Abstract 317: Transcriptional repressor DAXX promotes prostate cancer tumorigenicity via suppression of autophagy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objective: Cancer results from uncontrolled cell growth, in part due to mechanisms that block cell death, including autophagic cell death. The current study aims to understand the function of autophagy and its modulators in the context of prostate tumorigenesis and progression. Background: Long overlooked, the complex dynamics of the transcriptional repressor DAXX represents a new concept in tumor biology that promises to make a remarkable impact on cancer prevention and treatment. DAXX suppressess the expression of numerous autophagy-relevant genes, including tumor suppressors DAPK1/3. Through its ability to suppress tumor suppressors, DAXX would be expected to induce tumor growth or survival. Indeed, robust preliminary data show that DAXX has potent growth-enhancing effects on primary malignancies. However, the mechanism by which DAXX promotes tumorigenicity remains enigmatic. Through stable gene knockdown and subsequent subcutaneous xenograft models, one mechanism by which DAXX promotes tumorigenesis in PCa - autophagy suppression - has been elucidated. Study Design & Results: To elucidate the mechanism by which DAXX promotes tumorigenesis in PCa, retroviral-mediated shRNA was used to silence DAXX, DAPK3, and the essential autophagy gene ULK1 in the PCa cell line ALVA-31. A control shRNA was also included. Subcutaneous xenograft experiments, utilizing the above cells, were performed several times, involving subcutaneous injection of 10 million cells per mouse and six mice per group. At the conclusion of the experiment (4 weeks), excised tumors were weighed and processed for histological and molecular analyses. Histological analysis included immunostaining for p62, LC3, Beclin-1, Ubiquitin, CD31, and Ki-67. For molecular analysis, tumor lysates were analyzed for LC3-I/LC3-II ratios and p62 levels by immunoblotting. The results showed that (1) DAXX is required for tumor growth, because DAXX depletion in ALVA-31 cells results in profoundly reduced tumorigenicity of ALVA-31 cells, as determined by tumor wet weight, final volume, and growth kinetics. In addition, DAXX K/D tumors display increased autophagy markers LC3/Beclin-1 and decreased ubiquitin/CD31; (2) DAPK3 acts as a tumor suppressor, because tumor growth rate is faster for DAPK3 knockdown K/D, as determined by growth kinetics; and (3) Autophagy is directly involved in prostate tumorigenesis inhibition, because tumorigenesis of ALVA-31 DAXX K/D cells is significantly enhanced/restored when the essential autophagy gene ULK1 is depleted in these cells, as determined by tumor wet weight, final volume, growth kinetics, histological, and molecular analyses. The above results were confirmed using an additional PCa line, PC3. Conclusion: Autophagy suppresses early prostate tumorigenesis, while DAXX promotes prostate tumorigenesis by inhibiting autophagy. Citation Format: Lorena A. Puto, Tony Hunter. Transcriptional repressor DAXX promotes prostate cancer tumorigenicity via suppression of autophagy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 317. doi:10.1158/1538-7445.AM2014-317