Abstract 1334: Induction of autophagy by ormeloxifene and mevastatin through Protein Kinase D1 in prostate cancer cells
Authors: Gara Rishi K. , Kumari Sonam , Ganju Aditya , Yallapu Murali M. , Shah Ankita , Khan Sheema , Singh Man M. , Chauhan Subhash C. , Jaggi Meena 
Published: May 2015

Objective: Chemotherapy-induced emergence of drug resistance is one of the major limitations to treat prostate cancer. Numerous studies report that autophagic cell death functions as a suppressor of tumorigenesis in prostate cancer. Although chemotherapeutic drugs inhibit growth of cancer cells, they also activate chemokine, CCL2 which protects prostate cancer cells from autophagic cell death. Protein Kinase D1 (PKD1) plays multiple roles in the fundamental biological processes and is known as a tumor suppressor in prostate cancer. Here, we report for the first time that PKD1 inhibits CCL2 expression in prostate cancer cells. Therefore, we also investigated whether PKD1 modulation inhibits CCL2 and potentiates autophagic cell death. We have found a novel combination of mevastatin (MVS) and ormeloxifene (ORM), a non-steroidal, selective estrogen receptor inhibitor, effectively increases PKD1 expression and induces autophagic cell death in prostate cancer cells.

Methods: Prostate cancer cells (C4-2) stably overexpressing PKD1 (C4-2-PKD1-GFP) or vector control (C4-2-GFP) were treated with ORM (10µM) and MVS (20µM) and their combination for 48h. Cell viability was performed by CellTiter 96® AQueous One Solution cell proliferation assay. CCL2 expression levels were quantified by ELISA as per the manufacturer's instructions. Cell migration assays were performed with pre-incubation of 3-MA (5mM; 3-Methyladenine, an autophagy inhibitor) for 2h, or exogenously added CCL2 (100ng/mL) in the lower part of a Boyden chamber. The cells were treated with ORM alone or in combination with MVS for 18h for the analysis of proteins expression involved in autophagy. Immmunoblot experiments were performed using an autophagy sampler kit as per the manufacturer's instructions.

Results: Our results suggest that a combination of ORM and MVS increases PKD1 and inhibits CCL2 expression in prostate cancer cells. It inhibits cell proliferation, migration and metastasis of prostate cancer cells. Additionally, it was observed that ORM and MVS effectively inhibits CCL2 induced cell motility in PKD1 overexpressing cells, however the effect was not observed in PKD1 null cells. The PKD1 dependent inhibition of CCL2 expression activates autophagy as evidenced by activation of autophagy markers (LC3A, LC3B). It down-regulates survivin-1, Beclin-1 and NFκB-65, inducing autophagy in cancer cells. The cells treated with 3-MA and/or exogenous CCL2 abrogated the effects induced by MVS and ORM.

Conclusion: Here, we propose for the first time a link between PKD1 and autophagy. We have found a novel combination of MVS and ORM that effectively increases PKD1 expression and induces autophagic cell death in prostate cancer cells. This study prospects combined therapies targeting PKD1/CCL2 and the PI3K/Akt/mTOR/survivin pathway as a valid strategy to overcome the resistance of prostate cancer cells.

Citation Format: Rishi K. Gara, Sonam Kumari, Aditya Ganju, Murali M. Yallapu, Ankita Shah, Sheema Khan, Man M. Singh, Subhash C. Chauhan, Meena Jaggi. Induction of autophagy by ormeloxifene and mevastatin through Protein Kinase D1 in prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1334. doi:10.1158/1538-7445.AM2014-1334