Genomic Characterization of Prostatic Basal Cell Carcinoma.

Basal cell carcinoma (BCC) of the prostate is a rare tumor. Compared with the more common acinar adenocarcinoma (AAC) of the prostate, BCCs show features of basal cell differentiation and are thought to be biologically distinct from AAC. The spectrum of molecular alterations of BCC has not been comprehensively described, and genomic studies are lacking. Herein, whole genome sequencing was performed on archival formalin-fixed, paraffin-embedded specimens of two cases with BCC. Prostatic BCCs were characterized by an overall low copy number and mutational burden. Recurrent copy number loss of chromosome 16 was observed. In addition, putative driver gene alterations in KIT, DENND3, PTPRU, MGA, and CYLD were identified. Mechanistically, depletion of the CYLD protein resulted in increased proliferation of prostatic basal cells in vitro. Collectively, these studies show that prostatic BCC displays distinct genomic alterations from AAC and highlight a potential role for loss of chromosome 16 in the pathogenesis of this rare tumor type.

Single-cell atlas of epithelial and stromal cell heterogeneity by lobe and strain in the mouse prostate.

BACKGROUND: Evaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered and exposure-based mouse models in different mouse strains and prostate lobes within each mouse strain, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood. METHODS: In this study, we used single-cell RNA-sequencing (scRNA-seq) methods to assess the single-cell transcriptomes of 6-month-old mouse prostates from two commonly used mouse strains, friend virus B/NIH jackson (FVB/NJ) (N = 2) and C57BL/6J (N = 3). For each mouse, the lobes of the prostate were dissected (anterior, dorsal, lateral, and ventral), and individual scRNA-seq libraries were generated. In situ and pathological analyses were used to explore the spatial and anatomical distributions of novel cell types and molecular markers defining these cell types. RESULTS: Data dimensionality reduction and clustering analysis of scRNA-seq data revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Gene set enrichment analysis comparing luminal cells by strain showed enrichment of proto-Oncogene targets in FVB/NJ mice. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2 and Atp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressing Chga, Chgb, and Syp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate. CONCLUSIONS: Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes.

CCRL2 affects the sensitivity of myelodysplastic syndrome and secondary acute myeloid leukemia cells to azacitidine.

Better understanding of the biology of resistance to DNA methyltransferase (DNMT) inhibitors is required to identify therapies that can improve their efficacy for patients with high-risk myelodysplastic syndrome (MDS). CCRL2 is an atypical chemokine receptor that is upregulated in CD34+ cells from MDS patients and induces proliferation of MDS and secondary acute myeloid leukemia (sAML) cells. In this study, we evaluated any role that CCRL2 may have in the regulation of pathways associated with poor response or resistance to DNMT inhibitors. We found that CCRL2 knockdown in TF-1 cells downregulated DNA methylation and PRC2 activity pathways and increased DNMT suppression by azacitidine in MDS/sAML cell lines (MDS92, MDS-L and TF-1). Consistently, CCRL2 deletion increased the sensitivity of these cells to azacitidine in vitro and the efficacy of azacitidine in an MDS-L xenograft model. Furthermore, CCRL2 overexpression in MDS-L and TF-1 cells decreased their sensitivity to azacitidine. Finally, CCRL2 levels were higher in CD34+ cells from MDS and MDS/myeloproliferative neoplasm patients with poor response to DNMT inhibitors. In conclusion, we demonstrated that CCRL2 modulates epigenetic regulatory pathways, particularly DNMT levels, and affects the sensitivity of MDS/sAML cells to azacitidine. These results support CCRL2 targeting as having therapeutic potential in MDS/sAML.

Multiplex immunohistochemical phenotyping of T cells in primary prostate cancer.

BACKGROUND: Most prostate cancers are "immune cold" and poorly responsive to immune checkpoint inhibitors. However, the mechanisms responsible for the lack of a robust antitumor adaptive immune response in the prostate are poorly understood, which hinders the development of novel immunotherapeutic approaches. AIMS: Most inflammatory infiltrates in the prostate are centered around benign glands and stroma, which can confound the molecular characterization of the antitumor immune response. We sought to analytically validate a chromogenic-based multiplex immunohistochemistry (IHC) approach applicable to whole slide digital image analysis to quantify T cell subsets from the tumor microenvironment of primary prostatic adenocarcinomas. As an initial application, we tested the hypothesis that PTEN loss leads to an altered antitumor immune response by comparing matched regions of tumors within the same individual with and without PTEN loss. MATERIALS & METHODS: Using the HALO Image Analysis Platform (Indica Labs), we trained a classifier to quantify the densities of eight T cell phenotypes separately in the tumor epithelial and stromal subcompartments. RESULTS: The iterative chromogenic approach using 7 different antibodies on the same slide provides highly similar findings to results using individually stained slides with single antibodies. Our main findings in carcinomas (benign removed) include the following: i) CD4+ T cells are present at higher density than CD8+ T cells; ii) all T cell subsets are present at higher densities in the stromal compartment compared to the epithelial tumor compartment; iii) most CD4+ and CD8+ T cells are PD1+; iv) cancer foci with PTEN loss harbored increased numbers of T cells compared to regions without PTEN loss, in both stromal and epithelial compartments; and v) the increases in T cells in PTEN loss regions were associated with ERG gene fusion status. DISCUSSION: This modular approach can apply to any IHC-validated antibody combination and sets the groundwork for more detailed spatial analyses. CONCLUSION: Iterative chromogenic IHC can be used for whole slide analysis of prostate tissue samples and can complement transcriptomic results including those using single cell and spatial genomic approaches.

Reciprocal YAP1 loss and INSM1 expression in neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC) is a rare but aggressive histologic variant of prostate cancer that responds poorly to androgen deprivation therapy. Hybrid NEPC-adenocarcinoma (AdCa) tumors are common, often eluding accurate pathologic diagnosis and requiring ancillary markers for classification. We recently performed an outlier-based meta-analysis across a number of independent gene expression microarray datasets to identify novel markers that differentiate NEPC from AdCa, including up-regulation of insulinoma-associated protein 1 (INSM1) and loss of Yes-associated protein 1 (YAP1). Here, using diverse cancer gene expression datasets, we show that Hippo pathway-related genes, including YAP1, are among the top down-regulated gene sets with expression of the neuroendocrine transcription factors, including INSM1. In prostate cancer cell lines, transgenic mouse models, and human prostate tumor cohorts, we confirm that YAP1 RNA and YAP1 protein expression are silenced in NEPC and demonstrate that the inverse correlation of INSM1 and YAP1 expression helps to distinguish AdCa from NEPC. Mechanistically, we find that YAP1 loss in NEPC may help to maintain INSM1 expression in prostate cancer cell lines and we further demonstrate that YAP1 silencing likely occurs epigenetically, via CpG hypermethylation near its transcriptional start site. Taken together, these data nominate two additional markers to distinguish NEPC from AdCa and add to data from other tumor types suggesting that Hippo signaling is tightly reciprocally regulated with neuroendocrine transcription factor expression. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genomic profiles and clinical outcomes in primary versus secondary metastatic hormone-sensitive prostate cancer.

BACKGROUND: Clinical outcomes may differ among patients presenting with primary (de novo) metastatic hormone-sensitive prostate cancer (mHSPC) versus secondary (metachronous) mHSPC occurring after local therapy. It is unknown what molecular features distinguish these potentially distinct presentations. METHODS: A single-center retrospective study of mHSPC patients classified as primary mHSPC (n = 121) or secondary mHSPC (n = 106). A targeted set of genes was analyzed: BRCA2, PTEN, RB1, TP53, SPOP, CDK12, any two out of PTEN/RB1/TP53 alterations, and homologous recombination deficiency mutations. TP53 mutations were categorized as loss-of-function (LOF) versus dominant-negative (DN). The impacts of genetic features on progression-free survival (PFS) and overall survival (OS) were assessed using univariate and multivariate Cox proportional hazards regression. RESULTS: Median PFS was 15 and 30 months for men with primary and secondary mHSPC, respectively (hazard ratio: 0.57, 95% confidence interval: 0.41-0.78; p < .01). OS did not show a significant difference between groups. There were more men with Gleason 8-10 disease in the primary versus secondary mHSPC groups (83% vs. 68%; p < .01). In univariate and multivariate analyses, TP53 DN mutations showed a statistically significant association with OS for the entire mHSPC population. Conversely, SPOP mutations were associated with improved OS. Additionally, TP53 mutations (DN and LOF) were associated with worse OS for secondary mHSPC. A combination of PTEN/RB1/TP53 mutations was associated with worse OS and PFS for secondary mHSPC, while no genomic alteration affected outcomes for primary mHSPC. CONCLUSIONS: TP53 DN mutations, but not all TP53 alterations, were the strongest predictor of negative outcomes in men with mHSPC, while SPOP mutations were associated with improved outcomes. In subgroup analyses, specific alterations were prognostic of outcomes in secondary, but not primary, mHSPC.

Longitudinal measurement of subcutaneous and intratibial human prostate cancer xenograft growth and response to ionizing radiation by plasma Alu and LINE-1 ctDNA: A comparison to standard methods.

BACKGROUND: Current preclinical models of metastatic prostate cancer (PCa) require sophisticated technologies and/or genetically engineered cells for the noninvasive monitoring of tumors in remote sites, such as bone. Recent developments in circulating tumor DNA (ctDNA) analysis provide an alternative method for noninvasive tumor monitoring at a low cost. Here, we sought to evaluate human Alu and LINE-1 ctDNA for the longitudinal measurement of subcutaneous and intratibial human PCa xenograft growth and response to ionizing radiation (IR) through comparison with standard slide caliper and bioluminescence measurements. MATERIAL AND METHODS: Intratibial and subcutaneous xenografts were established in male athymic nude mice using LNCaP cells that stably express firefly luciferase. A subset of tumors was treated with a single dose of IR (CT-guided focal IR, 6 Gy). Tumor measurements were simultaneously taken by slide caliper (subcutaneous only), in vivo bioluminescence imaging, and quantitative real-time PCR (qPCR) of human-specific Alu and LINE-1 ctDNA for several weeks. RESULTS: Levels of ctDNA and bioluminescence increased concordantly with subcutaneous and intratibial tumor growth. A statistically significant correlation (Spearman) was observed between ctDNA and subcutaneous tumor volume (LINE-1, r = .94 and Alu, r = .95, p < .0001), ctDNA and bioluminescence (LINE-1, r = .66 and Alu, r = .60, p < .002), and bioluminescence and tumor volume (r = .66, p = .0003). Bioluminescence and ctDNA were also significantly correlated in intratibial tumors (LINE-1, r = .82 and Alu, r = .81, p < .0001). Following external beam IR, the tumor responses varied briefly by method of measurement, but followed a similar trend. Statistically significant correlations were maintained between ctDNA and slide caliper measurement in irradiated subcutaneous tumors (LINE-1, r = .64 and Alu, r = .44, p < .02), and ctDNA and bioluminescence in intratibial tumors (LINE-1, r = .55, p = .018). CONCLUSIONS: Real-time qPCR of circulating human Alu and LINE-1 DNA provides an accurate measurement of subcutaneous and intratibial xenograft burden that is comparable with conventional bioluminescence imaging and slide caliper measurement. Transient differences in measurements were observed following tumor-targeted IR, but overall all measurements mirrored tumor growth and response.

Phenotypic characterization of two novel cell line models of castration-resistant prostate cancer.

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.

An in Situ Atlas of Mitochondrial DNA in Mammalian Tissues Reveals High Content in Stem and Proliferative Compartments.

Mitochondria regulate ATP production, metabolism, and cell death. Alterations in mitochondrial DNA (mtDNA) sequence and copy number are implicated in aging and organ dysfunction in diverse inherited and sporadic diseases. Because most measurements of mtDNA use homogenates of complex tissues, little is known about cell-type-specific mtDNA copy number heterogeneity in normal physiology, aging, and disease. Thus, the precise cell types whose loss of mitochondrial activity and altered mtDNA copy number that result in organ dysfunction in aging and disease have often not been clarified. Here, an in situ hybridization approach to generate a single-cell-resolution atlas of mtDNA content in mammalian tissues was validated. In hierarchically organized self-renewing tissues, higher levels of mtDNA were observed in stem/proliferative compartments compared with differentiated compartments. Striking zonal patterns of mtDNA levels in the liver reflected the known oxygen tension gradient. In the kidney, proximal and distal tubules had markedly higher mtDNA levels compared with cells within glomeruli and collecting duct epithelial cells. In mice, decreased mtDNA levels were visualized in renal tubules as a function of aging, which was prevented by calorie restriction. This study provides a novel approach for quantifying species- and cell-type-specific mtDNA copy number and dynamics in any normal or diseased tissue that can be used for monitoring the effects of interventions in animal and human studies.

Integrated single-cell and bulk gene expression and ATAC-seq reveals heterogeneity and early changes in pathways associated with resistance to cetuximab in HNSCC-sensitive cell lines.

BACKGROUND: Identifying potential resistance mechanisms while tumour cells still respond to therapy is critical to delay acquired resistance. METHODS: We generated the first comprehensive multi-omics, bulk and single-cell data in sensitive head and neck squamous cell carcinoma (HNSCC) cells to identify immediate responses to cetuximab. Two pathways potentially associated with resistance were focus of the study: regulation of receptor tyrosine kinases by TFAP2A transcription factor, and epithelial-to-mesenchymal transition (EMT). RESULTS: Single-cell RNA-seq demonstrates heterogeneity, with cell-specific TFAP2A and VIM expression profiles in response to treatment and also with global changes to various signalling pathways. RNA-seq and ATAC-seq reveal global changes within 5 days of therapy, suggesting early onset of mechanisms of resistance; and corroborates cell line heterogeneity, with different TFAP2A targets or EMT markers affected by therapy. Lack of TFAP2A expression is associated with HNSCC decreased growth, with cetuximab and JQ1 increasing the inhibitory effect. Regarding the EMT process, short-term cetuximab therapy has the strongest effect on inhibiting migration. TFAP2A silencing does not affect cell migration, supporting an independent role for both mechanisms in resistance. CONCLUSION: Overall, we show that immediate adaptive transcriptional and epigenetic changes induced by cetuximab are heterogeneous and cell type dependent; and independent mechanisms of resistance arise while tumour cells are still sensitive to therapy.

Correction: Integrated single-cell and bulk gene expression and ATAC-seq reveals heterogeneity and early changes in pathways associated with resistance to cetuximab in HNSCC-sensitive cell lines.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Lactoferrin CpG Island Hypermethylation and Decoupling of mRNA and Protein Expression in the Early Stages of Prostate Carcinogenesis.

Lactoferrin (LTF) is an iron-binding protein canonically known for its innate and adaptive immune functions. LTF may also act as a tumor suppressor with antiproliferative action. LTF is inactivated genetically or epigenetically in various cancers, and a CpG island spanning the transcriptional start site of LTF is hypermethylated in prostate cancer cell lines. We, therefore, hypothesized that LTF expression is silenced via CpG island hypermethylation in the early stages of prostate tumorigenesis carcinogenesis. Targeted methylation analysis was performed using a combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes, and laser-capture microdissection followed by bisulfite sequencing on DNA isolated from prostate tissue samples, including both primary and metastatic disease. LTF mRNA in situ hybridization and LTF protein immunohistochemistry were also performed. We report that the LTF CpG island is frequently and densely methylated in high-grade prostatic intraepithelial neoplasia, primary prostate carcinoma, and metastases. We further report a decoupling of lactoferrin mRNA and protein expression, including in lesions where LTF mRNA has presumably been silenced via CpG island methylation. We conclude that LTF mRNA expression is silenced in prostate tumorigenesis via hypermethylation, supporting a role for LTF as a prostate cancer tumor suppressor gene. Likewise, the frequency at which the LTF CpG island is methylated across samples suggests it is an important and conserved step in prostate cancer initiation.

Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations.

The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next-generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild-type molecules.

Consequences of interleukin 1ß-triggered chronic inflammation in the mouse prostate gland: Altered architecture associated with prolonged CD4+ infiltration mimics human proliferative inflammatory atrophy.

BACKGROUND: Elevated expression of the proinflammatory cytokine interleukin 1ß (IL-1ß) has been observed in expressed prostatic secretions of patients with chronic prostatitis/chronic pelvic pain syndrome, and genetic polymorphisms associated with the IL1B gene are linked to increased risk for aggressive prostate cancer. METHODS: To study the role of IL-1ß expression in prostate inflammation, we examined IL1B expression in human prostatic proliferative inflammatory atrophy (PIA) lesions and developed a tetracycline-regulated human IL1B transgene in the mouse prostate. RESULTS: Here, we demonstrate that IL1B expression is a common finding in human PIA lesions, which harbored focal IL1B expression in epithelial and stromal compartments. Human IL1B expression in the mouse prostate elicited acute and chronic inflammation. Penetrance and expressivity were variable and tunable by altering transgene dosage and the presence of an exogenous inducible marker antigen (green fluorescent protein). Inflammation was characterized by infiltration of CD4+ T cells, demonstrating an adaptive immune response. Chronic inflammation persisted after doxycycline (Dox) withdrawal. Reactive epithelia increased expression of downstream cytokines, and altered glandular architecture was observed upon sustained induction of IL1B. Immunohistochemical analyses revealed a higher proliferative index and decreased Nkx3.1 expression in inflamed mouse prostates. CONCLUSIONS: These data implicate IL-1ß in human prostate pathology and this model provides a versatile platform to interrogate molecular mechanisms of inflammation-associated prostate pathologies associated with episodic or sustained IL-1ß expression.

Comprehensive Evaluation of Programmed Death-Ligand 1 Expression in Primary and Metastatic Prostate Cancer.

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.

MYC drives overexpression of telomerase RNA (hTR/TERC) in prostate cancer.

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.

ETS2 is a prostate basal cell marker and is highly expressed in prostate cancers aberrantly expressing p63.

BACKGROUND: Rare prostate carcinomas aberrantly express p63 and have an immunophenotype intermediate between basal and luminal cells. Here, we performed gene expression profiling on p63-expressing prostatic carcinomas and compared them to usual-type adenocarcinoma. We identify ETS2 as highly expressed in p63-expressing prostatic carcinomas and benign prostate basal cells, with lower expression in luminal cells and primary usual-type adenocarcinomas. METHODS: A total of 8 p63-expressing prostate carcinomas at radical prostatectomy were compared to 358 usual-type adenocarcinomas by gene expression profiling performed on formalin fixed paraffin embedded tumor tissue using Affymetrix 1.0 ST microarrays. Correlation between differentially expressed genes and TP63 expression was performed in 5239 prostate adenocarcinomas available in the Decipher GRID. For validation, ETS2 in situ hybridization was performed on 19 p63-expressing prostate carcinomas and 30 usual-type adenocarcinomas arrayed on tissue microarrays (TMA). RESULTS: By gene expression, p63-expressing prostate carcinomas showed low cell cycle activity and low Decipher prognostic scores, but were predicted to have high Gleason grade compared to usual-type adenocarcinomas by gene expression signatures and morphology. Among the genes over-expressed in p63-expressing carcinoma relative to usual-type adenocarcinoma were known p63-regulated genes, along with ETS2, an ETS family member previously implicated as a prostate cancer tumor suppressor gene. Across several cohorts of prostate samples, ETS2 gene expression was correlated with TP63 expression and was significantly higher in benign prostate compared to usual-type adenocarcinoma. By in situ hybridization, ETS2 gene expression was high in benign basal cells, and low to undetectable in benign luminal cells or usual-type adenocarcinoma. In contrast, ETS2 was highly expressed in 95% (18/19) of p63-expressing prostate carcinomas. CONCLUSIONS: ETS2 is a predominantly basally-expressed gene in the prostate, with low expression in usual-type adenocarcinoma and high expression in p63-expressing carcinomas. Given this pattern, the significance of ETS2 loss by deletion or mutation in usual-type adenocarcinomas is uncertain.

Characterization of novel cell lines derived from a MYC-driven murine model of lethal metastatic adenocarcinoma of the prostate.

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.

Redox-Responsive Nanoparticle-Mediated Systemic RNAi for Effective Cancer Therapy.

Biodegradable polymeric nanoparticles (NPs) have demonstrated significant potential to improve the systemic delivery of RNA interference (RNAi) therapeutics, such as small interfering RNA (siRNA), for cancer therapy. However, the slow and inefficient siRNA release inside tumor cells generally observed for most biodegradable polymeric NPs may result in compromised gene silencing efficacy. Herein, a biodegradable and redox-responsive NP platform, composed of a solid poly(disulfide amide) (PDSA)/cationic lipid core and a lipid-poly(ethylene glycol) (lipid-PEG) shell for systemic siRNA delivery to tumor cells, is developed. This newly generated NP platform can efficiently encapsulate siRNA under extracellular environments and can respond to the highly concentrated glutathione (GSH) in the cytoplasm to induce fast intracellular siRNA release. By screening a library of PDSA polymers with different structures and chain lengths, the optimized NP platform shows the unique features of i) long blood circulation, ii) high tumor accumulation, iii) fast GSH-triggered intracellular siRNA release, and iv) exceptionally effective gene silencing. Together with the facile polymer synthesis technique and robust NP formulation enabling scale-up, this new redox-responsive NP platform may become an effective tool for RNAi-based cancer therapy.

Profiling the Urinary Microbiome in Men with Positive versus Negative Biopsies for Prostate Cancer.

PURPOSE: Studies demonstrating bacterial DNA and cultivable bacteria in urine samples have challenged the clinical dogma that urine is sterile. Furthermore, studies now indicate that dysbiosis of the urinary microbiome is associated with pathological conditions. We propose that the urinary microbiome may influence chronic inflammation observed in the prostate, leading to prostate cancer development and progression. Therefore, we profiled the urinary microbiome in men with positive vs negative biopsies for prostate cancer. MATERIALS AND METHODS: Urine was collected from men prior to biopsy for prostate cancer. DNA was extracted from urine pellet samples and subjected to bacterial 16S rDNA Illumina® sequencing and 16S rDNA quantitative polymerase chain reaction. We determined the association between bacterial species and the presence or absence of cancer, cancer grade, and type and degree of prostate inflammation. RESULTS: Urine samples revealed diverse bacterial populations. There were no significant differences in α or ß diversity and no clear hierarchical clustering of benign or cancer samples. We identified a cluster of pro-inflammatory bacteria previously implicated in urogenital infections in a subset of samples. Many species, including known uropathogens, were significantly and differentially abundant among cancer and benign samples, in low vs higher grade cancers and in relation to prostate inflammation type and degree. CONCLUSIONS: To our knowledge we report the most comprehensive study to date of the male urinary microbiome and its relationship to prostate cancer. Our results suggest a prevalence of pro-inflammatory bacteria and uropathogens in the urinary tract of men with prostate cancer.