Abstract 3063: Over expression of protein kinase Nek1 and its modulation by Thioridazine in prostate cancer

Abstract

Abstract Introduction: Prostate Cancer (PCa) is one of the most common urological malignancies in men in the United States. We have identified NIMA kinase NEK1 through a proteomic approach, that interacts and co-localizes with Tousled Like kinases (TLKs) and has a role in the DDR, upstream of ATR and Chk1. In the present study we have checked the NEK1 activity in PCa patient samples and tested Thioridazine (THD), an anti-psychotic drug and inhibitor of TLKs in inhibiting the activity of NEK1 in human and mice derived prostate cancer cell lines and xenografts derived from LNCaP cells. Material & Methods: We have used tissue microarray (TMA) derived from patient samples and respective benign control. We have used a human prostate cancer cell lines: LNCaP, C4-2, C4-2b, 22Rv1, DU145 and PC3 and normal prostate cell line RWPE-1 as well as TRAMP-C2 cell lines derived from TRAMP mice. Clonogenic activity was measured by colony formation assay. Immunoblotting and immunohistochemistry was done for TLKs, NEK1, p-ATR, p-Chk1, cell cycle and apoptosis related protein in PCa cell lines and tumor tissues from xenografts. Xenografts derived from LNCaP and LNCaP-NEK1 mutant were used in present study. We have used THD (alone) or in combination with the anti-androgen drug bicalutamide in PCa cell lines as well as in xenografts mice model. Results: We found a significant over expression of NEK1 activity as measured by p-NEK1 with PCa samples as compare to benign control (p<0.001) and also correlated with high Gleason score patients. Treatment with THD significantly impaired the colony formation efficiency in human PCa cells in vitro (even of androgen independent lines) as well in TRAMP-C2 cells. THD treatment halts the PCa cells in subG0 phase and affects the cyclin D1, p27, p21 levels and leads to apoptosis with induction of cleaved PARP and Caspase-3 activity. Further p-ATR and p-Chk1 axis was inhibited by treatment with THD and in combination with bicalutamide. Combination therapy of THD and Bicalutamide remarkably inhibited the tumor growth in LNCaP xenografts model while tumor growth in LNCaP xenografts with NEK1 mutant were inhibited by Bicalutamide treatment alone. Following DNA damage, addition of the THD impaired ATR and Chk1 activation, that leads to TLK1>NEK1>ATR>Chk1 pathway activation in PCa. Conclusions: Our data indicated that NEK1 is over-expressed in PCa patients and its activity can be suppressed by treatment with THD (that inhibits its activating kinase, TLK1) in vitro and in vivo. Citation Format: Vibha Singh, Praveen K. Jaiswal, Hari K. Koul, Xiuping Yu, Arrigo Benedetti. Over expression of protein kinase Nek1 and its modulation by Thioridazine in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3063.