Impact of Lipoprotein(a) on Macrovascular Complications of Diabetes in a Multiethnic Population in the French Amazon.

Background and Aims: In French Guiana, the prevalence of diabetes is around 10%, and cardio and neurovascular pathologies are the first medical cause of early mortality. Lipoprotein(a) (Lp(a)) is described in the literature as a risk factor independent of other cardiovascular risk factors, but there are important interindividual differences, especially according to ethnicity. The objective of this study was to investigate the association of Lp(a) and macrovascular complications in a multiethnic population of patients with diabetes in the French Amazon. Materials and Methods: Since May 2019, 1243 patients were screened 806 of whom had Lp(a) determination. We compared the prevalence of macrovascular complications in three groups according to Lp(a) concentration: between 0 and 75 mg/mL, between 76 and 300 mg/mL, and >300 mg/mL. Results: 712 patients in the study had type 2 diabetes (88.34% of the sample). A history of hypertension was significantly associated with greater Lp(a) levels. Lp(a) concentration was greater among Creole ethnic groups. No association was found between Lp(a) levels and macrovascular complications in the Lp(a) > 300 mg/mL group. Conclusions: These results do not replicate findings in mostly Caucasian populations suggesting that the Lp(a) threshold for, or the link with, cardiovascular risk may be different given the predominantly African origin of the French Guianese population. Further studies should study genetic polymorphisms in our population.

Pre-malignant transformation by senescence evasion is prevented by the PERK and ATF6alpha branches of the Unfolded Protein Response.

The incidence of carcinomas highly increases with age. However, the initial steps of the age-related molecular carcinogenic processes remain poorly characterized. We previously showed that normal human epidermal keratinocytes spontaneously and systematically escape from senescence to give rise to preneoplastic emerging cells through a process called post-senescence neoplastic emergence (PSNE). To identify molecular pathways involved in the switch from senescence to pre-transformation, we performed Connectivity Map analyses and DAVID functional annotations followed by hierarchical clustering and multidimensional scaling of the gene expression signature of PSNE cells. We identified endoplasmic reticulum stress related pathways as key regulators of PSNE. Invalidation by RNA interference of the UPR sensors PERK, ATF6α, but not IRE1α, delayed the occurrence of senescence when performed in pre-senescent cells, and increased the PSNE frequency when performed in already senescent cells. Conversely, endoplasmic reticulum stress inducers applied to already senescent cells decreased the frequency of PSNE. In conclusion, these results indicate that the activation of the UPR could protect from the early carcinogenic steps by senescence evasion. This opens new avenues to explore therapeutics that could be useful in decreasing the age-associated tumor incidence.

The ATF6α arm of the Unfolded Protein Response mediates replicative senescence in human fibroblasts through a COX2/prostaglandin E2 intracrine pathway.

Senescence is recognized as a cellular state acquired in response to various stresses. It occurs in correlation with the activation of the Unfolded Protein Response (UPR) pathway. However, the UPR targets which might relay the establishment of the senescent phenotype are not known. Herein, we investigated whether the up-regulation of the COX2 (PTGS2) limiting enzyme in the prostaglandin biosynthesis pathway, known to mediate cellular senescence in normal human fibroblasts, could be controlled by the UPR sensors ATF6α, IRE1α and PERK. We found that UPR inducers cause premature senescence through an increase in COX2 expression, and an overproduction of prostaglandin E2 (PGE2) in wild type fibroblasts but not in ATF6α invalidated ones. In replicative senescent fibroblasts, ATF6α and IRE1α silencing abrogated COX2 up-regulation and PGE2 production. The expanded ER and the large cell size characteristics of senescent fibroblasts were both reduced upon the invalidation of COX2 as well as ATF6α. These effects of the ATF6α invalidation were prevented by favoring the import of PGE2, but not just by supplying extracellular PGE2. Taken together, our results support a critical role of ATF6α in the establishment and maintenance of cellular senescence in normal human fibroblasts via the up-regulation of a COX2/PGE2 intracrine pathway.

Expression and functional assessment of candidate type 2 diabetes susceptibility genes identify four new genes contributing to human insulin secretion.

OBJECTIVES: Genome-wide association studies (GWAS) have identified >100 loci independently contributing to type 2 diabetes (T2D) risk. However, translational implications for precision medicine and for the development of novel treatments have been disappointing, due to poor knowledge of how these loci impact T2D pathophysiology. Here, we aimed to measure the expression of genes located nearby T2D associated signals and to assess their effect on insulin secretion from pancreatic beta cells. METHODS: The expression of 104 candidate T2D susceptibility genes was measured in a human multi-tissue panel, through PCR-free expression assay. The effects of the knockdown of beta-cell enriched genes were next investigated on insulin secretion from the human EndoC-ßH1 beta-cell line. Finally, we performed RNA-sequencing (RNA-seq) so as to assess the pathways affected by the knockdown of the new genes impacting insulin secretion from EndoC-ßH1, and we analyzed the expression of the new genes in mouse models with altered pancreatic beta-cell function. RESULTS: We found that the candidate T2D susceptibility genes' expression is significantly enriched in pancreatic beta cells obtained by laser capture microdissection or sorted by flow cytometry and in EndoC-ßH1 cells, but not in insulin sensitive tissues. Furthermore, the knockdown of seven T2D-susceptibility genes (CDKN2A, GCK, HNF4A, KCNK16, SLC30A8, TBC1D4, and TCF19) with already known expression and/or function in beta cells changed insulin secretion, supporting our functional approach. We showed first evidence for a role in insulin secretion of four candidate T2D-susceptibility genes (PRC1, SRR, ZFAND3, and ZFAND6) with no previous knowledge of presence and function in beta cells. RNA-seq in EndoC-ßH1 cells with decreased expression of PRC1, SRR, ZFAND6, or ZFAND3 identified specific gene networks related to T2D pathophysiology. Finally, a positive correlation between the expression of Ins2 and the expression of Prc1, Srr, Zfand6, and Zfand3 was found in mouse pancreatic islets with altered beta-cell function. CONCLUSIONS: This study showed the ability of post-GWAS functional studies to identify new genes and pathways involved in human pancreatic beta-cell function and in T2D pathophysiology.

Identification of epigenetic factors regulating the mesenchyme to epithelium transition by RNA interference screening in breast cancer cells.

BACKGROUND: In breast cancer, the epithelial to mesenchyme transition (EMT) is associated to tumour dissemination, drug resistance and high relapse risks. It is partly controlled by epigenetic modifications such as histone acetylation and methylation. The identification of genes involved in these reversible modifications represents an interesting therapeutic strategy to fight metastatic disease by inducing mesenchymal cell differentiation to an epithelial phenotype. METHODS: We designed a siRNA library based on chromatin modification-related to functional domains and screened it in the mesenchymal breast cancer cell line MDA-MB-231. The mesenchyme to epithelium transition (MET) activation was studied by following human E-CADHERIN (E-CAD) induction, a specific MET marker, and cell morphology. Candidate genes were validated by studying the expression of several differential marker genes and their impact on cell migration. RESULTS: The screen led to the identification of 70 gene candidates among which some are described to be, directly or indirectly, involved in EMT like ZEB1, G9a, SMAD5 and SMARCD3. We also identified the DOT1L as involved in EMT regulation in MDA-MB-231. Moreover, for the first time, KAT5 gene was linked to the maintenance of the mesenchymal phenotype. CONCLUSIONS: A multi-parametric RNAi screening approach was developed to identify new EMT regulators such as KAT5 in the triple negative breast cancer cell line MDA-MB-231.

Identification of a gene signature of a pre-transformation process by senescence evasion in normal human epidermal keratinocytes.

BACKGROUND: Epidemiological data show that the incidence of carcinomas in humans is highly dependent on age. However, the initial steps of the age-related molecular oncogenic processes by which the switch towards the neoplastic state occurs remain poorly understood, mostly due to the absence of powerful models. In a previous study, we showed that normal human epidermal keratinocytes (NHEKs) spontaneously and systematically escape from senescence to give rise to pre-neoplastic emerging cells. METHODS: Here, this model was used to analyze the gene expression profile associated with the early steps of age-related cell transformation. We compared the gene expression profiles of growing or senescent NHEKs to post-senescent emerging cells. Data analyses were performed by using the linear modeling features of the limma package, resulting in a two-sided t test or F-test based on moderated statistics. The p-values were adjusted for multiple testing by controlling the false discovery rate according to Benjamini Hochberg method.The common gene set resulting of differential gene expression profiles from these two comparisons revealed a post-senescence neoplastic emergence (PSNE) gene signature of 286 genes. RESULTS: About half of these genes were already reported as involved in cancer or premalignant skin diseases. However, bioinformatics analyses did not highlight inside this signature canonical cancer pathways but metabolic pathways, including in first line the metabolism of xenobiotics by cytochrome P450. In order to validate the relevance of this signature as a signature of pretransformation by senescence evasion, we invalidated two components of the metabolism of xenobiotics by cytochrome P450, AKR1C2 and AKR1C3. When performed at the beginning of the senescence plateau, this invalidation did not alter the senescent state itself but significantly decreased the frequency of PSNE. Conversely, overexpression of AKR1C2 but not AKR1C3 increased the frequency of PSNE. CONCLUSIONS: To our knowledge, this study is the first to identify reprogrammation of metabolic pathways in normal keratinocytes as a potential determinant of the switch from senescence to pre-transformation.