cell lines were used, representing different stages of tumor progression: 21PT, non-tumorigenic; 21NT, tumorigenic; non-metastatic; and MDA-MB-435, tumorigenic, highly metastatic. Human recombinant OPN was found to induce the migration of all three lines. OPN-induced migration of 21PT and 21NT cells was alphavbeta5 and beta1-integrin dependent, and alphavbeta3-independent, while that of MDA-MB-435 cells was alphavbeta3-dependent. HGF also induced migration of all three cell lines, and a synergistic response was seen to HGF and OPN together. The increased migration response to OPN was found to be associated with an initial increase in Met kinase activity (within 30 min), followed by an increase in Met mRNA and protein expression. OPN-induced cell migration is thus mediated by different cell surface integrins in MEC lines representing different stages of progression, and involves activation of the HGF receptor, Met.

BIOLOGICAL ROLE OF HGF/MET PATHWAY IN RENAL CELL CARCINOMA

Purpose Several lines of evidence show that hepatocyte growth factor (HGF) and its receptor MET play a significant role in the progression of various cancers including renal cell carcinoma (RCC). Our objectives were to evaluate the gene expression of HGF and MET in RCC, and to examine the effect of HGF on the biological activities of cultured RCC cells. Materials and Methods We examined the gene expression of HGF and MET in 27 primary RCC tumors by quantitative competitive RT-PCR. The effects of HGF on in vitro chemoinvasion assay and the expression of matrix metalloproteinase-9 (MMP-9), and the induction of Fas-induced apoptosis were studied by transfection of HGF cDNA to cultured RCC cells, Caki-1. Results HGF mRNA and MET mRNA were detected in all surgical specimens. The level of expressed HGF mRNA was proportional with the volume of tumor (r = 0.50, p = 0.015). Caki-1 cells overexpressing HGF cells showed enhanced in vitro invasiveness in the chemoinvasion assay and increased activity of 92 kDa type IV collagenase (MMP-9). The sensitivity to Fas-induced cell death was reduced in HGF transfectants, which was reversed by the presence of anti-HGF antibody. Conclusions HGF enhanced the invasive properties of cultured RCC cells and inhibited Fas-induced apoptosis in vitro. Both HGF and MET mRNA were expressed in RCC tissues tested. Our results indicate that HGF/MET pathway may have a significant role in the progression of RCC.