The Effects of Cholesterol-Derived Oncometabolites on Nuclear Receptor Function in Cancer

A role for cholesterol in the etiology of cancers has long been suspected. However, epidemiologic data still remain contradictory as to the relationship between serum cholesterol levels and cancer risk or development, suggesting that the dosage of circulating cholesterol alone would be limiting. Indeed, recent data from animal and cell model experiments, as well as clinical data, indicate that cholesterol metabolism generates endogenous oxysterols with an important role in cancer promotion, resistance to therapy and tumor recurrence, as well as in the control of cancer development (1). These oxysterols have been shown to bind to and regulate different nuclear receptor activities and for some of these metabolites, a differential metabolism has been found between normal and cancerous tissues. From these studies, it appears that the cholesterol metabolism generates new classes of ligands for glucocorticoid, estrogen, or liver X receptors [glucocorticoid receptor (GR), estrogen receptor (ER), liver X receptors (LXR), respectively] with important functions in cancers (Fig. 1). Interestingly, these natural ligands reveal activities or regulations unknown to these receptors. Oxysterols in tissues and plasma are usually present at concentrations several orders of magnitude lower than cholesterol (10⁴- to 10⁶-fold times less) and depending on the oxysterol considered, their concentrations can increase with circulating cholesterol concentration and diet (2). Therefore, minor changes in cholesterol levels in plasma and tissues could impact the production of these ligands and nuclear receptor activity. Moreover, the consumption of commercial cholesterol-rich foods can directly influence the oxysterol content of plasma and tissues because their processing and storage can produce, by cholesterol autoxidation, various oxysterols, which will bind to these nuclear receptors (2). These different aspects will be discussed in the current review.

Cholesterol is a bioactive lipid with a key role in intracellular signal transduction and energy storage. It is essential for mammalian cell differentiation and growth and for membrane structuration. It is the precursor of steroid hormones and oxysterols. In normal cells, cholesterol biosynthesis is tightly regulated and is controlled by cholesterol itself and by some oxysterols (3). Oxysterols are products of cholesterol oxygenation on the steroid backbone rings or on the aliphatic side-chain that can be generated by enzymes or by autoxidation processes in the presence of free reactive oxygen species or indirectly through lipoperoxidation (2, 4). Endogenous oxysterols, including 22(R)-hydroxycholesterol [22(R)HC]; 24(S)-hydroxycholesterol [24(S)HC]; 25-hydroxycholesterol (25HC), 7α-hydroxycholesterol (7αHC), 27-hydroxycholesterol (27HC), 5,6α-epoxycholesterol (5,6α-EC); and 5,6β-epoxycholesterol (5,6β-EC) are known liver X receptor (LXR) ligands, activating LXR responses at physiologic range concentrations (μmol/L; refs. 5, 6). LXRs exist as two isoforms (α and β) that form heterodimers with retinoid X receptors (RXR) and are ligand-activated transcription factors (6, 7). Oxysterols also regulate cholesterol import and export according to the level of intracellular cholesterol by acting through the LXRs (3).

In cancer cells and tumors, cholesterol biosynthesis is generally deregulated and increased through an upregulation/activation of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and sterol regulatory element-binding protein, an increase in its import via the low-density lipoprotein receptor and scavenger receptor class B type I, an inhibition of its export by the ABC transporters, ABCA1, ABCG1, a greater amount of storage of cholesteryl esters and the activation of different receptors and signaling pathways such as PI3K/Akt/mTor, TP53, or Hedgehog (3, 8–11). Interestingly, while cholesterol and side-chain oxysterols activate Smoothened in Hedgehog signaling (12, 13), endogenous B-ring oxysterols inhibit this pathway (14).

Interestingly, an oncometabolism downstream of 5,6α-EC and 5,6β-EC (5,6EC) was identified in breast cancer in human and animal models (15). The cholesterol epoxide hydrolase (ChEH), formed by the D8D7I and 3β-hydroxysterol-Δ⁷-reductase (DHCR7) enzymes, is known to metabolize 5,6-ECs into cholestane-3β,5α,6β-triol (CT) in normal tissues (4, 16). In breast cancer, the ChEH transforms 5,6ECs into CT that is further transformed into 6-oxo-cholestan-3β,5α-diol (OCDO) by 11β-hydroxysteroid-dehydrogenase-type-2 (11βHSD2), the enzyme inactivating cortisol into cortisone. OCDO promotes proliferation of breast cancer cells and tumors independently of ERα expression by binding to the GR, a nuclear receptor, and a ligand-activated transcription factor (17). OCDO also binds to LXRβ but LXRβ is not involved in tumor proliferation and its role has to be explored. OCDO does not bind to the ER unlike 25HC and 27HC (18, 19). Interestingly, OCDO activates GR translocation into the nucleus, as do the glucocorticoid (GC) agonists, cortisol or dexamethasone, to regulate gene transcription; however, its action on GR is distinct from that of cortisol or dexamethasone on cell proliferation and GR transcriptional activity. Thus, OCDO appears as a new class of GR-regulatory ligands that can compete and modify GC actions on GR. Taking into account the controversial actions of GCs on cell proliferation, apoptosis, survival, and drug resistance in some cancers, it can be assumed that the levels of OCDO produced in cancers and its action on GR could influence GCs and GR functions on these different events that will be important to study in the future. Moreover, the effect of OCDO on inflammation through GR and/or LXRs needs to be explored because chronic low-grade inflammation is a hallmark of obesity, and has been proposed as a mechanistic link between obesity and cancer.

Patient breast cancer samples showed significant increase in OCDO levels and greater ChEH and 11βHSD2 protein expression compared with normal tissues (15). The analysis of several human breast cancer mRNA databases indicated that 11βHSD2 and ChEH subunit overexpression was correlated with a higher risk of patient death in breast cancer and that high GR expression or activation was correlated with poor therapeutic response or prognosis in many solid tumors including breast cancer (15). Interestingly, ChEH inhibition and 11βHSD2 silencing inhibited OCDO production and tumor growth and mifepristone, a GR antagonist, as well as GR silencing inhibits OCDO-induced tumor cell proliferation (15). Thus, targeting this oncometabolism and GR represents new opportunities for therapeutic interventions in breast cancer and potentially other cancers presenting such deregulations.

Interestingly, endogenous oxysterols can be secreted by various types of mouse and human tumor cells. These oxysterols mediate immunosuppressive functions and dampen antitumor responses by inhibiting CC chemokine receptor-7 (CCR7) expression on maturing dendritic cells (DC) and their migration to lymphoid organs by acting on the LXRα present on DC. Blocking LXRα signaling was able to restore growth control, tumor rejection, and animal survival. Similar results on DC were obtained with 22(R)HC and 25HC (20).

In addition to being LXR ligands, different endogenous oxysterols bind to and modulate the functions of the ER. Indeed, it was reported that 25HC binds to ERα and promotes breast and ovarian cancer cell growth in an ERα-dependent manner and upregulates different estrogen target genes (18). Similarly, 27HC was shown to bind to ERα and to be a selective estrogen receptor modulator (SERM; ref. 19). SERMs are ER ligands that display agonist or antagonist activity depending on the target genes or tissues. Tamoxifen was the first synthetic SERM to be used in the clinic for ER-positive breast cancer treatment (21). 27HC antagonizes the action of 17β-estradiol on vascular endothelial cells and shows a proestrogenic effect in hepatoma and colon cancers (19). In addition, it behaves as a partial agonist, in different models of breast cancer cells expressing ERα, on gene transcription and cell proliferation, associated with increased cyclin D1 expression and accumulation of cells in the S-phase of the cell cycle (22). 27HC induces a unique active conformation of ERα, which may explain its partial agonist/SERM activity (22). 27HC is not produced by an autoxidation process but only by an enzymatic reaction involving the cytochrome P450 (CYP) enzyme CYP27A1 and is catabolized by CYP7B1 enzyme.

27HC also promotes ER-positive breast tumor growth in various mouse models (23, 24). Moreover, 27HC mediates breast tumor progression induced by a high-cholesterol diet in mice, which increases circulating and local 27HC levels in peripheral tissues. The inhibition of CYP27A1 is sufficient to prevent high-cholesterol diet–induced tumor proliferation without changing cholesterol levels, indicating that the effect is due to 27HC elevation and is independent of total circulating cholesterol. These effects seem to have clinical relevance because 27HC levels are higher in ER-positive breast tumors compared with normal breast tissues in patients. Moreover, in patient tumors, increased 27HC has been found to be correlated with reduced expression of CYP7B1 and reduced expression of CYP7B1 in tumors was found to be associated with poorer patient survival rate (23, 24). Therefore, the production of 27HC and its estrogenic activity through the ERα could explain treatment failures in some patients with breast cancer treated with aromatase inhibitors that decrease estrogen production.

27HC was reported to increase lung metastasis in the MMTV-PyMT mouse model of spontaneous ER-positive breast tumors by acting through the LXR and this has been associated with an induction of the expression of several genes involved in epithelial-to-mesenchymal transition. No impact of this receptor on tumor growth has been reported (24). In MMTV-PyMT mice with a CYP7b1^−/− background, tumor metastases are increased while in a CYP27a1^−/− background, metastases are decreased and a treatment with 27HC reversed this effect. Treatment with the GW3965 synthetic LXR ligand also stimulated lung metastases but less efficiently than 27HC (24). Together, these data indicate that 27HC promotes ER-positive tumor progression and aggressiveness by its dual action on ERα and LXR, respectively. Because CYP7B1 metabolizes 25HC and 27HC, it would be of interest to determine whether increased levels of 25HC, through its action on ERα, display similar growth-promoting effects as 27HC on ER-positive breast tumors.

In a prospective clinical study in patients with breast cancer receiving tamoxifen or an aromatase inhibitor (AI) in adjuvant or in metastatic settings, tamoxifen treatment significantly reduced 25HC levels in serum while AI increased 27HC levels, suggesting that 25HC levels could reflect the antitumor efficacy of tamoxifen while 27HC could be a marker of AI resistance and of poor prognosis (25). Consistent with these results, the analysis of the BIG 1-98 prospective trial (26), revealed that cholesterol levels are reduced during tamoxifen therapy and initiation of cholesterol-lowering medication during adjuvant hormone therapy improved patient disease-free survival and may prevent recurrence of cancer, possibly by regulating the levels of oxysterols such as 27HC or 25HC (25). Further clinical studies are needed to address this hypothesis.

The cholesterol biosynthesis pathway was reported as a novel mechanism of resistance to estrogen deprivation in a panel of ER-positive breast cancer cells adapted to long-term estrogen deprivation. Indeed, several enzymes involved in cholesterol biosynthesis, in particular the 3β-hydroxysterol-Δ⁸-Δ⁷-isomerase (D8D7I or EBP), Lamin-B receptor, and squalene epoxidase, were found upregulated in these cellular models and their silencing significantly decreased tumor cell proliferation. In silico analysis of patients with primary ER-positive breast cancer treated with neoadjuvant AI revealed that the increased expression of these enzymes is associated with poor response to this therapy. Interestingly, increased levels of 25HC were measured in these cells and 25HC and 27HC treatment was shown to influence ER transcriptional activity (27).

Cholesterol is an important dietary component present in large amounts in meats, eggs, cheese, and dairy products including dried eggs and milk powders that are extensively used in a large number of commercial foods. The modes and lengths of storage as well as the processing methods (dehydration, heating treatment, contact with oxygen) can influence the generation of various oxysterols by cholesterol autoxidation (2).

5,6ECs are produced mainly by cholesterol autoxidation, although an enzymatic reaction has also been proposed, while CT is not a spontaneous product of oxidation (4). 25HC can also be produced by cholesterol autoxidation in addition to being produced by the 25-hydroxylase enzyme, whereas 27HC is only produced by an enzymatic transformation of cholesterol. Cholesterol-rich processed foods contain high amounts of 5,6ECs and CT (2, 4), the mandatory precursors of OCDO (15), while OCDO and 25HC, in lower amounts, have been reported in some studies (2, 4). This suggests that processed foods containing OCDO or its precursors, which can be transformed into OCDO when the OCDO biosynthetic pathway is upregulated in tumors, could lead to an increase in tumor growth (15). Processed meats have recently been reported to increase mortality following breast cancer (28), and ultra-processed foods or red meat intake, which contain high amounts of cholesterol, have been associated with a significant increase in breast cancer risk (29). The relationship between high intakes of red or processed meats and increased risk of colorectal cancer has also been reported (30).

Recent global statistics indicate that the incidence of obesity is constantly rising worldwide. Approximately 35% of the adult population (age 20+) is overweight (BMI ≥ 25 kg/m²) and obesity (BMI ≥ 30 kg/m²) affects around 12% of the population. Obesity has been associated with an increased risk of a variety of cancer types as well as tumor progression, aggressiveness, or death in some cases (31). High levels of cholesterol in the blood are common in obese individuals. Given that cholesterol-rich diets and cholesterol-rich processed foods can generate 27HC or OCDO, two tumor promoter metabolites, which are increased in patient breast cancer samples compared with normal tissues, the importance of these factors in the risk, progression, and outcome of breast cancer, and potentially other cancers, in overweight and obese people remains to be determined.

Tamoxifen inhibits cholesterol biosynthesis in breast cancer cells, expressing or not the ERα, by inhibiting the ChEH (15, 16, 21). 5,6ECs accumulation and their subsequent sulfation by the sulfotransferase SULT2B1b generates sulfated 5,6ECs (5,6ECS), which mediate tamoxifen cytotoxicity by acting through the LXRβ in ER-positive MCF7 cells and the genetic inhibition of SULT2B1b in MCF-7 leads to tamoxifen resistance (32). Interestingly, tamoxifen efficacy is limited in ER-negative MDA-MB-231 breast cancer cells that do not express SULT2B1b while the reexpression of the enzyme restores tamoxifen sensitivity (32). These data established the unsuspected importance of 5,6EC, 5,6ECS as antitumor metabolites and LXRβ targeting in tamoxifen sensitivity in breast cancer cells.

In normal mammalian tissues including human tissues, 5,6α-EC reacts with histamine via an enzymatic process to produce a B-ring alkyl amino oxysterol, named dendrogenin A (DDA; ref. 33). DDA levels were found to be significantly decreased in patients with breast cancer compared with normal breast tissues, indicating a deregulation of DDA metabolism in breast cancer. DDA is a new class of LXR ligand that does not bind to ER (33, 34). DDA exhibits a potent anticancer activity against various tumors cells and significantly inhibits tumor growth of mouse breast cancer cells implanted into immunocompetent mice and enhances animal survival (33). These effects were associated with an increased infiltration of CD11c⁺ dendritic cells and T lymphocyte cells into the tumors, indicating that DDA stimulates an antitumor immune response against breast cancer (33). DDA also inhibits tumor growth of different models of human melanoma and leukemia including patient-derived xenografts by inducing a cytotoxic autophagy driven under the transcriptional control of the LXRβ, whereas other canonical LXR ligands do not induce such a mechanism (34). Interestingly, DDA is a competitive inhibitor of the ChEH and the most potent natural inhibitor of this enzyme (33). DDA inhibits OCDO production, in breast cancer expressing or not the ERα, and the inhibition of OCDO production contributes to the efficacy of DDA in vivo, indicating that the targeting of OCDO metabolism could be relevant for cancer treatment (15). By its action on the LXRβ, DDA could also compete with some OCDO functions on this receptor that need to be explored. The effects of DDA, which inhibit breast cancer progression, are contrary to those of OCDO, which stimulate breast cancer progression. Both compounds arise from 5,6ECs; however, only 5,6α-EC generates DDA, whereas both 5,6α-EC and 5,6β-EC produce OCDO (15, 33). The study of OCDO and DDA levels in human breast cancer samples in relation to normal tissues indicates the existence of a metabolic balance between these two 5,6EC derivatives that may either stimulate or control breast cancer progression (1, 15, 33). In accordance with the fact that DDA levels are decreased in patients with breast cancer, and thus may be the sign of a pathologic condition, a recent meta-analysis of prospective cohort studies in women indicate a significant inverse correlation between total plasma cholesterol and HDL cholesterol levels and the risk of breast cancer (35).

From these and previous studies using LXRs knockout mice or synthetic agonist ligands in vivo, it appears that LXRs control tumor growth and invasion, highlighting that LXRs are important therapeutic targets in cancers (33, 34, 36–40). It should be noted that endogenous LXR ligands have either a protumor or antitumor activity and the diversity of these effects may be due, in a nonexhaustive manner, to their tumor-intrinsic or -extrinsic activity, the specific signaling pathways they activate and/or they repress, and their structure that will determine the receptor activation state varying from agonism, partial agonism, to antagonism. A better understanding of the functioning of the LXRs by these endogenous ligands will certainly help define the signaling pathways to target and to propose the best LXR ligands for the treatment of cancers. A good example is the endogenous DDA ligand (33, 34). DDA is a partial LXR agonist that drives LXR to trigger lethal autophagy in tumors by modulating its transcriptional activity (34). Lethal autophagy is not observed with others LXR agonists such as 22(R)HC, T0901317, and GW3965 (34).

More generally, it appears that cancer cells display multiple levels of cholesterol metabolism dysregulations. Among these, cancer cells can generate oncopromoter cholesterol metabolites at higher levels compared with normal tissues and/or the production of oncosuppressor cholesterol metabolites can be deregulated in an opposite manner. Unexpectedly, all these cholesterol metabolites are new classes of endogenous ligands of nuclear receptors, such as the ER, LXR, or GR. Some of these new ligands have revealed unknown activities of their cognate receptors, opening new horizons for a better understanding of their pathophysiologic functions and for their targeting to develop new therapies against cancer progression and drug resistance. Given that hypercholesterolemia and the high intake of cholesterol-rich foods as well as their processing and storage can generate these oxysterols, their importance in cancer risk or development in overweight and obese people are important areas to be explored.

The discovery of these cholesterol-derived metabolites and the identification of the nuclear receptors mediating their pro- or antitumor activity open new areas of research, which should have major implications in the diagnosis, prevention, and treatment of cancers, and will certainly help to better understand why the development of classic ligands of these receptors may not always work to treat cancers.

S. Silvente-Poirot has ownership interest (including stock, patents, etc.) in Affichem. M. Poirot has ownership interest (including stock, patents, etc.) in Affichem. No potential conflicts of interest were disclosed by the other author.

M. Poirot and S. Silvente-Poirot's team was funded by the Institut National de la Santé et de la Recherche Médicale (INSERM); the Université de Toulouse III; the Association pour la Recherche sur le Cancer (ARC; PJA 20131200342); the Agence Nationale pour la Recherche (ANR-11-RPIB-015-02 DAML, ANR-11-PHUC-0001), Onco SanTech (project DEMODA RMN13001BBA); the Fondation de France (00057930); The région Midi-Pyrénées CLE 2014 n°13053058; the Institut National du Cancer (INCA; PRTK-K15-118); the Initiatives d'Excellence (IDEX) Actions Thématiques Stratégiques (2016 057-ATS-2015 InnoVinBC); the Fondation Toulouse Cancer Santé (2017CS065); and the associations Céline and Flo.