Prognosis of hormone-dependent breast cancers: implications of the presence of dysfunctional transcriptional networks activated by insulin via the immune transcription factor T-bet

Abstract

Estrogen receptor alpha (ERalpha)-positive breast cancers that co-express transcription factors GATA-3 and FOXA1 have a favorable prognosis. These transcription factors form an autoregulatory hormonal network that influences estrogen responsiveness and sensitivity to hormonal therapy. Disruption of this network may be a mechanism whereby ERalpha-positive breast cancers become resistant to therapy. The transcription factor T-bet is a negative regulator of GATA-3 in the immune system. In this study, we report that insulin increases the expression of T-bet in breast cancer cells, which correlates with reduced expression of GATA-3, FOXA1, and the ERalpha:FOXA1:GATA-3 target gene GREB-1. The effects of insulin on GATA-3 and FOXA1 could be recapitulated through overexpression of T-bet in MCF-7 cells (MCF-7-T-bet). Chromatin immunoprecipitation assays revealed reduced ERalpha binding to GREB-1 enhancer regions in MCF-7-T-bet cells and in insulin-treated MCF-7 cells. MCF-7-T-bet cells were resistant to tamoxifen in the presence of insulin and displayed prolonged extracellular signal-regulated kinase and AKT activation in response to epidermal growth factor treatment. ERalpha-positive cells with intrinsic tamoxifen resistance as well as MCF-7 cells with acquired tamoxifen and fulvestrant resistance expressed elevated levels of T-bet and/or reduced levels of FOXA1 and GATA-3. Analysis of publicly available databases revealed ERalpha-positive/T-bet-positive breast cancers expressing lower levels of FOXA1 (P = 0.0137) and GATA-3 (P = 0.0063) compared with ERalpha-positive/T-bet-negative breast cancers. Thus, T-bet expression in primary tumors and circulating insulin levels may serve as surrogate biomarkers to identify ERalpha-positive breast cancers with a dysfunctional hormonal network, enhanced growth factor signaling, and resistance to hormonal therapy.

Figure 1. T-bet, GATA-3, and FOXA1 expression in primary breast cancer

A) Expression pattern of T-bet in ERα-positive and ERα-negative breast cancer. Gene expression levels from a published study (30) were extracted from Oncomine (www.oncomine.org). Difference in expression between two groups is statistically significant. B). ERα-positive breast cancers from the above study were classified into T-bet positive and T-bet negative subgroups based on significant differences in the expression levels. C). Expression levels of FOXA1 and GATA-3 in ERα+/T-bet- and ERα+/T-bet+ subgroups. D). T-bet expression negatively correlates with PR negativity. As in A, data was extracted from a published study (31).

Figure 2. Insulin alters T-bet, GATA-3, and FOXA1 expression in breast cancer cells

A) Insulin increases T-bet expression in MCF-7 cells. Cells were treated with insulin (50 nM) and/or E2 (0.1 nM) for indicated time and T-bet expression was measured by western blotting. B) The effect of insulin on GATA-3, FOXA1, and ERα expression. Cells were treated with insulin and/or E2 for 24 hours and the expression levels of different proteins were measured by western blotting. C) Densitometric scanning data of 3 or more experiments showing insulin-mediated significant increase in T-bet expression and reduction of GATA-3 and FOXA1 expression in MCF-7 cells. Mean and standard error of the mean are presented. D). Variable expression of T-bet, GATA-3, FOXA1, and ERα in luminal B cell lines. All luminal B cell lines (BT-474, MD-361, and ZR75-30) showed significantly lower levels of GATA-3 compared to the luminal A cell line T47-D. Note that insulin reduced FOXA1 expression in T47-D and ZR75-30 cell lines.

Figure 3. T-bet inhibits chromatin binding of ERα and E2-regulated gene expression

A) T-bet overexpression results in general reduction in GATA-3, FOXA1, and ERα expression. Expression levels of indicated transcription factors were measured in parental cells transduced with empty retrovirus (MCF-7p) or T-bet expressing virus (MCF-7-T-bet). Densitometric scanning data from two or more experiments normalized to the control β-Actin are presented. *p<0.001, MCF-7p vs MCF-7-T-bet. B) E2-inducible expression of XBP-1 and GREB-1 in MCF-7p and MCF-7-T-bet cells (left panel). Results of three or more experiments are presented (mean ±SEM). *p=0.01. The effect of insulin (INS) on basal and E2-inducible expression of GREB-1 is shown in the right panel. Cells were pre-treated with insulin overnight and then exposed to ethanol or E2 for four hours. *p=0.01 control versus insulin treatment; **p=0.02, E2 vs E2+ insulin (n=5). C) ERα binding to enhancer regions of XBP-1 was markedly lower in MCF-7-T-bet cells compared to MCF-7p cells. ERα and FOXA1 binding sites associated with XBP-1 from previous ChIP-on-Chip (13, 17) are shown on the top (black bars) along chromosomal location and direction of the gene (horizontal arrow). ERα binding in untreated and E2 treated MCF-7p and MCF-7-T-bet cells was determined by ChIP analysis followed by q-PCR. Asterisks denote statistically significant differences in ERα binding under identical treatment conditions between two cell types. D) ChIP assay was used to measure ERα binding to one of the ERα binding regions (black bars) associated with GREB-1 gene (indicated by inverted arrow on the top).

Figure 4. Insulin confers resistance to tamoxifen

A) The effect of insulin on proliferation and tamoxifen sensitivity of MCF-7p and MCF-7-T-bet cells. Cells were plated on 96 well plates and treated with E2 (0.1 nM), 4-hydroxy-tamoxifen (1 μM) and/or insulin (50 nM) as described in materials and methods. *p= 0.0001, MCF-7p vs MCF-7-T-bet cells under identical treatment condition. B) The effect of different concentrations of insulin on proliferation and tamoxifen sensitivity (1 μM) of MCF-7p and MCF-7-T-bet cells. *p<0.01 between MCF-7p and MCF-7-T-bet cells under identical treatment condition. C) The effect of insulin on proliferation under variable concentration of tamoxifen. Experiments are done as in B. D) T-bet is required for E2- and insulin-dependent proliferation of T47-D cells. T47-D cells were treated with siRNA against T-bet or control non-specific siRNA targeting luciferase gene for four days. T-bet siRNA reduced T-bet protein levels by 30% (left panel). Cells were treated with E2, insulin or both for six days. *p<0.01 control vs T-bet siRNA.

Figure 5. Changes in ERα:FOXA1:GATA-3 axis in MCF-7 cells that acquired resistance to tamoxifen (MCF-7-T) or fulvestrant (MCF-7-F)

A). Basal and insulin-regulated expression pattern of T-bet, ERα, FOXA1, and GATA-3 in MCF-7, MCF-7-T and MCF-7-F cells. Right panel displays densitometric scanning results of T-bet from three or more experiments. The difference in T-bet expression between different cell types is significant (*p=0.01, **p=0.03). Similarly, reduction in FOXA1 expression in MCF-7-T and MCF-7-F cells compared to parental cells is significant (p<0.05). B) E2 fails to induce ERα:FOXA1:GATA-3 target gene GREB-1 in MCF-7-T and MCF-7-F cells. GREB-1 expression was measured by qRT-PCR (n=3). C) T-bet siRNA inhibits growth of MCF-7-T cells. Cells were treated with siRNA as in Figure 4D and cell proliferation was measured by BrDU-ELISA. As in T47-D cells, T-bet siRNA reduced T-bet protein levels by 30% (left panel) and transcript levels by 50% (middle panel).

Figure 6. MCF-7p and MCF-7-T-bet cells show differential ERK and AKT activation in response to growth factor signaling

A) EGF-inducible ERK activation in MCF-7p and MCF-7-T-bet cells. Cells were treated with 20 ng/ml EGF for indicated time and ERK and AKT activation was measured using phospho-specific antibodies. Ratio between phosphorylated ERK and the loading control β-Actin is presented. B) Insulin-inducible (50 ng/ml) ERK and AKT activation in MCF-7p and MCF-7-T-bet cells. C) Heregulin-inducible (50 ng/ml) ERK and AKT activation in MCF-7p and MCF-7-T-bet cells.