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The TGF?-miR200-MIG6 pathway orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors.

ABSTRACT: Although specific mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) identify tumors that are responsive to EGFR tyrosine kinase inhibitors (TKI), these genetic alterations are present in only a minority of patients. Patients with tumors expressing wild-type EGFR lack reliable predictive markers of their clinical response to EGFR TKIs. Although epithelial-mesenchymal transition (EMT) has been inversely correlated with the response of cancers to EGFR-targeted therapy, the precise molecular mechanisms underlying this association have not been defined and no specific EMT-associated biomarker of clinical benefit has been identified. Here, we show that during transforming growth factor ? (TGF?)-mediated EMT, inhibition of the microRNAs 200 (miR200) family results in upregulated expression of the mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR. The MIG6-mediated reduction of EGFR occurs concomitantly with a TGF?-induced EMT-associated kinase switch of tumor cells to an AKT-activated EGFR-independent state. In a panel of 25 cancer cell lines of different tissue origins, we find that the ratio of the expression levels of MIG6 and miR200c is highly correlated with EMT and resistance to erlotinib. Analyses of primary tumor xenografts of patient-derived lung and pancreatic cancers carrying wild-type EGFR showed that the tumor MIG6(mRNA)/miR200 ratio was inversely correlated with response to erlotinib in vivo. Our data demonstrate that the TGF?-miR200-MIG6 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors, and identify a low ratio of MIG6 to miR200 as a promising predictive biomarker of the response of tumors to EGFR TKIs.

SUBMITTER: Izumchenko E

PROVIDER: S-EPMC4122100 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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The TGFβ-miR200-MIG6 pathway orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors.

Izumchenko Evgeny E Chang Xiaofei X Michailidi Christina C Kagohara Luciane L Ravi Rajani R Paz Keren K Brait Mariana M Hoque Mohammad O MO Ling Shizhang S Bedi Atul A Sidransky David D

Cancer research 20140515 14

Although specific mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) identify tumors that are responsive to EGFR tyrosine kinase inhibitors (TKI), these genetic alterations are present in only a minority of patients. Patients with tumors expressing wild-type EGFR lack reliable predictive markers of their clinical response to EGFR TKIs. Although epithelial-mesenchymal transition (EMT) has been inversely correlated with the response of cancers to EGFR-targeted thera ...[more]

PMID: 24830724

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Project description:Background/aimsA novel paradigm in tumor biology suggests that osteosarcoma (OS) chemo-resistance is driven by osteosarcoma stem cell-like cells (OSCs). As the sensitivity of only a few tumors to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations, there is a need to elucidate mechanisms of resistance to EGFR-targeted therapies in OS that do not harbor TK sensitizing mutations to develop new strategies to circumvent resistance to EGFR inhibitors.MethodsAs a measure of the characters of OSCs, serum-free cultivation, cell viability test with erlotinib, and serial transplantation in vivo was used. Western blot assays were used to detect the association between erlotinib resistance and transforming growth factor beta (TGFβ)-induced epithelial-to-mesenchymal transition (EMT) progression. By using TaqMan qPCR miRNA array, online prediction software, luciferase reporter assays and western blot analysis, we further elucidated the mechanisms.ResultsHere, CD166+ cells are found in 10 out of 10 tumor samples. We characterize that CD166+ cells from primary OS tissues bear hallmarks of OSCs and erlotinib-resistance. TGFβ-induced EMT-associated kinase switch is demonstrated to promote erlotinib-resistance of CD166+ OSCs. Further mechanisms study show that TGFβ-induced EMT decreases miR-499a expression through the direct binding of Snail1/Zeb1 to miR-499a promoter. Overexpression of miR-499a in CD166+ OSCs inhibits TGFβ-induced erlotinib-resistance in vitro and in vivo. SHKBP1, the direct target of miR-499a, regulates EGFR activity reduction occurring concomitantly with a TGFβ-induced EMT-associated kinase switch to an AKT-activated EGFR-independent state. TGFβ-induced activation of AKT co-opts an increased SHKBP1 expression, which further regulates EGFR activity. In clinic, the ratio of the expression levels of SHKBP1 and miR-499a is highly correlated with EMT and resistance to erlotinib.ConclusionTGFβ-miR-499a-SHKBP1 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors in CD166+ OSCs, implies that inhibition of TGFβ induced EMT-associated kinase switch may reverse the chemo-resistance of OSCs to EGFR inhibitors. We also suggest that an elevated SHKBP1/miR-499a ratio is a molecular signature that characterizes the erlotinib-resistant OS, which may have clinical value as a predictive biomarker.

Project description:Local invasion is the initial step towards metastasis, the main cause of cancer mortality. In human colorectal cancer (CRC), malignant cells predominantly invade as cohesive collectives and may undergo partial epithelial-mesenchymal transition (pEMT) at the invasive front. How this particular mode of stromal infiltration is generated is unknown. Here we investigated the impact of oncogenic transformation and the microenvironment on tumor cell invasion using genetically engineered organoids as CRC models. We found that inactivation of the Apc tumor suppressor combined with expression of oncogenic KrasG12D and dominant-negative Trp53R172H did not cell-autonomously induce invasion in vitro. However, oncogenic transformation primed organoids for activation of a collective invasion program upon exposure to the prototypical microenvironmental factor TGFβ1. Execution of this program co-depended on a permissive extracellular matrix which was further actively remodeled by invading organoids. Although organoids shed some epithelial properties particularly at the invasive edge, TGFβ1-stimulated organoids largely maintained epithelial gene expression while additionally implementing a mesenchymal transcription pattern, resulting in a pEMT phenotype that did not progress to a fully mesenchymal state. Notably, while TGFβ1 induced pEMT and promoted collective invasion, it abrogated self-renewal capacity of TKA organoids which correlated with the downregulation of intestinal stem cell (ISC) marker genes. Mechanistically, induction of the non-progressive pEMT required canonical TGFβ signaling mediated by Smad transcription factors (TFs), whereas the EMT master regulators Snail1 and Zeb1 were dispensable. Gene expression profiling provided further evidence for pEMT of TGFβ1-treated organoids and showed that their transcriptomes resemble those of human poor prognosis CMS4 cancers which likewise exhibit pEMT features. We propose that collective invasion in colorectal carcinogenesis is triggered by microenvironmental stimuli through activation of a novel, transcription-mediated form of non-progressive pEMT independently of classical EMT regulators.

Dataset Information

The TGF?-miR200-MIG6 pathway orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors.

Publications

The TGFβ-miR200-MIG6 pathway orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors.

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