Neuroendocrine cells in prostate cancer correlate with poor outcomes: a systematic review and meta-analysis.

OBJECTIVES: To perform a systematic review and meta-analysis of the literature to understand the variation in the reporting of neuroendocrine staining and determine the influence of reporting neuroendocrine staining at diagnosis on patient outcomes. METHODS: Medical databases were searched to identify studies in which adenocarcinoma specimens were stained with any of the following four neuroendocrine markers: chromogranin A (CgA), neuron-specific enolase (NSE), synaptophysin and CD56. The prevalence of neuroendocrine staining and correlation of the prevalence of neuroendocrine staining to patient outcomes were analysed using a random-effects model. All statistical tests were two-sided. RESULTS: Sixty-two studies spanning 7616 patients were analysed. The pooled prevalence for the most common marker, CgA (41%), was similar to that of NSE (39%) and higher than that of synaptophysin (31%). The prevalence of CgA staining was significantly influenced by reporting criteria, where objective thresholds reduced the variation in prevalence to 26%. No correlation was found between CgA prevalence and tumour grade. Patients positive for CgA staining using objective criteria had more rapid biochemical progression (hazard ratio [HR] 1.98, 95% confidence interval [CI] 1.49 to 2.65) and poorer prostate cancer-specific survival (HR 7.03, 95% CI 2.55 to 19.39) compared to negative patients, even among those with low-risk cancers. CONCLUSION: Discrepancies in the reported prevalence of neuroendocrine cells in adenocarcinoma are driven by the inconsistent scoring criteria. This study unequivocally demonstrates that when neuroendocrine cell staining is assessed with objective criteria it identifies patients with poor clinical outcomes. Future studies are needed to determine the exact quantifiable thresholds for use in reporting neuroendocrine cell staining to identify patients at higher risk of progression.

Prostate organogenesis: tissue induction, hormonal regulation and cell type specification.

Prostate organogenesis is a complex process that is primarily mediated by the presence of androgens and subsequent mesenchyme-epithelial interactions. The investigation of prostate development is partly driven by its potential relevance to prostate cancer, in particular the apparent re-awakening of key developmental programs that occur during tumorigenesis. However, our current knowledge of the mechanisms that drive prostate organogenesis is far from complete. Here, we provide a comprehensive overview of prostate development, focusing on recent findings regarding sexual dimorphism, bud induction, branching morphogenesis and cellular differentiation.

Human epithelial basal cells are cells of origin of prostate cancer, independent of CD133 status.

Normal prostatic epithelium is composed of basal and luminal cells. Prostate cancer can be initiated in both benign basal and luminal stem cells, but because basal cell markers are not expressed in patient tumors, the former result was unexpected. Since the cells of origin of prostate cancer are important therapeutic targets, we sought to provide further proof that basal stem cells have tumorigenic potential. Prostatic basal cells were enriched based on α2ß1integrin(hi) expression and further enriched for stem cells using CD133 in nontumorigenic BPH-1 cells. Human embryonic stem cells (hESCs) were also used as a source of normal stem cells. To test their tumorigenicity, we used two alternate stromal-based approaches; (a) recombination with human cancer-associated fibroblasts (CAFs) or (b) recombination with embryonic stroma (urogenital mesenchyme) and treated host mice with testosterone and 17ß-estradiol. Enriched α2ß1integrin(hi) basal cells from BPH-1 cells resulted in malignant tumor formation using both assays of tumorigenicity. Surprisingly, the tumorigenic potential did not reside in the CD133(+) stem cells but was consistently observed in the CD133(-) population. CAFs also failed to induce prostatic tumors from hESCs. These data confirmed that benign human basal cells include cells of origin of prostate cancer and reinforced their importance as therapeutic targets. In addition, our data suggested that the more proliferative CD133(-) basal cells are more susceptible to tumorigenesis compared to the CD133(+)-enriched stem cells. These findings challenge the current dogma that normal stem cells and cells of origin of cancer are the same cell type(s).

Estrogen receptor-beta activated apoptosis in benign hyperplasia and cancer of the prostate is androgen independent and TNFalpha mediated.

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are androgen-dependent diseases commonly treated by inhibiting androgen action. However, androgen ablation or castration fail to target androgen-independent cells implicated in disease etiology and recurrence. Mechanistically different to castration, this study shows beneficial proapoptotic actions of estrogen receptor-beta (ERbeta) in BPH and PCa. ERbeta agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells of estrogen-deficient aromatase knock-out mice. This occurs via extrinsic (caspase-8) pathways, without reducing serum hormones, and perturbs the regenerative capacity of the epithelium. TNFalpha knock-out mice fail to respond to ERbeta agonist, demonstrating the requirement for TNFalpha signaling. In human tissues, ERbeta agonist induces apoptosis in stroma and epithelium of xenografted BPH specimens, including in the CD133(+) enriched putative stem/progenitor cells isolated from BPH-1 cells in vitro. In PCa, ERbeta causes apoptosis in Gleason Grade 7 xenografted tissues and androgen-independent cells lines (PC3 and DU145) via caspase-8. These data provide evidence of the beneficial effects of ERbeta agonist on epithelium and stroma of BPH, as well as androgen-independent tumor cells implicated in recurrent disease. Our data are indicative of the therapeutic potential of ERbeta agonist for treatment of PCa and/or BPH with or without androgen withdrawal.

Brief report: a bioassay to identify primary human prostate cancer repopulating cells.

Cancer cells are heterogeneous in both their phenotypes and ability to promote tumor growth and spread. Xenografting is used to identify the most highly capable cells of regenerating tumors, referred to as cancer repopulating cells. Because prostate cancers (PCa's) rarely grow as xenografts, indentifying PCa repopulating cells has not been possible. Here, we report improved methods to xenograft localized primary PCa tissues using chimeric grafts with neonatal mouse mesenchyme. Xenograft survival of tumor tissue was significantly increased by neonatal mesenchyme (six of six patients, 66% of grafts, versus four of six patients, 41% of grafts) and doubled the proliferation index of xenografted cancer cells. When applied to isolated PCa cells, neonatal mesenchyme effectively reconstituted PCa's and increased xenograft survival (four of nine patients; 32% of grafts with mesenchyme and 0% without), and supported active cancer cell proliferation. Using this assay, we showed that unfractionated α2ß1integrin(hi) and α2ß1integrin(lo) cells from primary localized PCa's demonstrated tumor formation at comparable rates, similar to previous reports using metastatic specimens. Thus, this new protocol efficiently established tumors and enabled proliferative expansion of both intact tumor tissue and fractionated cancer cells, providing a bioassay to identify and therapeutically target PCa repopulating cells.

Hidden clues in prostate cancer - Lessons learned from clinical and pre-clinical approaches on diagnosis and risk stratification.

The heterogeneity of prostate cancer is evident at clinical, morphological and molecular levels. To aid clinical decision making, a three-tiered system for risk stratification is used to designate low-, intermediate-, and high-risk of disease progression. Intermediate-risk prostate cancers are the most frequently diagnosed, and even with common diagnostic features, can exhibit vastly different clinical progression. Thus, improved risk stratification methods are needed to better predict patient outcomes. Here, we provide an overview of the improvements in diagnosis/prognosis arising from advances in pathology reporting of prostate cancer, which can improve risk stratification, especially for patients with intermediate-risk disease. This review discusses updates to pathology reporting of morphological growth patterns, and proposes the utility of integrating prognostic biomarkers or innovative imaging techniques to enhance clinical decision-making. To complement clinical studies, experimental approaches using patient-derived tumors have highlighted important cellular and morphological features associated with aggressive disease that may impact treatment response. The intersection of urology, pathology and scientific disciplines is required to work towards a common goal of understanding disease pathogenesis, improving the stratification of patients with intermediate-risk disease and subsequently defining optimal treatment strategies using precision-based approaches.

The MURAL collection of prostate cancer patient-derived xenografts enables discovery through preclinical models of uro-oncology.

Preclinical testing is a crucial step in evaluating cancer therapeutics. We aimed to establish a significant resource of patient-derived xenografts (PDXs) of prostate cancer for rapid and systematic evaluation of candidate therapies. The PDX collection comprises 59 tumors collected from 30 patients between 2012-2020, coinciding with availability of abiraterone and enzalutamide. The PDXs represent the clinico-pathological and genomic spectrum of prostate cancer, from treatment-naïve primary tumors to castration-resistant metastases. Inter- and intra-tumor heterogeneity in adenocarcinoma and neuroendocrine phenotypes is evident from bulk and single-cell RNA sequencing data. Organoids can be cultured from PDXs, providing further capabilities for preclinical studies. Using a 1 x 1 x 1 design, we rapidly identify tumors with exceptional responses to combination treatments. To govern the distribution of PDXs, we formed the Melbourne Urological Research Alliance (MURAL). This PDX collection is a substantial resource, expanding the capacity to test and prioritize effective treatments for prospective clinical trials in prostate cancer.

A balancing act: PHLPP2 fine tunes AKT activity and MYC stability in prostate cancer.

PTEN loss stimulates prostate tumor progression by sustaining AKT activation. Nowak et al. (2019. J. Cell Biol https://doi.org/10.1083/jcb.201902048) surprisingly show that the AKT-suppressing phosphatase PHLPP2 promotes disease progression in the context of dual PTEN and p53 loss by increasing MYC stability.

Preclinical Models of Prostate Cancer: Patient-Derived Xenografts, Organoids, and Other Explant Models.

Prostate cancer remains a lethal disease. Preclinical cancer models that accurately represent the tumors of the patients they are intended to help are necessary to test potential therapeutic approaches and to better translate research discoveries. However, research in the prostate cancer field is hampered by the limited number of human cell lines and xenograft models, most of which do not recapitulate the human disease seen in the clinic today. This work reviews the recent advances in human patient-derived xenograft, organoid, and other explant models to address this need. In contrast to other tumor streams, the prostate cancer field is challenged by this approach, yet despite the limitations, patient-derived models remain an integral component of the preclinical testing pathway leading to better treatments for men with prostate cancer.

Patient-derived Models of Abiraterone- and Enzalutamide-resistant Prostate Cancer Reveal Sensitivity to Ribosome-directed Therapy.

BACKGROUND: The intractability of castration-resistant prostate cancer (CRPC) is exacerbated by tumour heterogeneity, including diverse alterations to the androgen receptor (AR) axis and AR-independent phenotypes. The availability of additional models encompassing this heterogeneity would facilitate the identification of more effective therapies for CRPC. OBJECTIVE: To discover therapeutic strategies by exploiting patient-derived models that exemplify the heterogeneity of CRPC. DESIGN, SETTING, AND PARTICIPANTS: Four new patient-derived xenografts (PDXs) were established from independent metastases of two patients and characterised using integrative genomics. A panel of rationally selected drugs was tested using an innovative ex vivo PDX culture system. INTERVENTION: The following drugs were evaluated: AR signalling inhibitors (enzalutamide and galeterone), a PARP inhibitor (talazoparib), a chemotherapeutic (cisplatin), a CDK4/6 inhibitor (ribociclib), bromodomain and extraterminal (BET) protein inhibitors (iBET151 and JQ1), and inhibitors of ribosome biogenesis/function (RNA polymerase I inhibitor CX-5461 and pan-PIM kinase inhibitor CX-6258). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Drug efficacy in ex vivo cultures of PDX tissues was evaluated using immunohistochemistry for Ki67 and cleaved caspase-3 levels. Candidate drugs were also tested for antitumour efficacy in vivo, with tumour volume being the primary endpoint. Two-tailed t tests were used to compare drug and control treatments. RESULTS AND LIMITATIONS: Integrative genomics revealed that the new PDXs exhibited heterogeneous mechanisms of resistance, including known and novel AR mutations, genomic structural rearrangements of the AR gene, and a neuroendocrine-like AR-null phenotype. Despite their heterogeneity, all models were sensitive to the combination of ribosome-targeting agents CX-5461 and CX-6258. CONCLUSIONS: This study demonstrates that ribosome-targeting drugs may be effective against diverse CRPC subtypes including AR-null disease, and highlights the potential of contemporary patient-derived models to prioritise treatment strategies for clinical translation. PATIENT SUMMARY: Diverse types of therapy-resistant prostate cancers are sensitive to a new combination of drugs that inhibit protein synthesis pathways in cancer cells.

Transdifferentiation as a Mechanism of Treatment Resistance in a Mouse Model of Castration-Resistant Prostate Cancer.

Current treatments for castration-resistant prostate cancer (CRPC) that target androgen receptor (AR) signaling improve patient survival, yet ultimately fail. Here, we provide novel insights into treatment response for the antiandrogen abiraterone by analyses of a genetically engineered mouse (GEM) model with combined inactivation of Trp53 and Pten, which are frequently comutated in human CRPC. These NPp53 mice fail to respond to abiraterone and display accelerated progression to tumors resembling treatment-related CRPC with neuroendocrine differentiation (CRPC-NE) in humans. Cross-species computational analyses identify master regulators of adverse response that are conserved with human CRPC-NE, including the neural differentiation factor SOX11, which promotes neuroendocrine differentiation in cells derived from NPp53 tumors. Furthermore, abiraterone-treated NPp53 prostate tumors contain regions of focal and/or overt neuroendocrine differentiation, distinguished by their proliferative potential. Notably, lineage tracing in vivo provides definitive and quantitative evidence that focal and overt neuroendocrine regions arise by transdifferentiation of luminal adenocarcinoma cells. These findings underscore principal roles for TP53 and PTEN inactivation in abiraterone resistance and progression from adenocarcinoma to CRPC-NE by transdifferentiation.Significance: Understanding adverse treatment response and identifying patients likely to fail treatment represent fundamental clinical challenges. By integrating analyses of GEM models and human clinical data, we provide direct genetic evidence for transdifferentiation as a mechanism of drug resistance as well as for stratifying patients for treatment with antiandrogens. Cancer Discov; 7(7); 736-49. ©2017 AACR.See related commentary by Sinha and Nelson, p. 673This article is highlighted in the In This Issue feature, p. 653.

Basal Progenitors Contribute to Repair of the Prostate Epithelium Following Induced Luminal Anoikis.

Contact with the extracellular matrix is essential for maintenance of epithelial cells in many tissues, while in its absence epithelial cells can detach and undergo anoikis. Here, we show that anoikis of luminal cells in the prostate epithelium is followed by a program of tissue repair that is mediated in part by differentiation of basal epithelial cells to luminal cells. We describe a mouse model in which inducible deletion of E-cadherin in prostate luminal cells results in their apoptotic cell death by anoikis, in the absence of phenotypic effects in the surrounding stroma. Quantitative assessments of proliferation and cell death in the luminal and basal compartments indicate that basal cells can rapidly generate luminal cells. Thus, our findings identify a role for basal-to-luminal differentiation in prostate epithelial repair, and provide a normal context to analogous processes that may occur during prostate cancer initiation.

Patient-derived xenografts reveal that intraductal carcinoma of the prostate is a prominent pathology in BRCA2 mutation carriers with prostate cancer and correlates with poor prognosis.

BACKGROUND: Intraductal carcinoma of the prostate (IDC-P) is a distinct clinicopathologic entity associated with aggressive prostate cancer (PCa). PCa patients carrying a breast cancer 2, early onset (BRCA2) germline mutation exhibit highly aggressive tumours with poor prognosis. OBJECTIVE: To investigate the presence and implications of IDC-P in men with a strong family history of PCa who either carry a BRCA2 pathogenic mutation or do not carry the mutation (BRCAX). DESIGN, SETTING, AND PARTICIPANTS: Patient-derived xenografts (PDXs) were generated from three germline BRCA2 mutation carriers and one BRCAX patient. Specimens were examined for histologic evidence of IDC-P. Whole-genome copy number analysis (WG-CNA) was performed on IDC-P from a primary and a matched PDX specimen. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The incidence of IDC-P and association with overall survival for BRCA2 and BRCAX patients were determined using Kaplan-Meier analysis. RESULTS AND LIMITATIONS: PDXs from BRCA2 tumours showed increased incidence of IDC-P compared with sporadic PCa (p=0.015). WG-CNA confirmed that the genetic profile of IDC-P from a matched (primary and PDX) BRCA2 tumour was similar. The incidence of IDC-P was significantly increased in BRCA2 carriers (42%, n=33, p=0.004) but not in BRCAX patients (25.8%, n=62, p=0.102) when both groups were compared with sporadic cases (9%, n=32). BRCA2 carriers and BRCAX patients with IDC-P had significantly worse overall and PCa-specific survival compared with BRCA2 carriers and BRCAX patients without IDC-P (hazard ratio [HR]: 16.9, p=0.0064 and HR: 3.57, p=0.0086, respectively). CONCLUSIONS: PDXs revealed IDC-P in patients with germline BRCA2 mutations or BRCAX classification, identifying aggressive tumours with poor survival even when the stage and grade of cancer at diagnosis were similar. Further studies of the prognostic significance of IDC-P in sporadic PCa are warranted. PATIENT SUMMARY: Intraductal carcinoma of the prostate is common in patients with familial prostate cancer and is associated with poor outcomes. This finding affects genetic counselling and identifies patients in whom earlier multimodality treatment may be required.

Stromal androgen receptor regulates the composition of the microenvironment to influence prostate cancer outcome.

Androgen receptor (AR) signaling in stromal cells is important in prostate cancer, yet the mechanisms underpinning stromal AR contribution to disease development and progression remain unclear. Using patient-matched benign and malignant prostate samples, we show a significant association between low AR levels in cancer associated stroma and increased prostate cancer-related death at one, three and five years post-diganosis, and in tissue recombination models with primary prostate cancer cells that low stromal AR decreases castration-induced apoptosis. AR-regulation was found to be different in primary human fibroblasts isolated from adjacent to cancerous and non-cancerous prostate epithelia, and to represent altered activation of myofibroblast pathways involved in cell cycle, adhesion, migration, and the extracellular matrix (ECM). Without AR signaling, the fibroblast-derived ECM loses the capacity to promote attachment of both myofibroblasts and cancer cells, is less able to prevent cell-matrix disruption, and is less likely to impede cancer cell invasion. AR signaling in prostate cancer stroma appears therefore to alter patient outcome by maintaining an ECM microenvironment inhibitory to cancer cell invasion. This paper provides comprehensive insight into AR signaling in the non-epithelial prostate microenvironment, and a resource from which the prognostic and therapeutic implications of stromal AR levels can be further explored.

Luminal cells are favored as the cell of origin for prostate cancer.

The identification of cell types of origin for cancer has important implications for tumor stratification and personalized treatment. For prostate cancer, the cell of origin has been intensively studied, but it has remained unclear whether basal or luminal epithelial cells, or both, represent cells of origin under physiological conditions in vivo. Here, we use a novel lineage-tracing strategy to assess the cell of origin in a diverse range of mouse models, including Nkx3.1(+/-); Pten(+/-), Pten(+/-), Hi-Myc, and TRAMP mice, as well as a hormonal carcinogenesis model. Our results show that luminal cells are consistently the observed cell of origin for each model in situ; however, explanted basal cells from these mice can generate tumors in grafts. Consequently, we propose that luminal cells are favored as cells of origin in many contexts, whereas basal cells only give rise to tumors after differentiation into luminal cells.

Single luminal epithelial progenitors can generate prostate organoids in culture.

The intrinsic ability to exhibit self-organizing morphogenetic properties in ex vivo culture may represent a general property of tissue stem cells. Here we show that single luminal stem/progenitor cells can generate prostate organoids in a three-dimensional culture system in the absence of stroma. Organoids generated from CARNs (castration-resistant Nkx3.1-expressing cells) or normal prostate epithelia exhibit tissue architecture containing luminal and basal cells, undergo long-term expansion in culture and exhibit functional androgen receptor signalling. Lineage-tracing demonstrates that luminal cells are favoured for organoid formation and generate basal cells in culture. Furthermore, tumour organoids can initiate from CARNs after oncogenic transformation and from mouse models of prostate cancer, and can facilitate analyses of drug response. Finally, we provide evidence supporting the feasibility of organoid studies of human prostate tissue. Our studies underscore the progenitor properties of luminal cells, and identify in vitro approaches for studying prostate biology.

A preclinical xenograft model identifies castration-tolerant cancer-repopulating cells in localized prostate tumors.

A lack of clinically relevant experimental models of human prostate cancer hampers evaluation of potential therapeutic agents. Currently, androgen deprivation therapy is the gold standard treatment for advanced prostate cancer, but inevitably, a subpopulation of cancer cells survives and repopulates the tumor. Tumor cells that survive androgen withdrawal are critical therapeutic targets for more effective treatments, but current model systems cannot determine when they arise in disease progression and are unable to recapitulate variable patient response to treatment. A model system was developed in which stromal-supported xenografts from multiple patients with early-stage localized disease can be tested for response to castration. The histopathology of these xenografts mimicked the original tumors, and short-term host castration resulted in reduced proliferation and increased apoptosis in tumor cells. After 4 weeks of castration, residual populations of quiescent, stem-like tumor cells remained. Without subsequent treatment, these residual cells displayed regenerative potential, because testosterone readministration resulted in emergence of rapidly proliferating tumors. Therefore, this model may be useful for revealing potential cellular targets in prostate cancer, which exist before the onset of aggressive incurable disease. Specific eradication of these regenerative tumor cells that survive castration could then confer survival benefits for patients.

A preclinical xenograft model of prostate cancer using human tumors.

Most cases of prostate cancer are now diagnosed as moderate-grade localized disease. These tumor specimens are important tools in the discovery and translation of prostate cancer research; however, unlike more advanced tumors, they are notoriously difficult to grow in the laboratory. We developed a system for efficiently xenografting localized human prostate cancer tissue, and we adapted this protocol to study the interactions between the specific subsets of epithelial and stromal cells. Fresh prostate tissues or isolated epithelial cells are recombined with mouse seminal vesicle mesenchyme (SVM) and grafted under the renal capsule of immunodeficient mice for optimum growth and survival. Alternatively, mouse mesenchyme can be replaced with human prostate fibroblasts in order to determine their contribution to tumor progression. Grafts can be grown for several months to determine the effectiveness of novel therapeutic compounds when administered to host mice, thereby paving the way for personalizing the treatment of individual prostate cancers.

Stem cells in prostate cancer: treating the root of the problem.

Prostate cancer is a hormone-dependent, epithelial-derived tumor, resulting from uncontrolled growth of genetically unstable transformed cells. Stem cells are therapeutic targets for prostate cancer, but as disease progression occurs over decades, the imperative is to identify and target the cancer-repopulating cell (CRC) that maintains malignant clones. In order to achieve this goal, we will review the current knowledge of three specific types of cells, their origins, and their differentiation potential. The first is the normal stem cell, the second is the cancer cell of origin, and the third is the CRC. Specifically, we review three proposed models of stem cell differentiation in normal tissues, including linear, bidirectional, and independent lineages. We consider evidence of the cancer cell of origin arising from both basal and luminal cells. Finally, we discuss the limited data available on the identity and characterization of CRCs in localized and castrate-resistant prostate cancer, which is where we believe the focus of future research efforts should be directed. Ultimately, understanding the intrinsic or extrinsic influences that dictate the behavior of these unique cells will be instrumental in facilitating the development of new therapeutic targets for prostate cancer.