Chronic oxidative stress promotes H2AX protein degradation and enhances chemosensitivity in breast cancer patients (2016)
Figure 5

Figure 5 - Chemotherapy reduces H2AX protein levels

A Gene Set Enrichment Analysis (GSEA) performed by using NRF2-specific gene signature showing significant enrichment of this gene set in TN patients after chemotherapy. P-value refers to false discovery rate (FDR) q-value.

B Left, Representative views of H2AX and γ-H2AX immunostaining in TN tumours (N = 22) before and after successive cycles of chemotherapy. Scale bars = 100 μm. Zoom views are focused on the epithelial cells. Scale bars = 25 μm. Right, Scatter plots of H2AX, γ-H2AX histological scores (Hscores) and γ-H2AX/H2AX ratio evaluated from immunostaining in the epithelium, before and after chemotherapy.

C Correlation plot between Cisplatin IC50 (half maximal inhibitory concentration) and H2AX protein level defined by western blots in BC cell lines (MCF7, BT474, MDA361, SKBr3, MDA453, MDA231, HCC70). Correlation coefficients σ and P-value are based on Spearman’s rank correlation test.

D Up, Representative western-blot showing H2AX protein levels from whole cell extracts in HCC70 TN BC cell line transfected with non-targeting siRNA (siCtrl) or an siRNA directed against H2AX (siH2AX). Actin is used as internal control for protein loading. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above).

E Bar plot showing the percentage (%) of apoptotic cells as assessed by flow-cytometry analysis of annexin V and DAPI staining in HCC70 cell line transfected with non-targeting siRNA (siCtrl) or an siRNA directed against H2AX (siH2AX), and next treated with cisplatin for 72 hrs.

F Box plot showing the impact of H2AX decrease on the therapeutic response of TN BC patients (N = 22). Reduction of H2AX protein level was assessed by the difference of H2AX Hscores before and after chemotherapy (as evaluated in B), for each patient (values in Log2). Only patients who have experienced an incomplete response have been taken into account. After treatment H2AX staining was evaluated only on residual tumour cells. The therapeutic response was monitored by pathologists through evaluation of tumour residual mass and detection of tumour cells inside lymph nodes.

G Kaplan-Meier curves showing specific overall survival (Left) and progression-free survival (Right) of TN patients with respect to major (N=15) or minor (N=7) H2AX protein level decrease, observed upon chemotherapy. Iterative analyses were performed on ΔLog2 H2AX Hscore to find optimal threshold that maximally discriminates the major and minor H2AX decrease (respectively 2/3 and 1/3 of patients).

H Scatter plots showing the number of cleaved caspase 3-positive cells / mm2 in TN BC, categorized according to major- or minor-H2AX protein level decrease after chemotherapy.

I Scatter plots showing variation of NRF2 histological score in patients with major-H2AX decrease before and after chemotherapy.

J Box plots showing expression levels of representative NRF2-target genes in TN tumours with minor- or major-H2AX decrease, as indicated. Lines indicate Means and whiskers show 10-90 percentiles. ACOT7: Acyl-CoA Thioesterase 7; PGD: Phosphogluconate Dehydrogenase. AHR: Aryl Hydrocarbon Receptor; JAG1: Jagged1. P-values are from t-test.

In all panels, data are shown as mean ± s.e.m. (B,D,E,F,H). P values are based on paired t-test (B,I), on Student’s t-test (D,E,H,J) on Kruskal-Wallis test (F) and on Log-rank test (G). NS stands for not significant.