Abstract NG15: Progesterone-mediated immune evasion in breast cancer
Authors: Hagan Christy R. , Werner Lauryn , Helm Emma , Axelrod Margaret , Balko Justin , Hartman Zachary , Hunter Kent , Yang Howard , Chalise Prabhakar , Markiewicz Mary 
Published: July 2021

Why are some early neoplastic lesions identified through immunosurveillance and subsequently destroyed, whereas other tumors evade this clearance step and progress to clinically-relevant tumors? The cancer immunoediting hypothesis highlights that the innate and adaptive immune responses work together to “flag” early neoplastic lesions for immune-mediated elimination. An early mediator of this “elimination” process is activation of type I interferon signaling. Thus, suppression of type I interferon signaling may help developing tumors evade the critical early steps of immune recognition and clearance. As such, female STAT1 knockout mice, lacking the key upstream activator of type I interferon signaling, develop mammary gland adenocarcinomas. Preliminary data from our lab suggest that progesterone, working through the progesterone receptor (PR), may be a key player in tumor immune evasion. The contribution of the ovarian steroid hormone, progesterone, to the development and progression of breast cancer has become increasingly evident, especially in light of recent clinical trial data linking the use of hormonal replacement therapy with breast cancer risk. These highly publicized data showed that women whose hormone replacement therapy (HRT) regimens included synthetic progesterone had a higher relative risk of developing invasive breast cancer. These data underscore the importance of understanding how hormones, particularly progesterone, affect breast cancer initiation and growth. Upon diagnosis, nearly 70% of breast cancers express PR and the estrogen receptor (ER). This is in sharp contrast to what is seen in the normal mammary gland, where only 7-10% of luminal epithelial cells express ER and PR. ER action in breast cancer has been well studied and as a result, ER has proven to be an excellent target for current endocrine-based therapies. However, despite convincing clinical evidence from the aforementioned trials, the role of PR in breast cancer has been largely understudied. Importantly, progesterone is emerging as a potent mitogen in the breast and a significant contributor to breast cancer progression. Despite maintaining receptor (ER/PR) expression, many breast cancer patients eventually progress to hormone-independence, failing current (largely ER/estrogen based) endocrine therapies. Therefore, ER-independent functions of PR are of great clinical interest. PR is a steroid-activated nuclear transcription factor known to regulate context-dependent gene programs in breast cancer. Using Gene Set Enrichment Analysis (GSEA), we analyzed microarray data sets from ligand-treated T47D breast cancer cells stably expressing wt PR (or PR-null). GSEA revealed that genes from multiple inflammatory and interferon-regulated gene sets were significantly enriched in our T47D-wt PR dataset, an effect that was lost in cells lacking PR expression. Leading Edge analysis identified many genes that are transcriptional targets of interferon signaling pathways and key mediators of inflammation (i.e. IFITs, IRF7, OAS1/2 and ISG15) whose regulation is lost in PR-null cells. These genes, classically activated by interferons, are collectively referred to as interferon-stimulated genes (ISGs). Interestingly, in response to treatment with various PR ligands, ISGs are potently transcriptionally repressed. Ligand-activated PR also repressed expression of these genes in response to their classical activator, interferon-alpha. In cells were PR expression has been reduced using si/shRNA, ISG transcriptional repression is lost. Of note, basal levels of ISGs are dramatically upregulated in cells where PR has been knocked-down, indicating that PR also has ligand-independent functions aimed at maintaining low levels of ISG expression. Moreover, we have shown using ChIP assays that all ISGs assayed thus far exhibited PR binding within their proximal regulatory regions, implying direct PR regulation of these genes. Finally, we have data to suggest that PR attenuates canonical (in response to type I interferons, such as interferon-alpha) interferon signaling. Pathway readouts in response to treatment with interferon-alpha, such as STAT1 phosphorylation, are reduced in cells expressing PR, and even further attenuated in PR-positive cells treated with ligand (progesterone). We have detected an interaction between PR and STAT1 that increases in response to ligand; this interaction may explain the observed decrease in phospho-STAT1 in response to interferon-alpha in PR-positive cells. Cumulatively, these data suggest a concerted effort aimed at reducing interferon-signaling through activation of PR. These preliminary data translate to the immune microenvironment in the murine mammary gland. In mice with transgenic overexpression of PR, we observed an immuno-suppressive microenvironment in the mammary gland and the inguinal (draining) lymph node through decreased antigen-presenting cell populations (i.e. dendritic cells and macrophages) and increased T-regulatory cells. We also observe decreases in MHC Class I presentation in PR+ mammary gland epithelial cells, a key step to tumor cell recognition by the immune system. These changes in the immune system suggest that PR can promote immunosuppressive changes in the mammary gland. Moreover, 80% of these mice in a multiparous cohort develop mammary gland tumors. Therefore, early changes in the immune microenvironment in the mammary gland may translate to the development of mammary gland tumors in PR transgenic mice. Together, these data support our hypothesis that PR attenuates the interferon response in breast cancer cells through manipulation of canonical interferon-responsive proteins and transcriptional downregulation of ISGs. This decrease in interferon signaling may be a key step in PR-positive breast tumors evading immune surveillance and clearance, allowing for progression of these tumors. STA1 knockout mouse data shows that inhibition of STAT1 signaling if sufficient to drive tumorigenesis. However, STAT1 knockout is not observed in human tumor. We propose that inhibition of STAT1, through the actions of PR/progesterone, are a driving force behind the development of hormone receptor-positive human tumors.

Citation Format: Christy R. Hagan, Lauryn Werner, Emma Helm, Margaret Axelrod, Justin Balko, Zachary Hartman, Kent Hunter, Howard Yang, Prabhakar Chalise, Mary Markiewicz. Progesterone-mediated immune evasion in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG15.