Evaluation of the Role of DAXX in Autophagy and Pathogenesis of Prostate Cancer

Lorena Puto

Appointment Period: 2012-2013, Grant Years: [28]

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. To this end, the following three aims will be undertaken:

Aim 1: Elucidate the mechanisms of DAPK1/3-DAXX axis in autophagy and prostate tumorigenesis. DAPK1, the founding member of the DAPK family (DAPK1-3), is a Ca2+/calmodulin Ser/Thr kinase that promotes cell death. DAPK1 is a tumor suppressor, and its expression is lost in many cancer types. Through phosphorylating Beclin-1 DAPK1 promotes autophagy, which can serve a tumor suppressor function. DAPK1 and DAPK3 are both target genes of the NF-B member RelB, and are potently repressed via epigenetic mechanisms by the transcriptional repressor DAXX, which interacts with RelB. Since DAPK1 triggers autophagy, suppression of DAPK1 by DAXX might block autophagic cell death. To test this hypothesis, I will stably deplete DAPK1 and DAPK3 individually in control and DAXX K/D ALVA-31 prostate carcinoma (PCa) cells to determine effects on tumorigenesis and autophagy in a mouse subcutaneous xenograft model. If increased DAPK1/3 expression plays a role in the decreased size of DAXX-depleted tumors, then we expect to observe an increase in tumor size upon depletion of DAPK1 and/or DAPK3, and a reduction in autophagy.

Aim 2: Determine the structure of DAXX bound to transcription factor RelB. DAXX physically interacts with RelB, but no other members of the Rel family. While the 3D structure of RelB has been solved, the DAXX structure remains poorly characterized. In collaboration with Gouri Ghosh (UCSD), who reported the RelB structure, I plan to solve the 3D structure of DAXX bound to RelB by X-ray crystallography, with the ultimate goal of understanding the functional significance of DAXX-RelB interaction through making noninteracting mutants and testing their function by expression in tumor cells.

Aim 3: Determine the potential of DAXX as an improved prognostic marker in prostate cancer. The extent to which DAXX expression is altered in human cancers and what impact functional inactivation of DAXX would have on cancer pathogenesis are unknown. To generate a suitable animal model to study the function of DAXX in tumorigenesis, I will generate tissue-specific Daxx knockout mice (the Daxx knockout is embryonic lethal). Following successful generation of prostate-specific Daxx-null mice, as confirmed by the lack of Daxx in the prostate, we will investigate their response to chemically-induced PCa, compared to wild type animals, by injecting NMU and testosterone into the dorsal prostate to induce PCa. If our model is correct, tumorigenesis will be decreased in the prostate-specific Daxx knockout mice compared to WT, because DAPK activation will induce autophagy in the conditional knockout mice. Relevant to this aim, I will also carry out correlative studies to see how DAXX expression in prostate tumor specimens correlates with disease in patients, using publicly available cancer gene expression databases, and by IHC staining for DAXX in primary PCa tissue sections obtained through our collaborator from the UCSD Moores Cancer Center Biorepository.

PUBLICATIONS (resulting from this training):

None yet – trainee just recently appointed

Other publications (prior to Training Grant appointment):

Puto, L.A., Brognard, J., Meisenhelder, J., Leblanc, M., and Hunter, T. Transcriptional repressor DAXX promotes tumorigenicity of prostate cancer cells via suppression of the autophagy regulator DAPK3. [in preparation]

Puto, L.A. and Reed, J.C. (2008) Daxx represses RelB target promoters via DNA methyltransferase recruitment and DNA hypermethylation. Genes Dev 222:998-1010. PMCID: PMC2335332

Brognard, J., Zhang, Y-W., Puto, L.A. and Hunter, T. (2011). Cancer-associated loss of function mutations implicate DAPK3 as a tumor suppressing kinase. Cancer Res 71:3152-61. PMCID: PMC3078168

Croxton, R., Puto, L.A., de Belle, I., Thomas, M., Torii, S., Hanaii, F., Cuddy, M. and Reed, J.C. (2006) Daxx represses expression of a subset of antiapoptotic genes regulated by nuclear factor-kappaB. Cancer Res 66:9026-35.

Puto, L.A., Pestonjamasp, K., King, C.C. and Bokoch, G.M. (2003) p21-activated kinase 1 (PAK1) interacts with the Grb2 adapter protein to couple to growth factor signaling. J Biol Chem 278:9388-93.