Epidemiologic and experimental animal studies have shown that stress can alter tumor growth and that adrenergic fibers from the sympathetic nervous system, acting through stromal beta2 adrenergic receptors, promote tumor cell survival during the initial phases of cancer development. Recent epidemiological data suggest that beta blocker intake improves survival of prostate cancer patients. Hypertension and high blood pressure have been suggested to increase likelihood of prostate cancer development, suggesting that the use of beta blockers in these patients could reduce prostate cancer risk. Signaling mediated by beta adrenergic receptors promotes neuroendocrine differentiation in the LNCaP prostate cancer cell line. Although neuroendocrine cells are a minor population in the epithelial compartment of normal prostate glands, the population of neuroendocrine-like cells that express markers such as SYN, Bcl-2, Dll-3, and AMACR correlates with cancer progression, androgen-independent state, and poor prognosis in prostate cancer patients. As recent studies show that an increase in neuroendocrine cell density occurs before development of benign prostatic hyperplasia, a non-lethal disease affecting the transition zone, in hypertensive rats, we investigated whether neuroendocrine differentiation is associated with benign prostatic hyperplasia. Our results showed that isobutyl-methyl-xanthine, which blocks cAMP degradation, and forskolin, an adenylate cyclase activator, triggered sustained activation of the cAMP/PKA pathway and led to an increase in neuroendocrine differentiation markers in BPH-1 cells, which are a benign prostatic hyperplasia cell line. Similar results were observed by combining the beta adrenergic receptors agonist isoproterenol with forskolin. The same treatments reduced BPH-1 cell proliferation rates by 40% and 60%, respectively. Effects on AMACR and SYN expression and cell proliferation were fully reversed by the β2 blocker carvedilol. These data suggest that beta adrenergic receptors are linked to the development of neuroendocrine-like cells and benign prostatic hyperplasia. As 20% of the prostate cancer tumors occur in this zone, this observation warrants further investigation. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Recent studies have shown that adrenergic fibers from the sympathetic nervous system (SNS), acting through stromal β2-adrenergic receptors, play an important role in the initial phases of cancer development by promoting tumor cell survival. Recent epidemiological data suggest that β-blocker intake is associated with improved survival of prostate cancer patients. Hypertension and high blood pressure have been suggested to increase prostate cancer development suggesting that the use of β-blockers in these patients could also reduce prostate cancer risk. β-adrenoceptors signal through the cAMP/PKA pathway that is known to promote neuroendocrine differentiation (NED) in LNCaP prostate cancer cell line. While neuroendocrine (NE) cells expressing synaptophysin (SYN) represent a minor cell population in the epithelial compartment of normal prostate glands, the population of NE-like cells, exhibiting NE phenotypes and expressing NE markers, is increased in, and correlates with, cancer progression, androgen-independent state and poor prognosis. Expression of the routine tissue biomarker of prostate cancer AMACR in NE-like cells discriminates them from NE cells. Our aim was to investigate whether NED could also be associated with benign prostatic hyperplasia (BPH) as recent studies showed that an increase in neuroendocrine cell density occurred before development of BPH in hypertensive rats. Our results showed that a 10-day treatment with 100µM IBMX (blocking cAMP degradation) and 10µM forskolin (FSK, adenylate cyclase activator) to trigger sustained activation of the cAMP/PKA pathway led to an increase in SYN and AMACR expression in BPH-1 cells (qPCR and WB). Similar results were observed by combining the effect of the β-adrenoceptor agonist isoproterenol (1µM) and FSK. The same treatments reduced BPH-1 cell proliferation rates by 40% and 60%, respectively, as expected for NED. Effects on AMACR and SYN expression and cell proliferation were fully reversed by the β-blocker carvedilol (5µM). This data suggests that β-adrenoceptors could be linked to the development of NE-like cells in BPH, a non-lethal disease affecting the transition zone (TZ). As 20% of the prostate cancer occur in this TZ, this observation may need further investigation. Citation Format: Natascha Pigat, Laura N. Garrido, Coline Lefevre, Manon Baures, Vincent Goffin, Anne-Sophie Armand, Thierry Capiod. In vitro neuroendocrine differentiation of BPH-1 cells under beta-adrenergic stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6276.

Prostate cancer (PCa) represents one of the most frequent diagnosed cancer in males worldwide. Due to routine screening tests and the efficiency of available treatments, PCa-related deaths have significantly decreased over the past decades. However, PCa remains a critical threat if detected at a late stage in which, cancer cells would have already detached from the primary tumor to spread and invade other parts of the body. Calcium (Ca2+) channels and their protein regulators are now considered as hallmarks of cancer and some of them have been well examined in PCa. Among these Ca2+ channels, isoform 3 of the ORAI channel family has been shown to regulate the proliferation of PCa cells via the Arachidonic Acid-mediated Ca2+ entry, requiring the involvement of STIM1 (Stromal Interaction Molecule 1). Still, no study has yet demonstrated a role of the "neglected" STIM2 isoform in PCa or if it may interact with ORAI3 to promote an oncogenic behavior. In this study, we demonstrate that ORAI3 and STIM2 are upregulated in human PCa tissues. In old KIMAP (Knock-In Mouse Prostate Adenocarcinoma) mice, ORAI3 and STIM2 mRNA levels were significantly higher than ORAI1 and STIM1. In vitro, we show that ORAI3-STIM2 interact under basal conditions in PC-3 cells. ORAI3 silencing increased Store Operated Ca2+ Entry (SOCE) and induced a significant increase of the cell population in G2/M phase of the cell cycle, consistent with the role of ORAI3 as a negative regulator of SOCE. Higher expression levels of CDK1-Y15/Cyclin B1 were detected and mitotic arrest-related death occurred after ORAI3 silencing, which resulted in activating Bax/Bcl-2-mediated apoptotic pathway and caspase-8 activation and cleavage. STIM2 and ORAI3 expression increased in M phase while STIM1 expression and SOCE amplitude significantly decreased. Taken together, ORAI3 -STIM2 complex allows a successful progression through mitosis of PCa cells by evading mitotic catastrophe.

This file contains the legends for the 7 supplementary figures

This file contains supplementary figures showing prostate tumor kinetics (Fig S1) and histology (Fig S2) for the two mouse models, the validation of regimens (Fig S3 and S4), the effects of diets (Fig S5) and aging (Fig S6) on cancer hallmarks, and the results of cell studies complementary to main Figures 3 and 4.

This file contains the list of antibodies (Table S1), and the sequences of primers (Table S2) and siRNAs (Table S3)

Rhabdomyolysis is the acute breakdown of skeletal myofibres in response to an initiating factor, most commonly toxins and over exertion. A variety of genetic disorders predispose to rhabdomyolysis through different pathogenic mechanisms, particularly in patients with recurrent episodes. However, most cases remain without a genetic diagnosis. Here we present six patients who presented with severe and recurrent rhabdomyolysis, usually with onset in the teenage years; other features included a history of myalgia and muscle cramps. We identified 10 bi-allelic loss-of-function variants in the gene encoding obscurin (OBSCN) predisposing individuals to recurrent rhabdomyolysis. We show reduced expression of OBSCN and loss of obscurin protein in patient muscle. Obscurin is proposed to be involved in sarcoplasmic reticulum function and Ca2+ handling. Patient cultured myoblasts appear more susceptible to starvation as evidenced by a greater decreased in sarcoplasmic reticulum Ca2+ content compared to control myoblasts. This likely reflects a lower efficiency when pumping Ca2+ back into the sarcoplasmic reticulum and/or a decrease in Ca2+ sarcoplasmic reticulum storage ability when metabolism is diminished. OSBCN variants have previously been associated with cardiomyopathies. None of the patients presented with a cardiomyopathy and cardiac examinations were normal in all cases in which cardiac function was assessed. There was also no history of cardiomyopathy in first degree relatives, in particular in any of the carrier parents. This cohort is relatively young, thus follow-up studies and the identification of additional cases with bi-allelic null OBSCN variants will further delineate OBSCN-related disease and the clinical course of disease.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.

Calcium stones and calculi are observed in numerous human tissues. They are the result of deposition of calcium salts and are due to high local calcium concentrations. Prostatic calculi are usually classified as endogenous or extrinsic stones. Endogenous stones are commonly caused by obstruction of the prostatic ducts around an enlarged prostate resulting from benign prostatic hyperplasia or from chronic inflammation. The latter occurs mainly around the urethra and is generally caused by reflux of urine into the prostate. Calcium concentrations higher than in the plasma at sites of infection may induce the chemotactic response that eventually leads to recruitment of inflammatory cells. The calcium sensing receptor (CaSR) may be crucial for this recruitment as its expression and activity are increased by cytokines such as IL-6 and high extracellular calcium concentrations, respectively. The links between calcium calculi, inflammation, calcium supplementation, and CaSR functions in prostate cancer patients will be discussed in this review.

Le pronostic du cancer de prostate varie en fonction des caractéristiques tumorales. La présence d’un contingent neuro-endocrine est associée à un pronostic péjoratif. Nous avons montré que le calcium peut accélérer la progression tumorale et que ses effets passent par un récepteur au calcium (CaSR), associé à un mauvais pronostic. Notre objectif a alors été de montrer l’implication du CaSR dans la différenciation neuro-endocrine. Nous avons analysé rétrospectivement les liquides de rinçage du pistolet à biopsies, des patients ayant bénéficié de biopsies prostatiques ciblées dans notre centre entre janvier 2013 et novembre 2016. Des qPCR ont été réalisées pour rechercher l’expression du CaSR et de la synaptophysine, un des marqueurs les mieux établis pour les cellules neuro-endocrines. Une analyse de régression logistique multivariée a été réalisée pour rechercher les facteurs associés à l’expression de CaSR et synaptophysine. En parallèle nous avons analysé par qPCR l’expression de CaSR et synaptophysine sur la lignée cellulaire tumorale prostatique humaine (LNCaP) chez lesquelles nous avons induit la différenciation neuro-endocrine. Nous avons analysé les liquides de rinçage de 214 patients et 164 qPCR ont pu être réalisées sur ce matériel. Sur les 164 liquides de rinçages, 100 (61,3 %) correspondaient à une biopsie positive. Le CaSR était exprimé chez 52 (32 %) patients. La synaptophysine était exprimée chez 116 (71 %) patients. Quatre-vingt-trois pour cent des patients qui exprimaient le CaSR exprimaient la synaptophysine. Chez les patients exprimant CaSR et synaptophysine, la prévalence du cancer était de 70 %. Chez les patients n’exprimant aucun des deux marqueurs, la prévalence du cancer était de 44,7 % (p = 0,0232). En analyse multivariée, l’expression de CaSR et synaptophysine n’étaient pas associées à la présence de cancer, ni aux caractéristiques tumorales. La différenciation neuro-endocrine des LNCaP a montré l’apparition et la co-localisation du CaSR et de la synaptophysine au niveau cellulaire. Le CaSR a été associé à une surmortalité dans un travail récent, mais n’est pas associé aux caractéristiques tumorales au moment du diagnostic. Le CaSR pourrait donc jouer un rôle tardif dans l’évolution du cancer en favorisant l’apparition d’un contingent neuro-endocrine. Le suivi de notre cohorte pourrait permettre de valider cette hypothèse.

Active surveillance (AS) is an attractive alternative to immediate treatment for men with low-risk prostate cancer. Thus, the identification of environmental factors that promote the progression of indolent disease towards aggressive stages is critical to optimize clinical management. Epidemiological studies suggest that calcium-rich diets contribute to an increased risk of developing prostate cancer and that vitamin D reduces this risk. However, the potential effect of these nutrients on the progression of early-stage prostate tumours is uncertain, as studies in this setting are scarce and have not provided unambiguous conclusions. By contrast, the results of a preclinical study from our own group demonstrate that a diet high in calcium dose-dependently accelerated the progression of early-stage prostate tumours and that dietary vitamin D prevented this effect. The extent to which the conclusions of preclinical and epidemiological studies support a role for calcium and vitamin D and the relevance of monitoring and adjustment of calcium and/or vitamin D intake in patients on AS require further investigation.

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8+ T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca2+-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs.

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.

Autophagy is an adaptation mechanism that is vital for cellular homeostasis in response to various stress conditions. Previous reports indicate that there is a functional interaction between the primary cilium (PC) and autophagy. The PC, a microtubule-based structure present at the surface of numerous cell types, is a mechanical sensor. Here we show that autophagy induced by fluid flow regulates kidney epithelial cell volume in vitro and in vivo . PC ablation blocked autophagy induction and cell-volume regulation. In addition, inhibition of autophagy in ciliated cells impaired the flow-dependent regulation of cell volume. PC-dependent autophagy can be triggered either by mTOR inhibition or a mechanism dependent on the polycystin 2 channel. Only the LKB1–AMPK–mTOR signalling pathway was required for the flow-dependent regulation of cell volume by autophagy. These findings suggest that therapies regulating autophagy should be considered in developing treatments for PC-related diseases.

Calcium channels and the two G-protein coupled receptors sensing extracellular calcium, calcium-sensing receptor (CaSR) and GPRC6a, are the two main means by which extracellular calcium can signal to cells and regulate many cellular processes including cell proliferation, migration and invasion of tumoral cells. Many intracellular signaling pathways are sensitive to cytosolic calcium rises and conversely intracellular signaling pathways can modulate calcium channel expression and activity. Calcium channels are undoubtedly involved in the former while the CaSR and GPRC6a are most likely to interfere with the latter. As for neurotransmitters, calcium ions use plasma membrane channels and GPCR to trigger cytosolic free calcium concentration rises and intracellular signaling and regulatory pathways activation. Calcium sensing GPCR, CaSR and GPRC6a, allow a supplemental degree of control and as for metabotropic receptors, they not only modulate calcium channel expression but they may also control calcium-dependent K+ channels. The multiplicity of intracellular signaling pathways involved, their sensitivity to local and global intracellular calcium increase and to CaSR and GPRC6a stimulation, the presence of membrane signalplex, all this confers the cells the plasticity they need to convert the effects of extracellular calcium into complex physiological responses and therefore determine their fate.

The 6th European Calcium Society (ECS) workshop "Calcium and Cell Fate" held in Seillac (France) on

Développer une méthode de détection combinée du variant du récepteur aux androgènes ARV7 dans les urines et le produit de rinçage du pistolet à biopsie prostatique. Évaluer la prévalence de l’ARV7 dans une population soumise au dépistage du cancer de la prostate. Nous avons inclus prospectivement 161 patients adressés pour biopsies de la prostate. Des prélèvements urinaires ont été réalisés avant et après biopsies, de même qu’un prélèvement du liquide de rinçage du pistolet à biopsies (LRB). La recherche de transcrits de ARV7 a été effectuée par RT-qPCR. L’analyse histologique des biopsies a précisé l’existence d’un cancer, et le score de Gleason. L’âge moyen (ds) était de 65 (8,5) ans. Le PSA moyen (ds) était de 17,3 (37,8) ng/mL. Le taux de détection biopsique était de 60,2 %. Au total, un transcrit de ARV7 a été détecté sur au moins un prélèvement chez 45 % des hommes ayant un cancer, et 39 % des hommes sans évidence de cancer. Les taux de détection de transcrit de ARV7 étaient respectivement de 25 %, 32 %, et 50 % sur le LRB seul, les urines seules, et les 2 tests combinés (Tableau 1). Les taux de détection des transcrits de ARV7 n’étaient pas statistiquement associés à l’âge, au taux de PSA, à la présence de cancer ou au score de Gleason biopsique. La prévalence d’expression du variant ARV7 est importante. Elle ne semble pas être associée à la présence de cancer. La sensibilité de détection pourrait être augmentée par une étude combinée des urines et du LRB.

Abstract Active surveillance has emerged as an alternative to immediate treatment for men with low-risk prostate cancer. Accordingly, identification of environmental factors that facilitate progression to more aggressive stages is critical for disease prevention. Although calcium-enriched diets have been speculated to increase prostate cancer risk, their impact on early-stage tumors remains unexplored. In this study, we addressed this issue with a large interventional animal study. Mouse models of fully penetrant and slowly evolving prostate tumorigenesis showed that a high calcium diet dramatically accelerated the progression of prostate intraepithelial neoplasia, by promoting cell proliferation, micro-invasion, tissue inflammation, and expression of acknowledged prostate cancer markers. Strikingly, dietary vitamin D prevented these calcium-triggered tumorigenic effects. Expression profiling and in vitro mechanistic studies showed that stimulation of PC-3 cells with extracellular Ca2+ resulted in an increase in cell proliferation rate, store-operated calcium entry (SOCE) amplitude, cationic channel TRPC6, and calcium sensing receptor (CaSR) expression. Notably, administration of the active vitamin D metabolite calcitriol reversed all these effects. Silencing CaSR or TRPC6 expression in calcium-stimulated PC3 cells decreased cell proliferation and SOCE. Overall, our results demonstrate the protective effects of vitamin D supplementation in blocking the progression of early-stage prostate lesions induced by a calcium-rich diet. Cancer Res; 77(2); 355–65. ©2016 AACR.

La surexpression de prolactine dans l’épithélium glandulaire prostatique de souris transgéniques induit, par l’intermédiaire d’une hyper-signalisation STAT5, l’apparition d’une hyperplasie bénigne de la prostate associée à des infiltrats inflammatoires. L’objectif était donc d’évaluer la prévalence de la signalisation STAT5 dans le tissu adénomyomateux prostatique humain, son association avec la présence d’infiltrats inflammatoires ainsi qu’avec la prise de certains traitements pharmacologiques. Cinquante-huit patients opérés d’une hyperplasie bénigne de prostate ont été sélectionnés dans trois centres en fonction de leurs traitements préalables à la chirurgie. Parmi eux, 18 et 15 avaient été traités pendant plus d’un an respectivement par finastéride seul ou alpha-bloquant seul. Vingt-cinq patients n’avaient eu aucun traitement avant chirurgie. Des recoupes ont été réalisées dans le tissu de la zone centrale. Une analyse immunohistochimique de STAT5 phosphorylé (activé) et de CD45 (marqueur leucocytaire) a été réalisée puis quantifiée à l’aide d’un logiciel d’analyse d’images numérisées. Enfin, l’analyse du tissu prostatique en zone périphérique a été réalisée sur matériel biopsique normal provenant de 2 patients. L’âge médian était de 71 [65–78] ans. Le volume prostatique médian était de 100 [70–120] mL. À ce stade, l’analyse a été réalisée chez 10 patients de chaque groupe. Un marquage phospho-STAT5 a été observé chez tous les patients au niveau nucléaire des cellules basales des glandes de la zone centrale. Le marquage phospho-STAT5 était significativement plus étendu chez les patients préalablement traités par Finastéride (Fig. 1). Au niveau de la zone périphérique, le marquage était sporadique. Le nombre et l’étendue des infiltrats leucocytaires, marqués au CD45, était significativement plus important après la prise de Finastéride (Fig. 1, Tableau 1). Nos résultats encore préliminaires suggèrent que la signalisation STAT5 est activée dans l’hyperplasie bénigne de la prostate humaine, et modulée par certains traitements comme le finastéride. Les mécanismes moléculaires impliqués devront être élucidés ultérieurement.