**3. Experimental and mechanistic evidence for role of LDL in cancer**

**Cholesterol homeostasis.** If cholesterol homeostasis is altered in cancer cells to meet a greater demand for cholesterol, an understanding of the mechanisms involved will open up new targets against cancer. In normal cells, free cholesterol in cells is closely regulated to maintain adequate membrane cholesterol but prevent free cholesterol toxicity. Excess cholesterol is stored in the form of neutral cholesteryl esters (CE) that are available to the cell through the CE cycle (Brown, Ho et al. 1980), or is effluxed to circulating HDL for transport back to the liver (Fielding and Fielding 2001). In cholesterol-accumulating tumors, there is more CE storage and less efflux of cholesterol to HDL. Is this cholesterol newly synthesized


**Table 1.** Large, prospective studies with a baseline total cholesterol measurement and long-term follow-up for cancer incidence. M, male; F, female; PrC, prostate cancer; BrC, breast cancer; CRC, colorectal cancer.

or obtained from LDL, and what determines this? Normal cells obtain cholesterol primarily through endocytosis of circulating LDL through the LDLR, but have the capacity for endogenous synthesis via the mevalonate pathway; both mechanisms are tightly controlled for cholesterol homeostasis (Goldstein, DeBose-Boyd et al. 2006). The expressions of both LDLR and HMGCR are regulated by the transcription factors sterol response element binding proteins (SREBP1/2), whose processing and maturation proceed in response to decreased intracellular cholesterol (Brown and Goldstein 1997). The observed accumulation of CE in some tumors, the positive association of total-C with the risk of some types of cancer, and the demand for cholesterol for membrane building in growing cells, all suggest that the expression of these proteins and other components of the cholesterol homeostatic response system are altered in cancer.

632 Lipoproteins – Role in Health and Diseases

**Years of followup**

Up to 14

11.9

Mean of 6.6

9.3

Mean of 7.0 - 8.3

Up to 7 18,018

Up to 7 5,586

>10 29,093

**n (n for cases)**

1,189,719 (M:53,944 F: 24,475)

6,816

2,118

29,364

200,660

6 - 12 79,994

Up to 10 170,374

11 - 14 24,329

521,448

Mean of 3.8

(2,041) M PrC

(43) M PrC

(269) F BrC

(242) F BrC

**Sex Type of cancer** 

M,F All

(698) M PrC Positive for high-

(1,251) M PrC Positive for high-

(438) M PrC Positive for high-

(650) M PrC Positive for high-

(5,112) M PrC None

(687) M PrC None Not

(3177) F BrC None Not

(714) F BrC Positive trend Not

(1238) M,F CRC None Not

**Table 1.** Large, prospective studies with a baseline total cholesterol measurement and long-term follow-up for cancer incidence. M, male; F, female; PrC, prostate cancer; BrC, breast cancer; CRC,

Positive for postmenopausal; none for premenopausal

Negative (premenopausal); none (post-menopausal)

**Association with risk of cancer for: Reference** 

Not measured

Not measured

Not measured

Negative trend

Not measured

Positive for nonaggressive PrC

measured None {Martin,

Not measured

Not

Not measured

Not measured

Positive for colon cancer

measured {Ha, 2009}

{Kitahara, 2011}

> {Platz, 2008}

> {Platz, 2009}

{Mondul, 2010}

{Mondul, 2011}

{Shafique, 2012}

{Kok, 2011}

2009}

{Van Hemelrijck, 2011}

{Eliassen, 2005}

{Manjer, 2001}

{Vatten, 1990}

{van Duijnhoven, 2011}

**Total-C LDL-C HDL-C** 

Not measured

Not measured

Not measured

Not measured

Not measured

Positive for total and aggressive PrC

measured

measured

Not measured

Not measured

measured

Positive for PrC (M), BrC (F), CRC (M,F); negative for stomach, liver (M,F), lung (M)

grade PrC

grade PrC

grade PrC

Positive for aggressive and advanced PrC

grade PrC

Positive for total and aggressive PrC

**Study (Country)** 

**Health Professionals Follow-Up (USA)** 

**(USA)** 

**Alpha-**

**Midspan** 

**Nijmegen Biomedical (Netherlands)** 

**Apolipoprotein MOrtality RISk (Sweden)** 

**Nurses' Health (self-reported serum cholesterol) (USA)** 

**Postmenopausal public servants (South Korea)** 

**Malmö Preventive** 

colorectal cancer.

**National Health Screening Service (Norway)** 

**EPIC and Nutrition (nested case-control)** 

**Project (Sweden)** Up to 20 9,738

**National Health Insurance Corp. enrollees (South Korea)** 

**Prostate Cancer Prevention Trial** 

**Tocopherol, Beta-Carotene Cancer Prevention (smokers, Finland)** 

**CLUE II (USA)** Mean of

**HUNT 2 (Norway)** Mean of

**(Scotland, UK)** Up to 37 12,926

**Cholesterol biosynthesis in cancer.** In order to obtain sufficient cholesterol, proliferating cells may accelerate the rate of cholesterol biosynthesis. Oncogenes that transform cells and dysregulate growth activate anabolic and biosynthetic pathways leading to *de novo* cholesterol and fatty acid synthesis. This is accomplished by a greatly increased flux of glucose into cells and through the glycolytic pathway to produce energy, and transport of TCA cycle citrate from the mitochondria to the cytosol for lipid biosynthesis (Vander Heiden, Cantley et al. 2009). The cytosolic enzyme ATP citrate lyase converts citrate to acetyl-CoA, the basic building block for both fatty acids and cholesterol. Growth factor activation of tyrosine kinase receptors and downstream PI3K/AKT and MAP-kinase signaling pathways increase expression and activation of the SREBPs (Kotzka, Muller-Wieland et al. 2000; Porstmann, Griffiths et al. 2005; Krycer, Sharpe et al. 2010), which control many lipid biosynthetic enzymes. Interesting, it was recently demonstrated that a mutated form of the cell cycle regulator p53, common in many tumors, bound to the promoter regions of the SREBPs and increased the expression of mevalonate pathway genes in BrC cells (Freed-Pastor, Mizuno et al. 2012).

A high enough rate of *de novo* biosynthesis may not always be possible; for example in solid tumors, expansion and insufficient vascularization may limit the delivery of glucose and oxygen. If oxygen is limited, activation of the hypoxia inducible factor 1 (HIF1) pathway can increase survival but divert pyruvate to lactate, reducing production of citrate (Gordan, Thompson et al. 2007). If glucose is limited, reducing ATP production, the AMP activated protein kinase (AMPK) pathway can inactivate key biosynthetic enzymes by phosphorylation (Shackelford and Shaw 2009). If biosynthesis becomes constrained, cells would have an advantage by being able to obtain lipids exogenously from circulating lipoproteins.

**Cholesterol uptake in cancer.** Uptake of cholesterol from LDL is primarily through the LDLR, although several scavenger receptors may also contribute. Over-expression of LDLR without feedback regulation by cholesterol has been observed in many types of cancer cells (Chen, Li et al. 1988; Hirakawa, Maruyama et al. 1991; Chen and Hughes-Fulford 2001; Antalis, Uchida et al. 2011). Although the role of SREBPs in feedback regulation of LDLR expression is well understood (Goldstein, DeBose-Boyd et al. 2006), there is evidence that

cell signaling pathways also contribute to LDLR up-regulation in cancer. In BrC cells, LDLR mRNA expression was 3-5-fold higher in ER- as compared to ER+ cell lines; PKC activation was strongly associated with increased LDLR expression in ER+ BrC cells, and to a lesser extent, even in ER- cells (Stranzl, Schmidt et al. 1997). Activation of the p42/44 (MAPK) cascade was sufficient to induce LDLR transcription in human hepatoma HepG2 cells expressing oncogenic Raf-1 kinase (Kapoor, Atkins et al. 2002). In glioblastoma cells, chronic activation of the EGF receptor tyrosine kinase, or other mechanisms which ultimately activated the PI3K/AKT pathway, led to increased expression of SREBP1 and the LDLR and to LDL-responsive proliferation (Guo, Reinitz et al. 2011).

Increased dietary cholesterol has been shown to promote tumorigenesis in animal models. A Western-type high cholesterol diet compared to a chow diet increased tumor incidence and metastasis in a mouse model of PrC (Llaverias, Danilo et al. 2010). The same group, using similar diets, showed an increase in tumor formation and more aggressive tumors in a mouse model of BrC (Llaverias, Danilo et al. 2011). In both studies, plasma total-C was reduced following tumor development, suggesting utilization of circulating cholesterol by the tumor and similarity to what is observed in people with cancer.

**Role of cholesterol esterification.** Whether tumor cells obtain the needed cholesterol endogenously or exogenously, it would be imperative to have a way to manage the increased flux of cholesterol so as to meet the dual goals of ensuring a ready supply and avoiding toxicity. Cholesterol toxicity is prevented by effluxing the excess free cholesterol to an extracellular acceptor or converting free cholesterol to non-toxic esters of fatty acids. The observed low HDL-C in cancer patients, combined with the observed increased cholesterol content in tumors suggest that efflux mechanisms are reduced and esterification is increased. Synthesis and storage of CE in lipid droplets not only reduces toxicity but provides an accessible depot of cholesterol for future cell needs.

The enzyme responsible for cholesterol esterification is acyl-CoA:cholesterol acyltransferase 1 (ACAT1/SOAT1), a constitutive resident of the endoplasmic reticulum. ACAT1 esterifies cholesterol obtained from LDL and also from endogenous synthesis (Chang, Li et al. 2009). ACAT1 is frequently found to be over-expressed in cancer vs. normal tissues in human tumor gene expression analyses, including cancers of brain, breast, cervix, esophagus, head and neck, kidney, and testis (P < 0.05, oncomine.org). Over-expression of ACAT1 has been specifically associated with cholesterol accumulation in renal clear cell carcinoma, a tumor type characterized by 35-fold more CE as compared to normal kidney (Gebhard, Clayman et al. 1987).

ACAT activity has been associated with proliferation in cancer cells. The CE content of lymphocytes from patients with acute or chronic lymphocytic leukemia (n = 30) was 6-fold higher as compared to lymphocytes from healthy age-matched controls (n = 15), and plasma HDL was >40% reduced in the leukemia patients compared to the controls (Mulas, Abete et al. 2011). Phytohemaglutinin (PHA)-stimulated proliferation of the isolated leukemic cells was positively correlated to esterification of oleate to cholesterol, and inhibition of ACAT greatly reduced PHA-induced proliferation (Mulas, Abete et al. 2011). Cholesterol esterification and ACAT1 expression were also studied in leukemia cell lines. Cells with a greater ability to esterify cholesterol and with lower cholesterol efflux (CEM) had a higher rate of proliferation as compared to cells with a greater ability to synthesize cholesterol *de novo* (MOLT4) (Dessi, Batetta et al. 1997). Further work demonstrated that the fastergrowing CEM cells expressed more ACAT1 and less HMGCR mRNA as compared to the slower-growing MOLT4 cells (Batetta, Pani et al. 1999).

634 Lipoproteins – Role in Health and Diseases

to LDL-responsive proliferation (Guo, Reinitz et al. 2011).

the tumor and similarity to what is observed in people with cancer.

provides an accessible depot of cholesterol for future cell needs.

al. 1987).

cell signaling pathways also contribute to LDLR up-regulation in cancer. In BrC cells, LDLR mRNA expression was 3-5-fold higher in ER- as compared to ER+ cell lines; PKC activation was strongly associated with increased LDLR expression in ER+ BrC cells, and to a lesser extent, even in ER- cells (Stranzl, Schmidt et al. 1997). Activation of the p42/44 (MAPK) cascade was sufficient to induce LDLR transcription in human hepatoma HepG2 cells expressing oncogenic Raf-1 kinase (Kapoor, Atkins et al. 2002). In glioblastoma cells, chronic activation of the EGF receptor tyrosine kinase, or other mechanisms which ultimately activated the PI3K/AKT pathway, led to increased expression of SREBP1 and the LDLR and

Increased dietary cholesterol has been shown to promote tumorigenesis in animal models. A Western-type high cholesterol diet compared to a chow diet increased tumor incidence and metastasis in a mouse model of PrC (Llaverias, Danilo et al. 2010). The same group, using similar diets, showed an increase in tumor formation and more aggressive tumors in a mouse model of BrC (Llaverias, Danilo et al. 2011). In both studies, plasma total-C was reduced following tumor development, suggesting utilization of circulating cholesterol by

**Role of cholesterol esterification.** Whether tumor cells obtain the needed cholesterol endogenously or exogenously, it would be imperative to have a way to manage the increased flux of cholesterol so as to meet the dual goals of ensuring a ready supply and avoiding toxicity. Cholesterol toxicity is prevented by effluxing the excess free cholesterol to an extracellular acceptor or converting free cholesterol to non-toxic esters of fatty acids. The observed low HDL-C in cancer patients, combined with the observed increased cholesterol content in tumors suggest that efflux mechanisms are reduced and esterification is increased. Synthesis and storage of CE in lipid droplets not only reduces toxicity but

The enzyme responsible for cholesterol esterification is acyl-CoA:cholesterol acyltransferase 1 (ACAT1/SOAT1), a constitutive resident of the endoplasmic reticulum. ACAT1 esterifies cholesterol obtained from LDL and also from endogenous synthesis (Chang, Li et al. 2009). ACAT1 is frequently found to be over-expressed in cancer vs. normal tissues in human tumor gene expression analyses, including cancers of brain, breast, cervix, esophagus, head and neck, kidney, and testis (P < 0.05, oncomine.org). Over-expression of ACAT1 has been specifically associated with cholesterol accumulation in renal clear cell carcinoma, a tumor type characterized by 35-fold more CE as compared to normal kidney (Gebhard, Clayman et

ACAT activity has been associated with proliferation in cancer cells. The CE content of lymphocytes from patients with acute or chronic lymphocytic leukemia (n = 30) was 6-fold higher as compared to lymphocytes from healthy age-matched controls (n = 15), and plasma HDL was >40% reduced in the leukemia patients compared to the controls (Mulas, Abete et al. 2011). Phytohemaglutinin (PHA)-stimulated proliferation of the isolated leukemic cells was positively correlated to esterification of oleate to cholesterol, and inhibition of ACAT greatly reduced PHA-induced proliferation (Mulas, Abete et al. 2011). Cholesterol In BrC, we showed that more aggressive basal-like ER- BrC cells had more lipid droplets and a much higher ratio of CE to TAG in stored neutral lipids as compared to less aggressive ER+ BrC cells; this was associated with higher expression of ACAT1 (Antalis, Arnold et al. 2010). The cell line differences were mirrored in gene expression analyses of human breast tumors, where higher expression of ACAT1/SOAT1 is characteristic of basallike ER- tumors (Antalis, Arnold et al. 2010). We further showed that ER- cells took up more LDL as compared to ER+ cells, and that LDL dose-responsively increased proliferation only of ER- cells and in an ACAT-sensitive manner. In a follow-up study, we examined the effect of lipoprotein deprivation on chemotactic migration of the highly motile basal-like ER- cell line MDA-MB-231. We showed that lipid droplets were depleted and migration was reduced 85% when cells were grown in medium without lipoproteins, and that adding back LDL or fatty acids restored migration in an ACAT-sensitive manner (Antalis, Uchida et al. 2011). In addition, LDLR expression in these cells was not affected by exogenous LDL but was reduced 75% in the presence of an ACAT inhibitor, suggesting that high ACAT1 expression permitted continued high expression of the LDLR.

What mediates the over-expression of ACAT1 in cancer is not completely understood. Although ACAT1 is a critical component of intracellular cholesterol homeostasis, its expression is not known to be regulated by the SREBPs (Goldstein, DeBose-Boyd et al. 2006). In monocytes and macrophages, ACAT1 expression was up-regulated by interferon γ and all-*trans*-retinoic acid via STAT1 (Yang, Duan et al. 2001) and by dexamethasone via a glucocorticoid response element in its promoter (Yang, Yang et al. 2004). ACAT1 has also been shown to have an NFκB binding element in its proximal promoter and to be upregulated in response to TNFα signaling through NFκB (Lei, Xiong et al. 2009). Cholesterol acts as an allosteric activator of ACAT1 activity (Liu, Chang et al. 2005).

**The LXR pathway.** The transcription factor LXR is a major regulator of fatty acid and cholesterol metabolism in cells. When cellular free cholesterol levels are high, some cholesterol is oxidized to form oxysterols, which act as endogenous ligands for LXR; thus LXRs are considered "cholesterol sensors"(Tontonoz 2011). LXR has an absolute requirement for RXR as a dimerization partner. RXR expression is highly regulated by both transcription and protein degradation (Boudjelal, Wang et al. 2000; Lefebvre, Benomar et al. 2010). RXR availability is also affected by competition with its other binding partners, including PPAR, RAR, VDR, TR and FXR. LXR/RXR is a permissive heterodimer, being stimulated by agonists of either partner (Tontonoz 2011).

LXR signaling is known to have dual roles: up-regulation of genes of fatty acid biosynthesis (including fatty acid synthase and stearoyl-CoA desaturase 1/2) and repression of NFκB controlled inflammatory genes (including IL-6, COX-2, and nitric oxide synthase) (Joseph, Castrillo et al. 2003). In addition, LXR/RXRα controls the transcription of key genes in cholesterol homeostasis: MYLIP/IDOL, the E3-ligase that ubiquitinates the LDLR leading to its degradation, ABCA1 and ABCG1, transporters involved in cholesterol efflux to APOA1 and HDL, and others (Tontonoz 2011). The demonstrated control of ACAT1 by NFκB suggests that its transcription could be antagonized by LXR activity. LXR signaling may have the ability to mediate the balance between lipid biosynthesis/efflux mechanisms and uptake/storage mechanisms. **Fig. 2** and **Fig. 3** illustrate how key factors in cellular cholesterol homeostasis may be affected by the activity of LXR and its target genes.

The uptake of exogenous LDL through LDLR leads to increased cellular free cholesterol, reduced maturation of SREBPs and reduced transcription of LDLR. When LXR/RXR is active (**Fig. 2**), LDLR protein is degraded by MYLIP and cholesterol efflux mechanisms are increased (Beltowski 2008). ACAT1 transcription may be reduced by the inhibitory effect of LXR/RXRon NFB transactivation activity, blocking cholesterol accumulation. Similarly ApoA1, the apolipoprotein acceptor for cholesterol efflux, which under some conditions is repressed by NFB, could be increased (Mogilenko, Dizhe et al. 2009). As a result, normal cellular cholesterol homeostasis is enforced.

When LXR/RXRα is less active (**Fig. 3**), and under the influence of cytokines, a different pattern of gene expression predominates. Cholesterol efflux is reduced and thus free cholesterol is maintained at a high enough level in bilayer membranes that maturation of SREBPs is not triggered. More free cholesterol is esterified and stored in lipid droplets, due to a possible induction of ACAT1. LDLR protein degradation is reduced, allowing the cell to maintain high LDLR expression and unrestrained uptake of LDL. In this way, cellular cholesterol homeostasis is perturbed in the direction of LDL uptake and cholesterol accumulation.

The pathways described in **Figs. 2 and 3** are hypothesized to explain the observed cholesterol accumulation in some tumors and cancer cell lines. LDLR is placed at the center of the process of LDL uptake and accumulation, with LXR pathway inactivation being the key factor allowing cholesterol accumulation. No doubt the situation is more complicated than shown, as it does not account for scavenger receptor participation. However, the central role of LXR makes it a potential target in cancer.

LXR agonists have been tested in experimental models of cancer. In glioblastoma cells overexpressing the EGFR, EGF stimulated PI3K/Akt-driven up-regulation of SREBP1 and LDLR (Guo, Reinitz et al. 2011). An LXR agonist induced MYLIP/IDOL-mediated degradation of LDLR, ABCA1-mediated cholesterol efflux, and cell death both *in vitro* and in an animal model. In OvC cells, oxLDL stimulated proliferation and secretion of the cytokine cardiotrophin 1 (Scoles, Xu et al. 2010). An LXR agonist blocked both the cytokine secretion and the proliferation induced by oxLDL; the authors attribute the response to increased cholesterol efflux and decreased inflammatory effects of the LXR agonist. In an athymic model of PrC, progression of androgen-dependent tumors to androgen-independent tumors after castration was accompanied by decreases in expression of LXR target genes in the tumor, and treatment with an LXR agonist delayed the progression for about 4 weeks (Chuu, Hiipakka et al. 2006).

636 Lipoproteins – Role in Health and Diseases

cellular cholesterol homeostasis is enforced.

central role of LXR makes it a potential target in cancer.

accumulation.

controlled inflammatory genes (including IL-6, COX-2, and nitric oxide synthase) (Joseph, Castrillo et al. 2003). In addition, LXR/RXRα controls the transcription of key genes in cholesterol homeostasis: MYLIP/IDOL, the E3-ligase that ubiquitinates the LDLR leading to its degradation, ABCA1 and ABCG1, transporters involved in cholesterol efflux to APOA1 and HDL, and others (Tontonoz 2011). The demonstrated control of ACAT1 by NFκB suggests that its transcription could be antagonized by LXR activity. LXR signaling may have the ability to mediate the balance between lipid biosynthesis/efflux mechanisms and uptake/storage mechanisms. **Fig. 2** and **Fig. 3** illustrate how key factors in cellular

cholesterol homeostasis may be affected by the activity of LXR and its target genes.

The uptake of exogenous LDL through LDLR leads to increased cellular free cholesterol, reduced maturation of SREBPs and reduced transcription of LDLR. When LXR/RXR is active (**Fig. 2**), LDLR protein is degraded by MYLIP and cholesterol efflux mechanisms are increased (Beltowski 2008). ACAT1 transcription may be reduced by the inhibitory effect of LXR/RXRon NFB transactivation activity, blocking cholesterol accumulation. Similarly ApoA1, the apolipoprotein acceptor for cholesterol efflux, which under some conditions is repressed by NFB, could be increased (Mogilenko, Dizhe et al. 2009). As a result, normal

When LXR/RXRα is less active (**Fig. 3**), and under the influence of cytokines, a different pattern of gene expression predominates. Cholesterol efflux is reduced and thus free cholesterol is maintained at a high enough level in bilayer membranes that maturation of SREBPs is not triggered. More free cholesterol is esterified and stored in lipid droplets, due to a possible induction of ACAT1. LDLR protein degradation is reduced, allowing the cell to maintain high LDLR expression and unrestrained uptake of LDL. In this way, cellular cholesterol homeostasis is perturbed in the direction of LDL uptake and cholesterol

The pathways described in **Figs. 2 and 3** are hypothesized to explain the observed cholesterol accumulation in some tumors and cancer cell lines. LDLR is placed at the center of the process of LDL uptake and accumulation, with LXR pathway inactivation being the key factor allowing cholesterol accumulation. No doubt the situation is more complicated than shown, as it does not account for scavenger receptor participation. However, the

LXR agonists have been tested in experimental models of cancer. In glioblastoma cells overexpressing the EGFR, EGF stimulated PI3K/Akt-driven up-regulation of SREBP1 and LDLR (Guo, Reinitz et al. 2011). An LXR agonist induced MYLIP/IDOL-mediated degradation of LDLR, ABCA1-mediated cholesterol efflux, and cell death both *in vitro* and in an animal model. In OvC cells, oxLDL stimulated proliferation and secretion of the cytokine cardiotrophin 1 (Scoles, Xu et al. 2010). An LXR agonist blocked both the cytokine secretion and the proliferation induced by oxLDL; the authors attribute the response to increased cholesterol efflux and decreased inflammatory effects of the LXR agonist. In an athymic model of PrC, progression of androgen-dependent tumors to androgen-independent tumors after castration was accompanied by decreases in expression of LXR target genes in the

**Figure 2.** LXR transcriptional targets control intracellular cholesterol concentrations. Dotted line indicates pathways not proven.

**Figure 3.** Reduced LXR signaling allows increased LDL uptake and intracellular cholesterol accumulation. Dotted line indicates pathways not proven.

**Cholesterol and tumorigenesis.** The question remains as to the role that CEs may play in the survival, proliferation and metastasis of cancer cells. We and others have proposed that accumulation of CE spares energy needed for *de novo* sterol synthesis, allowing greater proliferation and migration and perhaps a quicker return to growth after a period of stasis (Batetta, Pani et al. 1999; Antalis, Arnold et al. 2010; Antalis, Uchida et al. 2011). The process of cholesterol esterification was linked to proliferation in multiple studies in different cancer cell lines (Batetta, Pani et al. 1999; Peiretti, Dessi et al. 2007; Paillasse, de Medina et al. 2009; Antalis, Arnold et al. 2010; Mulas, Abete et al. 2011), implying a complex network of signaling pathways and gene expression that ties cholesterol accretion to tumorigenesis. However, the exact role of CE in tumorigenesis remains to be determined.

PrC is a unique case considering the slow growth characteristics of this malignancy. The lipid raft concept has been proposed to account for the tumorigenic effects of cholesterol (Freeman, Cinar et al. 2007), and a higher level of cholesterol in PrC cells has been linked to membrane lipid raft-induced oncogenic cell signaling (Hager, Solomon et al. 2006). A connection between LXR signaling and lipid raft-associated signaling was demonstrated in androgen-responsive LnCAP cells, where an LXR agonist down-regulated Akt signaling in a cholesterol- and lipid raft-dependent manner, resulting in apoptosis of cells and xenograft tumors (Pommier, Alves et al. 2010). In addition, a relationship between androgens and cholesterol metabolism was demonstrated in PrC cells. It was first noted that androgen stimulation caused a dramatic increase in lipid droplets in LNCap cells. The induced neutral lipids included both TAG (33-fold) and CE (7-fold increase), most of which originated from new lipid synthesis (Swinnen, Van Veldhoven et al. 1996). This was later found to be due to an up-regulation of the SREBPs and lipid biosynthetic genes (Nelson, Clegg et al. 2002). The androgen-independent PC-3 cells had a higher content of CE and but not higher ACAT1 activity or expression as compared to LNCap cells (Locke, Wasan et al. 2008). In both an androgen-independent cell line and a mouse xenograft model of PrC progression, changes in cholesterol metabolism and homeostasis were associated with initiation of tumoral androgen production and expression of the AR and PSA (Locke, Wasan et al. 2008; Leon, Locke et al. 2010). These data, along with the clinical data cited in **Section 2,** suggest that in PrC cholesterol accumulation may be important for androgen synthesis, which is closely involved with PrC progression even under castration therapy.

Another function of LDL and other lipoproteins is the provision of essential fatty acids. Mammalian cells are not able to make polyunsaturated fatty acids; the essential n-6 and n-3 fatty acids are derived from the diet and carried to cells by lipoproteins. Human glioma, one of the deadliest types of cancer, was found to contain up to 100-fold more CE compared to control tissue, and the fatty acid composition of the tumor CEs indicated an LDL origin (Nygren, von Holst et al. 1997). The n-6 fatty acid arachidonic acid is necessary for synthesis of second messengers such as the prostaglandin PGE2, a tumor promoter (Wang and Dubois 2006). In androgen-independent PrC PC-3 cells, PGE2 production increased >3-fold in response to LDL (Chen and Hughes-Fulford 2001). Thus the fatty acids esterified to cholesterol and other lipids may be important for the effect of LDL on cancer cells.

Finally, although lower plasma HDL-C in cancer patients may be due to reduced efflux of cholesterol to HDL from the tumor, there is evidence that some cancer cells can take up CE from circulating HDL, providing another explanation for low HDL. Recent investigations with the CEM-CCRF lymphoblastic cell line into the source of intracellular CE showed that HDL-CE were taken up and stored without hydrolysis and re-esterification, while LDL-CE were hydrolyzed and re-esterified (Uda, Accossu et al. 2012). Although the mechanism was not clear, the data implied that HDL as well as LDL could be a source of CE for leukemic cells. A previous study in BrC cells showed that either HDL or LDL dose-dependently stimulated proliferation of ER- cell lines, but only HDL had the effect on ER+ cells lines (Rotheneder and Kostner 1989). In an animal model of PrC, a diet high in fat and cholesterol resulted in increased tumor incidence and increased tumor expression of scavenger receptor B1, the receptor responsible for selective uptake of HDL-C (the major form of circulating cholesterol in mice) by cells (Llaverias, Danilo et al. 2010). The question of whether HDL can supply cholesterol to tumor cells *in vivo* in humans remains open.
