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UBE2C Is Upregulated by Estrogen and Promotes Epithelial–Mesenchymal Transition Via P53 in Endometrial Cancer

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Published OnlineFirst October 29, 2019; DOI: 10.1158/1541-7786.MCR-19-0561 MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS UBE2C Is Upregulated by Estrogen and Promotes Epithelial–Mesenchymal Transition via p53 in Endometrial Cancer Yan Liu1, Rong Zhao1, Shuqi Chi1, Wei Zhang1, Chengyu Xiao1, Xing Zhou1, Yingchao Zhao2, and Hongbo Wang1 ABSTRACT ◥ Ubiquitin-conjugating enzyme E2C (UBE2C) plays important inhibited endometrial cancer cell proliferation, migration, invasion, roles in tumor progression; nevertheless, its function in endometrial and epithelial–mesenchymal transition (EMT), whereas UBE2C cancer remains unclear. This study elucidated the impact of UBE2C overexpression exerted the opposite effects. UBE2C downregulation on endometrial cancer and its underlying mechanism. Human increased p53 and its downstream p21 expression, with p53 over- endometrial cancer and normal endometrial tissues were acquired expression reversing the EMT-promoting effects of UBE2C. UBE2C from patients at Wuhan Union Hospital and UBE2C expression was enhanced p53 ubiquitination to facilitate its degradation in endo- detected by Western blotting and qRT-PCR. Endometrial cancer metrial cancer cells. Estradiol (E2) induced UBE2C expression via cells were transfected with a UBE2C overexpression plasmid or estrogen receptor a, which binds directly to the UBE2C promoter UBE2C-specific short hairpin RNA (shRNA) to up- or downregu- element. Silencing of UBE2C inhibited E2-promoted migration, late UBE2C expression, respectively. CCK8 and transwell assays invasion, and EMT in vitro and in vivo. were applied to assess the effects of UBE2C on cell proliferation, migration, and invasion. We found a significant elevation of UBE2C Implications: UBE2C-mediated tumor EMT promotion by estro- expression in patients with endometrial cancer, and that UBE2C gen is a novel mechanism for the progression of estrogen-induced upregulation was associated with advanced histologic grade, FIGO endometrial cancer, which could offer new biomarkers for diagnosis stage, recurrence, and shorter overall survival. UBE2C knockdown and therapy of endometrial cancer in the future. Introduction upregulation or downregulation (7, 8). Studies of multiple cellular models have shown that estrogen receptor (ER) a may be involved Endometrial cancer is one of the most universal gynecologic in regulating endometrial cancer cell proliferation, invasion, and malignant tumors worldwide, and its incidence is increasing rapidly, apoptosis (9–11). Classically, ERa has been identified as a tran- with approximately 382,000 new cases in 2018 (1). In China, the scription factor that regulates estrogen-related molecular expres- number of deaths from endometrial cancer is rising, with endometrial sion and biological functions, through binding with estrogen cancer now the second most common gynecologic malignancy after response elements in the promoter of target genes. Numerous cervical carcinoma (2). The 5-year survival rate for patients with early- studies have concentrated on the mechanisms of estrogen-driven stage endometrial cancer is around 80% after standard surgery, endometrial tumorigenesis, revealing that estrogen enhances the chemotherapy, and radiotherapy (3, 4); however, the prognosis for ability of cell proliferation, invasion, and epithelial–mesenchymal those with advanced endometrial cancer remains poor (5, 6). Thus, it is transition (EMT) in endometrial cancer (12, 13). Moreover, estro- essential to find new diagnostic and prognostic markers for endome- gen can downregulate the expression of important cancer suppres- trial cancer and to further understand its molecular mechanisms sor gene, such as PTEN (8). underlying its progression. p53 is thought to be an important tumor suppressor that The important role of estrogen in endometrial cancer develop- influences multiple crucial biological processes, including apopto- ment and progression is supported by studies showing that nuclear sis, cell-cycle arrest, and DNA repair (14). More than 60% of transcription factors activation leads to related gene expression patients with endometrial cancer develop TP53 mutations (15, 16); mutant p53 proteins may not only abolish their tumor-suppressive functions, but also acquire oncogenic functions (17, 18). Endome- 1 Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical trial tissues from the majority of patients with endometrial cancer College, Huazhong University of Science and Technology, Wuhan 430022, stain positive for p53 protein, which is largely attributed to mutant China. 2Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. p53, whereas wild-type (WT) p53 expression becomes undetect- able (19). Thus, reactivation of p53 signaling is a striking strategy Note: Supplementary data for this article are available at Molecular Cancer for pharmaceutically interfering with tumor initiation and progres- Research Online (http://mcr.aacrjournals.org/). sion; yet, the mechanisms underlying p53 downregulation in endo- Corresponding Authors: Hongbo Wang, Union Hospital, Tongji Medical College, metrial cancer remain elusive. Huazhong University of Science and Technology, Wuhan 430022, China. Phone: 8615-0710-53765; E-mail: [email protected]; and Yingchao Zhao, Members of the ubiquitin-conjugating enzyme E2 (UBE2) protein [email protected] family are ubiquitin-related enzymes that determine ubiquitin chain topologyandcatalyzethe degradation ofseveraltarget proteins(20,21). Mol Cancer Res 2019;XX:XX–XX Their importance in tumorigenesis has been revealed by a series doi: 10.1158/1541-7786.MCR-19-0561 of studies. UBE2S was found to be involved in lung adenocarcinoma Ó2019 American Association for Cancer Research. and hepatocellular carcinoma by regulating the p53 signaling AACRJournals.org | OF1 Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 29, 2019; DOI: 10.1158/1541-7786.MCR-19-0561 Liu et al. pathway (22, 23), while UBE2D1, UBE2B, UBE2V1, and UBE2Q1 were gcGFP-IRES-puromycin vectors were used separately as control plas- shown to be involved in hepatocellular carcinoma, nasopharyngeal mids. The effective UBE2C-shRNA sequences were: shUBE2C-1, 50- carcinoma, and colorectal cancer (24–27) by regulating oncogenes or TGC AAG AAA CCT ACT CAA A-30; and shUBE2C-4, 50-TCT GGC tumor suppressors. This demonstrates the key roles of the UBE2 family GAT AAA GGG ATT T-30. Cells were transfected with the UBE2C in tumorigenesis. One UBE2 family member, UBE2C, has been overexpression plasmids or shRNA with Lipofectamine 2000 (Invi- implicated as a candidate oncogene in cancer progression, autophagy, trogen) and stable cell lines were obtained after 2 weeks of G418 or and drug resistance (28–31); however, its role in endometrial cancer puromycin screening. The p53, ESR1 overexpression plasmids, and and its underlying mechanisms are not fully elucidated. p53 shRNA were transiently transfected using Lipofectamine 2000. This study aimed to elucidate the role of UBE2C in endometrial Forty-eight hours after transfection, the cells were harvested for cancer by investigating its expression levels in endometrial cancer, Western blot analyses. association with patient prognosis, and effects on endometrial cancer cell migration, invasion, and EMT. In addition, we identified UBE2C as Western blot analysis an estrogen-induced mRNA that affects endometrial cancer in an Endometrial cancer samples or cells with indicated treatments were ERa-dependent manner. Our findings shed light on the relationship lysed using RIPA (Beyotime) with 1 mmol/L PMSF (MedChem between UBE2C and endometrial cancer, and contribute toward the Express) and a protease Cocktail Inhibitor (Pierce) at 4C for 30 development of new biomarkers for the diagnosis and treatment of minutes. The cells were subsequently collected by scraping, followed endometrial cancer. by centrifugation at 12,000 rpm for 15 minutes. The supernatant was harvested and the protein concentration was measured using a BCA Assay Kit (Beyotime). Protein samples were fractionated by SDS- Materials and Methods PAGE, transferred onto a polyvinylidene difluoride membrane (Milli- Human tissue samples and database pore) followed by incubation overnight at 4C with primary antibodies Human endometrial cancer and benign endometrial tissues specific to UBE2C (1:1,000, ABclonal), p53 (1:200, Santa Cruz Bio- were harvested from patients at Wuhan Union Hospital, Tongji technology), p21 (1:1,000, ABclonal), ERa (1:1,000, ABclonal), E- Medical College, Huazhong University of Science and Technology cadherin (1:200, Santa Cruz Biotechnology), Vimentin (1:200, Santa (Wuhan, China). Tissue samples were collected surgically, imme- Cruz Biotechnology), or GAPDH (1:10,000, Proteintech). After incu- diately deposited in liquid nitrogen until total RNA and protein bation with horseradish peroxidase (HRP)-conjugated secondary extraction. Two pathologists assessed all specimens in-line with the antibodies (1:8,000, Affinity), immune complexes were detected using World Health Organization guidelines. All experiments were enhanced chemiluminescence detection reagents (Thermo Fisher approved by the Institutional Review Board of the Tongji Medical Scientific) on Image Lab Software in Molecular Imager ChemiDoc College, Huazhong University of Science and Technology. All XRSþ (Bio-Rad). subjects have read written informed consent. Uterine corpus endo-
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  • The X-Linked Intellectual Disability Gene Product and E3 Ubiquitin Ligase KLHL15 Degrades Doublecortin Proteins to Constrain Neuronal Dendritogenesis

    The X-Linked Intellectual Disability Gene Product and E3 Ubiquitin Ligase KLHL15 Degrades Doublecortin Proteins to Constrain Neuronal Dendritogenesis

    bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.324285; this version posted October 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. KLHL15 degrades doublecortin proteins The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis Jianing Song1,2, Ronald A. Merrill1,2, Andrew Y. Usachev1, and Stefan Strack1* From the 1Department of Neuroscience and Pharmacology and the Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242 2 These authors contributed equally to the work. *To whom correspondence should be addressed: Stefan Strack: Dept. of Neuroscience and Pharmacology, The University of Iowa, Iowa City, IA 52242; [email protected]; Tel. (319) 384-4439; Fax. (319) 335-8930 Running Title: KLHL15 degrades doublecortin proteins Keywords: Kelch-like 15, KLHL15, E3 ubiquitin ligase, protein phosphatase 2A, signal transduction, doublecortin, doublecortin-like kinases, microtubule-associated protein, ubiquitination, proteasomal degradation, protein turnover, neurite outgrowth, dendritic complexity, pulse-chase, HaloTag ______________________________________________________________________________ ABSTRACT doublecortin (DCX), also an X-linked disease gene, and doublecortin-like kinases 1 and 2 Proper brain development and function (DCLK1/2) as bona fide KLHL15 interactors requires finely controlled mechanisms