Abstract 935: NEK1 phosphorylates YAP1 to promote prostate cancer tumorigenesis

Abstract Background: Prostate cancer (PCa) is the second leading cause of cancer death in men of western part of the world. The disease progression has been defined by substages (adenocarcinoma to castration resistant PCa to neuroendocrine PCa) both from mouse models and patients from clinical studies. The mechanism of the disease advancement and hence treatment modalities is still under investigation. Hippo pathway, the evolutionarily conserved developmental pathway, known to regulate organ size, proliferation, apoptosis, migration, stemness etc., is also implicated in PCa tumorigenesis. Transcriptional co-activator YAP1 which acts downstream of the canonical hippo kinases is known to interact with plethora of proteins. But how non-canonical hippo kinases can regulate YAP1 in this disease is yet to be unraveled. We find that NIMA-related kinase isoform 1, Nek1, interacts with YAP1 and phosphorylates Tyr-407 of YAP1. The significance of this post-translational modification is the focus of the study. We find that Nek1 phosphorylates YAP1 to stabilize YAP1 and promote tumorigenesis. Material and Methods: Human YAP1 cDNA encoding plasmid mutated at Tyr 407 to Phenylalanine using site-directed mutagenesis. YAP1-Y407F and WT expressed in HEK293A cells. For genetic analysis in mouse model, female TRAMP mice were bred with Nek1 -/+ male mouse (Jackson laboratory). Genotypic analysis confirmed Nek1-/+ TRAMP and Nek1+/+TRAMP which were used for this study. After 16weeks of age the male mice were castrated and sacrificed at 20weeks of age. Prostate tissues collected and histological sections done (paraffin embedded). Immunohistochemical (IHC) analysis was done using vector labs IHC kits, data analyzed and scored by our pathologist. Results: Immunoblots from YAP1-Y407F and WT cells treated with Cycloheximide (50ug/ml) at different time points (0.5-6hrs) show that YAP1-Y407F is less stable than WT with t1/2 of 0.5hrs whereas WT is stable till 2hrs. Very importantly, while intact TRAMP/Nek1+/- mice do develop PCa normally with much enlargement of the prostate at 18-20 weeks, if these mice are castrated, the tumors fail to grow and the prostate retains normal size and general morphology. Our IHC data confirms that firstly, castrated Nek1+/+TRAMP shows clearly cancerous acini whereas the Nek-/+ TRAMP shows mostly stromal fibrosis and hyperplasia (as shown in H&E and IHC). Secondly, when stained for activated Nek1 marker, pNek1 (T141), we find that Nek1 stain intensity is low in Nek1-/+ tissues compared to Nek1+/+. Most importantly, YAP1 level is lower in the same parallel tissue sections of Nek1-/+. From the above analysis, we conclude that Nek1 activity regulates stability of YAP1, and infer that YAP1 function is critical for the timely progression to adenocarcinoma following androgen deprivation therapy. Acknowledgements: Funding Resources: Department of Defense (DoD-PCRP grant W81XWH-17-1-0417) ; FWCC Bridge Award. Citation Format: Ishita Ghosh, Zobair Alam, Md. Imtiaz Khalil, Judy King, Arrigo DeBenedetti. NEK1 phosphorylates YAP1 to promote prostate cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 935.