Progesterone promotes immunomodulation and tumor development in the murine mammary gland.

BACKGROUND: Clinical studies have linked usage of progestins (synthetic progesterone [P4]) to breast cancer risk. However, little is understood regarding the role of native P4, signaling through the progesterone receptor (PR), in breast tumor formation. Recently, we reported a link between PR and immune signaling pathways, showing that P4/PR can repress type I interferon signaling pathways. Given these findings, we sought to investigate whether P4/PR drive immunomodulation in the mammary gland and promote tumor formation. METHODS: To determine the effect of P4 on immune cell populations in the murine mammary gland, mice were treated with P4 or placebo pellets for 21 days. Immune cell populations in the mammary gland, spleen, and inguinal lymph nodes were subsequently analyzed by flow cytometry. To assess the effect of PR overexpression on mammary gland tumor development as well as immune cell populations in the mammary gland, a transgenic mouse model was used in which PR was overexpressed throughout the entire mouse. Immune cell populations were assessed in the mammary glands, spleens, and inguinal lymph nodes of 6-month-old transgenic and control mice by flow cytometry. Transgenic mice were also monitored for mammary gland tumor development over a 2-year time span. Following development of mammary gland tumors, immune cell populations in the tumors and spleens of transgenic and control mice were analyzed by flow cytometry. RESULTS: We found that mice treated with P4 exhibited changes in the mammary gland indicative of an inhibited immune response compared with placebo-treated mice. Furthermore, transgenic mice with PR overexpression demonstrated decreased numbers of immune cell populations in their mammary glands, lymph nodes, and spleens. On long-term monitoring, we determined that multiparous PR-overexpressing mice developed significantly more mammary gland tumors than control mice. Additionally, tumors from PR-overexpressing mice contained fewer infiltrating immune cells. Finally, RNA sequencing analysis of tumor samples revealed that immune-related gene signatures were lower in tumors from PR-overexpressing mice as compared with control mice. CONCLUSION: Together, these findings offer a novel mechanism of P4-driven mammary gland tumor development and provide rationale in investigating the usage of antiprogestin therapies to promote immune-mediated elimination of mammary gland tumors.

Reciprocal fine-tuning of progesterone and prolactin-regulated gene expression in breast cancer cells.

Progesterone and prolactin are two key hormones involved in development and remodeling of the mammary gland. As such, both hormones have been linked to breast cancer. Despite the overlap between biological processes ascribed to these two hormones, little is known about how co-expression of both hormones affects their individual actions. Progesterone and prolactin exert many of their effects on the mammary gland through activation of gene expression, either directly (progesterone, binding to the progesterone receptor [PR]) or indirectly (multiple transcription factors being activated downstream of prolactin, most notably STAT5). Using RNA-seq in T47D breast cancer cells, we characterized the gene expression programs regulated by progestin and prolactin, either alone or in combination. We found significant crosstalk and fine-tuning between the transcriptional programs executed by each hormone independently and in combination. We divided and characterized the transcriptional programs into four broad categories. All crosstalk/fine-tuning shown to be modulated by progesterone was dependent upon the expression of PR. Moreover, PR was recruited to enhancer regions of all regulated genes. Interestingly, despite the canonical role for STAT5 in transducing prolactin-signaling in the normal and lactating mammary gland, very few of the prolactin-regulated transcriptional programs fine-tuned by progesterone in this breast cancer cell line model system were in fact dependent upon STAT5. Cumulatively, these data suggest that the interplay of progesterone and prolactin in breast cancer impacts gene expression in a more complex and nuanced manner than previously thought, and likely through different transcriptional regulators than those observed in the normal mammary gland. Studying gene regulation when both hormones are present is most clinically relevant, particularly in the context of breast cancer.

Progesterone Receptor Attenuates STAT1-Mediated IFN Signaling in Breast Cancer.

Why some tumors remain indolent and others progress to clinical relevance remains a major unanswered question in cancer biology. IFN signaling in nascent tumors, mediated by STAT1, is a critical step through which the surveilling immune system can recognize and destroy developing tumors. In this study, we have identified an interaction between the progesterone receptor (PR) and STAT1 in breast cancer cells. This interaction inhibited efficient IFN-induced STAT1 phosphorylation, as we observed a decrease in phospho-STAT1 in response to IFN treatment in PR-positive breast cancer cell lines. This phenotype was further potentiated in the presence of PR ligand. In human breast cancer samples, PR-positive tumors exhibited lower levels of phospho-STAT1 as compared with their PR-negative counterparts, indicating that this phenotype translates to human tumors. Breast cancer cells lacking PR exhibited higher levels of IFN-stimulated gene (ISG) RNA, the transcriptional end point of IFN activation, indicating that unliganded PR alone could decrease transcription of ISGs. Moreover, the absence of PR led to increased recruitment of STAT1, STAT2, and IRF9 (key transcription factors necessary for ISG transcription) to ISG promoters. These data indicate that PR, both in the presence and absence of ligand, attenuates IFN-induced STAT1 signaling, culminating in significantly abrogated activation of genes transcribed in response to IFNs. PR-positive tumors may use downregulation of STAT1-mediated IFN signaling to escape immune surveillance, leading to the development of clinically relevant tumors. Selective immune evasion of PR-positive tumors may be one explanation as to why over 65% of breast cancers are PR positive at the time of diagnosis.

O-GlcNAc-Dependent Regulation of Progesterone Receptor Function in Breast Cancer.

Emerging clinical trial data implicate progestins in the development of breast cancer. While the role for the progesterone receptor (PR) in this process remains controversial, it is clear that PR, a steroid-activated nuclear receptor, alters the transcriptional landscape of breast cancer. PR interacts with many different types of proteins, including transcriptional co-activators and co-repressors, transcription factors, nuclear receptors, and proteins that post-translationally modify PR (i.e., kinases and phosphatases). Herein, we identify a novel interaction between PR and O-GlcNAc transferase (OGT), the enzyme that catalyzes the addition of a single N-acetylglucosamine sugar, referred to as O-GlcNAc, to acceptor serines and threonines in target proteins. This interaction between PR and OGT leads to the post-translational modification of PR by O-GlcNAc. Moreover, we show that O-GlcNAcylated PR is more transcriptionally active on PR-target genes, despite the observation that PR messenger RNA and protein levels are decreased when O-GlcNAc levels are high. O-GlcNAcylation in breast cancer is clinically relevant, as we show that O-GlcNAc levels are higher in breast cancer as compared to matched normal tissues, and PR-positive breast cancers have higher levels of OGT. These data predict that under conditions where O-GlcNAc levels are high (breast cancer), PR, through an interaction with the modifying enzyme OGT, will exhibit increased O-GlcNAcylation and potentiated transcriptional activity. Therapeutic strategies aimed at altering cellular O-GlcNAc levels may have profound effects on PR transcriptional activity in breast cancer.

O-GlcNAcylation in women's cancers: breast, endometrial and ovarian.

O-GlcNAcylation is emerging as a critical regulatory post-translational modification, impacting proteins that regulate cell division, apoptosis, metabolism, cell signaling, and transcription. O-GlcNAc also affects biological homeostasis by integrating information coming from the environment, such as nutrient conditions and extracellular stimuli, with cellular response. Aberrant O-GlcNAc modulation has been linked to metabolic and neurodegenerative diseases, as well as cancers. While many studies have highlighted the significance of O-GlcNAc in cancer, a specific function for O-GlcNAc during tumorigenesis remains unclear and seems to differ according to cancer type. Herein, we review the impact of altered O-GlcNAcylation in breast, ovarian and uterine cancers.

Interferon-Stimulated Genes Are Transcriptionally Repressed by PR in Breast Cancer.

The progesterone receptor (PR) regulates transcriptional programs that drive proliferation, survival, and stem cell phenotypes. Although the role of native progesterone in the development of breast cancer remains controversial, PR clearly alters the transcriptome in breast tumors. This study identifies a class of genes, Interferon (IFN)-stimulated genes (ISGs), potently downregulated by ligand-activated PR which have not been previously shown to be regulated by PR. Progestin-dependent transcriptional repression of ISGs was observed in breast cancer cell line models and human breast tumors. Ligand-independent regulation of ISGs was also observed, as basal transcript levels were markedly higher in cells with PR knockdown. PR repressed ISG transcription in response to IFN treatment, the canonical mechanism through which these genes are activated. Liganded PR is robustly recruited to enhancer regions of ISGs, and ISG transcriptional repression is dependent upon PR's ability to bind DNA. In response to PR activation, key regulatory transcription factors that are required for IFN-activated ISG transcription, STAT2 and IRF9, exhibit impaired recruitment to ISG promoter regions, correlating with PR/ligand-dependent ISG transcriptional repression. IFN activation is a critical early step in nascent tumor recognition and destruction through immunosurveillance. As the large majority of breast tumors are PR positive at the time of diagnosis, PR-dependent downregulation of IFN signaling may be a mechanism through which early PR-positive breast tumors evade the immune system and develop into clinically relevant tumors.Implications: This study highlights a novel transcriptional mechanism through which PR drives breast cancer development and potentially evades the immune system. Mol Cancer Res; 15(10); 1331-40. ©2017 AACR.