Estudi dels exosomes com a mecanisme de comunicació intercel·lular en patologies pancreàtiques

[cat] La pancreatitis aguda és un procés inflamatori del pàncrees desencadenat per una activació inapropiada dels enzims pancreàtics. En la seva variant més severa, la malaltia progressa acompanyada d’afectacions no només a nivell local sino també sistèmic, essent la síndrome de resposta inflamatòria sistèmica (SIRS) i el dany pulmonar que se’n deriva una de les afectacions més primerenques i la principal causa de mortalitat dels pacients. Diferents estudis han identificat l’increment en l’expressió i secreció de múltiples mediadors inflamatoris, generats tant a nivell local com sistèmic. No obstant, el mecanisme patogènic de la malaltia encara es desconeix, de manera que en les fases inicials de la malaltia no és possible predir si un pacient desenvoluparà una variant lleu o severa de pancreatitis. A llarg dels últims anys, però, el descobriment dels exosomes ha revolucionat el camp de la comunicació intercel·lular. Aquestes vesícules extracel·lulars són capaces de transferir proteïnes, miRNAs i mRNAs de manera encapsulada, tot protegint-los del medi extracel·lular i transportant-los fins a òrgans i indrets del cos allunyats del seu focus de síntesi. En aquesta tesi, utilitzant un model experimental de pancreatitis aguda induïda per taurocolat sòdic en rata, hem avaluat el paper dels exosomes en la propagació de la resposta inflamatòria sistèmica. Els resultats obtinguts indiquen que posteriorment a la inducció de la malaltia hi ha un increment de la concentració d’exosomes circulants, els quals presenten un perfil proteic diferent dels exosomes circulants que hi ha en condicions control. Mitjançant experiments de marcatge i seguiment d’aquestes vesícules hem determinat que els exosomes circulants són capaços de creuar la barrera alveolocapil·lar i d’arribar fins l’espai aveolar, on són captats pels macròfags pulmonars residents. A més, en experiments in vitro es demostra com els exosomes secretats en condicions inflamatòries activen i polaritzen els macròfags cap a un fenotip de tipus proinflamatori. L’anàlisi proteòmic dels exosomes circulants mostra com, durant la pancreatitis aguda severa, aquestes vesícules presenten una gran quantitat de proteïnes sintetitzades majoritàriament pel teixit hepàtic, suggerint que el fetge podria estar secretant nous exosomes i alliberant-los al torrent sanguini en el transcurs de la malaltia. Aquesta hipòtesi va ser reforçada amb nous experiments de marcatge i seguiment dels exosomes, gràcies als quals es va observar com els exosomes generats a la cavitat peritoneal durant la pancreatitis aguda severa són filtrats i retinguts pel teixit hepàtic. En conjunt, els resultats obtinguts reforcen la idea que el fetge tindria un paper central durant la pancreatits aguda i que cal tenir-lo en compte per comprendre quin mecanisme regula l’extensió de la resposta inflamatòria en el transcurs de la malaltia. En tot aquest procés, a més, la lectina REG3-beta podria tenir un paper destacat. Aquesta proteïna d’estrès és una de les més sintetitzades pel pàncrees durant la pancreatitis aguda, la concentració de la qual pot arribar a augmentar fins a 100 vegades durant la fase aguda de la malaltia. La seva funció específica, però, a dia d’avui encara no s’ha descrit ben bé, tot i que se li han atribuït certes propietats antiinflamatòries, mitogèniques i d’opsonització de bacteris. En aquesta tesi es demostra com REG3-beta és capaç d’unir-se a la membrana dels exosomes circulants. A més, hem caracteritzat com aquesta unió depèn del seu domini lectina, i que comporta un bloqueig de la captació de les vesícules per part de les seves cèl·lules diana, impedint que aquestes alliberin els seus mediadors i duguin a terme la seva funció.[eng] Severe acute pancreatitis is an inflammatory disease of the pancreas which progresses accompanied by local and systemic alterations, being lung damage and SIRS the leading causes of death. Despite multiple inflammatory mediators have been detected and described, the pathogenic mechanism of the disease is still unknown. However, over the last few years the discovery of exosomes has revolutionized the field of intercellular communication, since they are able to transfer mediators such as proteins, miRNAs and mRNAs to distant organs. In this work, using an experimental model of taurocholate-induced acute pancreatitis in rats, we evaluated the role of exosomes in the extent of the systemic inflammatory response. Our results indicate that during acute pancreatitis, there is an increase in the concentration of circulating exosomes. By fluorescent exosome tracking in vivo experiments we demonstrate that circulating exosomes are able to cross the alveolocapillary barrier, be phagocyted by alveolar macrophages and polarize them to a proinflammatory phenotype in vitro. The proteomic analysis of circulating exosomes showed that, during acute pancreatitis, exosomes present a large number of proteins synthesized mainly by the hepatic tissue, suggesting that the liver is secreting new exosomes and releasing them into the bloodstream during the course of the disease. This hypothesis was reinforced with new tracking experiments, where we observed that that exosomes generated in the peritoneal cavity during severe acute pancreatitis are filtered and retained by the liver tissue. Overall, these results reveal that the liver would play a central role during severe acute pancreatitis and that it should be taken into account to understand which mechanism regulates the extent of the inflammatory response. The lectine REG3-beta could also play a prominent role in all this process. REG3-beta is the main protein synthesised by the pancreas during acute pancreatitis, the concentration of which can increase up to 100 times. Its specific function has not yet been described, although certain anti-inflammatory, mitogenic and opsonising properties have been attributed to it. In this thesis we demonstrate that REG3-beta binds to the membrane of circulating exosomes and that this interaction depends on its lectin domain. Consequently, this interaction blocks the exosome capture by target cells, preventing them from releasing their mediators and blocking their function

New roles for corticosteroid binding globulin and opposite expression profiles in lung and liver.

Corticosteroid-binding globulin (CBG) is the specific plasma transport glycoprotein for glu- cocorticoids. Circulating CBG is mainly synthesized in liver but, its synthesis has been located also in other organs as placenta, kidney and adipose tissue with unknown role. Using an experimental model of acute pancreatitis in cbg -/- mice we investigated whether changes in CBG affect the progression of the disease as well as the metabolism of gluco- corticoids in the lung. Lack of CBG does not modify the progression of inflammation associ- ated to pancreatitis but resulted in the loss of gender differences in corticosterone serum levels. In the lung, CBG expression and protein level were detected, and it is noteworthy that these showed a sexual dimorphism opposite to the liver, i.e. with higher levels in males. Reduced expression of 11 β -HSD2, the enzyme involved in the deactivation of corticoste- rone, was also observed. Our results indicate that, in addition to glucocorticoids transporter, CBG is involved in the gender differences observed in corticosteroids circulating levels and plays a role in the local regulation of corticosteroids availability in organs like lung

Approaches to functionally validate candidate genetic variants involved in colorectal cancer predisposition

Most next generation sequencing (NGS) studies identified candidate genetic variants predisposing to colorectal cancer (CRC) but do not tackle its functional interpretation to unequivocally recognize a new hereditary CRC gene. Besides, germline variants in already established hereditary CRC-predisposing genes or somatic variants share the same need when trying to categorize those with relevant significance. Functional genomics approaches have an important role in identifying the causal links between genetic architecture and phenotypes, in order to decipher cellular function in health and disease. Therefore, functional interpretation of identified genetic var iants by NGS platforms is now essential. Available approaches nowadays include bioinformatics, cell and mo lecular biology and animal models. Recent advances, such as the CRISPR-Cas9, ZFN and TALEN systems, have been already used as a powerful tool with this objective. However, the use of cell lines is of limited value due to the CRC heterogeneity and its close interaction with microenvironment. Access to tridimensional cultures or organoids and xenograft models that mimic the in vivo tissue architecture could revolutionize functional ana lysis. This review will focus on the application of state-of-the-art functional studies to better tackle new genes involved in germline predisposition to this neoplasm

Using linkage studies combined with whole-exome sequencing to identify novel candidate genes for familial colorectal cancer

Colorectal cancer (CRC) is a complex disorder for which the majority of the underlying germline predisposition factors remain still unidentified. Here, we combined whole‐exome sequencing (WES) and linkage analysis in families with multiple relatives affected by CRC to identify candidate genes harboring rare variants with potential high‐penetrance effects. Forty‐seven affected subjects from 18 extended CRC families underwent WES. Genome‐wide linkage analysis was performed under linear and exponential models. Suggestive linkage peaks were identified on chromosomes 1q22-q24.2 (maxSNP = rs2134095; LODlinear = 2.38, LODexp = 2.196), 7q31.2-q34 (maxSNP = rs6953296; LODlinear = 2.197, LODexp = 2.149) and 10q21.2-q23.1 (maxSNP = rs1904589; LODlinear = 1.445, LODexp = 2.195). These linkage signals were replicated in 10 independent sets of random markers from each of these regions. To assess the contribution of rare variants predicted to be pathogenic, we performed a family‐based segregation test with 89 rare variants predicted to be deleterious from 78 genes under the linkage intervals. This analysis showed significant segregation of rare variants with CRC in 18 genes (weighted p‐value > 0.0028). Protein network analysis and functional evaluation were used to suggest a plausible candidate gene for germline CRC predisposition. Etiologic rare variants implicated in cancer germline predisposition may be identified by combining traditional linkage with WES data. This approach can be used with already available NGS data from families with several sequenced members to further identify candidate genes involved germline predisposition to disease. This approach resulted in one candidate gene associated with increased risk of CRC but needs evidence from further studies

Germline Mutations in FAF1 Are Associated With Hereditary Colorectal Cancer

Background & aims: A significant proportion of colorectal cancer (CRC) cases have familial aggregation but little is known about the genetic factors that contribute to these cases. We performed an exhaustive functional characterization of genetic variants associated with familial CRC. Methods: We performed whole-exome sequencing analyses of 75 patients from 40 families with a history of CRC (including early-onset cases) of an unknown germline basis (discovery cohort). We also sequenced specific genes in DNA from an external replication cohort of 473 families, including 488 patients with colorectal tumors that had normal expression of mismatch repair proteins (validation cohort). We disrupted the Fas-associated factor 1 gene (FAF1) in DLD-1 CRC cells using CRISPR/Cas9 gene editing; some cells were transfected with plasmids that express FAF1 missense variants. Cells were analyzed by immunoblots, quantitative real-time polymerase chain reaction, and functional assays monitoring apoptosis, proliferation, and assays for Wnt signaling or nuclear factor (NF)-kappa-B activity. Results: We identified predicted pathogenic variant in the FAF1 gene (c.1111G>A; p.Asp371Asn) in the discovery cohort; it was present in 4 patients of the same family. We identified a second variant in FAF1 in the validation cohort (c.254G>C; p.Arg85Pro). Both variants encoded unstable FAF1 proteins. Expression of these variants in CRC cells caused them to become resistant to apoptosis, accumulate beta-catenin in the cytoplasm, and translocate NF-kappa-B to the nucleus. Conclusions: In whole-exome sequencing analyses of patients from families with a history of CRC, we identified variants in FAF1 that associate with development of CRC. These variants encode unstable forms of FAF1 that increase resistance of CRC cells to apoptosis and increase activity of beta-catenin and NF-kappa-B

Colorectal cancer genetic variants are also associated with serrated polyposis syndrome susceptibility

Background: Serrated polyposis syndrome (SPS) is a clinical entity characterised by large and/ormultiple serrated polyps throughout the colon and increased risk for colorectal cancer (CRC). The basis for SPS genetic predisposition is largely unknown. Common, low-penetrance genetic variants have been consistently associated with CRC susceptibility, however, their role in SPS genetic predisposition has not been yet explored. Objective: The aim of this study was to evaluate if common, low-penetrance genetic variants for CRC risk are also implicated in SPS genetic susceptibility. Methods: A case-control study was performed in 219 SPS patients and 548 asymptomatic controls analysing 65 CRC susceptibility variants. A risk prediction model for SPS predisposition was developed. Results: Statistically significant associations with SPS were found for seven genetic variants (rs4779584-GREM1, rs16892766-EIF3H, rs3217810-CCND2, rs992157-PNKD1/TMBIM1, rs704017-ZMIZ1, rs11196172-TCF7L2, rs6061231-LAMA5). The GREM1 risk allele was remarkably over-represented in SPS cases compared with controls (OR=1.573, 1.21-2.04, p value=0.0006). A fourfold increase in SPS risk was observed when comparing subjects within the highest decile of variants (≥65) with those in the first decile (≤50). Conclusions: Genetic variants for CRC risk are also involved in SPS susceptibility, being the most relevant ones rs4779584-GREM1, rs16892766-EIF3H and rs3217810-CCND2

Integrated Analysis of Germline and Tumor DNA Identifies New Candidate Genes Involved in Familial Colorectal Cancer

Colorectal cancer (CRC) shows aggregation in some families but no alterations in the known hereditary CRC genes. We aimed to identify new candidate genes which are potentially involved in germline predisposition to familial CRC. An integrated analysis of germline and tumor whole-exome sequencing data was performed in 18 unrelated CRC families. Deleterious single nucleotide variants (SNV), short insertions and deletions (indels), copy number variants (CNVs) and loss of heterozygosity (LOH) were assessed as candidates for first germline or second somatic hits. Candidate tumor suppressor genes were selected when alterations were detected in both germline and somatic DNA, fulfilling Knudson's two-hit hypothesis. Somatic mutational profiling and signature analysis were also performed. A series of germline-somatic variant pairs were detected. In all cases, the first hit was presented as a rare SNV/indel, whereas the second hit was either a different SNV (3 genes) or LOH affecting the same gene (141 genes). BRCA2, BLM, ERCC2, RECQL, REV3L and RIF1 were among the most promising candidate genes for germline CRC predisposition. The identification of new candidate genes involved in familial CRC could be achieved by our integrated analysis. Further functional studies and replication in additional cohorts are required to confirm the selected candidates

New roles for corticosteroid binding globulin and opposite expression profiles in lung and liver.

Corticosteroid-binding globulin (CBG) is the specific plasma transport glycoprotein for glu- cocorticoids. Circulating CBG is mainly synthesized in liver but, its synthesis has been located also in other organs as placenta, kidney and adipose tissue with unknown role. Using an experimental model of acute pancreatitis in cbg -/- mice we investigated whether changes in CBG affect the progression of the disease as well as the metabolism of gluco- corticoids in the lung. Lack of CBG does not modify the progression of inflammation associ- ated to pancreatitis but resulted in the loss of gender differences in corticosterone serum levels. In the lung, CBG expression and protein level were detected, and it is noteworthy that these showed a sexual dimorphism opposite to the liver, i.e. with higher levels in males. Reduced expression of 11 β -HSD2, the enzyme involved in the deactivation of corticoste- rone, was also observed. Our results indicate that, in addition to glucocorticoids transporter, CBG is involved in the gender differences observed in corticosteroids circulating levels and plays a role in the local regulation of corticosteroids availability in organs like lung