Abstract
Interactions between steroid hormone receptors and signal transducer and activator of transcription (Stat)-mediated signaling pathways have already been described. In the present study, we explored the capacity of progestins to modulate Stat3 transcriptional activation in an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in BALB/c mice and in the human breast cancer cell line T47D. We found that C4HD epithelial cells, from the MPA-induced mammary tumor model, expressed Stat3 and that MPA treatment of C4HD cells up-regulated Stat3 protein expression. In addition, MPA induced rapid, nongenomic Stat3, Jak1, and Jak2 tyrosine phosphorylation in C4HD and T47D cells. MPA treatment of C4HD cells also resulted in rapid c-Src tyrosine phosphorylation. These effects were completely abolished by the progestin antagonist RU486. Abrogation of Jak1 and Jak2 activity by transient transfection of C4HD cells with dominant negative (DN) Jak1 or DN Jak2 vectors, or inhibition of Src activity by preincubation of cells with the Src family kinase inhibitor PP2, blocked the capacity of MPA to induce Stat3 phosphorylation. Treatment of C4HD cells with MPA induced Stat3 binding to DNA. In addition, MPA promoted strong Stat3 transcriptional activation in C4HD and T47D cells that was inhibited by RU486 and by blockage of Jak1, Jak2, and Src activities. To investigate the correlation between MPA-induced Stat3 activation and cell growth, C4HD cells were transiently transfected with a DN Stat3 expression vector, Stat3Y705-F, or with a constitutively activated Stat3 mutant, Stat3-C. While expression of Stat3Y705-F mutant had an inhibitory effect on MPA-induced growth of C4HD cells, transfection with the constitutively activated Stat3-C vector resulted in MPA-independent proliferation. Finally, we addressed the effect of targeting Stat3 in in vivo growth of C4HD breast tumors. Blockage of Stat3 activation by transfection of C4HD cells with the DN Stat3Y705-F expression vector significantly inhibited these cells' ability to form tumors in syngeneic mice. Our results have for the first time demonstrated that progestins are able to induce Stat3 transcriptional activation, which is in turn an obligatory requirement for progestin stimulation of both in vitro and in vivo breast cancer growth.
Highlights
Progesterone regulates diverse biological effects in a broad range of tissues, mostly by interaction with the classical progesterone receptor (PR), a member of the nuclear receptor superfamily of ligand-dependent transcription factors
Functional interactions between progestins and signal transducer and activator of transcription (Stat) were demonstrated by Lange et al [26] and Richer et al [43], who found that treatment of T47D human breast cancer cells with the synthetic progestin R5020 resulted in up-regulation of Stat3 and Stat5 protein levels
On the other hand, when we transiently transfected LM3 cells with a human PRB expression vector, medroxyprogesterone acetate (MPA) treatment resulted in strong induction of Stat3 tyrosine phosphorylation, which was abolished by RU486 (Fig. 2C)
Summary
Progesterone regulates diverse biological effects in a broad range of tissues, mostly by interaction with the classical progesterone receptor (PR), a member of the nuclear receptor superfamily of ligand-dependent transcription factors. The mechanisms by which progestins stimulate growth of breast cancer cells have not been completely deciphered, several lines of evidence [6, 26, 33], including our own work [3, 4, 25, 44], have shown that convergence between progestins and growth factor (GF) signaling pathways mediates proliferative effects of progestins in mammary tumor cells In addition to their direct transcriptional effects, rapid or nongenomic biological effects of progestins have been described in several species, including fish, amphibian, and mammalian [27]. Constitutive activation of Stats, especially of Stat and Stat, has been found in transformed cells and in a variety of human tumor types, supporting the concept that Stats play a role in malignant transformation [55]
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