Identificació de gens associats a la transició epiteli-mesènquima induïda pels factors de transcripció E47 i Snail en les cèl·lules epitelials MDCK : mecanisme de l'activació transcripcional de MMP-9 i Id-1 induïda per E47 i Snail /

Consultable des del TDXTítol obtingut de la portada digitalitzadaEls carcinomes són tumors d'origen epitelial que constitueixen aproximadament el 90% dels tumors humans. La progressió tumoral implica diferents etapes: creixement del tumor primari, invasió local, intravasació, extravasació i proliferació de les cèl·lules tumorals en un nou òrgan on formen un tumor secundari o metàstasi. Són, precisament, les metàstasis la principal causa de mort dels malalts de càncer. La invasió local dels carcinomes requereix la pèrdua de l'expressió o de la funció de la molècula d'adhesió cadherina E, la qual és un supressor d'invasió. A més, el procés d'invasió també va acompanyat de la pèrdua d'altres marcadors epitelials, de l'adquisició de marcadors mesenquimals i de l'augment de les propietats migratòries i invasives. Aquests canvis tenen un gran paral·lelisme amb la conversió fenotípica que té lloc durant el desenvolupament embrionari i que s'anomena transició epiteli-mesènquima (TEM). El principal mecanisme que regula el silenciament de la cadherina E és la repressió transcripcional, i recentment s'han caracteritzat diversos factors de transcripció que a través de la seva interacció amb les caixes E (de seqüència CANNTG) del promotor de la cadherina E reprimeixen la seva expressió: Snail, Slug, E47, Twist, ZEB-1 i ZEB-2. L'expressió estable de Snail o E47 en les cèl·lules epitelials Madin Darby Canine Kidney (MDCK) indueix un procés de TEM complet, però no es coneix el mecanisme. Moltes de les alteracions que es donen en la TEM poden ser explicades com a conseqüència de la repressió de la cadherina E, però altres events cel·lulars, independents de la dissociació cel·lular, contribuirien també a la iniciació i completació del procés. Per aquest motiu, en aquesta Tesi s'ha realitzat l'anàlisi de l'expressió gènica diferencial mitjançant RAP-PCR i microarrays de cDNA per identificar gens implicats en el procés de TEM induït per Snail o E47. Així, s'han trobat gens que codifiquen per proteïnes relacionades amb diverses funcions cel·lulars com cicle cel·lular, apoptosi, metabolisme o senyalització, però el grup majoritari és el de gens implicats en migració i invasió (adhesió cel·lular, citoesquelet, matriu extracel·lular -MEC- i proteases de MEC). Aquest estudi mostra a més que l'expressió estable de Snail o E47 en les cèl·lules MDCK indueix programes genètics en part comuns i en part específics, suggerint el paper diferencial d'aquests dos factors de transcripció en la progressió tumoral. Per altra banda, s'ha estudiat el mecanisme que regula la sobreexpressió de la metal·loproteasa MMP-9 i del factor de transcripció Id-1 que es dóna en la TEM induïda tant per Snail com per E47. L'activació transcripcional de MMP-9 és induïda per Snail i E47 de forma indirecta i mediada per altres factors de transcripció tals com Ets-1 i Sp1 que s'uneixen a la regió proximal del promotor formant un multicomplexe, i NFkB/p65 que interacciona amb una regió més distal. En canvi, l'activació transcripcional d'Id-1 és regulada principalment a través de la segona caixa E del promotor humà que interacciona amb E47. Pel que fa a Snail, no hem pogut confirmar si s'uneix directament a aquest element o indueix l'expressió d'un altre factor de transcripció capaç d'unir-s'hi. També és important la caixa GC adjacent a la caixa E que recluta Sp1. Tant l'activació de MMP-9 com d'Id-1 és regulada per la via Erk/MAPK (activada per Snail i E47) que fosforila almenys Sp1. Aquests resultats juntament amb l'expressió coneguda de Snail i E47 en línies cel·lulars de carcinoma podrien explicar la sobreexpressió de MMP-9 i Id-1 en molts tumors.Carcinomas are tumors of epithelial origin that comprise approximately 90% of human tumors. Tumor progression is a multistep process: growth of primary tumor, local invasion, intravasation, extravasation and proliferation of malignant cells in a new organ where they form a secondary tumor or metastasis. Metastasis are the main cause of death of cancer patients. Local invasion of carcinomas requires loss of E-cadherin expression or function, which is an adhesion molecule and a well established invasion supressor. In addition, invasion process is accompanied by loss of other epithelial molecules, acquisition of mesenquimal markers and gain of migratory and invasive properties. These changes have many parallels with the phenotypic conversion that takes place during embryonic development known as epithelial-mesenquimal transition (EMT). The main mechanism that regulates E-cahderin silence is transcripcional repression, and recently several transcription factors have been characterized as E-cadherin repressors through their interaction with the E-boxes (of sequence CANNTG) of the promoter: Snail, Slug, E47, Twist, ZEB-1 and ZEB-2. Stable expression of Snail or E47 in Madin Darby Canine Kidney (MDCK) epithelial cells induce a complete EMT, but the mechanism that govern it is not known yet. Some of the alterations that occur during EMT can be explained as a consequence of E-cadherin repression, but other cellular events, independent from cellular dissociation, may contribute to the initiation and completion of the process. For this reason, in this Thesis we made a differential expression analysis by RAP-PCR and cDNA microarrays to identify genes implicated in Snail and E47 induced EMT. Thus we found genes related to different cellular functions such as cellular cycle, apoptosis, metabolism and signaling, but the great group was composed by genes implicated in migration and invasion (cellular adhesion, cytoskeleton, extracellular matrix -EMC- and EMC proteases). This study shows that Snail or E47 expression in MDCK cells induce common and specific genetic programs, suggesting a differential role for these transcription factors in tumor progression. On the other hand, we studied the mechanism that regulates metalloprotease MMP-9 and transcription factor Id-1 upregulation in Snail and E47 induced EMT. Snail and E47 induced activation of MMP-9 is indirect and mediated by other transcription factors such as Ets-1 and Sp1 that bind to the proximal region of promoter forming a multicomplex, and NFkB/p65 that interacts with a more distal region. On the contrary, Id-1 transcriptional activation is regulated mainly through the second E-box of human promoter that recruits E47. At the moment, we have not been able to confirm whether Snail binds directly to this element or it induces the expression of another factor that interacts with the E-box. It is also important the GC-box adjacent to this E-box that binds Sp1. Either MMP-9 or Id-1 activation are regulated by Erk/MAPK pathway(activated by Snail and E47) that phosphorilates at least Sp1. These results together with known expression of Snail and E47 in carcinoma cell lines may explain MMP-9 and Id-1 upregulation in tumors

The epigenetic landscape of Alu repeats delineates the structural and functional genomic architecture of colon cancer cells

Cancer cells exhibit multiple epigenetic changes with prominent local DNA hypermethylation and widespread hypomethylation affecting large chromosomal domains. Epigenome studies often disregard the study of repeat elements owing to technical complexity and their undefined role in genome regulation. We have developed NSUMA (Next-generation Sequencing of UnMethylated Alu), a cost-effective approach allowing the unambiguous interrogation of DNA methylation in more than 130,000 individual Alu elements, the most abundant retrotransposon in the human genome. DNA methylation profiles of Alu repeats have been analyzed in colon cancers and normal tissues using NSUMA and whole-genome bisulfite sequencing. Normal cells show a low proportion of unmethylated Alu (1%-4%) that may increase up to 10-fold in cancer cells. In normal cells, unmethylated Alu elements tend to locate in the vicinity of functionally rich regions and display epigenetic features consistent with a direct impact on genome regulation. In cancer cells, Alu repeats are more resistant to hypomethylation than other retroelements. Genome segmentation based on high/low rates of Alu hypomethylation allows the identification of genomic compartments with differential genetic, epigenetic, and transcriptomic features. Alu hypomethylated regions show low transcriptional activity, late DNA replication, and its extent is associated with higher chromosomal instability. Our analysis demonstrates that Alu retroelements contribute to define the epigenetic landscape of normal and cancer cells and provides a unique resource on the epigenetic dynamics of a principal, but largely unexplored, component of the primate genome

Quantification of unmethylated Alu (QUAlu): a tool to assess global hypomethylation in routine clinical samples

Hypomethylation of DNA is a hallmark of cancer and its analysis as tumor biomarker has been proposed, but its determination in clinical settings is hampered by lack of standardized methodologies. Here, we present QUAlu (Quantification of Unmethylated Alu), a new technique to estimate the Percentage of UnMethylated Alu (PUMA) as a surrogate for global hypomethylation. QUAlu consists in the measurement by qPCR of Alu repeats after digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation. QUAlu performance has been evaluated for reproducibility, trueness and specificity, and validated by deep sequencing. As a proof of use, QUAlu has been applied to a broad variety of pathological examination specimens covering five cancer types. Major findings of the preliminary application of QUAlu to clinical samples include: (1) all normal tissues displayed similar PUMA; (2) tumors showed variable PUMA with the highest levels in lung and colon and the lowest in thyroid cancer; (3) stools from colon cancer patients presented higher PUMA than those from control individuals; (4) lung squamous cell carcinomas showed higher PUMA than lung adenocarcinomas, and an increasing hypomethylation trend associated with smoking habits. In conclusion, QUAlu is a simple and robust method to determine Alu hypomethylation in human biospecimens and may be easily implemented in research and clinical settings

Overlapping DNA methylation dynamics in mouse intestinal cell differentiation and early stages of malignant progression

Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high), differentiated cells (EphB2negative), ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart

Effects of pre-operative isolation on postoperative pulmonary complications after elective surgery: an international prospective cohort study an international prospective cohort study

We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05–1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4–7 days or ≥ 8 days of 1.25 (1.04–1.48), p = 0.015 and 1.31 (1.11–1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care. We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05–1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4–7 days or ≥ 8 days of 1.25 (1.04–1.48), p = 0.015 and 1.31 (1.11–1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care