Alternative splicing of the dopamine D2 receptor directs specificity of coupling to G-proteins.

International audienceTwo isoforms of the dopamine D2 receptor have been characterized, D2L (long) and D2S (short), generated by alternative splicing from the same gene. They differ by an in-frame insert of 29 amino acids specific to D2L within the putative third intracytoplasmic loop of the receptor. We have previously demonstrated (Montmayeur, J.-P., Guiramand, J., and Borelli, E. (1993) Mol. Endocrinol. 7, 161-170) that D2S and D2L, although presenting very similar pharmacological profiles, couple differently to the alpha-subunit of guanine nucleotide-binding regulatory proteins (G-proteins). In particular, D2L, but not D2S, requires the presence of the alpha-subunit of the inhibitory G-protein (G alpha i2) to elicit greater inhibition of adenylyl cyclase activity. The insert present in D2L must therefore confer the specificity of interaction with G alpha i2. Thus, we introduced substitution mutations within the D2L insert. These mutant receptors were expressed in JEG3 cells, a G alpha i2-deficient cell line, scoring for those presenting an increased inhibition of adenylyl cyclase by dopamine. Our analysis identified two mutants, S259/262A and D249V, with these properties. These results clearly show that the insert present in D2L plays a critical role in the selectivity for the G-proteins interacting with the receptor

Mécanismes d'action d'une nouvelle classe de mutations du récepteur des androgènes dans les cancers de la prostate

Des mutations non-sens conduisant à la perte partielle ou totale de la région carboxy-terminale du récepteur des androgènes (RA) sont fréquemment détectées dans les cancers de la prostate (CaPs) localisés et les CaPs métastatiques en échappement hormonal. Par la formation d un complexe d activation transcriptionnelle différent due à une distribution intranucléaire et à un recrutement de cofacteurs particuliers, un de ces RA, le RA Q640X, exerce des activités transcriptionnelles différentes de celles du RA sauvage. Le RA Q640X conduit aussi à une interconnexion avec des facteurs de transcription comme AP-1 et NFAT. De plus, l expression du RA Q640X dans les cellules cancéreuses prostatiques conduit à la sécrétion de facteurs paracrines induisant à distance l activation du RA des cellules voisines, et ce en absence d hormone. Toutes ces données mettent en évidence un nouveau mécanisme de coopération clonale au cours de la progression du CaP au cours de l échappement hormonal.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Récepteurs des androgènes constitutivement actifs dans le cancer de la prostate (Interconnexions avec les voies de signalisation)

Un des mécanismes de résistance du cancer de la prostate (CaP) à l hormonothérapie est l altération de la voie de signalisation du récepteur des androgènes (RA). Mutations non sens ou épissages aberrants peuvent conduire à la perte partielle ou totale de la région CTE du RA. Ces RA mutés exhibant des activités transcriptionnelles ligand-indépendantes sont principalement retrouvées dans les CaPs métastatiques et en échappement hormonal. Nous montrons la coexistence de transcrits caractérisés par des mutations non sens et de transcrits issus d un épissage aberrant conduisant à des formes tronquées du RA dans la lignée 22Rv1. Nos resultats montrent que les voies voies de signalisation des récepteurs membranaires EGFR et HER2 et de leurs effecteurs intracellulaires PI3K/Akt, MEK1/2 et PKC sont fondamentales pour la transactivation du RA tronqué Q640X. Résultats preliminaires ont montré que le RA Q640X stimule les activités transcriptionnelles des facteurs de transcription NFAT et AP-1. Les RA tronqué pourraient stimuler l expression d un ou de plusieurs ligands des récepteurs membranaires, ou conduire à une augmentation de l expression d un effecteur intracellulaire ou à la libération de calcium intracellulaire; ou augmenter l expression d un récepteur membranaire. En conclusion, la perte de la région CTE conduit à un facteur de transcription ligand-indépendant dont les activités pourraient être liées à la progression du CaP et à l échappement à la privation androgénique. L ensemble des données de mes travaux suggère que la voie EGFR/PI3K pourrait représenter une cible thérapeutique conduisant à l inhibition des activités hormono-independentes des RA tronqués dans les CaP.The emergence of androgen receptor (AR) mutations is a key event in the progression of prostate cancer (PCa) toward androgen-independency. The AR protein consists in four functional domains: 1) the N-terminal region containing the activation function 1(AF-1) that is responsible for ligand-independent activity of the AR; 2) a central DNA binding domain (DBD) containing two zinc fingers; 3) the hinge region that encompasses a nuclear localization signal (NLS); 4) the C-terminal end (CTE) that includes the ligand binding domain (LBD) and AF-2. Mutations issued from clonal selection conferring new properties to the AR may be involved in PCa progression, bone metastasis and tumour growth under hormonal-treatment. The analysis of human tumour samples from localized and metastatic PCa allowed the identification of a new class of CTE-truncated mutant ARs. These mutant ARs the result of somatic nonsense mutations, and were twenty fold more frequent in hormone-refractory PCa that in hormone-naïve PCa. Furthermore, recent data proposed aberrant splicing as an alternative mechanism for CTE-truncated ARs. In the present study, we confirmed the coexistence of these two mechanisms, which offers a new preclinical model of hormoneresistant PCa. Investigations on the CTE-truncated Q640X AR have revealed in previous studies the constitutive nuclear localisation and the ligand-independent transcriptional activities of this CTE-truncated AR. Since the AR is a phosphoprotein, we explored the role of kinases signalling pathways in the activity of the Q640X AR. Our present results suggest that this AR mutant requires activating phosphorylation by PI3K/Akt and the MEK1-2 proteins, for full transactivation or transcriptional activities. Furthermore, we demonstrated that serine at position 213 in the AR sequence remains fundamental for signal transduction from PI3K/Akt kinases. In LNCaP cells, we have previously shown that the Q640X AR stimulates transcriptional activities of transcription factors such as NFAT and AP-1. We demonstrate that activation of Phospholipase C is a prerequisite for NFAT activity. In addition, we provide evidence that the crosstalk between truncated AR and NFAT or AP-1 factors depends on upstream signalling pathways. Altogether, our data indicate that Q640X AR requires activating phosphorylation by growth factors signalling pathways for full transcriptional activities. Moreover, this CTE-truncated AR may enhance some ligand/membrane receptors signalling to activate NFAT and AP-1. In conclusion, expression of CTE- truncated ARs may provide a mechanism for resistance to hormonal therapy. Understanding their functional properties ARs will allow the identification of new targeted therapies for the treatment of hormonal resistant PCa. Targeting the PI3K/Akt pathway may be a useful strategy for treating patients with hormone-refractory PCa positive for CTE truncated ARs.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceAustriaFRA

Osteoblasts promote castration-resistant prostate cancer by altering intratumoral steroidogenesis

The skeleton is the preferred site for prostate cancer (PC) metastasis leading to incurable castration-resistant disease. The increased expression of genes encoding steroidogenic enzymes found in bone metastatic tissue from patients suggests that up-regulated steroidogenesis might contribute to tumor growth at the metastatic site. Because of the overall sclerotic phenotype, we hypothesize that osteoblasts regulate the intratumoral steroidogenesis of castration resistant prostate cancer (CRPC) in bone. We here show that osteoblasts alter the steroidogenic transcription program in CRPC cells, closely mimicking the gene expression pattern described in CRPC. Osteoblast-stimulated LNCaP-19 cells displayed an increased expression of genes encoding for steroidogenic enzymes (CYP11A1, HSD3B1, and AKR1C3), estrogen signaling-related genes (CYP19A1, and ESR2), and genes for DHT-inactivating enzymes (UGT2B7, UGT2B15, and UGT2B17). The observed osteoblast-induced effect was exclusive to osteogenic CRPC cells (LNCaP-19) in contrast to osteolytic PC-3 and androgen-dependent LNCaP cells. The altered steroid enzymatic pattern was specific for the intratibial tumors and verified by immunohistochemistry in tissue specimens from LNCaP-19 xenograft tumors. Additionally, the overall steroidogenic effect was reflected by corresponding levels of progesterone and testosterone in serum from castrated mice with intratibial xenografts. A bi-directional interplay was demonstrated since both proliferation and Esr2 expression of osteoblasts were induced by CRPC cells in steroid-depleted conditions. Together, our results demonstrate that osteoblasts are important mediators of the intratumoral steroidogenesis of CRPC and for castration-resistant growth in bone. Targeting osteoblasts may therefore be important in the development of new therapeutic approaches

Dual effects of constitutively active androgen receptor and full-length androgen receptor for N-cadherin regulation in prostate cancer

Constitutively active androgen receptor (AR) variants have been involved in the expression of mesenchymal markers such as N-cadherin in prostate cancer (PCa). However, the underlying molecular mechanisms remain elusive. It remains unclear, whether N-cadherin gene (CDH2) is a direct transcriptional target of AR variants or whether the observed upregulation is due to indirect effects through additional regulatory factors. Moreover, the specific contribution of full-length AR and AR variants in N-cadherin regulation in PCa has never been explored deeply. To investigate this, we artificially mimicked the co-expression of AR variants together with a full-length AR and performed miRNA-seq, RNA-seq and ChIP assays. Our results were in favor of a direct AR variants action on CDH2. Our data also revealed a distinctive mode of action between full-length AR and AR variants to regulate N-cadherin expression. Both wild type AR and AR variants could interact with a regulatory element in intron 1 of CDH2. However, a higher histone H4 acetylation in this genomic region was only observed with AR variants. This suggests that full-length AR may play an occluding function to impede CDH2 upregulation. Our data further highlighted a negative effect of AR variants on the expression of the endogenous full-length AR in LNCaP. These differences in the mode of action of AR variants and full-length AR for the control of one key gene for prostate cancer progression could be worth considering for targeting AR variants in PCa

Androgen receptor-mediated transcriptional repression targets cell plasticity in prostate cancer

Androgen receptor (AR) signaling remains the key therapeutic target in the management of hormone-naive advanced prostate cancer (PCa) and castration-resistant PCa (CRPC). Recently, landmark molecular features have been reported for CRPC, including the expression of constitutively active AR variants that lack the ligand-binding domain. Besides their role in CRPC, AR variants lead to the expression of genes involved in tumor progression. However, little is known about the specificity of their mode of action compared with that of wild-type AR (AR-WT). We performed AR transcriptome analyses in an androgen-dependent PCa cell line as well as cross-analyses with publicly available RNA-seq datasets and established that transcriptional repression capacity that was marked for AR-WT was pathologically lost by AR variants. Functional enrichment analyses allowed us to associate AR-WT repressive function to a panel of genes involved in cell adhesion and epithelial-to-mesenchymal transition. So, we postulate that a less documented AR-WT normal function in prostate epithelial cells could be the repression of a panel of genes linked to cell plasticity, and that this repressive function could be pathologically abrogated by AR variants in PCa

Cross Modulation between the Androgen Receptor Axis and Protocadherin-PC in Mediating Neuroendocrine Transdifferentiation and Therapeutic Resistance of Prostate Cancer

Castration-resistant prostate cancers (CRPCs) that relapse after androgen deprivation therapies (ADTs) are responsible for the majority of mortalities from prostate cancer (PCa). While mechanisms enabling recurrent activity of androgen receptor (AR) are certainly involved in the development of CRPC, there may be factors that contribute to the process including acquired neuroendocrine (NE) cell-like behaviors working through alternate (non-AR) cell signaling systems or AR-dependent mechanisms. In this study, we explore the potential relationship between the AR axis and a novel putative marker of NE differentiation, the human male protocadherin-PC (PCDH-PC), in vitro and in human situations. We found evidence for an NE transdifferentiation process and PCDH-PC expression as an early-onset adaptive mechanism following ADT and elucidate AR as a key regulator of PCDH-PC expression. PCDH-PC overexpression, in turn, attenuates the ligand-dependent activity of the AR, enabling certain prostate tumor clones to assume a more NE phenotype and promoting their survival under diverse stress conditions. Acquisition of an NE phenotype by PCa cells positively correlated with resistance to cytotoxic agents including docetaxel, a taxane chemotherapy approved for the treatment of patients with metastatic CRPC. Furthermore, knockdown of PCDH-PC in cells that have undergone an NE transdifferentiation partially sensitized cells to docetaxel. Together, these results reveal a reciprocal regulation between the AR axis and PCDH-PC signals, observed both in vitro and in vivo, with potential implications in coordinating NE transdifferentiation processes and progression of PCa toward hormonal and chemoresistance

Cross modulation between the androgen receptor axis and protocadherin-PC in mediating neuroendocrine transdifferentiation and therapeutic resistance of prostate cancer.: PCDH-PC/AR cross-talk in driving NE differentiation

International audienceCastration-resistant prostate cancers (CRPCs) that relapse after androgen deprivation therapies (ADTs) are responsible for the majority of mortalities from prostate cancer (PCa). While mechanisms enabling recurrent activity of androgen receptor (AR) are certainly involved in the development of CRPC, there may be factors that contribute to the process including acquired neuroendocrine (NE) cell-like behaviors working through alternate (non-AR) cell signaling systems or AR-dependent mechanisms. In this study, we explore the potential relationship between the AR axis and a novel putative marker of NE differentiation, the human male protocadherin-PC (PCDH-PC), in vitro and in human situations. We found evidence for an NE transdifferentiation process and PCDH-PC expression as an early-onset adaptive mechanism following ADT and elucidate AR as a key regulator of PCDH-PC expression. PCDH-PC overexpression, in turn, attenuates the ligand-dependent activity of the AR, enabling certain prostate tumor clones to assume a more NE phenotype and promoting their survival under diverse stress conditions. Acquisition of an NE phenotype by PCa cells positively correlated with resistance to cytotoxic agents including docetaxel, a taxane chemotherapy approved for the treatment of patients with metastatic CRPC. Furthermore, knockdown of PCDH-PC in cells that have undergone an NE transdifferentiation partially sensitized cells to docetaxel. Together, these results reveal a reciprocal regulation between the AR axis and PCDH-PC signals, observed both in vitro and in vivo, with potential implications in coordinating NE transdifferentiation processes and progression of PCa toward hormonal and chemoresistance