MTHFD2於尿路上皮癌中的角色

許再臨¹、葉信志^1,2

¹高雄醫學大學醫學研究所、²高雄市立大同醫院/高雄醫學大學附設醫院/高雄醫學大學泌尿科

Investigation of the role of MTHFD2 in urothelial carcinoma

Zai-Lin Sheu¹; Hsin-Chih Yeh^1,2

¹Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; ²Department of Urology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

Purpose: Our study aims to investigate the role of MTHFD2 in tumor development, chemo-resistance and immune response in urothelial carcinomas (UC), including bladder cancer (BC) and upper tract urothelial carcinomas (UTUC), and to evaluate the anti-tumor effect of MTHFD2 inhibitor in UC.

Materials and Methods:

A. GSEA analyses: TCGA bladder cancer databases and KMUH UTUC cohort are analyzed through GSEA. Biological pathways that are co-regulated with MTHFD2 expression are identified.

B. In vitro experiments: We knocked down MTHFD2 in normal urothelium, BC and UTUC cell lines including SV-HUC-1, BFTC909, UMUC-3, and 5637. In addition, cancer cells are treated with MTHFD2 inhibitor and cisplatin. Cell proliferation is assessed through flow cytometry with carboxyfluorescein succinimidyl ester (CSFE). Migration and motility are evaluated through wound healing assay and time-lapse microscopy, respectively. Protein expression of epithelial-mesenchymal transition (EMT) and immune markers are analyzed through western blotting.

C. In vivo experiments: MTHFD2-knockdown cancer cells are injected subcutaneously in four-week-old nude mice. Tumor growth and size are assessed through IVIS spectrum twice a week. In addition, systemic toxicity and body weight are recorded. After one month, tumor tissue is harvested and protein expression including EMT and immune markers are evaluated through immunohistochemistry staining.

Results: GSEA analyses of TCGA bladder cancer databases show enrichment of several immune-related pathways including B-cell activation and NF-κB signaling in MTHFD2-high expression group. In vitro studies show that knockdown of MTHFD2 gene by shRNA in 5637 BC cells inhibits cell proliferation, migration, motility, increases E-cadherin expression and decreases N-cadherin, Vimentin, Slug, Snail as well as immune checkpoints expression. These effects can be exerted by MTHFD2 inhibitor. Moreover, knockdown of MTHFD2 sensitize tumor cells to cisplatin. In addition, MTHFD2-knockdown suppresses tumor growth in mice.

Conclusion: MTHFD2-knockdown impedes tumor development and reduces chemo-resistance of UC in vivo and in vitro. It may also be associated with less immune evasion of cancer cells. These effects can also be induced by treatment with MTHFD2 inhibitor, making MTHFD2 a novel target of advanced-stage UC.