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RNA sequencing to identify differentially-expressed genes in HER3-depleted KatoII MET-amplified cancer cells

ABSTRACT: Receptor tyrosine kinases (RTKs) are recognized as targets of precision medicine in human cancer upon their gene amplification or constitutive activation, resulting in increased downstream signal complexity including heterotypic crosstalk with other RTKs. The Met hepatocyte growth factor RTK exhibits such reciprocal crosstalk with several members of the human EGFR (HER) family of RTKs when amplified in cancer cells. We observed that Met signaling converges on HER3 tyrosine phosphorylation across a panel of seven MET-amplified cancer cell lines and that HER3 is required for cancer cell expansion and oncogenic capacity in vitro and in vivo. In order to identify genes required for cancer cell proliferation in the MET-amplified setting whose expression, we stably transduced KatoII MET-amplified cells with shRNA targeting ERBB3, the gene encoding HER3. Stably-transduced cells were selected with puromycin, assayed for proliferative phenotype, and lysed for RNA extraction. Pooled libraries of total RNA were then sequenced using the Illumina HiSeq4000 platform, and differentially-expressed genes were identified from read counts normalized across samples. Differentially-expressed genes were further assayed for dependence on HER3 in other MET-amplified cell lines to identify genes recurrently dependent on HER3 in the MET-amplified setting.

ORGANISM(S): Homo sapiens

PROVIDER: GSE189461 | GEO | 2021/11/23

REPOSITORIES: GEO

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RNA sequencing to identify differentially-expressed genes in HER3-depleted KatoII MET-amplified cancer cells

Project description:RNA sequencing to identify differentially-expressed genes in HER3-depleted KatoII MET-amplified cancer cells

| PRJNA783051 | ENA

MCF10A cells expressing HER2 and HER3 and grown in three-dimensional cultures

Project description:The tyrosine kinase receptors HER2 and HER3 play an important role in breast cancer. The HER2/HER3 heterodimer is a critical oncogenic unit associated with reduced relapse-free and decreased overall survival. We provide gene expression profile of the mammary epithelial cells MCF10A expressing HER2, HER3 or HER2/HER3 and grown in three-dimensional cultures for 15 days in the presence of heregulin, a known HER3-ligand that stabilizes and activates the HER2/HER3 heterodimer. The mammary epithelial cells MCF10A were transduced with retroviral vectors expressing HER2, HER3 or both. Cells from each group were grown for 15 days in three-dimensional cultures. At the end of the experiment, RNA was extracted for gene expression analysis.

2012-09-01 | E-GEOD-37009 | biostudies-arrayexpress

Homo sapiens

Project description:MET Amplified Gastric Cancer Cell Lines +/- Crizotinib

| PRJNA310017 | ENA

A receptor tyrosine kinase network comprised of FGFRs, EGFR, ERBB2, and MET drives growth and survival of HNSCC cell lines.

Project description:We have previously shown that some gefitinib insensitive head and neck squamous cell carcinoma (HNSCC) cell lines exhibit dominant autocrine fibroblast growth factor receptor (FGFR) signaling. Herein, we deployed a whole genome loss-of-function screen to identify genes whose knockdown potentiated the inhibitory effect of the FGFR inhibitor, AZ12908010, in HNSCC cell lines. Three HNSCC cell lines expressing a genome-wide shRNA library were treated with AZ8010 and the abundance of shRNA sequences was assessed by deep sequencing. Synthetic lethal hits were validated through use of specific inhibitors and independent shRNAs. We found that multiple alternate receptors provided protection from FGFR inhibition, including the receptor tyrosine kinases (RTKs), epidermal growth factor receptor 2 (ERBB2) and hepatocyte growth factor receptor (MET). We showed that specific knockdown of either ERBB2 or MET in combination with FGFR inhibition led to increased inhibition of growth relative to FGFR tyrosine kinase inhibitor (TKI) treatment alone. These results were confirmed using specific small molecule inhibitors of either ERBB family members or MET. Moreover, the combination of FGFR, MET and ERBB family inhibitors showed the largest inhibition of growth as compared to the double combinations. These results reveal a role for alternate RTKs in maintaining pro-growth and survival signaling in HNSCC cells in the setting of FGFR inhibition. Thus, improved therapies for HNSCC patients could involve rationally designed combinations of TKIs targeting FGFR, ERBB family members and MET. Using a genome-wide shRNA library in combination with deep sequencing, we screened for gene targets that were synthetic lethal with the FGFR inhibitor, AZ12908010 in HNSCC cells. Three HNSCC cell lines were screened in triplicate and the abundance of shRNA sequences in drug treated cells was compared to control treated cells.

2013-05-03 | E-GEOD-39305 | biostudies-arrayexpress

Acquired resistance mechanisms to capmatinib, a MET inhibitor in MET-amplified non-small cell lung cancer cells

Project description:Purpose: MET is a receptor tyrosine kinase (RTK) that has been considered a druggable target in non-small cell lung cancer (NSCLC). To understand the mechanisms of resistance to MET-TKIs and establish therapeutic strategies, we developed an in vitro model using capmatinib-resistant cell lines (EBC-CR1, CR2, and CR3) derived from the MET-amplified NSCLC cell line EBC-1. Methods: We established capmatinib-resistant NSCLC cell lines from the MET-amplified NSCLC cell line EBC-1 and identified alternative signaling pathways using 3’mRNA sequencing and human phospho-RTK arrays. Copy number alterations were evaluated by quantitative PCR and cell proliferation assay; activation of RTKs and downstream effectors were compared between the parental cell line EBC-1 and the EBC-CR1, -CR2, and -CR3 resistant cell lines. Results: We found that epidermal growth factor (EGFR) mRNA expression and protein activation were increased in EBC-CR1–3 cells compared to EBC-1 cells. EBC-CR1 cells showed EGFR-dependent growth and sensitivity to afatinib, an irreversible EGFR TKI. EBC-CR2 cells, which overexpressed the EGFR-MET heterodimer, responded dramatically to the combination of capmatinib and the phosphoinositide-3 kinase catalytic subunit α (PIK3CA) inhibitor afatinib. In addition, EBC-CR3 cells, which had activated EGFR along with amplified PIK3CA, were sensitive to the combination of afatinib and the PI3Kα inhibitor. Conclusions: Our in vitro studies suggested that activation of EGFR signaling and/or genetic alteration of downstream effectors like PIK3CA were alternative resistance mechanisms used by capmatinib-resistant NSCLC cell lines. In addition, combined treatments with MET, EGFR, and PI3Kα inhibitors may be an effective therapeutic strategy in MET-TKI-resistant NSCLC patients.

2017-05-01 | GSE94405 | GEO

A receptor tyrosine kinase network comprised of FGFRs, EGFR, ERBB2, and MET drives growth and survival of HNSCC cell lines.

2013-05-03 | GSE39305 | GEO

MET regulates expression of co-stimulatory and co-inhibitory molecules in tumor cells and contributes to suppression of immune cell function

Project description:MET-altered human cancer cell lines including Hs746T (MET-mutated/amplified), H596 (MET-mutated) and H1993 (MET-amplified) cells were used. In microarray analysis, MET suppression using MET inhibitor or siRNAs up-regulated variable co-stimulatory molecules, including 4-1BBL, OX40L and CD70, and down-regulated variable co-inhibitory molecules. Among co-inhibitory molecules, PD-L1 was one of the most markedly down-regulated genes by MET suppression, which was validated at the mRNA and total/surface protein levels in Hs746T and H1993 cells. Consistently, MET activation by HGF treatment increased PD-L1 expression in H596 cells. Co-culture of human peripheral blood mononuclear cells (PBMC) with Hs746T cells suppressed IFNg production from PBMC, which was restored by an MET inhibitor or PD-L1 blockade. A significant positive correlation between MET and PD-L1 expression was observed in The Cancer Genome Atlas (TCGA) analysis for variable cancer types and immunohistochemistry for lung cancer. MET overexpression in PD-L1-high tumor exhibited decreased expressions of T-cell effector molecules in TCGA analysis for lung cancer. In summary, MET overexpression/activation is related to immunosuppressive phenotype and is involved in immune escape of tumor by PD-L1 up-regulation. MET-targeted therapy combined with immunotherapy could be a potential therapeutic strategy in MET-dependent cancer.

2019-08-20 | GSE135976 | GEO

MCF10A cells expressing HER2 and HER3 and grown in three-dimensional cultures

2012-09-01 | GSE37009 | GEO

Cabozantinib and Panitumumab to Treat KRAS Wild-Type Metastatic Colorectal Cancer

Project description:There will be three parts to this phase I study: 1) the Combination Dose Finding cohort; 2) the Combination Expansion cohort; and 3) the Monotherapy MET Amplified cohort. In the Combination Dose Finding cohort and the Combination Expansion cohort, we will combine cabozantinib and panitumumab in patients with KRAS wild-type metastatic colorectal cancer (CRC). In the Monotherapy MET Amplified cohort, we will screen at least 50 patients for MET gene amplification ("MET amplification"). Patients with MET amplification will receive cabozantinib only (monotherapy). The primary objective of this open-label phase Ib trial are: 1. To determine the maximum tolerated dose and the recommended phase II dose for the combination of cabozantinib and panitumumab in patients with KRAS wild-type metastatic colorectal cancer and 2. To identify the objective response rate (ORR) of cabozantinib monotherapy in patients with prospectively identified MET amplified metastatic colorectal cancer. The secondary objectives are: 1. To describe the non-dose limiting toxicities of cabozantinib and panitumumab. 2. To describe the clinical activity (ORR, PFS, OS) of cabozantinib and panitumumab. 3. To describe the safety and tolerability of cabozantinib monotherapy in patients with MET amplified colorectal cancer. 4. To describe the clinical activity (PFS, OS) of cabozantinib monotherapy in patients with MET amplified colorectal cancer.

| 2156647 | ecrin-mdr-crc

The ShcA PTB Domain Functions as a Biological Sensor of Phospho-tyrosine Signaling During Breast Cancer Progression

Project description:The ShcA adaptor possesses two phosphotyrosine binding motifs, which include an SH2 and a PTB domain. In the majority of cases, ShcA utilizes its PTB domain to engage activated receptor tyrosine kinases (RTKs). To establish the mportance of this domain during mammary tumorigenesis, we employed a ShcA mutant (R175Q) that no longer binds phospho-tyrosine residues via its PTB domain. We demonstrate that the ShcR175Q mutant delays mammary tumor onset n MMTV/MT transgenic animals. Paradoxically, we observe a robust increase in the growth and angiogenesis of emerging mammary tumors. ShcR175Q-expressing breast cancer cells increase fibronectin secretion and possess elevated levels f integrin α5/β1, the principle fibronectin receptor. Sustained integrin engagement activates Src, which in turn phosphorylates pro-angiogenic RTKs, including PDGFR, FGFR and Met, leading to increased VEGF secretion from ShcR175Q-xpressing breast cancer cells. Finally, we describe a ShcR175Q-dependent gene signature that stratifies breast cancer patients with a high microvessel density. This is the first study to demonstrate that intracellular signaling pathways ownstream of the ShcA PTB domain both positively and negatively regulate tumorigenesis during the various stages of breast cancer progression ShcA mutant (R175Q) vs NT/ShcA cells

2013-08-07 | E-GEOD-41718 | biostudies-arrayexpress

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