Cell Plasticity in a Mouse Model of Benign Prostate Hyperplasia Drives Amplification of Androgen-Independent Epithelial Cell Populations Sensitive to Antioxidant Therapy.

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.

A rare castration-resistant progenitor cell population is highly enriched in Pten-null prostate tumours.

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 LSCmed according to their FACS profile (Lin- /Sca-1+ /CD49fmed ). Here, we investigated the prevalence and castration resistance of LSCmed in various mouse models of prostate tumourigenesis (Pb-PRL, Ptenpc-/- , and Hi-Myc mice). LSCmed 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 Ptenpc-/- prostates. LSCmed tolerate androgen deprivation and persist or are enriched 2-3 weeks after castration. The tumour-initiating properties of LSCmed from Ptenpc-/- mice were demonstrated by regeneration of tumours in vivo. Transcriptomic analysis revealed that LSCmed 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 LSCmed tolerate androgen deprivation. This also illuminates why Ptenpc-/- tumours are castration-resistant since LSCmed represent the most prevalent cell type in this model. We validated CK4 as a specific marker for LSCmed on sorted cells and prostate tissues by immunostaining, allowing for the detection of LSCmed in various mouse prostate specimens. In castrated Ptenpc-/- prostates, there was significant proliferation of CK4+ cells, further demonstrating their key role in castration-resistant prostate cancer progression. Taken together, this study identifies LSCmed 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.

AMPKα1 controls hepatocyte proliferation independently of energy balance by regulating Cyclin A2 expression.

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, AMPKα1, on hepatocyte proliferation. METHODS: Hepatocyte proliferation was determined in AMPKα1 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 AMPKα1 delayed liver regeneration by impacting on G1/S transition phase. The proliferative defect of AMPKα1-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 AMPKα1 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, AMPKα1 controlled cyclin A2 transcription mainly through the ATF/CREB element. CONCLUSIONS: Our study highlights a novel role for AMPKα1 as a positive regulator of hepatocyte division occurring independently of energy balance.

[Prostatic luminal progenitors: From tissue regeneration to therapeutic escape]. / Progéniteurs luminaux prostatiques - De la régénération tissulaire à la résistance thérapeutique.

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.

Title: Progéniteurs luminaux prostatiques - De la régénération tissulaire à la résistance thérapeutique. Abstract: 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.

Prostate luminal progenitor cells: from mouse to human, from health to disease.

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.

Transcriptomic Signature and Growth Factor Regulation of Castration-Tolerant Prostate Luminal Progenitor Cells.

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.

High Keratin-7 Expression in Benign Peri-Tumoral Prostatic Glands Is Predictive of Bone Metastasis Onset and Prostate Cancer-Specific Mortality.

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.

STAT5a/b Deficiency Delays, but does not Prevent, Prolactin-Driven Prostate Tumorigenesis in Mice.

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-PRLSTAT5 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-PRLSTAT5 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-PRLSTAT5 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.

Six1 and Eya1 expression can reprogram adult muscle from the slow-twitch phenotype into the fast-twitch phenotype.

Muscle fibers show great differences in their contractile and metabolic properties. This diversity enables skeletal muscles to fulfill and adapt to different tasks. In this report, we show that the Six/Eya pathway is implicated in the establishment and maintenance of the fast-twitch skeletal muscle phenotype. We demonstrate that the MEF3/Six DNA binding element present in the aldolase A pM promoter mediates the high level of activation of this promoter in fast-twitch glycolytic (but not in slow-twitch) muscle fibers. We also show that among the Six and Eya gene products expressed in mouse skeletal muscle, Six1 and Eya1 proteins accumulate preferentially in the nuclei of fast-twitch muscles. The forced expression of Six1 and Eya1 together in the slow-twitch soleus muscle induced a fiber-type transition characterized by the replacement of myosin heavy chain I and IIA isoforms by the faster IIB and/or IIX isoforms, the activation of fast-twitch fiber-specific genes, and a switch toward glycolytic metabolism. Collectively, these data identify Six1 and Eya1 as the first transcriptional complex that is able to reprogram adult slow-twitch oxidative fibers toward a fast-twitch glycolytic phenotype.

Conditional cell ablation by tight control of caspase-3 dimerization in transgenic mice.

Studying the effects of the loss of a specific cell type is a powerful approach in biology. Here we present a method based on the controlled activation of the apoptotic machinery. We expressed a modified caspase-3-containing chemical inducer of dimerization (CID)-binding sites in the livers of transgenic mice. In the absence of CID, no liver injury was detectable, underlining the absence of leakage in our system. In contrast, injection of the CID produced activation of the chimeric caspase-3, which led to a dose-dependent pure hepatocyte ablation with subsequent regeneration. This method is effective in both growing and nongrowing cells, and is therefore applicable to a wide range of cells and tissues. Moreover, because apoptosis has been described in numerous pathological circumstances, this system is useful for generating mouse models of human disorders as well as for studying the recovery or regeneration of tissues after cell loss.

Gain-of-function Prolactin Receptor Variants Are Not Associated With Breast Cancer and Multiple Fibroadenoma Risk.

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.

[Liver repopulation strategies]. / Strategies de repeuplement du foie.

Hepatocytes have the unique capacity to self-renew and repair the liver ad integrum when stimulated to proliferate by liver injury. However, transplantation of isolated hepatocytes is usually not sufficiently efficient for therapeutic purposes. We conferred a survival advantage on transplanted hepatocytes and showed that they were able to repopulate almost the entire mouse liver after repeated injury. In contrast, we found that bone marrow stem cell transdifferentiation was inadequate for therapeutic liver regeneration. Current data on liver stem cells will be discussed.

Minireview: prolactin regulation of adult stem cells.

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.

Residue 146 regulates prolactin receptor folding, basal activity and ligand-responsiveness: potential implications in breast tumorigenesis.

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.

GH administration rescues fatty liver regeneration impairment by restoring GH/EGFR pathway deficiency.

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.

EGFR: A Master Piece in G1/S Phase Transition of Liver Regeneration.

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.

Increased susceptibility to liver fibrosis with age is correlated with an altered inflammatory response.

It has been suggested that increasing age is correlated with an acceleration of the progression of liver fibrosis induced by various agents, such as hepatitis C virus or chronic alcohol consumption. However, the cellular and molecular changes underlying this predisposition are not entirely understood. In the context of an aging population, it becomes challenging to decipher the mechanisms responsible for this higher susceptibility of older individuals to this acquired liver disorder. To address this issue, we induced liver fibrosis by carbon tetrachloride (CCl(4)) chronic administration to 8-week- and 15-month-old mice. We confirmed that susceptibility to fibrosis development increased with age and showed that aging did not affect fibrosis resolution capacity. We then focused on the impairment of hepatocyte proliferation, oxidative stress, and inflammation as potential mechanisms accelerating the development of fibrosis in the elderly. We detected no inhibition of hepatocyte proliferation after CCl(4) injury in 15-month-old mice, whereas it was inhibited after a partial hepatectomy. Finally, we observed that, in a context in which liver oxidative stress was not differentially increased in both experimental groups, there was a higher recruitment of inflammatory cells, including mostly macrophages and lymphocytes, oriented toward a T helper 2 (T(H)2) response in older mice. Our data show that in conditions of equivalent levels of oxidative stress and maintained hepatocyte proliferative capacity, an increased inflammatory reaction mainly composed of CD4(+) lymphocytes and macrophages expressing T(H)2 cytokines is the main factor involved in the higher susceptibility to fibrosis with increasing age.

GH receptor plays a major role in liver regeneration through the control of EGFR and ERK1/2 activation.

GH is a pleiotropic hormone that plays a major role in proliferation, differentiation, and metabolism via its specific receptor. It has been previously suggested that GH signaling pathways are required for normal liver regeneration but the molecular mechanisms involved have yet to be determined. The aim of this study was to identify the mechanisms by which GH controls liver regeneration. We performed two thirds partial hepatectomies in GH receptor (GHR)-deficient mice and wild-type littermates and showed a blunted progression in the G(1)/S transition phase of the mutant hepatocytes. This impaired liver regeneration was not corrected by reestablishing IGF-1 expression. Although the initial response to partial hepatectomy at the priming phase appeared to be similar between mutant and wild-type mice, cell cycle progression was significantly blunted in mutant mice. The main defect in GHR-deficient mice was the deficiency of the epidermal growth factor receptor activation during the process of liver regeneration. Finally, among the pathways activated downstream of GHR during G(1) phase progression, namely Erk1/2, Akt, and signal transducer and activator of transcription 3, we only found a reduced Erk1/2 phosphorylation in mutant mice. In conclusion, our results demonstrate that GH signaling plays a major role in liver regeneration and strongly suggest that it acts through the activation of both epidermal growth factor receptor and Erk1/2 pathways.