Abstract 961: Genome– and proteome–wide analyses of HOXB13 and the G84E variant associated with prostate cancer

Abstract

Abstract HOXB13 is a prostate-specific transcription factor that plays a critical role in the development of the prostate. Recently, our group, in collaboration with Dr. Kathleen Cooney's group at the University of Michigan, discovered germline mutations in HOXB13 in families with hereditary prostate cancer. Among the HOXB13 mutations, the G84E variant has been shown in multiple independent studies to recur at an elevated frequency in prostate cancer patients. To gain molecular insights into prostate cancer development, it is crucial to investigate the mechanism behind HOXB13 G84E's association with prostate cancer. Our previous study demonstrated that HOXB13 G84E alone or with AR cannot transform prostate cells and that the G84E variant does not differ from wild type in cellular localization, interaction with cofactors (AR, MEIS2) and protein stability. To further search for G84E-induced alterations that link G84E to prostate cancer predisposition, we performed ChIP-seq analysis to study the genome-wide occupancy of HOXB13 WT and G84E in LAPC4, a prostate cell line derived from an AR-positive lymph node metastasis. The predominant DNA consensus sequence for both HOXB13 WT and G84E identified by the ChIP-seq data (TTTTAT) was in congruence with the literature, signifying the validity of the dataset. The enrichments for binding sites with a range of fold enrichment scores were validated using ChIP-qPCR to further assess the quality of the dataset. Many enriched sites were in proximity to genes with functional relevance to prostate cancer (SMYD2, HOXA7, PMEPA1, MAP3K5, SHH, etc.). Sites that were uniquely bound by either HOXB13 WT or G84E were absent, although currently it is unknown whether some sites are more strongly bound by WT or G84E. Next, to examine genes that are regulated directly by HOXB13 binding, we crossed our ChIP-seq dataset with an LAPC4 HOXB13 knockdown microarray dataset obtained from Dr. Donald McDonnell's lab at Duke University. Among ∼70 genes whose expression changed by at least 2.6-fold with HOXB13 knockdown, ∼45 genes had adjacent binding sites for HOXB13 WT and G84E. Pathway analysis of the ∼45 genes using g:Profiler suggested that they are involved in the MAPK cascade. Given the apparent lack of G84E-unique binding sites, we questioned whether changes in the protein interactome might affect the function of HOXB13. To this end, we performed mass spectrometry on proteins co-immunoprecipitated with HOXB13 WT or G84E. The experiment identified fatty acid synthase as a binding partner of HOXB13 WT but not G84E. Although our genome- and proteome-wide analyses of HOXB13 WT and G84E need further validation, our preliminary data suggests the possibility of identifying the potential source of quantitative and qualitative difference caused by the G84E mutation by studying gene-protein and protein-protein interaction on a global-scale. Citation Format: Dorhyun Johng, Michael C. Haffner, David M. Esopi, William B. Isaacs. Genome– and proteome–wide analyses of HOXB13 and the G84E variant associated with prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 961. doi:10.1158/1538-7445.AM2015-961