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Jérôme Bertherat, Aurélien de Reyniès and colleagues perform integrated genomic analyses of adrenocortical carcinomas. They discover recurrent alterations in several new driver genes, including ZNRF3 , DAXX , TERT and MED12 , and identify two distinct molecular subgroups with opposite clinical outcomes. Adrenocortical carcinomas (ACCs) are aggressive cancers originating in the cortex of the adrenal gland 1 . Despite overall poor prognosis, ACC outcome is heterogeneous 2 , 3 . We performed exome sequencing and SNP array analysis of 45 ACCs and identified recurrent alterations in known driver genes 4 , 5 ( CTNNB1 , TP53 , CDKN2A , RB1 and MEN1 ) and in genes not previously reported in ACC ( ZNRF3 , DAXX , TERT and MED12 ), which we validated in an independent cohort of 77 ACCs. ZNRF3 , encoding a cell surface E3 ubiquitin ligase 6 , was the most frequently altered gene (21%) and is a potential new tumor suppressor gene related to the β-catenin pathway. Our integrated genomic analyses further identified two distinct molecular subgroups with opposite outcome. The C1A group of ACCs with poor outcome displayed numerous mutations and DNA methylation alterations, whereas the C1B group of ACCs with good prognosis displayed specific deregulation of two microRNA clusters. Thus, aggressive and indolent ACCs correspond to two distinct molecular entities driven by different oncogenic alterations.

The triggering receptor expressed on myeloid cells 1 (TREM-1) drives inflammatory responses in several cardiovascular diseases but its role in abdominal aortic aneurysm (AAA) remains unknown. Our objective was to explore the role of TREM-1 in a mouse model of angiotensin II-induced (AngII-induced) AAA. TREM-1 expression was detected in mouse aortic aneurysm and colocalized with macrophages. Trem1 gene deletion (Apoe-/-Trem1-/-), as well as TREM-1 pharmacological blockade with LR-12 peptide, limited both AAA development and severity. Trem1 gene deletion attenuated the inflammatory response in the aorta, with a reduction of Il1b, Tnfa, Mmp2, and Mmp9 mRNA expression, and led to a decreased macrophage content due to a reduction of Ly6Chi classical monocyte trafficking. Conversely, antibody-mediated TREM-1 stimulation exacerbated Ly6Chi monocyte aorta infiltration after AngII infusion through CD62L upregulation and promoted proinflammatory signature in the aorta, resulting in worsening AAA severity. AngII infusion stimulated TREM-1 expression and activation on Ly6Chi monocytes through AngII receptor type I (AT1R). In human AAA, TREM-1 was detected and TREM1 mRNA expression correlated with SELL mRNA expression. Finally, circulating levels of sTREM-1 were increased in patients with AAA when compared with patients without AAA. In conclusion, TREM-1 is involved in AAA pathophysiology and may represent a promising therapeutic target in humans.

Gain-of-function mutations in with no lysine (K) 1 (WNK1) and WNK4 genes are responsible for familial hyperkalemic hypertension (FHHt), a rare, inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the Kelch-like 3-Cullin 3 (KLHL3-CUL3) E3 ubiquitin ligase complex have shed light on the importance of WNK's cellular degradation on renal ion transport. Using full exome sequencing for a 4-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants in the WNK1 gene clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early onset of a hyperkalemic hyperchloremic phenotype, but normal blood pressure values\"Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR/Cas9 engineered mouse model recapitulated both the clinical and biological phenotypes. Renal investigations showed increased activation of the Ste20 proline alanine-rich kinase-Na+-Cl- cotransporter (SPAK-NCC) phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Together, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.

Numerous clinical studies and experimental investigations using cell culture and animal models suggest that angiotensin II (AngII) via AT1 receptor activation might induce cardiovascular hypertrophy, fibrosis and atherosclerosis resulting in vascular events such as myocardial infarction, heart failure or stroke and in end-organ damages. However, a question still remains: which part of these damages is due to a direct effect of AngII on its target tissues and which is due to AngII-induced hypertension? In an attempt to answer this question, a new model of transgenic mice, expressing a constitutively activated AT1A receptor instead of the wild type receptor has been obtained by homologous recombination. These mice present with a moderate increase of blood pressure (20mm Hg), hypertrophy of the small kidney arteries but not cardiac hypertrophy. The major phenotypic trait of these mice is the early and progressive development of a cardiovascular fibrosis. In light of these results and those from the literature, there is more and more evidence that in human hypertension, activation of the renin angiotensin system plays a minor role in the development of cardiovascular hypertrophy, but clearly participates to the development of cardiovascular fibrosis.

Insulin-like growth factor 2 (IGF2) overexpression is an important molecular marker of adrenocortical carcinoma (ACC), which is a rare but devastating endocrine cancer. It is not clear whether IGF2 overexpression modifies the biology and growth of this cancer, thus more studies are required before IGF2 can be considered as a major therapeutic target. We compared the phenotypical, clinical, biological, and molecular characteristics of ACC with or without the overexpression of IGF2, to address these issues. We also carried out a similar analysis in an ACC cell line (H295R) in which IGF2 expression was knocked down with si- or shRNA. We found no significant differences in the clinical, biological and molecular (transcriptomic) traits between IGF2-high and IGF2-low ACC. The absence of IGF2 overexpression had little influence on the activation of tyrosine kinase pathways both in tumors and in H295 cells that express low levels of IGF2. In IGF2-low tumors, other growth factors (FGF9, PDGFA) are more expressed than in IGF2-high tumors, suggesting that they play a compensatory role in tumor progression. In addition, IGF2 knock-down in H295R cells substantially impaired growth (>50% inhibition), blocked cells in G1 phase, and promoted apoptosis (>2-fold). Finally, analysis of the 11p15 locus showed a paternal uniparental disomy in both IGF2-high and IGF2-low tumors, but low IGF2 expression could be explained in most IGF2-low ACC by an additional epigenetic modification at the 11p15 locus. Altogether, these observations confirm the active role of IGF2 in adrenocortical tumor growth, but also suggest that other growth promoting pathways may be involved in a subset of ACC with low IGF2 expression, which creates opportunities for the use of other targeted therapies.

The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT(1A)), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT(1A) leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension.

Cullin 3 (CUL3) is the scaffold of Cullin3 Ring E3-ligases (CRL3s), which use various BTB-adaptor proteins to ubiquitinate numerous substrates targeting their proteasomal degradation. CUL3 mutations, responsible for a severe form of familial hyperkalemia and hypertension (FHHt), all result in a deletion of exon 9 (amino-acids 403-459) (CUL3-∆9). Surprisingly, while CUL3-∆9 is hyperneddylated, a post-translational modification that typically activates CRL complexes, it is unable to ubiquitinate its substrates. In order to understand the mechanisms behind this loss-of function, we performed comparative label-free quantitative analyses of CUL3 and CUL3-∆9 interactome by mass spectrometry. It was observed that CUL3-∆9 interactions with COP9 and CAND1, both involved in CRL3 complexes’ dynamic assembly, were disrupted. These defects result in a reduction in the dynamic cycling of the CRL3 complexes, making the CRL3-∆9 complex an inactive BTB-adaptor trap, as demonstrated by SILAC experiments. Collectively, the data indicated that the hyperneddylated CUL3-∆9 protein is inactive as a consequence of several structural changes disrupting its dynamic interactions with key regulatory partners.

The constitutive activation of G-protein-coupled receptors is a major new approach to investigating their physiopathology and pharmacology. A large number of spontaneous and site-directed mutations resulting in constitutive activity have been identified, but systematic mapping of the amino acids involved for a given receptor would be extremely useful for complete elucidation of the molecular mechanisms underlying its activation. We carried out such mapping for the angiotensin II type 1A (AT1A) receptor by screening a randomly mutated cDNA library after expressing the mutated clones in eukaryotic cells. To test the AT1Amutants generated, we developed an original, specific, and highly sensitive assay based on the properties of CGP42112A. This classical AT2agonist is a weak partial agonist of the wild-type AT1Areceptor and becomes a full agonist for constitutively active AT1Amutants, as shown experimentally and in allostery-based theoretical models. Activation of the mutated receptors by CGP42112A was monitored by using the bioluminescent protein aequorin, a very sensitive and specific sensor of intracellular calcium mobilization. The screening of 4,800 clones, providing an exhaustive coverage of all of the mutations generated, led to the identification of 16 mutations in sequences encoding the transmembrane domains that were responsible for high sensitivity to CGP42112A. The constitutive activity was confirmed by agonist-independent production of inositol phosphates, which showed that at least half of the clones had significantly increased basal activity. These data demonstrate that this new type of approach is very efficient for the systematic identification of constitutively active mutants of G-protein-coupled receptors.

The authors describe a germ-line mutation in the gene for PRKAR1A in three unrelated patients with acrodysostosis and resistance to multiple hormones. The mutated protein subunit impairs the response of protein kinase A to cyclic-AMP stimulation. Numerous hormones activate heterotrimeric G-protein–coupled receptors, which then activate G protein and adenylyl cyclase, generating intracellular cAMP. 1 In turn, cAMP activates protein kinase A, resulting in the phosphorylation of specific proteins that mediate the physiological effects of these hormones. 2 – 4 Loss-of-function mutations in the gene encoding GNAS cause pseudohypoparathyroidism type 1a, a disease with characteristic developmental and skeletal abnormalities (collectively called Albright's hereditary osteodystrophy, i.e., short stature, brachydactyly most frequently affecting the fourth and fifth metacarpals, rounded facies, obesity, and heterotopic subcutaneous ossification) that are associated with hormone resistance. 1 , 5 Acrodysostosis (Online Mendelian Inheritance in Man number 101800) is a . . .

Purpose Polymerase proofreading-associated polyposis is a dominantly inherited colorectal cancer syndrome caused by exonuclease domain missense variants in the DNA polymerases POLE and POLD1 . Manifestations may also include malignancies at extracolonic sites. Cancer risks in this syndrome are not yet accurately quantified. Methods We sequenced POLE and POLD1 exonuclease domains in 354 individuals with early/familial colorectal cancer (CRC) or adenomatous polyposis. We assessed the pathogenicity of POLE variants with yeast fluctuation assays and structural modeling. We estimated the penetrance function for each cancer site in variant carriers with a previously published nonparametric method based on survival analysis approach, able to manage unknown genotypes. Results Pathogenic POLE exonuclease domain variants P286L, M294R, P324L, N363K, D368N, L424V, K425R, and P436S were found in ten families. The estimated cumulative risk of CRC at 30, 50, and 70 years was 11.1% (95% confidence interval [CI]: 4.2–17.5), 48.5% (33.2–60.3), and 74% (51.6–86.1). Cumulative risk of glioblastoma was 18.7% (3.2–25.8) at 70 years. Variants interfering with DNA binding (P286L and N363K) had a significantly higher mutagenic effect than variants disrupting ion metal coordination at the exonuclease site. Conclusion The risk estimates derived from this study provide a rational basis on which to provide genetic counseling to POLE variant carriers.