Theranostics 2020; 10(2):707-724. doi:10.7150/thno.39018 This issue

Research Paper

Amplification-driven BCL6-suppressed cytostasis is mediated by transrepression of FOXO3 and post-translational modifications of FOXO3 in urinary bladder urothelial carcinoma

Wen-Ren Wu1*, Jen-Tai Lin2*, Cheng-Tang Pan3,4*, Ti-Chun Chan5, Chen-Liang Liu6, Wen-Jeng Wu7,8,9,10,11, Jim Jinn-Chyuan Sheu1, Bi-Wen Yeh7,8,10, Steven K. Huang12, Jheng-Yan Jhung1, Meng-Shin Hsiao13, Chien-Feng Li14,15,16✉, Yow-Ling Shiue1,4,17✉

1. Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.
2. Surgery Department, Urology Division, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
3. Department of Mechanical and Electro-Mechanical Engineering.
4. Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, Taiwan.
5. Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan.
6. Division of Urology, Department of Surgery, Chi Mei Medical Center, Tainan, Taiwan.
7. Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
8. Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.
9. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
10. Regenerative Medicine and Cell Therapy Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan.
11. Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, Taiwan.
12. Division of Urology, Department of Surgery, Chi Mei Medical Center, Tainan, Taiwan.
13. Research Center, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
14. Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan.
15. Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan.
16. National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan.
17. Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.
*These authors contributed equally

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Citation:
Wu WR, Lin JT, Pan CT, Chan TC, Liu CL, Wu WJ, Sheu JJC, Yeh BW, Huang SK, Jhung JY, Hsiao MS, Li CF, Shiue YL. Amplification-driven BCL6-suppressed cytostasis is mediated by transrepression of FOXO3 and post-translational modifications of FOXO3 in urinary bladder urothelial carcinoma. Theranostics 2020; 10(2):707-724. doi:10.7150/thno.39018. Available from https://www.thno.org/v10p0707.htm

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Abstract

Graphic abstract

Muscle-invasive urinary bladder urothelial carcinoma (UBUC) is a lethal disease for which effective prognostic markers and potential therapy targets are still lacking. Previous array comparative genomic hybridization identified that 3q27 is frequently amplified in muscle-invasive UBUCs, one candidate proto-oncogene, B-cell CLL/lymphoma 6 (BCL6), mapped to this region. We therefore aimed to explore its downstream targets and physiological roles in UBUC progression.

Methods: Specimens from UBUC patients, NOD/SCID mice and several UBUC-derived cell lines were used to perform quantitative RT-PCR, fluorescence in situ hybridization immunohistochemistry, xenograft, gene stable overexpression/knockdown and a series of in vitro experiments.

Results: Amplification of the BCL6 gene lead to upregulation of BCL6 mRNA and protein levels in a substantial set of advanced UBUCs. High BCL6 protein level significantly predicted poor disease-specific and metastasis-free survivals. Knockdown of the BCL6 gene in J82 cells inhibited tumor growth and enhanced apoptosis in the NOD/SCID xenograft model. In vitro experiments demonstrated that BCL6 inhibited cytostasis, induced cell migration, invasion along with alteration of the expression levels of several related regulators. At molecular level, BCL6 inhibited forkhead box O3 (FOXO3) transcription, subsequent translation and upregulation of phosphorylated/inactive FOXO3 through phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase (AKT) and/or epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase 1/2 (MAP2K1/2) signaling pathway(s). Two BCL6 binding sites on the proximal promoter region of the FOXO3 gene were confirmed.

Conclusion: Overexpression of BCL6 served a poor prognostic factor in UBUC patients. In vivo and in vitro studies suggested that BCL6 functions as an oncogene through direct transrepression of the FOXO3 gene, downregulation and phosphorylation of the FOXO3 protein.

Keywords: urinary bladder urothelial carcinoma, BCL6, FOXO3, cytostasis.