The BH3-only protein NOXA serves as an independent predictor of breast cancer patient survival and defines susceptibility to microtubule targeting agents

Abstract

Breast cancer (BC) treatment frequently involves microtubule-targeting agents (MTAs), such as paclitaxel, that arrest cells in mitosis. Sensitivity to MTAs is defined by a subset of pro- and anti-apoptotic BCL2 family proteins controlling mitochondrial apoptosis. Here, we aimed to determine their prognostic value in primary tumour samples from 92 BC patients. Our analysis identified high NOXA/PMAIP mRNA expression levels as an independent prognostic marker for improved relapse-free survival (RFS) and overall survival (OS) in multivariate analysis in BC patients, independent of their molecular subtype. Analysis of available TCGA datasets of 1060 BC patients confirmed our results and added a clear predictive value of NOXA mRNA levels for patients who received MTA-based therapy. In this TCGA cohort, 122 patients received MTA-treatment and high NOXA mRNA levels correlated with their progression-free interval (PFI) and OS. Our follow-up analyses in a panel of BC cell lines of different molecular subtypes identified NOXA protein expression as a key determinant of paclitaxel sensitivity in triple-negative breast cancer (TNBC) cells. Moreover, we noted highest additive effects between paclitaxel and chemical inhibition of BCLX, but not BCL2 or MCL1, documenting dependence of TNBC cells on BCLX for survival and paclitaxel sensitivity defined by NOXA expression levels.

Fig. 1. mRNA expression analysis of BCL2 family members in 10 non neoplastic and 92 neoplastic breast tissues.

mRNA expression of A BCL2 (p = 0.039), B BCLX (p = 0.035), MCL1 (p = 0.001), BID (p = 0.020) and NOXA (p = 0.002) and C PUMA, BIM, BAX, BAK, BOK, BCLW and BCLB. Extreme values are marked with asterisks, outliers with circles.

Fig. 2. Kaplan–Meier survival analysis in the TCGA cohort.

A PFI and B OS based on BCL2 mRNA-expression in 471 BC patients treated with chemotherapy other than MTA. C DFI and D PFI based on MCL1 mRNA-expression in 112 BC patients treated with MTA chemotherapy. E PFI and F OS based on NOXA mRNA-expression in 112 BC patients treated with MTA chemotherapy.

Fig. 3. BCL2 protein family expression in different BC cell lines.

Different BC cell lines were subjected to western blotting using the indicated antibodies recognising A anti- and B pro-apoptotic BCL-2 proteins. HSP90 was used as a loading control.

Fig. 4. Paclitaxel increases the sensitivity towards BH3 mimetics.

A The indicated cell lines were treated with 50 nM paclitaxel alone or in combination with 1 µM of BH3mimetics for 48 h. B–E TNBC cell lines were treated with graded concentrations of BH3-mimetics in combination with a predetermined fixed concentration of paclitaxel for 48 h. Metabolic activity was calculated by setting the metabolic activity in relation to the DMSO control. Heatmaps show mean values of metabolic activity ranging from 0 to 100% as assessed by MTT-assay. A All cell lines (n = 3–4), B MDA-MB-231 (n = 6), C HS-578-T (n = 4), D Cal-51 (n = 6), E BT20 (n = 6).

Fig. 5. TNBC cell lines treated with paclitaxel show MCL1 and NOXA co-degradation.

A–C TNBC proficient (Parental) or deficient in NOXA (NOXA-KO) were left asynchronous (Asyn.) or synchronised with a double-thymidine block and released into 500 nM PTX. Cells were harvested in G2 and early M-phase (M); part of the M-phase cells was reseeded and harvested 5 h (M + 5 h) and 10 h (M + 10 h) later. Harvested cells were subjected to western analysis using the indicated antibodies.

Fig. 6. NOXA deletion protects TNBC from PTX and BH3-mimetic co-treatment.

Parental and two NOXA-KO clones of each TNBC cell line were treated with or without 5000 nM of the indicated BH3-mimetics alone (DMSO) or in combination with 20 or 50 nM PTX for 48 h. Heatmaps are showing the mean value of metabolic activity assessed with MTT-assay. A MDA-MB-231 (n = 5), B HS-578-T (n = 4), C Cal-51 (n = 5).