Post-translational inhibition of YAP oncogene expression by 4-hydroxynonenal in bladder cancer cells.

The transcriptional regulator YAP plays an important role in cancer progression and is negatively controlled by the Hippo pathway. YAP is frequently overexpressed in human cancers, including bladder cancer. Interestingly, YAP expression and activity can be inhibited by pro-oxidant conditions; moreover, YAP itself can also affect the cellular redox status through multiple mechanisms. 4-Hydroxynonenal (HNE), the most intensively studied end product of lipid peroxidation, is a pro-oxidant agent able to deplete GSH and has an anti-tumoral effect by affecting multiple signal pathways, including the down-regulation of oncogene expressions. These observations prompted us to investigate the effect of HNE on YAP expression and activity. We demonstrated that HNE inhibited YAP expression and its target genes in bladder cancer cells through a redox-dependent mechanism. Moreover, the YAP down-regulation was accompanied by an inhibition of proliferation, migration, invasion, and angiogenesis, as well as by an accumulation of cells in the G2/M phase of cell cycle and by an induction of apoptosis. We also established the YAP role in inhibiting cell viability and inducing apoptosis in HNE-treated cells by using an expression vector for YAP. Furthermore, we identified a post-translational mechanism for the HNE-induced YAP expression inhibition, involving an increase of YAP phosphorylation and ubiquitination, leading to proteasomal degradation. Our data established that HNE can post-translationally down-regulate YAP through a redox-dependent mechanism and that this modulation can contribute to determining the specific anti-cancer effects of HNE.

Crosstalk between Nrf2 and YAP contributes to maintaining the antioxidant potential and chemoresistance in bladder cancer.

Redox adaptation plays an important role in cancer cells drug resistance. The antioxidant response is principally mediated by the transcription factor Nrf2, that induces the transcriptional activation of several genes involved in GSH synthesis, chemoresistance, and cytoprotection. YAP is emerging as a key mediator of chemoresistance in a variety of cancers, but its role in controlling the antioxidant status of the cells is yet elusive. Here, we show that impairing YAP protein expression reduced GSH content and Nrf2 protein and mRNA expression in bladder cancer cells. Moreover, in YAP knocked down cells the expression of FOXM1, a transcription factor involved in Nrf2 transcription, was down-regulated and the silencing of FOXM1 reduced Nrf2 expression. On the other hand, the silencing of Nrf2, as well as the depletion of GSH by BSO treatment, inhibited YAP expression, suggesting that cross-talk exists between YAP and Nrf2 proteins. Importantly, we found that silencing either YAP or Nrf2 enhanced sensitivity of bladder cancer cells to cytotoxic agents and reduced their migration. Furthermore, the inhibition of both YAP and Nrf2 expressions significantly increased cytotoxic drug sensitivity and synergistically reduced the migration of chemoresistant bladder cancer cells. These findings provide a rationale for targeting these transcriptional regulators in patients with chemoresistant bladder cancer, expressing high YAP and bearing a proficient antioxidant system.

The Molecular Spectrum of ß- and α-Thalassemia Mutations in Non-Endemic Umbria, Central Italy.

The aim of this study was to describe the mutational spectrum of hemoglobinopathies during the period 1988-2015 in Umbria, Central Italy, which has never been considered endemic for these conditions. Twenty-four different ß-globin gene mutations were identified in 188 patients and eight different α-globin gene mutations in 74 patients. Sixty percent ß-thalassemia (ß-thal), 85.0% sickle cell disease, 44.0% Hb S (HBB: c.20A>T)/ß-thal and 85.0% compound heterozygotes for hemoglobin (Hb) variant-carrying patients were diagnosed or molecularly characterized in the last 3 years. Moreover, most homozygous or compound heterozygous patients (84.5%) came from foreign countries, while only 15.5% were of Italian origin. These data are in accordance with the increasing foreign resident population in Umbria, which has nearly doubled in 10 years (2004-2014). Different from ß-globin gene variations, no increasing trend in α defects was observed in our study cohort. Consistently, 58.0% of patients have an Italian origin, suggesting no broad influence of foreign migration in the α-globin genes genetic background. As few defects are prevalent in each country of origin or ethnic group, their knowledge may provide a proper strategy for the identification of mutations in immigrant individuals in a non-endemic region and be important for carrier identification and prenatal screening.

A comparative study of myocardial molecular phenotypes of two tfr2ß null mice: role in ischemia/reperfusion.

Transferrin receptor 2 (Tfr2) is an iron-modulator transcribed in two isoforms, Tfr2α and Tfr2ß. The latter is expressed in the heart. We obtained two mouse models with silencing of Tfr2ß: one with a normal systemic iron amount (SIA), i.e., Tfr2-KI, and the other, i.e., LCKO-KI, with high SIA due to hepatic Tfr2α silencing. We aimed to assess whether Tfr2ß might play a role in myocardial injury and whether Tfr2ß silencing might modify proteins of iron metabolism, antioxidant, apoptotic, and survival enzyme activities in the heart undergoing ischemia/reperfusion (I/R). Isolated hearts of wild-type (WT) and Tfr2-null mice were studied before or after an I/R protocol, and proteins/RNA analyzed by Western blot and/or quantitative PCR. Tfr2ß increased in WT hearts subject to I/R, and both Tfr2ß null mice hearts were protected against I/R injury (about 40% smaller infarct-size compared to WT hearts). RISK kinases (ERK1/2-AKT-PKCε) were found up-regulated after I/R in Tfr2-KI, whereas SAFE enzyme (Stat3) and GSK3ß resulted phosphorylated during I/R in LCKO-KI hearts. While HO-1 and HIF-2a were high in both Tfr2ß-null mice, Catalase, and proapoptotic factors were upregulated only in LCKO-KI. Finally, Tfr2-KI hearts presented an increased Ferritin-H and a decreased Ferroportin1, whereas LCKO-KI hearts displayed an upregulation of Ferritin-L chain and DMT1/Hamp-RNA. In conclusion, Tfr2ß isoform is involved in cardiac iron metabolism and its silencing leads to a protected phenotype (antioxidants, RISK, and/or SAFE upregulation) against I/R challenging. Iron-dependent signals involved in cardioprotection seem to be positively affected by Tfr2ß downregulation and subsequent Ferritins upregulation.