Co-targeting BET, CBP, and p300 inhibits neuroendocrine signalling in androgen receptor-null prostate cancer.

There are diverse phenotypes of castration-resistant prostate cancer, including neuroendocrine disease, that vary in their sensitivity to drug treatment. The efficacy of BET and CBP/p300 inhibitors in prostate cancer is attributed, at least in part, to their ability to decrease androgen receptor (AR) signalling. However, the activity of BET and CBP/p300 inhibitors in prostate cancers that lack the AR is unclear. In this study, we showed that BRD4, CBP, and p300 were co-expressed in AR-positive and AR-null prostate cancer. A combined inhibitor of these three proteins, NEO2734, reduced the growth of both AR-positive and AR-null organoids, as measured by changes in viability, size, and composition. NEO2734 treatment caused consistent transcriptional downregulation of cell cycle pathways. In neuroendocrine models, NEO2734 treatment reduced ASCL1 levels and other neuroendocrine markers, and reduced tumour growth in vivo. Collectively, these results show that epigenome-targeted inhibitors cause decreased growth and phenotype-dependent disruption of lineage regulators in neuroendocrine prostate cancer, warranting further development of compounds with this activity in the clinic. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Defining the challenges and opportunities for using patient-derived models in prostate cancer research.

BACKGROUND: There are relatively few widely used models of prostate cancer compared to other common malignancies. This impedes translational prostate cancer research because the range of models does not reflect the diversity of disease seen in clinical practice. In response to this challenge, research laboratories around the world have been developing new patient-derived models of prostate cancer, including xenografts, organoids, and tumor explants. METHODS: In May 2023, we held a workshop at the Monash University Prato Campus for researchers with expertise in establishing and using a variety of patient-derived models of prostate cancer. This review summarizes our collective ideas on how patient-derived models are currently being used, the common challenges, and future opportunities for maximizing their usefulness in prostate cancer research. RESULTS: An increasing number of patient-derived models for prostate cancer are being developed. Despite their individual limitations and varying success rates, these models are valuable resources for exploring new concepts in prostate cancer biology and for preclinical testing of potential treatments. Here we focus on the need for larger collections of models that represent the changing treatment landscape of prostate cancer, robust readouts for preclinical testing, improved in vitro culture conditions, and integration of the tumor microenvironment. Additional priorities include ensuring model reproducibility, standardization, and replication, and streamlining the exchange of models and data sets among research groups. CONCLUSIONS: There are several opportunities to maximize the impact of patient-derived models on prostate cancer research. We must develop large, diverse and accessible cohorts of models and more sophisticated methods for emulating the intricacy of patient tumors. In this way, we can use the samples that are generously donated by patients to advance the outcomes of patients in the future.

CDK9 inhibition inhibits multiple oncogenic transcriptional and epigenetic pathways in prostate cancer.

BACKGROUND: Cyclin-dependent kinase 9 (CDK9) stimulates oncogenic transcriptional pathways in cancer and CDK9 inhibitors have emerged as promising therapeutic candidates. METHODS: The activity of an orally bioavailable CDK9 inhibitor, CDKI-73, was evaluated in prostate cancer cell lines, a xenograft mouse model, and patient-derived tumor explants and organoids. Expression of CDK9 was evaluated in clinical specimens by mining public datasets and immunohistochemistry. Effects of CDKI-73 on prostate cancer cells were determined by cell-based assays, molecular profiling and transcriptomic/epigenomic approaches. RESULTS: CDKI-73 inhibited proliferation and enhanced cell death in diverse in vitro and in vivo models of androgen receptor (AR)-driven and AR-independent models. Mechanistically, CDKI-73-mediated inhibition of RNA polymerase II serine 2 phosphorylation resulted in reduced expression of BCL-2 anti-apoptotic factors and transcriptional defects. Transcriptomic and epigenomic approaches revealed that CDKI-73 suppressed signaling pathways regulated by AR, MYC, and BRD4, key drivers of dysregulated transcription in prostate cancer, and reprogrammed cancer-associated super-enhancers. These latter findings prompted the evaluation of CDKI-73 with the BRD4 inhibitor AZD5153, a combination that was synergistic in patient-derived organoids and in vivo. CONCLUSION: Our work demonstrates that CDK9 inhibition disrupts multiple oncogenic pathways and positions CDKI-73 as a promising therapeutic agent for prostate cancer, particularly aggressive, therapy-resistant subtypes.

Patient-derived castration-resistant prostate cancer model revealed CTBP2 upregulation mediated by OCT1 and androgen receptor.

BACKGROUND: Prostate cancer is dependent on androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) has proven effective in targeting prostate cancer. However, castration-resistant prostate cancer (CRPC) eventually emerges. AR signaling inhibitors (ARSI) have been also used, but resistance to these agents develops due to genetic AR alterations and epigenetic dysregulation. METHODS: In this study, we investigated the role of OCT1, a member of the OCT family, in an AR-positive CRPC patient-derived xenograft established from a patient with resistance to ARSI and chemotherapy. We conducted a genome-wide analysis chromatin immunoprecipitation followed by sequencing and bioinformatic analyses using public database. RESULTS: Genome-wide analysis of OCT1 target genes in PDX 201.1 A revealed distinct OCT1 binding sites compared to treatment-naïve cells. Bioinformatic analyses revealed that OCT1-regulated genes were associated with cell migration and immune system regulation. In particular, C-terminal Binding Protein 2 (CTBP2), an OCT1/AR target gene, was correlated with poor prognosis and immunosuppressive effects in the tumor microenvironment. Metascape revealed that CTBP2 knockdown affects genes related to the immune response to bacteria. Furthermore, TISIDB analysis suggested the relationship between CTBP2 expression and immune cell infiltration in prostate cancer, suggesting that it may contribute to immune evasion in CRPC. CONCLUSIONS: Our findings shed light on the genome-wide network of OCT1 and AR in AR-positive CRPC and highlight the potential role of CTBP2 in immune response and tumor progression. Targeting CTBP2 may represent a promising therapeutic approach for aggressive AR-positive CRPC. Further validation will be required to explore novel therapeutic strategies for CRPC management.

Translational offsetting as a mode of estrogen receptor α-dependent regulation of gene expression.

Estrogen receptor alpha (ERα) activity is associated with increased cancer cell proliferation. Studies aiming to understand the impact of ERα on cancer-associated phenotypes have largely been limited to its transcriptional activity. Herein, we demonstrate that ERα coordinates its transcriptional output with selective modulation of mRNA translation. Importantly, translational perturbations caused by depletion of ERα largely manifest as "translational offsetting" of the transcriptome, whereby amounts of translated mRNAs and corresponding protein levels are maintained constant despite changes in mRNA abundance. Transcripts whose levels, but not polysome association, are reduced following ERα depletion lack features which limit translation efficiency including structured 5'UTRs and miRNA target sites. In contrast, mRNAs induced upon ERα depletion whose polysome association remains unaltered are enriched in codons requiring U34-modified tRNAs for efficient decoding. Consistently, ERα regulates levels of U34-modifying enzymes and thereby controls levels of U34-modified tRNAs. These findings unravel a hitherto unprecedented mechanism of ERα-dependent orchestration of transcriptional and translational programs that may be a pervasive mechanism of proteome maintenance in hormone-dependent cancers.

New approaches to targeting epigenetic regulation in prostate cancer.

PURPOSE OF REVIEW: Many clinical trials are currently underway to target the epigenome of castration-resistant prostate cancer. In this review, we summarize the major epigenetic alterations that occur during prostate cancer progression, describe their biological consequences, and highlight potential of therapies that target epigenetic regulators for use in patients. RECENT FINDINGS: Epigenetic alterations frequently occur in tumour suppressor genes, DNA repair genes, and genes that regulate cell proliferation and differentiation. Unlike genetic alterations, epigenetic changes are reversible, making them promising targets for cancer therapy. Epigenetic regulators can be divided into three broad groups: writers, readers, and erasers , each with specific drug targets that are being assessed in phase I and II clinical trials for prostate cancer. CBP/p300, and BRD4 are coregulators of the androgen receptor and inhibit androgen signalling, making bromodomain extra-terminal inhibitors and CBP/p300 inhibitors attractive targets in prostate cancer. Enhancer of zeste homolog 2, a histone methyltransferase, is also a potential target in castrate-resistant prostate cancer. An emerging direction is to combine epigenetic inhibitors with other compounds to enhance their efficacy. SUMMARY: Preclinical studies indicate that the epigenome is a potential target in prostate cancer, and clinical trials are testing multiple agents that target the epigenome in different ways. However, the process of translating these therapies into the clinic is ongoing and none have yet been approved for castrate-resistant prostate cancer.

Androgen receptor enhancer amplification in matched patient-derived xenografts of primary and castrate-resistant prostate cancer.

Amplifications of the androgen receptor (AR) occur in up to 80% of men with castration-resistant prostate cancer (CRPC). Recent studies highlighted that these amplifications not only span the AR gene but usually encompass a distal enhancer. This represents a newly recognised, non-coding mechanism of resistance to AR-directed therapies, including enzalutamide. To study disease progression before and after AR amplification, we used tumour samples from a castrate-sensitive primary tumour and castrate-resistant metastasis of the same patient. For subsequent functional and genomic studies, we established serially transplantable patient-derived xenografts (PDXs). Whole genome sequencing showed that alterations associated with poor prognosis, such as TP53 and PTEN loss, existed before androgen deprivation therapy, followed by co-amplification of the AR gene and enhancer after the development of metastatic CRPC. The PDX of the primary tumour, without the AR amplification, was sensitive to AR-directed treatments, including castration, enzalutamide, and apalutamide. The PDX of the metastasis, with the AR amplification, had higher AR and AR-V7 expression in castrate conditions, and was resistant to castration, apalutamide, and enzalutamide in vivo. Treatment with a BET inhibitor outperformed the AR-directed therapies for the metastasis, resulting in tumour regression for some, but not all, grafts. Therefore, this study provides novel matched PDXs to test potential treatments that target the overabundance of AR in tumours with AR enhancer amplifications. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Enduring epigenetic landmarks define the cancer microenvironment.

The growth and progression of solid tumors involves dynamic cross-talk between cancer epithelium and the surrounding microenvironment. To date, molecular profiling has largely been restricted to the epithelial component of tumors; therefore, features underpinning the persistent protumorigenic phenotype of the tumor microenvironment are unknown. Using whole-genome bisulfite sequencing, we show for the first time that cancer-associated fibroblasts (CAFs) from localized prostate cancer display remarkably distinct and enduring genome-wide changes in DNA methylation, significantly at enhancers and promoters, compared to nonmalignant prostate fibroblasts (NPFs). Differentially methylated regions associated with changes in gene expression have cancer-related functions and accurately distinguish CAFs from NPFs. Remarkably, a subset of changes is shared with prostate cancer epithelial cells, revealing the new concept of tumor-specific epigenome modifications in the tumor and its microenvironment. The distinct methylome of CAFs provides a novel epigenetic hallmark of the cancer microenvironment and promises new biomarkers to improve interpretation of diagnostic samples.

Proteomic Profiling of Human Prostate Cancer-associated Fibroblasts (CAF) Reveals LOXL2-dependent Regulation of the Tumor Microenvironment.

In prostate cancer, cancer-associated fibroblasts (CAF) exhibit contrasting biological properties to non-malignant prostate fibroblasts (NPF) and promote tumorigenesis. Resolving intercellular signaling pathways between CAF and prostate tumor epithelium may offer novel opportunities for research translation. To this end, the proteome and phosphoproteome of four pairs of patient-matched CAF and NPF were characterized to identify discriminating proteomic signatures. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a hyper reaction monitoring data-independent acquisition (HRM-DIA) workflow. Proteins that exhibited a significant increase in CAF versus NPF were enriched for the functional categories "cell adhesion" and the "extracellular matrix." The CAF phosphoproteome exhibited enhanced phosphorylation of proteins associated with the "spliceosome" and "actin binding." STRING analysis of the CAF proteome revealed a prominent interaction hub associated with collagen synthesis, modification, and signaling. It contained multiple collagens, including the fibrillar types COL1A1/2 and COL5A1; the receptor tyrosine kinase discoidin domain-containing receptor 2 (DDR2), a receptor for fibrillar collagens; and lysyl oxidase-like 2 (LOXL2), an enzyme that promotes collagen crosslinking. Increased activity and/or expression of LOXL2 and DDR2 in CAF were confirmed by enzymatic assays and Western blotting analyses. Pharmacological inhibition of CAF-derived LOXL2 perturbed extracellular matrix (ECM) organization and decreased CAF migration in a wound healing assay. Further, it significantly impaired the motility of co-cultured RWPE-2 prostate tumor epithelial cells. These results indicate that CAF-derived LOXL2 is an important mediator of intercellular communication within the prostate tumor microenvironment and is a potential therapeutic target.

Head-to-head competition does not affect pacing or performance in 1 km cycling time trials.

Previous research has shown enhanced performance and altered pacing behaviour in the presence of a virtual opponent during middle-distance cycling time trials with a duration of 2 min and longer. The purpose of this study was to determine whether these effects are also present in cycling time trials of shorter duration. Twelve physically active men completed three 1-km time trials. After a familiarisation trial (FAM), participants performed two experimental conditions: one without opponent (NO) and one with a virtual opponent (OP). Repeated-measures ANOVAs were used to assess differences in pacing and performance using power output and duration (p<0.05). No differences in mean finishing times (FAM: 91.5 ± 7.7 s; NO: 91.6 ± 6.4 s; OP: 90.9 ± 4.9 s; p=0.907) or power output (FAM: 382 ± 111 W; NO: 363 ± 80 W; OP: 367 ± 67; p=0.564) were found between experimental conditions. Furthermore, no differences in pacing profiles between experimental conditions were found (p=0.199). Similarly, rate of perceived exertion did not differ between experimental conditions at any moment (p=0.831). In conclusion, unlike events of a more prolonged duration (>2 min), the presence of an opponent did not affect participants' pacing behaviour in short duration 1-km time trials.

Establishing a cryopreservation protocol for patient-derived xenografts of prostate cancer.

BACKGROUND: Serially transplantable patient-derived xenografts (PDXs) are invaluable preclinical models for studying tumor biology and evaluating therapeutic agents. As these models are challenging to establish from prostate cancer specimens, the ability to preserve them through cryopreservation has several advantages for ongoing research. Despite this, there is still uncertainty about the ability to cryopreserve PDXs of prostate cancer. This study compared three different cryopreservation protocols to identify a method that can be used to reproducibly cryopreserve a diverse cohort of prostate cancer PDX models. METHODS: One serially transplantable prostate cancer PDX from the Melbourne Urological Research Alliance cohort was used to compare three cryopreservation protocols: slow freezing in fetal calf serum (FCS) with 10% dimethyl sulfoxide (DMSO), FCS with 10% DMSO supplemented with the Rho-associated kinase (ROCK) inhibitor Y-27632 and vitrification. The efficiency of the slow freezing protocols was then assessed in 17 additional prostate cancer PDXs. Following cryopreservation, PDXs were re-established in host mice that were either intact and supplemented with testosterone or castrated. Graft take rate, tumor growth, histological features, and transcriptome profiles before and after cryopreservation were compared. RESULTS: Slow freezing maintained the viability and histological features of prostate cancer PDXs, and the addition of a ROCK inhibitor increased their growth following cryopreservation. Using the slow freezing method, we re-established 100% of PDXs grown in either testosterone-supplemented or castrated host mice. Importantly, the long-term tumor growth rate and transcriptome profile were maintained following cryopreservation. CONCLUSION: This study has identified a protocol to reliably cryopreserve and re-establish a diverse cohort of serially transplantable PDXs of prostate cancer. This study has the potential to significantly improve the practicality of maintaining PDX models. Cryopreservation may also increase the accessibility of these important resources and provide new opportunities for preclinical studies on a broader spectrum of prostate tumors.

Increased maternal abdominal subcutaneous fat thickness and body mass index are associated with increased cesarean delivery: A prospective cohort study.

INTRODUCTION: Early pregnancy body mass index (BMI) is known to predict adverse pregnancy outcomes but does not account for body fat distribution. This study aimed to determine prospectively whether maternal abdominal subcutaneous fat thickness (SCFT) measured by ultrasound at the fetal morphology scan is a better predictor than BMI of mode of delivery and other pregnancy outcomes. MATERIAL AND METHODS: This was a prospective cohort study of women delivering singleton neonates at a tertiary public hospital. Women were included if they had appropriate images at the routine fetal anomaly ultrasound scan and delivered in the facility. The primary outcome was mode of delivery categorized as cesarean section or vaginal delivery. The relation between maternal SCFT and BMI was described using the Pearson correlation coefficient. The association of maternal abdominal SCFT BMI at booking-in was compared with pregnancy outcomes using univariate linear and logistic regression. RESULTS: SCFT and BMI were obtained for 997 women. The median (interquartile range) SCFT was 15.3 mm (12.8-19.6) and median (interquartile range) BMI 24.3 kg/m2 (21.7-28.3). Maternal abdominal SCFT and BMI were highly correlated (R2  = 0.55). Both were significantly associated with cesarean delivery: SCFT per 5 mm (odds ratio [OR] 1.32, 95% confidence interval (CI) 1.18-1.48; BMI per 5 kg/m2 OR 1.29, 95% CI 1.15-1.44. CONCLUSIONS: Maternal abdominal SCFT and BMI were both significantly associated with cesarean delivery and other outcomes. More research is needed to define the strengths of maternal SCFT in predicting pregnancy outcomes.

Intraductal carcinoma of the prostate can evade androgen deprivation, with emergence of castrate-tolerant cells.

OBJECTIVE: To determine the relevance of intraductal carcinoma of the prostate (IDC-P) in advanced prostate cancer by first examining whether IDC-P was originally present in patients who later developed advanced prostate cancer and then using patient-derived xenografts (PDXs) to investigate the response of IDC-P to androgen deprivation therapy (ADT). MATERIALS AND METHODS: We conducted a retrospective pathology review of IDC-P in primary prostate biopsy or surgery specimens from 38 men who subsequently developed advanced prostate cancer. Overall survival was calculated using the Kaplan-Meier method. To demonstrate the response of IDC-P to ADT, we established PDXs from seven patients with familial and/or high-risk sporadic prostate cancer. After castration and testosterone restoration of host mice, we measured the volume and proliferation of IDC-P within PDX grafts. RESULTS: We found that IDC-P was a prominent feature in the primary prostate specimens, present in 63% of specimens and often co-existing with poorly differentiated adenocarcinoma. Overall survival was similar in patients with or without IDC-P. In the PDXs from all seven patients, IDC-P was identified and present at a similar volume to adenocarcinoma. Residual IDC-P lesions persisted after host castration and, similar to castrate-tolerant adenocarcinoma, testosterone restoration led to tumour regeneration. CONCLUSION: The study showed that IDC-P is prevalent in aggressive prostate cancer and contains cells that can withstand androgen deprivation. Thus, IDC-P appears functionally relevant in advanced prostate cancer. The presence of IDC-P may be a trigger to develop innovative clinical management plans.

Establishment of primary patient-derived xenografts of palliative TURP specimens to study castrate-resistant prostate cancer.

BACKGROUND: Fresh patient specimens of castrate-resistant prostate cancer (CRPC) are invaluable for studying tumor heterogeneity and responses to current treatments. They can be used for primary patient-derived xenografts (PDXs) or serially transplantable PDXs, but only a small proportion of samples grow successfully. To improve the efficiency and quality of PDXs, we investigated the factors that determine the initial engraftment of patient tissues derived from TURP specimens. METHODS: Fresh tissue was collected from castrate patients who required a TURP for urinary symptoms. Tissue was grafted under the renal capsule of immune-compromised mice for up to 14 weeks. The abundance of cancer in ungrafted and grafted specimens was compared using histopathology. Mice were castrated or implanted with testosterone pellets to determine the androgen-responsiveness of CRPC PDXs from TURP tissue. RESULTS: Primary PDXs were successfully established from 7 of 10 patients that underwent grafting. Of the 112 grafts generated from these 10 patients, 21% contained cancer at harvest. Grafts were most successful when the original patient specimens contained high amounts of viable cancer, defined as samples with (i) at least 50% cancer cells, (ii) no physical damage, and (iii) detectable Ki67 expression. PDX grafts survived in castrated hosts and proliferated in response to testosterone, confirming that they were castrate resistant but androgen-responsive. CONCLUSIONS: Primary PDXs of CRPC can be established from TURP specimens with modest success. The take rate can be increased if the original tissues contain sufficient numbers of actively proliferating cancer cells. Selecting specimens with abundant viable cancer will maximize the rate of engraftment and increase the efficiency of establishing PDXs that can be serially transplanted.

FOXA2 rewires AP-1 for transcriptional reprogramming and lineage plasticity in prostate cancer.

FOXA family proteins act as pioneer factors by remodeling compact chromatin structures. FOXA1 is crucial for the chromatin binding of the androgen receptor (AR) in both normal prostate epithelial cells and the luminal subtype of prostate cancer (PCa). Recent studies have highlighted the emergence of FOXA2 as an adaptive response to AR signaling inhibition treatments. However, the role of the FOXA1 to FOXA2 transition in regulating cancer lineage plasticity remains unclear. Our study demonstrates that FOXA2 binds to distinct classes of developmental enhancers in multiple AR-independent PCa subtypes, with its binding depending on LSD1. Moreover, we reveal that FOXA2 collaborates with JUN at chromatin and promotes transcriptional reprogramming of AP-1 in lineage-plastic cancer cells, thereby facilitating cell state transitions to multiple lineages. Overall, our findings underscore the pivotal role of FOXA2 as a pan-plasticity driver that rewires AP-1 to induce the differential transcriptional reprogramming necessary for cancer cell lineage plasticity.

Advances in preclinical models of prostate cancer for research discovery.

There is longstanding interest in the role of androgens in the aetiology of prostate cancer, one of the most common malignancies worldwide. In this review, we reflect on the ways that knowledge of prostate development and hormone action have catalysed advances in the management of patients with prostate cancer. The use of hormone therapies to treat prostate cancer has changed significantly over time, including the emergence of androgen receptor signalling inhibitors (ARSI). These compounds have improved outcomes for patients with castration-resistant prostate cancer, which was once considered 'androgen-independent' but is clearly still driven by androgen receptor signalling in many cases. There is also a need for new therapies to manage neuroendocrine prostate cancer, which is not responsive to hormonal agents. One of the major gaps is understanding how treatment-induced neuroendocrine prostate cancer emerges and whether it can be re-sensitised to treatment. Patient-derived models, including patient-derived xenografts (PDXs), will be instrumental in facilitating future discoveries in these areas. Developments in the use of PDXs have been fostered by lessons from the field of endocrinology, such as the role of stroma and hormones in normal and developmental tissues. Thus, there is ongoing reciprocity between the discoveries in endocrinology and advances in prostate cancer research and treatment.

The future of patient-derived xenografts in prostate cancer research.

Patient-derived xenografts (PDXs) are generated by engrafting human tumours into mice. Serially transplantable PDXs are used to study tumour biology and test therapeutics, linking the laboratory to the clinic. Although few prostate cancer PDXs are available in large repositories, over 330 prostate cancer PDXs have been established, spanning broad clinical stages, genotypes and phenotypes. Nevertheless, more PDXs are needed to reflect patient diversity, and to study new treatments and emerging mechanisms of resistance. We can maximize the use of PDXs by exchanging models and datasets, and by depositing PDXs into biorepositories, but we must address the impediments to accessing PDXs, such as institutional, ethical and legal agreements. Through collaboration, researchers will gain greater access to PDXs representing diverse features of prostate cancer.

The kallikrein 14 gene is down-regulated by androgen receptor signalling and harbours genetic variation that is associated with prostate tumour aggressiveness.

Kallikrein 14 (KLK14) has been proposed as a useful prognostic marker in prostate cancer, with expression reported to be associated with tumour characteristics such as higher stage and Gleason score. KLK14 tumour expression has also shown the potential to predict prostate cancer patients at risk of disease recurrence after radical prostatectomy. The KLKs are a remarkably hormone-responsive family of genes, although detailed studies of androgen regulation of KLK14 in prostate cancer have not been undertaken to date. Using in vitro studies, we have demonstrated that unlike many other prostatic KLK genes that are strictly androgen responsive, KLK14 is more broadly expressed and inversely androgen regulated in prostate cancer cells. Given these results and evidence that KLK14 may play a role in prostate cancer prognosis, we also investigated whether common genetic variants in the KLK14 locus are associated with risk and/or aggressiveness of prostate cancer in approximately 1200 prostate cancer cases and 1300 male controls. Of 41 single nucleotide polymorphisms assessed, three were associated with higher Gleason score (≥7): rs17728459 and rs4802765, both located upstream of KLK14, and rs35287116, which encodes a p.Gln33Arg substitution in the KLK14 signal peptide region. Our findings provide further support for KLK14 as a marker of prognosis in prostate cancer.

A variant of the KLK4 gene is expressed as a cis sense-antisense chimeric transcript in prostate cancer cells.

In humans, more than 30,000 chimeric transcripts originating from 23,686 genes have been identified. The mechanisms and association of chimeric transcripts arising from chromosomal rearrangements with cancer are well established, but much remains unknown regarding the biogenesis and importance of other chimeric transcripts that arise from nongenomic alterations. Recently, a SLC45A3-ELK4 chimera has been shown to be androgen-regulated, and is overexpressed in metastatic or high-grade prostate tumors relative to local prostate cancers. Here, we characterize the expression of a KLK4 cis sense-antisense chimeric transcript, and show other examples in prostate cancer. Using non-protein-coding microarray analyses, we initially identified an androgen-regulated antisense transcript within the 3' untranslated region of the KLK4 gene in LNCaP cells. The KLK4 cis-NAT was validated by strand-specific linker-mediated RT-PCR and Northern blotting. Characterization of the KLK4 cis-NAT by 5' and 3' rapid amplification of cDNA ends (RACE) revealed that this transcript forms multiple fusions with the KLK4 sense transcript. Lack of KLK4 antisense promoter activity using reporter assays suggests that these transcripts are unlikely to arise from a trans-splicing mechanism. 5' RACE and analyses of deep sequencing data from LNCaP cells treated +/-androgens revealed six high-confidence sense-antisense chimeras of which three were supported by the cDNA databases. In this study, we have shown complex gene expression at the KLK4 locus that might be a hallmark of cis sense-antisense chimeric transcription.