Differential regulation of Annexin A1, A2, A5 by p14ARF-p53 occurs in MCF-7 but not in U2OS osteosarcoma cells.

<p>p14ARF expression was induced in MCF-7p14ARF and U2OSp14ARF cells by the addition of 5mM IPTG at? 15h 24h and 72h, PBS was added to control cells. Expression and localization of p14ARF expression in (A) U2OS cells, (B) MCF-7 pre- and post IPTG induction using immunofluorescence microscopy. (C) Top panel shows a representative western blot analysis of annexin A1 and A2 protein expression in MCF-7 and U2OS cells at 24h and 72h time points after p14ARF induction. β-actin was used as a loading control. The MCF-7p14ARF and U2OSp14ARF protein samples shown are from the same western blot. Bottom panel, P21, a p53 transcriptional target, was used to confirm p53 activation in the cell lysates. β-actin was used as a loading control. (D) Gene expression of p14ARF was induced by the addition of 5mM IPTG for 15h in U2OSp14ARF cells. Vehicle control contained PBS in place of IPTG. Transcriptional regulation of ANXA1, ANXA2, ANXA5 and ANXA6 was analyzed at 15h post p14ARF induction using RT-qPCR. Data were normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data have been expressed as fold change in expression post p14ARF induction relative to the control (2^−ΔΔCt). Experiments were performed in duplicate in which each set of experiments contained technical triplicates. Statistical differences between groups were determined using a two tailed, paired t-test.</p

Differential regulation of Annexin A1, A2, A5 and A6 expression at the transcriptional level in MCF-7 breast cancer cells.

<p>p14ARF expression was induced by the addition of 5mM IPTG for 15h. Quantitation of ANXA1, ANXA2, ANXA5 and ANXA6 expression was analyzed at 15h post p14ARF induction using the Taqman fast master mix and pre-optimized primer and probe sets. Data were normalized to levels of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data have been expressed as fold change in expression post p14ARF induction by IPTG at the 15h time point relative to control (2^−ΔΔCt). Experiments were performed in duplicate in which each set of experiments contained technical triplicates. Statistical differences between groups were determined using a two tailed, paired t-test. *p < 0.02, **p < 0.003 respectively.</p

Schematic representation of the p53/p21/annexin/S100A interactome.

<p>TP53 and CDNK1A, and their association with ANXA A1, A2, A4, A5, A6, A7, A9, A11 and S100A -6, -10, -11, -13 and -14 were analyzed using the STRING database (v10.0) and the Cytoscape Mimi PLUGIN. (A) Each node represents all the proteins produced by a single protein-coding gene locus, whereby a small node signifies a protein of unknown 3D structure and a large node denotes a protein where some 3D structure is known or predicted. Colored nodes indicate query proteins and first shell interactors. Colored edges (lines) represent interactions between proteins: light blue—known interactions from curated databases; purple—experimentally determined interactions; green—predicted interactions between gene neighborhood; red—gene fusions; dark blue—gene co-occurrence; light green–represents text mining; black—co-expression; grey—protein homology. (B) and (C) Highlighted in yellow are the direct common interactions between CDNK1A and TP53. The annexins and S100A interactions are highlighted in grey. Linkages were analyzed using two methods. (B) TP53 interactions: black lines, CDKN1A red lines. (C) TP53 red lines and CDKN1A black lines (summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169925#pone.0169925.s002" target="_blank">S2 Table</a>).</p

Cytoscape (GeneMANIA) analysis of Annexin and associated S100A partner overexpression: defining the consequent physical interactions, co-localization and genetic interactions post p14ARF induction.

<p>Cytoscape (GeneMANIA) analysis of Annexin and associated S100A partner overexpression: defining the consequent physical interactions, co-localization and genetic interactions post p14ARF induction.</p

Effect of p53 induced upregulation of all annexin and S100A genes (annexin expression profile) on ER+ patient prognosis.

<p>Kaplan-Meier analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169925#pone.0169925.ref038" target="_blank">38</a>] was used to predict breast cancer patient survival (RFS, DMSF and OS) post p53-upregulation of all combined annexins/S100A genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169925#pone.0169925.g008" target="_blank">Fig 8A</a>). A comparison was made between ER+ patients (B), and a sub-set of patients with ER+/p53wt (C). A hazard ratio with 95% confidence intervals and the log-rank P-value (P<0.05) was determined for differences in survival for each treatment option outcome and the results are represented as a heat chart: green = positive prognosis; pink = negative prognosis and yellow neutral (the median was used as a cutoff). The number of patients in each treatment sample is shown. (D) and (E) show comparative representative Kaplan-Meier plots of relapse free survival (RFS) for ER+ (D) and ER+/p53wt+ (E) patients treated with Endo + chemo therapies. The x-axis shows the number survival months from diagnosis. The red line represents patients with high annexin/S100A expression, and the black line represents patients with low annexin/S100A expression.</p

Effect of p53 induced upregulation of individual annexins on patient prognosis.

<p>Kaplan-Meier analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169925#pone.0169925.ref038" target="_blank">38</a>] was used to predict breast cancer patient survival (RFS, DMSF and OS) post p53-upregulation of individual annexins: ANXA1, ANXA2, ANXA4, ANXA6, ANXA9 and S100A10, S100A11 and S100A13. A comparison was made between ER+ patients (A), and a sub-set of patients with ER+/p53wt (B). A hazard ratio of 95% confidence intervals and the log-rank P-value (P<0.05) was determined for differences in survival for each treatment option outcome and the results are represented as a heat chart: green = positive prognosis; pink = negative prognosis and yellow neutral (the median was used as a cutoff). The number of patients in each treatment sample is shown. Abbreviations: RFS = Relapse free survival; DMFS = Distant metastasis free survival; OS = Overall survival; UNTR = untreated; Endo = endocrine; Tam = Tamoxifen, C and chemo = chemotherapy.</p

Linear regression analysis of Annexin and S100A protein expression 24h and 72h post p14ARF-p53-p21 activation.

<p>MCF-7 cells were treated with IPTG (5mM) to induce the p14ARF-p53 signaling pathway. A filtered set of 1265 proteins was analyzed for two independent biological experiments with technical replicates at 24h and 72h post activation. The correlation coefficient value for the 24h and 72h data for biological duplicate experiments showed a strong correlation (0.79 and 0.72 respectively). The black box highlights proteins significantly over-expressed p<0.05) post p14ARF-p53-p21 activation at 24h and maintained at 72h. The green box highlights proteins significantly downregulated at 24h and maintained at 72h post treatment. The red box highlights proteins significantly upregulated at 72h. The red cubes represent annexin proteins (A1, A2, A4, A6) significantly upregulated at 24h and maintained at 72h; the black cube represents annexin A9 significantly upregulated at 72h (P<0.05). Yellow cubes represent S100A10, S100A11 and S100A13 proteins significantly upregulated at 24h and maintained at 72h. Annexins A5, A7, A11, S100A6 and S100A14 are expressed and not regulated (between ratios 0.8–1.1). Inset: Western blot shows the expression of p14ARF and ER status in MCF-7 cells pre- and post IPTG and β-estradiol treatment at 24 h.</p

p53/p21/annexin/S100A interactome scores for protein-protein interactions from the STRING database.

<p>p53/p21/annexin/S100A interactome scores for protein-protein interactions from the STRING database.</p

Annexin protein function(s) analyzed using the MetScape Plugin in Cytoscape.

<p>Annexin protein function(s) analyzed using the MetScape Plugin in Cytoscape.</p