Identificación de nuevos factores endocrinos-metabólicos y de la maquinaria de splicing con potencial diagnóstico, pronóstico y/o terapéutico en cáncer de próstata

Prostate cancer (PCa) is the tumor pathology with the highest incidence among men in developed countries and represents one of the main causes of cancer-related death in this group. Despite the advances achieved during the last years in PCa diagnosis and treatment, the diagnostic and therapeutic tools currently available are not sufficiently efficient. Specifically, prostate specific antigen (PSA) is the current “gold standard” used for PCa screening. Unfortunately, the PSA test has controversial specificity, since certain physiological conditions or non-tumor pathologies (e.g. benign prostatic hyperplasia, prostatitis) are also associated with high circulating PSA levels. Furthermore, the diagnostic capacity and clinical utility of the PSA test is especially low in patients with PSA levels between 3-10 ng/mL (the so-called grey zone). For these reasons, PSA test is associated with PCa overdiagnosis and unnecessary biopsies. Moreover, this invasive method required to subsequently confirm or discard the presence of PCa can cause side effects such as pain, bleeding and infections, which significantly impact patient’s quality of live. Therefore, there is an urgent need to identify new diagnostic tools that complement or replace PSA in clinical practice. Similarly, the therapeutic armamentarium in PCa is limited and clearly insufficient. Specifically, the pharmacological treatment used as the first option for the treatment of advanced-stage PCa or tumor recurrences consists of androgen deprivation (e.g. LHRH antagonists, abiraterone), due to the marked dependence of androgenic signaling of PCa cells. However, about 80% of patients treated with androgen deprivation therapy will develop resistance to this treatment, generating the so-called Castration Resistant PCa (CRPC), the most aggressive phenotype of this disease, which remains lethal nowadays. Therefore, the search for alternative therapeutic targets useful for the development of new, more efficient, treatments than those currently available continues to be one of the main objectives of the biomedical scientific community. PCa develops mainly in older patients, who suffer, with a high frequency, endocrinemetabolic pathologies (i.e. obesity, diabetes), which can influence the development and/or progression of PCa, increasing the complexity of this pathology. Specifically, obesity is associated with an increased risk of developing PCa, and with higher PCa aggressiveness. In this context, it has been described that metformin and statins (drugs commonly used in patients with endocrine-metabolic pathologies such as obesity or diabetes) exert antitumor effects in numerous tumor types, both in vitro and in vivo. However, although the combination of both drugs has been shown to exert additive antitumor actions in some cancer cell types, this possibility has not been systematically studied in the case of PCa. An important limitation in this field is that the information available regarding the exact molecular mechanisms underlying the pathophysiological interaction between obesity and PCa is very limited and fragmentary. In this sense, it is well-known that numerous components of different hormonal systems (i.e. androgen, ghrelin, etc.) are clearly dysregulated in PCa and obesity, which may constitute a source of biomarkers in these pathologies. The ghrelin system is a clear example in this context, since it is a pleiotropic hormonal system consisting of various ligands (e.g. Ghrelin, In1-ghrelin), enzymes (GOAT), and receptors (GHSR1a, GHSR1b). Specifically, In1-ghrelin (an alternative splicing variant derived as the result of the intron-1 retention) is overexpressed and plays an oncogenic role in PCa. Likewise, GOAT levels are higher in PCa tissues compared to nontumor prostate tissue, but the putative pathophysiological role of this enzyme in PCa is still unknown nowadays. Furthermore, it has been reported that circulating levels of both In1- ghrelin and GOAT are higher in PCa patients compared to healthy patients, suggesting that both elements could represent non-invasive diagnostic biomarkers of PCa. However, to date, it is unknown whether In1-ghrelin or GOAT can be detected and whether they are dysregulated in urine (which constitutes an enriched source of proteins derived from the prostate) in patients with PCa compared with patients without PCa. Finally, it is important to mention that the alteration of the splicing process has become a common hallmark of a large number of tumor pathologies, including PCa. Splicing is a physiological mechanism involved in the maturation of pre-mRNAs, which can generate different mature mRNAs from a single gene, thereby increasing the transcriptomic complexity of eukaryotic cells. However, increasing evidence demonstrates the existence of different splicing variants with oncogenic potential which are associated to the development, progression and/or resistance of pharmacological treatments of multiples tumor pathologies, including PCa. These dysregulations of the splicing process may be due to an alteration of the machinery that regulate this cellular process (spliceosome) and/or of the proteins that regulate this mechanism (splicing factors), suggesting that they could play a relevant role in the development and/or progression of PCa. Although certain studies have shown the specific dysregulation of some of these elements in PCa, the possible involvement of the alteration of this machinery and the putative implication in the development and/or progression of PCa has not been systematically explored yet. Based on the all the information mentioned above, the general aim of this Doctoral Thesis was to determine the potential role that certain splicing-related elements (spliceosome components and splicing factors) and metabolic modulators (endogenous elements and exogenous treatments) play and/or exert in the development, progression and/or aggressiveness of PCa, with the ultimate goal of discovering novel biomarkers (diagnostic and/or prognostic) and therapeutic tools that could improve the diagnosis, treatment and the clinical management of PCa patients. The first section of this Doctoral Thesis was focused on exploring the putative dysregulation and the clinical and functional implications of the splicing machinery in PCa. Results presented herein indicate that the splicing machinery is drastically dysregulated in PCa, including pivotal elements of this machinery such as SNRNP200, SRRM1, SRSF3, RBM22 or SF3B1. Among all the dysregulated spliceosome components and splicing factors, SNRNP200, SRRM1 and SRSF3 were selected initially to be further explored because they were associated with all the relevant aggressiveness features available in the cohort of patients used in this study. Mechanistically, silencing of SNRNP200, SRRM1 or SRSF3 inhibited oncogenic pathways and altered the splicing process of genes previously reported to be involved in tumor aggressiveness and/or CRPC development. Consistently, SNRNP200-, SRRM1- and SRSF3-silencing sensitized the CRPC cells to an androgen receptor (AR)-inhibitor (enzalutamide). Therefore, these data demonstrate that SNRNP200, SRRM1 and SRSF3 could represent attractive novel diagnostic and prognostic biomarkers as well as therapeutic targets for PCa and CRPC. This Doctoral Thesis was also focused on exploring the potential dysregulation and role of RBM22 in PCa, a splicing factor tightly involved in the activation of the spliceosome. Specifically, we found that RBM22 levels were lower (at mRNA and protein levels) in PCa tissues compared to non-tumor prostate tissues, and that these levels were inversely associated to key clinical parameters of aggressiveness in patients and in a preclinical mouse model. Moreover, we observed that the aggressiveness features of PCa cells were reduced in response to RBM22 overexpression in vitro and in vivo (i.e. reduced tumor growth, proliferation, migration, etc.) Mechanistically, results from high-throughput techniques (i.e. RNAseq, CLIP-seq and signaling array) performed in samples from in vitro and in vivo PCa models pointed out that the inhibition of MYC, MYCN and E2F, as well as a profound dysregulation of the splicing process could be the molecular mechanisms associated to the relevant findings previously observed after the RBM22 overexpression in PCa. Therefore, these results suggest that RBM22 plays a relevant antitumor role in PCa through the regulation of the splicing process and the reduction of MYC, MYCN and E2F levels/activity. In the case of SF3B1, a key component involved in the spliceosome assembly, we demonstrated that SF3B1 is overexpressed in PCa and that its levels were directly associated with important aggressiveness features of PCa. Moreover, the downregulation of SF3B1 and the inhibition of its activity (using the selective inhibitor pladienolide-B) significantly reduced functional aggressiveness features and increased the apoptotic rate of PCa cells. From a molecular perspective, SF3B1 inhibition altered PI3K/AKT and JNK oncogenic signaling pathways, down-regulated key oncogenic splicing variants (AR-v7 and In1-ghrelin), and dysregulated the expression of several elements involved in mRNA metabolism [splicing and non-sense mediated mRNA decay (NMD)]. Taken together, these results suggest that SF3B1 could represent a new prognostic biomarker and a therapeutic target in PCa, providing convincing evidence for the putative utility of pladienolide-B as novel therapeutic tool for the treatment of this devastating pathology. Once demonstrated the key role of several splicing-regulating factors in PCa, and since metformin has been found to exert antitumoral effects in various cancer types and that alter the expression of specific splicing factors in certain cell types, we next aimed to explore the potential involvement of the splicing dysregulation in the antitumor actions of metformin in PCa. Our data indicate that metformin produced a profound downregulation in the expression of different spliceosome components and splicing factors in PCa cells, being some of these changes (i.e. downregulation of SF3B1, SRRM1 and NOVA1) further validated in vivo using a PCa xenograft model. Moreover, the antiproliferative response of metformin treatment observed in PCa cells was completely blocked when the expression of SF3B1, SRRM1 or NOVA1 were silenced. Therefore, these results suggest that the dysregulation of some components of the splicing machinery, especially SF3B1, SRRM1 and NOVA1, might represent an additional molecular mechanism underlying the wellknown antitumor effects of metformin in PCa. The second section of this Doctoral Thesis was focused on the analysis of the antitumor actions of metformin, statins and their combination, as well as the underlying molecular mechanisms in PCa. Specifically, PCa patients treated with metformin and statins in combination were associated to more beneficial effects in key clinical parameters of aggressiveness compared to those receiving individual treatments. Consistently, although biguanides and statins significantly decreased the aggressiveness of PCa cells in vitro, these antitumor effects were more pronounced when combining both treatments. Mechanistically, we found that the treatment with the combination of metformin and simvastatin in vitro resulted in a hyper-inactivation of AR and mTOR, as well as in an upregulation of the expression of different cyclin-dependent kinase inhibitors (CKIs; CDKN1A, CDKN1B, CDKN2A and CDKN2D), being some of those changes further validated in samples from patients with PCa (i.e. upregulation of CDKN1B and CDKN2A). Altogether, this study demonstrates that metformin and simvastatin exert additive antitumor effects in PCa, thus suggesting that this combination might be a novel and attractive therapeutic approach to tackle PCa. Finally, in the third section of this Doctoral Thesis, we analysed specific components of the ghrelin system (specifically, GOAT enzyme and In1-ghrelin variant) to unveil their potential role as diagnostic, prognostic and/or therapeutic tools in PCa. These results demonstrated that GOAT could be detected in urine samples, wherein its levels outperformed the capacity of plasma PSA levels to diagnose PCa, especially clinically significant PCa (SigPCa; defined as Gleason score ≥7) in patients with PSA in the grey zone. Moreover, high urine GOAT levels were associated to increased risk of developing PCa and SigPCa, and were positively correlated to key clinical and molecular aggressiveness parameters in PCa patients. Furthermore, we demonstrated that GOAT overexpression increased PCa aggressiveness in vitro and in vivo, while its silencing and the blockade of its activity (using a specific GOAT inhibitor) significantly decreased the aggressiveness features of PCa cells in vitro. Therefore, these results demonstrated that GOAT enzyme may represent an additional non-invasive diagnostic biomarker, as well as a potential prognostic and therapeutic tool for PCa. Moreover, the analysis of In1-ghrelin in urine from patients with PSA in the grey zone indicated that its levels were able to distinguish between patients with and without PCa, were associated to higher risk of PCa, obesity and diabetes, and correlated with metabolic and aggressiveness parameters. In fact, we found that the capacity of urine In1-ghrelin levels to diagnose PCa was especially elevated when only obese patients were considered, suggesting its potential utility as a noninvasive diagnostic biomarker from a more personalized perspective. Altogether, the general conclusion derived from this Doctoral Thesis is that the alteration of the splicing process and the dysregulation of certain endocrine-metabolic systems could contribute to the development, progression and/or aggressiveness of PCa, representing a source of novel diagnostic and prognostic biomarkers, as well as therapeutic targets that could be exploited to improve the diagnosis of PCa patients, and to develop more effective prognostic and therapeutic tools to tackle this devastating pathology

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

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

Clinical Utility of Ghrelin-O-Acyltransferase (GOAT) Enzyme as a Diagnostic Tool and Potential Therapeutic Target in Prostate Cancer

Recent data suggested that plasma Ghrelin O-Acyl Transferase enzyme (GOAT) levels could represent a new diagnostic biomarker for prostate cancer (PCa). In this study, we aimed to explore the diagnostic and prognostic/aggressiveness capacity of GOAT in urine, as well as to interrogate its putative pathophysiological role in PCa. We analysed urine/plasma levels of GOAT in a cohort of 993 patients. In vitro (i.e., cell-proliferation) and in vivo (tumor-growth in a xenograft-model) approaches were performed in response to the modulation of GOAT expression/activity in PCa cells. Our results demonstrate that plasma and urine GOAT levels were significantly elevated in PCa patients compared to controls. Remarkably, GOAT significantly outperformed PSA in the diagnosis of PCa and significant PCa in patients with PSA levels ranging from 3 to 10 ng/mL (the so-called PSA grey-zone). Additionally, urine GOAT levels were associated to clinical (e.g., Gleason-score, PSA levels) and molecular (e.g., CDK2/CDK6/CDKN2A expression) aggressiveness parameters. Indeed, GOAT overexpression increased, while its silencing/blockade decreased cell-proliferation in PCa cells. Moreover, xenograft tumors derived from GOAT-overexpressing PCa (DU145) cells were significantly higher than those derived from the mock-overexpressing cells. Altogether, our results demonstrate that GOAT could be used as a diagnostic and aggressiveness marker in urine and a therapeutic target in PCa

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

Alternative splicing in bladder cancer: potential strategies for cancer diagnosis, prognosis, and treatment

Bladder cancer is the most common malignancy of the urinary tract worldwide.The therapeutic options to tackle this disease comprise surgery, intravesical orsystemic chemotherapy, and immunotherapy. Unfortunately, a wide number ofpatients ultimately become resistant to these treatments and develop aggressivemetastatic disease, presenting a poor prognosis. Therefore, the identification ofnovel therapeutic approaches to tacklethis devastating pathology is urgentlyneeded. However, a significant limitation is that the progression and drugresponse of bladder cancer is strongly associated with its intrinsic molecular het-erogeneity. In this sense, RNA splicing is recently gaining importance as a criticalhallmark of cancer since can have a significant clinical value. In fact, a profounddysregulation of the splicing process has been reported in bladder cancer, espe-cially in the expression of certain key splicing variants and circular RNAs with apotential clinical value as diagnostic/prognostic biomarkers or therapeutic targetsin this pathology. Indeed, some authorshave already evidenced a profound anti-tumor effect by targeting some splicing factors (e.g., PTBP1), mRNA splicing vari-ants (e.g., PKM2, HYAL4-v1), and circular RNAs (e.g., circITCH, circMYLK),which illustrates new possibilities to significantly improve the management ofthis pathology. This review represents the first detailed overview of the splicingprocess and its alterations in bladder cancer, and highlights opportunities for thedevelopment of novel diagnostic/prognostic biomarkers and their clinical poten-tial for the treatment of this devastating cancer type.This article is categorized under:RNA Processing > Splicing Regulation/Alternative SplicingRNA in Disease and Development > RNA in Diseas

Determination of the SO/PHI-HRT wavefront degradation using multiple defocused images

The Polarimetric and Helioseismic Imager on board the Solar Orbiter mission (SO/PHI) offers refocusing capabilities to cope with the strongly varying thermal environment of the optical system along the spacecraft's elliptical orbit. The series of images recorded during in-flight focus calibrations can be employed for phase diversity analyses. In this work we infer the wavefront degradation caused by the thermo-optical effects in the High Resolution Telescope (HRT) from images taken during the fine and coarse focus scans performed in the commissioning phase of the instrument. The difference between these two series of images are mainly related to the employed defocused step (smaller for the fine scans) and the signal-to-noise ratio (higher for the coarse scans). We use the retrieved wavefronts to reconstruct the original scene observed during the calibration of the instrument. We applied a generalized phase diversity algorithm that allowed us to use several images taken with different amounts of defocus to sense the wavefront degradation caused by the instrument. The algorithm also uses information from both the inferred wavefront and the series of images to restore the solar scene We find that most of the retrieved Zernike coefficients tend to converge to the same value when increasing the number of images employed for PD for both the fine and the coarse focusing scans. The restored scenes also show signs of convergence, and the merit function is minimized more as $K$ increases. Apart from a defocus, the inferred wavefronts are consistent for the two datasets ($For the fine scan images, the quiet-sun contrast improves from$4.5\,$for the original focused image up to about$10\,$. For the coarse scan images, the contrast of the restored scene is as high as$11\,$