Identificación de nuevos marcadores moleculares y contribución de mecanismos de splicing en patologías tumorales endocrinas

Cancer represents one of the main challenges for the human being, in that it encompasses some of the most severe and health-threatening pathologies worldwide. Although great efforts are being implemented and significant advances are being reached in basic, translational and clinical research over the last decades, the development of novel and more global and useful therapeutic strategies in Oncology is hampered by the heterogeneity and complexity of this disease. In order to tackle these difficulties, Hanahan and Weinberg proposed in 2000 and updated in 2011 a group of common alterations shared by most cancer types, which were defined as the hallmarks of cancer. Some of those cancer hallmarks are related with hormonal signaling, which is considered an important element in the control of malignant features. In this context, this Thesis has been mainly focused in the study of diverse endocrine-related cancers, such as prostate cancer (PCa), which is strongly influenced by the hormonal milieu, and different types of neuroendocrine tumors (NETs). Indeed, PCa is one of the tumor pathologies with highest incidence in men and one of the most common causes of cancer-related deaths among worldwide population. On the other hand, NETs comprise a markedly heterogeneous group of neoplasia originated from the diffuse neuroendocrine system that have been typically classified by their location. Among them, this Thesis have been focused on pancreatic tumors (PanNETs). Finally, we will also analyze a tumor type closely related, as it is the case of pituitary neuroendocrine tumors (PitNETs). One of the hormonal axes classically related to different types of tumors and that has represented the central interest of our group is the system comprised by somatostatin and its receptors (SST1-SST5). Particularly, this system has been classically linked to neuroendocrine tumors (NETs, PitNETs), wherein synthetic somatostatin agonists are widely used to act on several of its receptors (e.g. SST2, SST5), which represent useful therapeutic targets in these pathologies. In this context, it is remarkable the growing relevance of SST5 as putative therapeutic target of novel somatostatin analogs, and due to the discovery of novel truncated splicing variants (e.g. SST5TMD4), which are related with the aggressiveness of several cancer types. Nonetheless, very little is known about the biogenesis of SST5 from its gene, SSTR5, and about the role of other SST receptors in endocrine-related tumors. The growing identification of abnormal splicing variants that, similar to the above mentioned SST5TMD4, are overexpressed in different cancer types reinforces the idea that the alteration of the splicing process may be involved in the development and aggressiveness of tumor pathologies, through the dysregulation of the normal alternative splicing pattern and the generation of aberrant isoforms with oncogenic potential. In fact, over the last years, the alteration of the splicing process is being considered as a novel and transversal cancer hallmark, in that it seems to be affecting to all the hallmarks previously described. However, the information regarding the splicing process and its dysregulation is still scarce in some tumor pathologies, including NETs. Thus, for all the reasons indicated above, the general aim of this Thesis was to determine the role of somatostatin receptors and splicing machinery in different types of endocrine-related cancers and neuroendocrine tumors, as well as the underlying regulatory mechanisms, with the final purpose of discovering novel biomarkers and pharmacologic targets with potential to improve the diagnostic and therapeutic approaches in these pathologies.El cáncer es uno de los mayores retos que afronta la humanidad, pues comprende algunas de las patologías más graves que afectan a la salud a nivel mundial. Aunque se están realizado grandes esfuerzos y se han logrado avances significativos en investigación básica, clínica y traslacional durante las últimas décadas, el desarrollo de nuevas estrategias terapéuticas en oncología, más globales y a la vez precisas y eficientes, está limitado por la enorme heterogeneidad y complejidad de esta enfermedad. Para enfrentarse a dichas dificultades, Hanahan y Weinberg propusieron en el año 2000 y actualizaron en 2011 un grupo de alteraciones comunes compartidas por la mayoría de los tipos de cáncer a diferentes niveles, que se conocen como hallmarks o pilares del cáncer. Algunos de estos hallmarks están relacionados con la señalización hormonal, que se considera un componente importante en el control de la malignidad tumoral. En este contexto, la presente Tesis se ha enfocado fundamentalmente en el estudio de cánceres relacionados con el sistema endocrino, como el cáncer de próstata (CaP), fuertemente dependiente de la regulación hormonal, y distintos tipos de tumores neuroendocrinos (TNEs). El CaP es una de las patologías tumorales de mayor incidencia en hombres y una de las causas de muerte más comunes relacionadas con el cáncer en la población mundial. Por su parte, los TNEs son un grupo de neoplasias marcadamente heterogéneo, que surgen del sistema neuroendocrino difuso y se ha clasificado habitualmente según la localización del tumor. Concretamente, esta Tesis se enfoca en los tumores pancreáticos (PanNETs). Por último, analizaremos también un tipo tumoral estrechamente relacionado, los tumores neuroendocrinos hipofisarios (PitNETs)

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.Junta de AndalucíaMinisterio de EconomíaMinisterio de Ciencia e Innovació

Ghrelin-O-Acyltransferase (GOAT) Enzyme as a Novel Potential Biomarker in Gastroenteropancreatic Neuroendocrine Tumors

Objectives: The association between the presence and alterations of the components of the ghrelin system and the development and progression of neuroendocrine tumors (NETs) is still controversial and remains unclear. Methods: Here, we systematically evaluated the expression levels (by quantitative-PCR) of key ghrelin system components of in gastroenteropancreatic (GEP)-NETs, as compared to non-tumor adjacent (NTA; n = 42) and normal tissues (NT; n = 14). Then, we analyzed their putative associations with clinical-histological characteristics. Results: The results indicate that ghrelin and its receptor GHSR1a are present in a high proportion of normal tissues, while the enzyme ghrelin-O-acyltransferase (GOAT) and the splicing variants In1-ghrelin and GHSR1b were present in a lower proportion of normal tissues. In contrast, all ghrelin system components were present in a high proportion of tumor and NTA tissues. GOAT was significantly overexpressed (by quantitative-PCR (qPCR)) in tumor samples compared to NTA, while a trend was found for ghrelin, In1-ghrelin and GHSR1a. In addition, expression of these components displayed significant correlations with key clinical parameters. The marked overexpression of GOAT in tumor samples compared to NTA regions was confirmed by IHC, revealing that this enzyme is particularly overexpressed in gastrointestinal NETs, where it is directly correlated with tumor diameter. Conclusions: These results provide novel information on the presence and potential pathophysiological implications of the ghrelin system components in GEP-NETs, wherein GOAT might represent a novel diagnostic biomarker

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

The Caldera. No. 20

En las páginas de ésta edición encontrarán, en gran parte de los textos elaborados por nuestros educandos, un sencillo pero sentido homenaje a un ser humano que creyó, luchó y dio su vida, por causa de la libertad, de la igualdad, de la paz, por la defensa de sus profundas creencias y de una vida consagrada a su amada patria.PROYECTOS DE INVESTIGACIÓN.- PERFILES CALDISTAS.- LECTURA EN EL CALDAS Experiencias Significativas.- GRAN FINAL III Concurso Intercolegiado de Oratoria.-DEPORTES EN EL CALDAS.- BICENTENARIO FRANCISCO JOSÉ DE CALDAS.- ADIVINANZAS.- EXPRESIONES CALDISTAS.- GALERÍA DE IMÁGENES.-In the pages of this edition you will find, in a large part of the texts prepared by our students, a simple but a heartfelt tribute to a human being who believed, fought and gave his life, for the sake of freedom, equality, peace, for the defense of his deep beliefs and a life consecrated to his beloved homeland.Modalidad Presencia

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

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.This work has been supported by Spanish Ministry of Economy [MINECO; BFU2016-80360-R (to JPC)] and Ministry of Science and Innovation [MICINN; PID2019-105201RB-I00 (to JPC), PID2019-105564RB-I00 (to RML)]. Instituto de Salud Carlos III, co-funded by European Union (ERDF/ESF, “Investing in your future”) [FIS DTS Grant DTS20/00050 (to RML)); Postdoctoral Grant Sara Borrell CD19/00255 (to AIC); Predoctoral contract FI17/00282 (to EAP)]. Spanish Ministry of Universities [FPU20/03958 (to VGV); FPU18/02275 (to RBE); postdoctoral contract under the program María Zambrano funded by the European Union Next Generation-EU (to SPA)]. Asociación Cáncer de Páncreas and AESPANC 2022 (to JPC); Boehringer Ingelheim Fonds travel grant (EAP). Junta de Andalucía (BIO-0139). GETNE2016 and GETNE2019 Research grants (to JPC); and CIBERobn. Part of this work was supported by COST (European Cooperation in Science and Technology – www.cost.eu) through the COST Action TRANSPAN (CA21116).Peer reviewe

Calcitriol-Mediated Hypercalcemia, Somatostatin Receptors Expression and 25-Hydroxyvitamin D3-1α- Hydroxylase in GIST Tumors.

Hypercalcemia is a common complication in cancer patients Mainly caused by Parathyroid hormone-related protein (PTHrP) secretion and metastasis. Calcitriol secretion is a rare source of hypercalcemia in solid tumors, especially in gastrointestinal stromal tumors (GIST). We present a case report of a female patient with a 23 cm gastric GIST that expressed somatostatin-receptors and presented with severe hypercalcemia due to calcitriol secretion. Calcium control was achieved with medical treatment before the use of targeted-directed therapies. Surgery was performed and allowed complete tumor resection. Two years later, patient remains free of disease. Molecular analysis revealed the mRNA expression of 25-hydroxyvitamin D3-1-hydroxylase (1αOHase) and vitamin-D receptors in the tumor cells, confirming the calcitriol-mediated mechanism. Furthermore, the expression of the endotoxin recognition factors CD14 and TLR4 suggests an inflammatory mediated mechanism. Finally, the expression of somatostatin-receptors, especially SST2 might have been related with clinical evolution and prognosis in this patient

Splicing machinery dysregulation drives glioblastoma development/aggressiveness: oncogenic role of SRSF3.

Glioblastomas remain the deadliest brain tumour, with a dismal ∼12-16-month survival from diagnosis. Therefore, identification of new diagnostic, prognostic and therapeutic tools to tackle glioblastomas is urgently needed. Emerging evidence indicates that the cellular machinery controlling the splicing process (spliceosome) is altered in tumours, leading to oncogenic splicing events associated with tumour progression and aggressiveness. Here, we identify for the first time a profound dysregulation in the expression of relevant spliceosome components and splicing factors (at mRNA and protein levels) in well characterized cohorts of human high-grade astrocytomas, mostly glioblastomas, compared to healthy brain control samples, being SRSF3, RBM22, PTBP1 and RBM3 able to perfectly discriminate between tumours and control samples, and between proneural-like or mesenchymal-like tumours versus control samples from different mouse models with gliomas. Results were confirmed in four additional and independent human cohorts. Silencing of SRSF3, RBM22, PTBP1 and RBM3 decreased aggressiveness parameters in vitro (e.g. proliferation, migration, tumorsphere-formation, etc.) and induced apoptosis, especially SRSF3. Remarkably, SRSF3 was correlated with patient survival and relevant tumour markers, and its silencing in vivo drastically decreased tumour development and progression, likely through a molecular/cellular mechanism involving PDGFRB and associated oncogenic signalling pathways (PI3K-AKT/ERK), which may also involve the distinct alteration of alternative splicing events of specific transcription factors controlling PDGFRB (i.e. TP73). Altogether, our results demonstrate a drastic splicing machinery-associated molecular dysregulation in glioblastomas, which could potentially be considered as a source of novel diagnostic and prognostic biomarkers as well as therapeutic targets for glioblastomas. Remarkably, SRSF3 is directly associated with glioblastoma development, progression, aggressiveness and patient survival and represents a novel potential therapeutic target to tackle this devastating pathology

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) 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, TStage, 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.This work was funded by Instituto de Salud Carlos III, co-funded by European Union (ERDF/ESF, “Investing in your future”) [PI16/00264, PI17-02287, CM16/00180, FI17/00282, CD16/00092], FEDER (CCB.030PM), MINECO/MECD (BFU2016-80360-R; FPU16/06190,FPU18/02485, FPU16/05059, FPU17/00263, FPU14/04290), Junta de Andalucía (BIO-0139), Spanish Association Against Cancer (AECC; INVES18057YUBE) and CIBERobn. CIBER is an initiative of Instituto de Salud Carlos III, Ministerio de Sanidad, Servicios Sociales e Igualdad, Spain.Ye