The Exon Junction Complex component EIF4A3 plays a splicing-linked oncogenic role in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, underscoring the urgent need for in-depth biological research. The phenomenon of alternative RNA splicing dysregulation is a common hallmark in cancer, including PDAC, presenting new avenues for understanding and developing diagnostic and therapeutic tools. Our research focuses on EIF4A3, a core component of the Exon Junction Complex intimately linked to RNA splicing, and its role in PDAC. EIF4A3 is overexpressed in PDAC tissue and associated to clinical parameters of malignancy and poorer patient survival. Mechanistically, exploration of PDAC RNA-seq data unveiled the link of EIF4A3 to diverse malignancy processes, consistent with its association to key molecular pathways. EIF4A3 targeting in vitro decreased essential functional tumor features such as proliferation, migration, colony formation and sphere formation, while its in vivo targeting reduced tumor growth. EIF4A3 silencing in PDAC cell lines severely altered its transcriptional and spliceosomic landscapes, as shown by RNA-seq analyses, suggesting a role for EIF4A3 in maintaining RNA homeostasis. Our results indicate that EIF4A3 dysregulation in PDAC has a pleiotropic regulatory role on RNA biology, influencing key cellular functions. This paves the way to explore its potential as novel biomarker and actionable target candidate for this lethal cancer.

Spliceosomic dysregulation in pancreatic cancer uncovers splicing factors PRPF8 and RBMX as novel candidate actionable targets.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, characterized by late diagnosis and poor treatment response. Surgery is the only curative approach, only available to early-diagnosed patients. Current therapies have limited effects, cause severe toxicities, and minimally improve overall survival. Understanding of splicing machinery alterations in PDAC remains incomplete. Here, we comprehensively examined 59 splicing machinery components, uncovering dysregulation in pre-mRNA processing factor 8 (PRPF8) and RNA-binding motif protein X-linked (RBMX). Their downregulated expression was linked to poor prognosis and malignancy features, including tumor stage, invasion and metastasis, and associated with poorer survival and the mutation of key PDAC genes. Experimental modulation of these splicing factors in pancreatic cancer cell lines reverted their expression to non-tumor levels and resulted in decreased key tumor-related features. These results provide evidence that the splicing machinery is altered in PDAC, wherein PRPF8 and RBMX emerge as candidate actionable therapeutic targets.

Integrative clinical, hormonal, and molecular data associate with invasiveness in acromegaly: REMAH study.

INTRODUCTION: Growth hormone (GH)-secreting pituitary tumors (GHomas) are the most common acromegaly cause. At diagnosis, most of them are macroadenomas, and up to 56% display cavernous sinus invasion. Biomarker assessment associated with tumor growth and invasion is important to optimize their management. OBJECTIVES: The study aims to identify clinical/hormonal/molecular biomarkers associated with tumor size and invasiveness in GHomas and to analyze the influence of pre-treatment with somatostatin analogs (SSAs) or dopamine agonists (DAs) in key molecular biomarker expression. METHODS: Clinical/analytical/radiological variables were evaluated in 192 patients from the REMAH study (ambispective multicenter post-surgery study of the Spanish Society of Endocrinology and Nutrition). The expression of somatostatin/ghrelin/dopamine system components and key pituitary/proliferation markers was evaluated in GHomas after the first surgery. Univariate/multivariate regression studies were performed to identify association between variables. RESULTS: Eighty percent of patients harbor macroadenomas (63.8% with extrasellar growth). Associations between larger and more invasive GHomas with younger age, visual abnormalities, higher IGF1 levels, extrasellar/suprasellar growth, and/or cavernous sinus invasion were found. Higher GH1 and lower PRL/POMC/CGA/AVPR1B/DRD2T/DRD2L expression levels (P < .05) were associated with tumor invasiveness. Least Absolute Shrinkage and Selection Operator's penalized regression identified combinations of clinical and molecular features with areas under the curve between 0.67 and 0.82. Pre-operative therapy with DA or SSAs did not alter the expression of any of the markers analyzed except for DRD1/AVPR1B (up-regulated with DA) and FSHB/CRHR1 (down-regulated with SSAs). CONCLUSIONS: A specific combination of clinical/analytical/molecular variables was found to be associated with tumor invasiveness and growth capacity in GHomas. Pre-treatment with first-line drugs for acromegaly did not significantly modify the expression of the most relevant biomarkers in our association model. These findings provide valuable insights for risk stratification and personalized management of GHomas.

Altered CELF4 splicing factor enhances pancreatic neuroendocrine tumors aggressiveness influencing mTOR and everolimus response.

Pancreatic neuroendocrine tumors (PanNETs) comprise a heterogeneous group of tumors with growing incidence. Recent molecular analyses provided a precise picture of their genomic and epigenomic landscape. Splicing dysregulation is increasingly regarded as a novel cancer hallmark influencing key tumor features. We have previously demonstrated that splicing machinery is markedly dysregulated in PanNETs. Here, we aimed to elucidate the molecular and functional implications of CUGBP ELAV-like family member 4 (CELF4), one of the most altered splicing factors in PanNETs. CELF4 expression was determined in 20 PanNETs, comparing tumor and non-tumoral adjacent tissue. An RNA sequencing (RNA-seq) dataset was analyzed to explore CELF4-linked interrelations among clinical features, gene expression, and splicing events. Two PanNET cell lines were employed to assess CELF4 function in vitro and in vivo. PanNETs display markedly upregulated CELF4 expression, which is closely associated with malignancy features, altered expression of key tumor players, and distinct splicing event profiles. Modulation of CELF4 influenced proliferation in vitro and reduced in vivo xenograft tumor growth. Interestingly, functional assays and RNA-seq analysis revealed that CELF4 silencing altered mTOR signaling pathway, enhancing the effect of everolimus. We demonstrate that CELF4 is dysregulated in PanNETs, where it influences tumor development and aggressiveness, likely by modulating the mTOR pathway, suggesting its potential as therapeutic target.

Altered splicing machinery in lung carcinoids unveils NOVA1, PRPF8 and SRSF10 as novel candidates to understand tumor biology and expand biomarker discovery.

BACKGROUND: Lung neuroendocrine neoplasms (LungNENs) comprise a heterogeneous group of tumors ranging from indolent lesions with good prognosis to highly aggressive cancers. Carcinoids are the rarest LungNENs, display low to intermediate malignancy and may be surgically managed, but show resistance to radiotherapy/chemotherapy in case of metastasis. Molecular profiling is providing new information to understand lung carcinoids, but its clinical value is still limited. Altered alternative splicing is emerging as a novel cancer hallmark unveiling a highly informative layer. METHODS: We primarily examined the status of the splicing machinery in lung carcinoids, by assessing the expression profile of the core spliceosome components and selected splicing factors in a cohort of 25 carcinoids using a microfluidic array. Results were validated in an external set of 51 samples. Dysregulation of splicing variants was further explored in silico in a separate set of 18 atypical carcinoids. Selected altered factors were tested by immunohistochemistry, their associations with clinical features were assessed and their putative functional roles were evaluated in vitro in two lung carcinoid-derived cell lines. RESULTS: The expression profile of the splicing machinery was profoundly dysregulated. Clustering and classification analyses highlighted five splicing factors: NOVA1, SRSF1, SRSF10, SRSF9 and PRPF8. Anatomopathological analysis showed protein differences in the presence of NOVA1, PRPF8 and SRSF10 in tumor versus non-tumor tissue. Expression levels of each of these factors were differentially related to distinct number and profiles of splicing events, and were associated to both common and disparate functional pathways. Accordingly, modulating the expression of NOVA1, PRPF8 and SRSF10 in vitro predictably influenced cell proliferation and colony formation, supporting their functional relevance and potential as actionable targets. CONCLUSIONS: These results provide primary evidence for dysregulation of the splicing machinery in lung carcinoids and suggest a plausible functional role and therapeutic targetability of NOVA1, PRPF8 and SRSF10.

Splicing alterations in pancreatic ductal adenocarcinoma: a new molecular landscape with translational potential.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers worldwide, mainly due to its late diagnosis and lack of effective therapies, translating into a low 5-year 12% survival rate, despite extensive clinical efforts to improve outcomes. International cooperative studies have provided informative multiomic landscapes of PDAC, but translation of these discoveries into clinical advances are lagging. Likewise, early diagnosis biomarkers and new therapeutic tools are sorely needed to tackle this cancer. The study of poorly explored molecular processes, such as splicing, can provide new tools in this regard. Alternative splicing of pre-RNA allows the generation of multiple RNA variants from a single gene and thereby contributes to fundamental biological processes by finely tuning gene expression. However, alterations in alternative splicing are linked to many diseases, and particularly to cancer, where it can contribute to tumor initiation, progression, metastasis and drug resistance. Splicing defects are increasingly being associated with PDAC, including both mutations or dysregulation of components of the splicing machinery and associated factors, and altered expression of specific relevant gene variants. Such disruptions can be a key element enhancing pancreatic tumor progression or metastasis, while they can also provide suitable tools to identify potential candidate biomarkers and discover new actionable targets. In this review, we aimed to summarize the current information about dysregulation of splicing-related elements and aberrant splicing isoforms in PDAC, and to describe their relationship with the development, progression and/or aggressiveness of this dismal cancer, as well as their potential as therapeutic tools and targets.

The uprise of RNA biology in neuroendocrine neoplasms: altered splicing and RNA species unveil translational opportunities.

Neuroendocrine neoplasms (NENs) comprise a highly heterogeneous group of tumors arising from the diffuse neuroendocrine system. NENs mainly originate in gastrointestinal, pancreatic, and pulmonary tissues, and despite being rare, show rising incidence. The molecular mechanisms underlying NEN development are still poorly understood, although recent studies are unveiling their genomic, epigenomic and transcriptomic landscapes. RNA was originally considered as an intermediary between DNA and protein. Today, compelling evidence underscores the regulatory relevance of RNA processing, while new RNA molecules emerge with key functional roles in core cell processes. Indeed, correct functioning of the interrelated complementary processes comprising RNA biology, its processing, transport, and surveillance, is essential to ensure adequate cell homeostasis, and its misfunction is related to cancer at multiple levels. This review is focused on the dysregulation of RNA biology in NENs. In particular, we survey alterations in the splicing process and available information implicating the main RNA species and processes in NENs pathology, including their role as biomarkers, and their functionality and targetability. Understanding how NENs precisely (mis)behave requires a profound knowledge at every layer of their heterogeneity, to help improve NEN management. RNA biology provides a wide spectrum of previously unexplored processes and molecules that open new avenues for NEN detection, classification and treatment. The current molecular biology era is rapidly evolving to facilitate a detailed comprehension of cancer biology and is enabling the arrival of personalized, predictive and precision medicine to rare tumors like NENs.

Spliceosomic dysregulation unveils NOVA1 as a candidate actionable therapeutic target in pancreatic neuroendocrine tumors.

Dysregulation of the splicing machinery is emerging as a hallmark in cancer due to its association with multiple dysfunctions in tumor cells. Inappropriate function of this machinery can generate tumor-driving splicing variants and trigger oncogenic actions. However, its role in pancreatic neuroendocrine tumors (PanNETs) is poorly defined. In this study we aimed to characterize the expression pattern of a set of splicing machinery components in PanNETs, and their relationship with aggressiveness features. A qPCR-based array was first deployed to determine the expression levels of components of the major (n = 13) and minor spliceosome (n = 4) and associated splicing factors (n = 27), using a microfluidic technology in 20 PanNETs and non-tumoral adjacent samples. Subsequently, in vivo and in vitro models were applied to explore the pathophysiological role of NOVA1. Expression analysis revealed that a substantial proportion of splicing machinery components was altered in tumors. Notably, key splicing factors were overexpressed in PanNETs samples, wherein their levels correlated with clinical and malignancy features. Using in vivo and in vitro assays, we demonstrate that one of those altered factors, NOVA1, is tightly related to cell proliferation, alters pivotal signaling pathways and interferes with responsiveness to drug treatment in PanNETs, suggesting a role for this factor in the aggressiveness of these tumors and its suitability as therapeutic target. Altogether, our results unveil a severe alteration of the splicing machinery in PanNETs and identify the putative relevance of NOVA1 in tumor development/progression, which could provide novel avenues to develop diagnostic biomarkers and therapeutic tools for this pathology.

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.

Integrative Clinical, Radiological, and Molecular Analysis for Predicting Remission and Recurrence of Cushing Disease.

CONTEXT: Adrenocorticotropin (ACTH)-secreting pituitary tumors (ACTHomas) are associated with severe comorbidities and increased mortality. Current treatments mainly focus on remission and prevention of persistent disease and recurrence. However, there are still no useful biomarkers to accurately predict the clinical outcome after surgery, long-term remission, or disease relapse. OBJECTIVES: This work aimed to identify clinical, biochemical, and molecular markers for predicting long-term clinical outcome and remission in ACTHomas. METHODS: A retrospective multicenter study was performed with 60 ACTHomas patients diagnosed between 2004 and 2018 with at least 2 years' follow-up. Clinical/biochemical variables were evaluated yearly. Molecular expression profile of the somatostatin/ghrelin/dopamine regulatory systems components and of key pituitary factors and proliferation markers were evaluated in tumor samples after the first surgery. RESULTS: Clinical variables including tumor size, time until diagnosis/first surgery, serum prolactin, and postsurgery cortisol levels were associated with tumor remission and relapsed disease. The molecular markers analyzed were distinctly expressed in ACTHomas, with some components (ie, SSTR1, CRHR1, and MKI67) showing instructive associations with recurrence and/or remission. Notably, an integrative model including selected clinical variables (tumor size/postsurgery serum cortisol), and molecular markers (SSTR1/CRHR1) can accurately predict the clinical evolution and remission of patients with ACTHomas, generating a receiver operating characteristic curve with an area under the curve of 1 (P < .001). CONCLUSION: This study demonstrates that the combination of a set of clinical and molecular biomarkers in ACTHomas is able to accurately predict the clinical evolution and remission of patients. Consequently, the postsurgery molecular profile represents a valuable tool for clinical evaluation and follow-up of patients with ACTHomas.

Somatostatin Receptor Splicing Variant sst5TMD4 Overexpression in Glioblastoma Is Associated with Poor Survival, Increased Aggressiveness Features, and Somatostatin Analogs Resistance.

Glioblastoma (GBM) is the most malignant and lethal brain tumor. Current standard treatment consists of surgery followed by radiotherapy/chemotherapy; however, this is only a palliative approach with a mean post-operative survival of scarcely ~12-15 months. Thus, the identification of novel therapeutic targets to treat this devastating pathology is urgently needed. In this context, the truncated splicing variant of the somatostatin receptor subtype 5 (sst5TMD4), which is produced by aberrant alternative splicing, has been demonstrated to be overexpressed and associated with increased aggressiveness features in several tumors. However, the presence, functional role, and associated molecular mechanisms of sst5TMD4 in GBM have not been yet explored. Therefore, we performed a comprehensive analysis to characterize the expression and pathophysiological role of sst5TMD4 in human GBM. sst5TMD4 was significantly overexpressed (at mRNA and protein levels) in human GBM tissue compared to non-tumor (control) brain tissue. Remarkably, sst5TMD4 expression was significantly associated with poor overall survival and recurrent tumors in GBM patients. Moreover, in vitro sst5TMD4 overexpression (by specific plasmid) increased, whereas sst5TMD4 silencing (by specific siRNA) decreased, key malignant features (i.e., proliferation and migration capacity) of GBM cells (U-87 MG/U-118 MG models). Furthermore, sst5TMD4 overexpression in GBM cells altered the activity of multiple key signaling pathways associated with tumor aggressiveness/progression (AKT/JAK-STAT/NF-κB/TGF-ß), and its silencing sensitized GBM cells to the antitumor effect of pasireotide (a somatostatin analog). Altogether, these results demonstrate that sst5TMD4 is overexpressed and associated with enhanced malignancy features in human GBMs and reveal its potential utility as a novel diagnostic/prognostic biomarker and putative therapeutic target in GBMs.

Epigenetic and post-transcriptional regulation of somatostatin receptor subtype 5 (SST5 ) in pituitary and pancreatic neuroendocrine tumors.

Somatostatin receptor subtype 5 (SST5 ) is an emerging biomarker and actionable target in pituitary (PitNETs) and pancreatic (PanNETs) neuroendocrine tumors. Transcriptional and epigenetic regulation of SSTR5 gene expression and mRNA biogenesis is poorly understood. Recently, an overlapping natural antisense transcript, SSTR5-AS1, potentially regulating SSTR5 expression, was identified. We aimed to elucidate whether epigenetic processes contribute to the regulation of SSTR5 expression in PitNETs (somatotropinomas) and PanNETs. We analyzed the SSTR5/SSTR5-AS1 human locus in silico to identify CpG islands. SSTR5 and SSTR5-AS1 expression was assessed by quantitative real-time PCR (qPCR) in 27 somatotropinomas, 11 normal pituitaries (NPs), and 15 PanNETs/paired adjacent (control) samples. We evaluated methylation grade in four CpG islands in the SSTR5/SSTR5-AS1 genes. Results revealed that SSTR5 and SSTR5-AS1 were directly correlated in NP, somatotropinoma, and PanNET samples. Interestingly, selected CpG islands were differentially methylated in somatotropinomas compared with NPs. In PanNETs cell lines, SSTR5-AS1 silencing downregulated SSTR5 expression, altered aggressiveness features, and influenced pasireotide response. These results provide evidence that SSTR5 expression in PitNETs and PanNETs can be epigenetically regulated by the SSTR5-AS1 antisense transcript and, indirectly, by DNA methylation, which may thereby impact tumor behavior and treatment response.

Splicing machinery is impaired in rheumatoid arthritis, associated with disease activity and modulated by anti-TNF therapy.

OBJECTIVES: To characterise splicing machinery (SM) alterations in leucocytes of patients with rheumatoid arthritis (RA), and to assess its influence on their clinical profile and therapeutic response. METHODS: Leucocyte subtypes from 129 patients with RA and 29 healthy donors (HD) were purified, and 45 selected SM elements (SME) were evaluated by quantitative PCR-array based on microfluidic technology (Fluidigm). Modulation by anti-tumour necrosis factor (TNF) therapy and underlying regulatory mechanisms were assessed. RESULTS: An altered expression of several SME was found in RA leucocytes. Eight elements (SNRNP70, SNRNP200, U2AF2, RNU4ATAC, RBM3, RBM17, KHDRBS1 and SRSF10) were equally altered in all leucocytes subtypes. Logistic regressions revealed that this signature might: discriminate RA and HD, and anti-citrullinated protein antibodies (ACPAs) positivity; classify high-disease activity (disease activity score-28 (DAS28) >5.1); recognise radiological involvement; and identify patients showing atheroma plaques. Furthermore, this signature was altered in RA synovial fluid and ankle joints of K/BxN-arthritic mice. An available RNA-seq data set enabled to validate data and identified distinctive splicing events and splicing variants among patients with RA expressing high and low SME levels. 3 and 6 months anti-TNF therapy reversed their expression in parallel to the reduction of the inflammatory profile. In vitro, ACPAs modulated SME, at least partially, by Fc Receptor (FcR)-dependent mechanisms. Key inflammatory cytokines further altered SME. Lastly, induced SNRNP70-overexpression and KHDRBS1-overexpression reversed inflammation in lymphocytes, NETosis in neutrophils and adhesion in RA monocytes and influenced activity of RA synovial fibroblasts. CONCLUSIONS: Overall, we have characterised for the first time a signature comprising eight dysregulated SME in RA leucocytes from both peripheral blood and synovial fluid, linked to disease pathophysiology, modulated by ACPAs and reversed by anti-TNF therapy.

Dysregulated splicing factor SF3B1 unveils a dual therapeutic vulnerability to target pancreatic cancer cells and cancer stem cells with an anti-splicing drug.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, requiring novel treatments to target both cancer cells and cancer stem cells (CSCs). Altered splicing is emerging as both a novel cancer hallmark and an attractive therapeutic target. The core splicing factor SF3B1 is heavily altered in cancer and can be inhibited by Pladienolide-B, but its actionability in PDAC is unknown. We explored the presence and role of SF3B1 in PDAC and interrogated its potential as an actionable target. METHODS: SF3B1 was analyzed in PDAC tissues, an RNA-seq dataset, and publicly available databases, examining associations with splicing alterations and key features/genes. Functional assays in PDAC cell lines and PDX-derived CSCs served to test Pladienolide-B treatment effects in vitro, and in vivo in zebrafish and mice. RESULTS: SF3B1 was overexpressed in human PDAC and associated with tumor grade and lymph-node involvement. SF3B1 levels closely associated with distinct splicing event profiles and expression of key PDAC players (KRAS, TP53). In PDAC cells, Pladienolide-B increased apoptosis and decreased multiple tumor-related features, including cell proliferation, migration, and colony/sphere formation, altering AKT and JNK signaling, and favoring proapoptotic splicing variants (BCL-XS/BCL-XL, KRASa/KRAS, Δ133TP53/TP53). Importantly, Pladienolide-B similarly impaired CSCs, reducing their stemness capacity and increasing their sensitivity to chemotherapy. Pladienolide-B also reduced PDAC/CSCs xenograft tumor growth in vivo in zebrafish and in mice. CONCLUSION: SF3B1 overexpression represents a therapeutic vulnerability in PDAC, as altered splicing can be targeted with Pladienolide-B both in cancer cells and CSCs, paving the way for novel therapies for this lethal cancer.

A Somatostatin Receptor Subtype-3 (SST3) Peptide Agonist Shows Antitumor Effects in Experimental Models of Nonfunctioning Pituitary Tumors.

PURPOSE: Somatostatin analogues (SSA) are efficacious and safe treatments for a variety of neuroendocrine tumors, especially pituitary neuroendocrine tumors (PitNET). Their therapeutic effects are mainly mediated by somatostatin receptors SST2 and SST5. Most SSAs, such as octreotide/lanreotide/pasireotide, are either nonselective or activate mainly SST2. However, nonfunctioning pituitary tumors (NFPTs), the most common PitNET type, mainly express SST3 and finding peptides that activate this particular somatostatin receptor has been very challenging. Therefore, the main objective of this study was to identify SST3-agonists and characterize their effects on experimental NFPT models. EXPERIMENTAL DESIGN: Binding to SSTs and cAMP level determinations were used to screen a peptide library and identify SST3-agonists. Key functional parameters (cell viability/caspase activity/chromogranin-A secretion/mRNA expression/intracellular signaling pathways) were assessed on NFPT primary cell cultures in response to SST3-agonists. Tumor growth was assessed in a preclinical PitNET mouse model treated with a SST3-agonist. RESULTS: We successfully identified the first SST3-agonist peptides. SST3-agonists lowered cell viability and chromogranin-A secretion, increased apoptosis in vitro, and reduced tumor growth in a preclinical PitNET model. As expected, inhibition of cell viability in response to SST3-agonists defined two NFPT populations: responsive and unresponsive, wherein responsive NFPTs expressed more SST3 than unresponsive NFPTs and exhibited a profound reduction of MAPK, PI3K-AKT/mTOR, and JAK/STAT signaling pathways upon SST3-agonist treatments. Concurrently, SSTR3 silencing increased cell viability in a subset of NFPTs. CONCLUSIONS: This study demonstrates that SST3-agonists activate signaling mechanisms that reduce NFPT cell viability and inhibit pituitary tumor growth in experimental models that expresses SST3, suggesting that targeting this receptor could be an efficacious treatment for NFPTs.

Complement component 3 as biomarker of disease activity and cardiometabolic risk factor in rheumatoid arthritis and spondyloarthritis.

OBJECTIVE: To analyze the relationship between complement component 3 (C3) and the prevalence of cardiometabolic risk factors and disease activity in the rheumatic diseases having the highest rates of cardiovascular morbidity and mortality: rheumatoid arthritis (RA), psoriatic arthritis (PsA) and axial spondyloarthritis (axSpA). METHODS: This is a cross-sectional study including 200 RA, 80 PsA, 150 axSpA patients and 100 healthy donors. The prevalence of cardiometabolic risk factors [obesity, insulin resistance, type 2 diabetes mellitus, hyperlipidemia, apolipoprotein B/apolipoprotein A (apoB/apoA) and atherogenic risks and hypertension] was analyzed. Serum complement C3 levels, inflammatory markers and disease activity were evaluated. Cluster analysis was performed to identify different phenotypes. Receiver operating characteristic (ROC) curve analysis to assess the accuracy of complement C3 as biomarker of insulin resistance and disease activity was carried out. RESULTS: Levels of complement C3, significantly elevated in RA, axSpA and PsA patients, were associated with the prevalence of cardiometabolic risk factors. Hard clustering analysis identified two distinctive phenotypes of patients depending on the complement C3 levels and insulin sensitivity state. Patients from cluster 1, characterized by high levels of complement C3 displayed increased prevalence of cardiometabolic risk factors and high disease activity. ROC curve analysis showed that non-obesity related complement C3 levels allowed to identify insulin resistant patients. CONCLUSIONS: Complement C3 is associated with the concomitant increased prevalence of cardiometabolic risk factors in rheumatoid arthritis and spondyloarthritis. Thus, complement C3 should be considered a useful marker of insulin resistance and disease activity in these rheumatic disorders.

Oncogenic Role of Secreted Engrailed Homeobox 2 (EN2) in Prostate Cancer.

Engrailed variant-2 (EN2) has been suggested as a potential diagnostic biomarker; however, its presence and functional role in prostate cancer (PCa) cells is still controversial or unknown. Here, we analyzed 1) the expression/secretion profile of EN2 in five independent samples cohorts from PCa patients and controls (prostate tissues and/or urine) to determine its utility as a PCa biomarker; and 2) the functional role of EN2 in normal (RWPE1) and tumor (LNCaP/22Rv1/PC3) prostate cells to explore its potential value as therapeutic target. EN2 was overexpressed in our two cohorts of PCa tissues compared to control and in tumor cell lines compared with normal-like prostate cells. This profile was corroborated in silico in three independent data sets [The Cancer Genome Atlas(TCGA)/Memorial Sloan Kettering Cancer Center (MSKCC)/Grasso]. Consistently, urine EN2 levels were elevated and enabled discrimination between PCa and control patients. EN2 treatment increased cell proliferation in LNCaP/22Rv1/PC3 cells, migration in RWPE1/PC3 cells, and PSA secretion in LNCaP cells. These effects were associated, at least in the androgen-sensitive LNCaP cells, with increased AKT and androgen-receptor phosphorylation levels and with modulation of key cancer-associated genes. Consistently, EN2 treatment also regulated androgen-receptor activity (full-length and splicing variants) in androgen-sensitive 22Rv1 cells. Altogether, this study demonstrates the potential utility of EN2 as a non-invasive diagnostic biomarker for PCa and provides novel and valuable information to further investigate its putative utility to develop new therapeutic tools in PCa.

Biguanides Exert Antitumoral Actions in Pituitary Tumor Cells Through AMPK-Dependent and -Independent Mechanisms.

CONTEXT: Pituitary neuroendocrine tumors (PitNETs) are a commonly underestimated pathology in terms of incidence and associated morbimortality. Currently, an appreciable subset of patients are resistant or poorly responsive to the main current medical treatments [i.e., synthetic somatostatin analogs (SSAs) and dopamine agonists]. Thus, development and optimization of novel and available medical therapies is necessary. Biguanides (metformin, buformin, and phenformin) are antidiabetic drugs that exert antitumoral actions in several tumor types, but their pharmacological effects on PitNETs are poorly known. OBJECTIVE: We aimed to explore the direct effects of biguanides on key functions (cell viability, hormone release, apoptosis, and signaling pathways) in primary cell cultures from human PitNETs and cell lines. Additionally, we evaluated the effect of combined metformin with SSAs on cell viability and hormone secretion. DESIGN: A total of 13 corticotropinomas, 13 somatotropinomas, 13 nonfunctioning PitNETs, 3 prolactinomas, and 2 tumoral pituitary cell lines (AtT-20 and GH3) were used to evaluate the direct effects of biguanides on cell viability, hormone release, apoptosis, and signaling pathways. RESULTS: Biguanides reduced cell viability in all PitNETs and cell lines (with phenformin being the most effective biguanide) and increased apoptosis in somatotropinomas. Moreover, buformin and phenformin, but not metformin, reduced hormone secretion in a cell type-specific manner. Combination metformin/SSA therapy did not increase SSA monotherapy effectiveness. Effects of biguanides on PitNETs could involve the modulation of AMP-activated protein kinase-dependent ([Ca2+]i, PI3K/Akt) and independent (MAPK) mechanisms. CONCLUSION: Altogether, our data unveil clear antitumoral effects of biguanides on PitNET cells, opening avenues to explore their potential as drugs to treat these pathologies.

E-cadherin expression is associated with somatostatin analogue response in acromegaly.

Acromegaly is a rare disease resulting from hypersecretion of growth hormone (GH) and insulin-like growth factor 1 (IGF1) typically caused by pituitary adenomas, which is associated with increased mortality and morbidity. Somatostatin analogues (SSAs) represent the primary medical therapy for acromegaly and are currently used as first-line treatment or as second-line therapy after unsuccessful pituitary surgery. However, a considerable proportion of patients do not adequately respond to SSAs treatment, and therefore, there is an urgent need to identify biomarkers predictors of response to SSAs. The aim of this study was to examine E-cadherin expression by immunohistochemistry in fifty-five GH-producing pituitary tumours and determine the potential association with response to SSAs as well as other clinical and histopathological features. Acromegaly patients with tumours expressing low E-cadherin levels exhibit a worse response to SSAs. E-cadherin levels are associated with GH-producing tumour histological subtypes. Our results indicate that the immunohistochemical detection of E-cadherin might be useful in categorizing acromegaly patients based on the response to SSAs.

Association between radiological parameters and clinical and molecular characteristics in human somatotropinomas.

Acromegaly is a rare but severe disease, originated in 95% of cases by a growth hormone-secreting adenoma (somatotropinoma) in the pituitary. Magnetic resonance imaging (MRI) is a non-invasive technique used for the diagnosis and prognosis of pituitary tumours. The aim of this study was to determine whether the use of T2-weighted signal intensity at MRI could help to improve the characterisation of somatotropinomas, by analysing its relationship with clinical/molecular features. An observational study was implemented in a cohort of 22 patients (mean age = 42.1 ± 17.2 years; 59% women; 95% size>10 mm). Suprasellar-extended somatotropinomas presented larger diameters vs. non-extended tumours. T2-imaging revealed that 59% of tumours were hyperintense and 41% isointense adenomas, wherein hyperintense were more invasive (according to Knosp-score) than isointense adenomas. A higher proportion of hyperintense somatotropinomas presented extrasellar-growth, suprasellar-growth and invasion of the cavernous sinus compared to isointense adenomas. Interestingly, somatostatin receptor-3 and dopamine receptor-5 (DRD5) expression levels were associated with extrasellar and/or suprasellar extension. Additionally, DRD5 was also higher in hyperintense adenomas and its expression was directly correlated with Knosp-score and with tumour diameter. Hence, T2-weighted MRI on somatotropinomas represents a potential tool to refine their diagnosis and prognosis, and could support the election of preoperative treatment, when required.