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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

BACKGROUNDResistance to androgen deprivation therapies is a major driver of mortality in advanced prostate cancer. Therefore, there is a need to develop new pre-clinical models that allow the investigation of resistance mechanisms and the assessment of drugs for the treatment of castration resistant prostate cancer.METHODSWe 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 the novel cell line models.RESULTSThe 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.CONCLUSIONSThe 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.

DNA methylation can mediate epigenetic silencing of tumor suppressor and cancer protective genes. The protein ubiquitin-like containing PHD and ring finger domains 1 (UHRF1) is an essential component in cells for DNA methylation maintenance. The SET- and RING-associated (SRA) domain of UHRF1 can bind hemimethylated DNA, and mediate recruitment of DNA methyltransferases to copy the methylation pattern to the newly synthesized daughter strand. Loss of UHRF1 function can lead to demethylation and re-expression of epigenetically silenced tumor suppressor genes and can reduce cancer cell growth and survival. We created a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to screen for inhibitors capable of disrupting the interaction between the UHRF1-SRA domain and hemimethylated DNA. Using this assay (Z' factor of 0.74 in 384-well format) we screened the Library of Pharmacologically Active Compounds (LOPAC) for UHRF1-SRA inhibitors, and validated 7 hit compounds. These compounds included the anthracycline derivatives idarubicin and mitoxantrone, which are commonly used chemotherapeutic drugs known to mediate cytotoxicity by acting as class II topoisomerase (TOP2) poisons. In a panel of additional known topoisomerase poisons, only the anthracycline derivatives showed dose responsive inhibition of UHRF1-SRA. Additionally, mitoxantrone and doxorubicin showed dose-responsive global DNA demethylation and demonstrated a synergistic growth inhibition of multiple cancer cell lines when combined with the DNA methyltransferase (DNMT) inhibitor decitabine. These data validate a novel TR-FRET assay for identification of UHRF1 inhibitors, and revealed unexpected epigenetic properties of commonly used chemotherapeutic drugs that showed synergistic cytotoxicity of cancer cells when combined with a demethylating agent.

Background In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of TERT gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating TERT gene expression in cancer cells is as yet not fully understood. Methods Here, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines, including primary, immortalized and cancer cell types, as well as in control and reference samples. Results In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally found that hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells. Conclusions Our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to attenuation of TERT activation in cancer cells. This type of fine tuning of TERT expression may account for the modest activation of TERT expression in most cancers.

In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded byTERT. Yet, most cancers show only modest levels of telomerase gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating telomerase gene expression in cancer cells is not fully understood. Here, we have carried out the most comprehensive characterization to date ofTERTpromoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the coreTERTpromoter in 96 different human cell lines. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250TTERTpromoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally observe hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observedTERTexpression in cancer cells. Our findings suggest that hypermethylation of theTERTpromoter alleles signals transcriptional repression of those alleles, leading to the attenuation ofTERTactivation in cancer cells.SIGNIFICANCEHypermethylation of theTERTpromoter alleles to attenuateTERTactivation in cancer cells may account for the modest activation ofTERTexpression in most cancers.

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.

Background Androgen receptor (AR) splice variants have been clinically associated with progressive cancer, castration-resistance, and resistance to AR antagonists and androgen synthesis inhibitors. AR variants can be generated by genomic alterations and alternative splicing, and their expression is androgen-regulated. There has been a suggestion that AR variants bearing premature termination codons and coding for truncated proteins should be regulated by the nonsense-mediated decay (NMD) mRNA surveillance pathway, suggesting that either the NMD pathway is dysfunctional in variant-expressing cell lines or that variants are somehow able to evade degradation by NMD. Methods We first used siRNA knockdown of the NMD regulator, UPF1, in an NMD reporter assay to determine if this surveillance pathway is functioning normally in AR variant-expressing cell lines. We then used UPF1 knockdown to determine if expression of the AR variants ARV3 and ARV7 is affected by inhibition of NMD. Next, we analyzed androgen regulation of UPF1 and used transcript expression analysis to determine if there is any association between UPF1 expression, resistance, and ARV3 or ARV7 expression. Results We found that the NMD pathway functions normally in the AR variant-expressing cell line 22Rv1 and that inhibition of NMD does not increase expression of ARV3 or ARV7. Furthermore, we found that expression of UPF1 is not androgen-regulated. We also found that UFP1 expression levels do not differentiate castration-sensitive from resistant cell line and that UPF1 expression does not correlate with expression of ARV3 or ARV7 in cells in which these variants are highly expressed. Conclusion This study eliminates a possible mechanism of regulation of certain AR variants. Future research into the regulation of AR variants should focus on other mechanisms to better understand the origin of these variants and to possibly inhibit their expression for the resensitization of resistant cancers. Prostate 77:829-837, 2017. © 2017 Wiley Periodicals, Inc.

In cancers, the epigenetic regulation of telomerase expression is not fully characterized, with multiple conflicting reports. In particular, the role of DNA methylation of cytosine residues in the control of TERT expression remains unclear. Here, we have carried out the most comprehensive characterization of TERT promoter methylation using ultra-deep bisulfite sequencing spanning more than 310 CpGs within and surrounding the core TERT promoter in 96 different cell lines, including primary, immortalized, and cancer cell types, as well as in control and reference samples. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent G228A and G250A TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. Notably, in cancers, the hypermethylated alleles were associated with repressed expression while remaining unmethylated alleles were enriched with the open chromatin marks, H3K4me3 and H3K27ac. Furthermore, the hypomethylated alleles of the TERT promoter were responsible for the observed TERT expression in cancer. This association of hypomethylated alleles with active expression and hypermethylated alleles with repressed expression is particularly evident in cancers harboring TERT promoter mutations, and cancers displaying allele-specific expression. Reporter assays confirmed that DNA methylation of the TERT promoter greatly suppressed reporter expression in heterologous TERT promoter-reporter constructs. Overall, our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to the attenuation of TERT activation in cancer cells. Citation Format: Mindy K. Graham, David Esopi, Jacqueline A. Brosnan-Cashman, Anuj Gupta, Angelo De Marzo, William G. Nelson, Christopher M. Heaphy, Alan K. Meeker, Sarah J. Wheelan, Srinivasan Yegnasubramanian. Pervasive promoter hypermethylation of silenced TERT alleles in human cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 157.

Background Cancer cell metabolism requires sustained pools of intracellular nicotinamide adenine dinucleotide (NAD+) which is maintained by a balance of NAD+ hydrolase activity and NAD+ salvage activity. We recently reported that human prostate cancer can be initiated following oncogene expression in progenitor-like luminal cells marked by low expression of the NAD+-consuming enzyme CD38. CD38 expression is reduced in prostate cancer compared to benign prostate, suggesting that tumor cells may reduce CD38 expression in order to enhance pools of NAD+. However, little is known about how CD38 expression is repressed in advanced prostate cancer and whether CD38 plays a role in regulating NAD+ levels in prostate epithelial cells. Methods CD38 expression, its association with recurrence after prostatectomy for clinically localized prostate cancer, and DNA methylation of the CD38 promoter were evaluated in human prostate tissues representing various stages of disease progression. CD38 was inducibly over-expressed in benign and malignant human prostate cell lines in order to determine the effects on cell proliferation and levels of NAD+ and NADH. NAD+ and NADH were also measured in urogenital tissues from wild-type and CD38 knockout mice. Results CD38 mRNA expression was reduced in metastatic castration-resistant prostate cancer compared to localized prostate cancer. In a large cohort of men undergoing radical prostatectomy, CD38 protein expression was inversely correlated with recurrence. We identified methylation of the CD38 promoter in primary and metastatic prostate cancer. Over-expression of wild-type CD38, but not an NAD+ hydrolase-deficient mutant, depleted extracellular NAD+ levels in benign and malignant prostate cell lines. However, expression of CD38 did not significantly alter intracellular NAD+ levels in human prostate cell lines grown in vitro and in urogenital tissues isolated from wild-type and CD38 knockout mice. Conclusions CD38 protein expression in prostate cancer is associated with risk of recurrence. Methylation results suggest that CD38 is epigenetically regulated in localized and metastatic prostate cancer tissues. Our study provides support for CD38 as a regulator of extracellular, but not intracellular, NAD+ in epithelial cells. These findings suggest that repression of CD38 by methylation may serve to increase the availability of extracellular NAD+ in prostate cancer tissues.

Cancers must maintain their telomeres at lengths sufficient for cell survival. In several cancer subtypes, a recombination-like mechanism termed alternative lengthening of telomeres (ALT), is frequently used for telomere length maintenance. Cancers utilizing ALT often have lost functional ATRX, a chromatin remodeling protein, through mutation or deletion, thereby strongly implicating ATRX as an ALT suppressor. Herein, we have generated functional ATRX knockouts in four telomerase-positive, ALT-negative human glioma cell lines: MOG-G-UVW, SF188, U-251 and UW479. After loss of ATRX, two of the four cell lines (U-251 and UW479) show multiple characteristics of ALT-positive cells, including ultrabright telomeric DNA foci, ALT-associated PML bodies, and c-circles. However, telomerase activity and overall telomere length heterogeneity are unaffected after ATRX loss, regardless of cellular context. The two cell lines that showed ALT hallmarks after complete ATRX loss also did so upon ATRX depletion via shRNA-mediated knockdown. These results suggest that other genomic or epigenetic events, in addition to ATRX loss, are necessary for the induction of ALT in human 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.

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.

The family Lumbricidae is arguably the most well-known and well-studied earthworm group due to its dominance in the European earthworm fauna and its invasion in temperate regions worldwide. However, its North American members, especially the genus Bimastos Moore, 1893, are poorly understood. We revised the systematics of the genus Bimastos and tested the hypothesis of the monophyly of North American lumbricids using morphological characters and eight molecular markers. Phylogenetic analyses based on our extensive sampling of Bimastos and inclusion of Dendrodrilus and Allolobophoridella indicated a well-supported clade containing Bimastos and Eisenoides Gates, 1969, and provided the first evidence supporting that North American lumbricids are monophyletic. Assuming the available divergence time estimations and dating of land bridges are correct, it would suggest that the ancestor of this clade arrived North America through Beringia or the De Geer route during Late Cretaceous, and since then the clade has diverged from its Eurasian sister group, Eisenia. The peregrine genera Dendrodrilus and Allolobophoridella are nested within the Bimastos clade; we propose to treat them as junior synonyms of the genus Bimastos, and, contradictory to the commonly held belief of being European, they are indeed part of the indigenous North American earthworm fauna. Morphological characters, such as red-violet pigmentation, proclinate U-shaped nephridial bladders and calciferous diverticula in segment 10 further support this placement. The East Mediterranean-Levantine Spermophorodrilus Bouche, 1975 and Healyella Omodeo & Rota, 1989 are nested within the Dendrobaena sensu lato clade; therefore their close relationship with the North American Bimastos is refuted. Species fit the revised diagnosis of Bimastos are reviewed and keyed, and a new species, Bimastos schwerti sp. nov., is described.

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.

TMPRSS2-ERG rearrangements occur in approximately 50% of prostate cancers and therefore represent one of the most frequently observed structural rearrangements in all cancers. However, little is known about the genomic architecture of such rearrangements. We therefore designed and optimized a pipeline involving target capture of TMPRSS2 and ERG genomic sequences coupled with paired-end next-generation sequencing to resolve genomic rearrangement breakpoints in TMPRSS2 and ERG at nucleotide resolution in a large series of primary prostate cancer specimens (n = 83). This strategy showed > 90% sensitivity and specificity in identifying TMPRSS2-ERG rearrangements, and allowed identification of intra- and inter-chromosomal rearrangements involving TMPRSS2 and ERG with known and novel fusion partners. Our results indicate that rearrangement breakpoints show strong clustering in specific intronic regions of TMPRSS2 and ERG. The observed TMPRSS2-ERG rearrangements often exhibited complex chromosomal architecture associated with several intra- and inter-chromosomal rearrangements. Nucleotide resolution analysis of breakpoint junctions revealed that the majority of TMPRSS2 and ERG rearrangements (~88%) occurred at or near regions of microhomology or involved insertions of one or more base pairs. This architecture implicates non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) pathways in the generation of such rearrangements. These analyses have provided important insights into the molecular mechanisms involved in generating prostate cancer-specific recurrent rearrangements.

The persistence leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) despite tyrosine kinase inhibition (TKI) may explain relapse after TKI withdrawal. Here we performed genome-wide transcriptome analysis of highly refined CML and normal stem and progenitor cell populations to identify novel targets for the eradication of CML LSCs using exon microarrays. We identified 97 genes that were differentially expressed in CML versus normal stem and progenitor cells. These included cell surface genes significantly upregulated in CML LSCs: DPP4 (CD26), IL2RA (CD25), PTPRD, CACNA1D, IL1RAP, SLC4A4, and KCNK5. Further analyses of the LSCs revealed dysregulation of normal cellular processes, evidenced by alternative splicing of genes in key cancer signaling pathways such as p53 signaling (e.g. PERP, CDKN1A), kinase binding (e.g. DUSP12, MARCKS), and cell proliferation (MYCN, TIMELESS); downregulation of pro-differentiation and TGF-β/BMP signaling pathways; upregulation of oxidative metabolism and DNA repair pathways; and activation of inflammatory cytokines, including CCL2, and multiple oncogenes (e.g., CCND1). These data represent an important resource for understanding the molecular changes in CML LSCs, which may be exploited to develop novel therapies for eradication these cells and achieve cure.

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.

DNA hypermethylation can trigger silencing of tumor suppressor genes during cancer development and progression, partly through binding by methylated-DNA binding proteins (MBD), such as MBD2, that function as “epigenetic readers” and recruit co-repressor complexes to promote gene repression. Inhibiting MBD2-mediated repression represents an attractive cancer therapeutic strategy. Here, we used a cell-based screen to identify small molecules capable of reactivating hypermethylated promoter sequences. We used biochemical, molecular biologic, and pharmacologic approaches to characterize mechanism of action of identified MBD2 inhibitors. A subset of these compounds represent a new class of inhibitors capable of selectively antagonizing interactions between MBD2 and methylated DNA, leading to reactivation of the hypermethylated gene GSTP1 and the epigenetically silenced retinoic acid signaling pathway. Combinations of one of the newly identified MBD2 inhibitors, KCC-08, with the retinoic acid receptor agonist, isotretinoin, significantly reduced cancer cell growth/survival in vitro and in vivo. These novel MBD2 inhibitors are thus positioned for further pharmacologic lead development for use as probes to interrogate epigenetic gene silencing mechanisms and as cancer therapeutics. Citation Format: Hugh Giovinazzo, Zachary R. Reichert, Andries Bergman, Xiaohui Lin, Nicolas Wyhs, David Esopi, Ajay Vaghasia, Jianyong Liu, Yash Jain, Akshay Bhamidipati, Ruchama Steinberg, Traci Speed, Matthew Vaughn, Yonggang Zhang, Nate Brennen, Theodore Deweese, Srinivasan Yegnasubramanian, William G. Nelson. Novel inhibitors of the epigenetic reader protein MBD2 [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 5881.

DNA double-strand breaks (DSBs) can lead to the development of genomic rearrangements, which are hallmarks of cancer. Fusions between TMPRSS2, encoding the transmembrane serine protease isoform 2, and ERG, encoding the v-ets erythroblastosis virus E26 oncogene homolog, are among the most common oncogenic rearrangements observed in human cancer. We show that androgen signaling promotes co-recruitment of androgen receptor and topoisomerase II beta (TOP2B) to sites of TMPRSS2-ERG genomic breakpoints, triggering recombinogenic TOP2B-mediated DSBs. Furthermore, androgen stimulation resulted in de novo production of TMPRSS2-ERG fusion transcripts in a process that required TOP2B and components of the DSB repair machinery. Finally, unlike normal prostate epithelium, prostatic intraepithelial neoplasia cells showed strong coexpression of androgen receptor and TOP2B. These findings implicate androgen-induced TOP2B-mediated DSBs in generating TMPRSS2-ERG rearrangements.

Loss-of-function mutations in the nuclear factor erythroid-2–related factor 2 (Nrf2) inhibitor Kelch-like ECH-associated protein 1 (Keap1) result in increased Nrf2 activity in non–small cell lung cancer and confer therapeutic resistance. We detected point mutations in Keap1 gene, leading to nonconservative amino acid substitutions in prostate cancer cells. We found novel transcriptional and posttranscriptional mechanisms of Keap1 inactivation, such as promoter CpG island hypermethylation and aberrant splicing of Keap1, in DU-145 cells. Very low levels of Keap1 mRNA were detected in DU-145 cells, which significantly increased by treatment with DNA methyltransferase inhibitor 5-aza-deoxycytidine. The loss of Keap1 function led to an enhanced activity of Nrf2 and its downstream electrophile/drug detoxification pathway. Inhibition of Nrf2 expression in DU-145 cells by RNA interference attenuated the expression of glutathione, thioredoxin, and the drug efflux pathways involved in counteracting electrophiles, oxidative stress, and detoxification of a broad spectrum of drugs. DU-145 cells constitutively expressing Nrf2 short hairpin RNA had lower levels of total glutathione and higher levels of intracellular reactive oxygen species. Attenuation of Nrf2 function in DU-145 cells enhanced sensitivity to chemotherapeutic drugs and radiation-induced cell death. In addition, inhibition of Nrf2 greatly suppressed in vitro and in vivo tumor growth of DU-145 prostate cancer cells. Thus, targeting the Nrf2 pathway in prostate cancer cells may provide a novel strategy to enhance chemotherapy and radiotherapy responsiveness and ameliorate the growth and tumorigenicity, leading to improved clinical outcomes. Mol Cancer Ther; 9(2); 336–46

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.

Telomeres progressively shorten with each cell division, leading to critically short telomeres during tumorigenesis. While most cancers activate telomerase to maintain their telomeres, a subset of cancers utilize a recombination-based process known as alternative lengthening of telomeres, or ALT. ALT has been widely characterized mostly in the context of immortalized cells, and characteristic features include ultrabright telomeric foci, colocalization of telomere foci with PML (ALT-associated PML bodies, or APBs), extrachromosomal telomeric DNA (e.g. c-circles), and overall telomere length heterogeneity. While the overall rate of ALT in cancer is low (roughly 5%, overall), the prevalence of ALT is concentrated in certain cancer subtypes, including gliomas. Gliomas employing ALT have a common genomic signature: inactivating mutation of ATRX, a chromatin-modifying protein that functions at the telomeres. In order to study the role of ATRX loss in the biology of ALT in cancer, we have characterized a panel of six human cell lines derived from pediatric high grade glioma, two of which are ALT(+) and lack functional ATRX. Furthermore, in four pediatric and three adult high grade glioma cell lines that endogenously express functional ATRX, we knocked out ATRX using CRISPR/Cas9n and knocked down ATRX expression using a stable shRNA-mediated approach. Knockout or knockdown of ATRX was successful in these seven glioma cell lines. Overall, knockout and/or knockdown of ATRX induced the formation of ultrabright telomeric foci, as assessed by telomere FISH, in two out of seven glioma cell lines. However, the effect of ATRX loss on formation of c-circles and APBs was more variable: the presence of ultrabright telomeric foci was not predictive of the presence of c-circles or ALT-associated PML bodies. These results indicate that ATRX loss, alone, is not necessarily sufficient to activate the ALT pathway in gliomas. Further examination of ALT-competent and ALT-resistant cell lines from our cohort will yield clues as to additional ALT-associated gene expression changes. In addition, our results suggest that previously identified hallmarks of ALT (notably c-circles and APBs) may be more variable in cancer cells compared to immortalized normal cell lines. The ALT pathway is an intriguing target for synthetic lethality in the cancer subtypes that employ it; our hope is to better understand the ALT phenotype in cancer so that we may effectively translate ALT-specific therapeutics to the clinic. Citation Format: Jacqueline A. Brosnan-Cashman, Ming Yuan, Anthony J. Rizzo, Kaylar M. Myers, Walter T. Barry, Christine Davis, David M. Esopi, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. Context-dependent effects of ATRX loss on telomere biology in glioma cells [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 3465. doi:10.1158/1538-7445.AM2017-3465

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.

Introduction: Offspring born to obese mothers and/or mothers who gain excess weight during pregnancy are at increased risk of obesity and metabolic disease later in life, both of which are risk factors for certain cancers. While the biological mechanisms underlying these associations are not well understood, evidence from animal models and limited data from human studies suggests that DNA methylation may play an important role. Thus, we evaluated the association of maternal pre-pregnancy body mass index (BMI) and gestational weight gain (GWG) with DNA methylation in umbilical cord blood DNA using a candidate gene approach. Methods: This study includes 112 black and white mothers and their neonates in the Hormones in Umbilical Cord Blood Extended Study. Participants were enrolled in 2006-2007 from a prenatal clinic in Baltimore, MD. Self-reported, pre-pregnancy BMI was categorized as normal ( Results: A total of 29 CpG sites in 14 genes were differentially methylated with respect to pre-pregnancy BMI (FDR p-value ?0.05). A total of 30 CpG sites in 15 genes were differentially methylated with respect to GWG (FDR p-value ?0.05). Four CpG sites were significantly differentially methylated in the same direction with respect to both maternal pre-pregnancy BMI and GWG (ANAPC7, CAB39, HEYL and SAPCD2). We observed differences by race and sex. In particular, in males, pre-pregnancy overweight/obesity and excess GWG were associated with increased DNA methylation patterns at a majority of the significant CpG sites. Discussion: To our knowledge this study is one of the first to use targeted, next-generation bisulfite sequencing to quantify methylation in cord blood DNA. Our findings suggest maternal adiposity and GWG may influence DNA methylation of offspring genes, in a sex-specific manner. These findings warrant replication in large, prospective studies. Funding: NCI T32 CA009314, P30 CA006973, U54 CA091409, and the JHSPH Doctoral Thesis Research Fund. We wish to acknowledge Dr. Sarah Wheelan and Jennifer Meyers from the SKCCC Next Generation Sequencing Lab Citation Format: Megan A. Clarke, David Esopi, Srinivasan Yegnasubramanian, Elizabeth A. Platz, Corinne E. Joshu. A targeted, next-generation bisulfite sequencing approach to evaluate the influence of maternal pre-pregnancy body mass index and gestational weight gain on umbilical cord blood DNA methylation levels. [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 LB-379.

Prostate cancer (PCa) is one of the most heritable cancers. However, germline genetic variations that are consistently and highly associated with PCa have remained elusive. Recently, a variety of non-synonymous SNPs in HOXB13 have been identified in prostate cancer patients from distinct ethnic populations. Among the HOXB13 variants, HOXB13 G84E has been consistently shown to associate strongly with increased PCa risk in men of European descent. HOXB13 is a prostate-specific transcription factor that plays a role in prostate development. HOXB13 has also been implicated in various aspects of PCa biology. However, to this date, the exact role of HOXB13 in normal prostate physiology and in PCa biology remains obscure. Our lab previously reported that neither HOXB13 WT nor G84E alone can transform prostate cells and that the G84E variant does not behave differently from HOXB13 WT in interactions with cofactors (AR, MEIS2) and protein half-life. To further delineate the function of HOXB13 in the prostate and to identify G84E-induced alterations that subject G84E carriers to PCa susceptibility, we performed RNA-seq of the LAPC4 prostate cancer cell line overexpressing a control vector, HOXB13 WT or G84E. Comparisons of the RNA-seq datasets were made using Ingenuity Pathway Analysis (IPA) by selecting genes whose expression was altered by 2 standard deviations or higher. Among pathways altered by HOXB13 WT or G84E compared to the vector control dataset, the VDR/RXR activation pathway was negatively regulated by both WT and G84E. When the G84E dataset was compared to the WT dataset, the IPA analysis revealed that G84E can up-regulate the e-NOS signaling pathway. Interestingly, the top contributor to the difference imparted by HOXB13 G84E compared to WT was identified to be a set of genes regulated by HOXB13 itself. Overall, this preliminary analysis suggests that HOXB13 G84E may be a gain-of-function mutation whereby potential tumor-promoting functions of HOXB13 are over-activated. Finally, to further identify targets directly regulated by HOXB13 WT and G84E, we combined the RNA-seq datasets with HOXB13 overexpression ChIP-seq datasets we previously reported and a HOXB13 knockdown dataset in LAPC4 (Norris et al. 2009). We focused initially on genes that were shown to be up- or down-regulated in the same direction in the RNA-seq and HOXB13 knockdown datasets. Those genes were screened for nearby HOXB13 binding sites as annotated in our ChIP-seq analyses, followed by validation with droplet digital PCR. Among the genes that were down-regulated by HOXB13 WT and G84E include INPP4B, TNFSF10, IGFBP3, KLF5 and SOX9, which all have tumor suppressing functions in PCa. Among HOXB13-upregulated genes was BCHE, which is also implicated in the suppression of PCa initiation and progression. These results provide us with potential areas of further investigation in which HOXB13 G84E may differentially regulate transcription of these genes. Citation Format: Dorhyun Johng, Michael C. Haffner, Steven M. Mooney, David M. Esopi, Charles M. Ewing, Shuangling Chen, William B. Isaacs. Global analyses of HOXB13-regulated transcription reveal a potential link between HOXB13 G84E and prostate cancer risk. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2027.

miR-21 is the most commonly over-expressed microRNA (miRNA) in cancer and a proven oncogene. Hsa-miR-21 is located on chromosome 17q23.2, immediately downstream of the vacuole membrane protein-1 (VMP1) gene, also known as TMEM49. VMP1 transcripts initiate ∼130 kb upstream of miR-21, are spliced, and polyadenylated only a few hundred base pairs upstream of the miR-21 hairpin. On the other hand, primary miR-21 transcripts (pri-miR-21) originate within the last introns of VMP1, but bypass VMP1 polyadenylation signals to include the miR-21 hairpin. Here, we report that VMP1 transcripts can also bypass these polyadenylation signals to include miR-21, thus providing a novel and independently regulated source of miR-21, termed VMP1–miR-21. Northern blotting, gene-specific RT-PCR, RNA pull-down and DNA branching assays support that VMP1–miR-21 is expressed at significant levels in a number of cancer cell lines and that it is processed by the Microprocessor complex to produce mature miR-21. VMP1 and pri-miR-21 are induced by common stimuli, such as phorbol-12-myristate-13-acetate (PMA) and androgens, but show differential responses to some stimuli such as epigenetic modifying agents. Collectively, these results indicate that miR-21 is a unique miRNA capable of being regulated by alternative polyadenylation and two independent gene promoters. The ‘National Institutes of Health/National Cancer Institute’ [5R01CA143299 to S.E.L., 5P50CA058236 to S.E.L. (Project 1)]; Department of Defense Prostate Cancer Research Fund [W81XWH-08-13-5 to S.E.L.]; Patrick C. Walsh Prostate Cancer Research Fund (to S.E.L.); Spanish ‘Ministerio de Ciencia e Innovación’ [SAF2011-29730 to J.R.]. Funding for open access charge: National Institutes of Health/NCI R01CA143299.

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.

HOXB13 is a prostate-specific transcription factor that plays a critical role in the development of the prostate. Recently, our group, in collaboration with Dr. Kathleen Cooney's group at the University of Michigan, discovered germline mutations in HOXB13 in families with hereditary prostate cancer. Among the HOXB13 mutations, the G84E variant has been shown in multiple independent studies to recur at an elevated frequency in prostate cancer patients. To gain molecular insights into prostate cancer development, it is crucial to investigate the mechanism behind HOXB13 G84E's association with prostate cancer. Our previous study demonstrated that HOXB13 G84E alone or with AR cannot transform prostate cells and that the G84E variant does not differ from wild type in cellular localization, interaction with cofactors (AR, MEIS2) and protein stability. To further search for G84E-induced alterations that link G84E to prostate cancer predisposition, we performed ChIP-seq analysis to study the genome-wide occupancy of HOXB13 WT and G84E in LAPC4, a prostate cell line derived from an AR-positive lymph node metastasis. The predominant DNA consensus sequence for both HOXB13 WT and G84E identified by the ChIP-seq data (TTTTAT) was in congruence with the literature, signifying the validity of the dataset. The enrichments for binding sites with a range of fold enrichment scores were validated using ChIP-qPCR to further assess the quality of the dataset. Many enriched sites were in proximity to genes with functional relevance to prostate cancer (SMYD2, HOXA7, PMEPA1, MAP3K5, SHH, etc.). Sites that were uniquely bound by either HOXB13 WT or G84E were absent, although currently it is unknown whether some sites are more strongly bound by WT or G84E. Next, to examine genes that are regulated directly by HOXB13 binding, we crossed our ChIP-seq dataset with an LAPC4 HOXB13 knockdown microarray dataset obtained from Dr. Donald McDonnell's lab at Duke University. Among ∼70 genes whose expression changed by at least 2.6-fold with HOXB13 knockdown, ∼45 genes had adjacent binding sites for HOXB13 WT and G84E. Pathway analysis of the ∼45 genes using g:Profiler suggested that they are involved in the MAPK cascade. Given the apparent lack of G84E-unique binding sites, we questioned whether changes in the protein interactome might affect the function of HOXB13. To this end, we performed mass spectrometry on proteins co-immunoprecipitated with HOXB13 WT or G84E. The experiment identified fatty acid synthase as a binding partner of HOXB13 WT but not G84E. Although our genome- and proteome-wide analyses of HOXB13 WT and G84E need further validation, our preliminary data suggests the possibility of identifying the potential source of quantitative and qualitative difference caused by the G84E mutation by studying gene-protein and protein-protein interaction on a global-scale. Citation Format: Dorhyun Johng, Michael C. Haffner, David M. Esopi, William B. Isaacs. Genome– and proteome–wide analyses of HOXB13 and the G84E variant associated with prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 961. doi:10.1158/1538-7445.AM2015-961

TMPRSS2-ERG translocations occur in approximately 50% of prostate cancers and therefore represent one of the most frequently observed structural rearrangements in all cancers. However, little is known about the genomic architecture of such rearrangements. We therefore designed and optimized a pipeline involving target-capture of TMPRSS2 and ERG genomic sequences coupled with paired-end next generation sequencing to resolve genomic rearrangement breakpoints in TMPRSS2 and ERG at nucleotide resolution in a large series of primary prostate cancer specimens (n = 83). This strategy showed >90% sensitivity and specificity in identifying TMPRSS2-ERG rearrangements, and allowed identification of intra- and inter-chromosomal rearrangements involving TMPRSS2 and ERG with known and novel fusion partners. Our results indicate that rearrangement breakpoints show strong clustering in specific intronic regions of TMPRSS2 and ERG. The observed TMPRSS2-ERG rearrangements often exhibited complex chromosomal architecture associated with several intra- and inter-chromosomal rearrangements. Nucleotide resolution analysis of breakpoint junctions revealed that the majority of TMPRSS2 and ERG rearrangements (∼88%) occurred at or near regions of microhomology or involved insertions of one or more base pairs. This architecture implicates nonhomologous end joining (NHEJ) and microhomology mediated end joining (MMEJ) pathways in the generation of such rearrangements. These analyses have provided important insights into the molecular mechanisms involved in generating prostate cancer-specific recurrent rearrangements. Citation Format: Christopher Weier, Michael Haffner, Timothy Mosbruger, David Esopi, Jessica Hicks, Qizhi Zheng, William B. Isaacs, Angelo M. De Marzo, William G. Nelson, Srinivasan Yegnasubramanian. Nucleotide-resolution genomic breakpoint analysis of TMPRSS2-ERG rearrangements in prostate cancer by target-capture next-generation sequencing. [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 1773. doi:10.1158/1538-7445.AM2013-1773

After castration was shown by Charles Huggins in 1941 to be an effective treatment for advanced prostate cancer, androgenic hormones were assumed to be responsible in some way for disease development. Nonetheless, the manner in which androgens might contribute to prostatic carcinogenesis has remained elusive over the subsequent seven decades. Androgen levels decline steadily throughout adulthood in men, just as prostate cancers begin to appear (Rohrmann S et al. J Clin Endocrinol Metabol 92:2519-25, 2007). In addition, the action of testosterone, and its metabolite dihydrotestosterone, on prostate epithelial cells in adult men tends to be the promotion of terminal differentiation by activating transcription of target genes, such as PSA and TMPRSS2, which form the secretion elaborated by the prostate for the ejaculate. By driving terminal differentiation of normal prostate cells, androgens seem unlikely to cause neoplastic transformation. However, new insights into the generation of somatic genome rearrangements that create fusion oncogenes in prostate cells have revealed a new mechanism of androgen receptor-associated cancer pathogenesis. The fusion oncogene TMPRSS2-ERG, present in at least half of all prostate cancer cases, may be the most common somatic gene fusion event in all of human cancer (Tomlins SA et al. Science 310:644-8, 2005). We have found that androgen receptor-triggered transcription in prostate cells leads to the recruitment of topoisomerase II-beta (TOP2B) by the receptor to sites of TMPRSS2-ERG genomic breakpoints, promoting TOP2B-mediated DNA double strand breaks and de novo TMPRSS2-ERG fusion transcripts. The results implicate androgens in the creation of TMPRSS2-ERG rearrangements, via a mechanism involving the androgen receptor and TOP2B, and provide new hints as to how prostate cancers might be better prevented. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN05-04.