Aven blocks DNA damage-induced apoptosis by stabilising Bcl-xL

Induction of apoptosis by DNA-damaging agents involves the activation of mitochondrial apoptotic pathway. Aven has been identified as an antiapoptotic protein and has been shown to activate ATM in response to DNA damage. In this study, we demonstrated that enforced expression of Aven blocks UV-irradiation-, SN-38- or cisplatin-induced apoptosis upstream of mitochondria by stabilising Bcl-xL protein levels in breast cancer cells. Aven silencing by RNA interference markedly enhanced apoptotic response following treatment with DNA-damaging agents. Aven is complexed with Bcl-xL in untreated breast cancer cells and treatment with DNA-damaging agents led to decreased Aven/Bcl-xL interaction. Importantly, Bcl-xL was necessary for the prosurvival activity of Aven and depletion of Bcl-xL abrogated Aven-mediated protection against DNA damage-induced apoptosis. Analysis of breast cancer tissue microarrays revealed decreased Aven nuclear expression in breast cancer tissues compared with non-neoplastic breast tissues. In particular, we detected reduced nuclear expression of Aven in infiltrating ductal carcinoma and papillary carcinoma breast cancer subtypes compared with non-neoplastic breast tissues and infiltrating lobular breast cancer tissues. Our results suggest that Aven is an important mediator in DNA damage-induced apoptotic signalling in breast cancer cells and its nuclear expression is altered in breast cancer tissues, which may contribute to genomic instability in breast cancer tumours

Mitochondrial estrogen receptors alter mitochondrial priming and response to endocrine therapy in breast cancer cells

Breast cancer is the most common cancer with a high rate of mortality and morbidity among women worldwide. Estrogen receptor status is an important prognostic factor and endocrine therapy is the choice of first-line treatment in ER-positive breast cancer. However, most tumors develop resistance to endocrine therapy. Here we demonstrate that BH3 profiling technology, in particular, dynamic BH3 profiling can predict the response to endocrine therapy agents as well as the development of acquired resistance in breast cancer cells independent of estrogen receptor status. Immunofluorescence analysis and subcellular fractionation experiments revealed distinct ER-alpha and ER-beta subcellular localization patterns in breast cancer cells, including mitochondrial localization of both receptor subtypes. shRNA-mediated depletion of ER-beta in breast cancer cells led to resistance to endocrine therapy agents and selective reconstitution of ER-beta in mitochondria restored sensitivity. Notably, mitochondria-targeted ER-alpha did not restore sensitivity, even conferred further resistance to endocrine therapy agents. In addition, expressing mitochondria-targeted ER-beta in breast cancer cells resulted in decreased mitochondrial respiration alongside increased total ROS and mitochondrial superoxide production. Furthermore, our data demonstrated that mitochondrial ER-beta can be successfully targeted by the selective ER-beta agonist Erteberel. Thus, our findings provide novel findings on mitochondrial estrogen signaling in breast cancer cells and suggest the implementation of the dynamic BH3 technique as a tool to predict acquired endocrine therapy resistance

Clinical and molecular evaluation of MEFV gene variants in the Turkish population: a study by the National Genetics Consortium

Familial Mediterranean fever (FMF) is a monogenic autoinflammatory disorder with recurrent fever, abdominal pain, serositis, articular manifestations, erysipelas-like erythema, and renal complications as its main features. Caused by the mutations in the MEditerranean FeVer (MEFV) gene, it mainly affects people of Mediterranean descent with a higher incidence in the Turkish, Jewish, Arabic, and Armenian populations. As our understanding of FMF improves, it becomes clearer that we are facing with a more complex picture of FMF with respect to its pathogenesis, penetrance, variant type (gain-of-function vs. loss-of-function), and inheritance. In this study, MEFV gene analysis results and clinical findings of 27,504 patients from 35 universities and institutions in Turkey and Northern Cyprus are combined in an effort to provide a better insight into the genotype-phenotype correlation and how a specific variant contributes to certain clinical findings in FMF patients. Our results may help better understand this complex disease and how the genotype may sometimes contribute to phenotype. Unlike many studies in the literature, our study investigated a broader symptomatic spectrum and the relationship between the genotype and phenotype data. In this sense, we aimed to guide all clinicians and academicians who work in this field to better establish a comprehensive data set for the patients. One of the biggest messages of our study is that lack of uniformity in some clinical and demographic data of participants may become an obstacle in approaching FMF patients and understanding this complex disease

Psychomotor Delay in a Child with FGFR3 G380R Pathogenic Mutation Causing Achondroplasia

Achondroplasia (ACH) is a hereditary disorder of dwarfism that is caused by the aberrant proliferation and differentiation of chondrocyte growth plates. The common findings of macrocephaly and facial anomalies accompany dwarfism in these patients. Fibroblast growth factor receptor 3 (FGFR3) gene mutations are common causes of achondroplasia. The current study presents a case of 2-year-old male child patient presenting with phenotypic characteristics of ACH. The interesting finding of the case is the presence of psychomotor delay that is not very common in these patients. Clinical exome sequencing analyzing 4.813 disease causing genes revealed a de novo c.1138G > A mutation within the FGFR3 gene. In conclusion, the mutation confirms the clinical diagnosis of ACH, and it seems to be causing the psychomotor delay in this patient

BIK/BCL-2 and BIK/BCL-XL interactions were increased in HCT-116 wt cells in response to DNA damage.

<p>HCT-116 wt and HCT-116 p53 -/- cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 4h and the interaction of BIK with BCL-2, BCL-XL, and MCL-1 was detected by coimmunoprecipitation assays. Inputs for coimmunoprecipitation experiments were also subjected to immunoblot analysis and actin was probed as loading control.</p

Cisplatin induces early LMP in HCT-116 p53 -/- cells.

<p>(A) HCT-116 wt and HCT-116 p53-/- cells were treated with cisplatin (20 μM) for 1h. HCT-116 p53 -/- cells were TIRON (10 mM) for 2h and then treated with cisplatin for 1h to examine the effect of TIRON on cisplatin-induced LMP. Cells were stained for galectin-3 to evaluate the lysosomal membrane permeabilization. (B) HCT-116 wt and HCT-116 p53 -/- cells were transfected with BIK siRNA or scrambled siRNA for 24h. Cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 1h and the Cathepsin B/L activity was measured in untransfected and transfected cells (mean RFU±SEM, n = 3). (C) HCT-116 p53 -/- cells were pretreated with CA-074Me (100 μM) or Z-FA-FMK (10 μM) for 2h and then treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 48h. Cell viability was determined by CellTiterGlo assay and expressed as % of untreated control (mean±SEM, n = 3, *P<0.05, **P<0.01).</p

BIK mediates cisplatin-induced cell death in HCT-116 wt cellular spheroids, but not in HCT-116 p53 -/- spheroids.

<p>(A) HCT-116 wt and HCT-116 p53 -/- cells were transfected with BIK siRNA or scrambled siRNA for 24h. Cells were subsequently grown in 24-well 3D Algimatrix plates in the presence of RNAi duplexes. Microscopic evaluation of spheroids was done to verify that siRNA treatments did not interfere with the 3D growth of HCT-116 cells. (B) The efficiency of BIK depletion by RNA interference in HCT-116 wt and HCT-116 p53 -/- was determined by immunoblot analysis. Actin was probed as loading control. (C) Spheroids were treated with cisplatin (200 μM) for 48h and cell viability was assessed by using alamarBlue assay (mean±SEM, n = 3, *P<0.05).</p

RAB25 confers resistance to chemotherapy by altering mitochondrial apoptosis signaling in ovarian cancer cells

Ovarian cancer remains one of the most frequent causes of cancer-related death in women. Many patients with ovarian cancer suffer from de novo or acquired resistance to chemotherapy. Here, we report that RAB25 suppresses chemotherapy-induced mitochondrial apoptosis signaling in ovarian cancer cell lines and primary ovarian cancer cells. RAB25 blocks chemotherapy-induced apoptosis upstream of mitochondrial outer membrane permeabilization by either increasing antiapoptotic BCL-2 proteins or decreasing proapoptotic BCL-2 proteins. In particular, BAX expression negatively correlates with RAB25 expression in ovarian cancer cells. BH3 profiling assays corroborated that RAB25 decreases mitochondrial cell death priming. Suppressing RAB25 by means of RNAi or RFP14 inhibitory hydrocarbon-stapled peptide sensitizes ovarian cancer cells to chemotherapy as well as RAB25-mediated proliferation, invasion and migration. Our data suggest that RAB25 is a potential therapeutic target for ovarian cancer

Depletion of BIK led to decreased mitochondrial cell death priming in HCT-116 wt cells.

<p>(A) HCT-116 wt and (B) HCT-116 p53 -/- cells were untreated (upper panels) or transiently transfected with BIK siRNA (lower panels) and mitochondrial depolarization was measured following incubation of cells with DMSO, BIM, BID, BAD, PUMA, BMF and NOXA peptides at 100 μM and FCCP at 10 μM. Graphs are shown as mean±SEM, n = 3 and sample mitochondrial potential (ΔΨm) kinetic tracings are provided. (C) Microplate-based BH3 profiles were confirmed by ELISA-based cytochrome c release assays following incubation of isolated mitochondria with DMSO, BIM, BID, BAD, PUMA, BMF and NOXA peptides at 100 μM (mean±SEM, n = 3).</p

Analysis of ACE2 and TMPRSS2 coding variants as a risk factor for SARS-CoV-2 from 946 whole-exome sequencing data in the Turkish population

Heterogeneity in symptoms associated with COVID-19 in infected patients remains unclear. ACE2 and TMPRSS2 gene variants are considered possible risk factors for COVID-19. In this study, a retrospective comparative genome analysis of the ACE2 and TMPRSS2 variants from 946 whole-exome sequencing data was conducted. Allele frequencies of all variants were calculated and filtered to remove variants with allele frequencies lower than 0.003 and to prioritize functional coding variants. The majority of detected variants were intronic, only two ACE2 and three TMPRSS2 nonsynonymous variants were detected in the analyzed cohort. The main ACE2 variants that putatively have a protective or susceptibility effect on SARS-CoV-2 have not yet been determined in the Turkish population. The Turkish genetic makeup likely lacks any ACE2 variant that increases susceptibility to SARS-CoV-2 infection. TMPRSS2 rs75603675 and rs12329760 variants that were previously defined as common variants that have different allele frequencies among populations and may have a role in SARS-CoV-2 attachment to host cells were determined in the population. Overall, these data will contribute to the formation of a national variation database and may also contribute to further studies of ACE2 and TMPRSS2 in the Turkish population and differences in SARS-CoV-2 infection among other populations