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Background Resistance to androgen deprivation therapies is a major driver of mortality in advanced prostate cancer. Therefore, there is a need to develop new preclinical models that allow the investigation of resistance mechanisms and the assessment of drugs for the treatment of castration-resistant prostate cancer. Methods We generated two novel cell line models (LAPC4-CR and VCaP-CR) which were derived by passaging LAPC4 and VCaP cells in vivo and in vitro under castrate conditions. We performed detailed transcriptomic (RNA-seq) and proteomic analyses (SWATH-MS) to delineate expression differences between castration-sensitive and castration-resistant cell lines. Furthermore, we characterized the in vivo and in vitro growth characteristics of these novel cell line models. Results The two cell line derivatives LAPC4-CR and VCaP-CR showed castration-resistant growth in vitro and in vivo which was only minimally inhibited by AR antagonists, enzalutamide, and bicalutamide. High-dose androgen treatment resulted in significant growth arrest of VCaP-CR but not in LAPC4-CR cells. Both cell lines maintained AR expression, but exhibited distinct expression changes on the mRNA and protein level. Integrated analyses including data from LNCaP and the previously described castration-resistant LNCaP-abl cells revealed an expression signature of castration resistance. Conclusions The two novel cell line models LAPC4-CR and VCaP-CR and their comprehensive characterization on the RNA and protein level represent important resources to study the molecular mechanisms of castration resistance. ISSN:0270-4137 ISSN:1097-0045

Antibodies targeting the programmed cell death protein 1/programmed death-ligand 1 (PD-L1) interaction have shown clinical activity in multiple cancer types. PD-L1 protein expression is a clinically validated predictive biomarker of response for such therapies. Prior studies evaluating the expression of PD-L1 in primary prostate cancers have reported highly variable rates of PD-L1 positivity. In addition, limited data exist on PD-L1 expression in metastatic castrate-resistant prostate cancer (mCRPC). Here, we determined PD-L1 protein expression by immunohistochemistry using a validated PD-L1-specific antibody (SP263) in a large and representative cohort of primary prostate cancers and prostate cancer metastases. The study included 539 primary prostate cancers comprising 508 acinar adenocarcinomas, 24 prostatic duct adenocarcinomas, 7 small-cell carcinomas, and a total of 57 cases of mCRPC. PD-L1 positivity was low in primary acinar adenocarcinoma, with only 7.7% of cases showing detectable PD-L1 staining. Increased levels of PD-L1 expression were noted in 42.9% of small-cell carcinomas. In mCRPC, 31.6% of cases showed PD-L1-specific immunoreactivity. In conclusion, in this comprehensive evaluation of PD-L1 expression in prostate cancer, PD-L1 expression is rare in primary prostate cancers, but increased rates of PD-L1 positivity were observed in mCRPC. These results will be important for the future clinical development of programmed cell death protein 1/PD-L1-targeting therapies in prostate cancer.

Background Loss or mutation of PTEN alleles at 10q23 in combination with 8q24 amplification (encompassing MYC) are common findings in aggressive, human prostate cancer. Our group recently developed a transgenic murine model of prostate cancer involving prostate-specific Pten deletion and forced expression of MYC under the control of the Hoxb13 promoter. MYC overexpression cooperated with Pten loss to recapitulate lethal, human prostate cancer. Method We now report on the generation of two mouse prostate cancer cell lines, BMPC1 and BMPC2, derived from a lymph node, and liver metastasis, respectively. Results Both cell lines demonstrate a phenotype consistent with adenocarcinoma and grew under standard tissue culture conditions. Androgen receptor (AR) protein expression is minimal (BMPC1) or absent (BMPC2) consistent with AR loss observed in the BMPC mouse model of invasive adenocarcinoma. Growth in media containing charcoal-stripped serum resulted in an increase in AR mRNA in BMPC1 cells with no effect on protein expression, unless androgens were added, in which case AR protein was stabilized, and showed nuclear localization. AR expression in BMPC2 cells was not effected by growth media or treatment with androgens. Treatment with an anti-androgen/castration or androgen supplemented media did not affect in vitro or in vivo growth of either cell line, irrespective of nuclear AR detection. Discussion These cell lines are a novel model of androgen-insensitive prostatic adenocarcinoma driven by MYC over-expression and Pten loss.

Telomerase consists of at least two essential elements, an RNA component hTR or TERC that contains the template for telomere DNA addition and a catalytic reverse transcriptase (TERT). While expression of TERT has been considered the key rate-limiting component for telomerase activity, increasing evidence suggests an important role for the regulation of TERC in telomere maintenance and perhaps other functions in human cancer. By using three orthogonal methods including RNAseq, RT-qPCR, and an analytically validated chromogenic RNA in situ hybridization assay, we report consistent overexpression of TERC in prostate cancer. This overexpression occurs at the precursor stage (e.g. high-grade prostatic intraepithelial neoplasia or PIN) and persists throughout all stages of disease progression. Levels of TERC correlate with levels of MYC (a known driver of prostate cancer) in clinical samples and we also show the following: forced reductions of MYC result in decreased TERC levels in eight cancer cell lines (prostate, lung, breast, and colorectal); forced overexpression of MYC in PCa cell lines, and in the mouse prostate, results in increased TERC levels; human TERC promoter activity is decreased after MYC silencing; and MYC occupies the TERC locus as assessed by chromatin immunoprecipitation (ChIP). Finally, we show that knockdown of TERC by siRNA results in reduced proliferation of prostate cancer cell lines. These studies indicate that TERC is consistently overexpressed in all stages of prostatic adenocarcinoma and that its expression is regulated by MYC. These findings nominate TERC as a novel prostate cancer biomarker and therapeutic target. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Correlative human studies suggest that the pleiotropic cytokine IL6 contributes to the development and/or progression of prostate cancer. However, the source of IL6 production in the prostate microenvironment in patients has yet to be determined. The cellular origin of IL6 in primary and metastatic prostate cancer was examined in formalin-fixed, paraffin-embedded tissues using a highly sensitive and specific chromogenic in situ hybridization (CISH) assay that underwent extensive analytical validation. Quantitative RT-PCR showed that benign prostate tissues often had higher expression of IL6 mRNA than matched tumor specimens. CISH analysis further indicated that both primary and metastatic prostate adenocarcinoma cells do not express IL6 mRNA. IL6 expression was highly heterogeneous across specimens and was nearly exclusively restricted to the prostate stromal compartment—including endothelial cells and macrophages, among other cell types. The number of IL6-expressing cells correlated positively with the presence of acute inflammation. In metastatic disease, tumor cells were negative in all lesions examined, and IL6 expression was restricted to endothelial cells within the vasculature of bone metastases. Finally, IL6 was not detected in any cells in soft tissue metastases. These data suggest that, in prostate cancer patients, paracrine rather than autocrine IL6 production is likely associated with any role for the cytokine in disease progression. Cancer Immunol Res; 3(10); 1175–84. ©2015 AACR.

Recent controversies surrounding prostate cancer overtreatment emphasize the critical need to delineate the molecular features associated with progression to lethal metastatic disease. Here, we have used whole-genome sequencing and molecular pathological analyses to characterize the lethal cell clone in a patient who died of prostate cancer. We tracked the evolution of the lethal cell clone from the primary cancer to metastases through samples collected during disease progression and at the time of death. Surprisingly, these analyses revealed that the lethal clone arose from a small, relatively low-grade cancer focus in the primary tumor, and not from the bulk, higher-grade primary cancer or from a lymph node metastasis resected at prostatectomy. Despite being limited to one case, these findings highlight the potential importance of developing and implementing molecular prognostic and predictive markers, such as alterations of tumor suppressor proteins PTEN or p53, to augment current pathological evaluation and delineate clonal heterogeneity. Furthermore, this case illustrates the potential need in precision medicine to longitudinally sample metastatic lesions to capture the evolving constellation of alterations during progression. Similar comprehensive studies of additional prostate cancer cases are warranted to understand the extent to which these issues may challenge prostate cancer clinical management.

DNA methylation at the 5-position of cytosines (5 mC) represents an important epigenetic modification involved in tissue differentiation and is frequently altered in cancer. Recent evidence suggests that 5 mC can be converted to 5-hydroxymethylcytosine (5 hmC) in an enzymatic process involving members of the TET protein family. Such 5 hmC modifications are known to be prevalent in DNA of embryonic stem cells and in the brain, but the distribution of 5 hmC in the majority of embryonic and adult tissues has not been rigorously explored. Here, we describe an immunohistochemical detection method for 5 hmC and the application of this technique to study the distribution of 5 hmC in a large set of mouse and human tissues. We found that 5 hmC was abundant in the majority of embryonic and adult tissues. Additionally, the level of 5 hmC closely tracked with the differentiation state of cells in hierarchically organized tissues. The highest 5 hmC levels were observed in terminally differentiated cells, while less differentiated tissue stem/progenitor cell compartments had very low 5 hmC levels. Furthermore, 5 hmC levels were profoundly reduced in carcinoma of the prostate, breast and colon compared to normal tissues. Our findings suggest a distinct role for 5 hmC in tissue differentiation, and provide evidence for its large-scale loss in cancers.

A defining hallmark of primary and metastatic cancers is the migration and invasion of malignant cells. These invasive properties involve altered dynamics of the cytoskeleton and one of its major structural components β-actin. Here we identify AIM1 (absent in melanoma 1) as an actin-binding protein that suppresses pro-invasive properties in benign prostate epithelium. Depletion of AIM1 in prostate epithelial cells increases cytoskeletal remodeling, intracellular traction forces, cell migration and invasion, and anchorage-independent growth. In addition, decreased AIM1 expression results in increased metastatic dissemination in vivo. AIM1 strongly associates with the actin cytoskeleton in prostate epithelial cells in normal tissues, but not in prostate cancers. In addition to a mislocalization of AIM1 from the actin cytoskeleton in invasive cancers, advanced prostate cancers often harbor AIM1 deletion and reduced expression. These findings implicate AIM1 as a key suppressor of invasive phenotypes that becomes dysregulated in primary and metastatic prostate cancer., Invasion of malignant cells involves changes in cytoskeleton dynamics. Here the authors identify absent in melanoma 1 as an actin binding protein and show that it regulates cytoskeletal remodeling and cell migration in prostate epithelial cells, acting as a metastatic suppressor in cancer cells.

Prostate cancer often manifests as morphologically distinct tumour foci and is frequently found adjacent to presumed precursor lesions such as high-grade prostatic intraepithelial neoplasia (HGPIN). While there is some evidence to suggest that these lesions can be related and exist on a pathological and morphological continuum, the precise clonal and temporal relationships between precursor lesions and invasive cancers within individual tumours remain undefined. Here, we used molecular genetic, cytogenetic, and histological analyses to delineate clonal, temporal, and spatial relationships between HGPIN and cancer lesions with distinct morphological and molecular features. First, while confirming the previous finding that a substantial fraction of HGPIN lesions associated with ERG-positive cancers share rearrangements and overexpression of ERG, we found that a significant subset of such HGPIN glands exhibit only partial positivity for ERG. This suggests that such ERG-positive HGPIN cells either rapidly invade to form adenocarcinoma or represent cancer cells that have partially invaded the ductal and acinar space in a retrograde manner. To clarify these possibilities, we used ERG expression status and TMPRSS2-ERG genomic breakpoints as markers of clonality, and PTEN deletion status to track temporal evolution of clonally related lesions. We confirmed that morphologically distinct HGPIN and nearby invasive cancer lesions are clonally related. Further, we found that a significant fraction of ERG-positive, PTEN-negative HGPIN and intraductal carcinoma (IDC-P) lesions are most likely clonally derived from adjacent PTEN-negative adenocarcinomas, indicating that such PTEN-negative HGPIN and IDC-P lesions arise from, rather than give rise to, the nearby invasive adenocarcinoma. These data suggest that invasive adenocarcinoma can morphologically mimic HGPIN through retrograde colonization of benign glands with cancer cells. Similar clonal relationships were also seen for intraductal carcinoma adjacent to invasive adenocarcinoma. These findings represent a potentially undervalued indicator of pre-existing invasive prostate cancer and have significant implications for prostate cancer diagnosis and risk stratification.

Prostate cancer is a leading cause of cancer death in men due to the subset of cancers that progress to metastasis. Prostate cancers are thought to be hardwired to androgen receptor (AR) signaling, but AR-regulated changes in the prostate that facilitate metastasis remain poorly understood. We previously noted a marked reduction in secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) expression during invasive phases of androgen-induced prostate growth, suggesting that this may be a novel invasive program governed by AR. Herein, we show that SPARCL1 loss occurs concurrently with AR amplification or overexpression in patient-based data. Mechanistically, we demonstrate that SPARCL1 expression is directly suppressed by androgen-induced AR activation and binding at the SPARCL1 locus via an epigenetic mechanism, and these events can be pharmacologically attenuated with either AR antagonists or HDAC inhibitors. We establish using the Hi-Myc model of prostate cancer that in Hi-Myc/Sparcl1−/− mice, SPARCL1 functions to suppress cancer formation. Moreover, metastatic progression of Myc-CaP orthotopic allografts is restricted by SPARCL1 in the tumor microenvironment. Specifically, we show that SPARCL1 both tethers to collagen in the extracellular matrix (ECM) and binds to the cell's cytoskeleton. SPARCL1 directly inhibits the assembly of focal adhesions, thereby constraining the transmission of cell traction forces. Our findings establish a new insight into AR-regulated prostate epithelial movement and provide a novel framework whereby SPARCL1 in the ECM microenvironment restricts tumor progression by regulating the initiation of the network of physical forces that may be required for metastatic invasion of prostate cancer. Cancer Res; 75(20); 4322–34. ©2015 AACR.

We have described a rare group of prostate adenocarcinomas that show aberrant expression of p63, a protein strongly expressed in prostatic basal cells and absent from usual-type acinar prostate cancers. The partial basal-like immunophenotype of these tumors is intriguing in light of the persistent debate surrounding the cell-of-origin for prostate cancer; however, their molecular phenotype is unknown. We collected 37 of these tumors on radical prostatectomy and biopsy and assessed subsets for a diverse panel of molecular markers. The majority of p63-expressing tumors were positive for the ΔNp63 isoform (6/7) by immunofluorescence and p63 mRNA (7/8) by chromogenic in situ hybridization. Despite p63 positivity, these tumors uniformly expressed luminal-type cytokeratin proteins such as CK18 (13/13), CK8 (8/8), and markers of androgen axis signaling commonly seen in luminal cells, including androgen receptor (10/11), NKX3.1 (8/8), and prostein (12/13). Conversely, basal cytokeratins such as CK14 and CK15 were negative in all cases (0/8) and CK5/6 was weakly and focally positive in 36% (4/11) of cases. Pluripotency markers including β-catenin, Oct4, and c-kit were negative in p63-expressing tumors (0/11). Despite nearly universal expression of androgen receptor and downstream androgen signaling targets, p63-expressing tumors lacked ERG rearrangements by fluorescence in situ hybridization (0/14) and ERG protein expression (0/37). No tumors expressed SPINK1 or showed PTEN protein loss (0/19). Surprisingly, 74% (14/19) of p63-expressing tumors expressed GSTP1 protein at least focally, and 33% (2/6) entirely lacked GSTP1 CpG island hypermethylation by bisulfite sequencing. In contrast to usual prostatic adenocarcinomas, prostate tumors with p63 expression show a mixed luminal/basal immunophenotype, uniformly lack ERG gene rearrangement, and frequently express GSTP1. These data strongly suggest that p63-expressing prostate tumors represent a molecularly distinct subclass and further study of this rare tumor type may yield important insights into the role of p63 in prostatic biology and the prostate cancer cell-of-origin.

Human cancers nearly ubiquitously harbor epigenetic alterations. While such alterations in epigenetic marks, including DNA methylation, are potentially heritable, they can also be dynamically altered. Given this potential for plasticity, the degree to which epigenetic changes can be subject to selection and act as drivers of neoplasia has been questioned. Here, we carried out genome-scale analyses of DNA methylation alterations in lethal metastatic prostate cancer and created DNA methylation “cityscape” plots to visualize these complex data. We show that somatic DNA methylation alterations, despite showing marked inter-individual heterogeneity among men with lethal metastatic prostate cancer, were maintained across all metastases within the same individual. The overall extent of maintenance in DNA methylation changes was comparable to that of genetic copy number alterations. Regions that were frequently hypermethylated across individuals were markedly enriched for cancer and development/differentiation related genes. Additionally, regions exhibiting high consistency of hypermethylation across metastases within individuals, even if variably hypermethylated across individuals, showed enrichment of cancer-related genes. Interestingly, whereas some regions showed intra-individual metastatic tumor heterogeneity in promoter methylation, such methylation alterations were generally not correlated with gene expression. This was despite a general tendency for promoter methylation patterns to be strongly correlated with gene expression, particularly at regions that were variably methylated across individuals. These findings suggest that DNA methylation alterations have the potential for producing selectable driver events in carcinogenesis and disease progression and highlight the possibility of targeting such epigenome alterations for development of longitudinal markers and therapeutic strategies.

Purpose: Analytically validated assays to interrogate biomarker status in clinical samples are crucial for personalized medicine. PTEN is a tumor suppressor commonly inactivated in prostate cancer that has been mechanistically linked to disease aggressiveness. Though deletion of PTEN, as detected by cumbersome FISH spot counting assays, is associated with poor prognosis, few studies have validated immunohistochemistry (IHC) assays to determine whether loss of PTEN protein is associated with unfavorable disease. Experimental Design: PTEN IHC was validated by employing formalin fixed and paraffin-embedded isogenic human cell lines containing or lacking intact PTEN alleles. PTEN IHC was 100% sensitive and 97.8% specific for detecting genomic alterations in 58 additional cell lines. PTEN protein loss was then assessed on 376 prostate tumor samples, and PTEN FISH or high resolution single nucleotide polymorphism microarray analysis was done on a subset of these cases. Results: PTEN protein loss, as assessed as a dichotomous IHC variable, was highly reproducible, correlated strongly with adverse pathologic features (e.g., Gleason score and pathologic stage), detected between 75% and 86% of cases with PTEN genomic loss, and was found at times in the absence of apparent genomic loss. In a cohort of 217 high risk surgically treated patients, PTEN protein loss was associated with decreased time to metastasis. Conclusion: These studies validate a simple method to interrogate PTEN status in clinical specimens and support the utility of this test in future multicenter studies, clinical trials, and ultimately perhaps for routine clinical care. Clin Cancer Res; 17(20); 6563–73. ©2011 AACR.

Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.