Autophagy is associated with a robust specific transcriptional signature in breast cancer subtypes

Abstract

Previous works have described that autophagy could be associated to both pro- and anti-cancer properties according to numerous factors, such as the gene considered, the step of autophagy involved or the cancer model used. These data might be explained by the fact that some autophagy-related genes may be involved in other cellular processes and therefore differently regulated according to the type or the grade of the tumor. Indeed, using different approaches of transcriptome analysis in breast cancers, and further confirmation using digital PCR, we identified a specific signature of autophagy gene expression associated to Luminal A or Triple Negative Breast Cancers (TNBC). Moreover, we confirmed that ATG5, an autophagy gene specifically expressed in TNBC, favored cell migration, whereas BECN1, an autophagy gene specifically associated with ER-positive breast cancers, induced opposite effects. We also showed that overall inhibition of autophagy promoted cell migration suggesting that the role of individual ATG genes in cancer phenotypes was not strictly dependent of their function during autophagy. Finally, our work led to the identification of TXNIP1 as a potential biomarker associated to autophagy induction in breast cancers. This gene could become an essential tool to quantify autophagy levels in fixed biopsies, sort tumors according to their autophagy levels and determine the best therapeutic treatment.

Keywords: autophagy; breast cancer; gene expression signature; transcriptome.

Figure 1. Breast cancer subgroups are associated with a specific autophagy gene expression signature.

2691 and 2806 samples, respectively, and the most variable autophagy genes were hierarchically classified according to correlations between gene expressions normalized from HG-U133 plus2 arrays A. or microarray “A” B. and BC groups. The color Dark red means relative high expression while the color Dark blue means relative low expression. C. Venn diagrams comparing the identified genes identified in HG-U133 plus2 arrays and microarrays in regards to BC subtypes. D. Heatmap representing the most variable autophagy genes described from the data extracted from 1022 normalized BC RNA-seq (TCGA).

Figure 2. Confirmation of the specific autophagy gene expression signature using ddPCR in an independent cohort of BC.

A. Expression quantification boxes built from the HG-U133 plus2 data for LC3B, ATG5 and IRF1 gene expressions (top). Confirmation of the results obtained using ddPCR in an independent cohort (bottom). B. Expression quantification boxes built from the HG-U133 plus2 data for BECN1, ATG2B and ULK1 gene expressions (top). Confirmation of the results obtained using ddPCR in an independent cohort (bottom).

Figure 3. The restricted specific autophagy gene expression signature in BC groups efficiently discriminates BC cell lines.

A. Heatmap representing the selected autophagy signature (LC3B, ATG5, IRF1, ULK1, ATG2B, BECN1) which was obtained from data of normalized HG-U133 plus2 with BC cell lines. B. The score of -6 to +6 according to heatmap intensity (from blue to red) was calculated as follow: values of IRF1, LC3B, ATG5 minus values of ATG2B, BCN1. TNBC and ER cell lines were perfectly discriminated with this score (bottom). C. RT-qPCR confirmation of ATG2B, BECN1, LC3B, ATG5, IRF1 and ULK1 expressions.

Figure 4. The modulation of gene expression of genes from the specific autophagy signature in BC groups affects cancer-related phenotypes.

A. Overexpression or inhibition of LC3B, ATG5, BECN1, ATG2B expressions were confirmed using RT-qPCR, Western-blotting or flux cytometry analysis of MCF-7 or MDA-MB-231 cells. B-E. Cell proliferation monitored using the Incucyte technology in MCF-7 or MDA-MB-231 cells after overexpression or inhibition of the expression of the selected genes. F-G. Cell migration measured using a wound healing assay after 8 h in MCF-7 or MDA-MB-231 cells after overexpression or inhibition of the expression of the selected genes.

Figure 5. Autophagy directly affects cancer-related phenotypes.

A. Efficient basal autophagy and EBSS-induced autophagy weres confirmed using WB (LC3B-II accumulation) in both MCF-7 and MDA-MB-231 cells. B. Cell migration measured using a wound healing assay after 8 h in MCF-7 or MDA-MB-231 cells, previously treated, or not, with BafA1 (autophagy blockage) and/or EBSS (autophagy induction). C. Effect of the inhibition of the expression of the selected genes on autophagy flux.

Figure 6. ATG BC signature and TXNIP expressions are markers of autophagy induction.

A. Heatmap with the 40 most affected ATG-related genes in MDA-MB-231 cells treated with EBSS for 0 to 48 h to induce autophagy. B-E. B. Modulation of mRNA expression level of LC3B and ATG5 during autophagy in MDA-MB-231. C. A progressive increase in TXNIP expression was confirmed using RT-qPCR in MCF7 and MDA-MB-231. D. mRNA expression level of TXNIP demonstrating than TXNIP expression is independent of BC subgroup. E. Kaplan Meier data analysis revealed a significant better prognosis for patients presenting a high TXNIP expression.