Author ORCID Identifier

https://orcid.org/0000-0002-6650-7993

Defense Date

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Clinical and Translational Sciences

First Advisor

Charles Clevenger

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-tyrosine-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, specifically in luminal breast cancer. 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. 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 targeting Y694, S726 and S780. Characteristics of breast cancer examined in these mutation-carrying cells, including anchorage-independent growth and proliferation, show distinct phenotypes compared to controls. Further, PRL-inducible gene expression changes on the global transcriptomic level showed differential effects of the STAT5a mutations compared to wild type STAT5a, specifically enrichment of different oncogenic signatures and transcription factor programs. Mechanistic studies examine the ability of these STAT5a mutant proteins to undergo nuclear translocation and their regulatory role on phosphorylation of STAT5a tyrosine residue 694. Collectively, these studies provide novel insights into the role of the non-canonical pathway of STAT5a activation in breast cancer pathogenesis.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

3-29-2021

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