Abstract PR10: A mechanistic investigation into the dynamic alliance between autophagy and tumor suppression in prostate cancer
Authors: Puto Lorena A. , Hunter Tony 
Published: June 2012

Abstract
Background and Objective: Many types of cancers are slow-growing malignancies that are characterized by an imbalance in the rate of cell death. Several antiapoptotic genes have been discovered that are overexpressed in hormone refractory prostate cancers. One relevant example is DAXX, which encodes a transcriptional repressor. In prostate tissues, DAXX staining intensity is stronger in areas surrounding tumor glands compared to areas surrounding normal glands. DAXX levels are increased in various prostate cancer cell lines, relative to nontumorigenic human prostatic epithelial lines. Conversely, in many types of cancer, tumor suppressors are downregulated, a process that facilitates cancer metastasis. DAPK1 is a tumor suppressor and autophagy regulator that is downregulated in many cancers. One of the proteins that represses the DAPK1 gene (and its family member, DAPK3) is DAXX, in conjunction with the ReIB transcription factor. However, Daxx's mechanism of action in vivo has not been previously determined, constituting the objective of the current study.
Methodologies and Results: shRNA was used to silence DAXX in the hormone-refractory prostate cancer (PCa) cell line ALVA-31, investigating the role of DAXX in prostate tumorigenesis. In subcutaneous tumor xenograft experiments in mice (106 ALVA-31 DAXX knock-down (K/D) or ALVA-31 control shRNA (CNTL) PCa cells were injected per mouse, with each group consisting of six mice, DAXX K/D resulted in profoundly reduced tumorigenicity of ALVA-31 cells, despite decreased activation of apoptotic caspases. Interestingly, DAXX K/D tumors showed striking changes in the markers of autophagy: Beclin-1, LC3, and p62. In culture, DAXX K/D cells also exhibited increased basal and inducible (rapamycin; nutrient deprivation) autophagy. We observed an increase in the LC3-II:LC3-I ratio, decreased p62, and reduced sizes of p62 aggregates when cells were cultured under autophagy-inducing conditions. These alterations in autophagy markers seen in cultured cells are consistent with an increase in autophagy in DAXX K/D subcutaneous tumors, suggesting that DAXX represses the autophagy program. DAXX-depleted tumors exhibited elevated levels of DAPK1 (and DAPK3), and also an increase in the autophagy markers Beclin-1 and LC3, as determined via IHC. We found that DAXX has potent growth-enhancing effects on primary malignancies, and DAPK1 protein levels were strikingly elevated in vitro and in vivo in DAXX K/D prostate cancer cells, arguing that DAXX normally helps to reduce levels of this proautophagic protein kinase. Additionally, using shRNA, we stably depleted DAPK1 and DAPK3 individually in control and DAXX K/D ALVA-31 cells to determine their effects on tumorigenesis using a mouse subcutaneous xenograft model. The knockdown level was quantified by RT-PCR for mRNA and by immunoblotting for protein (90% knockdown efficiency). Male athymic nude mice were injected in the right flank subcutaneously with 10 million cells from each cell group indicated and tumor volumes were measured over the course of one month using external calipers. Tumor wet weights were measured at the conclusion of the experiment. Employing the required quantity of mice (six per group), we reached statistical significance. The tumor growth kinetics from these experiments showed that the tumor growth rate was faster—and the tumors grew bigger— in the case of DAPK3 K/D and DAXX/DAPK1 double K/D groups relative to the control or DAXX K/D groups, arguing that DAPKs are acting as tumor suppressors.
Conclusion: These observations suggest that DAXX has an inhibitory effect on autophagy in vivo. DAXX may promote prostate tumorigenesis by inhibiting autophagy.
Supported by National Cancer Institute grant CA082683-11A2 (TH) and a fellowship from The Department of Defense Prostate Cancer Research Program (PCRP), DoD W81XWH-08-1-0209 (LAP).
This abstract is also presented as Poster B6.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR10.