The Prostate

Benign prostatic hyperplasia (BPH) is a non-neoplastic proliferative disease producing lower urinary tract symptoms related to the enlarged prostate. BPH is pathologically characterized by hyperplastic growth in both epithelial and stromal compartments. Androgen signaling is essential for prostate function and androgen blockade is the second-line medical therapy to relieve symptoms of BPH. Here we examined the prostates of probasin promoter-driven prolactin (Pb-PRL) transgenic mice, a robust model of BPH that spontaneously develops prostate enlargement, to investigate prostate regression in response to surgical castration. Serial ultrasound imaging demonstrated very uniform self-limited growth of Pb-PRL prostate volume that is consistent with the benign, limited cellular proliferation characteristic of BPH and that contrasts with the highly variable, exponential growth of murine prostate cancer models. Castration elicited only a partial reduction in prostate volume, relative to castration-induced regression of the normal prostate gland. The anti-androgen finasteride induced a diminished reduction of Pb-PRL prostate volume versus castration alone. The limited extent of Pb-PRL mouse prostate volume regression correlated with the initial volume of the stromal compartment, suggesting a differential sensitivity to androgen withdrawal of the epithelial and stroma compartments. Indeed, two-dimensional morphometric analyses revealed a distinctly reduced rate of regression for the stromal compartment in Pb-PRL mice. The myofibroblast component of the Pb-PRL prostate stroma appeared normal, but contained more fibroblasts and extracellular collagen deposition. Like normal prostate, the rate of regression of the Pb-PRL prostate was partially dependent on TGF{beta} and TNF signaling, but unlike the normal prostate, the extent of castration-induced regression was not affected by TGF{beta} or TNF blockade. Our studies show that androgen deprivation can effectively reduce the overall volume of hyperplastic prostate, but the stromal compartment is relatively resistant, suggesting additional therapies might be required to offer an effective treatment for the clinical manifestations of BPH.

ContextSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry is facilitated by transmembrane protease serine 2 (TMPRSS2), which is regulated by the androgen receptor (AR). Androgen deprivation therapy (ADT), widely used in prostate cancer treatment, may potentially modulate TMPRSS2 expression, affecting SARS-CoV-2 infection susceptibility and severity. ObjectiveTo evaluate the impact of ADT on pulmonary TMPRSS2 expression in prostate cancer patients and analyze differences in expression patterns associated with specific ADT regimens. DesignWe examined TMPRSS2 immunohistochemical expression in lung tissue from 20 consecutive autopsy cases of men with prostate cancer (6 receiving ADT at time of death), compared with non-ADT prostate cancer patients and age-matched women controls. Histoscores were calculated by assessing percentage and intensity of pneumocyte TMPRSS2 expression. ResultsProstate cancer patients receiving ADT showed significantly reduced pulmonary TMPRSS2 expression compared to non-ADT patients (mean histoscores: 152.7 vs. 225.0, p=0.037) and age-matched women controls (mean histoscores: 152.7 vs. 238.0, p=0.024). Direct AR antagonists (apalutamide, bicalutamide) produced more pronounced TMPRSS2 suppression than GnRH modulators or androgen biosynthesis inhibitors. No significant correlation was observed between TMPRSS2 expression and Gleason score, PSA levels, or underlying lung pathology. ConclusionOur findings demonstrate that ADT significantly reduces pulmonary TMPRSS2 expression, with direct AR antagonists showing the strongest effect. This suggests a potential mechanistic explanation for differential COVID-19 susceptibility and provides rationale for investigating AR-targeted therapies as potential protective interventions against SARS-CoV-2 infection severity.

ABSTRACT/SUMMARYThe cellular composition and disease susceptibilities of the distinct zones of the human prostate remain incompletely understood. Through extensive single-cell RNA sequencing (scRNA-seq) of benign regions from prostatectomy specimens, we identified a basal cell population expressing WIF1, VCAN, and NRG1, among other genes, that was significantly enriched in the transition zone (TZ). Benign prostatic hyperplasia (BPH) is a common condition that causes widespread morbidity and is nearly exclusively localized to the TZ. Analysis of previously published scRNA-seq datasets further confirmed that WIF1+ basal cells were significantly enriched in BPH compared to normal prostate. Pathway and cell-cell communication analyses revealed that this basal subtype is associated with programs related to cell proliferation, epithelial-mesenchymal transition (EMT), angiogenesis, and hormone response. Together, the molecular signature, zonal distribution, and pathway enrichment suggest that TZ-enriched WIF1+ basal cells may contribute to BPH pathogenesis by promoting epithelial and stromal remodeling.

Benign prostatic hyperplasia (BPH) is a prevalent age-related condition often characterized by debilitating urinary symptoms. Its etiology is believed to stem from hormonal imbalance, particularly an elevated estradiol-to-testosterone ratio and chronic inflammation. Our previous studies using a mouse steroid hormone imbalance model identified a specific increase in macrophages that migrate and accumulate in the prostate lumen where they differentiate into lipid-laden foam cells in mice implanted with testosterone and estradiol pellets, but not in sham animals. The current study focused on further characterizing the cellular heterogeneity of the prostate in this model as well as identifying the specific transcriptomic signature of the recruited foam cells. Moreover, we aimed to identify the epithelia-derived signals that drive macrophage infiltration and luminal translocation. Male C57BL/6J mice were implanted with slow-release testosterone and estradiol pellets (T+E2) and harvested the ventral prostates two weeks later for scRNA-seq analysis, or performed sham surgery. We identified Ear2+ and Cd72+ macrophages that were elevated in response to steroid hormone imbalance, whereas a Mrc1+ resident macrophage population did not change. In addition, an Spp1+ foam cell cluster was almost exclusively found in T+E2 mice. Further markers of foam cells were also identified, including Gpnmb and Trem2, and GPNMB was confirmed as a novel histological marker with immunohistochemistry. Foam cells were also shown to express known pathological factors Vegf, Tgfb1, Ccl6, Cxcl16 and Mmp12. Intriguingly, a screen for chemokines identified the upregulation of epithelial-derived Cxcl17, a known monocyte attractant, in T+E2 prostates suggesting that it might be responsible for the elevated macrophage number as well as their translocation to the lumen. Our study identified macrophage subsets that respond to steroid hormone imbalance as well as further confirmed a potential pathological role of luminal foam cells in the prostate. These results underscore a pathological role of the identified prostate foam cells and suggests CXCL17-mediated macrophage migration as a critical initiating event.

Benign prostatic hyperplasia (BPH) is closely associated with obesity and metabolic syndrome. Although 5-reductase inhibitors (5ARIs) are widely used to treat BPH, their effectiveness varies significantly, particularly in obese patients. However, the mechanisms by which obesity affects prostate growth and modulates therapeutic responses remain poorly understood. In mice, highfat diet (HFD) increased prostate weight and induced SRD5A2-independent epithelial remodeling with expansion of proximal urethral luminal epithelial cells. ScRNA-seq of prostate tissues from Srd5a2null and control mice on HFD showed enrichment of proximal stem-like populations, reduced androgen signaling, and activation of WNT and NOTCH pathways via heightened stromal-epithelial crosstalk. Clinically, BMI gain, regardless of 5ARI use, correlated with transition zone growth, stronger activity of stem-cell signatures, reduced SRD5A2, and androgen signaling downregulation. Xenium spatial profiling indicated that BMI gain expands stem-like epithelial populations and a NOTCHenriched periepithelial stromal niche surrounding epithelial compartments, supporting stemness. Patients with substantial BMI gain were less responsive to 5ARI, whereas weight control plus 5ARI therapy synergistically improved outcomes. In conclusion, BMI gain promotes proximal prostate enlargement through an SRD5A2-independent stem-like cell mediated pathway that blunts 5ARI efficacy. Combining pharmacotherapy with weight control yields superior efficacy and supports individualized management of BPH.

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

Several groups recently published single-cell (sc) expression atlases of the adult mouse prostate cells based on RNA sequencing (scRNA-seq) data. All studies identified one computerized cluster of non-secretory luminal progenitor cells enriched in luminal and stemness-related gene transcripts. The actual correspondence between these luminal progenitor cell clusters has not been investigated. In addition, the presence of Krt4 (encoding cytokeratin 4) in these in silico-identified luminal progenitors suggested the overlap with FACS-enriched LSCmed luminal progenitor cells earlier identified as a stem-like, castration-tolerant and tumor-initiating cell population. Here, we used a unified bioinformatics pipeline to re-analyze published prostate scRNA-seq datasets and perform various pan-transcriptomic comparisons including the LSCmed cell signature. Our study demonstrates that i) the mouse prostate luminal progenitor cell clusters identified in the different scRNA-seq studies largely overlap and can be defined by a common 15-gene signature including Krt4, ii) mouse LSCmed cells match both mouse and human luminal progenitors identified by scRNA-seq analysis. Bridging these in silico-identified and ex vivo-characterized prostate luminal progenitor subsets should benefit our understanding of their actual involvement in prostate diseases.

BackgroundOne in eight men will be affected by prostate cancer (PCa) in their lives. While the current clinical standard prognostic marker for PCa is the Gleason score, it is subject to interreviewer variability. This study compares two machine learning methods for discriminating between high- and low-grade PCa on histology from 47 PCa patients. MethodsDigitized slides were annotated by a GU fellowship-trained pathologist. High-resolution tiles were extracted from annotated and unlabeled tissue. Glands were segmented and pathomic features were calculated and averaged across each patient. Patients were separated into a training set of 31 patients (Cohort A, n=9345 tiles) and a testing cohort of 16 patients (Cohort B, n=4375 tiles). Tiles from Cohort A were used to train a compact classification ensemble model and a ResNet model to discriminate tumor and were compared to pathologist annotations. ResultsThe ensemble and ResNet models had overall accuracies of 89% and 88%, respectively. The ResNet model was additionally able to differentiate Gleason patterns on data from Cohort B while the ensemble model was not. ConclusionsOur results suggest that quantitative pathomic features calculated from PCa histology can distinguish regions of cancer; how-ever, texture features captured by deep learning frameworks better differentiate unique Gleason patterns.

Castration-resistant prostate cancer represents a continuum of phenotypes, including tumors with high levels of androgen receptor (AR) expression and activity and those which do not express AR and rely on alternative pathways for survival. The process by which AR-positive prostate cancer cells and tumors lose AR expression and acquire neuroendocrine features is referred to as neuroendocrine differentiation. Numerous therapies and exposures have been demonstrated to induce neuroendocrine differentiation in vitro, including the pro-inflammatory cytokine, interleukin 1 beta (IL-1{beta}), encoded by the gene IL1B. The purpose of our studies was to determine the relationship between the expression and activity of AR in relationship to IL-1{beta} and IL1B in prostate cancer. We performed analysis of de-identified human clinical data and generated prostate cancer cell lines with overexpression or knockout of IL1B. In primary prostate cancer, higher expression of IL1B predicts longer time to biochemical recurrence. In metastatic castration-resistant prostate cancer, IL1B expression is decreased and inversely correlates with AR and AR-target gene expression and AR activity, while positively correlating with the neuroendocrine prostate cancer (NEPC) score and neuroendocrine marker gene expression. In vitro, we report that AR-positive castration-resistant prostate cancer cells (C4-2B, 22Rv1) secrete IL-1{beta}, and knockout of IL1B in these cells results in increased AR activity, in the presence and absence of dihydrotestosterone (DHT). Importantly, knockout of IL1B prevented AR attrition during androgen-deprivation. Taken together, our studies demonstrate that loss of IL1B in AR-positive castration-resistant prostate cancer cells can increase and maintain AR activity in the absence of androgens, suggesting another potential mechanism of high AR activity in castration-resistant prostate cancer.

BackgroundProstate cancer is a complex and heterogeneous disease with multiple tumor foci, each potentially harboring distinct driver molecular aberrations. This complexity poses challenges to effective management. We took an innovative approach to gain a comprehensive understanding of the genetic underpinnings of each tumor focus and avoid overlooking more minor yet clinically significant foci. Instead of relying solely on a systematic sampling of dominant foci, we conducted molecular analysis on whole-mount radical prostatectomy specimens. Our study aimed to find distinct molecular subsets of prostate cancer and assess their correlation with clinical outcomes, focusing on Caucasians (CA) and African Americans (AA). MethodWe randomly selected 2201 whole-mount radical prostatectomy cases, with 1207 (54.8%) from CA and 994 (45.1%) from AA patients evaluated for a 5-year biochemical recurrence-free survival rate (BCR). Of these 2201 cases, 834 (463 -56% were from CA and 371 -44% from AA patients) were subjected to molecular analysis using dual immunohistochemistry (IHC) for ERG and SPINK1, along with dual RNA in-situ hybridization (RNA-ISH) for ETV1 and ETV4 to evaluate tumor molecular heterogeneity on whole-mount specimens. The Chi-squared test examined racial disparities in aberrant oncogene expression. To assess BCR-free survival, we employed the Kaplan-Meier method and Cox-PH models for patients with distinct molecular subsets of prostate cancer. Additionally, Gleason Grade groups of prostate biopsies were summarized using a spaghetti plot and compared using linear mixed models. ResultsAnalysis of the 2201 cases revealed that AA with localized prostate cancer behaved differently with better 5-year BCR-free survival than CA after radical prostatectomy (AA: 0.82, 95% CI 0.80-0.85; CA: 0.71, 95% CI 0.68-0.75; p<.001). Molecular profiling of whole-mount specimens from 834 cases revealed that 16.4%, 58.4%, 21.7%, and 3.5% of patients with localized prostate cancer expressed none, one, two, and three of the four oncogenes, respectively. This finding identified new molecular subsets of prostate cancer with more than one driver mutation in a mutually exclusive manner within the multifocal disease. ERG and SPINK1 expression showed a negative correlation (p<.001). Notably, AA patients exhibited a lower incidence of ERG (38.8% vs. 60.3%) but a higher incidence of SPINK1 (63.3% vs. 35.6%) than CA patients. The incidences of ETV1 (9.4% vs. 9.3%) and ETV4 (4.6% vs. 3.9%) were not statistically significant between the two racial groups. However, significantly, ETV1 expression was associated with worse BCR-free survival in CA patients (hazard ratio [HR]=2.36, 95% CI 1.22-4.57, p=.02), while ETV4 expression was linked to worse BCR-free survival in AA patients (HR=2.65, 95% CI 1.15-6.09, p=.02). Moreover, ETV4 expression was associated with regional lymph node metastasis in AA patients (odds ratio [OR]=5.14, 95% CI 1.3-17.4, p=.01) but not in CA patients (OR=0.60, 95% CI 0.03-3.17, p=.63) at the time of radical prostatectomy. Additionally, in patients who underwent multiple biopsies before radical prostatectomy, the Gleason Grade group increased over time in AA patients (0.25 per year, p<.001) but remained unchanged in CA patients. ERG expression was associated with a lower Gleason Grade group (-0.20, p=.03), while ETV4 expression was linked to a higher Gleason Grade (0.54, p=.01). ConclusionsOur study reveals that AA with localized prostate cancer behaves differently and has better BCR-free survival than CA after radical prostatectomy, even after adjusting for known prognostic factors. Identification of new molecular subsets of prostate cancer with more than one ETS gene fusion within a multifocal prostate shows significant molecular heterogeneity between localized prostate cancer in CA and AA patients. Importantly, given the association of ETV1 and ETV4 expression with worse BCR-free survival in CA and AA, respectively, ETV1 and ETV4 emerge as potential prognostic markers, offering insights for clinical practice to predict prostate cancer recurrence after radical prostatectomy. Identification of new molecular subsets of prostate cancer with more than one ETS gene fusion and SPINK1 in a mutually exclusive pattern indicates the clonal origin of independent tumor foci, which is a rare and unique phenomenon in prostate cancer hitherto unidentified.

BackgroundDietary sugars, particularly fructose, are increasingly implicated in cancer progression through their impact on tumor metabolism. However, the specific metabolic adaptations driven by fructose in prostate cancer (PCa) remain unexplored. MethodsWe investigated the metabolic consequences of chronic fructose exposure in androgen-sensitive (LNCaP) and androgen-independent (PC-3) PCa cell lines. We evaluated the expression, localization, and function of the fructose transporter Glut-5 and assessed metabolic fluxes, enzyme expression, lipid accumulation, and global metabolite profiles using molecular, imaging, and metabolomic approaches. ResultsGlut-5 was primarily localized to early endosomes under basal and fructose-stimulated conditions, suggesting a non-canonical role potentially consistent with transceptor function. Chronic fructose exposure significantly upregulated Glut-5 expression and enhanced fructose uptake, but did not alter substantially its subcellular localization. Functionally, fructose reduced lactate production and mitochondrial ATP output, indicating a metabolic shift away from glycolysis and oxidative phosphorylation. In LNCaP cells, fructose induced robust activation of de novo lipogenesis (DNL), evidenced by upregulation of FASN and G6PD, increased lipid droplet accumulation, and enhanced levels of key fatty acid metabolites (e.g., TG, EPA, DHA). In contrast, PC-3 cells exhibited a distinct metabolic response, characterized by increased ceramide and amino acid metabolites. Notably, pharmacological inhibition of lipid metabolism using etomoxir abrogated proliferation in both cell lines under fructose stimulation. ConclusionsThese findings reveal that fructose promotes a metabolic reprogramming in PCa cells that is cell type- and AR-dependent, enhancing lipogenesis and potentially contributing to tumor progression. Our study identifies Glut-5-mediated fructolysis and lipid metabolic pathways as key vulnerabilities in PCa, offering potential avenues for metabolic intervention.

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.

We have dissected the role of Estrogen receptor beta (ER{beta}) in prostate cancer (PCa) with a novel ER{beta} ligand, OSU-ERb-12. Drug screens revealed additive interactions between OSU-ERB-12 and either epigenetic inhibitors or the androgen receptor antagonist, Enzalutamide (Enza). Clonogenic and cell biolody studies supported the potent additive effects of OSU-ERB-12 (100nM) and Enza (1{micro}M). The cooperative behavior was in PCa cell lines treated with either OSU- ERB-12 plus Enza or combinations involving 17{beta}-estradiol (E2). OSU-ERb-12 plus Enza uniquely impacted the transcriptiome, accessible chromatin, and the AR, MYC and H3K27ac cistromes. This included skewed transcriptional responses including suppression of the androgen and MYC transcriptomes, and repressed MYC protein. OSU-ERb-12 plus Enza uniquely impacted chromatin accessibility at approximately 3000 nucleosome-free sites, enriched at enhancers, enriched for basic Helix-Loop-Helix motifs. CUT&RUN experiments revealed combination treatment targeting of MYC, AR, and H3K27ac again shaping enhancer accessibility. Specifically, it repressed MYC interactions at enhancer regions enriched for bHLH motifs, and overlapped with publicly-available bHLH cistromes. Finally, cistrome-transcriptome analyses identified [~]200 genes that distinguished advanced PCa tumors in the SU2C cohort with high androgen and low neuroendocrine scores. Statement of ImplicationTargeting ER{beta} has potentially to augment AR antagonism to restrain MYC signaling and limit growth of advanced prostate cancer.

BackgroundProstate cancer diagnosis and prognosis is currently limited by the availability of sensitive and specific biomarkers. There is an urgent need to develop molecular biomarkers that allow for the distinction of indolent from aggressive disease, the sensitive detection of heterogeneous tumors, or the evaluation of micro-metastases. The availability of multi-omics datasets in publicly accessible databases provides a valuable foundation to develop computational workflows for the identification of suitable biomarkers for clinical management of cancer patients. ResultsWe combined transcriptomic data of primary localized and advanced prostate cancer from two cancer databases. Transcriptomic analysis of metastatic tumors unveiled a distinct overexpression pattern of genes encoding cell surface proteins intricately associated with cell-matrix components and chemokine signaling pathways. Utilizing an integrated approach combining machine learning and weighted gene correlation network modules, we identified the EZH2-TROAP axis as the main trajectory from initial tumor development to lethal metastatic disease. In addition, we identified and independently validated 58 promising biomarkers that were specifically upregulated in primary localized or metastatic disease. Among those biomarkers, 22 were highly significant for predicting biochemical recurrence. Notably, we confirmed TPX2 upregulation at the protein level in an independent cohort of primary prostate cancer and matched lymph node metastases. ConclusionsThis study demonstrates the effectiveness of using advanced bioinformatics approaches to identify the biological factors that drive prostate cancer progression. Furthermore, the targets identified show promise as prognostic biomarkers in clinical settings. Thus, integrative bioinformatics methods provide both deeper understanding of disease dynamics and open the doors for future personalized interventions.

Phenotypic plasticity is a recognized mechanism driving therapeutic resistance in prostate cancer (PCa) patients. While underlying molecular causations driving phenotypic plasticity have been identified, therapeutic success is yet to be achieved. To identify putative master regulator transcription factors (MR-TF) driving phenotypic plasticity in PCa, this work utilized a multiomic approach using genetically engineered mouse models of prostate cancer combined with patient data to identify MYBL2 as a significantly enriched transcription factor in PCa exhibiting phenotypic plasticity. Genetic inhibition of Mybl2 using independent murine PCa cell lines representing phenotypic plasticity demonstrated Mybl2 loss significantly decreased in vivo growth as well as cell fitness and repressed gene expression signatures involved in pluripotency and stemness. Because MYBL2 is currently not druggable, a MYBL2 gene signature was employed to identify cyclin-dependent kinase-2 (CDK2) as a potential therapeutic target. CDK2 inhibition phenocopied genetic loss of Mybl2 and significantly decreased in vivo tumor growth associated with enrichment of DNA damage. Together, this work demonstrates MYBL2 as an important MR-TF driving phenotypic plasticity in PCa. Further, high MYBL2 activity identifies PCa that would be responsive to CDK2 inhibition. SignificancePCa that escapes therapy targeting the androgen receptor signaling pathways via phenotypic plasticity are currently untreatable. Our study identifies MYBL2 as a MR-TF in phenotypic plastic PCa and implicates CDK2 inhibition as novel therapeutic target for this most lethal subtype of PCa.

Background: Men with incurable castration resistant prostate cancer (CRPC) are typically treated with taxanes; however, drug resistance rapidly develops. Thus, overcoming taxane resistant PC is a major clinical need. We previously identified a seven gene network in aggressive CRPC, which includes the mitotic serine threonine kinase BUB1, a major regulator of the spindle assembly checkpoint (SAC). Alterations in mitotic kinases (and SAC malfunction) are associated with advanced PC and taxane resistance development and thereby represent potential vulnerabilities. Methods: We evaluated BUB1 expression in publicly available data sets and in existing and newly generated taxane resistant PC cells. The effects of BUB1 depletion on the growth of a panel of PC and non-tumorigenic prostate epithelial cells was determined. We examined the capacity of pharmacologic inhibition of BUB1 kinase to reverse taxane-resistant PC growth. We evaluated the role of the prevalent androgen receptor variant AR-V7, in regulating BUB1 expression and taxane resistance. Results: BUB1 mRNA was over-expressed in PC, metastatic castration resistant prostate cancer (mCRPC) and in tumors of patients treated with taxane-based chemotherapeutics compared to benign prostate tissue. Furthermore, BUB1 levels were elevated in taxane resistant PC cell lines compared to their sensitive counterparts. BUB1 depletion decreased growth of CRPC cells through delayed mitosis but did not affect proliferation of androgen dependent (ADPC) or non-tumorigenic prostate epithelial cells. Furthermore, BUB1 inhibition with the specific kinase inhibitor, BAY1816032, re-sensitized taxane resistant CRPC cells to the clinically used drugs, docetaxel and cabazitaxel. Consistent with AR-V7 regulation of BUB1, we also found that AR-V7 was elevated in taxane resistant CRPC cells. Moreover, ectopic expression of AR-V7 in CRPC cells that lack this protein resulted in increased BUB1 and conferred docetaxel resistance. BUB1 pharmacologic inhibition in combination with taxanes sensitized AR-V7 expressing CRPC cells to docetaxel treatment. Conclusion: These data support BUB1 as an exploitable and therapeutically tractable vulnerability in taxane resistant CRPC including in AR variant driven CRPC.

BackgroundSpatial and single-cell transcriptomics have revealed significant heterogeneity in tumor and normal tissues. Each approach has its advantages: The Visium platform for spatial transcriptomics (ST) offers lower resolution than single-cell analysis, but histology enables the examination of cell morphology, tissue architecture, and potential cell-cell interactions. Single-cell transcriptomics (SC) provides high resolution, but manual cell-type annotation depends on incomplete scientific knowledge from heterogeneous experiments. When investigating poorly defined phenomena, such as the transition from normal tissue to cancer and metaplasia, researchers might overlook critical and unexpected findings in downstream analysis if they rely on pre-existing annotations to determine cell types, particularly in the context of phenotypic plasticity. ResultsWe employ our deep-transfer learning framework, DEGAS, to identify benign morphology glands in normal prostate tissue that are associated with poor progression-free survival in cancer patients and exhibit transcriptional signatures of carcinogenesis and de-differentiation. We confirm this finding in an additional ST dataset and use novel published methods to integrate SC data, showing that cells annotated as cancerous in the SC data map to regions of benign glands in another dataset. We pinpoint several genes, primarily MSMB, with expression closely correlated with progression-free survival scores, which are known markers of de-differentiation, and attribute their expression specifically to luminal epithelia, which are the presumed origin of most prostatic cancers. DiscussionOur work shows that morphologically normal epithelia can have transcriptional signatures like that of frank cancer, and that these tissues are associated with poor progression-free survival. We also highlight a critical gap in single-cell workflows: annotating continuous transitional phenomena like carcinogenesis with discrete labels can result in incomplete conclusions. Two approaches can help mitigate this issue: Tools like DEGAS and Scissor can provide a disease-association score for SC and ST data, independent of cell type and histology. Additionally, researchers should adopt a bidirectional approach, transferring histological labels from ST data to SC data using tools like RCTD, rather than only using SC cell-type assignments to annotate ST data. Employed together, these methods can offer valuable histology and disease-related information to better define tissue subtypes, especially epithelial cells in the process of carcinogenesis. ConclusionsDEGAS is a vital tool for generating clinically-oriented hypotheses from SC and ST data, which are heterogeneous, information-rich assays. In this study, we identify potential signatures of carcinogenesis in morphologically benign epithelia, which may be the precursors to cancer and high-grade pre-malignant lesions. Validating these genes as a panel may help identify patients at high risk for future cancer development, recurrence, and assist researchers in studying the biology of early carcinogenesis by detecting metaplastic changes before they are morphologically identifiable.

PDE4D7 expression is diminished during progression of PCa and the phosphodiesterase has been proposed as a prognostic biomarker. RNA sequencing of PCa tissue identified sequences in the PDE4D coding region on Chr5q12 exhibiting similarities in mRNA expression profile to PDE4D7. As previously identified, PART1 had a significantly similar expression pattern to PDE4D7 across samples. However, other genes also matched expression to PDE4D7, including miRNAs and lncRNAs. These novel PDE4D7 associated genes represent putative PCa biomarkers and could have mechanistic roles in PCa progression.

BackgroundMen with neuroendocrine prostate cancer (NEPC) have a poor prognosis. NEPC is commonly diagnosed by immunohistochemical markers (CHGA, SYP and NCAM1) and genomic features (mutations in RB1, PTEN, TP53). But by pathology, NEPC tumours are variable, leading to a classification of NE subtypes such as small cell and large cell neuroendocrine carcinomas, focal neuroendocrine differentiation (Focal NED), and Amphicrine. We postulated the diversity observed in NEPC pathologies might arise from differences in transcriptional profiles and the aim of this study is to utilize single-cell RNA sequencing to define the transcriptional differences between NEPC subtype pathologies. MethodsGene expression profiles were obtained for 18,632 individual tumour cells from 9 patient-derived xenograft (PDX) models representing five distinct neuroendocrine pathologies of prostate cancer. Integration and clustering of cell-level data demarked transcriptionally distinct sub-populations of cells. Differential gene expression, gene set enrichment and transcriptional factor regulon analysis identified expression signatures unique to specific neuroendocrine pathologies. Copy-number estimated from expression data revealed the clonal structure of PDXs with mixed adenocarcinoma and neuroendocrine pathologies. ResultsSignificant differences were observed in the transcriptional profiles of NEPC pathology subtypes. Focal NED cells maintain AR signaling, similar to the amphicrine subtype but different from small and large cell carcinomas. Cellular sub-populations enriched for expression of KRAS, IL2-STAT5 and TNF-signaling genes were found in focal NED and amphicrine pathologies, but not in small or large cell carcinomas. In contrast, sub-populations enriched for the YAP, Myc and E2F pathways were detected in small cell, large cell and amphicrine tumours, but not in focal NED cells. Each pathology showed unique patterns of master regulator activity as well, further implicating focal NED as a transcriptionally distinct entity. Based on copy number alterations within PDXs of mixed pathology, focal NED cells showed little clonal divergence from neighboring adenocarcinoma cells, whereas cells with small cell neuroendocrine pathology were clonally distinct. ConclusionsNeuroendocrine prostate cancer subtypes can be identified by pathology and our study shows that transcriptional features identified by single-cell RNA-sequencing also distinguish neuroendocrine subtypes pathologies from each other. In particular, our data redefine focal neuroendocrine differentiation as a pathology expressing androgen receptors (AR), exhibiting its distinctive composition of transcriptionally unique sub-populations. These findings advocate for differences in the treatment of NEPC tumors, particularly those displaying focal NED.

BackgroundThe histone chaperone complex, consisting of the death domain-associated protein (DAXX) and the alpha-thalassemia/mental retardation X-linked protein (ATRX), plays a pivotal role in maintaining chromatin through the deposition of the histone variant H3.3. Mutations leading to loss of ATRX or DAXX function are linked to the non-telomerase, alternative lengthening of telomeres (ALT) phenotype in certain cancers. Engineered ATRX mutations have previously been found to induce features of ALT in prostate cancer cell lines, notably in LAPC-4, but not in CWR22Rv1. This study determined the impact of DAXX mutations on ALT-associated characteristics in CWR22Rv1 and LAPC-4. MethodologyMutations were induced in CWR22Rv1 and LAPC-4 cells by targeting exon 2 of DAXX using the CRISPR-Cas9 genome editing strategy. The resulting mutant clones were then evaluated for ALT-associated characteristics, including the presence of ALT-associated PML bodies (APBs), C-circles, telomere length heterogeneity, and a lack of telomerase activity. ResultsFour CWR22Rv1 DAXX mutant clones (DAXXMut1-4) and five LAPC-4 clones (DAXXMut1-5) were evaluated. In CWR22Rv1, DAXXMut1, DAXXMut2, and DAXXMut4 were true knockout clones with frameshift mutations in both copies, while CWR22Rv1 DAXXMut3 had a frameshift mutation in one copy and an in-frame mutation in the other. Protein expression was undetectable in all the CWR22Rv1 clones, including CWR22Rv1 DAXXMut3. In LAPC-4, DAXXMut1 was a true knockout, while DAXXMut2, DAXXMut3, DAXXMut4, and DAXXMut5 clones had at least one in-frame mutation. Among these LAPC-4 clones, only DAXXMut1 had undetectable protein by western blotting. ALT-associated characteristics such as APBs, C-circles, and telomere length heterogeneity were observed only in CWR22Rv1 DAXXMut4. All the clones maintained telomerase activity, regardless of whether ALT-associated hallmarks were observed. ImplicationsThe CWR22Rv1 and LAPC-4 DAXX mutant clone models provide useful tools for future studies on telomere maintenance mechanisms and DAXX-related biology, particularly in prostate cancer.