ASPN expression is elevated in PCAFs.

Frequency of germline ASPN D-repeat-length in JHH prostate cancer cases and controls.

Supplementary Table S2 shows bivariate and multivariable analyses of germline ASPN D13/14 compared to ASPN D13/13 for lymph node involvement and metastatic recurrence. Supplementary Table S3 show bivariate analyses of ASPN D genotypes/alleles for biochemical recurrence. Supplementary Table S4 shows multivariable analyses of ASPN D13/14 for biochemical recurrence.

Comparison of prostate cancer characteristics of men undergoing radical prostatectomy at JHH with the most common germline ASPN D-repeat-length genotypes and alleles.

ASPN expression is associated with biochemical recurrence and metastatic recurrence.

Germline ASPN D-repeat-length is variable among the population.

Supplementary Tables S6 shows bivariate analyses of the most common germline ASPN D genotypes/alleles for adverse pathology. Supplementary Table S7 shows multivariable analyses of ASPN D13/14 and Any 14 with lymph node involvement.

BackgroundFew genes have germline mutations which predispose men to more aggressive prostate cancer (PCa). This study evaluated the contribution of germline loss of function (LOF) variants in PPFIBP2 to risk of lethal PCa.MethodsA case‐case study of 1414 PCa patients with lethal PCa and low‐risk localized PCa was performed. Germline DNA samples from these patients were sequenced for PPFIBP2. Mutation carrier rates and association with lethal PCa were analyzed using the Fisher exact test, logistic regression, and Kaplan‐Meier survival analysis.ResultsIn the entire study population, eight patients, all of European ancestry, were identified as carrying PPFIBP2 pathogenic or likely pathogenic mutations. Seven (1.52%) of 462 lethal PCa patients were carriers compared with only one (0.12%) carrier in 810 low‐risk PCa patients, P = 0.0029. The estimated Odds Ratio (OR) of carrying PPFIBP2 mutation for lethal PCa was 13.8 in European American population. The PPFIBP2 loss‐of‐function mutation carrier rate in lethal PCa cases was also higher than in 33 370 non‐Finnish European individuals from the Exome Aggregation Consortium (ExAC) (carrier rate of 0.17%, P = 1.92 × 10−5) and in 498 men with localized PCa from The Cancer Genome Atlas cohort (TCGA) cohort (carrier rate of 0%, P = 0.0058). Survival analysis in European American lethal cases revealed PPFIBP2 mutation status as an independent predictor of shorter survival after adjusting for age at diagnosis, PSA at diagnosis, and genetic background (hazard ratio = 2.62, P = 0.034).ConclusionsWhile larger studies are needed, germline mutations in a novel gene, PPFIBP2, differentiated risk for lethal PCa from low‐risk cases and were associated with shorter survival times after diagnosis.

Background The recurrent p.Gly84Glu germline mutation (G84E) in HOXB13 is consistently associated with prostate cancer (PCa), although the mechanisms underlying such linkage remain elusive. The majority of the PCa-associated HOXB13 mutations identified are localized to two conserved domains in HOXB13 that have been shown to mediate the interaction with MEIS cofactors belonging to the TALE family of homeodomain transcription factors. In this study, we sought to interrogate the biochemical and functional interactions between HOXB13 and MEIS in prostatic cells with a goal of defining how the HOXB13-MEIS complex impacts PCa pathobiology and define the extent to which the oncogenic activity of G84E is related to its effect on HOXB13-MEIS interaction/function. Methods HOXB13 and MEIS paralog expression in prostate epithelial cells and PCa cell lines was characterized by qPCR and immunoblot analyses. HOXB13 and MEIS1 co-expression in human prostate tissue was confirmed by IHC, followed by co-IP mapping of HOXB13-MEIS1 interactions. Proliferation of the PCa cell line LAPC4 following shRNA-mediated knockdown of each gene or both genes was assessed using DNA- and metabolic-based assays. Transcriptional targets of HOXB13 and MEIS1 were identified by gene expression profiling and qPCR. Finally, protein stability of HOXB13 in the context of MEIS1 was determined using pulse-chase assays. Results HOXB13 and MEIS1 are co-expressed and interact in prostate cells. Both of the putative MEIS interacting domains (MID) within HOXB13 were shown to be capable of mediating the interaction between HOXB13 and MEIS1 independently and such interactions were not influenced by the G84E mutation. The inhibitory effect of either HOXB13 or MEIS1 knockdown on cellular proliferation was augmented by knockdown of both genes, and MEIS1 knockdown abolished HOXB13-driven regulation of BCHE and TNFSF10 mRNA expression. Notably, we demonstrated that MEIS1 stabilized the HOXB13 protein in LAPC4 cells. Conclusions Our study provides evidence for functional HOXB13-MEIS1 interactions in PCa. MEIS1 may contribute to the cancer-promoting actions of HOXB13 in cellular proliferation and gene regulation by prolonging HOXB13 half-life. Our data demonstrates that G84E is not a loss-of-function mutation that interferes with HOXB13 stability or ability to interact with MEIS1.

Norway has one of the highest rates of death due to prostate cancer (PCa) in the world. To assess the contribution of both common and rare single nucleotide variants (SNPs) to the prostate cancer burden in Norway, we assessed the frequency of the established prostate cancer susceptibility allele, HOXB13 G84E, as well as a series of validated, common PCa risk SNPs in a Norwegian PCa population of 779 patients. The G84E allele was observed in 2.3% of patients compared to 0.7% of control individuals, OR = 3.8, P = 1 × 10‐4. While there was a trend toward an earlier age at diagnosis, overall the clinicopathologic features of PCa were not significantly different in G84E carriers and non‐carriers. Evaluation of 32 established common risk alleles revealed significant associations of risk alleles at 13 loci, including SNPs at 8q24, and near TET2, SLC22A3, NKX3‐1, CASC8, MYC, DAP2IP, MSMB, HNF1B, PPP1R14A, and KLK2/3. When the data for each SNP are combined into a genetic risk score (GRS), Norwegian men within the top decile of GRS have over 5‐fold greater risk to be diagnosed with PCa than men with GRS in the lowest decile. These results indicate that risk alleles of HOXB13 and common variant SNPs are important components of inherited PCa risk in the Norwegian population, although these factors appear to contribute little to the malignancy's aggressiveness.

Abstract Prostate cancer (PCa) is a commonly diagnosed disease with one of the highest heritability estimates among cancers. Among germline variations that account for PCa heritability, a recurrent mutation (G84E) in HOXB13 is associated with a 3 to 6 fold increase in PCa risk. HOXB13 is a prostate-specific transcription factor that plays a role in prostate development. Other mutations in HOXB13 in PCa patients have further implicated HOXB13 in PCa biology. The goal of this study is to generate HOXB13 cell line models of normal prostate to examine the role of HOXB13 in driving cellular differentiation and/or transformation. To this end, we utilized 957E/hTERT, a cell line derived from a radical prostatectomy specimen, as a model for normal prostate epithelial cells. HOXB13 WT or G84E was stably expressed in 957E/hTERT with a lentivirus system in the presence and absence of exogenous AR. HOXB13 caused a morphology change whereby an epithelial morphology transitioned to a fibroblastic-like structure. Cells adopted elongated bodies with a frequent appearance of lamellipodia. The morphologic conversion was transient in AR- cells but was maintained in AR+ cells. Analyses of epithelial cell markers using qPCR revealed a change in the epithelial cell marker profile (AR-: no change p63, CD49f↓, K5↓, K14↑, K18↓; AR+: no change p63, CD49f, K5, K14↑, K18↓). K14 upregulation was striking with a >10 fold increase. AR alone could upregulate K14 to a level comparable to HOXB13-induced K14 in AR- cells, which was surprising since AR mainly functions in luminal cells where K14 expression is turned off. Coexpression of HOXB13 and AR had a synergistic effect in K14 upregulation. Despite the morphology change suggestive of EMT, vimentin was downregulated by HOXB13 in AR- and AR+ cells. In vitro growth assays showed that HOXB13 reduced the proliferation rate of AR+ cells but not AR- cells. Gap closure assays revealed that HOXB13 does not induce a qualitative change in migration. Similar results were obtained with G84E models. Our study demonstrates that HOXB13 may have a potential function in dictating a cell fate in the epithelial lineage as evidenced by a change in the epithelial cell marker profile. Conclusively, HOXB13 and AR can drive K14 by themselves and in collaboration. This finding is exciting in light of the recent discovery (Cheung KJ et al. 2016) that K14 is not merely a basal cell marker but a major mediator of collective invasion in breast cancer. During breast development, K14 is expressed in the cap cells at the growing tips of mammary ducts invading into the fat pad. K14 driven by HOXB13 in collaboration with AR may regulate prostate epithelial budding into the surrounding mesenchyme during development. Hypothetical dysregulation of this embryonic programming by HOXB13 mutation could predispose a carrier to PCa. This study warrants the need for HOXB13 mouse models to test these fundamental questions. Citation Format: Dorhyun Johng, Charles M. Ewing, William B. Isaacs. HOXB13 collaborates with AR to alter the cellular phenotype of prostate epithelial cells through cytokeratin 14 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3524. doi:10.1158/1538-7445.AM2017-3524

Monocyte chemotactic protein-1 (MCP-1) is differentially expressed in benign prostatic hyperplasia (BPH) and is a potential clinical biomarker for BPH.We collected urine post-digital rectal examination (DRE) from 48 men who did not have a diagnosis of prostate cancer.We measured MCP-1 levels by enzyme-linked immunosorbent assay (ELISA) and compared them with prostate weight and lower urinary tract symptoms (LUTS) as measured by the International Prostate Symptom Score (I-PSS) utilizing correlation and regression analyses.In post-DRE urine, higher MCP-1 levels were associated with increased I-PSS but not with prostate volume.MCP-1 levels in men with prostate enlargement were significantly higher than those in men without enlargement.In multivariable regression adjusting for age, prostate volume, and PSA,higher urinary MCP-1 was associated with significantly higher I-PSS.Since MCP-1 is preferentially expressed in BPH tissue, these data suggest that MCP-1 may be a novel biomarker for clinically significant BPH.

A recurrent germline mutation (G84E) in the HOXB13 gene is associated with early onset and family history-positive prostate cancer in patients of European descent, occurring in up to 5% of prostate cancer families. To date, the molecular features of prostate tumors occurring in HOXB13 G84E carriers have not been studied in a large cohort of patients. We identified 101 heterozygous carriers of G84E who underwent radical prostatectomy for prostate cancer between 1985 and 2011 and matched these men by race, age and tumor grade to 99 HOXB13 wild-type controls. Immunostaining for HOXB13, PTEN, ERG, p53 and SPINK1 as well as RNA in situ hybridization for ETV1/4/5 were performed using genetically validated assays. Tumors from G84E carriers generally expressed HOXB13 protein at a level comparable to benign and wild-type glands. ETS gene expression (either ERG or ETV1/4/5) was seen in 36% (36/101) of tumors from G84E carriers compared to 68% (65/96) of the controls (p < 0.0001). PTEN was lost in 11% (11/101) of G84E carriers compared to 25% (25/99) of the controls (p = 0.014). PTEN loss was enriched among ERG-positive compared to ERG-negative tumors in both groups of patients. Nuclear accumulation of the p53 protein, indicative of underlying TP53 missense mutations, was uncommon in both groups, occurring in 1% (1/101) of the G84E carriers versus 2% (2/92) of the controls (p = NS). Taken together, these data suggest that genes other than ERG and PTEN may drive carcinogenesis/progression in the majority of men with germline HOXB13 mutations.

Background: Germline mutations in BRCA1/2 and ATM have been associated with prostate cancer (PCa) risk. Objective: To directly assess whether germline mutations in these three genes distinguish lethal from indolent PCa and whether they confer any effect on age at death. Design, setting, and participants: A retrospective case-case study of 313 patients who died of PCa and 486 patients with low-risk localized PCa of European, African, and Chinese descent. Germline DNA of each of the 799 patients was sequenced for these three genes. Outcome measurements and statistical analysis: Mutation carrier rates and their effect on lethal PCa were analyzed using the Fisher's exact test and Cox regression analysis, respectively. Results and limitations: The combined BRCA1/2 and ATM mutation carrier rate was significantly higher in lethal PCa patients (6.07%) than localized PCa patients (1.44%), p = 0.0007. The rate also differed significantly among lethal PCa patients as a function of age at death (10.00%, 9.08%, 8.33%, 4.94%, and 2.97% in patients who died <= 60 yr, 61-65 yr, 66-70 yr, 71-75 yr, and over 75 yr, respectively, p = 0.046) and time to death after diagnosis (12.26%, 4.76%, and 0.98% in patients who died <= 5 yr, 6-10 yr, and > 10 yr after a PCa diagnosis, respectively, p = 0.0006). Survival analysis in the entire cohort revealed mutation carriers remained an independent predictor of lethal PCa after adjusting for race and age, prostate-specific antigen, and Gleason score at the time of diagnosis (hazard ratio = 2.13, 95% confidence interval: 1.24-3.66, p = 0.004). A limitation of this study is that other DNA repair genes were not analyzed. Conclusions: Mutation status of BRCA1/2 and ATM distinguishes risk for lethal and indolent PCa and is associated with earlier age at death and shorter survival time. Patient summary: Prostate cancer patients with inherited mutations in BRCA1/2 and ATM are more likely to die of prostate cancer and do so at an earlier age. (C) 2016 European Association of Urology. Published by Elsevier B. V. All rights reserved.

The Androgen Receptor (AR) plays a key role in prostate biology and in the progression of prostate cancer (PCa) to castration resistance. The role of microRNAs (miRNAs) in aberrant AR signaling have not been fully characterized. Here we screened a library of 810 miRNA mimics to identify miRNAs that alter AR activity in complementary functional assays including protein lysate microarray (LMA) quantification of AR and PSA protein levels, AR transcriptional reporter activity, and AR-positive PCa cell viability. Candidate AR-regulating miRNAs were verified through AR transcriptional reporter and cell viability assays. MiRNA binding sites were found within the AR 3'-untranslated region (UTR) and within the AR and AR-V7 coding regions. MiRNA activity was characterized by western blotting, 3'-UTR reporter assay, and AR-GFP and AR-V7-GFP reporter assays. Results uncovered miR-30 family members as direct AR inhibitors. Inhibition of endogenous miR-30b-3p and miR-30d5p enhanced AR expression and androgen-independent cell growth. Droplet digital RT-PCR quantification of miR-30c-5p and miR-30d-5p revealed significantly reduced levels in metastatic castration resistant PCa (CRPC), when compared to healthy prostate tissues. MiR-30d-5p levels were inversely correlated with AR activity, as measured by PSA mRNA, in metastatic CRPC. Collectively, these studies provide a comprehensive evaluation of AR-regulating miRNAs in PCa.

Abstract Purpose: Prostate cancers incite tremendous morbidity upon metastatic growth. We previously identified Asporin (ASPN) as a potential mediator of metastatic progression found within the tumor microenvironment. ASPN contains an aspartic acid (D)-repeat domain and germline polymorphisms in D-repeat-length have been associated with degenerative diseases. Associations of germline ASPN D polymorphisms with risk of prostate cancer progression to metastatic disease have not been assessed. Experimental Design: Germline ASPN D-repeat-length was retrospectively analyzed in 1,600 men who underwent radical prostatectomy for clinically localized prostate cancer and in 548 noncancer controls. Multivariable Cox proportional hazards models were used to test the associations of ASPN variations with risk of subsequent oncologic outcomes, including metastasis. Orthotopic xenografts were used to establish allele- and stroma-specific roles for ASPN D variants in metastatic prostate cancer. Results: Variation at the ASPN D locus was differentially associated with poorer oncologic outcomes. ASPN D14 [HR, 1.72; 95% confidence interval (CI), 1.05–2.81, P = 0.032] and heterozygosity for ASPN D13/14 (HR, 1.86; 95% CI, 1.03–3.35, P = 0.040) were significantly associated with metastatic recurrence, while homozygosity for the ASPN D13 variant was significantly associated with a reduced risk of metastatic recurrence (HR, 0.44; 95% CI, 0.21–0.94, P = 0.035) in multivariable analyses. Orthotopic xenografts established biologic roles for ASPN D14 and ASPN D13 variants in metastatic prostate cancer progression that were consistent with patient-based data. Conclusions: We observed associations between ASPN D variants and oncologic outcomes, including metastasis. Our data suggest that ASPN expressed in the tumor microenvironment is a heritable modulator of metastatic progression. Clin Cancer Res; 22(2); 448–58. ©2015 AACR.

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.

Abstract In collaboration with Dr. K. Cooney's lab at the Univ of Michigan, our group recently discovered that a recurrent glycine-to-glutamic acid germline mutation (G84E) in HOXB13 is associated with an increased prostate cancer (PCa) risk. HOXB13 is an androgen receptor (AR)-independent prostate-specific transcription factor shown to play a critical role in the development of the mouse prostate. However, the exact function of HOXB13 in normal human prostate biology and the mechanism by which HOXB13 G84E predisposes carriers to PCa remain unknown. The purpose of this study is to test our hypothesis that HOXB13 G84E, by itself or together with other oncogenes, may reprogram normal prostate cells to be prone to transformation, possibly due to altered molecular characteristics that have downstream effects on transcriptional targets. To this end, using patient-derived normal prostate epithelial cells immortalized with hTERT in the presence or absence of transduced AR (957e/hTERT and 957e/hTERT/AR), we generated cell lines stably expressing HOXB13 WT or G84E to examine neoplastic phenotypes and differentiation. In the presence of AR, HOXB13 WT and G84E caused a change in cell morphology to a fibroblast-like appearance and suppressed proliferation without any significant differences between WT and G84E. In the absence of AR, the morphological and growth suppressive effects of HOXB13 were ablated, which indicates a cooperative relationship between HOXB13 and AR. In addition, we analyzed several molecular aspects of HOXB13 WT and G84E such as protein-interactions, subcellular localization and half-life which could hypothetically contribute to carcinogenesis when altered. Whether the HOXB13-MEIS and HOXB13-AR interactions are affected by G84E was studied for the following reasons. Most HOXB13 mutations identified in PCa lie in the MEIS-interacting domains (MID) of HOXB13 and the biological significance of HOX-MEIS complexes has been shown in leukemic transformation. HOXB13 mediates the transcription of a subset of AR-target genes and the HOXB13-AR interaction has been documented. Immunoprecipitation analysis in 957e/hTERT/AR demonstrated that HOXB13 and MEIS2 interact through the MIDs and G84E did not alter this interaction. Additionally, G84E did not disturb the HOXB13-AR interaction. Finally, HOXB13 G84E localized properly to the nucleus and no significant differences were found in the half-lives of HOXB13 WT and G84E, at least in the absence of AR. Taken together, our study suggests that HOXB13 G84E alone or with AR cannot initiate PCa in our cell line model with a caveat that our model does not accurately recapitulate human PCa since patients harbor the G84E mutation throughout development. Moreover, the lack of molecular differences between WT and G84E calls for further investigation of potential areas of G84E-induced alterations, such as gene-protein and protein-protein interactions on a global scale. Citation Format: Dorhyun Johng, Charles M. Ewing, Steven M. Mooney, Shuangling Chen, William B. Isaacs. HOXB13: Investigating mechanisms of G84E mutation associated with prostate carcinogenesis. [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 2948. doi:10.1158/1538-7445.AM2014-2948

Background: A rare nonconservative substitution (G84E) in the HOXB13 gene has been shown to be associated with risk of prostate cancer. DNA samples from male patients included in the Mayo Clinic Biobank (MCB) were genotyped to determine the frequency of the G84E mutation and its association with various cancers. Methods: Subjects were genotyped using a custom TaqMan (Applied Biosystems) assay for G84E (rs138213197). In addition to donating a blood specimen, all MCB participants completed a baseline questionnaire to collect information on medical history and family history of cancer. Results: Forty-nine of 9,012 male patients were carriers of G84E (0.5%). Thirty-one percent (n = 2,595) of participants had been diagnosed with cancer, including 51.1% of G84E carriers compared with just 30.6% of noncarriers (P = 0.004). G84E was most frequently observed among men with prostate cancer compared with men without cancer (P < 0.0001). However, the mutation was also more commonly observed in men with bladder cancer (P = 0.06) and leukemia (P = 0.01). G84E carriers were more likely to have a positive family history of prostate cancer in a first-degree relative compared to noncarriers (36.2% vs. 16.0%, P = 0.0003). Conclusions: Our study confirms the association between the HOXB13 G84E variant and prostate cancer and suggests a novel association between G84E and leukemia and a suggestive association with bladder cancer. Future investigation is warranted to confirm these associations in order to improve our understanding of the role of germline HOXB13 mutations in human cancer. Impact: The associations between HOXB13 and prostate, leukemia, and bladder suggest that this gene is important in carcinogenesis. Cancer Epidemiol Biomarkers Prev; 24(9); 1366–72. ©2015 AACR.

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2014MP31-03 ALTERED HOXB13 FUNCTION IN HUMAN PROSTATE CANCER Shuangling Chen, Dorhyun Johng, Charles Ewing, and William Isaacs Shuangling ChenShuangling Chen More articles by this author , Dorhyun JohngDorhyun Johng More articles by this author , Charles EwingCharles Ewing More articles by this author , and William IsaacsWilliam Isaacs More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.912AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our group, in collaboration with Dr. Kathleen Cooney’s lab at University of Michigan, recently identified a rare but recurrent germ line mutation (G84E) in HOXB13 as a high penetrance prostate cancer susceptibility allele (Ewing et al, NEJM 366:141). HOX genes are a family of homeodomain-containing transcription factors that play key roles in embryonic development, especially in the patterning of the anterior to posterior axis. HOX13 paralogs are particularly important in the development of the prostate gland in the mouse, but the function of HOXB13 in the adult human prostate and in prostate cancer development is unknown. To assess the role of wild-type (wt) HOXB13 in prostate biology and how this role might be altered by gene mutation to promote prostate carcinogenesis, we have initiated efforts to identify and characterize protein binding partners of HOXB13 in cell line models of prostate cancer, as well as to identify genes whose expression is modulated by varying expression levels of wt and mutant HOXB13. METHODS Mutant and wt HOXB13 were expressed by transduction in LNCaP cells. Endogenous HOXB13 was knocked down by siRNA targeting the 3’ UTR of the gene. Cells were treated with 10nM R1881 for 24 hours followed by gene expression microarray analysis. Gene Set Enrichment Analysis was performed to compare gene pathways differentially regulated by HOXB13 G84E. Co-immunoprecipitation (Co-IP) was performed to detect the interactions between AR, FOXA1, PBX2, PREP1, and HOXB13. RESULTS Cluster analysis of microarray gene expression data showed that over 250 genes were differentially regulated by HOXB13 G84E, including both androgen-dependent and -independent genes. Compared to wt HOXB13, G84E significantly down-regulated steroid metabolism pathway genes and up-regulated genes involved in polyamine metabolism. Co-IP results revealed that HOXB13 interacts with AR as previously demonstrated, and we identified the pioneer transcription factor FOXA1, and HOX cofactors, PBX2 and PREP1 as novel binding partners of HOXB13, at least in androgen responsive prostate cancer cell lines. CONCLUSIONS Differential expression of genes involved in steroid and polyamine metabolizing pathways might explain the ability of HOXB13 G84E to promote prostate carcinogenesis. Co-IP experiments demonstrate novel complex formation between HOXB13 and AR, FOXA1, PBX1, and PREP1. Further work is necessary to determine if alterations in this complex formation as a result of HOXB13 mutations are responsible for the altered gene expression patterns observed. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e323-e324 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Shuangling Chen More articles by this author Dorhyun Johng More articles by this author Charles Ewing More articles by this author William Isaacs More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...