Abstract
In breast cancer, prolactin-induced activation of the transcription factor STAT5a results from the phosphorylation of STAT5a tyrosine residue 694. However, its role in mammary oncogenesis remains an unsettled debate as STAT5a exhibits functional dichotomy with both pro-differentiative and pro-proliferative target genes. Phosphorylation of STAT5a serine residues, S726 and S780, may regulate STAT5a in such a way to underlie this duality. Given hematopoiesis studies showing phospho-serine STAT5a as necessary for transformation, we hypothesized that serine phosphorylation regulates STAT5a activity to contribute to its role in mammary oncogenesis, specifically in luminal breast cancer. Here, phosphorylation of S726-, S780-, and Y694-STAT5a in response to prolactin in MCF7 luminal breast cancer cells was investigated with STAT5a knockdown and rescue with Y694F-, S726A-, or S780A-STAT5a, where the phospho-sites were mutated. RNA-sequencing and subsequent Ingenuity Pathway Analysis predicted that loss of each phospho-site differentially affected both prolactin-induced gene expression as well as functional pathways of breast cancer (e.g. cell survival, proliferation, and colony formation). In vitro studies of anchorage-independent growth and proliferation confirmed distinct phenotypes: whereas S780A-STAT5a decreased clonogenicity, S726A-STAT5a decreased proliferation in response to prolactin compared to wild type STAT5a. Collectively, these studies provide novel insights into STAT5a activation in breast cancer pathogenesis.
Highlights
In mammary epithelium, the polypeptide prolactin (PRL) acts through its cognate receptor (PRLr), and is responsible for terminal maturation and differentiation of lactating glands during p regnancy[1,2,3,4,5,6,7]
Serine phosphorylation of signal transducer and activator of transcription 5a (STAT5a) has been observed in mouse mammary epithelial cells throughout mammary development, this has yet to be clinically characterized in human breast cancer[31,32]
STAT5a S780 phosphorylation was observed in the nucleus of tissue samples, there was no significant association of expression with either tumor grade or proliferative status (Ki67 staining; Fig. 1A)
Summary
The polypeptide prolactin (PRL) acts through its cognate receptor (PRLr), and is responsible for terminal maturation and differentiation of lactating glands during p regnancy[1,2,3,4,5,6,7]. The STAT family is highly conserved, each having a tyrosine residue near residue position 700 that is phosphorylated by JAK in the cytoplasm. These differing reports highlight the possibility of stimulus- and tissue-specific functions of upY-STAT vs pY-STAT Compounding this growing understanding of the regulation of STAT activity, STATs (with the exception of STATs 2 and 6) have serine residues in the Transactivation Domain (TAD) that are capable of being phosphorylated, with known function in regulating STAT activity[22,23,24,25,26,27]. PRL stimulation of the human breast cancer cell lines T47D (E), and MCF7 (F), show induction of pS726-STAT5a but constitutive pS780-STAT5a, quantified by densitometric analysis. Of phosphorylation of either S726 or S780 in PRL-responsive normal or malignant breast tissue, is less well understood
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