Abstract 2027: Global analyses of HOXB13-regulated transcription reveal a potential link between HOXB13 G84E and prostate cancer risk
Authors: Johng Dorhyun , Haffner Michael C. , Mooney Steven M. , Esopi David M. , Ewing Charles M. , Chen Shuangling , Isaacs William B. 
Published: July 2016

Abstract
Prostate cancer (PCa) is one of the most heritable cancers. However, germline genetic variations that are consistently and highly associated with PCa have remained elusive. Recently, a variety of non-synonymous SNPs in HOXB13 have been identified in prostate cancer patients from distinct ethnic populations. Among the HOXB13 variants, HOXB13 G84E has been consistently shown to associate strongly with increased PCa risk in men of European descent. HOXB13 is a prostate-specific transcription factor that plays a role in prostate development. HOXB13 has also been implicated in various aspects of PCa biology. However, to this date, the exact role of HOXB13 in normal prostate physiology and in PCa biology remains obscure. Our lab previously reported that neither HOXB13 WT nor G84E alone can transform prostate cells and that the G84E variant does not behave differently from HOXB13 WT in interactions with cofactors (AR, MEIS2) and protein half-life. To further delineate the function of HOXB13 in the prostate and to identify G84E-induced alterations that subject G84E carriers to PCa susceptibility, we performed RNA-seq of the LAPC4 prostate cancer cell line overexpressing a control vector, HOXB13 WT or G84E. Comparisons of the RNA-seq datasets were made using Ingenuity Pathway Analysis (IPA) by selecting genes whose expression was altered by 2 standard deviations or higher. Among pathways altered by HOXB13 WT or G84E compared to the vector control dataset, the VDR/RXR activation pathway was negatively regulated by both WT and G84E. When the G84E dataset was compared to the WT dataset, the IPA analysis revealed that G84E can up-regulate the e-NOS signaling pathway. Interestingly, the top contributor to the difference imparted by HOXB13 G84E compared to WT was identified to be a set of genes regulated by HOXB13 itself. Overall, this preliminary analysis suggests that HOXB13 G84E may be a gain-of-function mutation whereby potential tumor-promoting functions of HOXB13 are over-activated. Finally, to further identify targets directly regulated by HOXB13 WT and G84E, we combined the RNA-seq datasets with HOXB13 overexpression ChIP-seq datasets we previously reported and a HOXB13 knockdown dataset in LAPC4 (Norris et al. 2009). We focused initially on genes that were shown to be up- or down-regulated in the same direction in the RNA-seq and HOXB13 knockdown datasets. Those genes were screened for nearby HOXB13 binding sites as annotated in our ChIP-seq analyses, followed by validation with droplet digital PCR. Among the genes that were down-regulated by HOXB13 WT and G84E include INPP4B, TNFSF10, IGFBP3, KLF5 and SOX9, which all have tumor suppressing functions in PCa. Among HOXB13-upregulated genes was BCHE, which is also implicated in the suppression of PCa initiation and progression. These results provide us with potential areas of further investigation in which HOXB13 G84E may differentially regulate transcription of these genes.
Citation Format: Dorhyun Johng, Michael C. Haffner, Steven M. Mooney, David M. Esopi, Charles M. Ewing, Shuangling Chen, William B. Isaacs. Global analyses of HOXB13-regulated transcription reveal a potential link between HOXB13 G84E and prostate cancer risk. [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 2027.