MET mRNA Research Articles

Abstract 5867: Early activation of HGF-cMET serves as a bypass pathway to ALK and RET tyrosine kinase inhibitors in non-small cell lung cancer

Abstract Introduction: Alterations of the HGF-cMET axis, often through MET gene amplification, can act as resistance mechanism to tyrosine kinase inhibitors (TKIs) in patients with ALK and RET rearranged non-small cell lung cancer (NSCLC). The efficacy of targeting the cMET pathway through use of MET tyrosine kinase inhibitors continues to evolve. An unresolved question is whether the HGF-cMET pathway is activated early in cancer cells after ALK and RET TKI exposure. Here, we evaluate novel pre-clinical models to better characterize the onset of MET pathway-mediated resistance using murine cell lines and orthotopic murine models. Methods: Murine Eml4-Alk (EA1 and EA3) and Trim24-Ret (TR.1) cell lines were cultured with increasing doses of alectinib (EA1 and EA3) or pralestinib or selpercatinib (TR.1) until resistance was acquired. A cell line was established from an orthotopic TR.1 tumor that progressed on selpercatinib (TR.1-1092). Passage control cells and the TKI-resistant cultures were submitted to clonogenic growth assays in targeting TKI or crizotinib (MET TKI). Control and TKI-resistant cell lines were submitted to HGF ELISA, MET immunoblotting and RNAseq. Orthotopic TR.1 tumors were established in C57BL/6 mice and after ~2 weeks, daily treatment with selpercatinib was performed for 21 days with weekly mCT to monitor tumor size. At 21 days when the tumors were beginning to progress, half the mice were treated with selpercatinib and crizotinib (a MET TKI) while the other half continued on selpercatinib alone. Results: HGF levels (pg/μg) were significantly higher in alectinib-resistant EA1 cells vs. EA3 cells and was associated with retained sensitivity to crizotinib in alectinib-resistant EA1, but not EA3 cells. MET mRNA (but not gene copy number) and HGF mRNA/protein levels in pralsetinib and selpercatinib resistant TR.1 lines were significantly higher than controls and accompanied by acquired sensitivity to crizotinib. In C57BL/6 mice bearing orthotopic TR.1 tumors, the progression on selpercatinib occurring after 2-3 weeks of treatment was reversed in a durable manner by co-treatment with crizotinib. Conclusions: The HGF-cMET axis is activated early in response to ALK and RET TKI exposure through transcriptional mechanisms without evidence of MET gene amplification. These findings unveil a potential therapeutic window to disrupt the MET bypass pathway in ALK and RET rearranged NSCLC prior to overt clinical progression. Citation Format: Tejas Patil, Trista Hinz, Sharon Pine, Hatim Saabawy, Paul Bunn, Erin L. Schenk, Ross Camidge, Lynn Heasley. Early activation of HGF-cMET serves as a bypass pathway to ALK and RET tyrosine kinase inhibitors in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5867.

Abstract LB215: Transglutaminase 2 maintains hepatocyte growth factor signaling to enhance the epidermal cancer stem cell phenotype

Abstract Epidermal squamous cell carcinoma (SCC) is one of the most common cancers. Ultraviolet radiation exposure leads to DNA damage in keratinocyte stem cells leading to transformation. Epidermal cancer stem cells (ECS cells) display aggressive spheroid formation, invasion, and migration, and form rapidly growing and highly vascularized tumors. The transglutaminase 2 (TG2) cancer stem cell maintenance proteins is present at high levels in ECS cells and is required for maintenance of the ECS cell phenotype. Identifying how TG2 maintains the ECS cell phenotype is an important goal. RNA transcriptomic analysis, comparing gene expression in wild-type and TG2 knockout cells, reveals that hepatocyte growth factor (HGF) signaling is markedly reduced in TG2 knockout cells, a finding that was confirmed by showing that TG2 knockdown/knockout reduces HGF and MET mRNA and protein. HGF/MET signaling appears to play an important role in maintaining the cancer phenotype, as inhibition of HGF/MET reduces ECS cell spheroid formation, invasion and migration. Stem cell and EMT marker expression and MEK1/2 and ERK1/2 activity are also reduced. These changes phenocopy the response to TG2 knockdown. Treatment with MEK1/2 and ERK1/2 inhibitors suppresses the ECS cell phenotype indicating that MAPK signaling is a major HGF/MET target, and MET knockout reduces ECS cell tumor formation which is associated with reduced MEK1/2 and ERK1/2 activity. These findings suggest that TG2 maintains HGF/MET signaling which stimulates MEK1.2 and ERK1/2 activity to enhance the aggressive ECS cell cancer phenotype and drive tumor formation. Citation Format: Xi Chen, Gautam Adhikary, Suruchi Shrestha, Wen Xu, Richard Eckert. Transglutaminase 2 maintains hepatocyte growth factor signaling to enhance the epidermal cancer stem cell phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB215.

Continuing Cetuximab vs Bevacizumab plus chemotherapy after first progression in wild-type KRAS, NRAS and BRAF V600E metastatic colorectal cancer: a randomized phase II trial.

To evaluate the clinical efficacy of continuing cetuximab vs bevacizumab plus chemotherapy crossover after first progression to cetuximab regimen in wild-type KRAS, NRAS and BRAF V600E mCRC, we conducted this prospective, open-label and randomized phase 2 trial in three cancer centers from Oct 1, 2016 to July 1, 2020. Eligibility criteria included documented progressive disease during or after first-line treatment with cetuximab regimen; second biopsy confirmed as KRAS, NRAS and BRAF V600E wild-type mCRC. Patients were randomized to arm A (cetuximab+chemo) or arm B (bevacizumab+chemo) with second-line chemotherapy crossover. The primary end point was progression free survival (PFS). Secondary end points included objective response rate (ORR), overall survival (OS) and toxicity. Tissue VEGFA, ERBB2 and MET mRNA were examined by real time RT-PCR.A total of 104 patients (53 in arm A and 51 in arm B) were enrolled. Median PFS was 7.7 months (95% CI: 6.5-8.9) for arm A and 6.3 months (95% CI: 4.5-8.1) for arm B (p=0.931). Median OS was 18.2 months (95% CI: 14.5-21.9) for arm A and 16.4 months (95% CI: 14.2-18.6) for arm B (p=0.339). The ORR was 28.3% and 19.6% in arm A and arm B (p=0.31), respectively. MET mRNA was highly expressed in the cetuximab-progressed tumors, but treatment responsiveness to cetuximab or bevacizumab in each arm was not correlated with the MET expression level. The results showed no significant difference in PFS, OS and ORR between the two arms, but a trend in favor of the cetuximab continuation plus chemotherapy crossover was examined in all end points. High expression of MET in cetuximab-progressed tumors may indicate an existence of MET-dependent tumor cell population.

Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation

MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary.

A whole transcriptome sequencing (WTS)-based cMet dysregulation biomarker assay with correlates to cMet DNA mutations and cMet protein expression.

e15676 Background: Selective cMet kinase inhibitors (cMet-i) have demonstrated promising clinical activity as single-agents in non-small cell lung cancer (NSCLC) with cMet exon-14 skip mutations (MET∆14). APL-101 (PLB-1001) is a selective cMet-i and has demonstrated single-agent anti-tumor activity in secondary glioblastoma (GBM) harboring MET∆14 and/or MET fusion with evidence of blood-brain barrier permeability (Cell. 2018, 175: 1665-1678). A WTS-based cMet biomarker assay is being developed under Caris MI Transcriptome platform to predict clinical response to monotherapy APL-101 in tumors with cMet dysregulation (NCT03175224). Methods: In a pilot study, we used Caris WTS to analyze 202 patient samples of NSCLC (78%) and other tumor types (22%) mostly GBM and gastrointestinal tumors to establish key parameters of Caris WTS in detection of cMet dysregulation. These samples were also analyzed by DNA-seq and IHC to understand the correlations of Caris WTS to DNA-based cMet mutation detection and cMet protein expression. The samples included 63 (31%) MET∆14, 48 (24%) MET fusions based on WTS and covered a broad dynamic range in cMet expression and copy number variations. In order to establish a stable baseline against which to compare MET mRNA expression, 123 unique tissue samples from 93 unique non-tumor tissues covering 27 unique tissue types were analyzed for wild-type MET expression. Results: Concordance between WTS and DNA-seq in detection of MET∆14 was 84% based on 67 cases analyzed by both methods. WTS identified 45% positive MET∆14, compared to 35% by DNA-seq. Out of 11 discordant cases, 9 were positive by WTS and 2 positive by DNA-seq. The number of transcripts carrying MET∆14 variants correlated with cMet wild type mRNA expression (n = 63, r = 0.88, p< 0.0001). cMet mRNA expression correlated with total cMet protein by IHC in MET fusion subset (n = 43, ρ = 0.65, p= < 0.0001), MET∆14 subset (n = 55, ρ = 0.64, p< 0.0001), and in all samples (n = 175, ρ = 0.75, p< 0.0001). In 175 cases with both WTS and IHC results, 9 MET∆14 positive cases had no detectable cMet protein on plasma membrane albeit all showed a detectable level of total cMet protein. Conclusions: A cMet WTS assay has been established to identify patients with cMet dysregulation including MET∆14 and MET fusion. cMet IHC and DNA-seq may offer secondary utility to differentiate WTS-identified METΔ14 patients to further strengthen the prediction of clinical response to a cMet inhibitor.

Abstract 4905: Comprehensive characterization of MET alterations in a large cohort of 610 advanced non-small cell lung cancer patients

Abstract Background: A variety of alterations in MET have been described in advanced non-small cell lung cancer (NSCLC) patients, including gene amplification, protein overexpression, splicing variants and point mutations. MET alterations are receiving increasing attention as targets in precision medicine, and several clinical trials of anti-MET agents are ongoing in NSCLC. Methods: A cohort of 610 patients with stage IIIb-IV NSCLC from two institutions was retrospectively analyzed for MET alterations by next-generation sequencing (NGS) (Ion Torrent PGM® or GeneReader®), nCounter, reverse transcriptase polymerase chain reaction (RT-PCR) or immunohistochemistry (IHC) during a 2-year period. Patients positive for MET amplification by NGS or MET overexpression by nCounter and/or IHC were submitted to fluorescence in situ hybridization (FISH). Representative patients positive for MET exon 14-skipping (METΔex14) mutations by nCounter and/or RT-PCR were confirmed by sequencing exons 13-15 of the METgene. Results: Overall, MET alterations were found in 116/610 patients (19%). Some patients had ≥2 MET aberrations. The most frequent finding was MET overexpression (58/333; 17.6%), followed by METΔex14 (31/610; 5.1%) and MET point mutations (3/129; 2.3%). Regarding MET amplification, it was found in 24/157 patients (15.3%). MET positivity by IHC (3+, >50%) showed a 90.8% concordance with MET mRNA overexpression by nCounter, with a 0.768 Cohen’s kappa (confidence interval, CI 95% 0.575-0.961). A moderate agreement between RT-PCR and nCounter was found for METΔex14 (Cohen’s kappa 0.629; CI 95% 0.434-0.825). METamplification by FISH was found in the subset of MET-overexpressing patients with the highest mRNA levels by nCounter. Interestingly, a patient with a concomitant EGFR mutation and MET overexpression derived two years clinical benefit from crizotinib. Conclusions: MET aberrations are present in 19% of advanced NSCLC patients and represent one of the most frequent targetable alterations in this malignancy. A comprehensive testing of MET alterations in advanced NSCLC patients, including NGS and nCounter techniques, is needed to identify a broader number of patients’ candidates for targeted therapies. Citation Format: Cristina Aguado Esteban, Cristina Teixidó, Ruth Román, Ana María Gimenez Capitán, Carlos Cabrera, Mireia García, Clara D. Mayo De Las Casas, Ainara Arcocha, Sonia Rodriguez, Roxana Reyes, Elba Marín, Ana Perez Rosado, Niki Karachaliou, Aleix Prat, Rafael Rosell, Alejandro Martinez-Bueno, Miguel Angel Molina-Vila, Noemi Reguart. Comprehensive characterization of MET alterations in a large cohort of 610 advanced non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4905.

MicroRNA-206 Reduces Osteosarcoma Cell Malignancy In Vitro by Targeting the PAX3-MET Axis.

PurposeThis study was undertaken to explore how miR-206 represses osteosarcoma (OS) development.Materials and MethodsExpression levels of miR-206, PAX3, and MET mRNA were explored in paired OS and adjacent tissue specimens. A patient-derived OS cell line was established. miR-206 overexpression and knockdown were achieved by lentiviral transduction. PAX3 and MET overexpression were achieved by plasmid transfection. Treatment with hepatocyte growth factor (HGF) was utilized to activate c-Met receptor. Associations between miR-206 and PAX3 or MET mRNA in OS cells were verified by AGO2-RNA immunoprecipitation assay and miRNA pulldown assay. OS cell malignancy was evaluated in vitro by cell proliferation, metastasis, and apoptosis assays. PAX3 and MET gene expression in OS cells was assayed by RT-qPCR and Western blot. Activation of PI3K-AKT and MAPK-ERK in OS cells were assayed by evaluating Akt1 Ser473 phosphorylation and total threonine phosphorylation of Erk1/2, respectively.ResultsExpression levels of miR-206 were significantly decreased in OS tissue specimens, compared to adjacent counterparts, and were inversely correlated with expression of PAX3 and MET mRNA. miR-206 directly interacted with PAX3 and MET mRNA in OS cells. miR-206 overexpression significantly reduced PAX3 and MET gene expression in OS cells in vitro, resulting in significant decreases in Akt1 and Erk1/2 activation, cell proliferation, and metastasis, as well as increases in cell apoptosis, while miR-206 knockdown showed the opposite effects. The effects of miR-206 overexpression on OS cells were reversed by PAX3 or MET overexpression, but only partially attenuated by HGF treatment.ConclusionmiR-206 reduces OS cell malignancy in vitro by targeting PAX3 and MET gene expression.

LncRNA XIST/miR-34a axis modulates the cell proliferation and tumor growth of thyroid cancer through MET-PI3K-AKT signaling

BackgroundThyroid cancer is one of the most prevalent malignancies in endocrine system. Further understanding and revealing the molecular mechanism underlying thyroid cancer are indispensable for the development of effective diagnosis and treatments. In the present study, we attempted to provide novel basis for targeted therapy for thyroid cancer from the aspect of lncRNA-miRNA-mRNA interaction.MethodsThe expression and cellular function of XIST (X-inactive specific transcript) was determined. miRNAs which may be direct targets of XIST were screened for from online GEO database and miR-34a was selected. Next, the predicted binding between XIST and miR-34a, and the dynamic effect of XIST and miR-34a on downstream MET (hepatocyte growth factor receptor)-PI3K (phosphoinositide 3-kinase)-AKT (α-serine/threonine-protein kinase) signaling was evaluated.ResultsXIST was significantly up-regulated in thyroid cancer tissues and cell lines; XIST knockdown suppressed the cell proliferation in vivo and the tumor growth in vitro. Based on online database and online tool prediction results, miR-34a was underexpressed in thyroid cancer and might be a direct target of XIST. Herein, we confirmed the negative interaction between XIST and miR-34a; moreover, XIST knockdown could reduce the protein levels of MET, a downstream target of miR-34a, and the phosphorylation of PI3K and AKT. In thyroid cancer tissues, MET mRNA and protein levels of MET were up-regulated; MET was positively correlated with XIST while negatively correlated with miR-34a, further confirming that XIST serves as a ceRNA for miR-34a through sponging miR-34a, competing with MET for miR-34a binding, and finally modulating thyroid cancer cell proliferation and tumor growth.ConclusionIn the present study, we provided novel experimental basis for targeted therapy for thyroid cancer from the aspect of lncRNA-miRNA-mRNA interaction.

The expression of LINE1-MET chimeric transcript identifies a subgroup of aggressive breast cancers.

Demethylation of the long interspersed nuclear element (LINE-1; L1) antisense promoter can result in transcription of neighboring sequences as for the L1-MET transcript produced by the L1 placed in the second intron of MET. To define the role of L1-MET, we investigated the sequence and the transcription of L1-MET in vitro models and heterogeneous breast cancers, previously reported to show other L1-derived transcripts. L1-MET expressing cell lines were initially identified in silico and investigated for L1-MET promoter methylation, cDNA sequence and cell fraction mRNA. The transcriptional level of L1-MET and MET were then evaluated in breast specimens, including 9 cancer cell lines, 41 carcinomas of different subtypes, and 11 normal tissues. In addition to a L1-MET transcript ending at MET exon 21, six novel L1-MET splice variants were identified. Normal breast tissues were negative for the L1-MET expression, whereas the triple-negative breast cancer (TNBC) and the high-grade carcinomas were enriched with the L1-MET mRNA (p = 0.005 and p = 0.018, respectively). In cancer cells and tissues the L1-MET expression was associated with its promoter hypomethylation (ρ = -0.8 and -0.9, respectively). No correlation was found between L1-MET and MET mRNA although L1-MET expressing tumors with higher L1-MET/MET ratio were negative for the MET protein expression (p = 0.006). Besides providing the first identification and detailed description of L1-MET in breast cancer, we clearly demonstrate that higher levels of this transcript specifically recognize a subset of more aggressive carcinomas, mainly TNBC. We suggest the possible evaluation of L1-MET in the challenging diagnosis of early TNBCs.

Agonist-mediated assembly of the crustacean methyl farnesoate receptor

The methyl farnesoate receptor (MfR) orchestrates aspects of reproduction and development such as male sex determination in branchiopod crustaceans. Phenotypic endpoints regulated by the receptor have been well-documented, but molecular interactions involved in receptor activation remain elusive. We hypothesized that the MfR subunits, methoprene-tolerant transcription factor (Met) and steroid receptor coactivator (SRC), would be expressed coincident with the timing of sex programming of developing oocytes by methyl farnesoate in daphnids. We also hypothesized that methyl farnesoate activates MfR assembly. Met mRNA was expressed rhythmically during the reproductive cycle, with peak mRNA accumulation just prior period of oocytes programming of sex. Further, we revealed evidence that Met proteins self-associate in the absence of methyl farnesoate, and that the presence of methyl farnesoate stimulates dissociation of Met multimers with subsequent association with SRC. Results demonstrated that the Met subunit is highly dynamic in controlling the action of methyl farnesoate through temporal variation in its expression and availability for receptor assembly.

P2.06-017 Amethyst NSCLC Trial: Phase 2 Study of MGCD265 in Patients with Advanced or Metastatic NSCLC with Activating Genetic Alterations in MET: Topic: Phase II + NK

MGCD265 is a potent, orally available, small molecule RTK inhibitor of MET and Axl, both of which mediate signals for cell growth, survival, and migration. The Amethyst NSCLC trial is designed to evaluate the activity of MGCD265 in patients with NSCLC exhibiting genetic alterations involving MET. Alterations in MET, including gene amplification and/or genetic mutations, occur in approximately 7% of NSCLC cases converting MET to an oncogene capable of driving cancer development and progression. Amplification of MET has been associated with a poor prognosis in NSCLC. In addition, various genetic mutations result in the deletion of exon 14 in MET mRNA (METex14del) and the subsequent loss of the Y1003 regulatory binding site for CBL ubiquitin ligase, required for MET degradation and signal attenuation. Loss of the Y1003 binding site of MET results in sustained MET signaling, which has been implicated as an oncogenic driver in a subset of NSCLC. The importance of MET as a driver is demonstrated in xenograft models of NSCLC with METex14del and MET amplification, and where MGCD265 induces tumor regression. Additionally, confirmed partial responses have been observed in pts with NSCLC characterized by METex14del who were treated with MGCD265 in the Phase 1 setting. Pts with platinum pre-treated NSCLC characterized by activating genetic MET alterations identified in tumor tissue or circulating tumor DNA (ctDNA) are eligible for this multi-center, global, Phase 2 trial. Pts are assigned to one of four cohorts based on the type of MET dysregulation and detection method: 1) mutations in tissue, 2) amplification in tissue, 3) mutations in ctDNA, and 4) amplification in ctDNA. The primary endpoint is Objective Response Rate (ORR) in accordance with RECIST 1.1; a Bayesian Predictive Probability Design is applied independently to each cohort. Secondary objectives include safety, tolerability, response duration, survival, correlation between tissue and ctDNA testing, and PK/PD. This study is currently open globally, and recruitment is ongoing. Section not applicable. Section not applicable.

MiR-152 down-regulation is associated with MET up-regulation in leiomyosarcoma and undifferentiated pleomorphic sarcoma.

Highly aggressive adult soft tissue sarcomas (STS), i.e., leiomyosarcomas (LMS) and undifferentiated pleomorphic sarcomas (UPS), present complex genomic anomalies and overall 5-year survival rates of 20 to 40%. Here, we aimed to identify new biomarkers that may be employed to improve the treatment of non-translocation STS patients. We validated 12 miRNAs implicated in tumor development using primary STS samples and selected miR-152 for further analysis in STS-derived cell lines. 59 primary STS samples (27 LMS and 32 UPS) and 10 matched normal control tissues were included in the study, as well as 3 STS-derived cell lines (HT1080, SW872 and SKLMS1) and a normal control mesenchymal cell line (hMSC). miRNA expression analyses were performed using a TaqMan microRNA Array platform and qRT-PCR (miR-152), respectively. The expression levels of the putative miR-152 targets MET and KIT were assessed using qRT-PCR and immunohistochemistry on tissue microarrays, respectively. In addition, various functional analyses were performed before and after miR-152 transfection into SKLMS1 cells. We found that 12 pre-selected miRNAs were down-regulated in primary STS tumor samples compared to its normal control samples. A statistically significant miR-152 down-regulation was found to be accompanied by high MET and KIT mRNA levels in both the primary samples and the STS-derived cell lines tested. miR-152 transfection in SKLMS1 cells led to a reduction in KIT and MET mRNA and protein levels which, in turn, was associated with a transient down-regulation of the PI3K/AKT pathway, a transient decrease in cell growth, and a transient increase in both apoptotic and S-phase cells. Our data indicate that over-expression of MET and KIT in primary STS samples and its derived cell lines is associated with miR-152 down-regulation. This shift may play a role in STS development and, thus, may be used to identify patients at risk. The effect of MET down-regulation on downstream signaling pathways, such as the PI3K/AKT pathway, may provide a basis for the future design of novel STS treatment strategies.

Transcriptional upregulation of c-MET is associated with invasion and tumor budding in colorectal cancer.

c-MET and its ligand HGF are frequently overexpressed in colorectal cancer (CRC) and increased c-MET levels are found in CRC liver metastases. This study investigated the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Pre-clinically, we found marked upregulation of c-MET at both protein and mRNA levels in several invasive CRC cells. Down-regulation of c-MET using RNAi suppressed migration/invasion of parental and invasive CRC cells. Stimulation of CRC cells with rh-HGF or co-culture with HGF-expressing colonic myofibroblasts, resulted in significant increases in their migratory/invasive capacity. Importantly, HGF-induced c-MET activation promoted rapid downregulation of c-MET protein levels, while the MET transcript remained unaltered. Using RNA in situ hybridization (RNA ISH), we further showed that MET mRNA, but not protein levels, were significantly upregulated in tumor budding foci at the invasive front of a cohort of stage III CRC tumors (p < 0.001). Taken together, we show for the first time that transcriptional upregulation of MET is a key molecular event associated with CRC invasion and tumor budding. This data also indicates that RNA ISH, but not immunohistochemistry, provides a robust methodology to assess MET levels as a potential driving force of CRC tumor invasion and metastasis.

0782 In vivo knockdown of FGFR2 and MET mRNA in trophectoderm of ovine conceptuses retards their development via abrogation of MAPK and MTOR pathways

0782 In vivo knockdown of FGFR2 and MET mRNA in trophectoderm of ovine conceptuses retards their development via abrogation of MAPK and MTOR pathways

Synergistic Activation upon MET and ALK Coamplification Sustains Targeted Therapy in Sarcomatoid Carcinoma, a Deadly Subtype of Lung Cancer

IntroductionGenetic alterations suitable for targeted therapy are poorly known issues in pulmonary sarcomatoid carcinoma (PSC), an uncommon and life-threatening family of non–small cell lung cancers. MethodsNinety-eight PSCs were assessed for MNNG HOS Transforming gene (MET) and anaplastic lymphoma receptor tyrosine kinase gene (ALK) status by fluorescence in situ hybridization (FISH) and for relevant protein expression by immunohistochemical analysis, also taking advantage of phosphorylated (p-) antibodies. Moreover, levels of ALK and MET mRNA were also determined by real-time polymerase chain reaction and Western blot analysis for downstream activation pathways involving p-MET, p–protein kinase B, p–mitogen-activated protein kinase, p-SRC proto-oncogene tyrosine-protein kinase, and p–focal adhesion kinase (p-FAK). ResultsMET amplification was detected by FISH in 25 of 98 PSCs (25.6%) and ALK amplification (but not the relevant rearrangement) was found in 16 of 98 (16.3%), with all ALK-amplified tumors also showing MET amplification (p < 0.0001). Nine PSCs, however, showed MET amplification without any ALK gene alteration. ALK protein expression was always lacking, whereas MET and p-MET were confined to the relevant amplified tumors only. Increased levels of ALK and MET mRNA were detectable in tumors with no direct relationship between mRNA content, protein expression, or alterations detected by FISH. Western blot assays showed complete activation of downstream signal pathways up to p-SRC proto-oncogene tyrosine-protein kinase, and p–focal adhesion kinase recruitment in MET and ALK–coamplified tumors only, whereas isolated MET amplification, MET and ALK borderline amplification (5%–10% of tumor cells with ≥15 copies of the relevant gene), or negative tumors showing eusomy or chromosome polysomy were confined to p–mitogen-activated protein kinase, p–protein kinase B, and/or p-MET activation. Multivariate survival analysis pushed a higher percentage of MET altered cells or a higher value of MET copy gain per cell to marginally emerge for overall survival (p = 0.140) and disease-free survival (p = 0.060), respectively. ConclusionsALK and MET seemed to act as synergistic, nonrandom coactivators of downstream signal when coamplified in a subset of patients with PSC, thus likely suggesting a combined mechanism of oncogene addiction. These alterations could be a suitable target for therapy based on specific inhibitors.

Abstract 140: Oncogenic signaling by MET and other cabozantinib targets in cells derived from urothelial carcinoma of the bladder

Abstract Background: Mounting evidence supports MET as a target in urothelial carcinoma (UC) of the bladder. Activated MET can promote tumor growth and metastasis, as well as tumor angiogenesis by upregulating VEGF, and may play a role in UC pathogenesis. The effects of MET inhibitor cabozantinib on MET-driven UC cell growth, invasion and tumorigenicity were previously analyzed in biochemical, cell-based and animal models. Current known cabozantinib targets (MET, KDR, FLT3, KIT, RET, AXL and ROS) and possible targets based on structural similarity (MERTK, TYRO3, RON, RYK, CSF1R, ROR1, ROR2, ALK and LTK) were characterized in 13 UC-derived cell lines by determining the frequency and absolute level of mRNA expression, and ultimately, for targets where mRNA was present, encoded protein abundance, ligand expression and cabozantinib sensitivity. Methods: The effects and the targets of cabozantinib on human UC-derived cell lines were studied in vitro. Cells at 80% confluence were serum deprived 16h, then left untreated or treated with hepatocyte growth factor (HGF) and/or cabozantinib prior to analysis of MET, phospho- (p)MET, pAKT1, total AKT1, pMAPK1/2 and total MAPK1/2 by two-site immunoassay and/or immunoblotting. Cabozantinib effects on basal and HGF-induced UC cell invasion and proliferation were measured. Gene mRNA abundance (absolute copy number) of cabozantinib targets was measured using real time PCR from the extracted RNAs of 13 UC cell lines. Results: Basal pMET content was uniformly low, and was increased significantly by HGF; the latter was reversed by cabozantinib treatment. HGF-driven increases in pAKT1:AKT1 and pMAPK1/2:MAPK1/2 ratios in all cell lines were reversed by cabozantinib treatment, as were HGF-enhanced UC cell invasion, proliferation and tumorigenicity. UC cell lines displayed uniformly robust mRNA expression of MET, AXL, RYK, RON, CSF1R, ROR1 and ROR2, and frequent modest expression of RET and ROS, whereas KDR, ALK, KIT, LYK, TYRO3, MERTK and FLT3 showed little or no expression. MET mRNA and protein abundance were significantly positively correlated among these 13 UC cell lines. Conclusions: UC cell MET content was higher with higher UC grade. HGF was not expressed in any UC cell line tested, suggesting that this pathway is predominantly paracrine in vivo. Added HGF stimulated activation of MET and known effectors, and enhanced invasion, growth rate and anchorage-independent growth; cabozantinib effectively reversed these HGF-driven effects. Based on receptor expression, cabozantinib is most likely acting through MET, AXL and ROS, and potentially through RYK, ROR1, ROR2, and/or RET, in UC cells, all of which have been implicated in oncogenesis in other human malignancies. Note: This abstract was not presented at the meeting. Citation Format: Young H. Lee, Tiffany K. Wong, Andrea B. Apolo, Piyush K. Agarwal, Donald P. Bottaro. Oncogenic signaling by MET and other cabozantinib targets in cells derived from urothelial carcinoma of the bladder. [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 140. doi:10.1158/1538-7445.AM2015-140

Abstract 3121: Hypoxia-related microRNA, miR-199a-3p, displays tumor suppressor function in ovarian cancer

Abstract Purpose: Ovarian cancer has the fifth leading cause of death in women in the Western world. At diagnosis, over 60% of all ovarian cancer patients have advanced disease with peritoneal dissemination. During the transcoelomic metastasis of ovarian cancer cells, cells are floating in the peritoneal cavity without vascular supply and exposed in hypoxic conditions, suggesting that hypoxic stimuli affect cancer cell behavior and the cells acquire more aggressive malignancy potential. In this study, we screened miRNAs which expression were altered under hypoxic condition and investigated their function in vitro and in vivo. Methods: miRNA PCR arrays were performed on two ovarian cancer cells (CaOV3 and RMUG-S) cultured under 20% or 1% O2, revealing that miR-199a-3p was one of miRNAs, which expression drastically declined under hypoxia. In order to clarify the relationship between HIF-1α and miR-199a-3p, ovarian cancer cells were transfected with HIF-1A expression vector and the level of miR-199a-3p was determined by RT-real-time PCR. In silico analyses indicated that MET is one of target genes of miR-199a-3p. Thus, miR-199a-3p expression in ovarian cancer cell lines and clinical cancer tissues was assessed by RT-real-time PCR and compared with c-Met expression of those by Western blotting (cell lines) and immunohistochemistry (tissues). To analyze whether miR-199a-3p directly targets the 3′-UTR of MET mRNA, a luciferase reporter assay was performed. The effect of miR-199a-3p on c-Met expression as well as its downstream signaling was examined by the transfection of precursor miR-199a-3p. The inhibitory effect of miR-199a-3p on ovarian cancer cells was analyzed through in vitro anchorage-independent cell proliferation, adhesion and invasion assay. Using an ovarian cancer xenograft model, the therapeutic effect of miR-199a-3p against peritoneal dissemination of cancer cells was investigated. Results: miR-199a-3p expression was not altered by HIF-1A overexpression, suggesting that hypoxia-driven miR-199a-3p change was related to HIF-independent pathway. miR-199a-3p expression was inversely correlated with c-Met expression in both ovarian cancer cell lines and clinical samples. Transfection of precursor miR-199a-3p into SKOV3ip1 cells attenuated c-Met expression followed by the inhibition of phosphorylation of c-Met, ERK and AKT. In luciferase reporter assay, miR-199a-3p directly suppressed MET transcriptional activity. Furthermore, the proliferation, adhesion and invasion of cancer cells were inhibited by the transfection of miR-199a-3p. In an ovarian cancer xenograft model, the enforced expression of miR-199a-3p in SKOV3-13 cells significantly suppressed peritoneal dissemination. Conclusion: Hypoxia-decreased microRNA, miR-199a-3p, remarkably inhibited ovarian cancer progression through the suppression of c-Met. Thus, miR-199a-3p can be suggested as a potential target for new treatment of ovarian cancer. Note: This abstract was not presented at the meeting. Citation Format: Yasuto Kinose, Kenjiro Sawada, Koji Nakamura, Akihiko Yoshimura, Erika Nakatsuka, Seiji Mabuchi, Tadashi Kimura. Hypoxia-related microRNA, miR-199a-3p, displays tumor suppressor function in ovarian cancer. [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 3121. doi:10.1158/1538-7445.AM2015-3121

Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a

Therapeutic angiogenesis has been implicated in ischemic diseases and wound healing. Ginsenoside-Rg1 (Rg1), one of the most abundant active components of ginseng, has been demonstrated as an angiogenesis-stimulating compound in different models. There is increasing evidence implicating microRNAs (miRNAs), a group of non-coding RNAs, as important regulators of angiogenesis, but the role of microRNAs in Rg1-induced angiogenesis has not been fully explored. In this report, we found that stimulating endothelial cells with Rg1 could reduce miR-23a expression. In silico experiments predicted hepatocyte growth factor receptor (MET), a well-established mediator of angiogenesis, as the target of miR-23a. Transfection of the miR-23a precursor or inhibitor oligonucleotides validated the inverse relationship of miR-23a and MET expression. Luciferase reporter assays further confirmed the interaction between miR-23a and the MET mRNA 3′-UTR. Intriguingly, ginsenoside-Rg1 was found to increase MET protein expression in a time-dependent manner. We further demonstrated that ginsenoside-Rg1-induced angiogenic activities were indeed mediated through the down-regulation of miR-23a and subsequent up-regulation of MET protein expression, as confirmed by gain- and loss-of-function angiogenic experiments. In summary, our results demonstrated that ginsenoside-Rg1 could induce angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a. This study has broadened our understanding of the non-genomic effects of ginsenoside-Rg1, and provided molecular evidence that warrant further development of natural compound as novel angiogenesis-promoting therapy.

Abstract 4361: Hypoxia-related microRNA, miR-199a-3p, inhibits ovarian cancer progression through the suppression of c-Met expression

Abstract Purpose: Ovarian cancer has the highest mortality rate of all gynecologic tumors. It is often diagnosed at an advanced stage after tumor cells are disseminated within the peritoneal cavity. During the dissemination of ovarian cancer cells, cells are floating in the peritoneal cavity without vascular supply and exposed in hypoxic conditions, suggesting that hypoxic stimuli affect ovarian cancer cell behavior and the cells acquire more aggressive malignancy potential. In this study, we screened miRNAs which expression were altered under hypoxic condition and performed functional analysis of those in vitro and in vivo. Methods: miRNA PCR arrays were performed using two ovarian cancer cells (CaOV3, RMUG-S) incubated under 20% or 1% O2. miR-199a-3p, which expression decreased under hypoxic conditions, was extracted. In silico analyses led us to consider MET is one of the target genes of miR-199a-3p. Therefore, we determined to analyze the effect of miR-199a-3p on ovarian cancer cells. The expression of miR-199a-3p in various ovarian cancer cell lines was examined by RT-real-time PCR and the expression of c-Met was examined by Western blotting. In order to investigate whether miR-199a-3p directly targets the 3′-UTR of MET mRNA, a luciferase reporter assay was performed. Using clinical paraffin tissues, only the tumor part was collected by a laser capture microdissection and RNA extracted. The expression of miR-199a-3p was examined by miRNA RT-PCR assays and compared with c-Met protein expression by immunohistochemistry. The effect of miR-199a-3p on c-Met expression as well as its downstream signaling was examined by the transfection of precursor miR-199a-3p. The inhibitory effect of miR-199a-3p on ovarian cancer cells was analyzed through in vitro cell proliferation, adhesion assay and invasion assay. Using a xenograft model, the inhibitory effect of miR-199a-3p against peritoneal dissemination of ovarian cancer cells was analyzed. Results: miR-199a-3p expression was inversely correlated with c-Met expression in ovarian cancer cells and clinical samples. Transfection of precursor miR-199a-3p into SKOV3ip1 cells reduced c-Met expression followed by the inhibition of phosphorylation of c-Met, ERK and AKT. Accordingly, the proliferation, adhesion and invasion of cancer cells were inhibited by the transduction of miR-199a-3p. In luciferase reporter assay, miR-199a-3p directly suppresses MET transcriptional activity. In an ovarian cancer xenograft model, the enforced expression of miR-199a-3p in SKOV3-13 cells significantly suppressed peritoneal dissemination. Conclusion: Hypoxia related microRNA, miR-199a-3p, drastically inhibits ovarian cancer progression through the down-regulation of c-Met expression. miR-199a-3p can be suggested as a potential target for treating ovarian cancer dissemination. Citation Format: Yasuto Kinose, Kenjiro Sawada, Koji Nakamura, Seiji Mabuchi, Ken-ichirou Morishige, Tadashi Kimura. Hypoxia-related microRNA, miR-199a-3p, inhibits ovarian cancer progression through the suppression of c-Met expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4361. doi:10.1158/1538-7445.AM2014-4361

Comprehensive molecular profiling of lung adenocarcinoma.

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.