Abstract
The neuroendocrine hormone prolactin (PRL) and its cognate receptor (PRLr) have been implicated in the pathogenesis of breast cancer. PRL signaling relies on activating kinases such as the tyrosine kinase Jak2 and serine/threonine kinases ERK1/2, Nek3, PI3K, and AKT. In the canonical pathway of PRL signaling, Jak2 phosphorylates the transcription factor Stat5a at tyrosine residue 694 (pY694-Stat5a), preceding Stat5a nuclear translocation and transcriptional activity. However, Stat5a exists with functional duality as a transcription factor, having both pro-differentiative and pro-proliferative target genes. Other Stat family members (Stats 1, 3, and 6) have been shown to have transcriptional activity in the un-phosphorylated (upY) state, distinct from that of pY-Stat activity. This distinction (upY vs. pY) may underlie the duality of Stat5a, coupled with additional regulatory non-canonical post-translational modifications. Within this notion, Stat5a contains two serine residues, S726 and S780, whose phosphorylation are necessary for hematopoietic transformation. However, their functions in PRL-mediated breast cancer pathogenesis have not been examined. We hypothesize that Stat5a serine phosphorylation regulates Stat5a nuclear activity in a non-canonical fashion, contributing to its role in mammary oncogenesis. As shown in a tissue microarray (TMA), human breast cancer tissues express both pS726- and pS780-Stat5a. Nuclear Allred score for pS726-Stat5a increases two-fold with increasing tumor grade, with no difference in staining associated with estrogen or progesterone receptor (ER, PR) status, nor other clinical characteristics. Likewise, patient derived xenograft (PDX) tumors of various molecular subtypes express pS726- and pS780-Stat5a. Phosphorylation of S726-Stat5a is PRL-responsive in vitro. Pharmacologic inhibition of ERK1/2 prevents this phosphorylation, uncovering a novel pathway in which ERK1/2 mediates Stat5a activity in response to PRL in breast cancer. To examine the functional significance of Stat5a serine phosphorylation in vitro, we have performed Stat5a knockdown (KD) in the breast cancer cell line MCF7. Following Stat5a KD, cells were rescued with phospho-site specific Stat5a mutant constructs. Characteristics of breast cancer examined in these mutation-carrying cells, including anchorage-independent growth and proliferation, show distinct phenotypes compared to controls. PRL-induced expression of the CISH gene is significantly decreased up to 65% in the mutation-carrying cells compared to wild type Stat5a. Mechanistic studies will examine the ability of these Stat5a mutants to undergo nuclear translocation and interact with other transcription factors. Collectively, these studies have the potential to provide novel insights into the role of the non-canonical pathway of Stat5a activation in breast cancer pathogenesis.
Regulation of MEF2 by Histone Deacetylase 4- and SIRT1 Deacetylase-Mediated Lysine Modifications
