Antitumor Activity and Pharmacological Mechanisms of Pik3c3 Inhibitor in Bladder Cancer Cells

Bladder cancer is ranked as the ninth most common malignancy worldwide, with an estimated 429,800 new cases of bladder cancer and 165,100 deaths occurred. However, no molecular targeted therapies have been approved for treatment of this disease as yet. Autophagy flux was reportedly activated, and inhibition of autophagy induces apoptosis or enhances RAD001-induced cytotoxicity in human bladder cancer cells. PIK3C3, the only member of the class III PI3K family, generates phosphatidylinositol 3-phosphate (PtdIns3P), which recruits proteins containing FYVE or PX domains, thereby initiating various complexes at the membranes of endosomes, phagosomes and autophagosomes. From previous proposal entitled “Targeting class IIIphosphoinositide 3-Kinase (PI3K) for anticancer drug discovery”, we identified that compound 93 (compd. 93) is a highly selective and potent PIK3C3 inhibitor with Kd value of 0.53 nM. Recently, we obtained preliminary data suggesting that compd. 93 exhibits anticancer activity in bladder cancer cells and autophagy contributes to compd. 93-increased cytotoxicity. Compd. 93 increases the protein level of SQSTM1 and induces lucent vacuoles at the peri-nuclear region, suggesting that blocking PIK3C3 kinase activity affects vesicle trafficking and successfulness of autophagy. Moreover, compd. 93 shows the potential to overcome drug resistance of agent known to induce pro-survival autophagy. Based on the above rationale, we hypothesized that modulation of autophagy is a potential strategy for the discovery of novel therapeutic agents against bladder cancer. To address this hypothesis, we proposed three aims as listed below: Aim 1. The purpose of this aim will test the hypothesis that PIK3C3 is a potential target for discovering novel anticancer drugs in bladder cancer. Specifically, we will examine (a) the anticancer activity of compd. 93 in bladder cancer cell lines and cisplatin-resistant bladder cancer cells, (b) the combination of compd. 93 with the clinical therapeutic drugs, and (c) antitumor activity of compound 93 in xenograft models of RT-112 and PIK3C3 overexpressed RT-112 cells in vivo. The results will proof our hypothesis that PIK3C3 is a druggable target for the treatment of bladder cancer. Aim 2. To perform the mechanisms of action (MOA) study of PIK3C3 inhibitor in bladder cancer cells. We design experiments to validate whether this effect was resulted from on-target inhibition of PIK3C3 by (a) evaluating the effects of compound 93 on different types of cell death, (b) elucidating the mechanisms of compound 93-increased vacuolization, and (c) identifying the binding pattern of compound 93 in PIK3C3. The analysis will shed light on the action mechanisms of PIK3C3 inhibitor, provide pharmacodynamic markers, and guide structure-modification of compound 93 in the future. Aim 3. To explore new indications of PIK3C3 inhibitors in human diseases. PIK3C3 reportedly involves in the regulation of vesicle trafficking, endosome maturation and autophagy. Exosomes are generated from endosomes (multivescular bodies) and related to cancer cachexia. We therefore propose experiments to (a) evaluate the effects of compound 93 on tumor-derived exoxomes, and (b) investigate therapeutic potential of compound 93 in cancer-induced cachexia. The results will broaden therapeutic application of compound 93 by targeting autophagy as well as tumor microenvironment.