Visudyne™: a new therapeutic strategy for the treatment of ovarian cancer

Abstract

Objectives: Yes-associated protein (YAP) is a transcriptional coactivator regulated via the Hippo pathway that has been shown to be tumorigenic in ovarian cancer (OC). YAP has also been identified to be uniquely expressed within induced regulatory T cells (Tregs). A conditional knockout of YAP in T cells decreased Tregs within the tumor microenvironment and increased antitumor immune response in a subcutaneous mouse melanoma model. Visudyne™ is a liposomal derivative of verteporfin (VP), a known YAP inhibitor that is FDA approved for the treatment of adult macular degeneration. The objective of our study is to: 1) evaluate the effect of Visudyne™ on YAP and its transcriptional targets, 2) determine cytotoxic effects of Visudyne™ in platinum sensitive and resistant OC cell lines, and 3) to evaluate its effect on immune regulatory cells. Methods: Western blot analysis and immunofluorescence were performed to determine YAP expression and localization in R182 and MR182 OC cell lines using specific antibodies for YAP, phosphorylated YAP (pYAP), and connective tissue growth factor (CTGF), a transcriptional target of YAP. Cell proliferation was measured using Incucyte real-time imaging following treatment with increasing doses of cisplatin, VP and Visudyne™ in A2780 wildtype and platinum resistant OC cells. IC50 was calculated using GraphPad PRISM. Three C57/B6 mice were injected intraperitoneally (IP) with triple knockout p53mut/PTENmut/mCherry OC cells. Tumor progression was monitored by mCherry signal with fluorescence spectroscopy. Visudyne™ was administered IP at 2 mg/kg and 3 days later at 4 mg/kg. Flow cytometry was performed using antibodies for CD4, CD8, FOXP3, and interferon gamma to identify Treg and cytolytic T cells in peritoneal lavage and spleen of the control and Visudyne™ treated mice. Download : Download high-res image (329KB) Download : Download full-size image Results: Treatment of OC cells with 1uM Visudyne™ is associated with a significant decrease expression of total YAP, pYAP, and CTGF (Figure 1A). Immunofluorescence demonstrated loss of nuclear YAP and increased pYAP in the cytoplasm of R182 and MR182 cells after VP treatment (Figure 1B). Increasing doses of Visudyne™ significantly decreased cell growth in platinum resistant A2780 OC cells when compared to cells treated with cisplatin alone. In vivo treatment of tumor bearing mice with Visudyne™ is associated with a significant decrease of Treg CD4+/FOXP3+ cells in peritoneal lavage of treated mice compared to the control (85.5% vs. 92.9%, Figure IC). No changes were observed in the spleen in treated mice vs. control. Conclusions: We propose that Visudyne™ should be further investigated as a novel therapeutic agent for the treatment of OC. Its effect on immune function, specifically Tregs, suggests a promising effect either as a single agent or in combination with other checkpoint inhibitors in platinum sensitive and resistant OC.