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.
Highlights
The STAT (Signal Transducer and Activator of Transcription) family of proteins mediate cytokine and growth factor receptor signaling, which in turn regulate cell growth, survival, and differentiation [1,2,3,4,5]
STAT1 and STAT3 signaling pathways are differentially activated in endocrine sensitive and resistant breast cancer cell lines
In order to establish which pathways might influence estrogen signaling and endocrine therapy sensitivity and resistance, we initially carried out an unsupervised interrogation of biochemical signaling pathways using a phosphoprotein antibody array in MCF-7 sensitive and resistant breast cancer cell lines
Summary
The STAT (Signal Transducer and Activator of Transcription) family of proteins mediate cytokine and growth factor receptor signaling, which in turn regulate cell growth, survival, and differentiation [1,2,3,4,5]. A number of studies have implicated both oncogenic and tumor suppressor functions for STAT family members in breast cancer and it seems likely that individual STAT isoforms have pleiotropic functions at different stages of disease progression [6,7]. The roles of STAT1 and STAT3 in breast cancer remain controversial since multiple studies have reported variable results between STAT isoform expression and clinical outcome, suggesting a degree of complexity in STAT signaling which is poorly understood. Several reports describes both increased total and phospho-STAT3(-
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