Fasting in combination with the cocktail Sorafenib:Metformin blunts cellular plasticity and promotes liver cancer cell death via poly-metabolic exhaustion.

PURPOSE: Dual-Interventions targeting glucose and oxidative metabolism are receiving increasing attention in cancer therapy. Sorafenib (S) and Metformin (M), two gold-standards in liver cancer, are known for their mitochondrial inhibitory capacity. Fasting, a glucose-limiting strategy, is also emerging as chemotherapy adjuvant. Herein, we explore the anti-carcinogenic response of nutrient restriction in combination with sorafenib:metformin (NR-S:M). RESULTS: Our data demonstrates that, independently of liver cancer aggressiveness, fasting synergistically boosts the anti-proliferative effects of S:M co-treatment. Metabolic and Cellular plasticity was determined by the examination of mitochondrial and glycolytic activity, cell cycle modulation, activation of cellular apoptosis, and regulation of key signaling and metabolic enzymes. Under NR-S:M conditions, early apoptotic events and the pro-apoptotic Bcl-xS/Bcl-xL ratio were found increased. NR-S:M induced the highest retention in cellular SubG1 phase, consistent with the presence of DNA fragments from cellular apoptosis. Mitochondrial functionality, Mitochondrial ATP-linked respiration, Maximal respiration and Spare respiratory capacity, were all found blunted under NR-S:M conditions. Basal Glycolysis, Glycolytic reserve, and glycolytic capacity, together with the expression of glycogenic (PKM), gluconeogenic (PCK1 and G6PC3), and glycogenolytic enzymes (PYGL, PGM1, and G6PC3), were also negatively impacted by NR-S:M. Lastly, a TMT-proteomic approach corroborated the synchronization of liver cancer metabolic reprogramming with the activation of molecular pathways to drive a quiescent-like status of energetic-collapse and cellular death. CONCLUSION: Altogether, we show that the energy-based polytherapy NR-S:M blunts cellular, metabolic and molecular plasticity of liver cancer. Notwithstanding the in vitro design of this study, it holds a promising therapeutic tool worthy of exploration for this tumor pathology.

PRPF8 increases the aggressiveness of hepatocellular carcinoma by regulating FAK/AKT pathway via fibronectin 1 splicing.

Hepatocellular carcinoma (HCC) pathogenesis is associated with alterations in splicing machinery components (spliceosome and splicing factors) and aberrant expression of oncogenic splice variants. We aimed to analyze the expression and potential role of the spliceosome component PRPF8 (pre-mRNA processing factor 8) in HCC. PRPF8 expression (mRNA/protein) was analyzed in a retrospective cohort of HCC patients (n = 172 HCC and nontumor tissues) and validated in two in silico cohorts (TCGA and CPTAC). PRPF8 expression was silenced in liver cancer cell lines and in xenograft tumors to understand the functional and mechanistic consequences. In silico RNAseq and CLIPseq data were also analyzed. Our results indicate that PRPF8 is overexpressed in HCC and associated with increased tumor aggressiveness (patient survival, etc.), expression of HCC-related splice variants, and modulation of critical genes implicated in cancer-related pathways. PRPF8 silencing ameliorated aggressiveness in vitro and decreased tumor growth in vivo. Analysis of in silico CLIPseq data in HepG2 cells demonstrated that PRPF8 binds preferentially to exons of protein-coding genes, and RNAseq analysis showed that PRPF8 silencing alters splicing events in multiple genes. Integrated and in vitro analyses revealed that PRPF8 silencing modulates fibronectin (FN1) splicing, promoting the exclusion of exon 40.2, which is paramount for binding to integrins. Consistent with this finding, PRPF8 silencing reduced FAK/AKT phosphorylation and blunted stress fiber formation. Indeed, HepG2 and Hep3B cells exhibited a lower invasive capacity in membranes treated with conditioned medium from PRPF8-silenced cells compared to medium from scramble-treated cells. This study demonstrates that PRPF8 is overexpressed and associated with aggressiveness in HCC and plays important roles in hepatocarcinogenesis by altering FN1 splicing, FAK/AKT activation and stress fiber formation.

Spliceosomal profiling identifies EIF4A3 as a novel oncogene in hepatocellular carcinoma acting through the modulation of FGFR4 splicing.

INTRODUCTION: Altered splicing landscape is an emerging cancer hallmark; however, the dysregulation and implication of the cellular machinery controlling this process (spliceosome components and splicing factors) in hepatocellular carcinoma (HCC) is poorly known. This study aimed to comprehensively characterize the spliceosomal profile and explore its role in HCC. METHODS: Expression levels of 70 selected spliceosome components and splicing factors and clinical implications were evaluated in two retrospective and six in silico HCC cohorts. Functional, molecular and mechanistic studies were implemented in three cell lines (HepG2, Hep3B and SNU-387) and preclinical Hep3B-induced xenograft tumours. RESULTS: Spliceosomal dysregulations were consistently found in retrospective and in silico cohorts. EIF4A3, RBM3, ESRP2 and SRPK1 were the most dysregulated spliceosome elements in HCC. EIF4A3 expression was associated with decreased survival and greater recurrence. Plasma EIF4A3 levels were significantly elevated in HCC patients. In vitro EIF4A3-silencing (or pharmacological inhibition) resulted in reduced aggressiveness, and hindered xenograft-tumours growth in vivo, whereas EIF4A3 overexpression increased tumour aggressiveness. EIF4A3-silencing altered the expression and splicing of key HCC-related genes, specially FGFR4. EIF4A3-silencing blocked the cellular response to the natural ligand of FGFR4, FGF19. Functional consequences of EIF4A3-silencing were mediated by FGFR4 splicing as the restoration of non-spliced FGFR4 full-length version blunted these effects, and FGFR4 inhibition did not exert further effects in EIF4A3-silenced cells. CONCLUSIONS: Splicing machinery is strongly dysregulated in HCC, providing a source of new diagnostic, prognostic and therapeutic options in HCC. EIF4A3 is consistently elevated in HCC patients and associated with tumour aggressiveness and mortality, through the modulation of FGFR4 splicing.

Dysregulation of splicing variants and spliceosome components in breast cancer.

The dysregulation of the splicing process has emerged as a novel hallmark of metabolic and tumor pathologies. In breast cancer (BCa), which represents the most diagnosed cancer type among women worldwide, the generation and/or dysregulation of several oncogenic splicing variants have been described. This is the case of the splicing variants of HER2, ER, BRCA1, or the recently identified by our group, In1-ghrelin and SST5TMD4, which exhibit oncogenic roles, increasing the malignancy, poor prognosis, and resistance to treatment of BCa. This altered expression of oncogenic splicing variants has been closely linked with the dysregulation of the elements belonging to the macromolecular machinery that controls the splicing process (spliceosome components and the associated splicing factors). In this review, we compile the current knowledge demonstrating the altered expression of splicing variants and spliceosomal components in BCa, showing the existence of a growing body of evidence supporting the close implication of the alteration in the splicing process in mammary tumorigenesis.

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.

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.

Splicing factor SF3B1 is overexpressed and implicated in the aggressiveness and survival of hepatocellular carcinoma.

Splicing alterations represent an actionable cancer hallmark. Splicing factor 3B subunit 1 (SF3B1) is a crucial splicing factor that can be targeted pharmacologically (e.g. pladienolide-B). Here, we show that SF3B1 is overexpressed (RNA/protein) in hepatocellular carcinoma (HCC) in two retrospective (n = 154 and n = 172 samples) and in five in silico cohorts (n > 900 samples, including TCGA) and that its expression is associated with tumor aggressiveness, oncogenic splicing variants expression (KLF6-SV1, BCL-XL) and decreased overall survival. In vitro, SF3B1 silencing reduced cell viability, proliferation and migration and its pharmacological blockade with pladienolide-B inhibited proliferation, migration, and formation of tumorspheres and colonies in liver cancer cell lines (HepG2, Hep3B, SNU-387), whereas its effects on normal-like hepatocyte-derived THLE-2 proliferation were negligible. Pladienolide-B also reduced the in vivo growth and the expression of tumor-markers in Hep3B-induced xenograft tumors. Moreover, SF3B1 silencing and/or blockade markedly modulated the activation of key signaling pathways (PDK1, GSK3b, ERK, JNK, AMPK) and the expression of cancer-associated genes (CDK4, CD24) and oncogenic SVs (KLF6-SV1). Therefore, the genetic and/or pharmacological inhibition of SF3B1 may represent a promising novel therapeutic strategy worth to be explored through randomized controlled trials.

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.