The Cholesterol Metabolite 27HC Increases Secretion of Extracellular Vesicles Which Promote Breast Cancer Progression

Abstract

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 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 (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 include exosomes. The resulting EVs had a size distribution that was skewed slightly larger, compared to EVs generated by treating cells with vehicle. The increase in EV secretion and size was consistent across three 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 two 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 pro-tumor 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.

Neutrophil phenotypes and functions in cancer: A consensus statement

“…In cancer, both genetic- and diet-induced obesity models have elevated peripheral and pulmonary neutrophils that promote breast cancer metastasis to the lung in a GM-CSF–dependent manner ( McDowell et al, 2021 ; Quail et al, 2017 ). Similar findings have been reported in murine models of high fat diet–induced hypercholesterolemia, where the cholesterol metabolite 27-hydroxycholesterol supports neutrophil recruitment to distal sites to promote metastasis ( Baek et al, 2021 ; Baek et al, 2017 ; Ma et al, 2020 ). Consistently, it has been shown that a high-fat diet promotes the accumulation of Gr1 + myeloid cells within multiple tissues concomitant with suppressed CD8 + T cells and enhanced cancer progression ( Clements et al, 2018 ; Ringel et al, 2020 ; Xia et al, 2011 ).…”

Impacts of ezetimibe on risks of various types of cancers: a meta-analysis and systematic review

“…It seems that the conclusion contradicts with most of the results from animal or in-vitro experiments and the reason is far from clear. Similarly, many reports show that cholesterol promoted carcinogenesis, progression, proliferation and chemo/ radioresistance of breast cancer (Brindisi et al, 2020;Baek et al, 2021;González-Ortiz et al, 2021) while data of these studies showed that ezetimibe has a trend of increasing risk of breast cancer (Fig. 2b).…”

Regulation of intrinsic and extrinsic metabolic pathways in tumour‐associated macrophages

“…27‐HC is an abundant cholesterol metabolite in human circulation. Studies have demonstrated that 27‐HC functions as an endogenous oestrogen receptor agonist and its increased expression is associated with a poor prognosis of breast cancer [173,174]. A large‐scale clinical study by Borgquis et al.…”

“…27-HC is an abundant cholesterol metabolite in human circulation. Studies have demonstrated that 27-HC functions as an endogenous oestrogen receptor agonist and its increased expression is associated with a poor prognosis of breast cancer [173,174]. A large-scale clinical study by Borgquis et al demonstrated that patients treated with cholesterol-lowering drugs such as statins and non-statins showed a better response to Tamoxifen; the drug effect may be owing to the attenuated oestrogen receptor signalling by the reduction in 27-HC level.…”