PTP1B regulation of the transcription factor Stat5 in breast cancer
Thesis (Ph.D.)--Georgetown University, 2009.; Includes bibliographical references. Basal levels of active, nuclear localized and tyrosine phosphorylated Stat5 are present in healthy human breast epithelia. In contrast, Stat5 is frequently inactivated during breast cancer progression, a finding that correlates with loss of histological differentiation and poor patient prognosis. Identifying the mechanisms underlying Stat5 inactivation could provide novel targets for breast cancer therapy. Because total Stat5 protein remains detectable in an unphosphorylated form in a majority of breast cancers a hypothesis was formed that inactivation of Stat5 during breast cancer progression is the result of disregulated tyrosine phosphatase activity. Supporting this hypothesis, an experiment performed in human breast cancer cells using the tyrosine phosphatase inhibitor pervanadate revealed significant tyrosine phosphatase regulation of prolactin-induced phosphotyrosine-Stat5. Very little is known about tyrosine phosphatase regulation of Stat5 activation in human breast cancer. To explore this potential mechanism for Stat5 inactivation during breast cancer progression five candidate tyrosine phosphatases were chosen for investigation based on 1) reported regulation of Stat5 activity in non-breast cells and tissues, 2) modulation of Jak2/Stat5 homologue signaling in lower organisms, or 3) the ability to regulate Stat5 in in vitro overexpression systems and phosphatase assays. The five candidates were the classical tyrosine phosphatases PTP1B, TC-PTP, SHP1, and SHP2, and the dual-specificity phosphatase VHR. Lentiviral-mediated shRNA methodology allowed specific examination of the candidates in the context of human breast cancer. The results of these studies revealed enhanced and sustained prolactin-induced Stat5 tyrosine phosphorylation exclusively in response to PTP1B suppression; an effect that was mimicked by parallel enhanced and sustained prolactin-induced tyrosine phosphorylation of the upstream Stat5 tyrosine kinase, Jak2. The effect of PTP1B knockdown on Stat5 was found in both T47D and MCF7 cells and resulted in increased Stat5 reporter gene activity. Furthermore, PTP1B knockdown enhanced the prolactin-induced Stat5 sensitivity of breast cancer cells by reducing EC50 almost three fold, from 7.2 nM to 2.5 nM. Last, immunohistochemical analysis revealed a highly significant negative correlation between levels of phosphotyrosine-Stat5 and PTP1B in clinical breast cancer specimens. Collectively, these data implicate upregulation or activation of PTP1B as an important mechanism behind the suppression of Prolactin/Jak2/Stat5 signaling in breast cancer.
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