ZMIZ1 Variants Cause a Syndromic Neurodevelopmental Disorder.

ZMIZ1 is a coactivator of several transcription factors, including p53, the androgen receptor, and NOTCH1. Here, we report 19 subjects with intellectual disability and developmental delay carrying variants in ZMIZ1. The associated features include growth failure, feeding difficulties, microcephaly, facial dysmorphism, and various other congenital malformations. Of these 19, 14 unrelated subjects carried de novo heterozygous single-nucleotide variants (SNVs) or single-base insertions/deletions, 3 siblings harbored a heterozygous single-base insertion, and 2 subjects had a balanced translocation disrupting ZMIZ1 or involving a regulatory region of ZMIZ1. In total, we identified 13 point mutations that affect key protein regions, including a SUMO acceptor site, a central disordered alanine-rich motif, a proline-rich domain, and a transactivation domain. All identified variants were absent from all available exome and genome databases. In vitro, ZMIZ1 showed impaired coactivation of the androgen receptor. In vivo, overexpression of ZMIZ1 mutant alleles in developing mouse brains using in utero electroporation resulted in abnormal pyramidal neuron morphology, polarization, and positioning, underscoring the importance of ZMIZ1 in neural development and supporting mutations in ZMIZ1 as the cause of a rare neurodevelopmental syndrome.

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-naïve-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.

Fluorine NMR study of proline-rich sequences using fluoroprolines.

Proline homopolymer motifs are found in many proteins; their peculiar conformational and dynamic properties are often directly involved in those proteins' functions. However, the dynamics of proline homopolymers is hard to study by NMR due to a lack of amide protons and small chemical shift dispersion. Exploiting the spectroscopic properties of fluorinated prolines opens interesting perspectives to address these issues. Fluorinated prolines are already widely used in protein structure engineering - they introduce conformational and dynamical biases - but their use as 19F NMR reporters of proline conformation has not yet been explored. In this work, we look at model peptides where Cγ-fluorinated prolines with opposite configurations of the chiral Cγ centre have been introduced at two positions in distinct polyproline segments. By looking at the effects of swapping these (4R)-fluoroproline and (4S)-fluoroproline within the polyproline segments, we were able to separate the intrinsic conformational properties of the polyproline sequence from the conformational alterations instilled by fluorination. We assess the fluoroproline 19F relaxation properties, and we exploit the latter in elucidating binding kinetics to the SH3 (Src homology 3) domain.

Identification of novel truncated androgen receptor (AR) mutants including unreported pre-mRNA splicing variants in the 22Rv1 hormone-refractory prostate cancer (PCa) cell line.

Advanced prostate cancer (PCa) has emerged as a public health concern due to population aging. Although androgen deprivation has proven efficacy in this condition, most advanced PCa patients will have to face failure of androgen deprivation as a treatment. Mutations in the androgen receptor (AR) from tumor cells have been shown to induce androgen independency both in PCa cell lines and in the clinic. We have investigated the molecular events leading to androgen independency in the 22Rv1 cell line, a commonly used preclinical model of PCa. Besides AR mutants that have been described so far, including nonsense mutations, recent data have focused on AR pre-mRNA aberrant splicing as a new mechanism leading to constitutively active truncated AR variants. In this article, we describe two novel variants arising from aberrant splicing of AR pre-mRNA, characterized by long mRNA transcripts that encode truncated, constitutively active proteins. We also describe several new nonsense mutants that share ligand independency and transcriptional activity. Finally, we show that alongside these mutants, 22Rv1 cells also express a mutant AR lacking exon 3 tandem duplication, a major feature of this cell line. By describing unreported AR mutants in the 22Rv1 cell line, our data emphasize the complexity and heterogeneity of molecular events that occur in preclinical models, and supposedly in the clinic. Future work on the 22Rv1 cell line should take into account the concomitant expression of various AR mutants.

Specific properties of a C-terminal truncated androgen receptor detected in hormone refractory prostate cancer.

Mutations in the human androgen receptor (AR) gene that lead to C-terminus truncated AR variants are frequently detected in prostate cancer (PC). These AR variants lack both the ligand-binding domain (LBD) and the AF-2 region. The aim of this study was to delineate the alternative mechanisms that lead to the activation of such AR variants as they are unresponsive to hormone stimulation, and to outline consequences of the loss of the LBD/AF-2 region on their functional properties. By using an MMTV-luciferase reporter construct and LY294002, UO126, or ZD1839, inhibitor of PI3K, MEK1/2, and EGFR signaling pathway respectively, we demonstrated that phosphorylation was required for full transcriptional activities of one these AR variants, the Q640X mutant AR. Western-blot analyses confirmed that these inhibitors affect the phosphorylation status of this AR variant. Furthermore, studies of the intranuclear colocalization of the Q640X AR with cofactors, such as CBP, GRIP-1, and c-Jun, reveal that the transcriptional complex that forms around the mutant AR is different to that formed around the wild type AR. We demonstrated that CBP and c-Jun are highly recruited by the mutant AR, and this leads to an unexpected activation of AP-1, NFAT, and NFkappaB transcriptional activities. Similar enhanced activities of these transcription factors were not observed with the wild type AR. The importance of the LBD/AF-2 for the regulation of AR transcriptional activities, the impact of the presence of such AR variants on PC cells proliferation and survival, and on progression to androgen independence are discussed.

A splicing variant of the androgen receptor detected in a metastatic prostate cancer exhibits exclusively cytoplasmic actions.

The androgen receptor (AR) is a ligand-activated transcription factor that displays genomic actions characterized by binding to androgen-response elements in the promoter of target genes as well as nongenomic actions that do not require nuclear translocation and DNA binding. In this study, we report exclusive cytoplasmic actions of a splicing variant of the AR detected in a metastatic prostate cancer. This AR variant, named AR23, results from an aberrant splicing of intron 2, wherein the last 69 nucleotides of the intronic sequence are retained, leading to the insertion of 23 amino acids between the two zinc fingers in the DNA-binding domain. We show that the nuclear entry of AR23 upon dihydrotestosterone (DHT) stimulation is impaired. Alternatively, DHT-activated AR23 forms cytoplasmic and perinuclear aggregates that partially colocalize with the endoplasmic reticulum and are devoid of genomic actions. However, in LNCaP cells, this cytoplasmic DHT-activated AR23 remains partially active as evidenced by the activation of transcription from androgen-responsive promoters, the stimulation of NF-kappaB transcriptional activity and by the decrease of AP-1 transcriptional activity. Our data reveal novel cytoplasmic actions for this splicing AR variant, suggesting a contribution in prostate cancer progression.

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.

A yeast-based functional assay for the detection of the mutant androgen receptor in prostate cancer.

OBJECTIVE: Mutations in the ligand-binding domain of the human androgen receptor (AR) figure among the ways used by prostate adenocarcinoma (PCa) cells to escape androgen dependence. These mutations may broaden the specificity and/or affinity of the AR to other hormones, resulting in inappropriate receptor activation and thus affecting the PCa response to physiological stimuli and hormonal therapies. DESIGN: In order to clarify the impact of these mutations on disease progression and treatment, we have developed a yeast-based functional assay that allows the detection of mutant ARs and the analysis of their transactivation capacities in response to different ligands. METHODS: AR cDNA was directly cloned into an expression vector in a yeast strain that carries a reporter gene (ADE2) linked to an androgen-dependent promoter. The expression of the ADE2 gene and consequently the yeast cell growth in a selective medium depleted in adenine depends on the specificity of the AR for the ligand added to the medium. RESULTS: By analysing the transactivation capacities of different AR molecules in response to a broad range of steroid and non-steroid ligands, we have demonstrated that this assay can discriminate among wild-type AR, T877A, C685Y and L701H mutant ARs and that at least 1% of mutant ARs could be detected when mutant and wild-type ARs were mixed at the cDNA level. CONCLUSIONS: The data presented here show that this simple AR assay is convenient for the routine detection of mutant ARs in PCa and is also suitable to evaluate the antagonist activities of anti-androgen molecules.

Constitutively active androgen receptor variants upregulate expression of mesenchymal markers in prostate cancer cells.

Androgen receptor (AR) signaling pathway remains the foremost target of novel therapeutics for castration-resistant prostate cancer (CRPC). However, the expression of constitutively active AR variants lacking the carboxy-terminal region in CRPC may lead to therapy inefficacy. These AR variants are supposed to support PCa cell growth in an androgen-depleted environment, but their mode of action still remains unresolved. Moreover, recent studies indicate that constitutively active AR variants are expressed in primary prostate tumors and may contribute to tumor progression. The aim of this study was to investigate the impact of constitutively active AR variants on the expression of tumor progression markers. N-cadherin expression was analyzed in LNCaP cells overexpressing the wild type AR or a constitutively active AR variant by qRT-PCR, Western blot and immunofluorescence. We showed here for the first time that N-cadherin expression was increased in the presence of constitutively active AR variants. These results were confirmed in C4-2B cells overexpressing these AR variants. Although N-cadherin expression is often associated with a downregulation of E-cadherin, this phenomenon was not observed in our model. Nevertheless, in addition to the increased expression of N-cadherin, an upregulation of other mesenchymal markers expression such as VIMENTIN, SNAIL and ZEB1 was observed in the presence of constitutively active variants. In conclusion, our findings highlight novel consequences of constitutively active AR variants on the regulation of mesenchymal markers in prostate cancer.

Risk of hormone escape in a human prostate cancer model depends on therapy modalities and can be reduced by tyrosine kinase inhibitors.

Almost all prostate cancers respond to androgen deprivation treatment but many recur. We postulated that risk of hormone escape--frequency and delay--are influenced by hormone therapy modalities. More, hormone therapies induce crucial biological changes involving androgen receptors; some might be targets for escape prevention. We investigated the relationship between the androgen deprivation treatment and the risk of recurrence using nude mice bearing the high grade, hormone-dependent human prostate cancer xenograft PAC120. Tumor-bearing mice were treated by Luteinizing-Hormone Releasing Hormone (LHRH) antagonist alone, continuous or intermittent regimen, or combined with androgen receptor (AR) antagonists (bicalutamide or flutamide). Tumor growth was monitored. Biological changes were studied as for genomic alterations, AR mutations and protein expression in a large series of recurrent tumors according to hormone therapy modalities. Therapies targeting Her-2 or AKT were tested in combination with castration. All statistical tests were two-sided. Tumor growth was inhibited by continuous administration of the LH-RH antagonist degarelix (castration), but 40% of tumors recurred. Intermittent castration or complete blockade induced by degarelix and antiandrogens combination, inhibited tumor growth but increased the risk of recurrence (RR) as compared to continuous castration (RR(intermittent): 14.5, RR(complete blockade): 6.5 and 1.35). All recurrent tumors displayed new quantitative genetic alterations and AR mutations, whatever the treatment modalities. AR amplification was found after complete blockade. Increased expression of Her-2/neu with frequent ERK/AKT activation was detected in all variants. Combination of castration with a Her-2/neu inhibitor decreased recurrence risk (0.17) and combination with an mTOR inhibitor prevented it. Anti-hormone treatments influence risk of recurrence although tumor growth inhibition was initially similar. Recurrent tumors displayed genetic instability, AR mutations, and alterations of phosphorylation pathways. We postulated that Her-2/AKT pathways allowed salvage of tumor cells under castration and we demonstrated that their inhibition prevented tumor recurrence in our model.

Unexpected paracrine action of prostate cancer cells harboring a new class of androgen receptor mutation--a new paradigm for cooperation among prostate tumor cells.

The emergence of mutations in the androgen receptor (AR) gene is a recurrent event during progression of prostate cancer (PCa) on androgen ablation therapy. In this study, we show that nonsense mutations that lead to carboxyl-terminal end truncated ARs are found at high frequency in metastatic PCas. Transcriptional activities of the Q640X mutant AR in the androgen-sensitive LNCaP cell line differ to those of the wild-type AR. Indeed, this mutant AR exhibits strong and ligand-independent transcriptional activities from an artificial promoter construct containing two repeats of androgen-responsive elements, but is inactive on the human PSA gene promoter. Nevertheless, the expression of the Q640X mutant AR in LNCaP cells is accompanied by an increase in the level of PSA protein, and by an increase in the expression of the endogenous AR gene. This enhanced expression of the endogenous AR gene is not limited to the sole transfected cells, but is observed in non-transfected neighboring cells. Additionally, in co-cultures of transfected and non-transfected LNCaP cells, the Q640X mutant AR leads to an unpredicted nuclear localization of the endogenous AR protein in the two cellular populations and this, in the absence of androgen. These data indicate that cells expressing the Q640X mutant AR acquire the property to emit a signal that activates the AR in neighboring cells by a paracrine mechanism and in a ligand-independent manner. Our data strongly support the notion of cooperation among tumor cells in PCa and could be of relevance for the understanding of progression on androgen ablation therapy.

Constitutive activation of the androgen receptor by a point mutation in the hinge region: a new mechanism for androgen-independent growth in prostate cancer.

Androgen receptor (AR) mutations that modify both the ligand binding and the transactivation capacities of the AR represent one of the mechanisms involved in the transition of prostate cancer (PCa) from androgen-dependent to androgen-independent growth. We use a yeast-based functional assay to detect and analyze mutant ARs in PCa. We report the detection of 2 different mutant ARs within the same metastatic tumour sample harvested in a patient with advanced PCa who had escaped androgen deprivation. Concomitantly to the widely described T877A mutant AR, we identified an additional double mutant AR harboring the nonsense mutation Q640Stop just downstream the DNA binding domain together with the T877A point mutation. This type of mutation, which leads to a c-terminal truncated AR, has not been described yet in PCa. Using luciferase reporter assays we demonstrated that this truncated AR exhibited constitutive transactivation properties. In conclusion, our data suggest that mutation-induced constitutive activation of the AR could be a mechanism used by PCa cells to escape androgen deprivation.