Dysregulation of RNA-Exosome machinery is directly linked to major cancer hallmarks in prostate cancer: Oncogenic role of PABPN1.

Novel biomarkers and therapeutic strategies for prostate-cancer (PCa) are required to overcome its lethal progression. The dysregulation/implication of the RNA-Exosome-complex (REC; cellular machinery controlling the 3'-5'processing/degradation of most RNAs) in different cancer-types, including PCa, is poorly known. Herein, different cellular/molecular/preclinical approaches with human PCa-samples (tissues and/or plasma of 7 independent cohorts), and in-vitro/in-vivo PCa-models were used to comprehensively characterize the REC-profile and explore its role in PCa. Moreover, isoginkgetin (REC-inhibitor) effects were evaluated on PCa-cells. We demonstrated a specific dysregulation of the REC-components in PCa-tissues, identifying the Poly(A)-Binding-Protein-Nuclear 1 (PABPN1) factor as a critical regulator of major cancer hallmarks. PABPN1 is consistently overexpressed in different human PCa-cohorts and associated with poor-progression, invasion and metastasis. PABPN1 silencing decreased relevant cancer hallmarks in multiple PCa-models (proliferation/migration/tumourspheres/colonies, etc.) through the modulation of key cancer-related lncRNAs (PCA3/FALEC/DLEU2) and mRNAs (CDK2/CDK6/CDKN1A). Plasma PABPN1 levels were altered in patients with metastatic and tumour-relapse. Finally, pharmacological inhibition of REC-activity drastically inhibited PCa-cell aggressiveness. Altogether, the REC is drastically dysregulated in PCa, wherein this novel molecular event/mechanism, especially PABPN1 alteration, may be potentially exploited as a novel prognostic and therapeutic tool for PCa.

Tumor suppressor role of RBM22 in prostate cancer acting as a dual-factor regulating alternative splicing and transcription of key oncogenic genes.

Prostate cancer (PCa) is one of the leading causes of cancer-related deaths among men. Consequently, the identification of novel molecular targets for treatment is urgently needed to improve patients' outcomes. Our group recently reported that some elements of the cellular machinery controlling alternative-splicing might be useful as potential novel therapeutic tools against advanced PCa. However, the presence and functional role of RBM22, a key spliceosome component, in PCa remains unknown. Therefore, RBM22 levels were firstly interrogated in 3 human cohorts and 2 preclinical mouse models (TRAMP/Pbsn-Myc). Results were validated in in silico using 2 additional cohorts. Then, functional effects in response to RBM22 overexpression (proliferation, migration, tumorspheres/colonies formation) were tested in PCa models in vitro (LNCaP, 22Rv1, and PC-3 cell-lines) and in vivo (xenograft). High throughput methods (ie, RNA-seq, nCounter PanCancer Pathways Panel) were performed in RBM22 overexpressing cells and xenograft tumors. We found that RBM22 levels were down-regulated (mRNA and protein) in PCa samples, and were inversely associated with key clinical aggressiveness features. Consistently, a gradual reduction of RBM22 from non-tumor to poorly differentiated PCa samples was observed in transgenic models (TRAMP/Pbsn-Myc). Notably, RBM22 overexpression decreased aggressiveness features in vitro, and in vivo. These actions were associated with the splicing dysregulation of numerous genes and to the downregulation of critical upstream regulators of cell-cycle (i.e., CDK1/CCND1/EPAS1). Altogether, our data demonstrate that RBM22 plays a critical pathophysiological role in PCa and invites to suggest that targeting negative regulators of RBM22 expression/activity could represent a novel therapeutic strategy to tackle this disease.

Somatostatin, Cortistatin and Their Receptors Exert Antitumor Actions in Androgen-Independent Prostate Cancer Cells: Critical Role of Endogenous Cortistatin.

Somatostatin (SST), cortistatin (CORT), and their receptors (SSTR1-5/sst5TMD4-TMD5) comprise a multifactorial hormonal system involved in the regulation of numerous pathophysiological processes. Certain components of this system are dysregulated and play critical roles in the development/progression of different endocrine-related cancers. However, the presence and therapeutic role of this regulatory system in prostate cancer (PCa) remain poorly explored. Accordingly, we performed functional (proliferation/migration/colonies-formation) and mechanistic (Western-blot/qPCR/microfluidic-based qPCR-array) assays in response to SST and CORT treatments and CORT-silencing (using specific siRNA) in different PCa cell models [androgen-dependent (AD): LNCaP; androgen-independent (AI)/castration-resistant PCa (CRPC): 22Rv1 and PC-3], and/or in the normal-like prostate cell-line RWPE-1. Moreover, the expression of SST/CORT system components was analyzed in PCa samples from two different patient cohorts [internal (n = 69); external (Grasso, n = 88)]. SST and CORT treatment inhibited key functional/aggressiveness parameters only in AI-PCa cells. Mechanistically, antitumor capacity of SST/CORT was associated with the modulation of oncogenic signaling pathways (AKT/JNK), and with the significant down-regulation of critical genes involved in proliferation/migration and PCa-aggressiveness (e.g., MKI67/MMP9/EGF). Interestingly, CORT was highly expressed, while SST was not detected, in all prostate cell-lines analyzed. Consistently, endogenous CORT was overexpressed in PCa samples (compared with benign-prostatic-hyperplasia) and correlated with key clinical (i.e., metastasis) and molecular (i.e., SSTR2/SSTR5 expression) parameters. Remarkably, CORT-silencing drastically enhanced proliferation rate and blunted the antitumor activity of SST-analogues (octreotide/pasireotide) in AI-PCa cells. Altogether, we provide evidence that SST/CORT system and SST-analogues could represent a potential therapeutic option for PCa, especially for CRPC, and that endogenous CORT could act as an autocrine/paracrine regulator of PCa progression.

Dysregulation of the miRNome unveils a crosstalk between obesity and prostate cancer: miR-107 asa personalized diagnostic and therapeutic tool.

Prostate-specific antigen (PSA) is the gold-standard marker to screen prostate cancer (PCa) nowadays. Unfortunately, its lack of specificity and sensitivity makes the identification of novel tools to diagnose PCa an urgent medical need. In this context, microRNAs (miRNAs) have emerged as potential sources of non-invasive diagnostic biomarkers in several pathologies. Therefore, this study was aimed at assessing for the first time the dysregulation of the whole plasma miRNome in PCa patients and its putative implication in PCa from a personalized perspective (i.e., obesity condition). Plasma miRNome from a discovery cohort (18 controls and 19 PCa patients) was determined using an Affymetrix-miRNA array, showing that the expression of 104 miRNAs was significantly altered, wherein six exhibited a significant receiver operating characteristic (ROC) curve to distinguish between control and PCa patients (area under the curve [AUC] = 1). Then, a systematic validation using an independent cohort (135 controls and 160 PCa patients) demonstrated that miR-107 was the most profoundly altered miRNA in PCa (AUC = 0.75). Moreover, miR-107 levels significantly outperformed the ability of PSA to distinguish between control and PCa patients and correlated with relevant clinical parameters (i.e., PSA). These differences were more pronounced when considering only obese patients (BMI > 30). Interestingly, miR-107 levels were reduced in PCa tissues versus non-tumor tissues (n = 84) and in PCa cell lines versus non-tumor cells. In vitro miR-107 overexpression altered key aggressiveness features in PCa cells (i.e., proliferation, migration, and tumorospheres formation) and modulated the expression of important genes involved in PCa pathophysiology (i.e., lipid metabolism [i.e., FASN] and splicing process). Altogether, miR-107 might represent a novel and useful personalized diagnostic and prognostic biomarker and a potential therapeutic tool in PCa, especially in obese patients.

Comparative Cytotoxic Activity of Hydroxytyrosol and Its Semisynthetic Lipophilic Derivatives in Prostate Cancer Cells.

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.

Dysregulation of Components of the Inflammasome Machinery After Bariatric Surgery: Novel Targets for a Chronic Disease.

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 relation with clinical/biochemical parameters at baseline and 6 months after bariatric surgery. PATIENTS AND METHODS: Twenty-two 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 6 months after bariatric surgery. Inflammasome components and inflammatory-associated factors [nucleotide-binding oligomerization domain-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 6 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 NLRs and cell-cycle and DNA damage regulators. Several components were associated with baseline metabolic comorbidities including type 2 diabetes (C-C motif chemokine ligand 2/C-X-C motif chemokine receptor 1/sirtuin 1), hypertension (absent in melanoma 2/ASC/purinergic receptor P2X 7), and dyslipidemia [C-X-C motif chemokine ligand 3 (CXCL3)/NLR family pyrin domain containing (NLRP) 7) and displayed changes in their molecular profile 6 months after bariatric surgery. The gene expression fingerprint of certain factors NLR family CARD domain containing 4 (NLRC4)/NLRP12/CXCL3)/C-C motif chemokine ligand 8/toll-like receptor 4) accurately differentiated pre- and postoperative 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 the 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.

Clinical, Cellular, and Molecular Evidence of the Additive Antitumor Effects of Biguanides and Statins in Prostate Cancer.

CONTEXT: Prostate cancer (PCa) is one of the leading causes of cancer-related death among the 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: (1) the putative in vivo association between metformin and/or statins treatment and key tumor and clinical parameters and (2) 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 analyzed. 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 (ie, inhibition of proliferation/migration/tumorsphere formation) on PCa cells, and that their combination further decreased; in addition, these functional parameters compared with the individual treatments. These actions were mediated through modulation of key oncogenic and metabolic signalling pathways (ie, 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, with this effect being 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.

Influence of Obesity in the miRNome: miR-4454, a Key Regulator of Insulin Response Via Splicing Modulation in Prostate.

CONTEXT: Obesity is a major health problem associated with severe comorbidities, including type 2 diabetes and cancer, wherein microRNAs (miRNAs) might be useful as diagnostic/prognostic tools or therapeutic targets. OBJECTIVE: To explore the differential expression pattern of miRNAs 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/body mass index < 25) and obese subjects (n = 4/body mass index > 30). The main changes were validated in 2 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: A total of 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 (Homeostatic Model Assessment of Insulin Resistance/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.

Unleashing the Diagnostic, Prognostic and Therapeutic Potential of the Neuronostatin/GPR107 System in Prostate Cancer.

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.

Dysregulation of the splicing machinery is directly associated to aggressiveness of prostate cancer.

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) Clinically-localized 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.

Spliceosome component SF3B1 as novel prognostic biomarker and therapeutic target for prostate cancer.

Prostate cancer (PCa) is one of the most common cancers types among men. Development and progression of PCa is associated with aberrant expression of oncogenic splicing-variants (eg, AR-v7), suggesting that dysregulation of the splicing process might represent a potential actionable target for PCa. Expression levels (mRNA and protein) of SF3B1, one of the main components of the splicing machinery, were analyzed in different cohorts of PCa patients (clinically localized [n = 84], highly aggressive PCa [n = 42], and TCGA dataset [n = 497]). Functional and mechanistic assays were performed in response to pladienolide-B in nontumor and tumor-derived prostate cells. Our results revealed that SF3B1 was overexpressed in PCa tissues and its levels were associated with clinically relevant PCa-aggressive features (eg, metastasis/AR-v7 expression). Moreover, inhibition of SF3B1 activity by pladienolide-B reduced functional parameters of aggressiveness (proliferation/migration/tumorspheres-formation/apoptosis) in PCa cell lines, irrespective of AR-v7 expression, and reduced viability of primary PCa cells. Antitumor actions of pladienolide-B involved: (1) inhibition of PI3K/AKT and JNK signaling pathways, (2) modulation of tumor markers and splicing variants (AR-v7/In1-ghrelin), and (3) regulation of key components of mRNA homeostasis-associated machineries (spliceosome/SURF/EJC). Altogether, our results demonstrated that SF3B1 is overexpressed and associated with malignant features in PCa, and its inhibition reduces PCa aggressiveness, suggesting that SF3B1 could represent a novel prognostic biomarker and a therapeutic target in PCa.