Abstract A17: NUAK1 acts as a growth suppressor in epithelial ovarian cancer
Authors: Parima Saxena, Olga Collins, Yudith Ramos Valdes, Adrian Buensuceso, Kyle Francis, Kevin Brown, Karen Colwill, Anne-Claude Gingras, Robert Rottapel, Gabriel E. DiMattia, Trevor G. Shepherd
Journal: Clinical Cancer Research (2018)

Abstract Epithelial ovarian cancer (EOC) cells form multicellular aggregates, or spheroids, and enter a dormant state during intraperitoneal metastasis. Dormant spheroids reduce anabolic metabolism and cell proliferation, which are linked to chemo-resistance. Liver kinase B1 (LKB1), encoded by the STK11 gene, is a critical regulator of stress metabolic signaling; LKB1 phosphorylates the downstream substrates AMP-activated protein kinase (AMPK) and AMPK-related kinases (ARKs) to mediate stress signaling. We have demonstrated that LKB1 expression and activity is required for EOC spheroid cell survival, yet knockdown of AMPKα1/α2 has no effect on viability. In addition, we have preliminary data demonstrating that phosphorylated AMPK is still maintained in EOC spheroids generated from CRISPR-mediated STK11-deleted cells, which completely lack LKB1 expression. Taken together, these results implicate the importance of other AMPK-independent effectors of LKB1 signaling in spheroid cell viability. Therefore, to identify critical downstream targets of LKB1-mediated signaling in EOC spheroids, we employed multiplex inhibitor beads/mass spectrometry (MIB/MS) using OVCAR8 STK11-knockout cells and OVCAR8 control cells grown in adherent and spheroid culture conditions. Using this proteomic approach, we identified NUAK1 as the sole ARK out of 12 different family members that is negatively affected by LKB1 loss. In fact, both phosphorylated and total NUAK1 protein expression are decreased in STK11-knockout cells and spheroids. NUAK1 can negatively control cell growth and proliferation by direct regulation of cell cycle checkpoint proteins in several cell systems; however, it has been relatively understudied in EOC. In analysis of the serous ovarian carcinoma data from TCGA, NUAK1 is infrequently altered (&amp;lt;2% of tumors) and it has the highest mean mRNA expression level as compared with the other 11 ARK genes. However, using immunoblot analysis, we show that NUAK1 protein is largely underexpressed in many established (n=15) and new ascites-derived EOC cell lines (n=22). In fact, further NUAK1 knockdown increased spheroid cell viability, size, and reattachment capability, whereas knockdown of its closely-related family member NUAK2 had no effect compared with the non-targeting siRNA control. Likewise, treatment with the pharmacologic NUAK1/2 inhibitor WZ4003 increased EOC cell growth and clonogenicity. Conversely, ectopic overexpression of NUAK1 reduced EOC cell growth and clonogenicity. Collectively, our data indicate that NUAK1 acts as a newly identified growth suppressor downstream of LKB1 metabolic stress signaling in EOC. We are currently investigating mechanisms regulating NUAK1 protein stability and activity in EOC cells, and whether activated LKB1-NUAK1 signaling promotes stress metabolic signaling during tumor dormancy in the context of EOC metastasis. Citation Format: Parima Saxena, Olga Collins, Yudith Ramos Valdes, Adrian Buensuceso, Kyle Francis, Kevin Brown, Karen Colwill, Anne-Claude Gingras, Robert Rottapel, Gabriel E. DiMattia, Trevor G. Shepherd. NUAK1 acts as a growth suppressor in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A17.