BAP1 Malignant Pleural Mesothelioma Mutations in Caenorhabditis elegans Reveal Synthetic Lethality between ubh-4/BAP1 and the Proteasome Subunit rpn-9/PSMD13.

The deubiquitinase BAP1 (BRCA1-associated protein 1) is associated with BAP1 tumor predisposition syndrome (TPDS). BAP1 is a tumor suppressor gene whose alterations in cancer are commonly caused by gene mutations leading to protein loss of function. By CRISPR-Cas, we have generated mutations in ubh-4, the BAP1 ortholog in Caenorhabditis elegans, to model the functional impact of BAP1 mutations. We have found that a mimicked BAP1 cancer missense mutation (UBH-4 A87D; BAP1 A95D) resembles the phenotypes of ubh-4 deletion mutants. Despite ubh-4 being ubiquitously expressed, the gene is not essential for viability and its deletion causes only mild phenotypes without affecting 20S proteasome levels. Such viability facilitated an RNAi screen for ubh-4 genetic interactors that identified rpn-9, the ortholog of human PSMD13, a gene encoding subunit of the regulatory particle of the 26S proteasome. ubh-4[A87D], similarly to ubh-4 deletion, cause a synthetic genetic interaction with rpn-9 inactivation affecting body size, lifespan, and the development of germ cells. Finally, we show how ubh-4 inactivation sensitizes animals to the chemotherapeutic agent Bortezomib, which is a proteasome inhibitor. Thus, we have established a model to study BAP1 cancer-related mutations in C. elegans, and our data points toward vulnerabilities that should be studied to explore therapeutic opportunities within the complexity of BAP1 tumors.

Efficacy of CDK4/6 inhibitors in preclinical models of malignant pleural mesothelioma.

BACKGROUND: There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy. METHODS: We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM. RESULTS: Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models evaluated. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest, thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice. CONCLUSIONS: Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

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.

Imbalance in the Expression of Genes Associated with Purinergic Signalling in the Lung and Systemic Arteries of COPD Patients.

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.

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.

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.

Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the "adenosine hypothesis".

Schizophrenia (SZ) is a major chronic neuropsychiatric disorder characterized by a hyperdopaminergic state. The hypoadenosinergic hypothesis proposes that reduced extracellular adenosine levels contribute to dopamine D2 receptor hyperactivity. ATP, through the action of ecto-nucleotidases, constitutes a main source of extracellular adenosine. In the present study, we examined the activity of ecto-nucleotidases (NTPDases, ecto-5'-nucleotidase, and alkaline phosphatase) in the postmortem putamen of SZ patients (n = 13) compared with aged-matched controls (n = 10). We firstly demonstrated, by means of artificial postmortem delay experiments, that ecto-nucleotidase activity in human brains was stable up to 24 h, indicating the reliability of this tissue for these enzyme determinations. Remarkably, NTPDase-attributable activity (both ATPase and ADPase) was found to be reduced in SZ patients, while ecto-5'-nucleotidase and alkaline phosphatase activity remained unchanged. In the present study, we also describe the localization of these ecto-enzymes in human putamen control samples, showing differential expression in blood vessels, neurons, and glial cells. In conclusion, reduced striatal NTPDase activity may contribute to the pathophysiology of SZ, and it represents a potential mechanism of adenosine signalling impairment in this illness.

Ecto-nucleotidases distribution in human cyclic and postmenopausic endometrium.

Extracellular ATP and its hydrolysis product, adenosine, acting through specific receptors collectively named purinergic receptors, regulate female fertility by influencing the endometrial fluid microenvironment. There are four major groups of ecto-nucleotidases that control the levels of extracellular ATP and adenosine and thus their availability at purinergic receptors: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-nucleotide pyrophosphatase/phospho-diesterases (E-NPPs), ecto-5'-nucleotidase (5'NT), and alkaline phosphatases (APs). The aim of the present work is to characterize the expression and distribution of ecto-nucleotidases in human endometrium along the menstrual cycle and after menopause, to evaluate their potential utility as fertility markers. We examined proliferative, secretory and atrophic endometria from women without endometrial pathology undergoing hysterectomy. We show that the ecto-nucleotidases are mainly present at endometrial epithelia, both luminal and glandular, and that their expression fluctuates along the cycle and also changes after menopause. An important result was identifying NPP3 as a new biological marker of tubal metaplasia. Our results emphasize the relevance of the study of purinergic signaling in human fertility.

Ectonucleotidases in the digestive system: focus on NTPDase3 localization.

Extracellular nucleotides and adenosine are biologically active molecules that bind members of the P2 and P1 receptor families, respectively. In the digestive system, these receptors modulate various functions, including salivary, gastric, and intestinal epithelial secretion and enteric neurotransmission. The availability of P1 and P2 ligands is modulated by ectonucleotidases, enzymes that hydrolyze extracellular nucleotides into nucleosides. Nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5'-nucleotidase are the dominant ectonucleotidases at physiological pH. While there is some information about the localization of ecto-5'-nucleotidase and NTPDase1 and -2, the distribution of NTPDase3 in the digestive system is unknown. We examined the localization of these ectonucleotidases, with a focus on NTPDase3, in the gastrointestinal tract and salivary glands. NTPDase1, -2, and -3 are responsible for ecto-ATPase activity in these tissues. Semiquantitative RT-PCR, immunohistochemistry, and in situ enzyme activity revealed the presence of NTPDase3 in some epithelial cells in serous acini of salivary glands and mucous acini and duct cells of sublingual salivary glands, in cells from the stratified esophageal and forestomach epithelia, and in some enteroendocrine cells of the gastric antrum. Interestingly, NTPDase2 and ecto-5'-nucleotidase are coexpressed with NTPDase3 in salivary gland cells and stratified epithelia. In the colon, neurons express NTPDase3 and glial cells express NTPDase2. Ca(2+) imaging experiments demonstrate that NTPDases regulate P2 receptor ligand availability in the enteric nervous system. In summary, the specific localization of NTPDase3 in the digestive system suggests functional roles of the enzyme, in association with NTPDase2 and ecto-5'-nucleotidase, in epithelial functions such as secretion and in enteric neurotransmission.

High expression and activity of ecto-5'-nucleotidase/CD73 in the male murine reproductive tract.

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, steroidogenesis, and maintenance of fluid composition. Interestingly, adenosine might act as a key capacitative effector for mammalian spermatozoa to acquire the capacity for fertilisation. Extracellular nucleotide levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family regroups the most abundant and effective enzymes to hydrolyse ATP and ADP to AMP in physiological conditions. In the male reproductive tract three members of this family have been indentified: NTPDase1, NTPDase2 and NTPDase3 (Martín-Satué et al. in Histochem Cell Biol 131:615-628, 2009). The purpose of the present study was to characterize in the male reproductive tract the expression profile of the main enzyme responsible for the generation of adenosine from AMP, namely the ecto-5'-nucleotidase (CD73). The enzyme was identified by immunological techniques and by in situ enzymatic assays, including inhibition experiments with alpha,beta-methylene-ADP, a specific CD73 inhibitor. High levels of ecto-5'-nucleotidase were detected in testes in association with both germinal and somatic cells, in smooth muscle cells throughout the tract, in secretory epithelia from exocrine glands, and remarkably, in principal cells of epididymis, where co-localization with NTPDase3 was found. The relevance of this co-expression on nucleotide hydrolysis in these cells directly involved in the control of sperm fluid composition was addressed biochemically. This study suggests close regulation of extracellular nucleoside and nucleotide levels in the genital tract by ecto-5'-nucleotidase that, in concurrence with NTPDases, may impact male fertility.