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Metastatic estrogen receptor α (ERα)-positive breast cancer is presently incurable. Seeking to target these drug-resistant cancers, we report the discovery of a compound, called ErSO, that activates the anticipatory unfolded protein response (a-UPR) and induces rapid and selective necrosis of ERα-positive breast cancer cell lines in vitro. We then tested ErSO in vivo in several preclinical orthotopic and metastasis mouse models carrying different xenografts of human breast cancer lines or patient-derived breast tumors. In multiple orthotopic models, ErSO treatment given either orally or intraperitoneally for 14 to 21 days induced tumor regression without recurrence. In a cell line tail vein metastasis model, ErSO was also effective at inducing regression of most lung, bone, and liver metastases. ErSO treatment induced almost complete regression of brain metastases in mice carrying intracranial human breast cancer cell line xenografts. Tumors that did not undergo complete regression and regrew remained sensitive to retreatment with ErSO. ErSO was well tolerated in mice, rats, and dogs at doses above those needed for therapeutic responses and had little or no effect on normal ERα-expressing murine tissues. ErSO mediated its anticancer effects through activation of the a-UPR, suggesting that activation of a tumor protective pathway could induce tumor regression.

Cholesterol has been implicated in the clinical progression of breast cancer, a disease that continues to be the most commonly diagnosed cancer in women. Previous work has identified the cholesterol metabolite 27-hydroxycholesterol (27HC) as a major mediator of the effects of cholesterol on breast tumor growth and progression. 27HC can act as an estrogen receptor (ER) modulator to promote the growth of ERα+ tumors, and as a liver X receptor (LXR) ligand in myeloid immune cells to establish an immune-suppressive program. In fact, the metastatic properties of 27HC require the presence of myeloid cells with neutrophils (polymorphonuclear neutrophils; PMNs) being essential for the increase in lung metastasis in murine models. In an effort to further elucidate the mechanisms by which 27HC alters breast cancer progression, we made the striking finding that 27HC promoted the secretion of extracellular vesicles (EVs), a diverse assortment of membrane bound particles that includes exosomes. The resulting EVs had a size distribution that was skewed slightly larger than EVs generated by treating cells with vehicle. The increase in EV secretion and size was consistent across 3 different subtypes: primary murine PMNs, RAW264.7 monocytic cells, and 4T1 murine mammary cancer cells. Label-free analysis of 27HC-EVs indicated that they had a different metabolite composition to those from vehicle-treated cells. Importantly, 27HC-EVs from primary PMNs promoted tumor growth and metastasis in 2 different syngeneic models, demonstrating the potential role of 27HC-induced EVs in the progression of breast cancer. EVs from PMNs were taken up by cancer cells, macrophages, and PMNs, but not T cells. Since EVs did not alter proliferation of cancer cells, it is likely that their protumor effects are mediated through interactions with myeloid cells. Interestingly, RNA-seq analysis of tumors from 27HC-EV-treated mice do not display significantly altered transcriptomes, suggesting that the effects of 27HC-EVs occur early on in tumor establishment and growth. Future work will be required to elucidate the mechanisms by which 27HC increases EV secretion, and how these EVs promote breast cancer progression. Collectively, however, our data indicate that EV secretion and content can be regulated by a cholesterol metabolite, which may have detrimental effects in terms of disease progression, important findings given the prevalence of both breast cancer and hypercholesterolemia.

Triple negative breast cancer (TNBC) is challenging to treat successfully because targeted therapies do not exist. Instead, systemic therapy is typically restricted to cytotoxic chemotherapy, which fails more often in patients with elevated circulating cholesterol. Liver x receptors are ligand-dependent transcription factors that are homeostatic regulators of cholesterol, and are linked to regulation of broad-affinity xenobiotic transporter activity in non-tumor tissues. We show that LXR ligands confer chemotherapy resistance in TNBC cell lines and xenografts, and that LXRalpha is necessary and sufficient to mediate this resistance. Furthermore, in TNBC patients who had cancer recurrences, LXRalpha and ligands were independent markers of poor prognosis and correlated with P-glycoprotein expression. However, in patients who survived their disease, LXRalpha signaling and P-glycoprotein were decoupled. These data reveal a novel chemotherapy resistance mechanism in this poor prognosis subtype of breast cancer. We conclude that systemic chemotherapy failure in some TNBC patients is caused by co-opting the LXRalpha:P-glycoprotein axis, a pathway highly targetable by therapies that are already used for prevention and treatment of other diseases.

Triple negative breast cancer (TNBC) is challenging to treat successfully because targeted therapies do not exist. Instead, systemic therapy is typically restricted to cytotoxic chemotherapy, which fails more often in patients with elevated circulating cholesterol. Liver x receptors are ligand-dependent transcription factors that are homeostatic regulators of cholesterol, and are linked to regulation of broad-affinity xenobiotic transporter activity in non-tumor tissues. We show that LXR ligands confer chemotherapy resistance in TNBC cell lines and xenografts, and that LXRalpha is necessary and sufficient to mediate this resistance. Furthermore, in TNBC patients who had cancer recurrences, LXRalpha and ligands were independent markers of poor prognosis and correlated with P-glycoprotein expression. However, in patients who survived their disease, LXRalpha signaling and P-glycoprotein were decoupled. These data reveal a novel chemotherapy resistance mechanism in this poor prognosis subtype of breast cancer. We conclude that systemic chemotherapy failure in some TNBC patients is caused by co-opting the LXRalpha:P-glycoprotein axis, a pathway highly targetable by therapies that are already used for prevention and treatment of other diseases.

Breast cancer remains one of the leading causes of cancer mortality in the US. Elevated cholesterol is a major risk factor for breast cancer onset and recurrence, while cholesterol-lowering drugs, such as statins, are associated with a good prognosis. Previous work in murine models showed that cholesterol increases breast cancer metastasis, and the pro-metastatic effects of cholesterol were due to its primary metabolite, 27-hydroxycholesterol (27HC). In our prior work, myeloid cells were found to be required for the pro-metastatic effects of 27HC, but their precise contribution remains unclear. Here we report that 27HC impairs T cell expansion and cytotoxic function through its actions on myeloid cells, including macrophages, in a Liver X receptor (LXR) dependent manner. Many oxysterols and LXR ligands had similar effects on T cell expansion. Moreover, their ability to induce the LXR target gene ABCA1 was associated with their effectiveness in impairing T cell expansion. Induction of T cell apoptosis was likely one mediator of this impairment. Interestingly, the enzyme responsible for the synthesis of 27HC, CYP27A1, is highly expressed in myeloid cells, suggesting that 27HC may have important autocrine or paracrine functions in these cells, a hypothesis supported by our finding that breast cancer metastasis was reduced in mice with a myeloid specific knockout of CYP27A1. Importantly, pharmacologic inhibition of CYP27A1 reduced metastatic growth and improved the efficacy of checkpoint inhibitor, anti-PD-L1. Taken together, our work suggests that targeting the CYP27A1 axis in myeloid cells may present therapeutic benefits and improve the response rate to immune therapies in breast cancer.

Immune checkpoint blockade has had underwhelming responses in breast cancer, in part due to the highly immune suppressive microenvironment. As a result, breast cancer continues to be the second most common cancer-related mortality amongst women, providing strong rationale for the development of new therapeutic approaches. Elevated circulating cholesterol is a poor prognostic, while breast cancer patients taking cholesterol-lowering drugs display increased time to recurrence. We and others have previously demonstrated that cholesterol metabolites mediate these effects by promoting breast cancer growth and metastasis, in part by suppressing the immune system. Therefore, given the demonstrated importance of cholesterol and its metabolites in breast cancer pathophysiology and immunology, we hypothesized that proteins involved in the regulation of cholesterol homeostasis would influence cancer progression. Through informatics analysis of breast tumors, we found that elevated expression of Small Heterodimer Partner (SHP; NR0B2) was a favorable prognostic. Antigen presenting cells such as macrophages and dendritic cells were found to express SHP, and manipulation of SHP altered the expression of genes involved in cross-talk with T cells. Intriguingly, when activated T cells were co-cultured with macrophages overexpressing SHP, there was a decrease in the expansion of regulatory T cells (Tregs) and vice versa in the absence of SHP. Adoptive transfer studies confirmed that loss of SHP resulted in immune suppressive Tregs. We hypothesized that myeloid cell-expressed SHP would promote immune surveillance and tumor clearance. In support of this hypothesis, tumors in the MMTV-PyMT model of mammary cancer grew at an accelerated rate in SHP-knockout mice. Tumors from these mice had significantly more Tregs and fewer effector T cells. Furthermore, orthotopic mammary tumor grafts grew at an increased rate in mice lacking SHP expression in myeloid cells (SHPfl/fl;LysMCre), compared to controls. A small molecule agonist of SHP impaired primary and metastatic tumor growth, and significantly enhanced the efficacy of immune checkpoint blockade in murine models of mammary cancer. Therefore, SHP represents a potential target to decrease suppressive Tregs, thereby allowing for immune-clearance of tumors.

Breast cancer remains one of the leading causes of cancer mortality in the US. Elevated cholesterol is a major risk factor for breast cancer onset and recurrence, while cholesterol-lowering drugs, such as statins, are associated with a good prognosis. Previous work in murine models showed that cholesterol increases breast cancer metastasis, and the pro-metastatic effects of cholesterol were due to its primary metabolite, 27HC. In our prior work, myeloid cells were found to be required for the pro-metastatic effects of 27HC, but their precise contribution remains unclear. Here we report that 27HC impairs T cell expansion and cytotoxic function through its actions on myeloid cells, including macrophages, in an LXR-dependent manner. Many oxysterols and LXR ligands had similar effects on T cell expansion. Moreover, their ability to induce the LXR target gene ABCA1 was associated with their effectiveness in impairing T cell expansion. Interestingly, the enzyme responsible for the synthesis of 27HC, CYP27A1, is highly expressed in myeloid cells, suggesting that 27HC may have important autocrine or paracrine functions in these cells, a hypothesis supported by our finding that breast cancer metastasis was reduced in mice with a myeloid specific knockout of CYP27A1. Pharmacologic inhibition of CYP27A1 reduced metastatic growth and improved the efficacy of checkpoint inhibitor, anti-PD-L1. RNA sequencing of 27HC-treated macrophages and GSEA analysis provide further mechanistic insight, revealing an enrichment of MYC and NOTCH signaling pathways, both of which are documented pathways involved in tumor-associated macrophages. Taken together, our work suggests that targeting the CYP27A1 axis in myeloid cells may present therapeutic benefits and improve the response rate to immune therapies in breast cancer. Delineating the link between LXR and the known mechanisms of tumor-associated macrophages is important to fully understand 27HC-driven cancer metastasis. This work was supported by grants to ERN from the National Cancer Institute of the National Institutes of Health (R01CA234025) and METAvivor. This abstract is also being presented as PO050. Citation Format: Liqian Ma, Lawrence Wang, Adam T. Nelson, Chaeyeon Han, Sisi He, Madeline A. Henn, Karan Menon, Joy J. Chen, Amy E. Baek, Anna Vardanyan, Sayyed Hamed Shahoei, Sunghee Park, David J. Shapiro, Som G. Nanjappa, Erik R. Nelson. 27-Hydroxycholesterol acts on myeloid immune cells to induce T cell dysfunction, promoting breast cancer progression [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR006.

It has become clear that cholesterol metabolism and homeostasis play significant roles in the progression of breast cancer. Specifically, elevated circulating cholesterol is a poor prognostic, while patients taking cholesterol-lowering drugs such as statins display increased recurrence-free survival time. Preclinical and clinical work has established that in addition to cholesterol, various downstream metabolites play direct roles in promoting breast cancer growth and metastasis. Given the demonstrated importance of cholesterol and its metabolites in breast cancer pathophysiology, we hypothesized that proteins involved in the regulation of cholesterol homeostasis would play a role in cancer progression. Therefore, we performed an informatics screen to identify those regulatory proteins associated with breast cancer progression. We focused on nuclear receptors due to their well-defined ligand-binding pocket and thus their proclivity to drug intervention. Our screen revealed that increased expression of Small Heterodimer Partner (SHP; NR0B2) was associated with an increased time to recurrence. However, manipulation of SHP within breast cancer cells did not alter proliferation or migration, suggesting that its protective role is likely conveyed through the tumor microenvironment. Macrophages were found to express SHP, and manipulation of SHP within macrophages resulted in altered expression of molecules associated with antigen presentation. Considering the clinical data indicating a protective role for SHP, it was somewhat paradoxical that its loss within macrophages resulted in an increased expansion of T cells. Upon further investigation, we found that this expansion was skewed towards regulatory T cells (Tregs). On the other hand, overexpression of SHP resulted in decreased expansion of Tregs. The immune-suppressive activity of the resulting Tregs was confirmed in subsequent assays. While immune therapies have revolutionized the treatment of certain cancers, their utility in breast cancer has been limited, especially outside of triple-negative disease. It has been speculated that this may be due to the highly immune-suppressive activities of certain myeloid and T cell populations. Thus, reducing Treg infiltration or activity likely represents a rational way to enhance immune therapies. In this regard, SHP-knockout mice bred with the MMTV-PyMT model of mammary cancer displayed significantly enhanced tumor growth compared to SHP-replete mice. Likewise, orthotopic mammary tumor grafts grew at an increased rate in mice where SHP was selectively knocked out in cells of the myeloid lineage (SHPfl/fl;LysMCre), compared to controls. Importantly, treatment with a small molecule agonist of SHP significantly enhanced the efficacy of anti-PD-L1 therapy in blocking the growth of an orthotopically grafted tumor, as well as in a model of metastatic mammary cancer. Collectively, our data strongly support a role for SHP in reducing the progression of breast cancer by limiting Treg expansion, thereby facilitating an anti-cancer immune response. As this nuclear receptor is amenable to small molecule intervention, SHP may represent a unique way to enhance the efficacy of immune checkpoint blockade. This study was funded in part by awards to ERN from the DOD BCRP (BC171214) and NCI (R01CA234025), to LA from the French National Research Agency Lipstic Labex (ANR-11-LABX-0021), and a STEM Chateaubriand Fellowship to SHS from the Embassy of France in the United States. Citation Format: Erik R. Nelson, Sayyed Hamed Shahoei, Adam T Nelson, Madeline A Henn, Ashley E Mathews, Joy J Chen, Varsha Vembar, Liqian Ma, Lionel Apetoh. The small heterodimer partner in macrophages reduces expansion of regulatory T cells and enhances immune checkpoint inhibition in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-01.