Increased STAT1 signaling in endocrine-resistant breast cancer

Abstract

Proteomic profiling of the estrogen/tamoxifen-sensitive MCF-7 cell line and its partially sensitive (MCF-7/LCC1) and fully resistant (MCF-7/LCC9) variants was performed to identify modifiers of endocrine sensitivity in breast cancer. Analysis of the expression of 120 paired phosphorylated and non-phosphorylated epitopes in key oncogenic and tumor suppressor pathways revealed that STAT1 and several phosphorylated epitopes (phospho-STAT1(Tyr701) and phospho-STAT3(Ser727)) were differentially expressed between endocrine resistant and parental controls, confirmed by qRT-PCR and western blotting. The STAT1 inhibitor EGCG was a more effective inhibitor of the endocrine resistant MCF-7/LCC1 and MCF-7/LCC9 lines than parental MCF-7 cells, while STAT3 inhibitors Stattic and WP1066 were equally effective in endocrine-resistant and parental lines. The effects of the STAT inhibitors were additive, rather than synergistic, when tested in combination with tamoxifen in vitro. Expression of STAT1 and STAT3 were measured by quantitative immunofluorescence in invasive breast cancers and matched lymph nodes. When lymph node expression was compared to its paired primary breast cancer expression, there was greater expression of cytoplasmic STAT1 (∼3.1 fold), phospho-STAT3(Ser727) (∼1.8 fold), and STAT5 (∼1.5 fold) and nuclear phospho-STAT3(Ser727) (∼1.5 fold) in the nodes. Expression levels of STAT1 and STAT3 transcript were analysed in 550 breast cancers from publicly available gene expression datasets (GSE2990, GSE12093, GSE6532). When treatment with tamoxifen was considered, STAT1 gene expression was nearly predictive of distant metastasis-free survival (DMFS, log-rank p = 0.067), while STAT3 gene expression was predictive of DMFS (log-rank p<0.0001). Analysis of STAT1 and STAT3 protein expression in a series of 546 breast cancers also indicated that high expression of STAT3 protein was associated with improved survival (DMFS, p = 0.006). These results suggest that STAT signaling is important in endocrine resistance, and that STAT inhibitors may represent potential therapies in breast cancer, even in the resistant setting.

Figure 1. STAT protein (A) and mRNA (B) expression in the MCF-7, MCF-7/LCC1 (LCC1) and MCF-7/LCC9 (LCC9) breast cancer cell lines.

A. MCF-7 cells were double charcoal-stripped for 48 h. Protein lysates were run on a 10% SDS-gel and membranes were probed with phospho-STAT1(Tyr 701), STAT1, phospho-STAT3 (Ser 727), or STAT3 primary antibodies (1∶1000). Column charts show the relative expression level of protein normalized with loading control (tubulin). Data are presented as relative mean Integrated Intensity (correlated with the fluorescence intensity of secondary antibody) ratios of target protein over tubulin +/− SEM from quadruplicate samples. Statistical significance noted for multiple comparision where *P<0.05, ***P<0.001 (student's t-test). B. mRNA expression of STAT was measured by two step real time PCR. Total RNA was extracted from cells charcoal stripped for 48 h. The cDNA was synthesised by reverse transcription, and real time PCR was performed as described in Materials and Methods. Relative expression of the target gene was normalized to that of Beta-actin. Results are presented as mean ±SD from triplicate samples.

Figure 2. The effect of STAT inhibitors (A. EGCG, B. WP1066 and C. Stattic) on proliferation of Tamoxifen sensitive and resistant (MCF-7, MCF-7/LCC1 (LCC1), and MCF-7/LCC9 (LCC9)) cells.

All cells were incubated in double charcoal-stripped medium for another 48 h after seeding and treated with or without inhibitor. O.D values were measured on Day 5. Data were plotted as a mean of O.D values +/− SD from 6 replicate samples. Asterisks represent significant changes between treatment and no treatment, while hashes represent differences between resistant cell lines and MCF7. Error bars are standard deviations. Statistical significance noted for treatment groups compared with untreated controls were ^#/*P<0.05, ^##/**P<0.01, ^###/***P<0.001 (ANOVA followed by Tukey-Kramer).

Figure 3. The effect of STAT inhibitors (EGCG, Stattic, or WP1066) combined with tamoxifen on MCF-7, MCF-7/LCC1 (LCC1), and MCF-7/LCC9 (LCC9) cells in the presence of estrogen.

Cells were grown in charcoal-stripped serum, 48 h prior to treatment and were treated with control medium containing 1 nM E₂, tamoxifen (1 µM)+E₂ (1 nM), EGCG (25 µM)+E₂ (1 nM), tamoxifen (1 µm)+EGCG (25 µm)+1 nM E₂, Stattic (0.5 µM)+E₂ (1 nM), tamoxifen (1 µM)+Stattic (0.5 µm)+E₂ (1 nM), WP1066 (2 µM)+E₂ (1 nM), tamoxifen (1 µM)+WP1066 (2 µM)+E₂ (1 nM) for 5 days. O.D. values were measured on day 5. Data were plotted as a mean inhibition ratio of O.D. values over untreated control groups +/- SD from 4 replicate samples. Asterisks represent significant changes between combined treatment and the inhibitor alone, while hashes represent differences between combined treatment and tamoxifen treatment alone. ^#/*P<0.05, ^##/**P<0.01, ^###/***P<0.001 (ANOVA followed by Tukey-Kramer).

Figure 4. Comparison of STAT signaling in matched primary and metastatic tissue.

Representative immunofluorescence images of phosphorylated and total STAT1, STAT3 and STAT5 in TMA cores. Blue  =  DAPI nuclear counterstain, green  =  cytokeratin tumor mask and red  =  target protein.

Figure 5. STAT expression and prognosis.

Gene (A) and protein (B) expression of STAT1 and STAT3 in primary breast cancer (blue = low, green = high). Optimal cut-points were determined using the x-tile program while correcting for the use of minimum P statistics (31). Low STAT3 expression is associated with very poor outcomes for patients treated with tamoxifen (protein expression STAT3 high (78% fifteen-year survival) vs. STAT3 low (68% five year survival).