高雄醫學大學 泌尿學科1，高雄醫學大學附設醫院 泌尿科2，衛福部屏東醫院 泌尿科3，高雄市立大同醫院 病理科4，高雄榮民總醫院 醫學教育與研究科5，國立陽明醫學大學 解剖與細胞生物學研究所6，高雄醫學大學 再生醫學與細胞治療中心7，國立中山大學 醫學科學技術研究所8
Aquaporins are Involved in Tumor Formation and May Potentially Act as a Therapeutic Target in Urothelial Carcinoma
Bi-Wen Yeh1,2,7, Wei-Ming Li1,2,3, Ching-Chia Li1,2, Zi-Shian Chao1,2, Yu-Ching Wei4, Mei-Lang Kung5* , Hsueh-Te Lee6*, and Wen-Jeng Wu1,2,7,8*
Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University (KMU), Kaohsiung1; Department of Urology, KMU Hospital, Kaohsiung2; Department of Urology, Ministry of Health and Welfare Pingtung Hospital, Pingtung3; Department of Pathology, Kaohsiung Municipal Ta-Tung Hospital, KMU, Kaohsiung4; Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung5; Institute of Anatomy and Cell Biology, National Yang-Ming University, Taipei6; Regenerative Medicine and Cell Therapy Research Center, KMU, Kaohsiung7; Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, Taiwan8
Purposes: Aquaporins (AQPs) play a pivotal role in the cellular microenvironment and are responsible for maintaining water homeostasis and solute transfer. Due to their critical role in cell stability and integrity, it would make sense that AQPs may involve in urothelial carcinoma (UC). Searching for TCGA_BLCA database, we found that AQP4, AQP9 and AQP11 expression are significantly higher in UC than in normal tissue. GSE31684 and GSE13507 analysis also showed that AQP9 was related to cancer metastasis and patient prognosis. Besides, previous study using tumor-infiltrating immune subsets analyses showed that CD8+ T cells, macrophages, neutrophils, and dendritic cells were positively correlated with the expression of AQP9. In the present study, we explore the biological significances of AQPs in UC disease.
Materials and Methods: AQPs expression in UC cell lines were analyzed by RT-PCR and western blotting. In vitro characterizations of the cellular function of recombinant aquaporins protein in epithelial-mesenchymal transition (EMT) and tumorigenic behaviors were performed by trans-well assay and colony formation assay, respectively. The cross-talk between AQPs and various markers; such as EMT markers, cancer stemness, drug resistance related proteins expression, and immune markers (PD-L1, PD1) in UC cells were studied by Western blot and qPCR to identify the mechanisms underlying its action. C57BL/6 mice in vivo model was employed in the identification of possible effectors and inhibitors in this study.
Results: Our current results shown that overexpression of AQPs can led to ECM degradation and increase the migration/invasion capability in UC cells which were derived from Src/PI3K pathway activation and further induced their downstream effectors. Besides, overexpression of AQPs can increase side population characters in UC cell lines. By treating these UC cell lines with AQPs inhibitor (Furosemide), or WYC0209, or PD-1/PD-L1 inhibitor, the side population characters were all diminished. In addition, AQPs inhibitor, WYC0209 can decrease EMT markers and cancer stemness CD133 marker expression. PD-L1 inhibitor (Atezolizumab) can decrease AQP1, AQP4, PD-L1 and PD-1 expression. In vivo transient overexpression of Src increased tumor formation ability and induction of AQPs expression (AQP1, AQP4, AQP9) in C57BL/6 mice. Moreover, WYC0209 could inhibit the effect of Src over-expression related tumor formation ability and AQPs expression levels in C57BL/6 mice.
Conclusions: Our results demonstrated that AQPs are involved in UC disease. Block the activation of AQPs by inhibitor or combination block with its related pathways might have therapeutic effects for patients with AQPs overexpressed UC. Further identification of the molecular mechanisms involved and searching for specific targetable regimens that are related to AQPs overexpressed UC is warranted.