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Nkx2.8 Inhibits Epithelial-Mesenchymal Transition in Bladder Urothelial Carcinoma Via Transcriptional Repression of Twist1

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Nkx2.8 Inhibits Epithelial-Mesenchymal Transition in Bladder Urothelial Carcinoma Via Transcriptional Repression of Twist1 Author Manuscript Published OnlineFirst on January 8, 2018; DOI: 10.1158/0008-5472.CAN-17-1545 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Nkx2.8 inhibits epithelial-mesenchymal transition in bladder urothelial carcinoma via transcriptional repression of Twist1 Chunping Yu 1,2,3§, Zhuowei Liu1,2,3§, Qiuhong Chen1,2,3§, Yonghong Li1,2,3, Lijuan Jiang1,2,3, Zhiling Zhang1,2,3*, Fangjian Zhou1,2,3* 1 State Key Laboratory of Oncology in Southern China, Guangzhou, China 2 Department of Urology, Sun Yat-sen University Cancer Center, Guangzhou, China 3 Collaborative Innovation Center for Cancer Medicine, Guangzhou, China §Yu CP, Liu ZW and Chen QH contributed equally to this work. *To whom all correspondence should be addressed: Zhang ZL, Department of Urology, Cancer Center, Sun Yat-sen University, Guangzhou, China Phone: 86-20-87343860; Fax: 86-20-87343656; E-mail: [email protected] Or Zhou FJ, Department of Urology, Cancer Center, Sun Yat-sen University, Guangzhou, China Phone: 86-20-87343312; Fax: 86-20-87343656; E-mail: [email protected] Running title: Nkx2.8 regulates EMT through Twist1 Keywords: Nkx2.8, EMT, bladder urothelial carcinoma, Twist1 The authors declare no potential conflicts of interest. Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 8, 2018; DOI: 10.1158/0008-5472.CAN-17-1545 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 2 ABSTRACT Epithelial-to-mesenchymal transition (EMT) promotes metastasis which is the main cause of bladder urothelial carcinoma (UC)-related death. Loss of the candidate tumor suppressor gene Nkx2.8 has been associated with UC lymph node metastasis. Here we show that enforced expression of Nkx2.8 is sufficient to inhibit EMT, reduce motility and blunt invasiveness of UC cells. Mechanistic investigations showed that Nkx2.8 negatively regulated expression of the EMT inducer Twist1 in UC cells, at both the level of mRNA and protein accumulation. Nkx2.8 bound directly to the promoter region of this gene and transcriptionally repressed its expression. Twist1 upregulation reversed EMT inhibition by Nkx2.8, restoring the invasive phenotype of UC cells. In clinical UC specimens, expression of Nkx2.8 inversely correlated with Twist1 expression, and UC patients with Nkx2.8 positivity and low Twist1 expression displayed the best prognosis. Our findings highlight the Nkx2.8-Twist1 axis as candidate target for therapeutic intervention in advanced UC. Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 8, 2018; DOI: 10.1158/0008-5472.CAN-17-1545 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 3 INTRODUCTION The prognosis of metastatic bladder urothelial carcinoma (UC) is extremely poor, with a median survival time of less than 15 months, even with systematic therapy (1). However, the mechanism underlying UC metastasis is not clear. Epithelial-to-mesenchymal transition (EMT), a process of cell phenotypic change, is characterized by decreased E-cadherin expression and weakened adhesive cell-cell or cell-stroma attraction and subsequently facilitates cell migration (2-4). EMT has been implicated in the conversion of early stage tumors into invasive malignancies (2-4), which suggests that a better understanding of the mechanism underlying EMT may be important with respect to the clinical management of UC. Twist, a highly conserved transcriptional factor, has been well known as a key EMT inducer (5). Twist down-regulates E-cadherin expression, up-regulates fibronectin and vimentin expression, and subsequently facilitates EMT (5-7). Yang and colleagues (6) has found that forced expression of Twist results in decrease of E-cadherin-mediated adhesion and induction of cell motility, which suggests that Twist1 plays a promoting role in EMT. It is also well known that Twist1 participates in regulating the expression of Bmi-1, miR-200 and miR-205 (7-8), which are all involved in EMT. In UC, Twist has been found to be associated with tumor grade and progression and is inversely correlated with E-cadherin expression (9-11). Moreover, in bladder cancer tissues Twist is always been found to be negatively associated with the expression of E-cadherin (10-11). In addition, Twist is reported to be involved in UC invasiveness (9-10, 12). However, how Twist expression levels are regulated in UC remains a mystery. Human Nk2 homeobox 8 (Nkx2.8), which acts as a transcription factor, is a member of the NK-2 gene family (13). Nkx2.8 usually binds to DNA sequences Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 8, 2018; DOI: 10.1158/0008-5472.CAN-17-1545 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 4 containing 5'-(C/T)AAG-3' motifs (14-16). The findings of several studies have suggested that Nkx2.8 acts as a tumor suppressor in human carcinogenesis (17-19). In lung cancer, Harris and colleagues (17) found most tumors had low expression of Nkx2.8 and enforced expression of Nkx2.8 can inhibit proliferation of lung cancer cells. Lin et al (18) reported that down-regulation of Nkx2.8 can activate NF-B and promote angiogenesis in esophageal cancer cells. Qu and colleagues (19) also found a tumor suppressive role of Nkx2.8 in human liver cancer. Our previous study established that Nkx2.8 expression was markedly reduced in UC tissues and that Nkx2.8 negativity was associated with lymph node metastasis and prognosis in UC patients (20). However, the role of Nkx2.8 in metastasis and the regulatory mechanisms underlying this phenomenon are largely unknown. In the current study, we investigated the role of Nkx2.8 in EMT, the relationship between Nkx2.8 and Twist1 in UC and the mechanisms underlying the effects of Nkx2.8 in UC. Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 8, 2018; DOI: 10.1158/0008-5472.CAN-17-1545 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 5 Materials and Methods Cell lines The bladder cancer cell lines T24, 5637 and J82 were obtained from ATCC in 2013. The BIU87 and EJ were obtained from the institute of urology at the first affiliated hospital of Peking University as a gift in 2012. All cell lines were maintained in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), penicillin (100units/mL) , and streptomycin (100 units/mL) and tested to ensure mycoplasma free. All cell lines used in this study were authenticated three months before the beginning of the study (2013) based on viability, recovery, growth, morphology, and isoenzymology by the supplier and all the cell lines have not been in culture for greater than 2 months. The pBabe-Nkx2.8 and pSuper-retro-Nkx2.8 RNAi(s) were generated as described previously (20). Plasmids and retroviral infection The wild type human TWIST1 promoter and the TWIST1 promoter with a deletion or mutation of the Nkx2.8 binding sites were individually cloned into the pGL3 luciferase reporter plasmid (Promega). UC cells with endogenous silencing of Nkx2.8 and cells with the forced expression of exogenous Nkx2.8 were generated as previously described. The T24 cells exhibited no expression of Nkx2.8 and were infected with retroviruses carrying pBabe-Nkx2.8. The BIU87 cells showed high expression levels of Nkx2.8 and were infected with retroviruses carrying pSuper-retro-Nkx2.8-shRNAs. The 5637 cells showed moderate expression levels of Nkx2.8 and thus were infected with retroviruses carrying either pBabe-Nkx2.8 or pSuper-retro-Nkx2.8-shRNAs. Stable cell lines were selected with 0.5 g/ml puromycin for 10 days after transfection. Cell lysates, which were prepared from pooled populations of cells in sample buffer, were fractionated by sodium dodecyl Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 8, 2018; DOI: 10.1158/0008-5472.CAN-17-1545 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 6 sulfate-polyacrylamide gel electrophoresis to confirm Nkx2.8 protein levels. RNA extraction, quantitative real-time PCR Total RNA samples from cultured cells were extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The sequences of the primers are listed in Supplemental Table 1. Quantitative real-time PCR (qRT-PCR) was performed using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). The housekeeping gene glyceraldehyde-3 phosphate dehydrogenase (GAPDH) was used as an internal quantitative control. Western blot and immunofluorescence analyses Western blot and immunofluorescence analyses were performed according to standard methods as described previously(20) using anti-Nkx2.8 (Santa Cruz Boitechnology, Inc), anti-Twist1 (Abcam), anti-E-cadherin, anti-α-catenin, anti-fibronectin and anti-vimentin (BD Transduction Laboratories) antibodies. For the Western blot assays, anti-α-Tubulin antibody (Sigma) was used as a loading control. For immunofluorescence analysis, the coverslips were counterstained with 4', 6-diamidino-2-phenylindole and imaged with a confocal laser-scanning microscope (Olympus FV1000). The quantification of immunofluorescence staining was measured using Olympus FV10-ASW software. Patient information and immunohistochemistry The study has been approved by Institutional Review Board of Sun Yat-sen University Cancer Center and the study was performed in accordance with Declaration of Helsinki. Written informed consent was obtained from the patients before the study began.
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