Elucidation of the Role(s) of Estrogens in Regulating Natural Killer Cell Biology in Breast Cancer Metastasis

Breast cancer is the most diagnosed cancer and the leading cause of cancer-related deaths among women worldwide. Estrogens, acting through their cognate receptor(s) support the growth of most breast cancers and as such, endocrine therapies that target cancer cell intrinsic signaling by estrogens are the cornerstone of treatment for these patients. While these therapies have increased the survival of patients, resistance remains an impediment to durable clinical responses especially in patients with metastatic disease. Thus, there is an unmet medical need for new therapies to treat this disease. Recently, therapies to increase antitumor immunity have led to clinical success in various cancers, but their efficacy in ER-positive (ER+) breast cancers is still limited. Interestingly, studies outside the realm of cancer have established that estrogen signaling affects immunological responses. However, how estrogen signaling in ER-positive tumor infiltrating cells affects cancer progression and metastasis is underexplored. Consequently, the overarching goal of this thesis work was to decipher how estrogen signaling and endocrine therapy in the tumor immune microenvironment affect tumor progression. This work was undertaken with the goal of identifying processes and pathways, the targeting of which could improve antitumor immunity and increase the efficacy of endocrine therapies.

The first part of this work will describe our efforts to define the role of estrogens/estrogen signaling on the function of natural killer cells in breast cancer metastasis. In these studies, we utilized murine breast cancer cell lines that are themselves insensitive to estrogens and investigated how the most common estrogen, 17β-estradiol (E2) affected breast cancer metastasis of these cells in immunocompromised mice and when propagated syngeneically in immunocompetent mice. We report that much of the biology of estrogens in tumors/metastatic lesions can be attributed to their actions in immune cells. Notable was the observation that E2 decreases metastasis of breast cancer cells in immunocompromised mice, but these protective effects are lost or reversed in immunocompetent mice. Immune profiling of the estrogen-treated mice indicated that E2 reduced the numbers, activity of NK-cells and their ability to produce IFN. These activities reduced their antitumor potential. Using single-cell RNA-sequencing technology (scRNA-seq), we identified an immunosuppressive sub-population of NK cells characterized by high expression of transforming growth factor B (TGFβ) in metastasis-bearing lungs that is expanded upon estrogen treatment in mice. Additionally, we discovered that E2 inhibits NF-B signaling and suppresses the activities of transcription factors involved in IFN production. Using a mouse model of NK-specific ER-alpha (ER) ablation, we confirmed that the actions of estrogens in NK cell biology were mediated through direct actions of the receptor in these cells. We also report unexpected pharmacology of an ER degrader, fulvestrant in breast cancer metastasis in that, like E2, fulvestrant inhibited NK cell numbers and activity and increased breast cancer metastasis. Collectively, our data indicated that estrogen signaling in NK cells suppresses the activity of these immune cells and contributes to increased breast cancer metastasis. These findings beg a reevaluation of mechanisms by which ER-modulators/endocrine therapies impact NK cell function, work that we believe will enable the selection from among existing drugs those which have favorable activities in NK cells and/or inform approaches to develop the next generation of ER-modulators optimized for their activity in metastatic disease.

While most of this work focuses on estrogen signaling in NK cells, the second part of this thesis describes our studies investigating how estrogens affect the function of tumor-associated macrophages (TAMs) in the context melanoma primary tumor growth. Melanoma is not traditionally classified as a hormone-sensitive cancer, however, there exists a gender bias in response to immunotherapies in melanoma, with women experiencing worse outcomes than men. Our previous data indicated that estrogens increase the growth of primary melanoma tumors, decreasing the number of tumor-infiltrating T-cells, and skewing TAMs towards an immunosuppressive, M2-like phenotype. We undertook ex-vivo and in-vitro experiments to probe TAM- T cell interactions upon E2 treatment. Using this approach, it was determined that E2 did not directly affect T-cell activity but resulted in the suppression of T-cell activity secondary to its actions on TAMs. TAMs from E2-treated tumors suppressed T-cell proliferation, activation, and secretion of cytotoxic granules, and genetic ablation of ER in TAMs reversed the ability of TAMs to suppress T-cell activity upon E2 treatment. While this work discovered the clinical utility of targeting ER signaling in TAMs in melanoma, these findings are currently being translated to other cancer types, especially those with a high intratumoral macrophage infiltration.

Overall, the work performed has unraveled the importance of estrogen signaling in the tumor immune microenvironment, opening new avenues for increasing anti-tumor immunity through the targeting of estrogen signaling in immune cells.