Sex hormones, such as estrogens and androgens, regulate genomic and cellular processes that contribute to sex-specific disparities in the pathophysiology of various cancers. Sex hormones can modulate the immune signals and activities of tumor cells and tumor associated leukocytes to support or suppress cancer progression. Therefore, hormonal differences between males and females play a crucial role in cancer immunity and in the response to therapies that exploit the intrinsic immune system to eliminate malignant cells. In this review, we summarize the impact of sex hormones in the breast cancer microenvironment, with a focus on how the hormonal environment affects tumor immunity. We also discuss the potential benefits of endocrine therapy used in combination with immunotherapy to strengthen the antitumor immune response.

Breast cancer is the leading cause of cancer mortality in women. The breast cancer microenvironment supplies immune signals that influence tumor growth and metastatic behavior. The cytokine, Interleukin-4 (IL4), promotes increased survival, proliferation and invasion upon stimulation of the type II IL4 receptor alpha (IL4Rα) which is aberrantly expressed on epithelial cancer cells. Previously, we have shown that metastatic mouse mammary cancer cells have a strong dependence on IL4 signaling for colonization of metastatic sites in the lung and liver. Additionally, these cells uptake more glucose when treated with IL4 in vitro. Here, we assessed the importance of glucose consumption on pro-tumorigenic phenotypes induced by IL4/IL4Rα. In 4T1 cells, IL4/IL4Rα signaling increased protein expression of the glucose transporter, Glut1. IL4/IL4Rα activation also increased global glycosylation of Glut1. In human metastatic breast cancer cells (MDA-MB-231 and BT549), Crispr-mediated knockout (KO) of Glut1 significantly reduced glucose consumption when assayed by 2-NBDG uptake, a fluorescently labelled glucose analog. Additionally, Glut1 KO reversed the proliferative effects of IL4/IL4Rα stimulation on human cancer cells in vitro. Our data suggest that IL4-induced Glut1 expression supports the increased metabolic activity necessary for cancer cell proliferation. Targeting the IL4/IL4Rα signaling axis could potentially be a therapeutic strategy to impede tumor-associated metabolism and reduce tumor burden at primary and metastatic sites. Citation Format: Ebony Hargrove-Wiley, Daniel Valent, Demond Williams, Wendy Bindeman, Barbara Fingleton. IL4 receptor-induced proliferation is mediated via Glut1 activity in metastatic 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 2446.

Breast-to-brain metastasis is a devastating disease complication for which there are currently few treatment options. Among breast cancer subtypes, patients with triple-negative breast cancer (TNBC) and HER2+ breast cancer experience elevated rates of brain metastasis. The cytokines IL4 and IL13 are overexpressed by many human solid tumors. Both are associated with invasive and metastatic phenotypes; IL13 is additionally consequential in primary brain tumors. These cytokines signal through type I (IL4 only) and type II (IL4/IL13) IL4 receptors (IL4R). Importantly, expression of type II IL4R correlates with poor patient prognosis in basal-like (i.e., triple-negative) breast cancer, and HER2+ tumors display elevated type II IL4R expression. Our laboratory has shown that genetic loss of IL4R signaling in tumor cells attenuates lung and liver metastasis in mice. Additionally, we recently identified a role for IL4R signaling in modulating glycosylation in tumor cells. We hypothesize that IL4R signaling influences TNBC brain metastasis at least in part via modulating glycosylation. In a pilot mouse study, pharmacological blockade of IL4R after intracardiac injection of syngeneic TNBC cells resulted in attenuation of brain metastatic growth and circulating tumor cell burden. We have also observed significant changes in the expression of a sialyltransferase (ST8SIA1) that has been implicated in primary brain tumors, as well as in the abundance of several sialic acid-containing protein species in breast cancer cells following treatment with IL4 or IL13. Further, IL4 stimulation increases the overall glycosylation of several proteins of interest, including a glucose transporter and a glutamine transporter, without altering gene expression. Overall, our data suggest that type II IL4R signaling promotes metastasis, potentially through a mechanism involving protein glycosylation. Blockade of this receptor may be a useful therapeutic approach for patients with breast cancer brain metastases. Citation Format: Wendy Bindeman, Demond Williams, Ebony Hargrove-Wiley, Barbara Fingleton. Investigating the role of type II IL4 receptor in breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2401.

In the context of cancer biology Th2 cytokines such as IL4 and IL13 are most studied for their effects on immune cells such as macrophages. IL-4 receptor is established to cause polarization of macrophages to a pro-tumor M2 phenotype. While much is known about the signaling effects of IL-4 in immune cells there is relatively little known about its effects in cancer cells that express the receptor. Our lab has established a role of signaling in IL4 receptor expressing cancer cells for promoting metastasis. Many of the effects of IL-4 signaling in immune cells are accompanied by metabolic changes. Thus, we aimed to study the effects of IL-4 signaling on metabolism in breast cancer to better understand how these metabolic changes might induce metastatic phenotypes associated with IL-4 stimulation. IL-4 increased glucose uptake in multiple human TNBC cell lines assessed via NBDG uptake assay. Production of lactate was also significantly increased as assessed by colorimetric assay. We then became interested in possible alterations in metabolism downstream of increased glucose uptake. By Seahorse assay we found that IL-4 significantly increased glycolysis, as well as mitochondrial metabolism when assessed by ATP Rate assay. By metabolic flux using C13 labeled glucose we found that IL-4 increased incorporation of carbon from glucose into lactate while not having a significant effect on TCA cycle intermediates. This led us to question whether IL-4 may affect other arms of metabolism that feed into TCA cycle and mitochondrial metabolism. We looked at how IL4 impacts glutaminolysis and found that IL-4 stimulation leads to increased expression of glutamine transporter ASCT2, as well as increased glutamine uptake. We have begun to examine whether these metabolic changes affect epigenetic regulation. Initial data indicates that IL4 treatment leads to increased histone acetylation in TNBC cell lines. Citation Format: Demond Williams, Wendy Bindeman, Ebony Hargrove-Wiley, Barbara Fingleton. Interleukin-4 receptor signaling induces glycolysis in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2381.

Even when accounting for multiple clinical parameters, male breast cancer patients have significantly lower survival rates than females. Males and females differ in their levels of sex hormones androgens and estrogens. These sex hormones have varying influences on immune cell populations, which in turn may affect disease progression. Thus, the sex of the patient and the levels of sex hormones are likely important for anticancer immunity. Here, we assessed the importance of sex hormone signaling in the immune cells (host) versus the tumor cells for defining the immune landscape associated with primary and metastatic tumors. We used flow cytometry to determine the immune profile of mammary tumors, spleens, and lungs, and histochemistry to evaluate metastasis in transgenic MMTV-PyMT and in mice orthotopically injected with sex-specific MMTV-PyMT-derived cell lines in male and female cohorts. In transgenic mice, males had greater proportions of CD4+ T-helper and total myeloid cells in all organs analyzed. The splenic and pulmonary immune profiles of tumor-bearing males were more similar to cancer-free littermates than in females. Interestingly, males trended towards increased metastatic colonization in the lungs. Sex-associated immune profiles differed between transgenic and orthotopic mice. In mice bearing male-derived R221a PyMT tumors, females had decreased intratumoral neutrophils and elevated natural killer cells with increased splenic B-cells in comparison to males. Overall, our data supports a sex-dependent immune response in mammary carcinoma that is, in part, regulated by the hormonal environment of the host. With emerging therapeutics targeting the tumor immune microenvironment, characterization of immune profiles is critical for optimizing their use in all breast cancer patients. Citation Format: Ebony Hargrove-Wiley, Barbara Fingleton. The influence of sex on the immune microenvironment in male and female breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2506.