Background: Dysregulation of splicing variants (SVs) expression has recently emerged as a novel cancer hallmark. Although the generation of aberrant SVs (e.g. AR-v7/sst5TMD4/etc.) is associated to prostate-cancer (PCa) aggressiveness and/or castration-resistant PCa (CRPC) development, whether the molecular reason behind such phenomena might be linked to a dysregulation of the cellular machinery responsible for the splicing process [spliceosome-components (SCs) and splicing-factors (SFs)] has not been yet explored. Methods: Expression levels of 43 key SCs and SFs were measured in two cohorts of PCa-samples: 1) Clinicallylocalized formalin-fixed paraffin-embedded PCa-samples (n = 84), and 2) highly-aggressive freshly-obtained PCa-samples (n = 42). Findings: A profound dysregulation in the expression of multiple components of the splicing machinery (i.e. 7 SCs/19 SFs) were found in PCa compared to their non-tumor adjacent-regions. Notably, overexpression of SNRNP200, SRSF3 and SRRM1 (mRNA and/or protein) were associated with relevant clinical (e.g. Gleason score, T-Stage, metastasis, biochemical recurrence, etc.) and molecular (e.g. AR-v7 expression) parameters of aggressiveness in PCa-samples. Functional (cell-proliferation/migration) and mechanistic [gene-expression (qPCR) and protein-levels (western-blot)] assays were performed in normal prostate cells (PNT2) and PCa-cells (LNCaP/22Rv1/PC-3/ DU145 cell-lines) in response to SNRNP200, SRSF3 and/or SRRM1 silencing (using specific siRNAs) revealed an overall decrease in proliferation/migration-rate in PCa-cells through the modulation of key oncogenic SVs expression levels (e.g. AR-v7/PKM2/XBP1s) and alteration of oncogenic signaling pathways (e.g. p-AKT/p-JNK). Interpretation: These results demonstrate that the spliceosome is drastically altered in PCa wherein SNRNP200, SRSF3 and SRRM1 could represent attractive novel diagnostic/prognostic and therapeutic targets for PCa and CRPC.

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Context Obesity is a major health problem associated with severe comorbidities, including type-2 diabetes and cancer, wherein microRNAs might be useful as diagnostic/prognostic tools or therapeutic targets. Objective To explore the differential expression pattern of microRNAs in obesity and their putative role in obesity-related comorbidities such as insulin resistance. Methods An Affymetrix-miRNA array was performed in plasma samples from normoweight (n=4/BMI&25) and obese subjects (n=4/BMI>30). The main changes were validated in two independent cohorts (n=221/n=18). Additionally, in silico approaches were performed and in vitro assays applied in tissue samples and prostate (RWPE-1) and liver (HepG2) cell-lines. Results 26 microRNAs were altered (p&0.01) in plasma of obese subjects compared to controls using the Affymetrix-miRNA array. Validation in ampler cohorts revealed that miR-4454 levels were consistently higher in obesity, associated with insulin-resistance (HOMA-IR/insulin) and modulated by medical (metformin/statins) and surgical (bariatric surgery) strategies. miR-4454 was highly expressed in prostate and liver tissues and its expression was increased in prostate and liver cells by insulin. In vitro, overexpression of miR-4454 in prostate cells resulted in decreased expression levels of INSR, GLUT4, and phosphorylation of AMPK/AKT/ERK, as well as in altered expression of key spliceosome components (ESRP1/ESRP2/RBM45/RNU2) and insulin-receptor splicing variants. Conclusions Obesity was associated to an alteration of the plasmatic miRNA landscape, wherein miR-4454 levels were higher, associated with insulin-resistance and modulated by obesity-controlling interventions. Insulin regulated miR-4454, which, in turn may impair the cellular response to insulin, in a cell type-dependent manner (i.e. prostate gland), by modulating the splicing process.

Certain components of the somatostatin-system play relevant roles in Prostate Cancer (PCa), whose most aggressive phenotype (Castration-Resistant-PCa (CRPC)) remains lethal nowadays. However, neuronostatin and the G protein-coupled receptor 107 (GPR107), two novel members of the somatostatin-system, have not been explored yet in PCa. Consequently, we investigated the pathophysiological role of NST/GPR107-system in PCa. GPR107 expression was analyzed in well-characterized PCa patient′s cohorts, and functional/mechanistic assays were performed in response to GPR107-silencing and NST-treatment in PCa cells (androgen-dependent (AD: LNCaP) and androgen-independent (AI: 22Rv1/PC-3), which are cell models of hormone-sensitive and CRPC, respectively), and normal prostate cells (RWPE-1 cell-line). GPR107 was overexpressed in PCa and associated with key clinical parameters (e.g., advance stage of PCa, presence of vascular invasion and metastasis). Furthermore, GPR107-silencing inhibited proliferation/migration rates in AI-PCa-cells and altered key genes and oncogenic signaling-pathways involved in PCa aggressiveness (i.e., KI67/CDKN2D/MMP9/PRPF40A, SST5TMD4/AR-v7/In1-ghrelin/EZH2 splicing-variants and AKT-signaling). Interestingly, NST treatment inhibited proliferation/migration only in AI-PCa cells and evoked an identical molecular response than GPR107-silencing. Finally, NST decreased GPR107 expression exclusively in AI-PCa-cells, suggesting that part of the specific antitumor effects of NST could be mediated through a GPR107-downregulation. Altogether, NST/GPR107-system could represent a valuable diagnostic and prognostic tool and a promising novel therapeutic target for PCa and CRPC.

Context Prostate cancer (PCa) is one of the leading causes of cancer-related death among male population worldwide. Unfortunately, current medical treatments fail to prevent PCa progression in a high percentage of cases; therefore, new therapeutic tools to tackle PCa are urgently needed. Biguanides and statins have emerged as antitumor agents for several endocrine-related cancers. Objective To evaluate: i) the putative in vivo association between metformin and/or statins treatment and key tumor and clinical parameters, and ii) the direct effects of different biguanides (metformin/buformin/phenformin), statins (atorvastatin/simvastatin/lovastatin), and their combination, on key functional endpoints and associated signalling mechanisms. Methods An exploratory/observational retrospective cohort of patients with PCa (n=75) was analysed. Moreover, normal and tumor prostate cells [normal (RWPE-cells/primary prostate cell-cultures); tumor (LNCaP/22RV1/PC3/DU145 cell-lines)] were used to measure proliferation/migration/tumorsphere-formation/signalling pathways. Results The combination of metformin+statins in vivo was associated to lower Gleason-score and longer biochemical recurrence-free survival. Moreover, biguanides and statins exerted strong antitumor actions (i.e. inhibition of proliferation/migration/tumorspheres-formation) on PCa cells, and that their combination further decreased, additively, these functional parameters compared with the individual treatments. These actions were mediated through modulation of key oncogenic and metabolic signalling-pathways (i.e. AR/mTOR/AMPK/AKT/ERK) and molecular mediators (MKI67/cMYC/androgen-receptor/cell-cycle inhibitors). Conclusions Biguanides and statins significantly reduced tumor aggressiveness in PCa, being this effect more potent (in vitro and in vivo) when both compounds are combined. Therefore, given the demonstrated clinical safety of biguanides and statins, our results suggest a potential therapeutic role of these compounds, especially their combination, for the treatment of PCa.

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A high adherence to a Mediterranean diet has been related to numerous beneficial effects in human health, including a lower incidence and mortality of prostate cancer (PCa). Olive oil is an important source of phenolic bioactive compounds, mainly hydroxytyrosol (HT), of this diet. Because of the growing interest of this compound and its derivatives as a cancer chemopreventive agent, we aimed to compare the in vitro effect of HT isolated from olive mill wastewaters and five semisynthetic alkyl ether, ester, and nitro-derivatives against prostate cancer (PCa) cell lines. The effect in cell proliferation was determined in RWPE-1, LNCaP, 22Rv1, and PC-3 cells by resazurin assay, the effect in cell migration by wound healing assay, and tumorsphere and colony formation were evaluated. The changes in key signaling pathways involved in carcinogenesis were assessed by using a phosphorylation pathway profiling array and by Western blotting. Antiproliferative effects of HT and two lipophilic derivatives [hydroxytyrosyl acetate (HT-Ac)/ethyl hydroxytyrosyl ether (HT-Et)] were significantly higher in cancerous PC-3 and 22Rv1 cells than in non-malignant RWPE-1 cells. HT/HT-Ac/HT-Et significantly reduced migration capacity in RWPE-1 and PC-3 and prostatosphere size and colony formation in 22Rv1, whereas only HT-Ac and HT-Et reduced these functional parameters in PC-3. The cytotoxic effect in 22Rv1 cells was correlated with modifications in the phosphorylation pattern of key proteins, including ERK1/2 and AKT. Consistently, HT-Ac and HT-Et decreased p-AKT levels in PC-3. In sum, our results suggest that HT and its lipophilic derivatives could be considered as potential therapeutic tools in PCa.

Background Obesity is a metabolic-chronic disease with important associated morbidities and mortality. Bariatric-surgery is the most effective treatment for maintaining long-term weight-loss in severe obesity and consequently for decreasing obesity-related complications, including chronic inflammation. Aim To explore changes in components of the inflammasome-machinery after bariatric-surgery and their relations with clinical/biochemical-parameters at baseline and six-months after bariatric-surgery. Patients and methods 22 patients with morbid-obesity that underwent bariatric-surgery (sleeve-gastrectomy and roux-en-Y gastric bypass) were included. Epidemiological/clinical/anthropometric/biochemical evaluation was performed at baseline and six-months after bariatric-surgery. Inflammasome-components and inflammatory-associated factors [NOD-like-receptors (NLRs); inflammasome-activation-components; cytokines and inflammation/apoptosis-related components; and cell-cycle and DNA-damage regulators) were evaluated in peripheral-blood mononuclear-cells (PBMCs) at baseline and six-months after bariatric-surgery. Clinical-molecular correlations/associations were analyzed. Functional parameters (lipid-accumulation/viability/apoptosis) were analyzed in response to specific inflammasome-components silencing in liver HEPG2-cells-). Results A profound dysregulation of inflammasome-components after bariatric-surgery was found, especially in NOD-like-receptors, cell-cycle and DNA-damage regulators. Several components were associated to baseline metabolic comorbidities including type-2-diabetes (CCL2/CXCR1/SIRT1), hypertension (AIM2/ASC/P2RX7) and dyslipidemia (CXCL3/NLRP7), and displayed changes in their molecular profile six-months after bariatric-surgery. Gene-expression fingerprint of certain factors (NLRC4/NLRP12/CXCL3/CCL8/TLR4) accurately differentiated pre- and post-operative PBMCs. Most changes were independent of the performed surgical technique. Silencing of NLRC4/NLRP12- resulted in altered lipid-accumulation, apoptosis-rate and cell-viability in HEPG2-cells. Conclusion Bariatric-surgery induces a profound alteration in gene-expression pattern of components of the inflammasome-machinery in PBMCs. Expression and changes of certain inflammasome-components are associated to baseline metabolic comorbidities, including type-2-diabetes, and may be related to the improvement and reversion of some obesity-related comorbidities after bariatric-surgery.