Molecular Analysis of a Congenital Myasthenic Syndrome Due to a Pathogenic Variant Affecting the C-Terminus of ColQ.

Congenital Myasthenic Syndromes (CMSs) are rare inherited diseases of the neuromuscular junction characterized by muscle weakness. CMSs with acetylcholinesterase deficiency are due to pathogenic variants in COLQ, a collagen that anchors the enzyme at the synapse. The two COLQ N-terminal domains have been characterized as being biochemical and functional. They are responsible for the structure of the protein in the triple helix and the association of COLQ with acetylcholinesterase. To deepen the analysis of the distal C-terminal peptide properties and understand the CMSs associated to pathogenic variants in this domain, we have analyzed the case of a 32 year old male patient bearing a homozygote splice site variant c.1281 C > T that changes the sequence of the last 28 aa in COLQ. Using COS cell and mouse muscle cell expression, we show that the COLQ variant does not impair the formation of the collagen triple helix in these cells, nor its association with acetylcholinesterase, and that the hetero-oligomers are secreted. However, the interaction of COLQ variant with LRP4, a signaling hub at the neuromuscular junction, is decreased by 44% as demonstrated by in vitro biochemical methods. In addition, an increase in all acetylcholine receptor subunit mRNA levels is observed in muscle cells derived from the patient iPSC. All these approaches point to pathophysiological mechanisms essentially characterized by a decrease in signaling and the presence of immature acetylcholine receptors.

The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction.

Collagen Q (ColQ) is a nonfibrillar collagen that plays a crucial role at the vertebrate neuromuscular junction (NMJ) by anchoring acetylcholinesterase to the synapse. ColQ also functions in signaling, as it regulates acetylcholine receptor clustering and synaptic gene expression, in a manner dependent on muscle-specific kinase (MuSK), a key protein in NMJ formation and maintenance. MuSK forms a complex with low-density lipoprotein receptor-related protein 4 (LRP4), its coreceptor for the proteoglycan agrin at the NMJ. Previous studies suggested that ColQ also interacts with MuSK. However, the molecular mechanisms underlying ColQ functions and ColQ-MuSK interaction have not been fully elucidated. Here, we investigated whether ColQ binds directly to MuSK and/or LRP4 and whether it modulates agrin-mediated MuSK-LRP4 activation. Using coimmunoprecipitation, pull-down, plate-binding assays, and surface plasmon resonance, we show that ColQ binds directly to LRP4 but not to MuSK and that ColQ interacts indirectly with MuSK through LRP4. In addition, we show that the LRP4 N-terminal region, which contains the agrin-binding sites, is also crucial for ColQ binding to LRP4. Moreover, ColQ-LRP4 interaction was reduced in the presence of agrin, suggesting that agrin and ColQ compete for binding to LRP4. Strikingly, we reveal ColQ has two opposing effects on agrin-induced MuSK-LRP4 signaling: it constitutively reduces MuSK phosphorylation levels in agrin-stimulated myotubes but concomitantly increases MuSK accumulation at the muscle cell surface. Our results identify LRP4 as a major receptor of ColQ and provide new insights into mechanisms of ColQ signaling and acetylcholinesterase anchoring at the NMJ.

The Neuropilin-1/PKC axis promotes neuroendocrine differentiation and drug resistance of prostate cancer.

BACKGROUND: Neuroendocrine prostate cancer (NEPC) is a multi-resistant variant of prostate cancer (PCa) that has become a major challenge in clinics. Understanding the neuroendocrine differentiation (NED) process at the molecular level is therefore critical to define therapeutic strategies that can prevent multi-drug resistance. METHODS: Using RNA expression profiling and immunohistochemistry, we have identified and characterised a gene expression signature associated with the emergence of NED in a large PCa cohort, including 169 hormone-naïve PCa (HNPC) and 48 castration-resistance PCa (CRPC) patients. In vitro and preclinical in vivo NED models were used to explore the cellular mechanism and to characterise the effects of castration on PCa progression. RESULTS: We show for the first time that Neuropilin-1 (NRP1) is a key component of NED in PCa cells. NRP1 is upregulated in response to androgen deprivation therapies (ADT) and elicits cell survival through induction of the PKC pathway. Downmodulation of either NRP1 protein expression or PKC activation suppresses NED, prevents tumour evolution toward castration resistance and increases the efficacy of docetaxel-based chemotherapy in preclinical models in vivo. CONCLUSIONS: This study reveals the NRP1/PKC axis as a promising therapeutic target for the prevention of neuroendocrine castration-resistant variants of PCa and indicates NRP1 as an early transitional biomarker.

Overexpression of Nucleolin and Associated Genes in Prostate Cancer.

Prostate cancer (PCa) is the second most frequent cancer and the fifth leading cause of cancer death in men worldwide. If local PCa presents a favorable prognosis, available treatments for advanced PCa display limiting benefits due to therapeutic resistances. Nucleolin (NCL) is a ubiquitous protein involved in numerous cell processes, such as ribosome biogenesis, cell cycles, or angiogenesis. NCL is overexpressed in several tumor types in which it has been proposed as a diagnostic and prognostic biomarker. In PCa, NCL has mainly been studied as a target for new therapeutic agents. Nevertheless, little data are available concerning its expression in patient tissues. Here, we investigated the expression of NCL using a new cohort from Mondor Hospital and data from published cohorts. Results were then compared with NCL expression using in vitro models. NCL was overexpressed in PCa tissues compared to the normal tissues, but no prognostic values were demonstrated. Nine genes were highly co-expressed with NCL in patient tissues and tumor prostate cell lines. Our data demonstrate that NCL is an interesting diagnostic biomarker and propose a signature of genes co-expressed with NCL.

Correlative Imaging of Motoneuronal Cell Elasticity by Pump and Probe Spectroscopy.

Because of their role of information transmitter between the spinal cord and the muscle fibers, motor neurons are subject to physical stimulation and mechanical property modifications. We report on motoneuron elasticity investigated by time-resolved pump and probe spectroscopy. A dual picosecond geometry simultaneously probing the acoustic impedance mismatch at the cell-titanium transducer interface and acoustic wave propagation inside the motoneuron is presented. Such noncontact and nondestructive microscopy, correlated to standard atomic force microscopy or a fluorescent labels approach, has been carried out on a single cell to address some physical properties such as bulk modulus of elasticity, dynamical longitudinal viscosity, and adhesion.

AChR ß-Subunit mRNAs Are Stabilized by HuR in a Mouse Model of Congenital Myasthenic Syndrome With Acetylcholinesterase Deficiency.

Collagen Q (COLQ) is a specific collagen that anchors acetylcholinesterase (AChE) in the synaptic cleft of the neuromuscular junction. So far, no mutation has been identified in the ACHE human gene but over 50 different mutations in the COLQ gene are causative for a congenital myasthenic syndrome (CMS) with AChE deficiency. Mice deficient for COLQ mimic most of the functional deficit observed in CMS patients. At the molecular level, a striking consequence of the absence of COLQ is an increase in the levels of acetylcholine receptor (AChR) mRNAs and proteins in vivo and in vitro in murine skeletal muscle cells. Here, we decipher the mechanisms that drive AChR mRNA upregulation in cultured muscle cells deficient for COLQ. We show that the levels of AChR ß-subunit mRNAs are post-transcriptionally regulated by an increase in their stability. We demonstrate that this process results from an activation of p38 MAPK and the cytoplasmic translocation of the nuclear RNA-binding protein human antigen R (HuR) that interacts with the AU-rich element located within AChR ß-subunit transcripts. This HuR/AChR transcript interaction induces AChR ß-subunit mRNA stabilization and occurs at a specific stage of myogenic differentiation. In addition, pharmacological drugs that modulate p38 activity cause parallel modifications of HuR protein and AChR ß-subunit levels. Thus, our study provides new insights into the signaling pathways that are regulated by ColQ-deficiency and highlights for the first time a role for HuR and p38 in mRNA stability in a model of congenital myasthenic syndrome.

Erratum: CRIPTO overexpression promotes mesenchymal differentiation in prostate carcinoma cells through parallel regulation of AKT and FGFR activities.

[This corrects the article DOI: 10.18632/oncotarget.2740.].

Implication of NPM1 phosphorylation and preclinical evaluation of the nucleoprotein antagonist N6L in prostate cancer.

Despite the advent of several new treatment options over the past years, advanced/metastatic prostate carcinoma (PCa) still remains incurable, which justifies the search for novel targets and therapeutic molecules. Nucleophosmin (NPM1) is a shuttling nucleoprotein involved in tumor growth and its targeting could be a potential approach for cancer therapy. We previously demonstrated that the multivalent pseudopeptide N6L binds to NPM1 potently affecting in vitro and in vivo tumor cell growth of various tumor types as well as angiogenesis. Furthermore, NPM1 binds to androgen receptor (AR) and modulate its activity. In this study, we first investigated the implication of the NPM1 and its Thr199 and Thr234/237 phosphorylated forms in PCa. We showed that phosphorylated forms of NPM1 interact with androgen receptor (AR) in nucleoplasm. N6L treatment of prostate tumor cells led to inhibition of NPM1 phosphorylation in conjunction with inhibition of AR activity. We also found that total and phosphorylated NPM1 were overexpressed in castration-resistant PCa. Assessment of the potential therapeutic role of N6L in PCa indicated that N6L inhibited tumor growth both in vitro and in vivo when used either alone or in combination with the standard-of-care first- (hormonotherapy) and second-line (docetaxel) treatments for advanced PCa. Our findings reveal the role of Thr199 and Thr234/237 phosphorylated NPM1 in PCa progression and define N6L as a new drug candidate for PCa therapy.

Clinical value of ERG, TFF3, and SPINK1 for molecular subtyping of prostate cancer.

BACKGROUND: In view of the marked molecular heterogeneity of prostate cancer (PCa), clinical and pathologic parameters alone may be unreliable for predicting disease outcomes after surgical intervention. The development of biomarkers may be helpful to estimate tumor heterogeneity and stratify patients in terms of their risk of progression. Levels of v-ets avian erythroblastosis virus E26 oncogene homolog (ERG), trefoil factor 3 (TFF3), and serine peptidase inhibitor, Kazal type 1 (SPINK1) are commonly elevated in PCa, but it is unclear whether the evaluation of these 3 markers can help to discriminate patients who will have different clinical outcomes. The authors investigated whether assessment of ERG, TFF3, and SPINK1 expression could help to define clinically relevant, distinct subsets of patients with PCa. METHODS: The cohort consisted of 279 men with PCa who underwent radical prostatectomy at Henri Mondor Hospital. Expression levels of ERG, TFF3, and SPINK1 were evaluated immunohistochemically in the prostatectomy specimens. Potential associations of ERG, TFF3, and SPINK1 with age, prostate-specific antigen (PSA), tumor stage, Gleason score, and biochemical recurrence, defined by PSA failure, were investigated. RESULTS: Although prognostic significance was not observed for ERG or TFF3, an exclusive pattern of expression was demonstrated for TFF3 and ERG. SPINK1 expression was observed exclusively in a subgroup of cancers that expressed TFF3 (41 of 175 tumors). Moreover, SPINK1 positivity was identified as predictive of biochemical recurrence in univariate (P = .0009) and multivariate (P = .0003) analyses. CONCLUSIONS: The current results suggest that ERG and TFF3 characterize 2 distinct subsets of PCa, with a more aggressive subgroup of TFF3-expressing tumors that express SPINK1. Together, these findings support a rationale of screening for these biomarkers for prognostic purposes and molecular subtyping of the disease.

CRIPTO overexpression promotes mesenchymal differentiation in prostate carcinoma cells through parallel regulation of AKT and FGFR activities.

Members of the EGF-CFC (Cripto, FRL-1, Cryptic) protein family are increasingly recognized as key mediators of cell movement and cell differentiation during vertebrate embryogenesis. The founding member of this protein family, CRIPTO, is overexpressed in various human carcinomas. Yet, the biological role of CRIPTO in this setting remains unclear. Here, we find CRIPTO expression as especially high in a subgroup of primary prostate carcinomas with poorer outcome, wherein resides cancer cell clones with mesenchymal traits. Experimental studies in PCa models showed that one notable function of CRIPTO expression in prostate carcinoma cells may be to augment PI3K/AKT and FGFR1 signaling, which promotes epithelial-mesenchymal transition and sustains a mesenchymal state. In the observed signaling events, FGFR1 appears to function parallel to AKT, and the two pathways act cooperatively to enhance migratory, invasive and transformation properties specifically in the CRIPTO overexpressing cells. Collectively, these findings suggest a novel molecular network, involving CRIPTO, AKT, and FGFR signaling, in favor of the emergence of mesenchymal-like cancer cells during the development of aggressive prostate tumors.

FKBP12.6 mice display temporal gender differences in cardiac Ca(2+)-signalling phenotype upon chronic pressure overload.

Preventing Ca(2+)-leak during diastole may provide a means to improve overall cardiac function. The immunosuppressant FK506-binding protein 12.6 (FKBP12.6) regulates ryanodine receptor-2 (RyR2) gating and binds to and inhibits calcineurin (Cn). It is also involved in the pathophysiology of heart failure (HF). Here, we investigated the effects of FKBP12.6 over-expression and gender on Ca(2+)-handling proteins (RyR2, SERCA2a/PLB, and NCX), and on pro-(CaMKII, Cn/NFAT) and anti-hypertrophic (GSK3ß) signalling pathways in a thoracic aortic constriction (TAC) mouse model. Wild type mice (WT) and mice over-expressing FKBP12.6 of both genders underwent TAC or sham-operation (Sham). FKBP12.6 over-expression ameliorated post-TAC survival rates in both genders. Over time, FKBP12.6 over-expression reduced the molecular signature of left ventricular hypertrophy (LVH) and the transition to HF (BNP and ß-MHC mRNAs) and attenuated Cn/NFAT activation in TAC-males only. The gender difference in pro- and anti-hypertrophic LVH signals was time-dependent: TAC-females exhibited earlier pathological LVH associated with concomitant SERCA2a down-regulation, CaMKII activation, and GSK3ß inactivation. Both genotypes showed systolic dysfunction, possibly related to down-regulated RyR2, but only FK-TAC-males exhibited preserved diastolic LV function. Although FKBP12.6 over-expression did not impact the vicious cycle of TAC-induced HF, this study reveals some subtle sequential and temporal gender differences in Ca(2+)-signalling pathways of pathological LVH.