沃度保兒謨ニ因セル濕疹

Evaluation of CNA-IC50 correlations by known cancer driver mutations. Distributions of PCCs for cancer cell lines with mutated or wild-type proto-oncogenes or tumor suppressors as depicted in each figure. The rank position of negatively correlated drugs (p < 0.10) is shown. (EPS 975 kb

Breast Cancer Genes PSMC3IP and EPSTI1 Play a Role in Apoptosis Regulation

<div><p>A key element to delineate the biology of individual tumors is the regulation of apoptosis. In this work, we functionally characterize two breast cancer associated genes, the proteasome 26S subunit ATPase 3 interacting protein (PSMC3IP) and the epithelial-stromal interaction 1 (EPSTI1), to explore their potential apoptotic role in breast cancer. We first explore the existence of direct physical interactions with annotated BC-apoptotic genes. Based on the generated interaction network, we examine several apoptotic markers to determine the effect of PSMC3IP and EPSTI1 gene expression modulation in two different human breast cancer cell lines to suggest potential molecular mechanisms to unveil their role in the disease. Our results show that PSMC3IP and EPSTI1 are able to modulate the extrinsic apoptotic pathway in estrogen receptor positive and triple negative breast cancer cell lines, highlighting them as potential therapeutic targets.</p></div

Cell viability and recovery.

<p>Cell viability was determined by MTT absorbance assays <b>(A)</b> Histograms showing the viability of PSMC3IP or EPSTI1-overexpressing MDA-MB-231 cells and <b>(B)</b> MCF-7 cells under TRAIL-induced conditions. Based on empty vector (MYC-tag) as a negative control, we do not observe a significant recovery of apoptosis-induced cells after gene overexpression. <b>(C)</b> Viability measurement of gene-depleted MDA-MB-231 cells reveals that EPSTI1 depletion reduces about 50% of viability as compared to siLUC negative control. <b>(D)</b> Intriguingly, in MCF-7 cells, both PSMC3IP and EPSTI1 depletion lead to a decreased viability under basal but not under TRAIL-treated conditions. XIAP was used as an anti-apoptotic reference in all experiments. <i>EPSTI1</i>-depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, cells were treated with TRAIL for 24h, at 80 or 100ng/mL respectively. Each bar represents the mean ±SD of three experiments performed in duplicate (*<i>P</i> <0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001 vs siLUC).</p

Expression of PSMC3IP and EPSTI1 in normal and breast cancer cell lines.

<p>We inspected the endogenous expression of PSMC3IP <b>(A)</b> and EPSTI1 <b>(B)</b> in two types of breast cancer cell lines, MDA-MB-231 and MCF-7, as compared to a normal breast epithelial cell line, MCF-10A. Estimated protein levels based on densitometry (right) of the immunoblots (left) show a PSMC3IP 19- and 15-fold expression in MDA-MB-231 and MCF-7 cells, while EPSTI1 only shows 1.9- and 1.3-fold in each cell line, respectively. Protein levels were normalized based on the loading control protein β-actin. (*<i>P</i> <0.05, **P<0.01, ***<i>P</i> <0.001 vs MCF-10A cells).</p

Caspase-8 activity modulation.

<p>Caspase-8 activity was quantified by measuring the chromophore levels released from caspase-8 cleaved substrates. Overexpression of PSMC3IP or EPSTI1 in TRAIL-treated MDA-MB-231 <b>(A)</b> and MCF-7 cells <b>(B)</b> decrease caspase-8 activity based on the MYC-tag empty transfection vector (Vector) as control. Caspase-8 activity was also measured after gene silencing in MDA-MB-231 <b>(C)</b> and MCF-7 <b>(D)</b> cells, under basal or TRAIL-treated conditions. Genes were silenced using specific siRNAs targeting <i>XIAP</i>, <i>PSMC3IP</i> or <i>EPSTI1</i> and siRNA against luciferase expression (siLUC) was used as a negative control. <i>EPSTI1</i>-depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. MDA-MB-231 and MCF-7 cells were treated with TRAIL for 24h at 80 or 100ng/mL, respectively. XIAP was used as an anti-apoptotic reference in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (*<i>P</i> <0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001 vs MYC-tag vector in overexpression assays and vs siLUCIFERASE in silencing).</p

TRAIL-induced apoptosis in breast cancer cells.

<p><b>(A)</b> MDA-MB-231 cells treated with the apoptosis inducing ligand TRAIL at 80ng/mL for 24h show a moderate decrease in cell viability while <b>(B)</b> MCF-7 cells treated with TRAIL at 100ng/mL.for 24h show a more pronounced decrease in viability. Each bar represents the mean ±SD of three experiments performed in duplicate (*<i>P</i> <0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001 vs untreated cells).</p

Caspase-3 activity modulation and analysis of cleaved PARP protein levels.

<p><b>(A)</b> Caspase-3 activity was measured by colorimetric quantification of fluorescent products released from caspase-3 cleaved <a href="http://substrates.in" target="_blank">substrates.in</a> TRAIL-treated MDA-MB-231 cells overexpressing XIAP, PSMC3IP or EPSTI1. None of the overexpressed genes was able to significantly decrease the activity relative to MYC-tag empty transfection vector (Vector) as control. <b>(B)</b> Caspase-3 activity was also measured in MDA-MB-231 cells under basal or TRAIL-treated conditions after gene silencing using specific siRNA targeting <i>XIAP</i>, <i>PSMC3IP</i> or <i>EPSTI1</i>. <b>(C)</b> Immunoblot analysis of cleaved PARP protein levels in gene-overexpressing MDA-MB-231 cells under TRAIL conditions, using MYC-tag transfection vector (Vector) as a negative control, reveals an attenuation of downstream apoptotic cascades. <b>(D)</b> This effect is much more pronounced in TRAIL-treated MCF-7 cells. <b>(E)</b> Analysis of cleaved PARP protein levels after gene silencing in MDA-MB-231 cells. siLUC was used as a negative control. <b>(F)</b> The same analysis in MCF-7 cells shows a more pronounced effect, even under basal conditions. <i>EPSTI1</i>-depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, MDA-MB-231 and MCF-7 cells were treated with TRAIL for 24h at 80 or 100ng/mL, respectively. XIAP was used as an anti-apoptotic reference in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (*<i>P</i> <0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001 vs MYC-tag vector in overexpression assays and vs siLUCIFERASE in silencing).</p

Additional file 5: Figure S3. of Cancer network activity associated with therapeutic response and synergism

The CNA-IC50 correlation differences are not observed when a random, null network model that preserves degree distribution and connectedness is analyzed. (PDF 148 kb

Additional file 2: Table S2. of Cancer network activity associated with therapeutic response and synergism

Normalized CNA values for all cancer cell lines. (XLSX 55 kb

Additional file 8: Table S4. of Cancer network activity associated with therapeutic response and synergism

Correlations of drug target gene expression and CNA values. (XLSX 42 kb