Abstract 4970: Phosphorylation of the intrinsically disordered cancer/testis antigen PAGE4 by HIPK1 and CLK2 in prostate cancer
Authors: Kagohara Luciane T. , Mooney Steven , Chen Yihong , Orban John , Kulkarni Prakash , Veltri Robert W. 
Published: July 2016

Prostate-associated gene 4 (PAGE4) is an intrinsically disordered cancer/testis antigen that is typically restricted to the normal testis but is aberrantly expressed in prostate cancer (PCa). Furthermore, PAGE4 is developmentally regulated with dynamic expression patterns in the fetal prostate and is also a stress-response protein that is up-regulated in response to cellular stress. PAGE4 interacts with c-Jun and plays an important role in the development and pathology of the prostate gland. HIPK1, a component of the stress-response pathway, and CLK2, a dual specificity kinase are kinases that phosphorylate PAGE4 at predominantly at T51 and at multiple S/T residues, respectively. While phosphorylation at T51 is critical in potentiating c-Jun, the functional significance of the multisite phosphorylation by CLK2 is not known. The aim of this study was to determine the expression levels of HIPK1 and CLK2 in PCa cell lines, and in a set of 80 paired cases of PCa and benign adjacent tissue. Protein levels of HIPK1 and CLK2 were evaluated in cell lines and tissue samples by Western Blotting (WB) and immunohistochemistry (IHC), respectively. WB was performed with whole cell lysates from BPH1, DU145, LNCAP and PC3 cells using a fluorescence approach. IHC was performed with TMAs containing paired tumor and benign samples and after scanning (Aperio ScanScope), data were quantified using the ImageScope software. WB showed that HIPK1 is expressed in all cell lines selected with no significant changes in expression levels, while CLK2 was expressed only in BPH1 and LNCAP, suggesting this protein is expressed in cells displaying a less aggressive phenotype. Protein expression analysis by IHC in PCa and benign samples showed that CLK2 is more commonly expressed in PCa than HIPK1 when compared to non-cancer cases. CLK2 expression was positive in 81.7% of the cancer cases and HIPK1 was expressed in 72.2%. When compared to benign cases these differences are significant with p&lt;0.0001. To further evaluate the importance of these two proteins as biomarkers for PCa, we performed ROC curve analysis to verify if the expression profile could be used to separate cancer from non-cancer cases. Both, CLK2 and HIPK1 can discriminate PCa from the benign tissue (AUC = 0.81 and 0.70, respectively). CLK2 as a biomarker for PCa is more specific than HIPK1, with specificity of 78.4% for the first vs. 64.4% for the last. Since CLK2 is expressed in cell lines with a less aggressive phenotype, we evaluated whether CLK2 expression correlates with Gleason score (GS) in an attempt to determine their utility as outcome predictors. However, no association was observed with GS and CLK2 and HIPK1 expression patterns. In summary, our results suggest that CLK2 may be a potential biomarker for PCa.

Citation Format: Luciane T. Kagohara, Steven Mooney, Yihong Chen, John Orban, Prakash Kulkarni, Robert W. Veltri. Phosphorylation of the intrinsically disordered cancer/testis antigen PAGE4 by HIPK1 and CLK2 in prostate cancer. [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 4970.