Down-regulation of Aquaporins Sensitizes Immune Check-point inhibitors -induced Cytotoxicity of urothelial carcinoma

水通道蛋白的下調使免疫檢查點抑制劑誘導的尿路上皮癌細胞毒性敏感

葉碧雯^1,2,8、李威明^1,2,3、李經家^1,2,3、趙子翔^1,2、韋又菁⁴、李健逢⁵、

孔美蘭^6*、李學德^7*、吳文正^1,2,8,9*

¹高雄醫學大學 泌尿學科，²高雄醫學大學附設醫院 泌尿科，³高雄醫學大學岡山醫院 泌尿科，⁴高雄市立大同醫院 病理科，⁵奇美醫院 醫學研究部，⁶高雄榮民總醫院 醫學教育與研究科，⁷國立陽明交通大學 解剖與細胞生物學研究所，⁸高雄醫學大學 再生醫學與細胞治療中心，⁹國立中山大學 醫學科學技術研究所

Bi-Wen Yeh^1,2,8、Wei-Ming Li^1,2,3、Ching-Chia Li^1,2,3、Zi-Shian Chao^1,2、Yu-Ching Wei⁴、

Chien-Feng Li⁵、 Mei-Lang Kung⁶*、Hsueh-Te Lee⁷*、and Wen-Jeng Wu^1,2,8,9*

¹Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University (KMU), Kaohsiung; ²Department of Urology, KMU Hospital, Kaohsiung; ³Department of Urology, Kaohsiung Medical University Gangshan Hospital, Kaohsiung; ⁴Department of Pathology, Kaohsiung Municipal Ta-Tung Hospital, KMU, Kaohsiung; ⁵Department of Medical Research, Chi-Mei Medical Center, Tainan; ⁶Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung; ⁷Institute of Anatomy and Cell Biology, National Yang Ming Chiao Tung University, Taipei; ⁸Regenerative Medicine and Cell Therapy Research Center, KMU, Kaohsiung; ⁹Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, Taiwan

Purposes: The combination of gemcitabine and cisplatin (GC) has been widely used as a standard chemotherapeutic remedy for advanced/metastatic urothelial carcinoma (UC). However, outcomes with cisplatin-based chemotherapy remain unchanged and resistance to this remedy has been noticed. Atezolizumab is an engineered fully humanized anti-PD-L1 monoclonal antibody that designed to interfere with the binding of PD-L1 ligand to PD-1 and B7.1 receptor. Immune check point inhibitors (ICI) reduce immunosuppressive signals found within the tumor microenvironment by blocking the PD-L1/PD-1 and enhancing immune-mediated tumor killing. 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 UC. Searching for TCGA_BLCA database, we found that AQP4, AQP9 and AQP11 expression are significantly higher in UC than in normal tissue. Moreover, knock down separately of various aquaporins all results in induced PD-L1 expressed declined. In the present study, we explore the biological significances of down-regulation AQPs in UC disease and the effect of PD-L1 inhibitors (Atezolizumab and Pembrolizumab) in the AQPs and the downstream proteins expression.

Materials and Methods: AQPs expression in UC cell lines were analyzed by qPCR and western blotting. In vitro characterizations of the cellular function of recombinant AQPs shRNA plasmids, AQPs inhibitors or PD-L1 inhibitors in epithelial-mesenchymal transition (EMT) and tumorigenic behaviors were performed by trans-well assay and side population 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 knock down of AQPs can reduced ECM degradation and decrease the migration/invasion capability in UC cells which were derived from Src/PI3K pathway inhibition and further decline their downstream effectors. Besides, down-regulation of AQPs can decrease side population characters in UC cell lines. By treating these UC cell lines with AQPs inhibitor (TGN020), or transient transfected with AQPs shRNA plasmids, or PD-1/PD-L1 inhibitors, the side population characters were all diminished. In addition, AQPs inhibitor, shRNA of AQPs or PD-L1 inhibitors all can decrease EMT markers and cancer stemness CD133 or CD44 marker expression. Moreover, both of PD-L1 inhibitors can decrease AQP1, AQP4, AQP9, PD-L1 and PD-1 expression. In vivo transient knock down of AQP9 or stable clone of shAQP9 cell line both shown that decreased tumor formation ability in C57BL/6 mice. Pembrolizumab decreased the malignant cancer protein expression in mice bladder tissue by western blotting assay resulted in the inhibition of tumor formation ability. Besides, AQP1, AQP4 and AQP9 upregulation were observed in 124 human UTUC by ELISA.

Conclusions: Our results demonstrated that AQPs are involved in UC disease processes. Block the activation of specific AQPs by inhibitors 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 (for example, combination with ICI) that are related to AQPs overexpressed UC is undergoing.