Role of de novo cholesterol synthesis enzymes in cancer

Despite extensive research in the cancer field, cancer remains one of the most prevalent diseases. There is an urgent need to identify specific targets that are safe and effective for the treatment of cancer. In recent years, cancer metabolism has come into the spotlight in cancer research. Lipid metabolism, especially cholesterol metabolism, plays a critical role in membrane synthesis as well as lipid signaling in cancer. This review focuses on the contribution of the de novo cholesterol synthesis pathway to tumorigenesis, cancer progression and metastasis. In conclusion, cholesterol metabolism could be an effective target for novel anticancer treatment.


Introduction
Over the past few decades, numerous published studies have focused on cancer cell metabolism and have determined that metabolic reprogramming is a hallmark of cancer [1][2][3][4][5][6] . It is thought that reprogramming of catabolic and anabolic metabolism to harvest energy and synthesize biomass is critical for the survival and growth of cancer cells. Nearly a century ago, Otto Warburg observed that cancer cells tend to use glucose extensively through aerobic glycolysis 7 . Highly proliferative cancer cells also tend to have a high lipid content (fatty acids and cholesterol), which is important for providing energy, membrane synthesis, and lipid signaling 8 . Cancer cells often exhibit an enhanced ability to synthesize lipids and have a higher lipid uptake 9 . Most studies have reported that the upregulation of fatty acid and cholesterol related enzymes is required for tumor progression [9][10][11][12][13][14] . Lipid metabolism involves lipid synthesis, storage and degradation. In mammals, cholesterol is either absorbed from dietary sources or synthesized de novo. The liver and intestinal mucosa are the main sites of cholesterol synthesis. Up to 70-80% of cholesterol in humans is synthesized de novo by the liver, and 10% is synthesized de novo by the small intestine. Accumulating evidence demonstrates that cholesterol plays a critical role in cancer progression [15][16][17][18][19] . Furthermore, intracellular cholesterol homeostasis is different among various cancer types, and cholesterol itself plays varying roles among different cancer types 17 . In this review, we describe normal cholesterol synthesis and cholesterol metabolic changes in cancer cells. Cholesterol biosynthesis pathways could be an attractive therapeutic target for cancer therapeutics.

Total cholesterol and cancer
Cholesterol is a primary lipid that is essential for membrane biogenesis, cell proliferation, and differentiation. Cholesterol is also the precursor of steroid hormones and sterols that induce specific biological responses. Cholesterol is mainly synthesized by the liver in humans, and is distributed throughout the body via high-density lipoprotein (HDL) and low-density lipoprotein (LDL) transporters. Acetyl-CoA is a key precursor of de novo cholesterol synthesis 20

Transcription factor and cholesterol de novo synthesis enzymes
Several steps are required to convert acetyl-CoA to cholesterol, which then is involved in numerous biological roles. These steps involve cholesterol synthase (ACAT, HMGCR, SQLE, OSC), acyl coenzyme A, cholesterol acyltransferases (SOAT), and ATP-binding cassette transporter A-1 (ABCA1). In a situation of decreasing cholesterol availability, inhibiting these enzymes could influence cancer cell growth. Interestingly, many inhibitors of these enzymes have effects on cancer treatment ( Figure 1). SREBPs, which were reported the most transcription factors (sterol regulatory element binding proteins,) regulate cholesterol de novo synthesis. Also, KLF14 22 , ChREBP 23,24 , LXRα 25 and LRH-1 26 have very important roles in cholesterol metabolism. Due to the limitation of words, we just reviewed the role SREBP played on it.

SREBP
Lipid homeostasis in vertebrate cells is regulated by a series of membrane-bound transcription factors, the sterol regulatory element-binding proteins (SREBPs). SREBPs directly activate more than 30 genes specific to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids, as well as the nicotinamide adenine dinucleotide phosphate cofactor required to synthesize these molecules 27 .
In 2016, Zhao et al. demonstrated that the hepatitis B X-interacting protein (HBXIP) upregulates SREBP-1c/SREBF1, which activates the transcription of fatty acid synthase by directly interacting with nuclear receptor coactivators and LXR. Overexpression of SREBP-1c can also activate HBXIP transcription. HBXIP enhances fat production, leading to the growth of breast cancer cells in vitro and in vivo 28 . Therefore, direct inhibition of SREBPs, resulting in cholesterol depletion, may be an effective cancer therapeutic strategy 29 .

HMGCR
The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) enzyme is the rate-limiting enzyme of the cholesterol synthesis pathway. In 2016, two reports showed that autoantibody-positive anti-HMGCR myopathies and HMGCR-positive inflammatory myopathies were association with cancer 30,31 . In a clinical study, positive cytoplasmic HMGCR expression was associated with colon cancer with distant metastasis-free disease at diagnosis. Positive HMGCR expression was significantly associated with prolonged cancer-specific survival in an unadjusted Cox regression analysis in the entire cohort and in stage III-IV disease 32 . Increased HMGCR expression was also observed in gastric cancer tissues. Overexpression of HMGCR promoted the growth and migration of gastric cancer cells, while HMGCR knockdown inhibited growth, migration and tumorigenesis 33 . Upregulation of HMGCR was also observed in clinical glioblastoma samples. Forced expression of HMGCR promoted the growth and migration of U251 and U373 cells, while knockdown of HMGCR inhibited their growth, migration and metastasis 34 . HMGCR was overexpressed in prostate cancer (PC) stroma, especially in early-stage PC. These results provide insights into the molecular mechanisms underlying PC invasion 35 . Another study found that ectopic expression of hsa-miR-195 in MCF-7 and MDA-MB-231 cells, resulting from targeting HMGCR, significantly altered cellular cholesterol and triglyceride levels as well as reduced proliferation, invasion and migration 36 . Statins are often used to lower cholesterol by inhibiting HMGCR. The multipotency of statins has been associated with cancer risk. Several studies have also found that statins play an important role in the treatment of several cancers 37 . In 2012, Gazzerro et al. reviewed statin pharmacology, elucidating the prospect of utilizing statins in cancer treatment for hepatocellular carcinoma (HCC), colorectal carcinoma (CRC), and acute myelocytic leukemia 38 . A recent Danish study correlated the use of statins in cancer patients. They observed reduced cancer-related mortality for 13 cancer types among statin users. Statin use in patients with cancer is also associated with reduced cancer-related mortality 39 . In epithelial ovarian cancer, a retrospective study reported that the use of statins was associated with improved clinical outcomes 40 . Statin use prior to a cancer diagnosis was correlated to a reduction in all-cause and cancer-specific mortality. Consistent with these data, patients with CRC with pre-diagnosis statin use had prolonged cancer-specific survival, but no benefits were observed for patients with post-diagnosis statin use 41 . Additionally, the risk of CRC was lower in statin users versus nonusers 42 . The anticancer effect of simvastatin through the induction of apoptosis is related to AKT signaling-dependent downregulation of survival in A549 lung cancer cells 43 . In an analysis of 999 colon cancer patients, statin use was correlated with a reduced risk of death from any cause or from cancer. The benefit of statin use was greater for patients whose tumors had intact bone morphogenetic protein signaling independent of KRAS mutation status 44 . Simvastatin also affected OCM-1 cell growth, apoptosis and cell cycle. In addition, simvastatin resulted in increased ROS levels and significantly increased apoptosis and the expression of the mitochondrion-related apoptosis protein p53 in OCM-1 cells 45 . In 2016, a surprising report found that statins preferentially inhibited the growth of cancer cells that express mutations, and p53 status impacted statin-dependent efficacy of cancer therapy 46 .
ACAT Acetyl-CoA acetyltransferase 1 (ACAT1) is a tetrameric enzyme in the ketogenesis pathway that converts two acetyl-CoA molecules into acetyl-CoA and CoA 47-49 . Fan et al. found that knockdown of ACAT1 attenuated tumor growth 50 . The inhibition of tetrameric ACAT1 by abolishing Y407 phosphorylation, or eliminating arecoline hydrobromide treatment, resulted in suppressed ACAT1 activity 51 . This lead to increased pyruvate dehydrogenase complex flux and oxidative phosphorylation, with attenuated cancer cell proliferation and tumor growth. These findings suggested that ACAT1 could be an effective anticancer target. Acetyl-CoA acetyltransferase 2 (ACAT2) also plays an important role in lipid metabolism. ACAT2 downregulation has been associated with a poorer cancer-specific survival prognosis in clear cell renal cell carcinoma 52 .

SOAT
Sterol-o-acyltransferase (SOAT), also called Acyl-CoA cholesterol acyltransferase (ACAT), is an integral membrane protein of the rough ER that catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acids. The solubility characteristics of cholesteryl esters make them the ideal molecule for cholesterol storage, which occurs mostly in cytoplasmic lipid droplets inside cells. Thus, SOAT is a key enzyme involved in the control of intracellular cholesterol storage and in determining free cholesterol levels 53 . SOAT plays an important role in the homeostasis of cell cholesterol metabolism and is a drug target for treatment intervention in various diseases, such as atherosclerosis, Alzheimer's disease and cancer 54 .
In 2019, through proteomic and phosphorproteomic profiling, he's group found that SOAT1 expression was higher in HCC and was associated with a poor prognosis. In addition, inhibiting SOAT1 markedly suppressed cell proliferation and migration 55 . SOAT1 was also overexpressed in human castration-resistant metastatic PC tissues. These results indicate that the enzymes involved in the ketogenic pathway are upregulated in high-grade PC and could act as potential tissue biomarkers for the diagnosis or prognosis of high-grade disease 56 . Li et al. found an abnormal accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines, which was mediated by SOAT1. SOAT1 expression was associated with poor outcomes. By using an SOAT1 inhibitor or shRNA knockdown, abrogated cholesterol esterification significantly suppressed tumor growth and metastasis in an orthotopic pancreatic cancer mouse model. SOAT1 inhibition was found to increase intracellular free cholesterol levels, which was associated with ER stress and apoptosis 57 . Furthermore, the availability of cholesteryl esters increased the proliferation and invasiveness of normal cells, indicating that cholesteryl esters may contribute to a tumorpromoting phenotype. Another study showed that breast cancer cell lines treated with auraptene, a naturally occurring SOAT1 inhibitor, also had decreased cellular proliferation, invasion, and colony formation 58 . Treatment of human prostate, pancreatic, lung, and colon cancer cell lines with avasimin, a potent SOAT1 inhibitor, significantly reduced cholesteryl ester storage in lipid droplets and increased intracellular free cholesterol levels. This led to apoptosis and the suppression of proliferation. Systemic treatment of avasimin notably suppressed tumor growth in mice and prolonged survival time 59 . Similarly, SOAT1 inhibition decreased cell proliferation and invasion in two tumor cell lines 60 .
The SOAT1 inhibitor CP-113818 reduced proliferation of breast cancer cells and specifically inhibited LDL-induced proliferation of ERα-cells. A greater ability to take up, store and utilize exogenous cholesterol conferred a proliferative advantage in basal-like ERα-breast cancer cells as well 61 .
Revitalizing the cytotoxic potential of CD8+ T cells is of great clinical interest in the cancer immunotherapy field. A novel mechanism to enhance the antitumor response of CD8+ T cells in mice by regulating cholesterol metabolism has been reported. The inhibition of cholesterol esterification in T cells, either by genetic ablation or by pharmacological inhibition of SOAT1, resulted in enhanced effects and proliferation of CD8+ T cells but not CD4+ T cells. In the absence of SOAT1, CD8+ T cells performed better than wild-type CD8+ T cells in controlling the growth and metastasis of melanoma in mice. In addition, combination therapy with avasimibe, SOAT inhibitors and anti-PD-1 antibodies had a superior effect on controlling tumor progression than single agent therapy 62 .
Human SOAT2 is mainly expressed in the intestine and fetal liver. Inhibiting SOAT2 leads to the intracellular accumulation of unesterified oxysterols and suppresses the growth of both HCC cell lines in vitro and as xenograft tumors. Further mechanistic studies have revealed that HCC-linked promoter hypomethylation is a major mechanism for SOAT2 gene expression induction. Specifically blocking the cholesterol metabolism pathway established in HCC may have therapeutic effects in HCC patients 63 . The inhibition of SOAT2 expression significantly decreased leptin-induced proliferation, migration and invasion of MCF-7 and T47D cells. Additionally, leptin may enhance the proliferation, migration and invasion of breast cancer cells in an SOAT2dependent manner through the PI3K/AKT/SREBP2 signaling pathway 64 .

SQLE
Squalene epoxidase (SQLE) is involved in the first oxygenation step in the cholesterol synthesis pathway, and is therefore a good target for controlling the cholesterol synthesis process 65 . Liu et al. sequenced RNA from nonalcoholic fatty liver disease (NAFLD)-induced HCC samples and revealed SQLE as the top metabolic gene overexpressed in NAFLDinduced HCC patients. In human NAFLD-induced HCC and HCC, SQLE was overexpressed and its expression was associated with poor patient outcomes. Terbinafine, an SQLE-targeting drug, markedly inhibited SQLE-induced cell growth in NAFLD-induced HCC and HCC cells and attenuated tumor development in xenograft models and SQLE transgenic mice 66,67 . These findings are consistent with observations from Stopsack et al. and Brown and his colleagues. In breast cancer and PC, SQLE overexpression is more common and is an independent prognostic factor for poor prognosis 68,69 . Given that SQLE may be oncogenic in a growing number of cancers, molecules targeting the SQLE signaling axis may be effective therapeutics for the treatment of malignancies.

OSC
Oxidative squalene cyclase (OSC) catalyzes the transformation of the linear triterpene (3S)-2,3-oxidosqualene into cyclic compounds 70 . A previous study investigated the role of this post-squalene enzyme, which is involved in the cholesterol biosynthesis pathway, in regulating tumor angiogenesis and metastatic dissemination in mouse models of cancer. The authors showed that Ro 48-8071, a selective inhibitor of OSC 71 , inhibited tumor growth in a spontaneous mouse model of pancreatic cancer and two metastatic mouse models. Remarkably, OSC inhibition suppressed metastasis formation in both the HCT116 and HPAF-II models. OSC inhibition specifically interfered with the PI3K pathway 72 . Ro 48-8071 also potently reduced breast cancer cell viability, especially in human breast cancer cells. The administration of Ro 48-8071 to mice with tumor xenografts prevented tumor growth with no apparent toxicity. Ro 48-8071 had no effect on the viability of normal human mammary cells 73 . In vivo, Ro 48-8071 effectively inhibited the growth of human PC xenograft cells in an anti-castration setting, without any signs of toxicity in experimental animals. Importantly, Ro 48-8071 did not decrease the survival rate of normal prostate cells in vitro 74 . Ro 48-8071 is a potent inhibitor of cancer cell proliferation.

ABCA1
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are drug efflux pumps that can lead to multidrug resistance and to tumor treatment failure 75,76 . ATP-binding cassette transporter A-1 (ABCA1) mediates the export of cholesterol and phospholipids to apolipoprotein A-I for HDL assembly 77 . In addition, ABCA1 is involved in bidirectional sterol movement through the plasma membrane, and regulates cell cholesterol homeostasis 78 .
Compared with the sensitive MDA-MB-231 breast cancer cells, the overexpression of ABCA1, a gene involved in the lipid removal pathway in drug-resistant M14 melanoma 79 . In human PC biopsy specimens, the expression of ABCA1 mRNA was approximately twice as high in the androgen-depleted treatment group than in benign prostatic hyperplasia or PC 80 . Suppression of the cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro. Additionally, the expression of ABCA transporters is correlated with poor outcomes in serous ovarian cancer 81 . JNJ-26854165 is a new type of chemotherapy with p53 activation abilities 82,83 . Interestingly, ABCA1 depletion increased sensitivity to JNJ-26854165 84 .
ABCA1 exhibits anticancer activity that inhibits the expression of the ABCA1 gene through functional mutations in oncogenes or cancer-specific ABCA1. In 2012, one excellent study by Smith et al. revealed that the anticancer activity of ABCA1 efflux is impaired after ABCA1 gene expression is inhibited by either an oncogenic mutation or cancer-specific ABCA1 functional deletion mutation. ABCA1 deficiency in conjunction with the high cholesterol synthesis found in cancer cells can lead to increased mitochondrial cholesterol, thereby promoting cancer cell survival 19 . In LNCaP cells, a DNA methylome analysis revealed that the promoter of ABCA1 is markedly hypermethylated. These findings suggest that the loss of cancer-specific ABCA1 hypermethylation and protein expression directly leads to elevated levels of cholesterol in cells, thereby contributing to tumor development 85 . ABCA1 is also aberrantly expressed in colon cancer tissues and cells. Silencing ABCA1 or miR-183 promoted proliferation and inhibited apoptosis in colon cancer cells 86 .

Conclusions
In summary, cancer cells rely on cholesterol as a cellular building block for membrane formation and for the production of signaling molecules. This review highlights the importance of cholesterol, cholesterol transporters and metabolites, and key enzymes of cholesterol metabolism in cancer ( Figure 1). The requirement of cholesterol for cancer cell proliferation reveals a potential cancer therapeutic target at multiple points within the cholesterol metabolism pathway to inhibit proliferation. Numerous chemical inhibitors for specific steps in the metabolism pathway already exist. Targeting cholesterol metabolism could be more a selective therapeutic modality for highly proliferative cells. Alternatively, cholesterol metabolism inhibitors could be utilized in a cell-specific and targeted manner. Cancer is a diverse set of diseases with various genetic changes. Cholesterol metabolism is also complex, with many different feedback mechanisms and regulatory points. In addition, most cholesterol metabolism enzymes have multiple isoforms, which may be connected to different lipid metabolism processes, cellular localization, or tissue distribution.
Successful treatments may depend on understanding specific metabolic abnormalities in certain types of cancer 87,88 (Figure 1). While some cholesterol metabolites contribute to cancer development and resistance, others have therapeutic potential. Further insight on cholesterol metabolism in cancer cells will allow us to take advantage of new and effective targets to improve the survival rate of cancer patients.