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.
Project description:Resistance to adjuvant chemotherapy is a major clinical problem in the treatment of colorectal cancer (CRC). The aim of this study was to elucidate the role of an epithelial to mesenchymal transition (EMT)-inducing protein, ZEB2, in chemoresistance of CRC, and to uncover the underlying mechanism. We performed IHC for ZEB2 and association analyses with clinical outcomes on primary CRC and matched CRC liver metastases in compliance with observational biomarker study guidelines. ZEB2 expression in primary tumours was an independent prognostic marker of reduced overall survival and disease-free survival in patients who received adjuvant FOLFOX chemotherapy. ZEB2 expression was retained in 96% of liver metastases. The ZEB2-dependent EMT transcriptional programme activated nucleotide excision repair (NER) pathway largely via upregulation of the ERCC1 gene and other components in NER pathway, leading to enhanced viability of CRC cells upon oxaliplatin treatment. ERCC1-overexpressing CRC cells did not respond to oxaliplatin in vivo, as assessed using a murine orthotopic model in a randomised and blinded preclinical study. Our findings show that ZEB2 is a biomarker of tumour response to chemotherapy and risk of recurrence in CRC patients. We propose that the ZEB2-ERCC1 axis is a key determinant of chemoresistance 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.
Project description:<h4>Simple Summary</h4> 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. <h4>Abstract</h4> (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:FBXW7 mutations occur in a variety of human cancers including colorectal cancer (CRC). Elucidating its mechanism of action has become crucial for cancer therapy; however, it is also complicated by the fact that FBXW7 can influence many pathways due to its role as an E3-ubiquitin ligase in proteasome degradation. FBXW7 and TP53 are tumour suppressors intensively implicated in colorectal carcinogenesis. Deletion mutations in these two genes in animal models mark the progression from adenoma to carcinoma. Although still largely unknown, the last defense mechanism against CRC at the molecular level could be through a synergistic effect of the two genes. The underlying mechanism requires further investigation. In our laboratory, we have used a phospho-kinase profiler array to illustrate a potential molecular link between FBXW7 and p53 in CRC cells. In vitro and in vivo assessments demonstrated aberrant induction of phosphorylated p53 at Serine 15 [phospho-p53(Ser15)] in human FBXW7-deficient CRC cells as compared to their FBXW7-wild-type counterparts. FBXW7 loss in HCT116 cells promoted resistance to oxaliplatin. Immunoblotting data further confirmed that reduction of phospho-p53(Ser15) may contribute to the decreased efficacy of therapy in FBXW7-mutated CRC cells. The findings may suggest the applicability of phospho-p53(Ser15) as an indicative marker of FBXW7-mutations. Phospho-p53(Ser15) regulation by FBXW7 E3-ligase activity could provide important clues for understanding FBXW7 behavior in tumour progression and grounds for its clinical applicability thereafter.
Project description:<b>Purpose:</b> Tumor metastasis seriously affects the survival of patients. In recent years, some studies confirmed that long non-coding RNA (lncRNA) played an essential role in tumor progression. A few studies reported that LINC01296 acted as an oncogenic regulator of cancer. However, its in-depth specific biological mechanism in tumor metastasis is still unknown. <b>Methods:</b> Real-time quantitative PCR (qPCR) was performed to detect the expression of LINC01296 and miR-141-3p in NSCLC, CRC tissues and cell lines, and the dual luciferase report was used to evaluate the relationship between LINC01296, miR-141-3p and ZEB1/ZEB2 relationship. Western blot experiments are used to detect changes in protein levels. Transwell and wound healing measures migration and invasion capabilities. <b>Results:</b> In this study, we used non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) as the research objects, LINC01296 was found to be highly expressed in NSCLC and CRC tissues and positively related to poor prognosis. We also demonstrated LINC01296 regulated NSCLC and CRC invasion and metastasis by modulating epithelial-mesenchymal transition (EMT) by up-regulating ZEB1 and ZEB2. Consequently, LINC01296 acted as a sponge of miR-141-3p, which negatively regulates EMT process. <b>Conclusions:</b> The report revealed a new mechanism by which LINC01296 regulates the EMT process through miR-141-3p/ZEB1-ZEB2 axis and affects cancer metastasis.
Project description:Although the cancer stem cell (CSC) concept has provided a reasonable explanation for cancer recurrence following chemotherapy, the relationship between CSCs and chemotherapy resistance has not been thoroughly investigated, especially in solid tumors. We aimed to identify the mechanism underlying colorectal cancer (CRC) chemoresistance focusing on the cell cycle mediator F-Box/WD repeat domain-containing 7 (FBXW7). From 55 consecutive CRC cases who underwent neoadjuvant chemotherapy (NAC) or neoadjuvant chemoradiotherapy (NACRT) at Kyoto University Hospital, pre-treatment endoscopic biopsy specimens were collected and divided into two groups upon immunohistochemical (IHC) analysis: 21 cases of FBXW7 high expression (FBXW7-high group) and 34 cases of low expression (FBXW7-low group). High FBXW7 expression in pre-treatment biopsy specimen was significantly associated with poor pathological therapeutic effect (<i>p</i> = 0.019). The proportion of FBXW7-positive cells in surgically resected CRC specimens from patients who underwent NAC or NACRT was significantly higher than that in the pre-treatment biopsy specimens (<i>p</i> < 0.001). The expression of FBXW7 was inversely correlated with that of Ki67 in both pre-treatment biopsy specimens and surgically resected specimens. <i>FBXW7</i> expression in the EpCAM<sup>high</sup>/CD44<sup>high</sup> subpopulation isolated by flow cytometry from CRC samples was significantly higher than that in the EpCAM<sup>high</sup>/CD44<sup>low</sup> subpopulation. Cell-cycle analysis in CRC cell lines revealed that, upon <i>FBXW7</i> silencing, the proportion of G0/G1 cells was significantly lower than that in control cells. Moreover, knockdown of <i>FBXW7</i> in CRC cell lines increased the sensitivity to anti-cancer drugs in vitro and in vivo. A subset of CRC stem cells possesses chemoresistance through FBXW7 expression. Cell cycle arrest induced by FBXW7 expression should be considered as a potential therapeutic target to overcome chemoresistance in CRC stem cell subsets.
Project description:The long noncoding RNAs (lncRNAs) have been increasingly appreciated as key players underlying tumourigenesis and hold great potentials as prognostic biomarkers and therapeutic targets. However, their roles in head neck squamous cell carcinoma (HNSCC) have remained incompletely known. Here, we sought to reveal the oncogenic roles and clinical significance of a tumour-associated lncRNA, zinc finger E-box binding homeobox 2 antisense RNA 1 (ZEB2-AS1), in HNSCC. ZEB2-AS1 was aberrantly overexpressed in a fraction of HNSCC samples. Its overexpression significantly associated with large tumour size, cervical node metastasis and reduced overall and disease-free survival. Antisense oligonucleotides (ASO)-mediated ZEB2-AS1 depletion markedly inhibited cell proliferation, migration and invasion while triggered apoptosis in HNSCC cells in part via modulating ZEB2 mRNA stability. Enforced overexpression of ZEB2 largely attenuated the phenotypic changes resulted from ZEB2-AS1 inhibition except the impaired cell proliferation. In addition, ZEB2-AS1 was required for TGF-?1-induced epithelial-mesenchymal transition (EMT) in vitro. Significantly reduced tumour growth and lung metastasis were observed in ZEB2-AS1-depleted cells in HNSCC xenograft animal models. Taken together, our findings reveal that overexpression of ZEB2-AS1 associates with tumour aggressiveness and unfavourable prognosis by serving as a putative oncogenic lncRNA and a novel prognostic biomarker in HNSCC.
Project description:To explore the role of long-chain non-coding RNA (lncRNA) taurine up-regulated gene 1 (TUG1) in the development of colorectal cancer (CRC) via the miR-138-5p/zinc finger E-box-binding homeobox 2 (ZEB2) axis. Eighty-four CRC tissue specimens and 84 corresponding paracancerous tissue specimens were sampled from 84 patients with CRC admitted to the First Hospital of Jilin University from January 2018 to September 2019. The TUG1 expression in the specimens was determined, and its value in diagnosis and prognosis of CRC was analyzed. Additionally, constructed stable and transient overexpresison vectors and inhibition vectors were transfected into CRC cells. The MTT, transwell, and flow cytometry were adopted for analysis on the proliferation, invasion, and apoptosis of transfected cells, respectively, and a dual luciferase reporter (DLR) assay was carried out for correlation determination between TUG1 and miR-138-5p and between miR-138-5p and ZEB2. TUG1 was up-regulated in CRC, and serum TUG1 could be adopted as a diagnostic marker of CRC, with area-under-the-curve (AUC) larger than 0.8. In addition, siRNA-TUG1, shRNA-TUG1, miR-138-5p-mimics, and miR-138-5p-inhibitor were transfected into cells, and it turned out that overexpressing miR-138-5p and inhibiting ZEB2 exerted the same effects. The DLR assay revealed that TUG1 was able to targetedly regulate miR-138-5p, and miR-138-5p could targetedly regulate ZEB2, and in vitro experiments revealed that TUG1 could affect the epithelial-to-mesenchymal transition (EMT) of CRC via the miR-138-5p/ZEB2 axis. TUG1 could promote the development of CRC via the miR-138-5p/ZEB2 axis.
Project description:Epithelial-mesenchymal transition (EMT) is an early event in tumour invasion and metastasis, and widespread and distant metastasis at early stages is the typical biological behaviour in small cell lung cancer (SCLC). Our previous reports showed that high expression of the transcription factor E2F1 was involved in the invasion and metastasis of SCLC, but the role of E2F1 in the process of EMT in SCLC is unknown.Immunohistochemistry was performed to evaluate the expressions of EMT related markers. Immunofluorescence was used to detect the expressions of cytoskeletal proteins and EMT related markers when E2F1 was silenced in SCLC cell lines. Adenovirus containing shRNA against E2F1 was used to knock down the E2F1 expression, and the dual luciferase reporter system was employed to clarify the regulatory relationship between E2F1 and ZEB2.In this study, we observed the remodelling of cytoskeletal proteins when E2F1 was silenced in SCLC cell lines, indicating that E2F1 was involved in the EMT in SCLC. Depletion of E2F1 promoted the expression of epithelial markers (CDH1 and CTNNB1) and inhibited the expression of mesenchymal markers (VIM and CDH2) in SCLC cell lines, verifying that E2F1 promotes EMT occurrence. Next, the mechanism by which E2F1 promoted EMT was explored. Among the CDH1 related inhibitory transcriptional regulators ZEB1, ZEB2, SNAI1 and SNAI2, the expression of ZEB2 was the highest in SCLC tissue samples and was highly consistent with E2F1 expression. ChIP-seq data and dual luciferase reporter system analysis confirmed that E2F1 could regulate ZEB2 gene expression.Our data supports that E2F1 promotes EMT by regulating ZEB2 gene expression in SCLC.
Project description:Colorectal cancer (CRC) is one of the leading cancers worldwide, accounting for high morbidity and mortality. The mechanisms governing tumor growth and metastasis in CRC require detailed investigation. The results of the present study indicated that the transcription factor (TF) myocyte enhancer factor 2A (MEF2A) plays a dual role in promoting proliferation and metastasis of CRC by inducing the epithelial-mesenchymal transition (EMT) and activation of WNT/β-catenin signaling. Aberrant expression of MEF2A in CRC clinical specimens was significantly associated with poor prognosis and metastasis. Functionally, MEF2A directly binds to the promoter region to initiate the transcription of ZEB2 and CTNNB1. Simultaneous activation of the expression of EMT-related TFs and Wnt/β-catenin signaling by MEF2A overexpression induced the EMT and increased the frequency of tumor formation and metastasis. The present study identified a new critical oncogene involved in the growth and metastasis of CRC, providing a potential novel therapeutic target for CRC intervention.