Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

Discovery of Genetic Variation on Chromosome 5q22 Associated with Mortality in Heart Failure

Failure of the human heart to maintain sufficient output of blood for the demands of the body, heart failure, is a common condition with high mortality even with modern therapeutic alternatives. To identify molecular determinant

Novel delivery system for T-oligo using a nanocomplex formed with an alpha helical peptide for melanoma therapy

Oligonucleotides homologous to 3'-telomere overhang (T-oligos) trigger inherent telomere-based DNA damage responses mediated by p53 and/or ATM and induce senescence or apoptosis in various cancerous cells. However, T-oligo has limited stability in vivo due to serum and intracellular nucleases. To develop T-oligo as an innovative, effective therapeutic drug and to understand its mechanism of action, we investigated the antitumor effects of T-oligo or T-oligo complexed with a novel cationic alpha helical peptide, PVBLG-8 (PVBLG), in a p53 null melanoma cell line both in vitro and in vivo. The uptake of T-oligo by MM-AN cells was confirmed by immunofluorescence, and fluorescence-activated cell sorting analysis indicated that the T-oligo-PVBLG nanocomplex increased uptake by 15-fold. In vitro results showed a 3-fold increase in MM-AN cell growth inhibition by the T-oligo-PVBLG nanocomplex compared with T-oligo alone. Treatment of preformed tumors in immunodeficient mice with the T-oligo-PVBLG nanocomplex resulted in a 3-fold reduction in tumor volume compared with T-oligo alone. This reduction in tumor volume was associated with decreased vascular endothelial growth factor expression and induction of thrombospondin-1 expression and apoptosis. Moreover, T-oligo treatment downregulated procaspase-3 and procaspase-7 and increased catalytic activity of caspase-3 by 4-fold in MM-AN cells. Furthermore, T-oligo induced a 10-fold increase of senescence and upregulated the melanoma tumor-associated antigens MART-1, tyrosinase, and thrombospondin-1 in MM-AN cells, which are currently being targeted for melanoma immunotherapy. Interestingly, siRNA-mediated knockdown of p73 (4–10-fold) abolished this upregulation of tumor-associated antigens. In summary, we suggest a key role of p73 in mediating the anticancer effects of T-oligo and introduce a novel nanoparticle, the T-oligo-PVBLG nanocomplex, as an effective anticancer therapeutic

Alternative Signaling Pathways as Potential Therapeutic Targets for Overcoming EGFR and c-Met Inhibitor Resistance in Non-Small Cell Lung Cancer

<div><p>The use of tyrosine kinase inhibitors (TKIs) against EGFR/c-Met in non-small cell lung cancer (NSCLC) has been shown to be effective in increasing patient progression free survival (PFS), but their efficacy is limited due to the development of resistance and tumor recurrence. Therefore, understanding the molecular mechanisms underlying development of drug resistance in NSCLC is necessary for developing novel and effective therapeutic approaches to improve patient outcome. This study aims to understand the mechanism of EGFR/c-Met tyrosine kinase inhibitor (TKI) resistance in NSCLC. H2170 and H358 cell lines were made resistant to SU11274, a c-Met inhibitor, and erlotinib, an EGFR inhibitor, through step-wise increases in TKI exposure. The IC₅₀ concentrations of resistant lines exhibited a 4–5 and 11–22-fold increase for SU11274 and erlotinib, respectively, when compared to parental lines. Furthermore, mTOR and Wnt signaling was studied in both cell lines to determine their roles in mediating TKI resistance. We observed a 2–4-fold upregulation of mTOR signaling proteins and a 2- to 8-fold upregulation of Wnt signaling proteins in H2170 erlotinib and SU11274 resistant cells. H2170 and H358 cells were further treated with the mTOR inhibitor everolimus and the Wnt inhibitor XAV939. H358 resistant cells were inhibited by 95% by a triple combination of everolimus, erlotinib and SU11274 in comparison to 34% by a double combination of these drugs. Parental H2170 cells displayed no sensitivity to XAV939, while resistant cells were significantly inhibited (39%) by XAV939 as a single agent, as well as in combination with SU11274 and erlotinib. Similar results were obtained with H358 resistant cells. This study suggests a novel molecular mechanism of drug resistance in lung cancer.</p></div

MTT assay showing differential response between parental and resistant NSCLC cell lines.

<p>H2170 and H358 cells were treated with tivantinib (0.01–0.4 µM) for 24 hours, tivantinib was removed, and cells were incubated for 72 hours, after which MTT viability assay was performed. SR H2170 cells showed a 3.2-fold decrease in sensitivity to the anti-proliferative effect of tivantinib at 0.1 µM tivantinib compared with parental cells. A 3.7-fold decrease in growth inhibition was also observed in SR H358 cells with 0.2 µM tivantinib compared to parental cells. Data shown are representative of three independent experiments showing similar results (n = 6, p<0.01).</p

IC₅₀ of RTKIs and Combinations for Parental and Resistant NSCLC cell lines.

<p>IC₅₀ of RTKIs and Combinations for Parental and Resistant NSCLC cell lines.</p

Differences in protein expression between parental and erlotinib resistant cell lines by western blotting.

<p>A. EGFR is autophosphorylated in ER H2170 and downregulated in H358-E4 resistant cell lines. p-mTOR (S2448) and its downstream signaling protein phospho-p70S6K (T389) are upregulated in both resistant cell lines. H2170 and H358 parental and resistant cell lines were starved overnight in 0.5% BSA and then treated with or without 7.0 µM of erlotinib for 24 hours and cells were stimulated with 10 ng/mL of EGF for 2.5 minutes. Higher concentrations of erlotinib were used since these NSCLC cell lines have no EGFR TK mutation. Autophosphorylation of EGFR on Y1068 was seen in the absence of EGF in ER H2170 cells which was not seen in ER H358-E4 cells. Upregulation of p-mTOR and its downstream protein phosho-p70S6K (T389) is seen in H2170 resistant lines +/− erlotinib. ER H2170 cells show increased EGFR phosphorylation +/− EGF. Upregulation of p-ERK (2–5-fold) was also seen in ER H2170 and H358 cells in +/− erlotinib B. To confirm autophosphorylation of EGFR, cells were plated on chamber slides, allowed to adhere for 24 hours and then starved overnight. Cells were then treated with +/− EGF for 15 minutes, fixed with acetone: methanol and visualized with p-EGFR (Y1068) primary antibody and anti rabbit DyLight secondary antibody (Thermo Fisher Scientific) (green) or Hoechst dye for nuclear staining (blue) on a Zeiss Axio Observer Z1 fluorescent microscope. Graph showing relative average total cell fluorescence units per 8 microscopic fields. There was a 3.8-fold increase in fluorescence when comparing parental to resistant cells in the absence of EGF in H2170 cells.</p