AF1q: A Novel Mediator of Basal and 4-HPR-Induced Apoptosis in Ovarian Cancer Cells

<div><h3>Background</h3><p>Fenretinide (4-HPR) is a synthetic retinoid that exhibits potent antitumor and chemopreventive activities against different malignancies, including ovarian tumors. We previously showed that in ovarian cancer cells, 4-HPR induces apoptosis through a signaling cascade starting from reactive oxygen species (ROS) generation and involving endoplasmic reticulum (ER) stress response, Jun N-terminal Kinase (JNK) activation, and induction of the proapoptotic PLAcental Bone morphogenetic protein (PLAB). Since recent studies have shown that the oncogene ALL1-fused from chromosome 1q (AF1q), a retinoic acid target gene, is implicated in apoptosis induction by several therapeutic agents, we investigated its possible involvement in the apoptosis induced by 4-HPR in ovarian cancer cells.</p> <h3>Methodology/Principal Findings</h3><p>Protein expression analysis, performed in ovarian cancer cells and extended to other histotypes (breast, neuroblastoma, and cervical), revealed that 4-HPR enhanced AF1q expression in cancer cells sensitive to the retinoid but not in resistant cells. Through gene silencing, AF1q was found functionally involved in 4-HPR-induced apoptosis in A2780, an ovarian cancer cell line highly sensitive to retinoid growth inhibitory and apoptotic effects. Inhibition of the signaling intermediates of the 4-HPR apoptotic cascade showed that AF1q upregulation was depended on prior generation of ROS, induction of ER stress response, JNK activation, and PLAB upmodulation. Finally, we found that direct overexpression of AF1q, in the absence of external stimuli, increased apoptosis in ovarian cancer cell lines.</p> <h3>Conclusions/Significance</h3><p>The study expands the knowledge of the 4-HPR mechanism of action, which has not yet been completely elucidated, identifying AF1q as a novel mediator of retinoid anticancer activity. In addition, we demonstrate, for the first time, that AF1q plays a role in the onset of basal apoptosis in ovarian cancer cells, thus providing new information about the activity of this protein whose biologic functions are mostly unknown.</p> </div

Relationship between AF1q upregulation and 4-HPR-induced signaling cascade.

<p>Western blot analysis for AF1q expression in A2780 cells treated for 24 hours with 5 µM 4-HPR, with or without 100 µM vitamin C (A), 10 µM salubrinal (B), or 10 µM SP600125 (C). (D) Western blot analysis for AF1q expression in A2780 cells stably transfected with a plasmid containing a PLAB siRNA or a scrambled nonsilencing siRNA following addition of 5 µM 4-HPR for 24 hours. As a control for loading, the blots were incubated with actin antibody.</p

Effects of different retinoids on AF1q expression in A2780 cells.

<p>Western blot analysiso for AF1q expression and caspase-3 cleavage in A2780 cells treated for 24 hours with 10 µM RA or 4-MPR, or 3 µM 4-oxo-4-HPR. As a control for loading, the blot was incubated with actin antibody.</p

Effects of 4-HPR on AF1q expression in A2780 and A2780/HPR cells.

<p>Western blot analysis for AF1q expression in A2780 (A) and A2780/HPR (B) cells treated for 24 hours with 5 and 10 µM 4-HPR. As a control for loading, the blots were incubated with actin antibody.</p

Role of AF1q upmodulation in 4-HPR-induced apoptosis.

<p>Western blot analysiso for AF1q expression, caspase-3 cleavage, and BAD expression in A2780 cells transiently transfected with a plasmid containing a AF1q siRNA or a scrambled nonsilencing siRNA following addition of 5 µM 4-HPR for 24 hours. As a control for loading, the blot was incubated with actin antibody.</p

Effect of AF1q overexpression in the onset of basal apoptosis.

<p>(A) Immunofluorescence analysis of A2780 cells transiently transfected with GFP alone (upper panels) or GFP-tagged AF1q vector (AF1q-GFP) (lower panels). After 48 hours, GFP and AF1q-GFP cells were stained with Hoechst 33342 and nuclear morphology was examined with a fluorescent microscope. Cells of interest are marked by arrows. Cells with condensed and fragmented nuclei were identified and scored as apoptotic cells. One experiment representative of three is shown. The scale bar represents 10 µm. (B) Apoptosis was represented as percentage of apoptotic cells per 100 green fluorescent cells (at least 200 cells per sample) in A2780 (left panel) and OVCAR-3 (right panel) cells transfected as in (A). Data represent the mean±S.D. of three independent experiments. Asterisks indicate significant difference (P<0.05).</p

Scheme showing proposed cascade of events involved in 4-HPR-induced growth inhibitory effect.

<p>4-HPR induces apoptosis through a signaling cascade involving oxidative stress, ER stress response, JNK activation, overexpression of the proapoptotic protein PLAB, and AF1q upregulation.</p

Boletín de Segovia: Número 127 - 1865 octubre 20

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Additional file 3: Figure S1. of Proposal of supervised data analysis strategy of plasma miRNAs from hybridisation array data with an application to assess hemolysis-related deregulation

Workflow of the strategy used for sample processing, data pre-processing and supervised data analyses. (TIF 148 kb

Additional file 5: Table S2. of Proposal of supervised data analysis strategy of plasma miRNAs from hybridisation array data with an application to assess hemolysis-related deregulation

Class comparison results in the training set with raw and ratio data. List of miRNAs and miRNA ratios significantly differentially expressed according to t- or AD test. The table also shows the class comparison results obtained on the validation set for the same miRNAs and miRNA ratios. Abbreviations: 'med': median of miRNA expression values; 'FC': miRNA fold change; 't.p': p-value at t-test; 'AD.p': p-value at AD test; 't.pFDR': False Discovery Rate (FDR)-adjusted p-value at t-test; 'AD.pFDR': FDR-adjusted p-value at AD test. (XLS 2059 kb