Tuberous sclerosis complex protein 1 expression is affected by VHL Gene alterations and HIF-1α production in sporadic clear-cell renal cell carcinoma.

Alterations in von Hippel-Lindau gene (VHL) do not determine deregulation of hypoxia-inducible factors (HIFs) in clear-cell renal carcinoma (ccRCC). Their effects on tuberous sclerosis proteins (TSC1/2) and heat shock protein 90 (Hsp90) expressions in sporadic ccRCC are unknown. Therefore, we analyze the impact of VHL alterations and HIF-α production on the expression of TSC proteins and Hsp90 in these tumors. Alterations in VHL gene region exhibited 37/47 (78.7%) tumors. Monoallelic inactivation (intragenic mutation or LOH) was found in 10 (21.3%) and biallelic inactivation (intragenic mutation plus LOH) in 27 (57.4%) ccRCCs. Tumorous expression of HIF-α mRNAs, HIF-α, Hsp90 and TSC2 were VHL independent; TSC2 was underexpressed in all tumors by immunostaining (P<0.001). Immunoblotting revealed that TSC1 production was lower in tumors with monoallelic VHL inactivation than in control (P=0.01) and tissues with biallelic VHL inactivation (P=0.019), while tumors lacking HIF-1α (16/47) concurrently overexpressed HIF-2α and underexpressed TSC1 in comparison to controls (P=0.01 for both) and HIF-1α positive tumors (P=0.015 and P=0.050). Significant portion of variability (56.4%) in tumor diameter was explained by oscillations in nuclear grade, and TSC1 and HIF-2α expression in VHL altered tumors. In conclusion, while TSC2 is broadly downregulated in sporadic ccRCC, TSC1 expression is reduced in two subsets of these tumors, those with monoallelic VHL gene inactivation and those with concurrent low HIF-1α and high HIF-2α expression. Hence, the involvement of nuclear grade, TSC1 and HIF-2α in the progression of VHL altered tumors, implies the interplay between pVHL and TSC1.

Glucocorticoid receptor and molecular chaperones in the pathogenesis of adrenal incidentalomas: potential role of reduced sensitivity to glucocorticoids.

Glucocorticoid (GC) sensitivity depends on glucocorticoid receptor (GR) and heat shock proteins (Hsps). We investigated whether common GR genes (ER22/23EK, N363S, Bcl I, and 9ß) and adrenocorticotropin receptor promoter polymorphisms influence susceptibility for unilateral adrenal incidentaloma (AI), plus GR and Hsp expression in tumorous (n = 19), peritumorous (n = 13) and normal adrenocortical (n = 11) tissues. Patients (n = 112), population-matched controls (n = 100) and tumor tissues (n = 32) were genotyped for these polymorphisms. Postdexamethasone serum cortisol was higher in patients (p < 0.001). GR gene variants, larger allele of Bcl I (odds ratio [OR] 2.9; 95% confidence interval [CI] 1.7-5.1; p < 0.001] and minor allele of 9ß (OR 3.0; 95% CI 1.6-5.7; p < 0.001) were independent predictors of AI. In patients, the first allele is linked with larger tumors (p = 0.002) and the latter with higher postdexamethasone cortisol levels (p = 0.025). Both allele carriers had lesser waist circumference (p = 0.02), similar adrenocorticotropin and higher basal (p = 0.024) and postdexamethasone cortisol concentrations (p < 0.001). Tumorous and constitutional genotypes were similar. GR-D is the major receptor isoform in normal adrenal cortex by Western blotting. Loss of other receptor isoforms, decrease in immunostaining for GR (p < 0.0001), underexpression of chaperones (p ≤ 0.01) and the presence of inducible Hsp70 were found in adenomas. In conclusion, GR gene variants, C allele of Bcl I and minor allele of 9ß, are associated with AIs. Their concurrent presence in patients reduces GC sensitivity. Normal adrenal cortex preferentially expresses GR-D. In adenomas, the lack of other GR isoforms and underexpression of heat shock proteins perhaps permanently impair GC signaling, which could promote dysregulated cortisol production and tumor growth. The innate GC sensitivity probably modifies these effects.

ARMC5 Alterations in Patients With Sporadic Neuroendocrine Tumors and Multiple Endocrine Neoplasia Type 1 (MEN1).

CONTEXT: Adrenal lesions are frequent among patients with sporadic neuroendocrine tumors (spNETs) or multiple endocrine neoplasia type 1 (MEN1). Armadillo repeat-containing 5 (ARMC5)-inactivating variants cause adrenal tumors and possibly other neoplasms. OBJECTIVE: The objective of this work is to investigate a large cohort spNETs or MEN1 patients for changes in the ARMC5 gene. PATIENTS AND METHODS: A total of 111 patients, 94 with spNET and 17 with MEN1, were screened for ARMC5 germline alterations. Thirty-six tumors (18 spNETs and 18 MEN1 related) were collected from 20 patients. Blood and tumor DNA samples were genotyped using Sanger sequencing and microsatellite markers for chromosomes. ARMC5 and MEN1 expression were assessed by immunohistochemistry. RESULTS: In 76 of 111 (68.4%) patients, we identified 16 different ARMC5 germline variants, 2 predicted as damaging. There were no differences in the prevalence of ARMC5 variants depending on the presence of MEN1-related adrenal lesions. Loss of heterozygosity (LOH) at chromosome 16p and ARMC5 germline variants were present together in 23 or 34 (67.6%) tumors; in 7 of 23 (30.4%) their presence led to biallelic inactivation of the ARMC5 gene. The latter was more prevalent in MEN1-related tumors than in spNETs (88.9% vs 38.9%; P = .005). LOH at the chromosome 16p (ARMC5) and 11q (MEN1) loci coexisted in 16/18 MEN1-related tumors, which also expressed lower ARMC5 (P = .02) and MEN1 (P = .01) proteins compared to peritumorous tissues. CONCLUSION: Germline ARMC5 variants are common among spNET and MEN1 patients. ARMC5 haploinsufficiency or biallelic inactivation in spNETs and MEN1-related tumors suggests that ARMC5 may have a role in modifying the phenotype of patients with spNETs and/or MEN1 beyond its known role in macronodular adrenocortical hyperplasia.

VHL Dependent Expression of REDD1 and PDK3 Proteins in Clear-cell Renal Cell Carcinoma.

BACKGROUND: Sporadic clear-cell renal cell carcinoma (ccRCC) is associated with mutations in the VHL gene, upregulated mammalian target of rapamycin (mTOR) activity and glycolytic metabolism. Here, we analyze the effect of VHL mutational status on the expression level of mTOR, eIF4E-BP1, AMPK, REDD1, and PDK3 proteins. METHODS: Total proteins were isolated from 21 tumorous samples with biallelic inactivation, 10 with monoallelic inactivation and 6 tumors with a wild-type VHL (wtVHL) gene obtained from patients who underwent total nephrectomy. The expressions of target proteins were assessed using Western blot. RESULTS: Expressions of mTOR, eIF4EBP1 and AMPK were VHL independent. Tumors with monoallelic inactivation of VHL underexpressed REDD1 in comparison to wtVHL tumors (P = 0.042), tumors with biallelic VHL inactivation (P < 0.005) and control tissue (P = 0.004). Additionally, REDD1 expression was higher in tumors with VHL biallelic inactivation than in control tissue (P = 0.008). Only in wt tumor samples PDK3 was overexpressed in comparison to tumors with biallelic inactivation of VHL gene (P = 0.012) and controls (P = 0.016). In wtVHL ccRCC, multivariate linear regression analysis revealed that 97.4% of variability in PDK3 expression can be explained by variations in AMPK amount. CONCLUSION: Expressions of mTOR, eIF4EBP1 and AMPK were VHL independent. We have shown for the first time VHL dependent expression of PDK3 and we provide additional evidence that VHL mutational status affects REDD1 expression in sporadic ccRCC.

Phenylpropiophenone derivatives as potential anticancer agents: synthesis, biological evaluation and quantitative structure-activity relationship study.

Series of twelve chalcone and propafenone derivatives has been synthesized and evaluated for anticancer activities against HeLa, Fem-X, PC-3, MCF-7, LS174 and K562 cell lines. The 2D-QSAR and 3D-QSAR studies were performed for all compounds with cytotoxic activities against each cancer cell line. Partial least squares (PLS) regression has been applied for selection of the most relevant molecular descriptors and QSAR models building. Predictive potentials of the created 2D-QSAR and 3D-QSAR models for each cell line were compared, by use of leave-one-out cross-validation and external validation, and optimal QSAR models for each cancer cell line were selected. The QSAR studies have selected the most significant molecular descriptors and pharmacophores of the chalcone and propafenone derivatives and proposed structures of novel chalcone and propafenone derivatives with enhanced anticancer activity on the HeLa, Fem-X, PC-3, MCF-7, LS174 and K562 cells.