A critical knowledge gap in prostate cancer research is understanding whether castration-tolerant progenitor-like cells that reside in treatment-naïve tumors play a direct role in therapy resistance and tumor progression. Herein, we reveal that the castration tolerance of LSCmed (Lin-, Sca-1+, CD49fmed) progenitor cells, the mouse equivalent of human prostatic Club cells, arises not from intrinsic properties, but from significant transcriptional reprogramming. Utilizing single-cell RNA sequencing of LSCmed cells isolated from prostate-specific Pten-deficient (Ptenpc−/−) mice, we identify the emergence of castration-resistant LSCmed cells enriched in stem-like features, driven by the transcription factor FOSL1/AP-1. We demonstrate that cells exhibiting Ptenpc−/− LSCmed characteristics are prevalent in aggressive double-negative prostate cancer (DNPC) subtypes recently identified in human castration-resistant prostate cancer (CRPC). Furthermore, our findings show that the dual-targeting agents JQ-1 and CX-6258—focused on FOSL1/AP-1 and PIM kinases, respectively—effectively suppress both the progenitor properties and the growth of mouse and human DNPC surrogates in vitro and in vivo. Thus, early eradication of castration-tolerant Club-like cells presents a promising therapeutic strategy to mitigate prostate cancer progression toward CRPC. Although castration is the primary treatment for advanced prostate cancer, castration-resistant prostate cancer (CRPC) eventually develops in all patients. Here, we identify a therapeutic strategy that enhances the castration efficacy by targeting castration-tolerant cells in prostate tumors. Although castration is the primary treatment for advanced prostate cancer, castration-resistant prostate cancer (CRPC) eventually develops in all patients. Here, we identify a therapeutic strategy that enhances the castration efficacy by targeting castration-tolerant cells in prostate tumors.

Benign prostate hyperplasia (BPH) is caused by the nonmalignant enlargement of the transition zone of the prostate gland, leading to lower urinary tract symptoms. Although current medical treatments are unsatisfactory in many patients, the limited understanding of the mechanisms driving disease progression prevents the development of alternative therapeutic strategies. The probasin-prolactin (Pb-PRL) transgenic mouse recapitulates many histopathological features of human BPH. Herein, these alterations parallel urodynamic disturbance reminiscent of lower urinary tract symptoms. Single-cell RNA-sequencing analysis of Pb-PRL mouse prostates revealed that their epithelium mainly includes low-androgen signaling cell populations analogous to Club/Hillock cells enriched in the aged human prostate. These intermediate cells are predicted to result from the reprogramming of androgen-dependent luminal cells. Pb-PRL mouse prostates exhibited increased vulnerability to oxidative stress due to reduction of antioxidant enzyme expression. One-month treatment of Pb-PRL mice with anethole trithione (ATT), a specific inhibitor of mitochondrial ROS production, reduced prostate weight and voiding frequency. In human BPH-1 epithelial cells, ATT decreased mitochondrial metabolism, cell proliferation, and stemness features. ATT prevented the growth of organoids generated by sorted Pb-PRL basal and LSCmed cells, the two major BPH-associated, androgen-independent epithelial cell compartments. Taken together, these results support cell plasticity as a driver of BPH progression and therapeutic resistance to androgen signaling inhibition, and identify antioxidant therapy as a promising treatment of BPH.

Inhibition of androgen signaling is the gold standard treatment of benign prostate hyperplasia and prostate cancer. Despite the initial response to these treatments, therapeutic resistance is ultimately observed in most patients. Single cell RNAseq studies have shown that castration-tolerant luminal cells share several molecular and functional features with cells identified as luminal progenitor in physiological conditions. The increased prevalence of luminal progenitor-like cells in tumor contexts might result from their intrinsic androgen-independence and from the reprogramming of differentiated luminal cells into a castration-tolerant state. Thus, it is currently hypothesized that the luminal progenitor molecular profile might constitute a functional hub for cell survival in androgen deprivation context, a prerequisite for tumor regrowth. Therapeutic intervention interfering with luminal lineage plasticity is a promising approach to prevent prostate cancer progression.

Les traitements médicaux de l’hyperplasie bénigne et du cancer de la prostate reposent essentiellement sur l’inhibition de la signalisation androgénique. Bien qu’initialement efficaces, ces traitements sont tôt ou tard confrontés à une résistance thérapeutique. Des données récentes de séquençage d’ARN sur cellules uniques montrent que les cellules luminales survivant à la déprivation androgénique dans ces contextes pathologiques présentent un profil moléculaire semblable à celui de cellules luminales progénitrices, présentes en faible quantité dans un contexte physiologique. Ce profil moléculaire pourrait constituer un hub de résistance à la castration et résulter, en partie, de la reprogrammation des cellules luminales tumorales. L’inhibition thérapeutique de cette plasticité cellulaire constitue une piste prometteuse pour limiter la progression du cancer prostatique.

Background: New predictive biomarkers are needed to accurately predict metastasis-free survival (MFS) and cancer-specific survival (CSS) in localized prostate cancer (PC). Keratin-7 (KRT7) overexpression has been associated with poor prognosis in several cancers and is described as a novel prostate progenitor marker in the mouse prostate. Methods: KRT7 expression was evaluated in prostatic cell lines and in human tissue by immunohistochemistry (IHC, on advanced PC, n = 91) and immunofluorescence (IF, on localized PC, n = 285). The KRT7 mean fluorescence intensity (MFI) was quantified in different compartments by digital analysis and correlated to clinical endpoints in the localized PC cohort. Results: KRT7 is expressed in prostatic cell lines and found in the basal and supra-basal compartment from healthy prostatic glands and benign peri-tumoral glands from localized PC. The KRT7 staining is lost in luminal cells from localized tumors and found as an aberrant sporadic staining (2.2%) in advanced PC. In the localized PC cohort, high KRT7 MFI above the 80th percentile in the basal compartment was significantly and independently correlated with MFS and CSS, and with hypertrophic basal cell phenotype. Conclusion: High KRT7 expression in benign glands is an independent biomarker of MFS and CSS, and its expression is lost in tumoral cells. These results require further validation on larger cohorts.

Background: The molecular and cellular mechanisms that drive castration-resistant prostate cancer (CRPC) remain poorly understood. LSCmed cells defines an FACS-enriched population of castration-tolerant luminal progenitor cells that has been proposed to promote tumorigenesis and CRPC in Pten-deficient mice. The goals of this study were to assess the relevance of LSCmed cells through the analysis of their molecular proximity with luminal progenitor-like cell clusters identified by single-cell (sc)RNA-seq analyses of mouse and human prostates, and to investigate their regulation by in silico-predicted growth factors present in the prostatic microenvironment. Methods: Several bioinformatic pipelines were used for pan-transcriptomic analyses. LSCmed cells isolated by cell sorting from healthy and malignant mouse prostates were characterized using RT-qPCR, immunofluorescence and organoid assays. Results: LSCmed cells match (i) mouse luminal progenitor cell clusters identified in scRNA-seq analyses for which we provide a common 15-gene signature including the previously identified LSCmed marker Krt4, and (ii) Club/Hillock cells of the human prostate. This transcriptional overlap was maintained in cancer contexts. EGFR/ERBB4, IGF-1R and MET pathways were identified as autocrine/paracrine regulators of progenitor, proliferation and differentiation properties of LSCmed cells. The functional redundancy of these signaling pathways allows them to bypass the effect of receptor-targeted pharmacological inhibitors. Conclusions: Based on transcriptomic profile and pharmacological resistance to monotherapies that failed in CRPC patients, this study supports LSCmed cells as a relevant model to investigate the role of castration-tolerant progenitor cells in human prostate cancer progression.

Stem and progenitor cells of the adult prostate epithelium have historically been believed to reside mainly or exclusively within the basal cell compartment and to possess basal-like phenotypic characteristics. Within the past decade, evidence of the existence of luminal epithelial cells exhibiting stem/progenitor properties has been obtained by lineage tracing and by functional characterization of sorted luminal-like cells. In 2020, the boom of single-cell transcriptomics led to increasingly exhaustive profiling of putative mouse luminal progenitor cells and, importantly, to the identification of cognate cells in the human prostate. The enrichment of luminal progenitor cells in genetically modified mouse models of prostate inflammation, benign prostate hypertrophy and prostate cancer, and the intrinsic castration tolerance of these cells, suggest their potential role in prostate pathogenesis and in resistance to androgen deprivation therapy. This Review bridges different approaches that have been used in the field to characterize luminal progenitor cells, including the unification of multiple identifiers employed to define these cells (names and markers). It also provides an overview of the intrinsic functional properties of luminal progenitor cells, and addresses their relevance in mouse and human prostate pathophysiology.

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 LSC med 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 LSC med 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 LSC med 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.

The canonical prolactin (PRL) Signal Transducer and Activator of Transcription (STAT) 5 pathway has been suggested to contribute to human prostate tumorigenesis via an autocrine/paracrine mechanism. The probasin (Pb)-PRL transgenic mouse models this mechanism by overexpressing PRL specifically in the prostate epithelium leading to strong STAT5 activation in luminal cells. These mice exhibit hypertrophic prostates harboring various pre-neoplastic lesions that aggravate with age and accumulation of castration-resistant stem/progenitor cells. As STAT5 signaling is largely predominant over other classical PRL-triggered pathways in Pb-PRL prostates, we reasoned that Pb-Cre recombinase-driven genetic deletion of a floxed Stat5a/b locus should prevent prostate tumorigenesis in so-called Pb-PRLΔSTAT5 mice. Anterior and dorsal prostate lobes displayed the highest Stat5a/b deletion efficiency with no overt compensatory activation of other PRLR signaling cascade at 6 months of age; hence the development of tumor hallmarks was markedly reduced. Stat5a/b deletion also reversed the accumulation of stem/progenitor cells, indicating that STAT5 signaling regulates prostate epithelial cell hierarchy. Interestingly, ERK1/2 and AKT, but not STAT3 and androgen signaling, emerged as escape mechanisms leading to delayed tumor development in aged Pb-PRLΔSTAT5 mice. Unexpectedly, we found that Pb-PRL prostates spontaneously exhibited age-dependent decline of STAT5 signaling, also to the benefit of AKT and ERK1/2 signaling. As a consequence, both Pb-PRL and Pb-PRLΔSTAT5 mice ultimately displayed similar pathological prostate phenotypes at 18 months of age. This preclinical study provides insight on STAT5-dependent mechanisms of PRL-induced prostate tumorigenesis and alternative pathways bypassing STAT5 signaling down-regulation upon prostate neoplasia progression.

Introduction Castration-resistant prostate cancer (CRPC) is a lethal disease. Therefore the identification and understanding of castration-tolerant cell(s) is mandatory. While there is strong evidence for the existence of castration-tolerant cells within the basal layer of the prostate epithelium, most of human prostate cancers (hormone-naïve and CRPC) exhibit a luminal phenotype devoid of basal cell features. The nature of the putative castration-resistant luminal cell(s) is unknown. We recently identified in the mouse prostate a population of luminal progenitor cells that we called LSCmed (Lin−/Sca-1+/CD49fmed). This population is rare (5%) in healthy prostates but significantly enriched (30%) in premalignant prostates. The aim of this study was to investigate the relevance of LSCmed in prostate cancer. Material and methods We compared 6–8 month-old wild type (WT), probasin-prolactin (Pb-PRL) and Pb-Cre4/PTEN floxed (Pten-KO) mice exhibiting healthy, premalignant or malignant prostates, respectively. We used cell sorting (Lin, CD49f, Sca-1) to quantify or enrich the various prostate cell subpopulations from intact versus castrated mice. Transcriptomic profiling of prostate cell subpopulations was performed using GeneChip Mouse Transcriptome Arrays 1.0 (Affymetrix). Tumor-initiating properties of LSCmed cells was determined using the in vivo regeneration assay. Results and discussions Transcriptomic profiling showed that LSCmed is a distinct cell entity that exhibits a specific gene expression signature, among which cytokeratin 4 (CK4) was validated as a specific marker to track them on tissue sections. Using CK4 immunohistochemistry and cell sorting we discovered that LSCmed represent the major cell component (80%) of aggressive prostate tumours harboured by Pten-KO mice. LSCmed are castration-tolerant in the three genotypes, which correlates their intrinsically low androgen signalling. According to their progenitor properties, Pten-KO LSCmed exhibit cancer-initiating properties in transplantation assays, and generate more aggressive tumours than basal cells used in control. Finally, in Pten-KO prostate tumours, several clusters of LSCmed continue to proliferate after castration. Conclusion LSCmed represent a newly-identified luminal prostatic cell subpopulation. The combination of progenitor, castration-resistance and tumor-initiating properties makes them strong candidates for mediating prostate cancer recurrence under androgen deprivation therapy (CRPC).

Castration‐resistant prostate cancer is a lethal disease. The cell type(s) that survive androgen deprivation remain poorly described, despite global efforts to understand the various mechanisms of therapy resistance. We recently identified in wild‐type (WT) mouse prostates a rare population of luminal progenitor cells that we called LSC med according to their FACS profile (Lin − /Sca‐1 + /CD49f med ). Here, we investigated the prevalence and castration resistance of LSC med in various mouse models of prostate tumourigenesis (Pb‐PRL, Pten pc−/− , and Hi‐Myc mice). LSC med prevalence is low (∼8%, similar to WT) in Hi‐Myc mice, where prostatic androgen receptor signalling is unaltered, but is significantly higher in the two other models, where androgen receptor signalling is decreased, rising up to more than 80% in Pten pc−/− prostates. LSC med tolerate androgen deprivation and persist or are enriched 2–3 weeks after castration. The tumour‐initiating properties of LSC med from Pten pc−/− mice were demonstrated by regeneration of tumours in vivo . Transcriptomic analysis revealed that LSC med represent a unique cell entity as their gene expression profile is different from luminal and basal/stem cells, but shares markers of each. Their intrinsic androgen signalling is markedly decreased, explaining why LSC med tolerate androgen deprivation. This also illuminates why Pten pc−/− tumours are castration‐resistant since LSC med represent the most prevalent cell type in this model. We validated CK4 as a specific marker for LSC med on sorted cells and prostate tissues by immunostaining, allowing for the detection of LSC med in various mouse prostate specimens. In castrated Pten pc−/− prostates, there was significant proliferation of CK4 + cells, further demonstrating their key role in castration‐resistant prostate cancer progression. Taken together, this study identifies LSC med as a probable source of prostate cancer relapse after androgen deprivation and as a new therapeutic target for the prevention of castrate‐resistant prostate cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

CONTEXT:In a cohort of 95 women with multiple breast fibroadenomas (MFAs), we recently identified patients harboring germline heterozygous variants of the prolactin receptor (PRLR) exhibiting constitutive activity (PRLRI146L and PRLRI176V).OBJECTIVE:This study sought to better delineate the potential role of PRLR gain-of-function variants in benign and malignant mammary tumorigenesis.DESIGN:This was an observational study and transgenic mouse model analysis.SETTING:The study took place at the Department of Endocrinology, Reproductive Disorders and Rare Gynecologic Diseases, Pitié Salpêtrière, Paris, and Inserm Unit 1151, Paris.PATIENTS OR OTHER PARTICIPANTS:We generated a second MFA cohort (n = 71) as well as a group of control subjects (n = 496) and a cohort of women with breast cancer (n = 119). We also generated two transgenic mouse models carrying the coding sequences of human PRLRI146L or PRLRWT.INTERVENTION:We aimed to determine the prevalence of PRLR variants in these three populations and to uncover any association of the latter with specific tumor pattern, especially in patients with breast cancer.RESULTS:This study did not highlight a higher prevalence of PRLR variants in the MFA group and in the breast cancer group compared with control subjects. Transgenic mice expressing PRLRI146L exhibited very mild histological mammary phenotype but tumors were never observed.CONCLUSION:PRLRI146L and PRLRI176V variants are not associated with breast cancer or MFA risk. However, one cannot exclude that low but sustained PRLR signaling may facilitate or contribute to pathological development driven by oncogenic pathways. Long-term patient follow-up should help to address this issue.

Adult stem/progenitor cells are found in many tissues, where their primary role is to maintain homeostasis. Recent studies have evaluated the regulation of adult stem/progenitor cells by prolactin in various target tissues or cell types, including the mammary gland, the prostate, the brain, the bone marrow, the hair follicle, and colon cancer cells. Depending on the tissue, prolactin can either maintain stem cell quiescence or, in contrast, promote stem/progenitor cell expansion and push their progeny towards differentiation. In many instances, whether these effects are direct or involve paracrine regulators remains debated. This minireview aims to overview the current knowledge in the field.

PRLR(I146L) is the first identified gain-of-function variant of the prolactin receptor (PRLR) that was proposed to be associated with benign breast tumorigenesis. Structural investigations suggested this hydrophobic core position in the extracellular D2 domain to be linked to receptor dimerization. Here, we used a mutational approach to address how the conservative I-to-L substitution induced constitutive activity. Using cell-based assays of different I146-PRLR variants in combination with spectroscopic/nuclear magnetic resonance analyses we found that chemical manipulation of position 146 profoundly altered folding, PRL-responsiveness, and ligand-independent activity of the receptor in a mutation-specific manner. Together, these data further add to the critical role of position 146, showing it to also be crucial to structural integrity thereby imposing on the biological PRLR properties. When stably introduced in MCF-7 (luminal) and MDA-MB231 (mesenchymal) breast cancer cells, the most potent of the PRL-insensitive mutants (PRLR(I146D)) had minimal impact on cell proliferation and cell differentiation status.

L’activation du signal PRL/STAT5 (prolactine/signal transducer and activator of transcription 5) dans le cancer de la prostate est proportionnelle au grade histologique de la tumeur. Cette voie stimule la survie, la prolifération et l’invasion des cellules tumorales luminales [1]. Par ailleurs, les cellules basales/souches de l’épithélium prostatique sont des cellules initiatrices de tumeurs et potentiellement la source de la résistance aux thérapies anti-androgène [2]. Les souris transgéniques Pb-PRL, qui expriment la PRL spécifiquement dans la prostate, développent des lésions prostatiques pré-cancéreuses caractérisées par une activation luminale de STAT5 et une amplification des cellules basales/souches. Nous cherchons à comprendre les mécanismes reliant ces deux événements. Les prostates de souris et des échantillons de tissu prostatique humain sont analysés par cytométrie de flux, cultures tridimensionnelles (prostasphères) et immunohistochimie. Les essais fonctionnels et les analyses de la voie PRL/STAT5 indiquent que les cellules basales/souches (humaines, souris) ne sont pas des cibles directes de la PRL. Par contre, l’analyse des prostates de souris Pb-PRL révèle que les foyers de cellules basales/souches amplifiées sont souvent proches de cellules luminales montrant une forte activation de STAT5. Ces observations suggèrent une régulation paracrine des cellules basales/souches en aval de la voie PRL/STAT5. Les analyses en cours visent à étudier le dialogue entre les cellules basales/souches et les autres compartiments cellulaires (luminal, stromal) afin d’identifier les facteurs paracrines impliqués.

GH pathway has been shown to play a major role in liver regeneration through the control of epidermal growth factor receptor (EGFR) activation. This pathway is down-regulated in nonalcoholic fatty liver disease. Because regeneration is known to be impaired in fatty livers, we wondered whether a deregulation of the GH/EGFR pathway could explain this deficiency. Hepatic EGFR expression and triglyceride levels were quantified in liver biopsies of 32 obese patients with different degrees of steatosis. We showed a significant inverse correlation between liver EGFR expression and the level of hepatic steatosis. GH/EGFR down-regulation was also demonstrated in 2 steatosis mouse models, a genetic (ob/ob) and a methionine and choline-deficient diet mouse model, in correlation with liver regeneration defect. ob/ob mice exhibited a more severe liver regeneration defect after partial hepatectomy (PH) than methionine and choline-deficient diet-fed mice, a difference that could be explained by a decrease in signal transducer and activator of transcription 3 phosphorylation 32 hours after PH. Having checked that GH deficiency accounted for the GH signaling pathway down-regulation in the liver of ob/ob mice, we showed that GH administration in these mice led to a partial rescue in hepatocyte proliferation after PH associated with a concomitant restoration of liver EGFR expression and signal transducer and activator of trnascription 3 activation. In conclusion, we propose that the GH/EGFR pathway down-regulation is a general mechanism responsible for liver regeneration deficiency associated with steatosis, which could be partially rescued by GH administration.

Background: AMP-activated protein kinase (AMPK) is an evolutionarily conserved sensor of cellular energy status that contributes to restoration of energy homeostasis by slowing down ATP-consuming pathways and activating ATP-producing pathways. Unexpectedly, in different systems, AMPK is also required for proper cell division. In the current study, we evaluated the potential effect of the AMPK catalytic subunit, AMPK alpha 1, on hepatocyte proliferation.Methods: Hepatocyte proliferation was determined in AMPK alpha 1 knockout and wild-type mice in vivo after two thirds partial hepatectomy, and in vitro in primary hepatocyte cultures. The activities of metabolic and cell cycle-related signaling pathways were measured.Results: After partial hepatectomy, hepatocytes proliferated rapidly, correlating with increased AMPK phosphorylation. Deletion of AMPK alpha 1 delayed liver regeneration by impacting on G1/S transition phase. The proliferative defect of AMPK alpha 1-deficient hepatocytes was cell autonomous, and independent of energy balance. The priming phase, lipid droplet accumulation, protein anabolic responses and growth factor activation after partial hepatectomy occurred normally in the absence of AMPK alpha 1 activity. By contrast, mRNA and protein expression of cyclin A2, a key driver of S phase progression, were compromised in the absence of AMPK activity. Importantly, AMPK alpha 1 controlled cyclin A2 transcription mainly through the ATF/CREB element.Conclusions: Our study highlights a novel role for AMPK alpha 1 as a positive regulator of hepatocyte division occurring independently of energy balance. (C) 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Unraveling the molecular clues of liver proliferation has become conceivable thanks to the model of two-third hepatectomy. The synchronicity and the well-scheduled aspect of this process allow scientists to slowly decipher this mystery. During this phenomenon, quiescent hepatocytes of the remnant lobes are able to reenter into the cell cycle initiating the G1-S progression synchronously before completing the cell cycle. The major role played by this step of the cell cycle has been emphasized by loss-of-function studies showing a delay or a lack of coordination in the hepatocytes G1-S progression. Two growth factor receptors, c-Met and EGFR, tightly drive this transition. Due to the level of complexity surrounding EGFR signaling, involving numerous ligands, highly controlled regulations and multiple downstream pathways, we chose to focus on the EGFR pathway for this paper. We will first describe the EGFR pathway in its integrity and then address its essential role in the G1/S phase transition for hepatocyte proliferation. Recently, other levels of control have been discovered to monitor this pathway, which will lead us to discuss regulations of the EGFR pathway and highlight the potential effect of misregulations in pathologies.