Fusion-negative Prostate Cancer Research Articles

Loss of feedback regulation between FAM3B and androgen receptor driving prostate cancer progression.

Although the fusion of the transmembrane serine protease 2 gene (TMPRSS2) with the erythroblast transformation-specific-related gene (ERG), or TMPRSS2-ERG, occurs frequently in prostate cancer, its impact on clinical outcomes remains controversial. Roughly half of TMPRSS2-ERG fusions occur through intrachromosomal deletion of interstitial genes and the remainder via insertional chromosomal rearrangements. Because prostate cancers with deletion-derived TMPRSS2-ERG fusions are more aggressive than those with insertional fusions, we investigated the impact of interstitial gene loss on prostate cancer progression. We conducted an unbiased analysis of transcriptome data from large collections of prostate cancer samples and employed diverse in vitro and in vivo models combined with genetic approaches to characterize the interstitial gene loss that imposes the most important impact on clinical outcome. This analysis identified FAM3B as the top-ranked interstitial gene whose loss is associated with a poor prognosis. The association between FAM3B loss and poor clinical outcome extended to fusion-negative prostate cancers where FAM3B downregulation occurred through epigenetic imprinting. Importantly, FAM3B loss drives disease progression in prostate cancer. FAM3B acts as an intermediator of a self-governing androgen receptor feedback loop. Specifically, androgen receptor upregulates FAM3B expression by binding to an intronic enhancer to induce an enhancer RNA and facilitate enhancer-promoter looping. FAM3B, in turn, attenuates androgen receptor signaling. Loss of FAM3B in prostate cancer, whether through the TMPRSS2-ERG translocation or epigenetic imprinting, causes an exit from this autoregulatory loop to unleash androgen receptor activity and prostate cancer progression. These findings establish FAM3B loss as a new driver of prostate cancer progression and support the utility of FAM3B loss as a biomarker to better define aggressive prostate cancer.

Upregulation of the heterogeneous nuclear ribonucleoprotein hnRNPA1 is an independent predictor of early biochemical recurrence in TMPRSS2:ERG fusion-negative prostate cancers

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a ubiquitous RNA splicing factor that is overexpressed and prognostically relevant in various human cancer types. To study the impact of hnRNPA1 expression in prostate cancer, we analyzed a tissue microarray containing 17,747 clinical prostate cancer specimens by immunohistochemistry. hnRNPA1 was expressed in normal prostate glandular cells but often overexpressed in cancer cells. hnRNPA1 immunostaining was interpretable in 14,258 cancers and considered strong in 33.4%, moderate in 45.9%, weak in 15.3%, and negative in 5.4%. Moderate to strong hnRNPA1 immunostaining was strongly linked to adverse tumor features including high classical and quantitative Gleason score, lymph node metastasis, advanced tumor stage, positive surgical margin, and early biochemical recurrence (p < 0.0001 each). The prognostic impact of hnRNPA1 immunostaining was independent of established preoperatively or postoperatively available prognostic parameters (p < 0.0001). Subset analyses revealed that all these associations were strongly driven by the fraction of cancers lacking the TMPRSS2:ERG gene fusion. Comparison with other key molecular data that were earlier obtained on the same TMA showed that hnRNPA1 overexpression was linked to high levels of androgen receptor (AR) expression (p < 0.0001) as well as presence of 9 of 11 chromosomal deletions (p < 0.05 each). A strong association between hnRNPA1 upregulation and tumor cell proliferation that was independent from the Gleason score supports a role for tumor cell aggressiveness. In conclusion, hnRNPA1 overexpression is an independent predictor of poor prognosis in ERG-negative prostate cancer. hnRNPA1 measurement, either alone or in combination, might provide prognostic information in ERG-negative prostate cancer.

Distinct transcriptional repertoire of the androgen receptor in ETS fusion-negative prostate cancer

BackgroundProstate cancer (PCa) tumors harboring translocations of ETS family genes with the androgen responsive TMPRSS2 gene (ETS+ tumors) provide a robust biomarker for detecting PCa in approximately 70% of patients. ETS+ PCa express high levels of the androgen receptor (AR), yet PCa tumors lacking ETS fusions (ETS−) also express AR and demonstrate androgen-regulated growth. In this study, we evaluate the differences in the AR-regulated transcriptomes between ETS+ and ETS− PCa tumors.Methods10,608 patient tumors from three independent PCa datasets classified as ETS+ (samples overexpressing ERG or other ETS family members) or ETS− (all other PCa) were analyzed for differential gene expression using false-discovery-rate adjusted methods and gene-set enrichment analysis (GSEA).ResultsBased on the expression of AR-dependent genes and an unsupervised Principal Component Analysis (PCA) model, AR-regulated gene expression alone was able to separate PCa samples into groups based on ETS status in all PCa databases. ETS status distinguished several differentially expressed genes in both TCGA (6.9%) and GRID (6.6%) databases, with 413 genes overlapping in both databases. Importantly, GSEA showed enrichment of distinct androgen-responsive genes in both ETS− and ETS+ tumors, and AR ChIP-seq data identified 131 direct AR-target genes that are regulated in an ETS-specific fashion. Notably, dysregulation of ETS-dependent AR-target genes within the metabolic and non-canonical WNT pathways was associated with clinical outcomes.ConclusionsETS status influences the transcriptional repertoire of the AR, and ETS− PCa tumors appear to rely on distinctly different AR-dependent transcriptional programs to drive and sustain tumorigenesis.

Systematic analysis reveals molecular characteristics of ERG-negative prostate cancer

The TMPRSS2:ERG gene fusion is the most prevalent early driver gene activation in prostate cancers of European ancestry, while the fusion frequency is much lower in Africans and Asians. The genomic characteristics and mechanisms for patients lacking ERG fusion are still unclear. In this study, we systematically compared the characteristics of gene fusions, somatic mutations, copy number alterations and gene expression signatures between 201 ERG fusion positive and 296 ERG fusion negative prostate cancer samples. Both common and group-specific genomic alterations were observed, suggesting shared and different mechanisms of carcinogenesis in prostate cancer samples with or without ERG fusion. The genomic alteration patterns detected in ERG-negative group showed similarities with 77.5% of tumor samples of African American patients. These results emphasize that genomic and gene expression features of the ERG-negative group may provide a reference for populations with lower ERG fusion frequency. While the overall expression patterns were comparable between ERG-negative and ERG-positive tumors, we found that genomic alterations could affect the same pathway through distinct genes in the same pathway in both groups of tumor types. Altogether, the genomic and molecular characteristics revealed in our study may provide new opportunities for molecular stratification of ERG-negative prostate cancers.

Abstract B016: MicroRNA drivers of TMPRSS2 fusion-negative prostate cancer in African Americans

Abstract TMPRSS2:ERG fusions are being pursued as genomic biomarkers to define disease state and predict treatment responses in prostate cancer (PCa). Successful biomarker exploitation depends on the frequency of TMPRSS2 fusions, which are very common in European American (EA) men with both high-grade PIN, and in at least 50% PCa patients. However, the incidence is far lower in men from other ethnicities, and as low as 10% among African American (AA) patients. Therefore in AA patients, TMPRSS2:ERG fusion events are unlikely to either explain the elevated incidence of PCa or be relevant as a biomarker. To seek novel biomarkers of TMPRSS2 fusion-negative PCa we have considered a role for miRNA drivers. Previously, we analyzed miRNA expression in various PCa cohorts, to identify differentially expressed miRNA in TMPRSS2 fusion-negative disease in AA men. These cohorts included PCa in AA men, PCa progression in EA men following surgery, and mining of TCGA-PRAD and MSKCC cohorts to identify TMPRSS2 fusion-negative associated miRNA. We have now complemented this by a genome-wide miRNA screen in serum samples using the NanoString platform of 97 aged matched EA and AA men with HGPIN, ~30% of whom progressed to PCa (SWOG S9917). Serum levels of 22 miRNA significantly distinguished AA men who progressed to PCa, from those whose HGPIN remained stable; these miRNA included miR-25-3p, miR-93-5p, and miR-423-5p. Combining the findings identified 38 miRNA, and we sought to identify shared and unique links between these miRNA associated with PCa progression and TMPRSS2 fusion-negative status. Although the PCa drivers in AA men remain obscured, strong links are emerging between vitamin D receptor (VDR) signaling and AA PCa. Epidemiologic studies have established links between low serum vitamin D3 levels and PCa incidence and progression in AA men. VDR signaling has also been linked to altered local immune responses in AA PCa patients. Remarkably, we found strong evidence from our studies and those of others that 29 of the 38 miRNA identified were regulated by 1,25(OH)2D3 in prostate cells, including the miR-25-miR-93-miR-106b cluster, miR-152 and miR-423-5p. As this regulation includes both direct and indirect effects, we examined how these miRNA genes, or host genes, were targeted by the vitamin D receptor (VDR). Previously, we identified VDR binding at the MCM7 host gene for the miR-25-miR-93-miR-106b cluster used ChIP-PCR in nonmalignant RWPE-1 cells. We have now mined publicly available VDR ChIP-Seq in LNCaP cells (GSE64656); these cells are TMPRSS2 fusion negative. Selecting all binding sites within +/- 7.5 kb of a TSS, we identified VDR associated with 8/29 miRNA, including NSRP1 (miR-423-5p host gene) and COPZ2 (miR-152 host gene). To measure associations with TMPRSS2 fusion-negative PCa, we identified the most commonly altered of the 38 miRNA in the TCGA-PRAD cohort. Specifically, we identified 18 miRNA altered by more than 2 Z scores in more than 25% tumors, which included miR-25-3p, miR-93-5p, miR-152, and miR-423-5p. Expression of these miRNA clustered tumors into two groups that significantly distinguished TMPRSS2 fusion status (p=0.007) and race (p=0.01). 15/18 miRNA were known to be regulated by 1,25(OH)2D3, and 5/8 were bound by VDR in LNCaP cells. Currently, we are measuring how these 18 miRNA are regulated by 1,25(OH)2D3 in AA and EA nonmalignant and malignant prostate cell models: AA-RC77T, RC43T; EA-HPr-1AR, LNCaP, VCaP (TMPRSS2 fusion positive), PC-3. Together these data suggest that a distinct PCa subtype, independent of TMPRSS2 fusion, arises more frequently in AA men due to reduced 1,25(OH)2D3 levels that disrupts VDR regulation of miRNA, including miR-25-3p, miR-93, miR-152, and miR-423-5p. Understanding the miRNA regulated gene networks has the potential to illuminate the cancer drivers in TMPRSS2 fusion-negative AA PCa, and expression of these miRNA may predict progression risks. Citation Format: Manjunath Siddappa, Jason White, Honghe Wang, Lara E. Sucheston-Campbell, Clayton Yates, Moray J. Campbell. MicroRNA drivers of TMPRSS2 fusion-negative prostate cancer in African Americans [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B016.

Abstract 4332: Meta-analysis of molecular features associated with aggressive prostate cancer

Abstract The TMPRSS2:ERG fusion is the most common genomic alteration in prostate cancer, occurring in 40-50% of primary tumors. The frequency of the ERG fusion remains between 40-50% in metastatic tumors, supporting previous findings that the fusion can drive tumor development, but is not sufficient to drive aggressive, lethal disease. Large -omic datasets collected over thousands of patient tumors, such as The Cancer Genome Atlas (TCGA), paired with outcomes present the raw data needed to associate genetic aberrations with markers of aggressive disease. Accordingly, we performed a meta-analysis of over 2,000 patient samples to identify combinations of -omic features associated with aggressive ERG fusion-positive and ERG fusion-negative prostate cancer. The data were collected from 8 independent, publicly available prostate cancer cohorts and cover range of disease phenotypes (e.g., neuroendocrine, castration resistance). Our analysis consisted of training univariate Cox regression models separately for single genomic or pairs of genomic features within each independent dataset. Based on the availability, we explored gene expression, mutation, and copy number alterations, all used to build separate models. We then combined models for a genomic feature across all datasets by weighting individual coefficients by the inverse of their squared error in a fixed effects model; final results are reported as corrected p-values. We compared and contrasted results from combined models within data type (i.e., only gene expression) to models integrating multiple data types. Interestingly, we find that the genomic features associated with aggressive disease differed based on ERG status. We report genomic loci that are altered in all patients, but associate with aggressive disease only in patients with an ERG fusion. We also report loci that co-occur or are mutually exclusive of the ERG fusion and associated with aggressiveness. For example, SPOP mutations are known to be mutually exclusive of ERG fusions. We report a finer stratification of patients with SPOP mutations based on biochemical relapse. Our results present a compressive meta-analysis of molecular features in prostate cancer and identify novel molecular subtypes associated with aggressive disease. Citation Format: James C. Costello, Rani Powers, Andrew Goodspeed, Scott D. Cramer. Meta-analysis of molecular features associated with aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4332.

Abstract B72: DNA methylation and genetic alterations contribute to aggressive prostate cancer in African American men

Abstract Background: African-American (AA) men are more often diagnosed with early-onset aggressive prostate cancer than European American (EA) men. Prostate cancer health disparities between AA and EA men are attributed to socioeconomic as well as biologic differences existing between the two groups. The purpose of this study is to investigate how genetic and epigenetic alterations, specifically DNA methylation, contribute to early-onset aggressive prostate cancer in AA men. Methods: We performed 450K methylation array and RNA-sequencing on radical prostatectomy specimens from AA (n=32) and EA (n=5) men treated at Roswell Park Cancer Institute (RPCI). To increase analytical power, we included AA (n=22) and EA (n=172) patients from The Cancer Genome Atlas (TCGA) database. We found no significant difference in the average age between EA and AA men in the RPCI cohort, but the TCGA cohort consisted of AA men who had significantly lower average and median age compared to EA men (57 and 56 vs 61 and 62; p=0.019). We assessed baseline androgen receptor (AR) protein levels in matched benign and malignant AA (n=95) and EA (n=93) radical prostatectomy tissues from RPCI using immunohistochemistry. Gleason score &amp;lt;3+4 and &amp;gt;4+3 were classified as low and high aggressive tumors, respectively. Results: Unsupervised hierarchical clustering of DNA methylation levels in prostate cancers revealed 9 clusters. We focused on the 2 largest clusters, Cluster 1 (n=133) and Cluster 3 (n=73). Cluster 1 consisted predominantly of low aggressive disease (p=0.00002) with lower serum prostate specific antigen (PSA) values (8.97 vs 13.35 ng/ml) compared to Cluster 3. Following this trend, AA patients, but not EA patients, in Cluster 1 had better overall (57 vs 50 months, p=0.48) and disease-free time (47 vs 22 months, p=0.01) as compared to Cluster 3. ERG (ETS-related gene) fusion-positive prostate cancer is believed to be more aggressive than fusion-negative prostate cancer due to increased ERG gene transcription. We did not observe a difference in fusion status between the two DNA methylation clusters. However, overall DNA methylation was higher in fusion-negative samples as compared to fusion-positive samples in the TCGA dataset. ERG is known to bind to and reduce PSA, a direct downstream AR target. In the TCGA dataset, AA fusion-negative patients had higher average and median preoperative PSA values (18.38, 20.15 ng/ml) as compared with AA fusion-positive (7.51, 6.70 ng/ml), EA fusion-positive (9.43, 7.00 ng/ml), and EA fusion-negative patients (11.47, 7.50 ng/ml). There was no significant difference in the AR transcript and AR protein levels between fusion-positive and -negative AA and EA men. AR protein expression in RPCI cohort tissue microarrays showed that adjacent nontumor tissues from AA men had higher percent AR positive nuclei (p&amp;lt;0.05) compared to adjacent nontumor tissues from EA men. Conclusions: Analysis of the RPCI and TCGA cohorts indicated that DNA methylation separated low and high aggressive prostate cancer in AA men. Further analysis will include more patients as well as identify differences in specific CpG loci between Cluster 1 and Cluster 3. Our results suggest that DNA methylation alterations can be important in patients with ERG fusion-negative disease since these prostate cancers exhibit higher levels of overall DNA methylation. Lastly, our observations of higher AR expression in adjacent nontumor tissues suggest that there are underlying genetic variations that contribute to early-onset high aggressive prostate cancer in AA men compared to EA men. Citation Format: Swathi Ramakrishnan, Xuan Peng, Qianya Qi, Qiang Hu, Gissou Azabdaftari, Elena Pop, James Mohler, Kristopher Attwood, Li Yan, Jianmin Wang, Anna Woloszynska-Read. DNA methylation and genetic alterations contribute to aggressive prostate cancer in African American men [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B72.

The untold stories of the speech gene, the FOXP2 cancer gene.

FOXP2 encodes a transcription factor involved in speech and language acquisition. Growing evidence now suggests that dysregulated FOXP2 activity may also be instrumental in human oncogenesis, along the lines of other cardinal developmental transcription factors such as DLX5 and DLX6 [1–4].Several FOXP familymembers are directly involved during cancer initiation, maintenance and progression in the adult [5–8]. This may comprise either a pro-oncogenic activity or a deficient tumor-suppressor role, depending upon cell types and associated signaling pathways. While FOXP2 is expressed in numerous cell types, its expression has been found to be down-regulated in breast cancer [9], hepatocellular carcinoma [8] and gastric cancer biopsies [10]. Conversely, overexpressed FOXP2 has been reported in multiple myelomas, MGUS (Monoclonal Gammopathy of Undetermined Significance), several subtypes of lymphomas [5,11], as well as in neuroblastomas [12] and ERG fusion-negative prostate cancers [13]. According to functional evidences reported in breast cancer [9] and survey of recent transcriptomic and proteomic analyses of different tumor biopsies, we postulate that FOXP2 dysregulation may play a main role throughout cancer initiation and progression. In some cancer conditions, FOXP2 levels are now considered as a critical diagnostic marker of neoplastic cells, and in many situations, they even bear strong prognostic value [5]. Whether FOXP2 may further become a therapeutic target is an actively explored lead. Knowledge reviewed here may help improve our understanding of FOXP2 roles during oncogenesis and provide cues for diagnostic, prognostic and therapeutic analyses.

FABP5 RNA expression as potential marker in TMPRSS2:ERG fusion negative prostate cancer

FABP5 RNA expression as potential marker in TMPRSS2:ERG fusion negative prostate cancer

Circ-SHKBP1 Regulates the Angiogenesis of U87 Glioma-Exposed Endothelial Cells through miR-544a/FOXP1 and miR-379/FOXP2 Pathways.

Circular RNAs (circRNAs) are a type of endogenous non-coding RNAs, which have been considered to mediate diverse tumorigenesis including angiogenesis. The present study aims to elucidate the potential role and molecular mechanism of circ-SHKBP1 in regulating the angiogenesis of U87 glioma-exposed endothelial cells (GECs). The expression of circ-SHKBP1, but not linear SHKBP1, was significantly upregulated in GECs compared with astrocyte-exposed endothelial cells (AECs). circ-SHKBP1 knockdown inhibited the viability, migration, and tube formation of GECs dramatically. The expressions of miR-379/miR-544a were downregulated in GECs, and circ-SHKBP1 functionally targeted miR-544a/miR-379 in an RNA-induced silencing complex (RISC) manner. Dual-luciferase reporter assay demonstrated that forkhead box P1/P2 (FOXP1/FOXP2) were targets of miR-544a/miR-379. The expressions of FOXP1/FOXP2 were upregulated in GECs, and silencing of FOXP1/FOXP2 inhibited the viability, migration, and tube formation of GECs. Meanwhile, FOXP1/FOXP2 promoted angiogenic factor with G patch and FHA domains 1 (AGGF1) expression at the transcriptional level. Furthermore, knockdown of AGGF1 suppressed the viability, migration, and tube formation of GECs via phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK)1/2 pathways. Taken together, the present study demonstrated that circ-SHKBP1 regulated the angiogenesis of GECs through miR-544a/FOXP1 and miR-379/FOXP2 pathways, and these findings might provide a potential target and effective strategy for combined therapy of gliomas.

Abstract 5883: CHD1 as regulator of cell adhesion in Ets fusion negative prostate cancer

Abstract Prostate cancer (PCa) develops into lethal disease in about 10% of men diagnosed with this malignancy. Common genomic changes are seen in PCa such as alterations in CDKN1B, RB1, TP53, PTEN, NKX3-1, MYC and androgen receptor. Oncogenic fusions involving the Ets family are seen in 50-70% PCa patients, the most common of which is TMPRSS2-ERG. TMPRSS2-ERG influences cell migration, particularly when PTEN is co-deleted or PIK3CA activating mutations are present. However, in the absence of ERG fusions, molecular drivers of the PCa phenotype are less clear but include alterations in SPOP (6-15% of PCa), SPINK1 (10%), MAP3K7 (18-38%) and CHD1 (15-27%). CHD1 (Chromo-domain Helicase DNA Binding Protein-1) is a chromatin remodeling factor with many roles including homologous recombination mediated DNA repair of double strand breaks and maintenance of genomic stability and therefore it is hypothesized to cause the predominant intrachromosomal rearrangements observed in Ets fusion negative tumors. This study was undertaken to evaluate the cellular effects that follow CHD1 deletion that could aid PCa initiation and progression. CHD1 was knocked out in the non tumorigenic, HPV16 immortalised prostate epithelial cell line, RWPE-1, using CRISPR/Cas9. Compared to parental RWPE-1 and non target cells (NT2), the CHD1 KO cells had a smaller and rounder morphology and were less adherent to tissue culture plates. To study expression of extracellular matrix and adhesion molecules, RT profiler assay was performed using RWPE-1 parental and CHD1 KO cells. CHD1 KO cells showed a decrease in the expression of SPARC, MMP2, ITGA2, ITGA5, ITGA6, FN1, LAMB3, collagen, tenascin and vitronectin as compared to parentals and NT2. Reduction in extracellular matrix and adhesion molecules and altered morphology of CHD1 KO cells as compared to parentals and NT2 cells suggest that loss of CHD1 reduces cell-cell and cell-matrix adhesion. These results suggest that in the absence of PTEN loss in Ets negative tumors, deletion of CHD1 gene could change cell adhesion dynamics ascribing a new role for CHD1 in the development and progression of PCa. Citation Format: Aparna Kareddula, Whitney Petrosky, Irina Tereshchenko, Daniel Medina, Hana Aviv, Eric Singer, Robert S. DiPaola, Kim M. Hirshfield. CHD1 as regulator of cell adhesion in Ets fusion negative prostate cancer [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 5883. doi:10.1158/1538-7445.AM2017-5883

Abstract 5317: Association of physical activity with risk of prostate cancer defined by TMPRSS2:ERG

Abstract Background There is growing evidence of heterogeneity in risk factor associations for prostate cancer defined by clinical and molecular characteristics. Physical activity has moderate protective effects on development of prostate cancer, in particular of advanced and fatal disease. We hypothesized that the influence of physical activity on hormonal and anti-inflammatory pathways may differentially affect the development of TMPRSS2:ERG-positive compared to TMPRSS2:ERG-negative tumors. Our objective was to examine the association between physical activity and risk of prostate cancer defined by the TMPRSS2:ERG fusion. Materials &amp; Methods We followed 49,160 men in the prospective Health Professionals Follow-up Study from 1986 to 2010. Participants were age 40 years or older, had no previous cancer diagnosis, and reported physical activity by questionnaire. Men self-reported the average number of hours per week spent engaged in various forms of recreational physical activity at baseline and every two years thereafter with greater than 90% follow-up rates. We examined long-term physical activity type and intensity in units of metabolic equivalent of task (MET)-hours per week. Presence of the TMPRSS2:ERG fusion in tumor tissue was estimated by ERG protein expression using immunohistochemistry. We used competing risks models to obtain subtype-specific hazard ratios (HRs) and 95% confidence intervals (CIs) for incidence of fusion-positive and fusion-negative prostate cancer. Results During 23 years of follow-up, 5,819 men were diagnosed with prostate cancer. Of the 2,389 men who underwent radical prostatectomy, 910 were assayed for ERG. Men with higher cumulative average vigorous activity had a lower risk of developing TMPRSS2:ERG-positive prostate cancer (top vs. bottom quintile multivariate HR: 0.64, 95% CI: 0.46-0.90, P-value for trend=0.01). There was no significant association observed between vigorous activity and risk of TMPRSS2:ERG-negative prostate cancer (HR: 0.93, 95% CI: 0.67-1.28, P-value for trend=0.46). However, the test for heterogeneity in these associations was not significant (P-value for heterogeneity=0.55). No association was observed between total activity and risk of either TMPRSS2:ERG subtype. Conclusions Physical activity of vigorous intensity is inversely associated with risk of TMPRSS2:ERG-positive but not TMPRSS2:ERG-negative prostate cancer. These findings suggest that regular vigorous activity may lower risk of prostate cancer through mechanisms specific to development of this fusion in prostate tumors. Future studies are needed to further elucidate these mechanisms and translate these findings into recommendations for prostate cancer prevention. Citation Format: Claire H. Pernar, Ericka M. Ebot, Andreas Pettersson, Rebecca E. Graff, Thomas U. Ahearn, Amparo G. Gonzalez-Feliciano, Sarah C. Markt, Kathryn M. Wilson, Stephen Finn, Michelangelo Fiorentino, Edward L. Giovannucci, Lorelei A. Mucci. Association of physical activity with risk of prostate cancer defined by TMPRSS2:ERG [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 5317. doi:10.1158/1538-7445.AM2017-5317

Prostate cancer risk regions at 8q24 and 17q24 are differentially associated with somatic TMPRSS2:ERG fusion status.

Molecular and epidemiological differences have been described between TMPRSS2:ERG fusion-positive and fusion-negative prostate cancer (PrCa). Assuming two molecularly distinct subtypes, we have examined 27 common PrCa risk variants, previously identified in genome-wide association studies, for subtype specific associations in a total of 1221 TMPRSS2:ERG phenotyped PrCa cases. In meta-analyses of a discovery set of 552 cases with TMPRSS2:ERG data and 7650 unaffected men from five centers we have found support for the hypothesis that several common risk variants are associated with one particular subtype rather than with PrCa in general. Risk variants were analyzed in case-case comparisons (296 TMPRSS2:ERG fusion-positive versus 256 fusion-negative cases) and an independent set of 669 cases with TMPRSS2:ERG data was established to replicate the top five candidates. Significant differences (P < 0.00185) between the two subtypes were observed for rs16901979 (8q24) and rs1859962 (17q24), which were enriched in TMPRSS2:ERG fusion-negative (OR = 0.53, P = 0.0007) and TMPRSS2:ERG fusion-positive PrCa (OR = 1.30, P = 0.0016), respectively. Expression quantitative trait locus analysis was performed to investigate mechanistic links between risk variants, fusion status and target gene mRNA levels. For rs1859962 at 17q24, genotype dependent expression was observed for the candidate target gene SOX9 in TMPRSS2:ERG fusion-positive PrCa, which was not evident in TMPRSS2:ERG negative tumors. The present study established evidence for the first two common PrCa risk variants differentially associated with TMPRSS2:ERG fusion status. TMPRSS2:ERG phenotyping of larger studies is required to determine comprehensive sets of variants with subtype-specific roles in PrCa.

Abstract 1763: Body size and incidence of TMPRSS2:ERG fusion-positive and fusion-negative prostate cancer

Abstract Background: Evidence suggests that genetic, lifestyle, and hormonal factors are differentially associated with the incidence of TMPRSS2:ERG fusion-positive and fusion-negative tumors, suggesting that these molecular subtypes have distinct etiologies. There is scant literature on whether anthropometrics are differentially associated the risk of developing fusion-positive and fusion-negative prostate cancer. Methods: We examined the associations between adult height, recalled BMI at age 21, and updating BMI over time with risk of prostate cancer defined by TMPRSS2:ERG subtype using a prospective cohort of 49,372 men from the Health Professionals Follow-up Study. Tumor TMPRSS2:ERG status was assessed by ERG immunohistochemistry on tissue microarrays constructed from radical prostatectomy of the prostate specimens. We utilized multivariable competing risks models to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for risk of fusion-positive disease and, separately, fusion-negative disease. Results: During 23 years of follow-up, we identified 5,847 total prostate cancer cases. Among them, 2,402 were treated with RP, of whom 913 (15.6% of all cases) were assayed for ERG status. Height (per 5 inches) was positively associated with the risk of developing ERG-positive prostate cancer (HR = 1.24, 95% CI 1.03-1.50), but not associated with ERG-negative disease (HR = 0.98, 95% CI = 0.82-1.18, Pheterogenity = 0.07). Updated BMI (per 5 kg/m2) was inversely associated with the risk of developing ERG-positive disease (HR = 0.86, 95% CI = 0.74-1.00), but not associated with ERG-negative disease (HR = 1.03, 95% CI = 0.90-1.18, Pheterogenity = 0.07). BMI at age 21 was not differentially associated with the risk of developing ERG-positive or ERG-negative disease. Conclusion: Our results suggest that height and obesity are differentially associated with the risk of developing fusion-positive and fusion-negative prostate cancer. Citation Format: Thomas Ahearn, Rebecca E. Graff, Andreas Pettersson, Claire Pernar, Sarah C. Markt, Kathryn M. Wilson, Michelangelo Fiorentino, Massimo Loda, Edward L. Giovannucci, Lorelei A. Mucci. Body size and incidence of TMPRSS2:ERG fusion-positive and fusion-negative prostate cancer. [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 1763.

Serine protease inhibitor Kazal type 1 promotes epithelial-mesenchymal transition through EGFR signaling pathway in prostate cancer

Overexpression of serine protease inhibitor Kazal type 1 (SPINK1) defines an aggressive molecular subtype of ETS fusion-negative prostate cancer (PCa) patients in western countries. However, how SPINK1 contributes to PCa invasion and metastasis is largely unknown. Fluorescence in situ hybridization and immunohistochemistry were utilized to detect ERG rearrangement, SPINK1 expression, and EGFR aberrations in a cohort of 211 PCa patients with radical prostatectomy. Real-time quantitative PCR and Western blotting were used to study the transcript and protein expression levels. Cellular distribution of E-cadherin and vimentin were observed by immunofluorescence. Cellular function was evaluated by siRNA, transwell, and wound healing assay, respectively. SPINK1-induced Epithelial-mesenchymal transition (EMT) in benign prostate RWPE cells, manifested by acquisition of mesenchymal morphology, alternation of EMT markers as well as migration and invasion capabilities. Knockdown of SPINK1 in 22RV1 PCa cells results in up-regulation of E-cadherin and down-regulation of vimentin. SPINK1-induced EMT is mediated by EGFR, in which MAPK/MEK/ERK pathway is mainly involved. Connective tissue growth factor (CTGF) might be an important down-stream molecule of SPINK1-EGFR axis. Clinically, SPINK1 and EGFR were significantly co-overexpressed in a cohort of Chinese PCa patients (n > 200). SPINK1 is an unfavorable prognostic factor in Chinese PCas (P = 0.025). These findings suggest that SPINK1 promotes EMT through EGFR signaling pathway in PCa and SPINK1 could be a new prognostic marker in Chinese PCas.

Roles of Serine Protease Inhibitor Kazal type 1 (SPINK1) in Prostate Cancer

Altered genes that play a driving role in cancer development can often serve as specific diagnostic markers, criteria of molecular classification and therefore potential therapeutic targets. Serine protease inhibitor Kazal type 1 (SPINK1), also known as pancreatic secretory trypsin inhibitor or tumor-associated trypsin inhibitor, encodes a 56 amino acid secreted peptide, and its normal function is thought to be the inhibition of serine proteases such as trypsin. Recent studies have indicated marked overexpression of SPINK1 defines an aggressive molecular subtype of ETS (erythroblastosis virus E26 transformation-specific) fusion-negative prostate cancer ((PCa) patients. SPINK1 may act as an autocrine growth factor and promotes PCa growth and invasion. Most recently, we suggested that SPINK1 induces epithelial-mesenchymal transition (EMT) through EGFR signaling pathway in PCa. The association between SPINK1 overexpression and poor prognosis in PCa has been reported. Notably, SPINK1 might be a novel extracellular therapeutic target in a subset of high-grade PCa patients. In this review, we will summarize the current understanding of SPINK1 involving its role in PCa biology, association with prognosis as well as perspective in therapy from the pathologist's point of view.

Loss of CDKN1B/p27Kip1 expression is associated with ERG fusion-negative prostate cancer, but is unrelated to patient prognosis

The cyclin-dependent kinase inhibitor p27Kip1 has been suggested as a prognostic marker in prostate cancer. The aim of this study was to determine the clinical and prognostic role of p27 expression in hormone-naive prostate cancers. A tissue microarray containing samples from 4,699 prostate cancers with attached pathological, clinical follow-up and molecular data was analyzed for nuclear p27 expression by immunohistochemistry. p27 staining was negative in 18.6%, weak in 33.5%, moderate in 28.4% and strong in 19.5% of 3,701 interpretable cancer spots. Loss of p27 immunostaining was linked to tumors of low Gleason grade (P<0.0001) and ERG fusion-negative cancers (P<0.0001). p27 levels were not associated with other parameters, including tumor stage, nodal stage, preoperative prostate-specific antigen (PSA) levels, surgical margin status and cell proliferation (as measured by the Ki67 labeling index). p27 expression was also unrelated to clinical outcome in all cancers, as well as in the subsets of ERG fusion-positive and -negative cancers. Overall, the present data demonstrated that elevated p27 expression was often unrelated to prostate cancer phenotype. Furthermore, the lack of an effect of the p27 protein levels on PSA recurrence following radical prostatectomy indicated that factors other than p27 expression are likely to be the major determinants of prostate cancer recurrence. However, a subset of ERG-negative, low-grade tumors was frequently characterized by loss of p27, suggesting a role of this alteration for the development of these tumors.

Abstract A18: Pre-diagnostic serum antioxidant levels and prediction of TMPRSS2:ERG fusion status in prostate cancer

Abstract Background: The TMPRSS2:ERG gene fusion is a distinct molecular subtype of prostate cancer characterized by chromosomal rearrangements joining the androgen responsive TMPRSS gene with ERG, an oncogenic member of the ETS gene family. TMPRSS2:ERG is thought to be an early pathologic event occurring in 50% of prostate tumors and resulting in more than 100,000 fusion positive cancer cases each year in the US. No studies to date have examined underlying risk factors associated with development of the fusion. Oxidative stress and the production of free radicals may result in chromosomal instability and predispose to the formation of TMPRSS2:ERG positive prostate cancer. Higher levels of serum antioxidants prior to prostate cancer diagnosis may therefore be protective against later development of fusion positive cancer. Methods: We undertook a case-control analysis nested within the prospective Physicians' Health Study and the Health Professionals Follow-up Study among men with prostate cancer (n=331) and age-matched controls (n=2139). The cohorts include pre-diagnostic bloods with antioxidant levels at baseline as well as tumor tissue to characterize TMPRSS2:ERG status among prostate cancer cases. We used multivariate logistic regression analysis to determine if plasma antioxidant levels influenced the development of the TMPRSS2:ERG fusion. Results: Of seven antioxidants examined (alpha-tocopherol, alpha-carotene, beta-carotene, beta-cryptoxanthin, gamma-tocopherol, lutein, and lycopene), levels of beta-carotene above the lowest quartile were protective against fusion positive vs. fusion negative prostate cancer (OR 0.49, 95% CL 0.28-0.86, p = 0.01). This association was driven by a non-significantly decreased risk of fusion positive cancer vs. controls (OR 0.75, 95% CL 0.51-1.09, p = 0.13) and a significantly increased risk of fusion negative cancer vs. controls (OR 1.58, 95% CL 1.03-2.41, p = 0.03). None of the six other antioxidants were associated with fusion status, and none of the antioxidants, including beta-carotene, predicted overall prostate cancer risk. Discussion: Previous studies examining antioxidant status and overall prostate cancer risk have been inconclusive. This is the first study to examine pre-diagnostic serum antioxidant levels in relationship to fusion status. We find that beta-carotene levels may alter prostate cancer risk based on molecular subtype. Citation Format: Gregory Judson, Andreas Pettersson, Allison Meisner, Lorelei Mucci. Prediagnostic serum antioxidant levels and prediction of TMPRSS2:ERG fusion status in prostate cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A18.

Distinct Epigenetic Mechanisms DistinguishTMPRSS2–ERG Fusion-Positive and -Negative Prostate Cancers

This issue of Cancer Discovery features an article that describes distinct epigenetic mechanisms that operate in TMPRSS2-ERG fusion-negative prostate cancers. This finding clarifies molecular features of these TMPRSS2-ERG fusion-negative tumors and may have implications for how to treat this prostate cancer subtype.

Genome-wide DNA Methylation Events in TMPRSS2–ERG Fusion-Negative Prostate Cancers Implicate an EZH2-Dependent Mechanism with miR-26a Hypermethylation

Prostate cancer is the second most common cancer among men worldwide. Alterations in the DNA methylation pattern can be one of the leading causes for prostate cancer formation. This study is the first high-throughput sequencing study investigating genome-wide DNA methylation patterns in a large cohort of 51 tumor and 53 benign prostate samples using methylated DNA immunoprecipitation sequencing. Comparative analyses identified more than 147,000 cancer-associated epigenetic alterations. In addition, global methylation patterns show significant differences based on the TMPRSS2-ERG rearrangement status. We propose the hypermethylation of miR-26a as an alternative pathway of ERG rearrangement-independent EZH2 activation. The observed increase in differential methylation events in fusion-negative tumors can explain the tumorigenic process in the absence of genomic rearrangements. In contrast to TMPRSS2-ERG -rearranged tumors, the pathomechanism for gene fusion-negative tumors is completely unclear. Using a sequencing-based approach, our work uncovers significant global epigenetic alterations in TMPRSS2-ERG gene fusion-negative tumors and provides a mechanistic explanation for the tumor formation process.