Abstract 184: The interaction of TLK1 and NEK1 in Prostate cancer as key regulators of the DDR and in mitosis
Authors: Connelly Zachary M. , DeBenedetti. Arrigo 
Published: July 2016

Background: The Tousled Like Kinases (TLKs) are involved in DNA repair, the DDR, and also in mitotic segregation. However, only a few substrates of TLK1 have been identified thus far, such that the mechanism of action of these kinases remains largely unknown. We have recently identified the proteome target of TLK1 that consists of 165 proteins identified with high confidence. Among these, stands out prominently NEK1, and member of the NIMA family of protein kinases that is specifically involved in the DDR and in mitotic segregation, making NEK1 and ideal target to explain several of the functions of TLK1. Specifically, NEK1 is involved in the early response of the DDR such that it regulates the activity of the effector kinases Chk1 and Chk2, while specific inhibitors of TLK1 lead to prolonged S phase arrest and Chk1 activation after treatment of cells with doxorubicin or hydroxyurea (HU). We have now begun to investigate the relationship between TLK1 and NEK1.

Results: The strong interaction between TLK1 and NEK1 was established by reciprocal coIP. We also found that the interaction is cell cycle regulated in DU145 cells, with strongest interaction in S and G2/M phases after synchronization of cells with HU. The expression of NEK1 in the most common human PCa cell lines was also established, and the protein was found to be variably expressed and phosphorylated. The activity of NEK1 was also found to be cell cycle regulated using immunoprecipitated NEK1 and casein as the substrate. Again maximal activity was found in S and phases of the cell cycle. The activity of NEK1 and TLK1 is regulated by the DDR, and NEK1 activity was increase after treatment of cells with doxorubicin. However, an inhibitor of TLK1 suppressed the increased activation, reinforcing a strong relation between TLK1 and NEK1 activity. Recombinant TLK1B stimulates the activity of recombinant NEK1 using casein as a substrate. Further suggesting that TLK1B is a regulator of NEK1 activity in cells. LNCaP cells when treated for 4 hours in charcoal stripped medium, have much higher NEK1 activity.

Conclusions: We have identified, first through a proteomic screen, a strong interaction between TLK1 and NEK1. The activity of the two proteins throughout the cell cycle and in response to DNA damage is exactly coregulated, and we propose that TLK1 is a direct regulator of NEK1 activity. TLK1 and NEK1 are highly expressed in PCa samples compared to normal prostate cells (Atlas of human cancers), and we propose a novel axis that is upregulated in PCa that leads to resistance to DNA damaging agents/treatments (including androgen deprivation) and to the induction of mitotic aberrations that are a common occurrence in cancer cells. Since TLK1B expression is induced by androgen deprivation (ADT), we suggest that NEK1 activity is also increased following ADT via the PI3K/AKT/ mTOR/eIF4E pathway.

Citation Format: Zachary M. Connelly, Arrigo DeBenedetti. The interaction of TLK1 and NEK1 in Prostate cancer as key regulators of the DDR and in mitosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 184.