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An FBXW7-ZEB2 axis links EMT and tumour microenvironment to promote colorectal cancer stem cells and chemoresistance.

ABSTRACT: Colorectal cancer (CRC) patients develop recurrence after chemotherapy owing to the survival of stem cell-like cells referred to as cancer stem-like cells (CSCs). The origin of CSCs is linked to the epithelial-mesenchymal transition (EMT) process. Currently, it remains poorly understood how EMT programmes enable CSCs residing in the tumour microenvironment to escape the effects of chemotherapy. This study identifies a key molecular pathway that is responsible for the formation of drug-resistant CSC populations. Using a modified yeast-2-hybrid system and 2D gel-based proteomics methods, we show that the E3-ubiquitin ligase FBXW7 directly binds and degrades the EMT-inducing transcription factor ZEB2 in a phosphorylation-dependent manner. Loss of FBXW7 induces an EMT that can be effectively reversed by knockdown of ZEB2. The FBXW7-ZEB2 axis regulates such important cancer cell features, as stemness/dedifferentiation, chemoresistance and cell migration in vitro, ex vivo and in animal models of metastasis. High expression of ZEB2 in cancer tissues defines the reduced ZEB2 expression in the cancer-associated stroma in patients and in murine intestinal organoids, demonstrating a tumour-stromal crosstalk that modulates a niche and EMT activation. Our study thus uncovers a new molecular mechanism, by which the CRC cells display differences in resistance to chemotherapy and metastatic potential.

SUBMITTER: Li N

PROVIDER: S-EPMC6381143 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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An FBXW7-ZEB2 axis links EMT and tumour microenvironment to promote colorectal cancer stem cells and chemoresistance.

Li Ningning N Babaei-Jadidi Roya R Lorenzi Federica F Spencer-Dene Bradley B Clarke Philip P Domingo Enric E Tulchinsky Eugene E Vries Robert G J RGJ Kerr David D Pan Yihang Y He Yulong Y Bates David O DO Tomlinson Ian I Clevers Hans H Nateri Abdolrahman S AS

Oncogenesis 20190219 3

Colorectal cancer (CRC) patients develop recurrence after chemotherapy owing to the survival of stem cell-like cells referred to as cancer stem-like cells (CSCs). The origin of CSCs is linked to the epithelial-mesenchymal transition (EMT) process. Currently, it remains poorly understood how EMT programmes enable CSCs residing in the tumour microenvironment to escape the effects of chemotherapy. This study identifies a key molecular pathway that is responsible for the formation of drug-resistant ...[more]

PMID: 30783098

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Project description:ObjectiveTumor metastasis and resistance to chemotherapy are two critical factors that contribute to the high death rate of colorectal cancer (CRC) patients. Metastasis is facilitated by the epithelial-mesenchymal transition (EMT) of tumor cells, which has emerged not only as a fundamental process during metastasis, but is also a key process leading to chemoresistance of cancer cells. However, the underlying mechanisms of EMT in CRC cell remain unknown. Here, we aim to assess the role of dual serine/threonine and tyrosine protein kinase (DSTYK) in CRC metastasis and chemoresistance.MethodsTo study the role of DSTYK in TGF-β-induced EMT, we employed techniques including Crispr/Cas9 knockout (KO) to generate DSTYK KO cell lines, RT-PCR to detect the mRNA expression, immunofluorescence analyses, and western blots to detect protein levels of DSTYK in the following 4 cell lines: control LS411N-TβRII and LS411N-TβRII/DSTYK KO, control LS513 and LS513/DSTYK KO cells, treated with/without TGF-β. The effects of DSTYK on apoptosis were investigated by MTT assays, flow cytometry assays, and TUNEL assays. The expression of DSTYK in CRC patients and its correlation with EMT markers were determined by bioinformatics analysis. For in vivo analysis, both xenograft and orthotopic tumor mouse models were employed to investigate the function of DSTYK in chemoresistance and metastasis of tumors.ResultsIn this study, we demonstrate that the novel kinase DSTYK promotes both TGF-β-induced EMT and the subsequent chemoresistance in CRC cells. DSTYK KO significantly attenuates TGF-β-induced EMT and chemoresistance in CRC cells. According to the Gene Expression Omnibus (GEO) database, the expression of DSTYK is not only positively correlated to the expression of TGF-β, but proportional to the death rate of CRC patients as well. Evidently, the expression of DSTYK in the metastatic colorectal cancer samples from patients was significantly higher than that of primary colorectal cancer samples. Further, we demonstrate in mouse models that chemotherapeutic drug treatment suppresses the growth of DSTYK KO tumors more effectively than control tumors.ConclusionOur findings identify DSTYK as a novel protein kinase in regulating TGF-β-mediated EMT and chemoresistance in CRC cells, which defines DSTYK as a potential therapeutic target for CRC therapy.

Project description:Simple Summary The epithelial-mesenchymal transition (EMT) program plays a central role in the metastasis of patients with colorectal carcinoma (CRC). However, few studies have investigated the dominant regulatory factors and underlying mechanisms during the EMT process in CRC metastasis. Here we have characterized a novel transcriptionally repressive complex, ZEB2/TWIST1/PRMT5/NuRD, which epigenetically silences E-cadherin, leading to the invasion and metastasis of CRC cells. Our work provides a comprehensive understanding of the epigenetic mechanisms by which the key EMT metastasis driver, E-cadherin, is regulated in CRC, thereby suggesting PRMT5 and HDAC2 inhibitors as potentially therapeutic agents in CRC therapy. Abstract (1) Background: The EMT plays a crucial role in tumor metastasis, which is the major cause for colorectal carcinoma-related mortality. However, the underlying regulators and mechanisms of EMT in CRC metastasis are still poorly understood; (2) Methods: The transcriptional regulators of EMT in CRC and their functions were examined using RT2212PCR, Western blotting, and luciferase reporter assay. The components of ZEB2/TWIST1 complex and their mutual interactions were identified via affinity purification, mass spectrometry, co-immunoprecipitation, and pull-down experiments. The functional mechanisms of ZEB2/TWIST1/PRMT5/NuRD axis were determined by chromatin immunoprecipitation and luciferase reporter assay. The contribution of ZEB2/TWIST1/PRMT5/NuRD complex in the CRC metastasis was investigated using wound healing, transwell assay, and in vivo xenograft mouse model; (3) Results: We found that ZEB2 and TWIST1 were both significantly upregulated in CRC tissues and EMT of CRC cells. ZEB2 could recruit TWIST1 to the E-cadherin promoter and synergistically repressed its transcription. In addition, ZEB2 physically interacted with TWIST1, PRMT5, and the nucleosome remodeling and deacetylase (NuRD) complex to form a novel repressive multicomplex, leading to epigenetic silencing of E-cadherin in CRC cells. Notably, the combined inhibition of ZEB2 and TWIST1 and epigenetic inhibition markedly reduced CRC metastasis in mice; (4) Conclusions: We revealed for the first time that ZEB2 could recruit TWIST1, PRMT5, and NuRD to form a repressive multicomplex and epigenetically suppresses the transcription of E-cadherin, thereby inducing the EMT process and metastasis in CRC. Our results also confirmed the therapeutic potential of epigenetic inhibitors in CRC.

Project description:BACKGROUND:Quiescent/slow cycling cells have been identified in several tumors and correlated with therapy resistance. However, the features of chemoresistant populations and the molecular factors linking quiescence to chemoresistance are largely unknown. METHODS:A population of chemoresistant quiescent/slow cycling cells was isolated through PKH26 staining (which allows to separate cells on the basis of their proliferation rate) from colorectal cancer (CRC) xenografts and subjected to global gene expression and pathway activation analyses. Factors expressed by the quiescent/slow cycling population were analyzed through lentiviral overexpression approaches for their ability to induce a dormant chemoresistant state both in vitro and in mouse xenografts. The correlation between quiescence-associated factors, CRC consensus molecular subtype and cancer prognosis was analyzed in large patient datasets. RESULTS:Untreated colorectal tumors contain a population of quiescent/slow cycling cells with stem cell features (quiescent cancer stem cells, QCSCs) characterized by a predetermined mesenchymal-like chemoresistant phenotype. QCSCs expressed increased levels of ZEB2, a transcription factor involved in stem cell plasticity and epithelial-mesenchymal transition (EMT), and of antiapototic factors pCRAF and pASK1. ZEB2 overexpression upregulated pCRAF/pASK1 levels resulting in increased chemoresistance, enrichment of cells with stemness/EMT traits and proliferative slowdown of tumor xenografts. In parallel, chemotherapy treatment of tumor xenografts induced the prevalence of QCSCs with a stemness/EMT phenotype and activation of the ZEB2/pCRAF/pASK1 axis, resulting in a chemotherapy-unresponsive state. In CRC patients, increased ZEB2 levels correlated with worse relapse-free survival and were strongly associated to the consensus molecular subtype 4 (CMS4) characterized by dismal prognosis, decreased proliferative rates and upregulation of EMT genes. CONCLUSIONS:These results show that chemotherapy-naive tumors contain a cell population characterized by a coordinated program of chemoresistance, quiescence, stemness and EMT. Such population becomes prevalent upon drug treatment and is responsible for chemotherapy resistance, thus representing a key target for more effective therapeutic approaches.

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An FBXW7-ZEB2 axis links EMT and tumour microenvironment to promote colorectal cancer stem cells and chemoresistance.

Publications

An FBXW7-ZEB2 axis links EMT and tumour microenvironment to promote colorectal cancer stem cells and chemoresistance.

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