Background ERG rearrangements and PTEN loss are two of the most common genetic alterations in prostate cancer. However, there is still significant controversy regarding the order of events of these two changes during the carcinogenic process. We used IHC to determine ERG and PTEN status and calculated the fraction of cases with homogeneous/heterogeneous ERG and PTEN staining in a given tumor. Methods and Results Using a single standard tissue section from the index tumor from radical prostatectomies (N= 77), enriched for relatively high grade and stage tumors, we examined ERG and PTEN status by IHC. We determined whether ERG or PTEN staining was homogeneous (all tumor cells staining positive) or heterogeneous (focal tumor cell staining) in a given tumor focus. 57% (N=44/77) of tumor foci showed ERG positivity, with 93% of these (N=41/44) cases showing homogeneous ERG staining in which all tumor cells stained positively. 53% (N=41/77) of tumor foci showed PTEN loss, and of these, 66% (N=27/41) showed heterogeneous PTEN loss. In ERG homogeneously positive cases, any PTEN loss occurred in 56 % (N=23/41) of cases, and of these, 65% (N=15/23) showed heterogeneous loss. In ERG negative tumors, 51.5% (N=17/33) showed PTEN loss, and of these, 64.7% (N=11/17) showed heterogeneous PTEN loss. In a subset of cases, genomic deletions of PTEN were verified by FISH in regions with PTEN protein loss as compared to regions with intact PTEN protein, which did not show PTEN genomic loss. Conclusions These results support the concept that PTEN loss tends to occur as a subclonal event within a given established prostatic carcinoma clone after ERG gene fusion. The combination of ERG and PTEN IHC staining can be used as a simple test to ascertain PTEN and ERG gene rearrangement status within a given prostate cancer in either a research or clinical setting.

Purpose Small cell neuroendocrine differentiation in prostatic carcinoma is an increasingly common resistance mechanism to potent androgen deprivation therapy (ADT), but can be difficult to identify morphologically. We investigated whether cyclin D1 and p16 expression can inform on Rb functional status and distinguish small cell carcinoma from adenocarcinoma. Experimental Design We used gene expression data and immunohistochemistry to examine cyclin D1 and p16 levels in patient-derived xenografts (PDX), and prostatic small cell carcinoma and adenocarcinoma specimens. Results Using PDX, we show proof-of-concept that a high ratio of p16 to cyclin D1 gene expression reflects underlying Rb functional loss and distinguishes morphologically identified small cell carcinoma from prostatic adenocarcinoma in patient specimens (n=13 and 9, respectively). At the protein level cyclin D1, but not p16, was useful to distinguish small cell carcinoma from adenocarcinoma. Overall, 88% (36/41) of small cell carcinomas showed cyclin D1 loss by immunostaining compared to 2% (2/94) of Gleason score 7–10 primary adenocarcinomas at radical prostatectomy, 9% (4/44) of Gleason score 9–10 primary adenocarcinomas at needle biopsy, and 7% (8/115) of individual metastases from 39 patients at autopsy. Though rare adenocarcinomas showed cyclin D1 loss, many of these were associated with clinical features of small cell carcinoma, and in a cohort of men treated with adjuvant ADT who developed metastasis, lower cyclin D1 gene expression was associated with more rapid onset of metastasis and death. Conclusions Cyclin D1 loss identifies prostate tumors with small cell differentiation and may identify a small subset of adenocarcinomas with poor prognosis.

Consumption of tomato products containing the carotenoid lycopene is associated with a reduced risk of prostate cancer. To identify gene expression patterns associated with early testosterone-driven prostate carcinogenesis, which are impacted by dietary tomato and lycopene, wild type (WT) and transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were fed control or tomato- or lycopene-containing diets from 4-10 wk-of-age. Eight-week-old mice underwent sham surgery, castration, or castration followed by testosterone-repletion (2.5 mg/kg/d initiated 1 wk after castration). Ten-wk-old intact TRAMP mice exhibit early multifocal prostatic intraepithelial neoplasia (PIN). Of the 200 prostate cancer-related genes measured by quantitative NanoString®, 189 are detectable, 164 significantly differ by genotype, 179 by testosterone status, and 30 by diet type (P<0.05). In TRAMP, expression of Birc5, Mki67, Aurkb, Ccnb2, Foxm1, and Ccne2 is greater compared to WT and is decreased by castration. In parallel, castration reduces Ki67-positive staining (P<0.0001) compared to intact and testosterone-repleted TRAMP mice. Expression of genes involved in androgen metabolism/signaling pathways are reduced by lycopene feeding (Srd5a1) and by tomato-feeding (Srd5a2, Pxn, and Srebf1). Additionally, tomato-feeding significantly reduced expression of genes associated with stem cell features, Aldh1a and Ly6a, while lycopene-feeding significantly reduced expression of neuroendocrine differentiation-related genes, Ngfr and Syp. Collectively, these studies demonstrate a profile of testosterone-regulated genes associated with early stages of prostate carcinogenesis that are potential mechanistic targets of dietary tomato components. Future studies on androgen signaling/metabolism, stem cell features, and neuroendocrine differentiation pathways may elucidate the mechanisms by which dietary tomato and lycopene impact prostate cancer risk.

There is considerable evidence supporting the hypothesis that tomato carotenoids may contribute to prostate cancer prevention however the mechanisms remain uncertain. We propose that bioactive tomato components, such as lycopene, may impact testosterone signaling to inhibit prostate carcinogenesis. Four week-old C57/BL6 WT and TRAMP male mice were fed either control (AIN93G), 10% tomato powder or 0.25% lycopene beadlets. At eight weeks of age, mice were randomized among treatments: intact, castration, or castration + 2.5mg/kg testosterone repletion and animals sacrificed at 10 weeks of age. Prostate gene expression was quantified by Nanostring nCounter was used to quantify prostate gene expression with a 200 gene murine prostate carcinogenesis custom codeset and miRNA with codeset v1.1. Carotenoid analysis is by HPLC and proliferation (Ki67) by immunohistochemical evaluation. Feeding dietary tomato or lycopene increased serum lycopene, for example, in WT mice concentrations reached 335 +/- 33 nmol/L and 337 +/- 34 nmol/L in tomato and lycopene fed animals, respectively. As expected, the TRAMP genotype and testosterone stimulate proliferation with diet as a modulating factor. In the WT intact prostate, the tomato powder and lycopene diet decreased proliferation (p&lt;0.009). The TRAMP genotype and endocrine status impacted the expression of multiple genes and miRNAs in several distinct patterns. For example, Birc5, Ki67, PCNA, BRCA1, ATM, Aurora kinase A / B, Cdc25c, Cyclin B2, cyclin E2, Fox M1,and miRs 15b, 16, 25, 200a, 200b, and 429 are increased in the TRAMP prostate, only when testosterone is present. Alternatively, TMPRSS2, CD31, CD34, Cox 2, nCad, Cyclin D1, Keratin 5, Shh, and p63, and miRs 145, 205, 328, and 423-5p are decreased in the TRAMP prostate in the presence of testosterone. Nkx3.1, miR 148a, and miR 375 are decreased upon castration whereas beta catenin, FGFR1 and TNF alpha are increased. Tomato and / or Lycopene modestly decreased expression of eCad, HIF 1 alpha, Paxillin, RxR alpha, Sca 1 and Vimentin and increased expression of CD133 and miR 22. These highly controlled in vivo studies document the strong interactions between genetic defects in the prostate, the hormonal environment, and diet in modulating early prostate carcinogenesis. Citation Format: Jennifer M. Thomas-Ahner, Lei Wan, Hsueh-Li Tan, Nancy E. Moran, Amy C. Elsen, Dennis K. Pearl, John W. Erdman, Steven K. Clinton. Tomato carotenoids and testosterone modulate mRNA and miRNA profiles during prostate carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3701. doi:10.1158/1538-7445.AM2013-3701