Increased macroH2A1.1 expression correlates with poor survival of triple-negative breast cancer patients

Abstract

Purpose: Epithelial-Mesenchymal Transition (EMT) features appear to be key events in development and progression of breast cancer. Epigenetic modifications contribute to the establishment and maintenance of cancer subclasses, as well as to the EMT process. Whether histone variants contribute to these transformations is not known. We investigated the relative expression levels of histone macroH2A1 splice variants and correlated it with breast cancer status/prognosis/types.

Methods: To detect differential expression of macroH2A1 variant mRNAs in breast cancer cells and tumor samples, we used the following databases: GEO, EMBL-EBI and publisher databases (may-august 2012). We extracted macroH2A1.1/macroH2A1 mRNA ratios and performed correlation studies on intrinsic molecular subclasses of breast cancer and on molecular characteristics of EMT. Associations between molecular and survival data were determined.

Results: We found increased macroH2A1.1/macroH2A1 mRNA ratios to be associated with the claudin-low intrinsic subtype in breast cancer cell lines. At the molecular level this association translates into a positive correlation between macroH2A1 ratios and molecular characteristics of the EMT process. Moreover, untreated Triple Negative Breast Cancers presenting a high macroH2A1.1 mRNA ratio exhibit a poor outcome.

Conclusion: These results provide first evidence that macroH2A1.1 could be exploited as an actor in the maintenance of a transient cellular state in EMT progress towards metastatic development of breast tumors.

Figure 1. Expression levels of macroH2A1 splice variants in breast cancer cell lines.

A-B- MacroH2A1.1, macroH2A1.2, and total macroH2A1 protein expression levels in five breast cancer cell lines. Quantification of each macroH2A1 splice variants, global macroH2A1 (A) and macroH2A1.1/macroH2A1, macroH2A1.2/macroH2A1 ratios (B) for each cell line normalized to GADPH is shown relative to the ZR-75 cell line. C-D- qPCR analysis of mRNA expression levels of macroH2A1, macroH2A1.1, macroH2A1.2 and macroH2A1 splice variants/macroH2A1 mRNA ratio. Each quantification was performed at minimum in biological triplicate. Expression levels were normalized to expression of RPLP0, and referred to the cell line ZR-75 as a sample calibrator. E- Analysis of expression data of macroH2A1 variants in 51 breast cancer cell lines on the basis of U133A array hybridization . Log2 macroH2A1, MacroH2A1.1 and macroH2A1.1/macroH2A1 values are determined and classified according to luminal or basal molecular subtype as defined in . Data of DU4475, HCC1008 and HCC1599 are included in the analysis with the molecular subtype assigned in the synthesis part of Table S1. The median of each subgroup is shown (grey bar). The reported p-values are the results of a two-tailed Mann-Whitney test.

Figure 2. High macroH2A1.1 expression level in breast cancer cell lines characterizes Claudin-low molecular subtype.

In two independent analyses, macroH2A1, macroH2A1.1 and macroH2A1.1/macroH2A1 mRNA ratios were determined for each cell line and classified according to claudin-low or non claudin-low molecular subtype assigned in the synthesis part of Table S1. The median of each subgroup are specified. In E-TABM-827 analysis, GI-101, HB4A, PMC42 and VP229 cell lines were omitted as the subgroup Basal A or B was not specified; HCC1509, MT3 and VP267 cell lines are omitted as their subtype were not assigned. The reported p-values are the result of a two-tailed Mann-Whitney test.

Figure 3. Analysis of expression data of macroH2A1 variants in breast cancer cell lines on the basis of Affymetrix Human Junction technology (E-MTAB-183[11]).

A- Log2 expression values of exons of H2AFY gene are represented by molecular subtype non claudin-low (non CL) and claudin-low (CL). The analysis for exons 6a/b are highlight in B. C- Log2 expression values of exons of H2AFY gene normalized to the one of exon 9 are represented by molecular subtype non claudin-low (non CL) and claudin-low (CL). The analysis for exons 6a/b are highlight in D. E- Splicing index values for exon 6a included in macroH2A1.1 splice variant are represented by molecular subtype. The median of each subgroup is shown (grey bar). The reported p-values are the results of a two-tailed Mann-Whitney test.

Figure 4. Overrepresentation of macroH2A1.1 correlated with mesenchymal features and induction of EMT features in HMLE cells is accompanied by an increase in macroH2A1.1/macroH2A1 mRNA ratio.

A–B Analysis of GSE16795 data set . MacroH2A1.1/macroH2A1 mRNA ratio are determined for each cell line and classified depending of the level of expression of E-cadherin (A); of the level of expression of Vimentin or N-cadherin (B). The median of macroH2A1.1/macroH2A1 values of each subgroup are specified. The reported p-values are the result of a two-tailed Mann-Whitney test. C- macroH2A1.1/macroH2A1 mRNA ratios were determined in immortalized human mammary epithelial cells with inhibiting E-cadherin function either shRNA-mediated (GSE9691 and E-MTAB-884 [45]) or by expression of a truncated form of E-cadherin (ΔN-Ecad) (GSE9691 [34]) and compared. D- MacroH2A1.1/macroH2A1 mRNA ratios were determined for different breast cancer stem cell-like lines which overexpressed one EMT inducer, i.e. TGFβ, Twist, Gsc or Snail, and compared (GSE24202 [6]). The median of each subgroup is shown (grey bar). The reported p-values are the results of a two-tailed Mann-Whitney test. E- Upper panel- genomic sequence of H2AFY gene encompassing exon 6a. Potential ESRP1 and RBFox2 binding sites are represented with grey background and white letters. Bottom panel- Hypothetical schema for alternative splicing of exon 6a included in the macroH2A1.1 splice variant. Two cellular backgrounds are represented, epithelial with exon skipping of exon 6a related to the inhibitory binding of ESRP1 to EBSI, and mesenchymal with exon inclusion of exon 6a potentiated by binding of RBFox2 to RBSE.

Figure 5. Kaplan Meier analysis according to the macroH2A1.1 mRNA ratio.

A- The 383 TNBC samples from the GSE31519 cohort were stratified according to the macroH2A1.1/macroH2A1 mRNA ratio. Kaplan Meier analysis of event free survival of 383 samples with follow up information is shown. Positive and negative predictive values (PPV and NPV) of macroH2A1.1/macroH2A1 mRNA ratio in the cohort are specified. B- Multivariate Cox proportional hazards models of disease-free survival.

Figure 6. Kaplan Meier analysis according to the macroH2A1.1 mRNA ratio in untreated and treated sub-cohort.

A- The 259 TNBC untreated samples from the GSE31519 cohort were stratified according to the macroH2A1.1/macroH2A1 mRNA ratio. Kaplan Meier analysis of event free survival of 259 samples with follow up information is shown. PPV and NPV of macroH2A1.1/macroH2A1 mRNA ratio in the untreated cohort are specified. B- The 87 TNBC adjuvant chemotherapy treated samples from the GSE31519 cohort were stratified according to the highest macroH2A1.1/macroH2A1 mRNA ratio. Kaplan Meier analysis of event free survival of 87 samples with follow up information is shown. PPV and NPV of macroH2A1.1/macroH2A1 mRNA ratio in the treated cohort are specified.