Pooled CRISPR Screening Identifies P-Bodies as Repressors of Cancer Epithelial-Mesenchymal Transition
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE224779 | GEO | 2023/12/21
REPOSITORIES: GEO
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Project description:Epithelial-mesenchymal transition (EMT) is a fundamental cellular process frequently hijacked by cancer cells to promote tumor progression, especially metastasis. EMT is orchestrated by a complex molecular network acting at different layers of gene regulation. In addition to transcriptional regulation, post-transcriptional mechanisms may also play a role in EMT. Here, we performed a pooled CRISPR screen analyzing the influence of 1547 RNA binding proteins (RBPs) on cell motility in colon cancer cells and identified multiple core components of P-bodies (PBs) as negative modulators of cancer cell migration. Further experiments demonstrated that PB depletion by silencing DDX6 or EDC4 could activate hallmarks of EMT thereby enhancing cell migration in vitro as well as metastasis formation in vivo. Integrative multi-omics analysis revealed that PBs could repress the translation of the EMT-driver gene HMGA2, which contributed to PB-meditated regulation of EMT. This mechanism is conserved in other cancer types. Furthermore, endoplasmic reticulum (ER) stress was an intrinsic signal that induced PB disassembly and translational derepression of HMGA2. Taken together, this study has identified a function of PBs in the regulation of EMT in cancer.
2023-12-21 | GSE224778 | GEO
Project description:Epithelial-mesenchymal transition (EMT) is a fundamental cellular process frequently hijacked by cancer cells to promote tumor progression, especially metastasis. EMT is orchestrated by a complex molecular network acting at different layers of gene regulation. In addition to transcriptional regulation, post-transcriptional mechanisms may also play a role in EMT. Here, we performed a pooled CRISPR screen analyzing the influence of 1547 RNA binding proteins (RBPs) on cell motility in colon cancer cells and identified multiple core components of P-bodies (PBs) as negative modulators of cancer cell migration. Further experiments demonstrated that PB depletion by silencing DDX6 or EDC4 could activate hallmarks of EMT thereby enhancing cell migration in vitro as well as metastasis formation in vivo. Integrative multi-omics analysis revealed that PBs could repress the translation of the EMT-driver gene HMGA2, which contributed to PB-meditated regulation of EMT. This mechanism is conserved in other cancer types. Furthermore, endoplasmic reticulum (ER) stress was an intrinsic signal that induced PB disassembly and translational derepression of HMGA2. Taken together, this study has identified a function of PBs in the regulation of EMT in cancer.
2023-12-21 | GSE224773 | GEO
Project description:The overall description of the project: Epithelial-mesenchymal transition (EMT) is a fundamental cellular process frequently hijacked by cancer cells to promote tumor progression, especially metastasis formation. Molecularly, EMT is orchestrated by various molecular networks acting at different layers of gene regulation. Compared to transcriptional regulation that has been extensively studied in the context of EMT, post-transcriptional mechanisms remain relatively underexplored. Here, by taking advantage of pooled CRISPR screen, we analyzed the influence of 1547 RNA binding proteins (RBPs) on cell motility and identified multiple core P-body (PB) components as negative modulators of cancer cell migration. Further experiments demonstrated that depletion of the PB via silencing DDX6 or EDC4 could activate hallmarks of EMT thereby enhancing cell migration in vitro as well as metastasis formation in vivo. Integrative multi-omics analysis revealed that the PB could repress the translation of its target genes, including an EMT-driver gene, HMGA2. Furthermore, we demonstrated that endoplasmic reticulum (ER) stress is an intrinsic signal that can induce PB disassembly and translational derepression of HMGA2. Finally, we used mouse genetics to demonstrate that knockout of Ddx6 resulted in EMT-related defects in embryonic development. Taken together, our study has put forward a novel function of the PB as an EMT regulator in both pathological and physiological conditions. The description of the MS part: Recent studies have suggested that the PB is mainly involved in translational regulation. Therefore, we performed mass spectrometry analysis on DDX6- and EDC4-KO clones and parental cells to measure changes at the protein level upon PB perturbation. After filtering and normalization, 3,762 and 3,000 peptides corresponding to 3,721 and 2,991 proteins were identified in DDX6- and EDC4-KO cells, respectively, and their abundance was then compared to parental HCT116 cells. A good correlation between two independent gene KO clones was observed in terms of protein level changes. In total, DDX6-KO induced up- and down-regulation of 230 and 291 proteins, respectively, while EDC4-KO induced up- and down-regulation of 73 and 22 proteins, respectively. To assess whether the PB mainly contributes to the RNA degradation or translational repression of its mRNA targets in HCT116 cells, we compared the RNA level and translation efficiency changes of DDX6-bound genes with other genes upon PB loss. As a result, both DDX6 and EDC4 KO led to a lower RNA, but higher translational efficiency of DDX6-bound genes compared to other unbound genes, suggesting PBs mainly function as a translational repressor of stored mRNAs in HCT116 cells.
2023-12-22 | PXD040524 | Pride
Project description:The molecular events that mediate the epithelial to mesenchymal transition (EMT) in endometrial cancer remain poorly understood. Using cDNA microarrays, we analyzed a group of endometrial carcinosarcomas (ECS), a true example of EMT in vivo, and we compared their gene expression profiles with those obtained from a group of endometrioid endometrial carcinomas (EEC). The HMGA2 gene (High Mobility Group AT-hook 2), an embryonic nuclear factor that mediates EMT in various tumour models, was among the genes overexpressed in ECS, and HMGA2 overexpression was confirmed in 54% of ECSs by qRT-PCR and immunohistochemistry. Moreover, we found a significant inverse correlation between the expression of HMGA2 and let-7b, a member of the let-7 family of miRNAs that represses HMGA2 expression. These changes were also associated with overexpression of Lin28B, a suppressor of microRNA biogenesis implicated in cancer progression and metastasis. Finally, HMGA2 overexpression, which was detected in less than 3% of EECs, was observed in many non-endometrioid carcinomas (46%). For the first time, we describe a role for HMGA2 in both the process of EMT that contributes to endometrial carcinogenesis and in the acquisition of aggressive phenotypes by this neoplasia. Moreover, we demonstrate changes in the expression of genes modulating processes such as EMT, muscle differentiation, the expression of cancer testis antigens (CTAs) and the immune response. Identification of new molecular markers in endometrial carcinogenesis 15 endometrial carcinosarcomas and 23 endometrioid endometrial carcinoma
2012-11-01 | E-GEOD-33723 | biostudies-arrayexpress
Project description:We have reported that the nuclear protein high mobility group A2 (HMGA2), is required for the induction of EMT by TGF-beta (Thuault et al. J. Cell Biol. 2006). HMGA2 regulates a cohort of transcriptional regulators of the EMT process, such as Snail1 and Twist1 (Thuault et al. J. Biol. Chem. 2008, E-Jean Tan et al. J. Biol. Chem. 2012). Since HMGA2 is a chromatin-associated protein, we expect that it should regulate many genes in addition to the EMT-related factors. The aim of our study is to decipher novel molecular mechanisms that explain how EMT links to the process of tumor-initiating cell self-renewal via the chromatin factor HMGA2. In this experiment, we stably transfected human breast cancer MDA-MB-231 cells with shRNA against HMGA2 and investigated effects of the knock-down on global gene expression using Affymetrix arrays, in order to identify novel downstream players involved in this network. In parallel experiments we also analyzed the phenotypic effects of the HMGA2 knock-down on MDA-MB-231 cell proliferation, survival and inavasiveness.
2012-02-13 | E-MEXP-3547 | biostudies-arrayexpress
Project description:The relatively poor sensitivity of clear cell renal cell carcinoma (ccRCC) to conventional chemotherapy and radiotherapy makes its treatment challenging. The Ndc80 kinetochore complex component (NUF2) is involved in the development and progression of several cancers. However, its role in ccRCC remains unclear. In an effort to identify novel treatment targets and prognostic markers for ccRCC, we investigated the role of NUF2 in ccRCC progression. We found that the expression of NUF2 was increased in ccRCC tissues and cells, and the elevated NUF2 levels were significantly associated with worse clinicopathological parameters and shorter survival. In addition, NUF2 was an independent prognostic biomarker for ccRCC. Knocking down NUF2 expression affected the proliferation, migration, and invasion of ccRCC cells and the expression of epithelial–mesenchymal transition (EMT)-related markers. Moreover, RNA sequencing and in vitro experiments identified the oncogene high-mobility group AT-hook 2 (HMGA2) as the target factor regulated by NUF2 in ccRCC. Restoring HMGA2 levels ameliorated the inhibitory effects of NUF2 depletion on cell proliferative, migratory, and invasive capacities and recovered the expression of EMT-related markers to basal levels.These results suggest that NUF2 promotes proliferation, migration, and invasion of ccRCC cells, at least partly by regulating HMGA2, and that the NUF2-HMGA2 axis could be an ideal therapeutic target and a promising prognostic indicator for ccRCC.
2022-06-30 | GSE192754 | GEO
Project description:PI3K (phosphoinositide 3-kinase)/AKT and RAS/MAPK (mitogen-activated protein kinase) pathway coactivation in the prostate epithelium promotes both epithelial–mesenchymal transition (EMT) and metastatic castration-resistant prostate cancer (mCRPC), which is currently incurable. To study the dynamic regulation of the EMT process, we developed novel genetically defined cellular and in vivo model systems from which epithelial, EMT and mesenchymal-like tumor cells with Pten deletion and Kras activation can be isolated. When cultured individually, each population has the capacity to regenerate all three tumor cell populations, indicative of epithelial–mesenchymal plasticity. Despite harboring the same genetic alterations, mesenchymal-like tumor cells are resistant to PI3K and MAPK pathway inhibitors, suggesting that epigenetic mechanisms may regulate the EMT process, as well as dictate the heterogeneous responses of cancer cells to therapy. Among differentially expressed epigenetic regulators, the chromatin remodeling protein HMGA2 is significantly upregulated in EMT and mesenchymal-like tumors cells, as well as in human mCRPC. Knockdown of HMGA2, or suppressing HMGA2 expression with the histone deacetylase inhibitor LBH589, inhibits epithelial–mesenchymal plasticity and stemness activities in vitro and markedly reduces tumor growth and metastasis in vivo through successful targeting of EMT and mesenchymal-like tumor cells. Importantly, LBH589 treatment in combination with castration prevents mCRPC development and significantly prolongs survival following castration by enhancing p53 and androgen receptor acetylation and in turn sensitizing castration-resistant mesenchymal-like tumor cells to androgen deprivation therapy. Taken together, these findings demonstrate that cellular plasticity is regulated epigenetically, and that mesenchymal-like tumor cell populations in mCRPC that are resistant to conventional and targeted therapies can be effectively treated with the epigenetic inhibitor LBH589.
2015-10-12 | GSE67872 | GEO
Project description:PI3K (phosphoinositide 3-kinase)/AKT and RAS/MAPK (mitogen-activated protein kinase) pathway coactivation in the prostate epithelium promotes both epithelial–mesenchymal transition (EMT) and metastatic castration-resistant prostate cancer (mCRPC), which is currently incurable. To study the dynamic regulation of the EMT process, we developed novel genetically defined cellular and in vivo model systems from which epithelial, EMT and mesenchymal-like tumor cells with Pten deletion and Kras activation can be isolated. When cultured individually, each population has the capacity to regenerate all three tumor cell populations, indicative of epithelial–mesenchymal plasticity. Despite harboring the same genetic alterations, mesenchymal-like tumor cells are resistant to PI3K and MAPK pathway inhibitors, suggesting that epigenetic mechanisms may regulate the EMT process, as well as dictate the heterogeneous responses of cancer cells to therapy. Among differentially expressed epigenetic regulators, the chromatin remodeling protein HMGA2 is significantly upregulated in EMT and mesenchymal-like tumors cells, as well as in human mCRPC. Knockdown of HMGA2, or suppressing HMGA2 expression with the histone deacetylase inhibitor LBH589, inhibits epithelial–mesenchymal plasticity and stemness activities in vitro and markedly reduces tumor growth and metastasis in vivo through successful targeting of EMT and mesenchymal-like tumor cells. Importantly, LBH589 treatment in combination with castration prevents mCRPC development and significantly prolongs survival following castration by enhancing p53 and androgen receptor acetylation and in turn sensitizing castration-resistant mesenchymal-like tumor cells to androgen deprivation therapy. Taken together, these findings demonstrate that cellular plasticity is regulated epigenetically, and that mesenchymal-like tumor cell populations in mCRPC that are resistant to conventional and targeted therapies can be effectively treated with the epigenetic inhibitor LBH589.
2015-10-07 | GSE67681 | GEO
Project description:Regulation of cell-cell junction formation and regulation of cell migration were enriched among EMT (Epithelial-Mesenchymal Transition)-associated alternatively splicing events. Our analysis suggested that most EMT-associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMTassociated splicing pattern. Expression of EMT-associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT-dependent splicing changes occur commonly in human tumors. The functional significance of EMT-associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT-associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression. Examination of transcriptomes of HMLE/Twist-ER before and after induction of EMT by tamoxifen
2011-07-15 | E-GEOD-30290 | biostudies-arrayexpress
Project description:Dynamic regulation of glucose flux between aerobic glycolysis and the pentose phosphate pathway (PPP) during epithelial-mesenchymal transition (EMT) is not well-understood. Here we show that Snail (SNAI1), a key transcriptional repressor of EMT, regulates glucose flux toward PPP, allowing cancer cell survival under metabolic stress. Mechanistically, Snail regulates glycolytic activity via repression of PFKP (phosphofructokinase, platelet), a major isoform of cancer-specific phosphofructokinase-1 (PFK-1), an enzyme involving the first rate-limiting step of glycolysis. The suppression of PFKP switches the glucose flux towards PPP, generating NADPH with increased metabolites of oxidative PPP. Functionally, dynamic regulation of PFKP significantly potentiates cancer cell survival under metabolic stress and increases metastatic capacities in vivo. Further, knockdown of PFKP rescues metabolic reprogramming and cell death induced by loss of Snail. Thus, the Snail-PFKP axis plays an important role in cancer cell survival via regulation of glucose flux between glycolysis and PPP.Dynamic regulation of glucose flux between aerobic glycolysis and the pentose phosphate pathway (PPP) during epithelial-mesenchymal transition (EMT) is not well-understood. Here we show that Snail (SNAI1), a key transcriptional repressor of EMT, regulates glucose flux toward PPP, allowing cancer cell survival under metabolic stress. Mechanistically, Snail regulates glycolytic activity via repression of PFKP (phosphofructokinase, platelet), a major isoform of cancer-specific phosphofructokinase-1 (PFK-1), an enzyme involving the first rate-limiting step of glycolysis. The suppression of PFKP switches the glucose flux towards PPP, generating NADPH with increased metabolites of oxidative PPP. Functionally, dynamic regulation of PFKP significantly potentiates cancer cell survival under metabolic stress and increases metastatic capacities in vivo. Further, knockdown of PFKP rescues metabolic reprogramming and cell death induced by loss of Snail. Thus, the Snail-PFKP axis plays an important role in cancer cell survival via regulation of glucose flux between glycolysis and PPP.
2017-11-07 | MTBLS387 | MetaboLights