High expression of ecto-nucleotidases CD39 and CD73 in human endometrial tumors

One of the strategies used by tumors to evade immunosurveillance is the accumulation of extracellular adenosine, which has immunosupressive and tumor promoting effects. The study of the mechanisms leading to adenosine formation at the tumor interstitium are therefore of great interest in oncology. The dominant pathway generating extracellular adenosine in tumors is the dephosphorylation of ATP by ecto-nucleotidases. Two of these enzymes acting sequentially, CD39 and CD73, efficiently hydrolyze extracellular ATP to adenosine. They have been found to play a crucial role in a variety of tumors, but there were no data concerning endometrial cancer, the most frequent of the invasive tumors of the female genital tract. The aim of the present work is to study the expression of CD39 and CD73 in human endometrial cancer. We have analyzed protein and gene expression, as well as enzyme activity, in type I endometrioid adenocarcinomas and type II serous adenocarcinomas and their nonpathological endometrial counterparts. High levels of both enzymes were found in tumor samples, with significantly increased expression of CD39 in type II serous tumors, which also coincided with the higher tumor grade. Our results reinforce the involvement of the adenosinergic system in cancer, emphasizing the relevance of ecto-nucleotidases as emerging therapeutic targets in oncology

Vascular disease in COPD: systemic and pulmonary expression of PARC (Pulmonary and Activation-Regulated Chemokine)

Introduction: The role of Pulmonary and Activation-Regulated Chemokine (PARC) in the physiopathology of Chronic Obstructive Pulmonary Disease (COPD) is not fully understood. The aim of the present study is to analyze the expression of PARC in lung tissue and its relationship with the vascular remodeling of the systemic and pulmonary arteries of COPD subjects. Methods: To achieve this objective, protein and gene expression experiments, together with ELISA assays, were performed on the lung tissue, intercostal arteries and serum samples from COPD patients, non-obstructed smokers (NOS) and never-smokers (NS). Results: A total of 57 subjects were included in the analysis (23 COPD, 18 NOS and 16 NS). In the comparisons between groups, a significantly increased lung protein expression of PARC was observed in the COPD group compared to the NOS group (1.96±0.22 vs. 1.29±0.27, P-adjusted = 0.038). PARC was located predominantly in the smooth muscle cells of the remodeled pulmonary muscular arteries and the macrophage-rich area of the alveolar parenchyma. No differences were detected in PARC gene expression analyses. The protein content of PARC in the intercostal arteries were similar between groups, though little remodeling was observed in these arteries. Circulating levels of PARC were numerically higher in patients with COPD compared to NOS and NS. Conclusion: The results of the present study suggest an increased lung protein expression of PARC in COPD subjects. This protein was mainly localized in the smooth muscle cells of the pulmonary muscular arteries and was associated with the severity of intimal thickening, indicating its possible role in this remodeling process

Imbalance in the expression of genes associated with purinergic signalling in the lung and systemic arteries of COPDpatients.

Growing evidence indicates that purinergic signalling is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and in the vascular remodelling that occurs in other disorders; however, its role in initial vascular changes of COPD is not entirely known. We hypothesised that expression of genes regulating extracellular ATP and adenosine levels would be altered in the lung and systemic arteries of COPD patients. Quantitative real-time PCR was performed to analyse the relative expression of 17 genes associated with purinergic signalling and inflammation in lungs and intercostal arteries of never smokers (NS) (n = 16), non-obstructed smokers (NOS) (n = 17) and COPD patients (n = 21). Gene expression of ATP-degrading enzymes was decreased in both tissues of NOS and COPD patients compared to NS. NT5E expression (gene transcribing for an AMP hydrolyzing ectonucleotidase) was increased in both tissues in NOS compared to the other groups. P1 and P2 receptors did not show changes in expression. Expression of genes associated with inflammation (interleukin-13) was upregulated only in lung tissues of COPD. These findings suggest that the expression of different extracellular ATP-degrading enzymes is altered in smokers (NOS and COPD patients), promoting inflammation. However, the high NT5E expression found only in NOS could compensate this inflammatory environment

Is the purinergic pathway involved in the pathology of COPD? Decreased lung CD39 expression at initial stages of COPD

Background: Extracellular adenosine triphosphate (ATP) is up-regulated in the airways of patients with chronic obstructive pulmonary disease (COPD), resulting in increased inflammation, bronchoconstriction, and cough. Although extracellular ATP levels are tightly controlled by nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; also known as CD39) in the lungs, the role of CD39 in the pathology of COPD is unknown. We hypothesized that alterations in the expression and activity of CD39 could be part of the mechanisms for initiating and perpetuating the disease. Methods: We analyzed CD39 gene and protein expression as well as ATPase enzyme activity in lung tissue samples of patients with COPD (n = 17), non-obstructed smokers (NOS) (n = 16), and never smokers (NS) (n = 13). Morphometry studies were performed to analyze pulmonary vascular remodeling. Results: There was significantly decreased CD39 gene expression in the lungs of the COPD group (1.17 [0.85-1.81]) compared with the NOS group (1.88 [1.35-4.41]) and NS group (3.32 [1.23-5.39]) (p = 0.037). This attenuation correlated with higher systemic inflammation and intimal thickening of muscular pulmonary arteries in the COPD group. Lung CD39 protein levels were also lower in the COPD group (0.34 [0.22-0.92]) compared with the NOS group (0.67 [0.32-1.06]) and NS group (0.95 [0.4-1.1) (p = 0.133). Immunohistochemistry showed that CD39 was downregulated in lung parenchyma, epithelial bronchial cells, and the endothelial cells of pulmonary muscular arteries in the COPD group. ATPase activity in human pulmonary structures was reduced in the lungs of patients with COPD. Conclusion: An attenuation of CD39 expression and activity is presented in lung tissue of stable COPD patients, which could lead to pulmonary ATP accumulation, favoring the development of pulmonary inflammation and emphysema. This may be a mechanism underlying the development of COPD

Integrative transcriptome analysis of malignant pleural mesothelioma reveals a clinically relevant immune-based classification

Background: Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasia affecting the lung mesothelium. Immune checkpoint inhibitors (ICI) in MPM have not been extremely successful, likely due to poor identification of suitable candidate patients for the therapy. We aimed to identify cellular immune fractions associated with clinical outcome and classify patients with MPM based on their immune contexture. For each defined group, we sought for molecular specificities that could help further define our MPM classification at the genomic and transcriptomic level, as well as identify differential therapeutic strategies based on transcriptional signatures predictive of drug response. Methods: The abundance of 20 immune cell fractions in 516 MPM samples from 7 gene expression datasets was inferred using gene set variation analysis. Identification of clinically relevant fractions was performed with Cox proportional-hazards models adjusted for age, stage, sex, and tumor histology. Immune-based groups were defined based on the identified fractions. Results: T-helper 2 (TH2) and cytotoxic T (TC) cells were found to be consistently associated with overall survival. Three immune clusters (IG) were subsequently defined based on TH2 and TC immune infiltration levels: IG1 (54.5%) was characterized by high TH2 and low TC levels, IG2 (37%) had either low or high levels of both fractions, and IG3 (8.5%) was defined by low TH2 and high TC levels. IG1 and IG3 groups were associated with worse and better overall survival, respectively. While no differential genomic alterations were identified among immune groups, at the transcriptional level, IG1 samples showed upregulation of proliferation signatures, while IG3 samples presented upregulation of immune and inflammation-related pathways. Finally, the integration of gene expression with functional signatures of drug response showed that IG3 patients might be more likely to respond to ICI. Conclusions: This study identifies a novel immune-based signature with potential clinical relevance based on TH2 and TC levels, unveiling a fraction of patients with MPM with better prognosis and who might benefit from immune-based therapies. Molecular specificities of the different groups might be used to tailor specific potential therapies in the future

La senyalització purinèrgica al sistema reproductor. Paper de les ecto-nucleotidases a l’endometri no tumoral i amb adenocarcinoma

[cat] Els nucleòtids extracel·lulars, com l’ATP, i els nucleòsids que deriven de la seva hidròlisi, com l’adenosina, estan àmpliament distribuïts en tots els òrgans i sistemes animals i estan considerats com a potents molècules senyalitzadores que, a través de receptors purinèrgics o purinoceptors (de tipus P1 i P2), intervenen en diverses funcions fisiològiques i fisiopatològiques de l’aparell reproductor. La concentració extracel·lular de nucleòtids i nucleòsids ve regulada per enzims de membrana anomenats ecto-nucleotidases, els quals hidrolitzen seqüencialment l’ATP fins a adenosina, controlant així, els seus nivells extracel·lulars i en conseqüència les vies de senyalització derivades de la unió d’aquests lligands als purinoceptors. Existeixen quatre famílies d’ecto-nucleotidases: a) la família de les ecto­nucleòsid trifosfat difosfohidrolases (E-NTPDases) o família CD39 consta de vuit membres (NTPDasa 1­8) quatre dels quals s’expressen a la membrana plasmàtica (NTPDasa 1, 2, 3 i 8) i hidrolitzen amb diferents afinitats l’ATP i l’ADP fins a AMP, b) la família de les ecto-nucleòtid pirofosfatases/fosfodiesterases (-E-NPPs) conté set membres (NPP 1-7) dels quals tres (NPP 1, 2 i 3) hidrolitzen l’ATP i l’ADP fins AMP, c) les fosfatases alcalines, catalitzen la degradació de nucleòtids tri­, di-i monofosfats indistintament, i d) la família de les 5’-nucleotidases o CD73 com a únic membre de membrana que actua extracel·lularment hidrolitzant l’AMP fins adenosina. S’entén per senyalització purinèrgica el conjunt d’efectes biològics (autocrins i paracrins) produïts per les purines i les pirimidines en actuar com a lligands extracel·lulars. I, al complex molecular implicat en l’activació, la conservació i la terminació de la senyalització purinèrgica se li ha donat el nom de Purinoma, i inclou tant els lligands, com els receptors, els transportadors /canals i els enzims de degradació. Estudis previs del nostre grup demostren expressió d’algunes de les ecto-nucleotidases més importants al sistema reproductor masculí. No es coneixia encara, però, el paper de les ecto-nucleotidases al sistema reproductor femení ni en les patologies relacionades amb aquest aparell com és el càncer d’endometri, aspectes que han estat estudiats en aquesta tesi doctoral. Així, a través de tècniques histològiques, immunològiques i moleculars, hem estudiat l’expressió gènica i proteica així com l’activitat de les ecto­nucleotidases a l’aparell reproductor femení, en el endometri funcional i atròfic humà, i en el càncer d’endometri. A més, hem posat a punt dos models cel·lulars (línies cel·lulars d’adenocarcinoma endometrial humà i cultius primaris de cèl·lules de l’estroma endometrial humà) on estudiar la regulació de les ecto-nucleotidases. Els nostres resultats mostren expressió i activitat d’aquests enzims a l’endometri amb marcades diferències al llarg del cicle i després de la menopausa, indicant una possible regulació hormonal d’aquestes. També hem detectat una elevada expressió de dues de les ecto-nucleotidases que controlen directament els nivells extracel·lulars d’adenosina, CD39 i CD73, en el carcinoma endometrial Tipus I i Tipus II evidenciant així el seu potencial us com a dianes terapèutiques en oncologia. Per tot això, proposem les ecto-nucleotidases com a possibles biomarcadors de fertilitat i amb utilitat pronòstica i diagnòstica de l’adenocarcinoma endometrial.[eng] Extracellular ATP and its hydrolysis product adenosine, acting through specific receptors collectively named purinergic receptors, modulate various reproductive functions. There are four major groups of ecto-nucleotidases that control the levels of extracellular ATP and adenosine and thus their availability at purinergic receptors: a) ecto-nucleoside triphosphate diphosphohydrolases, b) ecto-nucleotide pyrophosphatase/phosphodiesterase, c) ecto-5’­nucleotidase, and d) alkaline phosphatase. The Purinome is the molecular complex implicated in the triggering, maintenance and termination of the purinergic signalling which is the group of biological effects (autocrine or paracrine) produced by the purines and pyrimidines acting as a extracellular ligand and it is form by the ligands, receptors, transporters/channels and degrading enzymes. Previous studies of our group demonstrate expression of some of the most important ecto-nucleotidases in the male reproductive tract, but the role of the ecto-nucleotidases in the female reproductive system in health and disease were unknown. In this thesis, we have studied both aspects. We studied the gen and protein expression and the enzyme histochemistry of the main ecto-nucleotidases in the female reproductive tract, in the human cyclic (proliferative and secretory) and atrophic endometria, and in endometrial cancer. In addition, we have set up two cellular models (human endometrial adenocarcinoma cell lines and primary cultures of endometrial stromal cells) to study the regulation of the ecto-nucleotidases. Our results show expression and activity of these enzymes in the endometrium with marked changes along the cycle and after the menopause, indicating a hormonal regulation. Also we have detected an elevated expression of two of the ecto-nucleotidases that directly control the adenosine extracellular levels (CD39 and CD73), in the endometrial cancer type I and type II. Our results support the growing body of evidence that CD39 is a potential therapeutic target for cancer immunotherapy. And also reinforce the relevance of CD73 in tumors. We suggest that the ecto­nucleotidases can be used as a biological markers of fertility with prognostic and diagnostic utility in endometrial cancer

High expression of ecto-nucleotidases CD39 and CD73 in human endometrial tumors

One of the strategies used by tumors to evade immunosurveillance is the accumulation of extracellular adenosine, which has immunosupressive and tumor promoting effects. The study of the mechanisms leading to adenosine formation at the tumor interstitium are therefore of great interest in oncology. The dominant pathway generating extracellular adenosine in tumors is the dephosphorylation of ATP by ecto-nucleotidases. Two of these enzymes acting sequentially, CD39 and CD73, efficiently hydrolyze extracellular ATP to adenosine. They have been found to play a crucial role in a variety of tumors, but there were no data concerning endometrial cancer, the most frequent of the invasive tumors of the female genital tract. The aim of the present work is to study the expression of CD39 and CD73 in human endometrial cancer. We have analyzed protein and gene expression, as well as enzyme activity, in type I endometrioid adenocarcinomas and type II serous adenocarcinomas and their nonpathological endometrial counterparts. High levels of both enzymes were found in tumor samples, with significantly increased expression of CD39 in type II serous tumors, which also coincided with the higher tumor grade. Our results reinforce the involvement of the adenosinergic system in cancer, emphasizing the relevance of ecto-nucleotidases as emerging therapeutic targets in oncology

Vascular disease in COPD: systemic and pulmonary expression of PARC (Pulmonary and Activation-Regulated Chemokine)

Introduction: The role of Pulmonary and Activation-Regulated Chemokine (PARC) in the physiopathology of Chronic Obstructive Pulmonary Disease (COPD) is not fully understood. The aim of the present study is to analyze the expression of PARC in lung tissue and its relationship with the vascular remodeling of the systemic and pulmonary arteries of COPD subjects. Methods: To achieve this objective, protein and gene expression experiments, together with ELISA assays, were performed on the lung tissue, intercostal arteries and serum samples from COPD patients, non-obstructed smokers (NOS) and never-smokers (NS). Results: A total of 57 subjects were included in the analysis (23 COPD, 18 NOS and 16 NS). In the comparisons between groups, a significantly increased lung protein expression of PARC was observed in the COPD group compared to the NOS group (1.96±0.22 vs. 1.29±0.27, P-adjusted = 0.038). PARC was located predominantly in the smooth muscle cells of the remodeled pulmonary muscular arteries and the macrophage-rich area of the alveolar parenchyma. No differences were detected in PARC gene expression analyses. The protein content of PARC in the intercostal arteries were similar between groups, though little remodeling was observed in these arteries. Circulating levels of PARC were numerically higher in patients with COPD compared to NOS and NS. Conclusion: The results of the present study suggest an increased lung protein expression of PARC in COPD subjects. This protein was mainly localized in the smooth muscle cells of the pulmonary muscular arteries and was associated with the severity of intimal thickening, indicating its possible role in this remodeling process

Vascular disease in COPD: systemic and pulmonary expression of PARC (Pulmonary and Activation-Regulated Chemokine)

Introduction: The role of Pulmonary and Activation-Regulated Chemokine (PARC) in the physiopathology of Chronic Obstructive Pulmonary Disease (COPD) is not fully understood. The aim of the present study is to analyze the expression of PARC in lung tissue and its relationship with the vascular remodeling of the systemic and pulmonary arteries of COPD subjects. Methods: To achieve this objective, protein and gene expression experiments, together with ELISA assays, were performed on the lung tissue, intercostal arteries and serum samples from COPD patients, non-obstructed smokers (NOS) and never-smokers (NS). Results: A total of 57 subjects were included in the analysis (23 COPD, 18 NOS and 16 NS). In the comparisons between groups, a significantly increased lung protein expression of PARC was observed in the COPD group compared to the NOS group (1.96±0.22 vs. 1.29±0.27, P-adjusted = 0.038). PARC was located predominantly in the smooth muscle cells of the remodeled pulmonary muscular arteries and the macrophage-rich area of the alveolar parenchyma. No differences were detected in PARC gene expression analyses. The protein content of PARC in the intercostal arteries were similar between groups, though little remodeling was observed in these arteries. Circulating levels of PARC were numerically higher in patients with COPD compared to NOS and NS. Conclusion: The results of the present study suggest an increased lung protein expression of PARC in COPD subjects. This protein was mainly localized in the smooth muscle cells of the pulmonary muscular arteries and was associated with the severity of intimal thickening, indicating its possible role in this remodeling process