Retinoid orphan nuclear receptor alpha (RORα) suppresses the epithelial–mesenchymal transition (EMT) by directly repressing Snail transcription
Gaofeng Xiong, Ren Xu
Abstract
Retinoid orphan nuclear receptor alpha (RORα) is a member of the orphan nuclear factor family and regulates gene expression by binding to ROR response elements (ROREs). RORα has been identified as a potential tumor suppressor; however, how downregulation of RORα promotes cancer progression is not fully understood. Here, we showed that protein levels of RORα were downregulated during the Snail-, Twist-, or transforming growth factor-β–induced epithelial–mesenchymal transition (EMT). We found that silencing of RORα induced expression of mesenchymal markers in MCF10A cells, accompanied by enhanced cell invasion, migration, and mammosphere formation. Furthermore, ectopic expression of RORα suppressed transforming growth factor-β–induced EMT processes in MCF10A and HMLE cells. These results indicate that downregulation of RORα is crucial for the induction of EMT in mammary epithelial cells. By analyzing gene expression profiles in control and RORα-expressing cells, we also identified Snail, a key regulator of EMT, as a potential target of RORα. We show that RORα expression significantly inhibits Snail transcription in breast cancer cells. Chromatin immunoprecipitation analysis demonstrated that RORα bound to the ROREs in promoter region of SNAI1 gene, and using the luciferase reporter assay, we showed that binding to the ROREs was critical for RORα to repress Snail transcription. Finally, rescue experiments substantiated that Snail mediates RORα function in suppressing EMT and mammosphere formation. These results reveal a novel function of RORα in suppressing EMT and identify Snail as a direct target of RORα in mammary epithelial cells. Retinoid orphan nuclear receptor alpha (RORα) is a member of the orphan nuclear factor family and regulates gene expression by binding to ROR response elements (ROREs). RORα has been identified as a potential tumor suppressor; however, how downregulation of RORα promotes cancer progression is not fully understood. Here, we showed that protein levels of RORα were downregulated during the Snail-, Twist-, or transforming growth factor-β–induced epithelial–mesenchymal transition (EMT). We found that silencing of RORα induced expression of mesenchymal markers in MCF10A cells, accompanied by enhanced cell invasion, migration, and mammosphere formation. Furthermore, ectopic expression of RORα suppressed transforming growth factor-β–induced EMT processes in MCF10A and HMLE cells. These results indicate that downregulation of RORα is crucial for the induction of EMT in mammary epithelial cells. By analyzing gene expression profiles in control and RORα-expressing cells, we also identified Snail, a key regulator of EMT, as a potential target of RORα. We show that RORα expression significantly inhibits Snail transcription in breast cancer cells. Chromatin immunoprecipitation analysis demonstrated that RORα bound to the ROREs in promoter region of SNAI1 gene, and using the luciferase reporter assay, we showed that binding to the ROREs was critical for RORα to repress Snail transcription. Finally, rescue experiments substantiated that Snail mediates RORα function in suppressing EMT and mammosphere formation. These results reveal a novel function of RORα in suppressing EMT and identify Snail as a direct target of RORα in mammary epithelial cells. The epithelial–mesenchymal transition (EMT) is a process characterized by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. This process is crucial for normal development such as embryogenesis and organ development, and for pathologic conditions such as wound healing and tumor progression (1Kalluri R. Weinberg R.A. The basics of epithelial-mesenchymal transition.J. Clin. Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (7861) Google Scholar, 2Yang J. Weinberg R.A. Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis.Dev. Cell. 2008; 14: 818-829Abstract Full Text Full Text PDF PubMed Scopus (2540) Google Scholar, 3Yang J. Antin P. Berx G. Blanpain C. Brabletz T. Bronner M. et al.Guidelines and definitions for research on epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2020; 21: 341-352Crossref PubMed Scopus (992) Google Scholar). It is well established that EMT dynamics drive cancer progression and metastasis by enhancing cancer cell migration, invasion, and stemness (4Dongre A. Weinberg R.A. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer.Nat. Rev. Mol. Cell Biol. 2019; 20: 69-84Crossref PubMed Scopus (2014) Google Scholar, 5Wilson M.M. Weinberg R.A. Lees J.A. Guen V.J. Emerging mechanisms by which EMT programs control stemness.Trends Cancer. 2020; 6: 775-780Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Therefore, inhibition of the EMT is considered a potential strategy for suppressing cancer progression. Given the important function and dynamic nature of EMT, this cellular event is controlled by a number of EMT inducers, such as transforming growth factor-β (TGF-β), Snail, Slug, ZEBs, and Twists (6Nieto M.A. Huang R.Y. Jackson R.A. Thiery J.P. Emt: 2016.Cell. 2016; 166: 21-45Abstract Full Text Full Text PDF PubMed Scopus (3150) Google Scholar). The Snail family are DNA-binding zinc finger proteins and play a fundamental role in EMT by suppressing E-cadherin expression in epithelial cells (7Carver E.A. Jiang R. Lan Y. Oram K.F. Gridley T. The mouse snail gene encodes a key regulator of the epithelial-mesenchymal transition.Mol. Cell. Biol. 2001; 21: 8184-8188Crossref PubMed Scopus (528) Google Scholar). Three Snail family proteins (Snail, Slug, and Snail3) have been identified in vertebrates (8Wang Y. Shi J. Chai K. Ying X. Zhou B.P. The role of snail in EMT and tumorigenesis.Curr. Cancer Drug Targets. 2013; 13: 963-972Crossref PubMed Scopus (666) Google Scholar). The expression of Snail in breast carcinomas is associated with tumor recurrence, metastasis, and poor prognosis (9Foubert E. De Craene B. Berx G. Key signalling nodes in mammary gland development and cancer. The Snail1-Twist1 conspiracy in malignant breast cancer progression.Breast Cancer Res. 2010; 12: 206Crossref PubMed Scopus (76) Google Scholar, 10Wu Y. Zhou B.P. Snail: more than EMT.Cell Adh. Migr. 2010; 4: 199-203Crossref PubMed Scopus (210) Google Scholar). To maintain epithelial structure and function, the expression or activity of Snail and other EMT inducers is normally repressed, which suggests the presence of a potential EMT suppressor in normal mammary epithelial cells. Despite recent progress in studying function of EMT in cancer progression, we know little about these suspected EMT suppressors and how these EMT suppressors and inducers regulate the dynamic EMT process in a coordinated fashion. Nuclear receptors, a family of ligand-dependent transcription factors, regulate gene expression by directly binding to the cis response elements in the regulatory regions. Retinoid orphan nuclear receptor alpha (RORα) is considered a member of the orphan nuclear factor family because its ligand has not been well characterized (11Jetten A.M. Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism.Nucl. Recept. Signal. 2009; 7e003Crossref PubMed Scopus (527) Google Scholar, 12Jetten A.M. Kurebayashi S. Ueda E. The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes.Prog. Nucleic Acid Res. Mol. Biol. 2001; 69: 205-247Crossref PubMed Google Scholar). RORα regulates gene transcription by binding to ROR response elements (ROREs) (11Jetten A.M. Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism.Nucl. Recept. Signal. 2009; 7e003Crossref PubMed Scopus (527) Google Scholar, 12Jetten A.M. Kurebayashi S. Ueda E. The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes.Prog. Nucleic Acid Res. Mol. Biol. 2001; 69: 205-247Crossref PubMed Google Scholar). It plays critical roles in many physiological processes, including cell differentiation, metabolism, inflammation, transformation, and circadian rhythm (13Dzhagalov I. Giguere V. He Y.W. Lymphocyte development and function in the absence of retinoic acid-related orphan receptor alpha.J. Immunol. 2004; 173: 2952-2959Crossref PubMed Scopus (70) Google Scholar, 14Sato T.K. Panda S. Miraglia L.J. Reyes T.M. Rudic R.D. 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The RORA gene maps to 15q22.2, a region that is often deleted in cancer (18Zhu Y. McAvoy S. Kuhn R. Smith D.I. RORA, a large common fragile site gene, is involved in cellular stress response.Oncogene. 2006; 25: 2901-2908Crossref PubMed Scopus (112) Google Scholar). We and others have identified RORα as a potential tumor suppressor in colon cancer, hepatocellular carcinoma, prostate cancer, glioma, and breast cancer (16Lee J.M. Kim I.S. Kim H. Lee J.S. Kim K. Yim H.Y. et al.RORalpha attenuates Wnt/beta-catenin signaling by PKCalpha-dependent phosphorylation in colon cancer.Mol. Cell. 2010; 37: 183-195Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 17Xiong G. Wang C. Evers B.M. Zhou B.P. Xu R. RORalpha suppresses breast tumor invasion by inducing SEMA3F expression.Cancer Res. 2012; 72: 1728-1739Crossref PubMed Scopus (95) Google Scholar, 19Du J. Xu R. RORalpha, a potential tumor suppressor and therapeutic target of breast cancer.Int. J. Mol. 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We showed that downregulation of RORα is associated with poor clinical outcomes and that reduced RORα expression promotes tumor growth and cancer cell invasion (17Xiong G. Wang C. Evers B.M. Zhou B.P. Xu R. RORalpha suppresses breast tumor invasion by inducing SEMA3F expression.Cancer Res. 2012; 72: 1728-1739Crossref PubMed Scopus (95) Google Scholar). A recent study suggests a potential function of RORα in suppressing EMT phenotypes of glioblastoma cells (23Zhao J. Jiang Y. Chen L. Ma Y. Zhang H. Zhou J. et al.The EIF4A3/CASC2/RORA feedback loop regulates the aggressive phenotype in glioblastomas.Front Oncol. 2021; 11: PubMed Scopus Google Scholar). the by which RORα suppresses EMT to the we show that RORα expression is suppressed during the EMT, and that this is to mesenchymal phenotypes in mammary epithelial cells. We also found that RORα EMT and mammosphere by directly Snail transcription. These results identified RORα as a novel EMT suppressor and into roles of RORα in breast cancer progression. and Snail are that EMT in mammary epithelial cells (8Wang Y. Shi J. Chai K. Ying X. Zhou B.P. The role of snail in EMT and tumorigenesis.Curr. Cancer Drug Targets. 2013; 13: 963-972Crossref PubMed Scopus (666) Google Scholar, Y. J. Epithelial-mesenchymal in development and 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. Ying X. Zhou B.P. epithelial-mesenchymal transition promotes breast tumor cell invasion inhibition of 2016; 6: PubMed Scopus Google Scholar). expression of or in HMLE cells or MCF10A cells the EMT phenotype Y. J. Ying X. Zhou B.P. epithelial-mesenchymal transition promotes breast tumor cell invasion inhibition of 2016; 6: PubMed Scopus Google Scholar). RORα protein levels were reduced in MCF10A and HMLE cells during the or EMT and induced EMT phenotypes and induced EMT marker expression in MCF10A and HMLE cells RORα protein levels were also in cells with a of receptor M. C. R. J. et regulates signaling and promotes metastasis of breast cancer Sci. S. A. 2010; PubMed Scopus Google the RORα expression in cells These indicate that reduced RORα protein expression is associated with the EMT To the in RORα expression is for the EMT we RORα expression with and the EMT phenotypes in cells. of epithelial cell marker E-cadherin was reduced in MCF10A cells expression of mesenchymal cell markers and and the EMT inducers and were RORα that silencing RORα in mammary epithelial cells enhanced expression and induced a mesenchymal phenotype MCF10A cells aggressive phenotype with control MCF10A cells and for showed that RORα also the of MCF10A cells in It is well established that EMT cell invasion and we found that cell invasion was in MCF10A cells A and We also using a of RORα expression in MCF10A cells significantly enhanced cell To we MCF10A cells in a and a wound healing of cell were by with a RORα in MCF10A cells significantly enhanced cell and with control cells These that reduced RORα expression promotes EMT phenotypes in mammary epithelial cells. To the role of RORα in of EMT, we RORα in MCF10A cells We control MCF10A cells and RORα-expressing MCF10A cells with for cells into RORα-expressing MCF10A cells epithelial cell We also found that RORα expression suppressed the downregulation of E-cadherin and of mesenchymal markers and was induced in control MCF10A cells protein levels in RORα-expressing MCF10A cells results were also in HMLE cells These results that a RORα protein in mammary epithelial cells suppresses the It has been that EMT is accompanied with enhanced potential and cancer cell stemness (4Dongre A. Weinberg R.A. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer.Nat. Rev. Mol. Cell Biol. 2019; 20: 69-84Crossref PubMed Scopus (2014) Google Scholar, 5Wilson M.M. Weinberg R.A. Lees J.A. Guen V.J. Emerging mechanisms by which EMT programs control stemness.Trends Cancer. 2020; 6: 775-780Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The has been to cancer cells and the of cancer cells E. C. J. P. et cancer cell functional cancer cells with and a Res. 2009; 69: PubMed Scopus Google Scholar). RORα protein levels were in with levels in cells which suggests a potential function of RORα in cancer cell the assay, we showed that RORα significantly the of mammosphere in MCF10A cells and ectopic expression of RORα suppressed in cells, cells, and cells and cells expression of RORα A and with the RORα enhanced in and cells, the RORα in breast cancer cells and and These results that of RORα expression to cancer cell To how RORα suppresses the EMT we gene expression profiles control and RORα-expressing cells G. Y. Wang C. D. Li et al.RORalpha suppresses by J. Mol. Sci. 2021; PubMed Scopus Google Scholar). the downregulated Snail has been identified as a EMT (6Nieto M.A. Huang R.Y. Jackson R.A. Thiery J.P. Emt: 2016.Cell. 2016; 166: 21-45Abstract Full Text Full Text PDF PubMed Scopus (3150) Google Scholar). Therefore, we on SNAI1 as a potential RORα target in EMT analysis that SNAI1 levels were in RORα-expressing cells, RORα SNAI1 transcription and and with a RORα reduced SNAI1 levels in cells, RORα induced SNAI1 expression in a in cells and Snail protein levels were also significantly in RORα-expressing mammary epithelial cells showed that with RORα a Snail in cells that Snail is with RORα. with the not rescue Snail expression in RORα-expressing cells which suggests that the is not protein By analyzing the of the SNAI1 gene, we identified potential to to and to Chromatin immunoprecipitation analysis showed that RORα bound to the ROREs in these and To these ROREs are important for RORα of Snail we luciferase reporter these regulatory The luciferase reporter showed that RORα suppressed transcription by to to in the SNAI1 gene To the binding of RORα to this region is we deleted the to to in the reporter of ROREs to to the luciferase reporter a region of SNAI1 gene to which the of It is that of potential ROREs the luciferase that ROREs in this these results indicate that RORα Snail expression by directly binding to ROREs in the regulatory of To of Snail by RORα is for the function of RORα in suppressing we Snail expression with in MCF10A cells and results showed that Snail expression in cells E-cadherin expression and reduced the protein levels of and A and We also cell invasion and stemness in these cells. of Snail significantly cell invasion and stemness in MCF10A cells and These results that RORα suppresses EMT phenotypes by Snail transcription in mammary epithelial cells. the we identified RORα as a EMT suppressor in mammary epithelial cells by directly Snail transcription. nuclear receptors, the receptor of and the receptor in epithelial-mesenchymal transition and invasion of prostate cancer J. 2010; PubMed Scopus Google Scholar, M. 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PubMed Scopus Google Scholar). the protein Snail expression is factor B. T. J. et of promotes EMT and metastasis control of and PubMed Scopus Google binding protein A.M. H. et of by promotes Cell. Full Text Full Text PDF PubMed Scopus Google and protein Y. S. Chen H. H. H. et promotes of the Snail family of transcription in cancer progression and PubMed Scopus Google Scholar, E. J.S. et promotes metastasis of aggressive breast of Cell Biol. PubMed Scopus Google Scholar). We found that RORα directly SNAI1 transcription by binding to in the SNAI1 The rescue experiments indicate that RORα suppresses EMT in mammary epithelial cells at by Snail analysis in breast cancer showed that was RORα expression and levels of Snail in breast cancer potential is that Snail expression in breast cancer is by multiple RORα is of these we found that the of in breast cancer was associated with These results that the of Snail expression by RORα is associated with breast cancer progression. It has been that also to M.A. The orphan receptor alpha transcription a novel response Cell. Biol. 13: PubMed Google and often the of RORα in many physiological processes and ROR nuclear receptors as Rev. Drug 2014; 13: PubMed Scopus Google Scholar). the important to function in Snail in the We showed that RORα expression was downregulated during the EMT These results that RORα expression is suppressed by Snail directly or Therefore, the Snail and RORα a feedback is EMT identified in mammary epithelial cells M.A. B. S. A. H. et is for epithelial-mesenchymal transition and metastasis in a of breast cancer Clin. Invest. 2004; PubMed Scopus Google Scholar, P. A. S. H. NF-kappaB E-cadherin expression and epithelial to mesenchymal transition of mammary epithelial cells: potential of and PubMed Scopus Google Scholar). It has been that RORα inhibits and transcription. RORα suppresses the signaling by induction of nuclear factor of gene in alpha a protein of the P. D. G. J. J. et al.The orphan nuclear receptor ROR alpha is a regulator of the 2001; PubMed Scopus Google Scholar). study showed that RORα to target genes and activity in epithelial cells Kim D. Kim K. K. Kim I.S. et al.RORalpha is crucial for response to maintain Sci. S. A. 2019; PubMed Scopus Google Scholar). glioma cell and glioma cells, RORα inhibits proliferation and tumorigenesis by the signaling Y. Zhou J. Zhao J. Hou D. Zhang H. Li L. et al.MiR-18a-downregulated RORA inhibits the proliferation and tumorigenesis of glioma using the TNF-alpha-mediated NF-kappaB signaling pathway.EBioMedicine. 2020; 52: 102651Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). 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