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.

A high-throughput screen of pharmacologically active compounds for inhibitors of UHRF1 reveals epigenetic activity of anthracycline derivative chemotherapeutic drugs.

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.

ATRX loss induces multiple hallmarks of the alternative lengthening of telomeres (ALT) phenotype in human glioma cell lines in a cell line-specific manner.

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.

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.

AIM1 is an actin-binding protein that suppresses cell migration and micrometastatic dissemination.

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 ß-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.

Molecular evidence that invasive adenocarcinoma can mimic prostatic intraepithelial neoplasia (PIN) and intraductal carcinoma through retrograde glandular colonization.

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 adenocarcinomas aberrantly expressing p63 are molecularly distinct from usual-type prostatic adenocarcinomas.

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 ß-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.

Tracking the clonal origin of lethal prostate cancer.

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.

Nucleotide resolution analysis of TMPRSS2 and ERG rearrangements in prostate cancer.

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.

Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers.

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.

Identification of replication competent murine gammaretroviruses in commonly used prostate cancer cell lines.

A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV, Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral "contamination", much like routine mycoplasma testing.

Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements.

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.