MET Expression Research Articles

Inhibition of NF-κB improves sensitivity to irradiation and EGFR-TKIs and decreases irradiation-induced lung toxicity.

Resistance to radiotherapy and to EGFR tyrosine kinase inhibitors (EGFR-TKIs), as well as therapy-related lung toxicity, are serious problems in the treatment of lung cancer. NF-κB has been reported to be associated with radioresistance. Therefore, we evaluated its effects on sensitivity to irradiation and to EGFR-TKIs; irradiation-induced lung toxicity; and the effects of irradiation on sensitivity to EGFR-TKIs. We used IKKβ inhibitor IMD 0354 or p65 depletion to explore their effects on sensitivity to irradiation and to EGFR-TKIs in vitro and in vivo. We evaluated the efficacy of IMD 0354 in a radiation-induced pulmonary-fibrosis mouse model. Irradiation enhanced activation and expression of MET and therefore suppressed the sensitivity of lung cancer cells to irradiation or EGFR-TKIs. Inhibition of NF-κB by IMD 0354 or by p65 depletion reversed irradiation-induced MET activation and increased the sensitivity of lung cancer cells to irradiation, to EGFR-TKIs and to the combination thereof in vitro and in vivo. In addition, IMD 0354 significantly reduced lung toxicity in a murine model of irradiation-induced pneumonia and lung fibrosis. These findings indicated that NF-κB inhibition can improve sensitivity to irradiation and to EGFR-TKIs and can decrease irradiation-induced lung toxicity in lung cancer.

An integrated approach of bioinformatic prediction and in vitro analysis identified that miR-34a targets MET and AXL in triple-negative breast cancer

BackgroundBreast cancer is the most prevalent cancer among women, and AXL and MET are the key genes in the PI3K/AKT/mTOR pathway as critical elements in proliferation and invasion of cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs regulating the expression of genes.MethodsBioinformatic approaches were used to find a miRNA that simultaneously targets both AXL and MET 3′-UTRs. The expression of target miRNA was evaluated in triple-negative (MDA-MB-231) and HER2-overexpressing (SK-BR-3) breast cancer cell lines as well as normal breast cells, MCF-10A, using quantitative real-time PCR. Then, the miRNA was overexpressed in normal and cancer cell lines using a lentiviral vector system. Afterwards, effects of overexpressed miRNA on the expression of AXL and MET genes were evaluated using quantitative real-time PCR.ResultsBy applying bioinformatic software and programs, miRNAs that target the 3′-UTR of both AXL and MET mRNAs were determined, and according to the scores, miR-34a was selected for further analyses. The expression level of miR-34a in MDA-MB-231 and SK-BR-3 was lower than that of MCF-10A. Furthermore, AXL and MET expression in SK-BR-3 and MDA-MB-231 was lower and higher, respectively, than that of MCF-10A. After miR-34a overexpression, MET and AXL were downregulated in MDA-MB-231. In addition, MET was downregulated in SK-BR-3, while AXL was upregulated in this cell line. ConclusionsThese findings may indicate that miR-34a is an oncogenic miRNA, downregulated in the distinct breast cancer subtypes. It also targets MET and AXL 3′-UTRs in triple-negative breast cancer. Therefore, it can be considered as a therapeutic target in this type of breast cancer.

HER2 inhibition in gastro-oesophageal cancer: A review drawing on lessons learned from breast cancer.

Human epidermal growth factor receptor 2 (HER2)-inhibition is an important therapeutic strategy in HER2-amplified gastro-oesophageal cancer (GOC). A significant proportion of GOC patients display HER2 amplification, yet HER2 inhibition in these patients has not displayed the success seen in HER2 amplified breast cancer. Much of the current evidence surrounding HER2 has been obtained from studies in breast cancer, and we are only recently beginning to improve our understanding of HER2-amplified GOC. Whilst there are numerous licensed HER2 inhibitors in breast cancer, trastuzumab remains the only licensed HER2 inhibitor for HER2-amplified GOC. Clinical trials investigating lapatinib, trastuzumab emtansine, pertuzumab and MM-111 in GOC have demonstrated disappointing results and have not yet changed the treatment paradigm. Trastuzumab deruxtecan may hold promise and is currently being investigated in phase II trials. HER2 amplified GOC differs from breast cancer due to inherent differences in the HER2 amino-truncation and mutation rate, loss of HER2 expression, alterations in HER2 signalling pathways and differences in insulin-like growth factor-1 receptor and MET expression. Epigenetic alterations involving different microRNA profiles in GOC as compared to breast cancer and intrinsic differences in the immune environment are likely to play a role. The key to effective treatment of HER2 amplified GOC lies in understanding these mechanisms and tailoring HER2 inhibition for GOC patients in order to improve clinical outcomes.

Abstract 2389: Role of MET in head and neck cancer

Abstract Introduction c-MET is a well-known target due to its amplification and overexpression in gastric cancer and NSCLC. Overexpression has also been reported for Head and Neck Cancer (HNSCC), but initial clinical trials with MET inhibitors in HNSCC have not been successful. Methods We investigated the expression level of MET in HNSCC and also characterized the molecular activity level based on the phosphorylation of the intracellular adaptor protein docking site, Tyrosine-1349. In a second larger cohort, we tested expression of MET and correlate it with follow-up data in order to test the prognostic relevance of MET expression in gastric cancer. All clinical samples were all obtained in compliance with clinical regulations and informed consent of every patient. Results We found MET clearly overexpressed in HNSCC. However, the signaling activity of MET was not elevated compared to normal adjacent tissue. To test the relevance of MET for growth of HNSCC cells, we tested the activity of BAY 853474 in a panel of HNSCC cell lines. In contrast to gastric cancer control cell lines which had low nanomolar IC50 values, none of the 12 HNSCC cell lines was sensitive to MET inhibition. This was in contrast to their sensitivity to Cisplatin/Fluoruracil as positive control. Compared to MET dependent cell lines from gastric cancer, HNSCC cell lines had 10 fold less MET expression. The phosphorylation was two orders of magnitude below that of responder cell lines. On the molecular level, we also compared the properties of the HNSCC cell lines with that of fresh frozen tumor biopsies from 50 patients. In this analysis, it became evident that clinical samples had strikingly lower MET expression and phosphorylation even compared to the HNSCC cell lines. Based on these findings, a clinical response to MET inhibitors cannot be expected. In a larger cohort of several hundred patients, we are currently testing whether the elevated MET expression may still be prognostic for progression and/or survival. It appears possible that MET expression may give a growth advantage to tumor cells although it is clearly not fulfilling the criteria for an oncogenic driver in this indication. The significant overexpression of MET in tumor tissue and the very strong overexpression in cell lines compared to clinical samples show that MET expression gives a selective advantage to the tumor cells. We will show whether this advantage results in shorter time to progression or overall survival. Conclusion We show that MET expression in HNSCC cell lines is not representative for the clinical situation. HNSCC does not overexpress Met and the molecular activity is low. MET is devalidated as therapeutic target in HNSCC. An analysis of the prognostic value of MET expression in HNSCC will follow and presented at the AACR meeting in 2018. Citation Format: Thomas Schlange, Martin Khan, Sami Khaznadar, arndt schmitz, Thomas Krahn, Oliver von Ahsen. Role of MET in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2389.

Abstract 4156: Inhibition of NF-kB improves sensitivity to radiation and EGFR inhibitor and decreases radiation-induced lung toxicity in lung cancer

Abstract Combination with EGFR-TKIs and radiation seems to be a good strategy against lung cancer, since radiation could enhance EGFR expression. However, the results of clinical trials were controversial. MET activation was involved in resistance to both radiation and EGFR inhibitor, while NF-kB was reported to closely relate to HGF/MET signaling axis. Furthermore, NF-κB has been proved to play a role in bleomycin-induced pulmonary fibrosis, which could be ameliorated by IKKB inhibitor (IMD-0354). Therefore, we evaluated the influence of NF-kB/MET inhibition to the sensitivity of radiation and EGFR-TKIs in lung cancer cells in vitro and in vivo. we also examined the role of NF-κB in radiation-induced lung toxicity. Our data showed that radiation enhanced activation and expression of MET and EGFR, whereas only activation of MET could be reversed by IMD-0354 and p65 depletion, suggesting that radiation-induced MET activation was mediated by NF-kB signaling. Furthermore, NF-kB inhibition increased radiation-induced DNA damage and apoptosis in lung cancer cells. On the other hand, radiation decreased sensitivity to EGFR inhibitor in EGFR mutant lung cancer cells, while inhibition of NF-kB or MET overcame the resistance. Importantly, IMD-0354 increased sensitivity to radiation, EGFR-TKIs, or their combination in mouse xenograft tumors by arresting cell proliferation and inducing cell apoptosis. Interestingly, IMD-0354 significantly reduced the lung toxicity in a mouse model of radiation-induced pneumonia and lung fibrosis. Our preclinical findings indicated that inhibition of NF-kB/MET could improve sensitivity to radiation and EGFR inhibitor and decrease radiation-induced lung toxicity in lung cancer. Citation Format: Wei Wang, Rong Wang, Shunli Peng, Qi Li, Xiaojuan Zhang, Yueyun Ma. Inhibition of NF-kB improves sensitivity to radiation and EGFR inhibitor and decreases radiation-induced lung toxicity in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4156.

Abstract 2519: Identification of novel MET fusion transcript amplification in glioblastoma

Abstract Glioblastoma (GBM) is the most common and aggressive primary CNS malignant tumor with a 15.2% 2 year survival rate for tumors diagnosed between 2008 and 2012. Amplification of MET proto-oncogene has been identified in 4% of glioblastomas, leading to high expression and ligand independent activation in some cases. Our goal was to develop MET-amplified GBM models to study signaling and investigate response to MET inhibitors as a therapy for GBMs. We identified 2 GBM patients (HF3035 and HF3077) with MET gene amplifications after low-pass whole genome DNA sequencing of 13 cases. Fluorescent in situ hybridization (FISH) analyses confirmed heterogeneous MET amplification in HF3035 and HF3077 tumors, in 63.5% and 83.0% of nuclei, respectively. In vitro neurosphere cultures derived from these tumors showed drastic depletion of MET amplicons, to 15.5% MET in HF3035 (P7) and 1.5% in HF3077 (P11). FISH in the metaphase neurosphere spreads showed that MET amplification was extrachromosomal. Interestingly, MET-amplified neurospheres were strongly selected for after intracranial (IC) implant in immunocompromised mice. HF3035 and HF3077 PDX presented MET amplified in high frequency: 79.5-86.5% for HF3035 and 47-65% for HF3077. Met expression levels by RNAseq were congruent with the oscillating gene amplification pattern. In depth RNA sequencing analysis using PRADA has revealed genomic rearrangements involving MET, yielding three novel MET-CAPZA2 fusion transcripts. For both cell lines exon 1 of CAPZA2 was fused to exon 2 of MET, resulting in full length MET coding region, with altered 5' cis-regulatory sequences. For HF3035 samples, we observed an additional in frame fusion of exon 1 of CAPZA2 to exon 6 of MET, resulting in a truncated MET transcript with 13 codons from CAPZA2. Co-expression of the wild-type and fusion MET transcripts in the tumors and PDXs were validated using PCR. MET and p-MET levels were high thoughout the parental and PDX tumors. Capmatinib, which is a selective c-MET inhibitor was administered to the PDXs orally 5days/week. The treatment was effective in improving survival of HF3077 IC PDXs (p=0.028) and decreasing subcutaneous tumor size to 30% of the controls after 2 week treatment (t-test, p=0.017). However, treatment of HF3035 IC PDXs did not significantly improve survival (p=0.313). Kaplan-Meier survival curves were compared by log-rank (Mantel-Cox) test, sig. set at p<0.05. MET and p-MET detection by IHC of control and capmatinib treated xenografts show complete inhibition of p-MET, but did not affect MET overexpression in HF3035 PDX. Our results show that highly amplified regions are susceptible to genomic arrangements and the formation of fusion genes. Under investigation, is the basis for the strong selection for MET expressing cells in vivo and potential novel roles for MET in tumor progression. Citation Format: Oluwademilade Nuga, Ana deCarvalho, Yuling Meng, Laura Hasselbach, Kevin Nelson, Susan Irtenkauf, Andrea Transou. Identification of novel MET fusion transcript amplification in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2519.

Abstract 3906: Inhibition ofNF-kB Inducing Kinase (NIK) to supressNF-kB alternative pathway and cell migration in HNSCC

Abstract Nuclear factor-κB (NF-κB) activation through the alternative pathway has been implicated in cancer progression and survival, however the regulatory signaling and biological significances are not well understood, especially in head and neck squamous cell carcinoma (HNSCC). NF-κB-inducing kinase (NIK) is a key molecule in the alternative NF-κB pathway, which receives signals from Lymphotoxin beta/Lymphotoxin beta receptor (LTβ/LTβR) to activate Inhibitor-κB kinase α (IKKα) and transcription factors RELB/NF-κB2. In a panel of HNSCC cell lines, we observed that dysregulation of the alternative NF-κB pathway occurred primarily through increased LTβ receptor signaling via NIK and the non-canonical NF-κB subunits. We identified a NIK inhibitor 1, 3[2H, 4H]-Iso-Quinoline Dione, which inhibited cell migration at a low dose range, but exhibited minimal anti-proliferative effect in HPV(-) HNSCC cell lines. The biological effects of the inhibitor were through suppression of the alternative NF-κB signaling. Using Western blot and immunofluorescence staining, we showed that the inhibitor reduced the protein expression and/or phosphorylation of NIK, IKKα, and RELB/NF-κB2 (p52) in the cytoplasm, and/or the protein translocation in the nucleus. The NIK inhibitor also blocked LTβ induced translocation of RELB to the nucleus. The effects of NIK inhibitor were validated by knockdown of NIK using siRNA, which inhibited cell migration and LTβ-induced expression of MET. Met is a hepatocyte growth factor receptor implicated in promoting metastasis, and knockdown of MET slowed down HNSCC cell migration. We further extended the study on a murine oral metastasis cell line MOC2, which is a syngeneic mouse model suitable for testing drug effects on tumor growth and migration in vivo. We found that the NIK inhibitor decreased cell proliferation, migration, and invasion through an extracellular matrix barrier at low concentration ranges. Currently, we have identified a potent and selective small molecule NIK inhibitor, which is orally bioavailable and active in vivo. We plan to test the effects of this NIK inhibitor on HNSCC tumor growth and metastasis in the syngeneic MOC2 model. Our study may shed light on how HNSCC tumors migrate and metastasize to local lymph nodes, where the microenvironment promotes alternative NF-κB activation by LTβ through NIK signaling. Targeting NIK mediated alternative NF-κB activation could be a potential therapeutic strategy for preventing tumor migration and metastasis of HNSCC. (Supported by NIDCD intramural projects ZIA-DC-000016, 74) Citation Format: Rita Das, Paul E. Clavijo, Clint Allen, Anthony Saleh, Zhong Chen, Carter Van Waes. Inhibition ofNF-kB Inducing Kinase (NIK) to supressNF-kB alternative pathway and cell migration in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3906.

Abstract 2937: Crizotinib inhibits growth, migration, and invasion of breast cancer cells in vitro and synergizes with chemotherapeutic agents

Abstract MET is a receptor tyrosine kinase known to be associated with cancer development and progression. Dysregulations of MET expression and/or signaling have been reported in breast cancer patients and determined to be associated with inferior outcomes rendering MET an appealing target for therapeutic interventions. Crizotinib is a multi-targeted small-molecule kinase inhibitor for MET, ALK, and ROS1 kinases. In this study, the anti- proliferative, cytotoxic, anti-migratory, and anti-invasive effects of crizotinib have been evaluated in breast cancer cells in vitro. Cell viability was assessed by 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay. In vitro wound- healing assay was used to examine the effect of crizotinib on breast cancer cell migration. Anti-invasive effect of crizotinib treatment in MDA-MB-231 breast cancer cells was measured using Trevigen Cultrex® BME Cell Invasion Assay. The expressions of Ki-67, MET, and phospho-MET receptors were characterized using immunofluorescence staining. Results showed that crizotinib has significant anti-proliferative activity on all mammary tumor cells with IC50 values of 5.16, 1.5, and 3.85 μM in MDA-MB-231, MCF- 7, and SK-BR-3 cells, respectively. Crizotinib induced cytotoxic effects in all breast cancer cells examined after acute exposure in culture. Combined treatment of small dose of crizotinib with the chemotherapeutic agent paclitaxel or doxorubicin exhibited a highly synergistic inhibition of growth of MDA-MB-231 and MCF-7 cells with combination index values of less than 1. However, no significant effect was observed for the combined treatment of crizotinib with chemotherapy in SK-BR-3 cells. Treatment with crizotinib demonstrated a remarkable reduction in the total levels of the proliferation marker Ki-67 protein in all tested cell lines. Crizotinib inhibited migration and invasion of MDA-MB-231 cells in a dose-dependent fashion. Crizotinib reduced MET receptor activation in MDA-MB-231 cells when treated at effective concentrations. In conclusion, crizotinib suppressed proliferation, migration, and invasion of breast cancer cells in vitro. The results of this study demonstrated that combined treatment of crizotinib with chemotherapeutic agents resulted in a synergistic growth inhibition of specific breast cancer cell lines. Combination treatment of chemotherapy with novel targeted therapy could be a promising approach to overcome the limitations of conventional chemotherapeutic regimens in breast cancer management. Citation Format: Nehad M. Ayoub, Kamal M. Al-Shami, Mohammad A. Alqudah, Nizar M. Mhaidat. Crizotinib inhibits growth, migration, and invasion of breast cancer cells in vitro and synergizes with chemotherapeutic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2937.

MET in gastric cancer with liver metastasis: The relationship between MET amplification and Met overexpression in primary stomach tumors and liver metastasis.

Although MET amplification/overexpression was observed in a subset of gastric cancer (GC) patients, the relationship between MET amplification/overexpression in primary GC and liver metastasis was unclear. GC samples and matched liver metastases (N = 47) were analyzed by fluorescence/silver in-situ hybridization (FISH/SISH) and by immunohistochemistry for MET amplification and MET expression, respectively. MET-copy number (CN) and Met expression data from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD, N = 356) were also analyzed. Significant overlap existed between MET amplification and Met expression in both primary stomach tumors (P = 0.013) and liver metastasis (P = 0.001). In TCGA-STAD, MET-CN (≥4 copies) and MET expression were also positively correlated (r = 0.761; P = 0.017). Comparative analysis revealed a strong association between MET expression and MET amplification (85% concurrence) in primary stomach tumors and matched liver metastasis. MET status in synchronous liver metastasis (N = 36) was correlated with primary stomach tumors. However, a significant correlation between primary tumors and liver metastases was not observed in patients with metachronous liver metastasis. Survival analyses revealed that both MET amplification and MET overexpression were prognostic of poor outcomes. MET amplification and Met overexpression were positively correlated in GC. MET status should be re-evaluated in GC patients with liver metastasis, especially for metachronous metastasis.

The association among HER2, MET and FOXP3 expression and tumor regression grading in gastric adenocarcinoma.

Although the introduction of the perioperative chemotherapy on the management of gastric cancer has improved patients survival, heterogeneity of clinical outcomes has been evidenced in parallel to different histopathological regression pattern of gastric cancer cells. Thus, this study evaluated the tumor regression grading (TRG) in a series of post-treatment gastric tumors and its associations with HER2, MET, and FOXP3 expression. Material of 54 gastric cancer samples was available for TRG evaluation and immunohistochemistry. We found that total and subtotal pathologic response were significantly associated to the intestinal subtype (p=0.04) and that well-differentiated tumors were significantly correlated with total or partial response (p=0.019). Although not associated with the TRG, FOXP3 expression in gastric tumors was associated to poorly differentiated tumors (p=0.03), to the diffuse and mixed subtypes together (p=0.04) and to the presence of vascular infiltration (p=0.04), while HER2 overexpression was associated to better differentiated cases (p=0.04) and to the absence of vascular infiltration (p=0.02). MET expression, however, showed no association with the analyzed clinicopathological factors. This study highlights the role of tissue differentiation on pathological response to neoadjuvant chemotherapy in gastric cancer and shows no impact between FOXP3, HER2 and MET expression in terms of TRG.

A subgroup of pleural mesothelioma expresses ALK protein and may be targetable by combined rapamycin and crizotinib therapy.

Malignant pleural mesothelioma (MPM) is a neoplasm with inferior prognosis and notorious chemotherapeutic resistance. Targeting aberrantly overexpressed kinases to cure MPM is a promising therapeutic strategy. Here, we examined ALK, MET and mTOR as potential therapeutic targets and determined the combinatorial efficacy of ALK and mTOR targeting on tumor cell growth in vivo.First, ALK overexpression, rearrangement and mutation were studied in primary MPM by qRT-PCR, FISH, immunohistochemistry and sequence analysis; mTOR and MET expression by qRT-PCR and immunohistochemistry. Overexpression of full-length ALK transcripts was observed in 25 (19.5%) of 128 primary MPM, of which ten expressed ALK protein. ALK overexpression was not associated with gene rearrangement, amplification or kinase-domain mutation. mTOR protein was detected in 28.7% MPM, co-expressed with ALK or MET in 5% and 15% MPM, respectively. The ALK/MET inhibitor crizotinib enhanced the anti-tumor effect of the mTOR-inhibitor rapamycin in a patient-derived MPM xenograft with co-activated ALK/mTOR: combined therapy achieved tumor shrinkage in 4/5 tumors and growth stagnation in one tumor. Treatment effects on proliferation, apoptosis, autophagy and pathway signaling were assessed using Ki-67 immunohistochemistry, TUNEL assay, LC3B immunofluorescence, and immunoblotting. Co-treatment significantly suppressed cell proliferation and induced autophagy and caspase-independent, necrotic cell death. Rapamycin/crizotinib simultaneously inhibited mTORC1 (evidenced by S6 kinase and RPS6 dephosphorylation) and ALK signaling (ALK, AKT, STAT3 dephosphorylation), and crizotinib suppressed the adverse AKT activation induced by rapamycin.In conclusion, co-treatment with rapamycin and crizotinib is effective in suppressing MPM tumor growth and should be further explored as a therapeutic alternative in mesothelioma.

Elucidating the Regulatory Mechanism of the Transcription Factor Krüppel homolog 1 in Mosquito Reproduction

Insect juvenile hormone (JH) plays a crucial role during reproduction in adult mosquitoes. Krüppel homolog 1 (Kr‐h1) is a transcription factor that is upregulated by JH. However, the molecular action of AaKr‐h1 in the JH‐regulated gene expression has not yet been well studied in adult mosquitoes. Our recent ChIP‐seq study has indicated that Kr‐h1 in Aedes aegypti binds to the promoter regions of genes that are under the control of JH or another crucial insect hormone, 20‐hydroxyecdysone (20E). Therefore, we hypothesize that AaKr‐h1 modulates the JH and 20E responses in adult mosquitoes. We have performed RNAi mediated gene silencing to examine the regulatory function of AaKr‐h1 on the selected JH (Met, Early Trypsin) and 20E (EcRA, HR3, E75A, βFTZ‐F1, and E93) responsive genes. Successful knockdown at the mRNA and protein levels was confirmed by Quantitative PCR (qPCR) and Western blot analyses respectively. In the AaKr‐h1 depleted mosquitoes, most of the selected JH and 20E responsive genes showed considerable alteration in their expression. Knockdown of AaKr‐h1 led to an increase in the expression of Early Trypsin and a significant reduction in the expression of Met, EcRA, HR3, and E93. In conclusion, our study showed that AaKr‐h1 regulates the expression of diverse genes that are crucial for egg maturation in adult female mosquitoes. Ae. aegypti is a vector of several tropical diseases including yellow fever, dengue, chikungunya and zika fever. The growing resistance to many of the currently used insecticides in mosquitoes creates an urgent need to develop novel insecticides with unique modes of action. This study will advance our understanding of the regulatory mechanism of AaKr‐h1 during reproduction and might provide a new target for novel insecticides to suppress mosquito population.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long noncoding RNA LINC01510 promotes the growth of colorectal cancer cells by modulating MET expression

BackgroundAbnormal expression of long non-coding RNA (lncRNAs) often facilitates unrestricted growth of cancer cells. Long intergenic non-protein coding RNA 1510, an enhancer lncRNA (LINC01510), a lncRNA enhancer is upregulated in colorectal cancer (CRC), and its expression might relate to MET as revealed by lncRNA microarray data. However, the potential biological role of LINC01510 and its regulatory mechanism in CRC remain unclear. Therefore, we investigated the involvement of LINC01510 in the proliferation of CRC cells.MethodsMicroarray analysis, In situ hybridization, colony formation assay, MTT assay, Western blotting, quantitative RT-PCR and flow cytometry were applied. The two-tailed Student’s t test and analysis of variance or general linear model of single factor variable was used for statistical analyse.ResultsIn the present study, we found that LINC01510 was significantly upregulated in CRC tissues and cell lines. The LINC01510 expression level were associated with the clinicopathological grade and stage. Meanwhile, gain- and loss-of-function assays demonstrated that LINC01510 overexpression increased CRC cell proliferation, and promoted cell cycle progression from the G1 phase to the S phase. Further study indicated that LINC01510 was positively correlated with the expression of MET, and its effects were most likely at the transcriptional level.ConclusionsTaken together, our findings suggested that upregulation of LINC01510 contributes to the proliferation of CRC cells, at least in part, through the regulation of MET protein. LINC01510 could be a candidate prognostic biomarker and a target for new therapies in CRC patients.

Perioperative chemotherapy with or without epidermal growth factor receptor blockade in unselected patients with locally advanced oesophagogastric adenocarcinoma: Randomized phase II study with advanced biomarker program of the German Cancer Society (AIO/CAO STO-0801)

BackgroundPerioperative chemotherapy significantly improves survival in patients with locally advanced oesophagogastric cancer (EGC). However, as approximately 60% of patients will die from their disease, new therapeutic agents such as molecular-targeted drugs are needed. Patients and methodsTo evaluate the role of panitumumab with perioperative chemotherapy, previously untreated patients with locally advanced EGC received, in an open-label randomised phase II study (NEOPECX), standard epirubicin, cisplatin, capecitabine (ECX) chemotherapy with or without panitumumab. The primary end-point was the histological response rate after neoadjuvant therapy. The expression status and gene copy number of EGFR, HER2, and MET were determined by immunohistochemistry and fluorescence in situ hybridization (FISH). Plasma samples were collected before the first cycle of neoadjuvant chemotherapy. ResultsOverall, 160 patients (80 versus 80) were eligible. The majority (82% versus 80%) showed lymph node involvement. Rate of R0-resection, percentage of patients with downstaging to ypT0-2 at pathohistological evaluation, and rate of major histological response was equal in both arms. Toxicity was increased by panitumumab with regard to thromboembolic events and skin toxicity. Patients with tumour EGFR, HER2 or MET expression had shorter progression-free and overall survival. FISH positivity for these markers was associated with shorter survival independent of therapy. High levels of soluble EGFR in particular predicted poor survival in the panitumumab arm. ConclusionThe addition of panitumumab to ECX did not improve downstaging of locally advanced EGC. Low plasma levels of pathway-associated proteins such as sEGFR may identify a group of patients that benefit from EGFR-directed therapy. ClinicalTrials.govNCT01234324.

Abstract A008: Establishment of novel gastric cancer PDX models and cell lines for developing new therapeutics

Abstract It is essential to have appropriate preclinical models for developing new drugs for gastric cancer (GC). Cell lines have been the most commonly used model for this purpose. However, the numbers of gastric cancer cell lines established and available for this purpose are limited and not enough to deal with variety of gastric cancer molecular subtypes such as recently proposed by The Cancer Genome Atlas (TCGA). In addition, patient-derived xenograft (PDX) models have been considered more appropriate models for evaluating the efficacy of new drugs in vivo. For contributing to develop new drugs as well as to seek more effective biomarkers, we have been conducting an intramural project, called DEF study, for establishing new GC PDXs as well as new GC cell lines. So far, 30 multi-passage-competent new PDXs have been successfully established from surgically removed gastric cancer tissues or ascites. Twenty-two (22) new GC cell lines have also been established from PDX tissue (16 lines) or directly from ascites (6 lines). All GC PDXs have been examined by immunohistochemistry for HER2, EGFR, and Met expressions, and we identified 5 cases overexpressing HER2 (corresponding to Score3+) and 1 case overexpressing both EGFR and MET. Comprehensive gene mutation analysis has been performed in 21 PDX tissues, and repetitive mutations (SNV/Indel) have been observed in ERBB2, ERBB3, BRAF, APC, ARID1A, and p53. Copy number alternations have also been identified; examples include KRAS and ERBB2 for amplification and PTEN and CDKN2A for deletion. Citation Format: Takeshi Kuwata, Kazuyoshi Yanagihara. Establishment of novel gastric cancer PDX models and cell lines for developing new therapeutics [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A008.

MET Expression and Genetic Aberrations in Non-Small Cell Lung Cancer

MET Expression and Genetic Aberrations in Non-Small Cell Lung Cancer

Distinct MET Protein Localization Associated With MET Exon 14 Mutation Types in Patients With Non–small-cell Lung Cancer

BackgroundThe MET gene has been recognized as a potential important therapeutic target in non–small-cell lung cancer (NSCLC). We sought to investigate the MET exon 14 mutations in a cohort of Chinese patients with NSCLC. MethodsWe tested 461 NSCLCs for MET exon 14 mutations by sequencing whole exon 14 and its flanking introns. The protein expression was determined by immunohistochemical analysis. ResultsIn this study, we identified MET exon 14 mutations in 9 (2.0%) of 461 NSCLCs. Of these 9 mutations, 7 (77.8%) were located in the splice sites of MET exon 14, with MET overexpression in 6. One point mutation c.3010C>T (p.Arg1004Ter) was nonsense mutation with no MET expression. One insertion mutation was within exon 14 of MET with MET overexpression. MET protein localization in tumor cells with MET exon 14 mutations was different between mutation types. Three point mutations that disrupted the splice donor site of intron 14 were membranous staining, whereas the other mutations were cytoplasmic staining. Patients with MET exon 14 splice site mutations were significantly older. The incidence of MET exon 14 mutations in sarcomatoid carcinoma was significantly higher than in other histologic types (P = .034). ConclusionDistinct MET protein localization is associated with MET exon 14 mutation types in patients with NSCLC. Different MET exon 14 mutation types were identified in a subset of Chinese patients with NSCLC who could possibly benefit from MET targeted therapy.

A non-randomized, open-label, single-arm, Phase 2 study of emibetuzumab in Asian patients with MET diagnostic positive, advanced gastric cancer

PurposeMesenchymal–epithelial transition factor (MET) is expressed in gastric cancer and associated with poor clinical outcomes. We assessed activity, safety, and pharmacokinetics of emibetuzumab, a bivalent monoclonal anti-MET antibody that blocks ligand-dependent and ligand-independent MET signaling.MethodsThis non-randomized, single-arm, Phase 2 study enrolled Asian patients with MET diagnostic positive advanced gastric adenocarcinoma. Emibetuzumab (2000 mg, intravenous) was given on days 1 and 15 (28-day cycle). The primary endpoint was 8-week progression-free survival rate. Secondary objectives included safety, pharmacokinetics, overall survival, and change in tumor size.ResultsTumors from 65 patients were immunohistochemically screened to enroll 15 MET diagnostic positive patients (23% positivity; 8 Japanese, 7 Korean; 10 male). Eight-week progression-free survival rate was 0.47 (70% CI, 0.33–0.59). Disease control rate was 40% (target lesion decreases, three patients; no complete/partial responses according to RECIST). Median overall survival was 17.1 weeks (95% CI, 6.3–not achievable). No serious emibetuzumab-related adverse events or new safety signals emerged. Grade ≥ 3 possibly drug-related adverse events were hyperkalemia, hyponatremia, and hyperuricemia (one each). Emibetuzumab’s pharmacokinetics profile was similar to that observed previously. MET expression and clinical outcomes were not obviously associated.ConclusionEmibetuzumab was well tolerated with limited single-agent activity in advanced gastric adenocarcinoma.

Clinical significance and mechanism of upregulation of PI3Kp110α in non-small cell lung carcinoma

Objective: To investigate the clinical significance and mechanism of upregulation of phosphoinositide 3-kinase p110α(PI3Kp110α)in non-small cell lung carcinoma (NSCLC). Methods: Expressions of PI3Kp110α and other components in PI3K signaling pathway (including phospho-Akt (p-Akt, Ser 473), MET, ROS1, HER-2, ALK, total EGFR and mutant EGFR) and p53 (the transcription factor of PIK3CA) mutation in NSCLC were detected by immunohistochemistry. The relationships between PI3Kp110α expression and clinicopathological characteristics, expressions of other proteins in PI3K pathway and p53 mutation were analyzed. Results: In 170 NSCLC patients, 72 cases (42.4%) showed lower expression and 98 cases (57.6%) showed higher expression of PI3Kp110α. Upregulation of PI3Kp110α was not significantly associated with gender, age, T stage and pathologic grade (P>0.05). While upregulation of PI3Kp110α was significantly associated with smoking status of patients, pathologic classification, N stage, TNM stage and Ki-67 index (P<0.05). Expression of PI3Kp110α was positively correlated with expressions of MET (P<0.05) and mutant EGFR (P=0.018), while not significantly related with expressions of p-Akt(Ser473), HER-2, ALK, ROS1, total EGFR or p53 mutation (P>0.05). Conclusions: Upregulation of PI3Kp110α is closely related with tumorigenesis of non-smoking lung adenocarcinoma. MET overexpression and EGFR mutation may be crucial to upregulate expression of PI3Kp110α in NSCLC. Overexpression of PI3Kp110α may inhibit tumor cell proliferation in NSCLC through a different pathway other than classical PI3K pathway. Upregulation of PI3Kp110α may predict favorable prognosis of NSCLC patients.

Distinct projection targets define subpopulations of mouse brainstem vagal neurons that express the autism‐associated MET receptor tyrosine kinase

AbstractDetailed anatomical tracing and mapping of the viscerotopic organization of the vagal motor nuclei has provided insight into autonomic function in health and disease. To further define specific cellular identities, we paired information based on visceral connectivity with a cell‐type specific marker of a subpopulation of neurons in the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (nAmb) that express the autism‐associated MET receptor tyrosine kinase. As gastrointestinal disturbances are common in children with autism spectrum disorder (ASD), we sought to define the relationship between MET‐expressing (MET+) neurons in the DMV and nAmb, and the gastrointestinal tract. Using wholemount tissue staining and clearing, or retrograde tracing in a METEGFP transgenic mouse, we identify three novel subpopulations of EGFP+ vagal brainstem neurons: (a) EGFP+ neurons in the nAmb projecting to the esophagus or laryngeal muscles, (b) EGFP+ neurons in the medial DMV projecting to the stomach, and (b) EGFP+ neurons in the lateral DMV projecting to the cecum and/or proximal colon. Expression of the MET ligand, hepatocyte growth factor (HGF), by tissues innervated by vagal motor neurons during fetal development reveal potential sites of HGF‐MET interaction. Furthermore, similar cellular expression patterns of MET in the brainstem of both the mouse and nonhuman primate suggests that MET expression at these sites is evolutionarily conserved. Together, the data suggest that MET+ neurons in the brainstem vagal motor nuclei are anatomically positioned to regulate distinct portions of the gastrointestinal tract, with implications for the pathophysiology of gastrointestinal comorbidities of ASD.