Epigenetic control of the basal-like gene expression profile via Interleukin-6 in breast cancer cells

Abstract

Background: Basal-like carcinoma are aggressive breast cancers that frequently carry p53 inactivating mutations, lack estrogen receptor-α (ERα) and express the cancer stem cell markers CD133 and CD44. These tumors also over-express Interleukin 6 (IL-6), a pro-inflammatory cytokine that stimulates the growth of breast cancer stem/progenitor cells.

Results: Here we show that p53 deficiency in breast cancer cells induces a loss of methylation at IL-6 proximal promoter region, which is maintained by an IL-6 autocrine loop. IL-6 also elicits the loss of methylation at the CD133 promoter region 1 and of CD44 proximal promoter, enhancing CD133 and CD44 gene transcription. In parallel, IL-6 induces the methylation of estrogen receptor (ERα) promoter and the loss of ERα mRNA expression. Finally, IL-6 induces the methylation of IL-6 distal promoter and of CD133 promoter region 2, which harbour putative repressor regions.

Conclusion: We conclude that IL-6, whose methylation-dependent autocrine loop is triggered by the inactivation of p53, induces an epigenetic reprogramming that drives breast carcinoma cells towards a basal-like/stem cell-like gene expression profile.

Figure 1

p53 inactivation up-regulates IL-6 expression and induces the loss of methylation at IL-6 proximal promoter. a) IL-6 ELISA assay and RT-PCR analysis of IL-6 mRNA level in MCF-7 and MDA-MB231 cells; b) quantitative evaluation of IL-6prox methylation-specific PCR analysis (MS-PCR) in MCF-7 and MDA-MB231 cells. "U" stands for Unmethylated and "M" Methylated specific PCR product; c) Western blot analysis of p53 protein level, RT-PCR analysis of Bnip3 and p21Waf1 mRNA level and luciferase assay of p53 responsive reporter plasmid (p53-Luc) in MCF-7 cells transduced with empty (pBabe) or p53 mini-protein (p53D) retroviral vector; d) IL-6 ELISA assay, RT-PCR analysis of IL-6 mRNA level and quantitative evaluation of IL-6prox MS-PCR analysis in pBabe/p53 D cells. β-Actin was assessed as quantitative control for RT-PCR and Western Blot. Student t test, *p < 0.05, ^#p < 0.005. NSF: Non Specific Fragment

Figure 2

Reduced methylation at IL-6prox and induction of IL-6 expression by exogenous or autocrine IL-6. a) IL-6 ELISA assay, RT-PCR analysis of IL-6 mRNA level and quantitative evaluation of IL-6prox MS-PCR analysis in MCF-7 cells in presence/absence of IL-6 (10 ng/ml, 48 h) or 5AzadCytidine (5azadC, 15 μM, 48 h); b) IL-6 ELISA assay, RT-PCR analysis of IL-6 mRNA level, quantitative evaluation of IL-6prox MS-PCR analysis and IL-6 promoter driven luciferase assay (IL6P-Luc) in MDA-MB231cells and p53 D cells in presence/absence to αIL-6 (1.5 μg/ml, 48 h); C) IL-6 ELISA assay, RT-PCR analysis of IL-6 mRNA level and quantitative evaluation of IL-6prox MS-PCR analysis in MCF-7 derived mammospheres (MCF-7S) or MCF-7 adherent cells; β-Actin was assessed as quantitative control for RT-PCR analysis. Student t test, *p < 0.05; ^#p < 0.005. n.a.: not assessed.

Figure 3

Regulation of CD133, CD44 and ERα mRNA by exogenous or autocrine IL-6. RT-PCR analysis of CD133, CD44, ERα mRNA level in: a) MCF-7 cells in presence/absence of IL-6 (10 ng/ml, 48 h) or 5azadC (15 μM, 48 h), b) p53 D cells and MDA-MB231 cells in presence/absence of αIL-6 (1.5 μg/ml, 48 h) or transfected with scr/siIL-6 (1 μg, 48 h). β-Actin of panel a) and b) are reported in panels 2a and 2b, respectively.

Figure 4

Decrease in methylation at CD133 promoter 1 (CD133p1) by exogenous or autocrine IL-6. Quantitative evaluation of CD133p1 MS-PCR analysis in: a) MCF-7 cells in presence/absence of IL-6 (10 ng/ml, 48 h) or 5azadC (15 μM, 48 h); b) pBabe/p53 D and scr/sip53 transfected (1 μg, 48 h) MCF-7 cells, c) p53 D cells in presence/absence of αIL-6 (1.5 μg/ml, 48 h) or transfected with scr/siIL-6 (1 μg, 48 h). NSF Non Specific Fragment.

Figure 5

Reduction of methylation at CD44 proximal promoter (CD44p) by exogenous or autocrine IL-6. Quantitative evaluation of CD44p MS-PCR analysis in: a) MCF-7 cells in presence/absence of IL-6 (10 ng/ml, 48 h) or 5azadC (15 μM, 48 h); b) pBabe/p53 D and scr/sip53 transfected (1 μg, 48 h) cells, c) p53 D cells in presence/absence of αIL-6 (1.5 μg/ml, 48 h) or transfected with scr/siIL-6 (1 μg, 48 h).

Figure 6

Gain of ERα promoter (ERαp) methylation by autocrine or exogenous IL-6. Quantitative evaluation of ERαp MS-PCR analysis in: a) MCF-7 cells in presence/absence of IL-6 (10 ng/ml, 48 h) or 5azadC (15 μM, 48 h); b) p53 D and MDA-MB231 cells in presence/absence of αIL-6 (1.5 μg/ml, 48 h) or scr/siIL-6 (1 μg, 48 h). PD: Primer Dimers, NSF Non Specific Fragment.

Figure 7

IL-6dist gains methylation in response to IL-6 and contains putative repressive IRF-1/IRF-2 binding sites. Quantitative evaluation of IL-6dist MS-PCR analysis in: a) MCF-7 cells in presence/absence of IL-6 (10 ng/ml, 48 h) or 5azadC (15 μM, 48 h), b) p53 D and MDA-MB231cells in presence/absence of αIL-6 (1.5 μg/ml, 48 h); c) Luciferase assay of MCF-7 cells transfected with IL6P-Luc in presence/absence of IL-6 (10 ng/ml), and pIRF-1 or pIRF-2 (1 μg each, 24/48 h) in presence/absence of IL-6 (10 ng/ml, lower panel). Student t test, *p < 0.05; ^#p < 0.005.

Figure 8

CD133p2 gains methylation in response to IL-6 and contains putative repressive ERα binding sites. Quantitative evaluation of CD133p2 MS-PCR analysis in: a) MCF-7 cells in presence/absence of IL-6 or 5azadC (10 ng/ml and 15 μM, 48 h, respectively), b) p53 D and MDA-MB231 cells in presence/absence of αIL-6 (1.5 μg/ml, 48 h); c) RT-PCR analysis of ERα and CD133 mRNA level and quantitative evaluation of CD133p2 MS-PCR analysis in MCF-7 cells exposed to Tamoxifen (4OHT, 3 μM, 48 h or vehicle) or to scr or ERα specific siRNA (siERα, 1 μg, 48 h); β-Actin was assessed as quantitative control in RT-PCR analysis. PD: primer Dimers.