TRPA1 Contributes to FGFR2c Signaling and to Its Oncogenic Outcomes in Pancreatic Ductal Adenocarcinoma-Derived Cell Lines.

Fibroblast growth factor receptor (FGFR) signaling is a key modulator of cellular processes dysregulated in cancer. We recently found that the high expression of the mesenchymal FGFR2c variant in human pancreatic ductal adenocarcinoma (PDAC)-derived cells triggers the PKCε-mediated improvement of EMT and of MCL-1/SRC-dependent cell invasion. Since other membrane proteins can affect the receptor tyrosine kinase signaling, including transient receptor potential channels (TRPs), in this work, we investigated the role of TRPs in the FGFR2c/PKCε oncogenic axis. Our results highlighted that either the FGFR2c/PKCε axis shut-off obtained by shRNA or its sustained activation via ligand stimulation induces TRPA1 downregulation, suggesting a channel/receptor dependence. Indeed, biochemical molecular and immunofluorescence approaches demonstrated that the transient depletion of TRPA1 by siRNA was sufficient to attenuate FGFR2c downstream signaling pathways, as well as the consequent enhancement of EMT. Moreover, the biochemical check of MCL1/SRC signaling and the in vitro assay of cellular motility suggested that TRPA1 also contributes to the FGFR2c-induced enhancement of PDAC cell invasiveness. Finally, the use of a selective channel antagonist indicated that the contribution of TRPA1 to the FGFR2c oncogenic potential is independent of its pore function. Thus, TRPA1 could represent a putative candidate for future target therapies in PDAC.

FGFR2c Upregulation Contributes to Cancer-Associated Fibroblast Program Activation and to Enhanced Autophagy in Actinic Keratosis-Derived Dermal Fibroblasts: A Possible Role in Precancerous Cell/Stromal Cell Crosstalk.

Actinic keratosis (AK) is a preneoplastic skin disorder which can rapidly progress to cutaneous squamous cell carcinomas (SCCs). In light of our previous findings, indicating a possible oncogenic role of the mesenchymal isoform of FGFR2 (FGFR2c) aberrantly expressed in AK keratinocytes, we analyzed the possible tumor-promoting role of this receptor in the stromal AK counterpart in this work. Molecular analysis showed that, particularly in early AK lesions, FGFR2c dermal upregulation is accompanied by the downregulation of the cancer-associated fibroblasts (CAF) transcription repressor CSL, the upregulation of the CAF activator ULK3, and the consequent CAF gene induction. Immunofluorescence and molecular analysis, coupled with silencing approaches by siRNA, applied on primary cultures of KIN I-derived fibroblasts, indicated that FGFR2c upregulation contribute to CAF signature and the increased autophagy in response to FGF2. Magnetic bead-based multiplex assay, combined with FGFR2 signaling shut-off approaches, indicated that, especially in response to FGF2, IL-6 secretion could depend on FGFR2c high expression and signaling, suggesting the possible establishment of FGFR2c-dependent secretory autophagy, contributing to tumor-promoting factor release. Overall, our results identified FGFR2c as a signaling molecule involved in controlling precancerous/stromal cell oncogenic crosstalk, pointing to this receptor as a possible early molecular marker predictive for AK's rapid malignant progression.

NPPA/atrial natriuretic peptide is an extracellular modulator of autophagy in the heart.

NPPA/atrial natriuretic peptide (natriuretic peptide type A) exerts critical pleiotropic effects in the cardiovascular system, limiting cardiomyocyte hypertrophy and death, reducing cardiac fibrosis and promoting vascular integrity. However, the molecular mechanisms underlying these beneficial effects still need to be clarified. We demonstrated for the first time that macroautophagy/autophagy is involved in the local protective effects of NPPA in cardiomyocytes (CMs), both in vitro and in vivo. Exogenous NPPA rapidly activates autophagy in CMs through NPR1/type A natriuretic peptide receptor and PRKG/protein kinase G signaling and also increases cardiac autophagy in mice. Remarkably, endogenous NPPA is secreted by CMs in response to glucose deprivation or hypoxia, thereby stimulating autophagy through autocrine/paracrine mechanisms. NPPA preserves cell viability and reduces hypertrophy in response to stress through autophagy activation. In vivo, we found that Nppa knockout mice undergoing ischemia-reperfusion (I/R) show increased infarct size and reduced autophagy. Reactivation of autophagy by Tat-Beclin D11 limits I/R injury. We also found that the protective effects of NPPA in reducing infarct size are abrogated in the presence of autophagy inhibition. Mechanistically, we found that NPPA stimulates autophagy through the activation of TFEB (transcription factor EB). Our data suggest that NPPA is a novel extracellular regulator of autophagy in the heart.

The FGFR2c/PKCε Axis Controls MCL-1-Mediated Invasion in Pancreatic Ductal Adenocarcinoma Cells: Perspectives for Innovative Target Therapies.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy whose main characterizations are Kirsten Rat Sarcoma-activating mutations (KRAS) and a highly aggressive phenotype. Based on our recent findings demonstrating that the highly aberrant expression of the mesenchymal isoform of Fibroblast Growth Factor Receptor 2 (FGFR2c) in PDAC cells activates Protein-Kinase C Epsilon (PKCε), which in turn controls receptor-mediated epithelial to mesenchymal transition (EMT), here we investigated the involvement of these signaling events in the establishment of additional tumorigenic features. Using PDAC cell lines expressing divergent levels of the FGFR2c and stable protein depletion approaches by short hairpin RNA (shRNA), we found that FGFR2c expression and its PKCε downstream signaling are responsible for the invasive response to Fibroblast Growth Factor 2 (FGF2) and for anchorage-independent growth. In addition, in vitro clonogenic assays, coupled with the check of the amount of cleaved Poly Adenosine Diphosphate-Ribose Polymerase 1 (PARP1) by Western blot, highlighted the involvement of both FGFR2c and PKCε in cell viability. Finally, monitoring of Myeloid Cell Leukemia 1 (MCL-1) expression and Sarcoma kinase family (SRC) phosphorylation suggested that the FGFR2c/PKCε axis could control cell migration/invasion possibly via MCL-1/SRC-mediated reorganization of the actin cytoskeleton. Being PKCs RAS-independent substrates, the identification of PKCε as a hub molecule downstream FGFR2c at the crossroad of signaling networks governing the main malignant tumor hallmarks could represent an important advance towards innovative target therapies overcoming RAS.

Genotype-Phenotype Correlations in Monogenic Parkinson Disease: A Review on Clinical and Molecular Findings.

Parkinson disease (PD) is a complex neurodegenerative disorder, usually with multifactorial etiology. It is characterized by prominent movement disorders and non-motor symptoms. Movement disorders commonly include bradykinesia, rigidity, and resting tremor. Non-motor symptoms can include behavior disorders, sleep disturbances, hyposmia, cognitive impairment, and depression. A fraction of PD cases instead is due to Parkinsonian conditions with Mendelian inheritance. The study of the genetic causes of these phenotypes has shed light onto common pathogenetic mechanisms underlying Parkinsonian conditions. Monogenic Parkinsonisms can present autosomal dominant, autosomal recessive, or even X-linked inheritance patterns. Clinical presentations vary from forms indistinguishable from idiopathic PD to severe childhood-onset conditions with other neurological signs. We provided a comprehensive description of each condition, discussing current knowledge on genotype-phenotype correlations. Despite the broad clinical spectrum and the many genes involved, the phenotype appears to be related to the disrupted cell function and inheritance pattern, and several assumptions about genotype-phenotype correlations can be made. The interest in these assumptions is not merely speculative, in the light of novel promising targeted therapies currently under development.

Role of FGFR2c and Its PKCε Downstream Signaling in the Control of EMT and Autophagy in Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a treatment-resistant malignancy characterized by a high malignant phenotype including acquired EMT signature and deregulated autophagy. Since we have previously described that the aberrant expression of the mesenchymal FGFR2c and the triggering of the downstream PKCε signaling are involved in epidermal carcinogenesis, the aim of this work has been to assess the contribution of these oncogenic events also in the pancreatic context. Biochemical, molecular and immunofluorescence approaches showed that FGFR2c expression impacts on PDAC cell responsiveness to FGF2 in terms of intracellular signaling activation, upregulation of EMT-related transcription factors and modulation of epithelial and mesenchymal markers compatible with the pathological EMT. Moreover, shut-off via specific protein depletion of PKCε signaling, activated by high expression of FGFR2c resulted in a reversion of EMT profile, as well as in a recovery of the autophagic process. The detailed biochemical analysis of the intracellular signaling indicated that PKCε, bypassing AKT and directly converging on ERK1/2, could be a signaling molecule downstream FGFR2c whose inhibition could be considered as possible effective therapeutic approach in counteracting aggressive phenotype in cancer.

Trehalose, a natural disaccharide, reduces stroke occurrence in the stroke-prone spontaneously hypertensive rat.

Cerebrovascular disease, a frequent complication of hypertension, is a major public health issue for which novel therapeutic and preventive approaches are needed. Autophagy activation is emerging as a potential therapeutic and preventive strategy toward stroke. Among usual activators of autophagy, the natural disaccharide trehalose (TRE) has been reported to be beneficial in preclinical models of neurodegenerative diseases, atherosclerosis and myocardial infarction. In this study, we tested for the first time the effects of TRE in the stroke-prone spontaneously hypertensive rat (SHRSP) fed with a high-salt stroke permissive diet (JD). We found that TRE reduced stroke occurrence and renal damage in high salt-fed SHRSP. TRE was also able to decrease systolic blood pressure. Through ex-vivo studies, we assessed the beneficial effect of TRE on the vascular function of high salt-fed SHRSP. At the molecular level, TRE restored brain autophagy and reduced mitochondrial mass, along with the improvement of mitochondrial function. The beneficial effects of TRE were associated with increased nuclear translocation of TFEB, a transcriptional activator of autophagy. Our results suggest that TRE may be considered as a natural compound efficacious for the prevention of hypertension-related target organ damage, with particular regard to stroke and renal damage.

Expression of the E5 Oncoprotein of HPV16 Impacts on the Molecular Profiles of EMT-Related and Differentiation Genes in Ectocervical Low-Grade Lesions.

Infection with human papillomavirus type 16 (HPV16) is one of the major risk factors for the development of cervical cancer. Our previous studies have demonstrated the involvement of the early oncoprotein E5 of HPV16 (16E5) in the altered isoform switch of fibroblast growth factor receptor 2 (FGFR2) and the consequent expression in human keratinocytes of the mesenchymal FGFR2c isoform, whose aberrant signaling leads to EMT, invasiveness, and dysregulated differentiation. Here, we aimed to establish the possible direct link between these pathological features or the appearance of FGFR2c and the expression of 16E5 in low-grade squamous intraepithelial lesions (LSILs). Molecular analysis showed that the FGFR2c expression displayed a statistically significant positive correlation with that of the viral oncoprotein, whereas the expression values of the epithelial FGR2b variant, as well as those of the differentiation markers keratin 10 (K10), loricrin (LOR) and involucrin (INV), were inversely linked to the 16E5 expression. In contrast, the expression of EMT-related transcription factors Snail1 and ZEB1 overlapped with that of 16E5, becoming a statistically significant positive correlation in the case of Snail2. Parallel analysis performed in human cervical LSIL-derived W12 cells, containing episomal HPV16, revealed that the depletion of 16E5 by siRNA was able to counteract these molecular events, proving to represent an effective strategy to identify the specific role of this viral oncoprotein in determining LSIL oncogenic and more aggressive profiles. Overall, coupling in vitro approaches to the molecular transcript analysis in ectocervical early lesions could significantly contribute to the characterization of specific gene expression profiles prognostic for those LSILs with a greater probability of direct neoplastic progression.

Effects of Sorafenib, a Tyrosin Kinase Inhibitor, on Adrenocortical Cancer.

The lack of an effective medical treatment for adrenocortical carcinoma (ACC) has prompted the search for better treatment protocols for ACC neoplasms. Sorafenib, a tyrosine kinase inhibitor has exhibited effectiveness in the treatment of different human tumors. Therefore, the aim of this study was to understand the mechanism through which sorafenib acts on ACC, especially since treatment with sorafenib alone is sometimes unable to induce a long-lasting antiproliferative effect in this tumor type. The effects of sorafenib were tested on the ACC cell line H295R by evaluating cell viability, apoptosis and VEGF receptor signaling which was assessed by analyzing VE-cadherin and ß-catenin complex formation. We also tested sorafenib on an in vitro 3D cell culture model using the same cell line. Apoptosis was observed after sorafenib treatment, and coimmunoprecipitation data suggested that the drug prevents formation VEGFR-VE-cadherin and ß-catenin proteins complex. These results were confirmed both by ultrastructural analysis and by a 3D model where we observed a disaggregation of spheres into single cells, which is a crucial event that represents the first step of metastasis. Our findings suggest that although sorafenib induces apoptotic cell death a small portion of cells survive the treatment and have characteristics of a malignancy. Based on our data we recommend against the use of sorafenib in patients with ACC.

Expression Profile of Fibroblast Growth Factor Receptors, Keratinocyte Differentiation Markers, and Epithelial Mesenchymal Transition-Related Genes in Actinic Keratosis: A Possible Predictive Factor for Malignant Progression?

Actinic keratosis (AK) is the ultra violet (UV)-induced preneoplastic skin lesion clinically classified in low (KIN I), intermediate (KIN II), and high (KIN III) grade lesions. In this work we analyzed the expression of Fibroblast Growth Factor Receptors (FGFRs), as well as of keratinocyte differentiation and epithelial-to-mesenchymal transition (EMT)-related markers in differentially graded AK lesions, in order to identify specific expression profiles that could be predictive for direct progression of some KIN I lesions towards squamous cell carcinoma (SCC). Our molecular analysis showed that the keratinocyte differentiation markers keratin 1 (K1), desmoglein-1 (DSG1), and filaggrin (FIL) were progressively downregulated in KIN I, II, and III lesions, while the modulation of epithelial/mesenchymal markers and the induction of the transcription factors Snail1 and Zinc finger E-box-binding homeobox 1 (ZEB1) compatible with pathological EMT, even if observable, did not appear to correlate with AK progression. Concerning FGFRs, a modulation of epithelial isoform of FGFR2 (FGFR2b) and the mesenchymal FGFR2c isoform compatible with an FGFR2 isoform switch, as well as FGFR4 upregulation were observed starting from KIN I lesions, suggesting that they could be events involved in early steps of AK pathogenesis. In contrast, the increase of FGFR3c expression, mainly appreciable in KIN II and KIN III lesions, suggested a correlation with AK late progression. Interestingly, the strong modulation of FIL, Snail1, as well as of FGFR2c, FGFR4, and of their ligand FGF2, observed in some of the KIN I samples, may indicate that they could be molecular markers predictive for those low graded lesions destined to a direct progression to SCC. In conclusion, our data point on the identification of molecular markers predictive for AK rapid progression through the "differentiated" pathway. Our results also represent an important step that, in future, will help to clarify the molecular mechanisms underlying FGFR signaling deregulation in epithelial tissues during the switch from the pre-neoplastic to the oncogenic malignant phenotype.

A protective role for autophagy in vitiligo.

A growing number of studies supports the existence of a dynamic interplay between energetic metabolism and autophagy, whose induction represents an adaptive response against several stress conditions. Autophagy is an evolutionarily conserved and a highly orchestrated catabolic recycling process that guarantees cellular homeostasis. To date, the exact role of autophagy in vitiligo pathogenesis is still not clear. Here, we provide the first evidence that autophagy occurs in melanocytes and fibroblasts from non-lesional skin of vitiligo patients, as a result of metabolic surveillance response. More precisely, this study is the first to reveal that induction of autophagy exerts a protective role against the intrinsic metabolic stress and attempts to antagonize degenerative processes in normal appearing vitiligo skin, where melanocytes and fibroblasts are already prone to premature senescence.

TNNI3 and KCNQ1 co-inherited variants in a family with hypertrophic cardiomyopathy and long QT phenotypes: A case report.

QTc prolongation is reported in patients with hypertrophic cardiomyopathy (HCM). However, the causes of the QTc interval increase remain unclear. The main contribution to QTc prolongation in HCM is attributed to the myocardial hypertrophy and related structural damage. In a 24-year-old male proband, affected by HCM and long QTc, we identified by Next Generation Sequencing a pathogenic variant in gene TNNI3 co-inherited with a damaging variant in KCNQ1 gene. This evidence suggests the possibility that QTc interval prolongation and its dispersion in HCM could be associated not only to the severity of left ventricular hypertrophy but also to the co-inheritance of pathogenic variants related to both long QT Syndrome (LQTS) and HCM. Although the simultaneous presence of pathogenic variants in genes related to different heart diseases is extremely rare, counseling and genetic testing appear crucial for the clinical diagnosis. Screening of LQTS genes should be considered in HCM patients to clarify the origin of long QTc, to provide more information about the clinical presentation and to evaluate the incidence of the co-existence of LQTS/HCM gene variants that could occur more frequently than so far reported.

The aberrant expression in epithelial cells of the mesenchymal isoform of FGFR2 controls the negative crosstalk between EMT and autophagy.

Signalling of the epithelial splicing variant of fibroblast growth factor receptor 2 (FGFR2b) triggers both differentiation and autophagy, while the aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells induces impaired differentiation, inhibition of autophagy as well as the induction of the epithelial-mesenchymal transition (EMT). In light of the widely proposed negative loop linking autophagy and EMT in the early steps of carcinogenesis, here we investigated the possible involvement of FGFR2c aberrant expression and signalling in orchestrating this crosstalk in human keratinocytes. Biochemical, molecular, quantitative immunofluorescence analysis and in vitro invasion assays, coupled to the use of specific substrate inhibitors and transient or stable silencing approaches, showed that AKT/MTOR and PKCε are the two hub signalling pathways, downstream FGFR2c, intersecting with each other in the control of both the inhibition of autophagy and the induction of EMT and invasive behaviour. These results indicate that the expression of FGFR2c, possibly resulting from FGFR2 isoform switch, could represent a key upstream event responsible for the establishment of a negative interplay between autophagy and EMT, which contributes to the assessment of a pathological oncogenic profile in epithelial cells.

Beyond BRCA1 and BRCA2: Deleterious Variants in DNA Repair Pathway Genes in Italian Families with Breast/Ovarian and Pancreatic Cancers.

The 5-10% of breast/ovarian cancers (BC and OC) are inherited, and germline pathogenic (P) variants in DNA damage repair (DDR) genes BRCA1 and BRCA2 explain only 10-20% of these cases. Currently, new DDR genes have been related to BC/OC and to pancreatic (PC) cancers, but the prevalence of P variants remains to be explored. The purpose of this study was to investigate the spectrum and the prevalence of pathogenic variants in DDR pathway genes other than BRCA1/2 and to correlate the genotype with the clinical phenotype. A cohort of 113 non-BRCA patients was analyzed by next-generation sequencing using a multigene panel of the 25 DDR pathways genes related to BC, OC, and PC. We found 43 unique variants in 18 of 25 analyzed genes, 14 classified as P/likely pathogenic (LP) and 28 as variants of uncertain significance (VUS). Deleterious variants were identified in 14% of index cases, whereas a VUS was identified in 20% of the probands. We observed a high incidence of deleterious variants in the CHEK2 gene, and a new pathogenic variant was detected in the RECQL gene. These results supported the clinical utility of multigene panel to increase the detection of P/LP carriers and to identify new actionable pathogenic gene variants useful for preventive and therapeutic approaches.

Risk Stratification in Hypertrophic Cardiomyopathy. Insights from Genetic Analysis and Cardiopulmonary Exercise Testing.

The role of genetic testing over the clinical and functional variables, including data from the cardiopulmonary exercise test (CPET), in the hypertrophic cardiomyopathy (HCM) risk stratification remains unclear. A retrospective genotype-phenotype correlation was performed to analyze possible differences between patients with and without likely pathogenic/pathogenic (LP/P) variants. A total of 371 HCM patients were screened at least for the main sarcomeric genes MYBPC3 (myosin binding protein C), MYH7 (ß-myosin heavy chain), TNNI3 (cardiac troponin I) and TNNT2 (cardiac troponin T): 203 patients had at least an LP/P variant, 23 patients had a unique variant of uncertain significance (VUS) and 145 did not show any LP/P variant or VUS. During a median 5.4 years follow-up, 51 and 14 patients developed heart failure (HF) and sudden cardiac death (SCD) or SCD-equivalents events, respectively. The LP/P variant was associated with a more aggressive HCM phenotype. However, left atrial diameter (LAd), circulatory power (peak oxygen uptake*peak systolic blood pressure, CP%) and ventilatory efficiency (C-index = 0.839) were the only independent predictors of HF whereas only LAd and CP% were predictors of the SCD end-point (C-index = 0.738). The present study reaffirms the pivotal role of the clinical variables and, particularly of those CPET-derived, in the HCM risk stratification.

Role of PKCε in the epithelial-mesenchymal transition induced by FGFR2 isoform switch.

BACKGROUND: The epithelial isoform of the fibroblast growth factor receptor 2 (FGFR2b) controls the entire program of keratinocyte differentiation via the sequential involvement of protein kinase C (PKC) δ and PKCα. In contrast, the FGFR2 isoform switch and the aberrant expression of the mesenchymal FGFR2c isoform leads to impairment of differentiation, epithelial-mesenchymal transition (EMT) and tumorigenic features. Aim of our present study was to contribute in clarifying the complex network of signaling pathways involved in the FGFR2c-mediated oncogenic outcomes focusing on PKCε, which appears to be involved in the induction of EMT and tumorigenesis in several epithelial contexts. METHODS: Biochemical and molecular analysis, as well as in vitro invasion assays, combined with the use of specific small interfering RNA (siRNA), were performed in human keratinocytes stably expressing FGFR2c or FGFR2b isoforms. RESULTS: Our results showed that aberrant expression and signaling of FGFR2c, but not those of FGFR2b, in human keratinocytes induced a strong phosphorylation/activation of PKCε. The use of siRNA approach showed that PKCε is the hub signaling downstream FGFR2c responsible for the modulation of EMT markers and for the induction of the EMT-related transcription factors STAT3, Snail1 and FRA1, as well as for the acquisition of the invasive behavior. Moreover, experiments of depletion of ESRP1, responsible for FGFR2 splicing in epithelial cells, indicated that the activation of PKCε is the key molecular event triggered by FGFR2 isoform switch and underlying EMT induction. CONCLUSIONS: Overall, our results point to the identification of the downstream PKC isoform responsible for the FGFR signaling deregulation occurring in epithelial tissues from the physiological oncosoppressive to the pathological oncogenic profile. Video Abstract.

Pharmacological restoration of autophagy reduces hypertension-related stroke occurrence.

The identification of the mechanisms predisposing to stroke may improve its preventive and therapeutic strategies in patients with essential hypertension. The role of macroautophagy/autophagy in the development of hypertension-related stroke needs to be clarified. We hypothesized that a defective autophagy may favor hypertension-related spontaneous stroke by promoting mitochondrial dysfunction. We studied autophagy in the stroke-prone spontaneously hypertensive (SHRSP) rat, which represents a clinically relevant model of stroke associated with high blood pressure. We assessed autophagy, mitophagy and NAD+:NADH levels in brains of SHRSP and stroke-resistant SHR fed with high salt diet. Vascular smooth muscle cells silenced for the mitochondrial complex I subunit Ndufc2 gene (NADH:ubiquinone oxidoreductase subunit C2) and cerebral endothelial cells isolated from SHRSP were also used to assess autophagy/mitophagy and mitochondrial function in response to high salt levels. We found a reduction of autophagy in brains of high salt-fed SHRSP. Autophagy impairment was associated with NDUFC2 downregulation, mitochondrial dysfunction and NAD+ depletion. Restoration of NAD+ levels by nicotinamide administration reactivated autophagy and reduced stroke development in SHRSP. A selective reactivation of autophagy/mitophagy by Tat-Beclin 1 also reduced stroke occurrence, restored autophagy/mitophagy and improved mitochondrial function. Endothelial progenitor cells (EPCs) from subjects homozygous for the thymine allele variant at NDUFC2/rs11237379, which is associated with NDUFC2 deficiency and increased stroke risk, displayed an impairment of autophagy and increased senescence in response to high salt levels. EPC senescence was rescued by Tat-Beclin 1. Pharmacological activation of autophagy may represent a novel therapeutic strategy to reduce stroke occurrence in hypertension. ABBREVIATIONS: 10 VSMCs: aortic vascular smooth muscle cells; COX4I1/COX IV: cytochrome c oxidase subunit 4I1; ECs: endothelial cells; EPCs: endothelial progenitor cells; JD: Japanese-style diet; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NAD: nicotinamide adenine dinucleotide; NDUFC2: NADH:ubiquinone oxidoreductase subunit C2; NMN: nicotinamide mononucleotide; RD: regular diet; SHRSP: stroke-prone spontaneously hypertensive rat; SHRSR: stroke-resistant spontaneously hypertensive rat.

The Aberrant Expression of the Mesenchymal Variant of FGFR2 in the Epithelial Context Inhibits Autophagy.

Signaling of the epithelial splice variant of fibroblast growth factor receptor 2 (FGFR2b) triggers both differentiation and autophagy, while the aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells induces impaired differentiation, epithelial mesenchymal transition (EMT) and tumorigenic features. Here we analyzed in the human keratinocyte cell line, as well as in primary cultured cells, the possible impact of FGFR2c forced expression on the autophagic process. Biochemical and quantitative immunofluorescence analysis, coupled to the use of autophagic flux sensors, specific substrate inhibitors or silencing approaches, showed that ectopic expression and the activation of FGFR2c inhibit the autophagosome formation and that AKT/MTOR is the downstream signaling mainly involved. Interestingly, the selective inhibition of AKT or MTOR substrates caused a reversion of the effects of FGFR2c on autophagy, which could also arise from the imbalance of the interplay between AKT/MTOR pathway and JNK1 signaling in favor of JNK1 activation, BCL-2 phosphorylation and possibly phagophore nucleation. Finally, silencing experiments of depletion of ESRP1, responsible for FGFR2 splicing and consequent FGFR2b expression, indicated that the switching from FGFR2b to FGFR2c isoform could represent the key event underlying the inhibition of the autophagic process in the epithelial context. Our results provide the first evidence of a negative impact of the out-of-context expression of FGFR2c on autophagy, suggesting a possible role of this receptor in the modulation of the recently proposed negative loop between autophagy and EMT during carcinogenesis.

The reduction of NDUFC2 expression is associated with mitochondrial impairment in circulating mononuclear cells of patients with acute coronary syndrome.

BACKGROUND: Deficiency of NADH dehydrogenase [ubiquinone], the mitochondrial complex I, represents an emerging mechanism of cardiovascular diseases. Ndufc2, a subunit of mitochondrial complex I, is involved in stroke development. We aimed to gain some insights on the role of Ndufc2 into acute coronary syndrome (ACS) through the assessment of its gene expression, along with that of anti-oxidant proteins and of mitochondrial function parameters, in circulating mononuclear cells (PBMCs) of ACS versus stable angina (SA) patients. The impact of NDUFC2 silencing in human endothelial and vascular smooth muscle cells was assessed in vitro. METHODS AND RESULTS: One hundred twenty-three patients presenting with SA (n = 41) or ACS (n = 82) were enrolled. PBMCs were used to assess the gene expression level of: NDUFC2, uncoupling protein 2 (UCP2), superoxide dysmutases 1 and 2 (SOD1, SOD2), levels of ROS and ATP. The mitochondrial dysfunction was assessed by cytofluorimetry; the structural damage by transmission electron microscopy. Cell viability, angiogenesis, markers of atherogenesis were evaluated in NDUFC2-silenced vascular cells. NDUFC2 mRNA level was significantly downregulated, along with UCP2, SOD1, SOD2 expression, in ACS patients. We found significant increases of ROS levels, reduced ATP levels, higher degree of mitochondrial structural damage and dysfunction in ACS patients. In vitro, NDUFC2 silencing favored mechanisms involved in atherogenesis and plaque vulnerability. CONCLUSIONS: A significant reduction of NDUFC2 expression is detected in ACS. In vitro, NDUFC2 silencing affects vascular cell viability and angiogenesis while stimulating the expression of markers of plaque rupture. Our observations suggest that these mechanisms may contribute to ACS development.