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(ROC1) Negatively Regulates the Gli2 Regulator SUFU to Activate the Hedgehog Pathway in Bladder Cancer

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(ROC1) Negatively Regulates the Gli2 Regulator SUFU to Activate the Hedgehog Pathway in Bladder Cancer Regulator of cullins-1 (ROC1) negatively regulates the Gli2 regulator SUFU to activate the hedgehog pathway in bladder cancer Wei Wang Jiangsu Province Hospital and Nanjing Medical University First Aliated Hospital Jianxin Qiu Shanghai Jiaotong University First People's Hospital Pin Qu Yancheng rst people's hospital Hui Chen yancheng rst people's hospital Jianyun Lan yancheng rst people's hospital Haitao Chen yancheng rst People's Hospital Lei Li yancheng rst people's hospital Min Gu ( [email protected] ) Jiangsu Province Hospital and Nanjing Medical University First Aliated Hospital https://orcid.org/0000-0002-9110-8054 Primary research Keywords: Bladder cancer, ROC1, Sonic Hedgehog signaling, SUFU, Gli2 Posted Date: June 24th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-31507/v2 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on January 26th, 2021. See the published version at https://doi.org/10.1186/s12935-021-01775-5. Page 1/19 Abstract Background The regulator of cullins-1 (ROC1) is an essential subunit in the Cullin-RING ligase (CRL) protein complex and was shown to be critical in bladder cancer cell survival and malignant progression. This study aimed to explore the regulatory mechanism of ROC1 in bladder cancer malignant progression. Methods This study explored the underlying mechanisms using both in vitro and in vivo experiments. The expression of the components of Sonic Hedgehog (SHH) pathway was determined by western blotting analysis. ROC1 expression in human tumours was evaluated by immunohistochemical analysis. Results The data showed that ROC1 overexpression promoted growth of bladder cancer cells, whereas knockdown of ROC1 expression had an opposite effect in bladder cancer cells. Mechanistically, ROC1 was able to target SUFU for ubiquitin-dependent degradation, allowing the Gli2 release from the SUFU complex to activate SHH pathway. Furthermore, knockdown of SUFU expression partially rescue the ROC1 knockdown-suppressed SHH activity as well as cancer cell growth inhibition. At ex vivo, tissue microarray analysis of human bladder cancer specimens revealed an positive association of ROC1 expression with the SHH pathway activity. Conclusion The current study demonstrated the dysregulation of ROC1-SUFU-GLI2 axis played an important role in bladder cancer progression and targeting of ROC1 expression is warranted further investigation as a novel strategy for future control of bladder cancer. Background Bladder cancer is a common malignancy, accounting for the 4th most commonly diagnosed cancer globally and the 8th leading cause of cancer-related deaths in male worldwide [1, 2]. Approximately 75% to 80% bladder cancer patients are initially diagnosed as non-muscle invasive bladder cancer (NMIBC) and a half of such patients will recur within ve years, and up to 30% of NMIBC patients will progress to muscle-invasive bladder cancer (MIBC), and the latter initially occurs in approximately 25% of bladder cancer patients and has a poor prognosis [3]. Treatment of MIBC patients is usually to surgical tumor resection and then cisplatin-based chemotherapy [3, 4]. Surgery alone is dicult to effectively control tumor recurrence, while the cisplatin-based chemotherapy leads to the severe toxicity and relatively low anticancer eciency; for example, only 40-65% of patients with metastatic bladder cancer showed clinical response [3, 4]. Thus, better understanding of the molecular mechanisms underlying bladder cancer progression and recurrence, will lead to identication of novel anticancer targets for bladder cancer therapy. The Cullin-RING ligases (CRL) are the enzymes to target proteins for ubiquitin-mediated degradation and altered CRL activity contributed to development and progression of human cancers, including bladder cancer [5]. CRL, also known as Skp1, Cullin, or the F-box protein, belongs to the largest family of the E3 ubiquitin ligases which mediate the proteasome-targeted degradation of 20% of ubiquitinated protein substrates, like the cell cycle-related, DNA replication, and signal transduction proteins as well as transcription factors [5] . Experimentally, the small molecule inhibitors, like MLN4924 were able to suppress CRL activation by inhibition of the Cullin activity, and in turn to effectively reduce growth of various human cancer cells in vitro [6]. Furthermore, the regulator of cullins-1 (ROC1), or namely as the Page 2/19 RING box protein-1 (RBX1), is a key CRL subunit and heterodimerizes with other Cullins to form the CRL catalytic cores [5] . ROC1 contains a small zinc-binding domain (the RING nger), which is evolutionarily conserved and is essential in embryonic development, while aberrant ROC1 expression led to CRL dysfunction and embryonic lethality [7, 8]. ROC1 is also essential in maintenance of the genome integrity and cancer development [8-12]. Our previous studies demonstrated that ROC1 protein was overexpressed in bladder cancer tissues, while knockdown of ROC1 expression reduced the CRL activity, triggered accumulation of its specic substrates (such as p21, p27, and DEPTOR), leading to suppression of tumor progression [13, 14]. In this study, we further investigated and explored the underlying molecular mechanisms, by which ROC1 regulates the Sonic Hedgehog (SHH) pathway in bladder cancer using our in vitro and in vivo experiments. We expected to elucidate the molecular basis for the future development of a novel ROC1-based targeted therapy against bladder cancer. Materials And Methods Cell culture and reagents Human bladder cancer 5637 and T24 cell lines were obtained from Chinese Academy of Science (Shanghai, China) and maintained in Roswell Park Memorial Institute medium-1640 (RPMI-1640; Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and1% penicillin- streptomycin in a humidied 5% CO2 environment at 37°C. Cycloheximide (CHX), the SHH signaling activator SAG (an SMO agonist), the SMO antagonist GDC0449, and the NAE (Nedd8-activating enzyme) inhibitor MLN4924 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored at -20°C as stock solutions. G418 was also from Sigma- Aldrich. The recombinant plasmid carrying HA-ubiquitin cDNA was purchased from Invitrogen (Shanghai, China). Establishment of stable ROC1 overexpressed or silenced bladder cancer cell lines To establish a stable ROC1 overexpressed bladder cancer cell sublines, we subcloned the full-length wild- type human ROC1 cDNA into the pcDNA3.1 vector (Invitrogen, Shanghai, China), named it as pcDNA3.1- ROC1. After DNA sequence conrmation, this recombinant plasmid or pcDNA3.1 vector-only plasmid was transfected into bladder cancer cells using Lipofectamine 2000 (Invitrogen) for 48 h and the cells were then cultured in G418-selecting cell culture medium at 100 μg/ml for 14 days. After that, individual of the G418-resistant monoclonal cells were selected and expanded under the 100 μg/ml G418-selective medium. The stable cell sublines were named as p-ROC1 or p-CONT. Furthermore, to knockdown ROC1 or SUFU expression, we purchased siRNA oligonucleotides targeting ROC1 or SUFU from Invitrogen (Shanghai, China) and transfected them into bladder cancer cells according to the manufacturer’s instructions. ROC1 siRNA sequences were 5'-GACTTTCCCTGCTGTTACCTAA-3'; SUFU siRNA sequences were 5'-GCCATGACAATCGGAAATCTA-3'; and scrambled control siRNA sequences were 5'- ACGTGACACGTTCGGAGAA-3'. Page 3/19 Cell viability and colonogenic assays Changed cell viability was assessed by using the Cell Counting Kit-8 kit (Beyotime, China) and carried out as described previously [13]. For the colonogenic assay, tumor cells were seeded in triplicate into 35-mm culture dishes at a density of 400 cells (for 5637 tumor cells) or 1000 cells (for T24 cells) per well and cultured for 9 days. The cells were xed and stained with crystal violet in 50% methanol and the number of cell colonies with more than 50 cells was counted. Flow cytometry cell cycle distribution assay Both ROC1-overexpressed and siRNA-transfected bladder cancer cells were detached from cell culture dishes and xed in ice-cold 70% ethanol overnight. One day after, cells were washed twice with ice-cold phosphate buffered saline (PBS) and then stained with propidium iodide (PI; Sigma-Aldrich) solution (20 mg/ml) for 5 min and then analyzed by using a BD FACScan ow cytometer (San Diego, CA, USA). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) Total RNA was isolated with the Trizol reagent (Invitrogen) and reversely transcribed into cDNA with a PrimeScript Reverse Transcription kit (Takara, China) according to the manufacturers’ protocols. The resulted cDNA samples were then amplified in the 7300 Real Time PCR system (Applied Biosystems, Foster City, CA) with the SYBR Green master mix kit (Takara, Dalian, China) for detection of different genes using gene-specic primers (the detailed DNA sequences of each primer used in this study are available upon request). All measurements were performed in triplicate and quantied using the 2-∆∆Ct method. Western blotting and co-immunoprecipitation After cells were subjected gene transfection or drug treatments, cell lysates were prepared and quantied according to a previous study [14]. The Western blot was carried out as described previously [13], while the co-immunoprecipitation (Co-IP) Kit (Cat. #26419) from Thermo Scientic
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