**3. De novo lipogenesis in metabolic disease**

192 Lipid Metabolism

the main MUFA in olive oil, oleic acid, and to certain minor compounds such as squalene and phenolic compounds [82]. Recent studies have also shown that canola oil, with high MUFA, oleic acid, can decrease colon and breast cancer incidence significantly [83, 84]. Although the authors suggested that such effect may be caused by omega-3 fatty acids, ALA, as high as 10% in canola oil, however, the role of oleic acid, as high as 61% in canola oil, cannot be excluded. So far, no epidemiological studies or animal studies can clearly demonstrate the preventive effect of MUFA on cancer, However, *in vivo* analysis of the fatty acid composition of the adipose tissue of breast cancer and healthy women showed that elevated adipose MUFA, oleic acid, are associated with reduced odds of breast cancer [85]. Although the mechanism underling the protective function of oleic acid on cancer is, so far, not clear, it has been found that oleic acid, when complexed with the molten globule form of alpha-lactalbumin (α-LA), acquires tumoricidal activity [86]. Carrillo et al found that oleic acid can inhibit store-operated Ca(2+) entry (SOCE), a Ca(2+) influx pathway, involved in the control of multiple cellular and physiological processes including cell proliferation,

Increasing evidences from animal and in vitro studies indicate that populations who ingest high amounts of omega-3 fatty acids in their diets have lower incidences of breast, colon, and, perhaps, prostate cancers. Paola et al. [88] used MTT viability test and expression of apoptotic markers to evaluate the effect of PUFAs on cancer growth, and their results indicated that EPA and DHA might induce modifications of tumor cell membrane structure leading to an obviously decreased induction rate of breast cancer. Menéndez et al. [89] also reported that omega-3 PUFAs ALA suppresses the overexpression of HER2, which plays an important role in aetiology, progression and chemosensitivity of various types of human cancers, suggesting that ALA is a potential anticancer agent. However, the clinical roles of omega-3 PUFAs may rely not only on the absolute content but also on the proportion of omega-3 PUFAs to omega-6 PUFAs in the cells due to the inverse biological functions of these two series of PUFAs. A higher omega-6/omega-3 PUFAs ratio contributes to many diseases including cancer, cardiovascular and inflammation. Reducing the omega-6/omega-3 PUFAs ratio can help lower the risk of initiation and development of cancer. Berquin et al. established a prostate-specific phosphatase and tension homolog (PTEN) knockout mouse model, and the result demonstrated that a dietary ratio of omega-6/omega-3 PUFA lower than 5 was effective in suppressing tumor growth, and extending animal lifespan [90] The recent research suggested that a balanced ratio of omega-6/omega-3 PUFAs (1:1) exerts a

thus regulating the growth of colon carcinoma cells [87].

beneficial effects on cell function and physiology [91].

*2.2.4. Potential mechanisms of the association of dietary lipids with cancer* 

Although it has been generally accepted that dietary lipids are associated with carcinogenesis and the development of cancer, the detailed mechanism is still far from clear. When lipids are digested and absorbed by small intestine mucosa cells, they can be transported to adipocytes for storage, or used for energy production by peripheral cells

*2.2.3. Polyunsaturated fatty acids* 

#### **3.1. De novo lipogenesis in metabolic syndrome**

De novo fatty acid biosynthesis occurs in essentially all cells, but adipose tissue and liver are the major sites. The first committed step in fatty acid synthesis is catalyzed by fatty acid synthase (FAS), a multifunctional cytosolic protein that primarily synthesizes palmitate. Variations in FAS expression and enzyme activity have been implicated in insulin resistance and obesity in humans [98]. A circulating form of FAS has been reported as a biomarker of metabolic stress and insulin sensitivity. In humans it changes with weight loss and may reflect improved insulin sensitivity [99].

Fatty acid elongation is catalyzed by Elovl (elongation of very long-chain fatty acid) proteins. Elovl6 is thought to be involved in de novo lipogenesis and is regulated by dietary,

#### 194 Lipid Metabolism

hormonal and developmental factors. Mice with Elov6 deficiency are obese but protected from insulin resistance [100, 101].

Lipid Metabolism, Metabolic Syndrome, and Cancer 195

metabolism, and lipogenic genes in conjunction with ChREBP [113]. ChREBP knockout mice show decreased liver triglyceride but increased liver glycogen content indicating that ChREBP may regulate metabolic gene expression to convert excess carbohydrate into triglyceride rather than glycogen [114]. In addition, complete inhibition of ChREBP in ob/ob mice reduces the effects of the MS such as obesity, fatty liver, and glucose intolerance,

Enhanced flux of glucose derivatives through glycolysis, which sustain the redirection of mitochondrial ATP to glucose phosphorylation, and de novo FA synthesis is a hallmark of aggressive cancers. Although most normal cells use FA from dietary lipids, tumor cells de novo synthesize more than 95% of lipids required for cell proliferation despite having enough nutritional supply of lipids. Lipogenic enzymes such as, FAS, ACC, and ACL involved in FA biosynthesis, glycerol-3-phosphate dehydrogenase involved in lipid biosynthesiss, and SREBP1, the master regulator of lipogenic gene expression, are found to be overexpressed in a number of cancer or cancer cells, such as prostate cancer [116], ovarian cancer [117], breast cancer [118], lung cancer [119], colon cancer [120], and etc. Some research has been carried out to provide insights into the molecular mechanism of the association of lipogenesis and cancer. In this chapter we focused on three main lipogenic

High levels of FAS expression have been found in many human cancers, including prostate cancer [121], ovarian cancer [117, 122], breast cancer [123], bladder cancer [124], colon cancer [125] mantle cell lymphoma [126], and etc. However, the cellular mechanism by which FAS is up-regulated in cancer cells is not fully understood. Nevertheless a few studies suggested that steroid hormones and human epidermal growth factor receptor (HER) family ligands, especially HER2 could increase FAS expression via the PI3K/Akt or mitogen-activated protein kinases (MAPK) pathways[117]. Recently Mukherjee et al [117] demonstrated that bioactive lipid lysophosphatidic acid (LPA) induces FAS expression and lipogenesis through LPA2-G12/13-Rho-SREBP signaling pathway in ovarian cancer cells. Moreover over-expression of FAS in non-cancerous epithelial cells is sufficient to induce a cancer-like phenotype through the induction of HER1/HER2 [127], therefore FAS over-expression may play a role in carcinogenesis. Furthermore FAS overexpression is found to be associated with the advanced stage of colorectal cancer and liver metastasis, thus it may also play a role in the progression of cancer [128]. So far abundant evidences have shown that FAS contributes to both tumorigenesis and metastasis, and it becomes an ideal target for cancer therapy. In deed inhibition of FAS activity by FAS specific inhibitors or siRNA can significantly inhibit cancer or cancer cell growth, induce cancer cell apoptosis, and reduce the metastasis of several cancers[124, 129]. Both synthetic chemicals and natural products of

indicating it as a potential target for treatment of MS [115].

**3.2. De novo lipogenesis in cancer** 

genes: FAS, ACC, and ACL.

*3.2.1. Fatty acid synthase* 

Citrate produced by the tricarboxylic acid cycle in mitochondria is converted by ATP-citrate lyase (ACL) to acetyl-CoA, which is next converted to malonyl-CoA by acetyl CoA carboxylase (ACC). Malonyl-CoA is a potent inhibitor of carnitine- palmitoyl transferase 1 (CPT1), which transports FAs into the mitochondria for oxidation, thus plays a key role in the regulation of both mitochondrial fatty acid oxidation and fat synthesis. ACC catalyzes a key rate-controlling step in both de novo lipogenesis and fatty acid oxidation. The absence of ACC decreases the cellular concentration of malonyl-CoA, removes the inhibition of CPT1 and maintains FA oxidation. In rats with NAFLD, suppression or knockdown of ACC isoforms significantly reduced hepatic malonyl-CoA levels, lowered hepatic lipids including long-chain acyl-CoAs, DAG, and triglycerides, and improved hepatic insulin sensitivity [102].

Lipogenesis and FA oxidation are highly integrated processes. Studies in genetically modified mice have demonstrated that inhibition of FA synthesis and storage is associated with upregulation of FA oxidation [103]. For examples, knockout the diacylglycerol acyltransferase (DGAT), an enzyme that catalyses the final acylation step of TAG synthesis, reduced fat deposition and protected mice against diet- induced obesity and, in the meanwhile, elevated mice energy expenditure and increased activity, suggesting a correlation of disrupted FA storage and increased FA oxidation [104, 105]. Similarly, deletion of acetyl-CoA carboxylase 2 (ACC2), an isoform of ACC and key enzyme for de novo FA synthesis, leads to a lean mouse with increased FA oxidation [106].

As a major component of the metabolic syndrome, NAFLD characterizes with the accumulation of TAGs in hepatocytes, and development of steatohepatitis, cirrhosis, and hepatocellular carcinoma. FAs stored in adipose tissue and newly made through liver de novo lipogenesis are the major sources of TAGs in the liver [107].

Lipogenesis is also an insulin- and glucose-dependent process that is under the control of specific transcription factors. SREBP1 is such a transcription factor and activates most genes involved in FA synthesis. It occurs in two isoforms, SREBP1a and 1c, through alternative splicing. SREBP-1c is highly expressed in the WAT, liver, adrenal gland, brain, and muscle and regulates the expression of many of the genes involved in de novo FA and TAG synthesis including ACC and FAS [108, 109]. Insulin increases lipogenesis through activating SREBP-1c that is dependent on the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) [110]. SREBP1 gene expression is decreased in adipose tissue of obese subjects and the aberrant activation of SREBPs may contribute to obesity-related pathophysiology in various organs, including cardiac arrhythmogenesis and hepatic insulin resistance.

Lipogenesis is also regulated by glucose activated carbohydrate response element-binding protein (ChREBP), which induces gene expression of liver-type pyruvate kinase, a key regulatory enzyme in glycolysis; this enzyme in turn provides the precursors for lipogenesis [111]. ChREBP also stimulates expression of genes involved in lipogenesis [112] including SREBP-1c, which in turn activates glycolytic gene expression, promoting glucose metabolism, and lipogenic genes in conjunction with ChREBP [113]. ChREBP knockout mice show decreased liver triglyceride but increased liver glycogen content indicating that ChREBP may regulate metabolic gene expression to convert excess carbohydrate into triglyceride rather than glycogen [114]. In addition, complete inhibition of ChREBP in ob/ob mice reduces the effects of the MS such as obesity, fatty liver, and glucose intolerance, indicating it as a potential target for treatment of MS [115].

#### **3.2. De novo lipogenesis in cancer**

194 Lipid Metabolism

from insulin resistance [100, 101].

hormonal and developmental factors. Mice with Elov6 deficiency are obese but protected

Citrate produced by the tricarboxylic acid cycle in mitochondria is converted by ATP-citrate lyase (ACL) to acetyl-CoA, which is next converted to malonyl-CoA by acetyl CoA carboxylase (ACC). Malonyl-CoA is a potent inhibitor of carnitine- palmitoyl transferase 1 (CPT1), which transports FAs into the mitochondria for oxidation, thus plays a key role in the regulation of both mitochondrial fatty acid oxidation and fat synthesis. ACC catalyzes a key rate-controlling step in both de novo lipogenesis and fatty acid oxidation. The absence of ACC decreases the cellular concentration of malonyl-CoA, removes the inhibition of CPT1 and maintains FA oxidation. In rats with NAFLD, suppression or knockdown of ACC isoforms significantly reduced hepatic malonyl-CoA levels, lowered hepatic lipids including long-chain

Lipogenesis and FA oxidation are highly integrated processes. Studies in genetically modified mice have demonstrated that inhibition of FA synthesis and storage is associated with upregulation of FA oxidation [103]. For examples, knockout the diacylglycerol acyltransferase (DGAT), an enzyme that catalyses the final acylation step of TAG synthesis, reduced fat deposition and protected mice against diet- induced obesity and, in the meanwhile, elevated mice energy expenditure and increased activity, suggesting a correlation of disrupted FA storage and increased FA oxidation [104, 105]. Similarly, deletion of acetyl-CoA carboxylase 2 (ACC2), an isoform of ACC and key enzyme for de

As a major component of the metabolic syndrome, NAFLD characterizes with the accumulation of TAGs in hepatocytes, and development of steatohepatitis, cirrhosis, and hepatocellular carcinoma. FAs stored in adipose tissue and newly made through liver de

Lipogenesis is also an insulin- and glucose-dependent process that is under the control of specific transcription factors. SREBP1 is such a transcription factor and activates most genes involved in FA synthesis. It occurs in two isoforms, SREBP1a and 1c, through alternative splicing. SREBP-1c is highly expressed in the WAT, liver, adrenal gland, brain, and muscle and regulates the expression of many of the genes involved in de novo FA and TAG synthesis including ACC and FAS [108, 109]. Insulin increases lipogenesis through activating SREBP-1c that is dependent on the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) [110]. SREBP1 gene expression is decreased in adipose tissue of obese subjects and the aberrant activation of SREBPs may contribute to obesity-related pathophysiology in various organs,

Lipogenesis is also regulated by glucose activated carbohydrate response element-binding protein (ChREBP), which induces gene expression of liver-type pyruvate kinase, a key regulatory enzyme in glycolysis; this enzyme in turn provides the precursors for lipogenesis [111]. ChREBP also stimulates expression of genes involved in lipogenesis [112] including SREBP-1c, which in turn activates glycolytic gene expression, promoting glucose

acyl-CoAs, DAG, and triglycerides, and improved hepatic insulin sensitivity [102].

novo FA synthesis, leads to a lean mouse with increased FA oxidation [106].

novo lipogenesis are the major sources of TAGs in the liver [107].

including cardiac arrhythmogenesis and hepatic insulin resistance.

Enhanced flux of glucose derivatives through glycolysis, which sustain the redirection of mitochondrial ATP to glucose phosphorylation, and de novo FA synthesis is a hallmark of aggressive cancers. Although most normal cells use FA from dietary lipids, tumor cells de novo synthesize more than 95% of lipids required for cell proliferation despite having enough nutritional supply of lipids. Lipogenic enzymes such as, FAS, ACC, and ACL involved in FA biosynthesis, glycerol-3-phosphate dehydrogenase involved in lipid biosynthesiss, and SREBP1, the master regulator of lipogenic gene expression, are found to be overexpressed in a number of cancer or cancer cells, such as prostate cancer [116], ovarian cancer [117], breast cancer [118], lung cancer [119], colon cancer [120], and etc. Some research has been carried out to provide insights into the molecular mechanism of the association of lipogenesis and cancer. In this chapter we focused on three main lipogenic genes: FAS, ACC, and ACL.

#### *3.2.1. Fatty acid synthase*

High levels of FAS expression have been found in many human cancers, including prostate cancer [121], ovarian cancer [117, 122], breast cancer [123], bladder cancer [124], colon cancer [125] mantle cell lymphoma [126], and etc. However, the cellular mechanism by which FAS is up-regulated in cancer cells is not fully understood. Nevertheless a few studies suggested that steroid hormones and human epidermal growth factor receptor (HER) family ligands, especially HER2 could increase FAS expression via the PI3K/Akt or mitogen-activated protein kinases (MAPK) pathways[117]. Recently Mukherjee et al [117] demonstrated that bioactive lipid lysophosphatidic acid (LPA) induces FAS expression and lipogenesis through LPA2-G12/13-Rho-SREBP signaling pathway in ovarian cancer cells. Moreover over-expression of FAS in non-cancerous epithelial cells is sufficient to induce a cancer-like phenotype through the induction of HER1/HER2 [127], therefore FAS over-expression may play a role in carcinogenesis. Furthermore FAS overexpression is found to be associated with the advanced stage of colorectal cancer and liver metastasis, thus it may also play a role in the progression of cancer [128]. So far abundant evidences have shown that FAS contributes to both tumorigenesis and metastasis, and it becomes an ideal target for cancer therapy. In deed inhibition of FAS activity by FAS specific inhibitors or siRNA can significantly inhibit cancer or cancer cell growth, induce cancer cell apoptosis, and reduce the metastasis of several cancers[124, 129]. Both synthetic chemicals and natural products of

#### 196 Lipid Metabolism

FAS inhibitors have been developed [123[130], and the recent progress in developing FAS inhibitors as cancer drugs has been reviewed by Pandey et al [131].

Lipid Metabolism, Metabolic Syndrome, and Cancer 197

SREBPs may play critical roles in phospholipid homeostasis and lipotoxic cardiomyopathy. Dysregulated phospholipid signaling that alters SREBP activity has been reported to contribute to the progression of impaired heart function in flies and also act as a potential link to lipotoxic cardiac diseases in humans [143]. Thus the role of SREBPs in modulating heart function and its associated phospholipid signaling maybe a candidate target for future

An aberrant choline phospholipid metabolism is another major hallmark of cancer cells. In deed alterations of choline phospholipid metabolism have been reported in ovarian cancer and also in breast cancer [144, 145]. Altered choline phospholipid metabolism in ovarian cancer has been found to be linked with the regulation of FAS. Because the drop in the level of PC (59%) was significantly correlated with a drop in *de novo* synthesized FA levels, PC was identified as a potential noninvasive magnetic resonance spectroscopy–detectable biomarker of FAS inhibition *in vivo* [146]. Phospholipids and their metabolism have been found to be involved in ovarian cancer in several forms, including LPA, PLA2, PLD, and autotoxin (ATX). Although aberrant phospholipid metabolism has been found in other cancers, the most detailed research work has been carried out using ovarian cancer as a model, so in this section we summarized the recent advances in the research of

The LPAs, with their various FA side chains, are the constituents of a growth-stimulating factor—ovarian cancer activating factor—that has been identified from ascites in patients with ovarian cancer [147]. As a bioactive compound, LPA works to induce cell proliferation or differentiation, prevents apoptosis induced by environmental stress or stimuli, induce platelet aggregation and smooth muscle contraction, and stimulate morphological changes, adhesion and migration of cells. It thus is involved in a broad range of biologic processes in a variety of cellular systems [148, 149]. As an established mitogen, LPA also promotes the invasiveness of hepatoma cells into monolayers of mesothelial cells, and stimulates proliferation of ovarian and breast cancer cell lines even in the absence of other growth promoters such as serum. Furthermore, LPA stimulates rapid neurite retraction and rounding of the cell body in serum-deprived neuroblastoma cells [150], and plays a critical role in regulation of gene expression in normal and neoplastic cells. It is a potent modulator of the expression of genes involved in inflammation, angiogenesis, and carcinogenesis such as interleukin [151-154], vascular endothelial growth factor (VEGF) [155], urokinase plasminogen activator [156], and cyclooxygenase-2 [157]. Thus LPA may contribute to cancer progression by triggering expression of those target genes, resulting in a more invasive and metastatic microenvironment for tumor cells [152, 158]. A significant increase in the expression of LPA receptors (LPA2 and LPA3) with VEGF was found by Fujita *et al.*  [159], who suggested that LPA receptors might be involved in VEGF expression mediated

therapies for obesity- and diabetes- related cardiac dysfunction.

**4.2. Phospholipid metabolism in cancer** 

phospholipid metabolism and ovarian cancer.

*4.2.1. Lysophosphatidic acid* 

#### *3.2.2. ATP-Citrate lyase and acetyl CoA carboxylase*

Apart from FAS, other key lipogenic enzymes for de novo FA biosynthesis include ACL and ACC. While ACL produce the substrate acetyl-CoA from glycolytic product citrate, ACC activates the substrate to generate malanyl-CoA, the building block for fatty acid synthesis. Both ACC and ACL have been found to be over-expressed in many cancers such as breast, liver, lung, ovarian, prostate and leukemia cancers [132, 133]. Inhibition of either ACL or ACC induces growth arrest and apoptosis in several cancer cell lines [134-136]. The potential mechanism of ACL overexpression in tumorigenesis is through PI3K/AKT and MAPK signaling pathway [135, 137]. Yoon et al [138] found that the major mechanism of HER2 mediated induction of ACC alpha in breast cancer cells is translational regulated primarily through mTOR signaling pathway. While Mukherjee et al [117] found that LPA induced induction of ACC in ovarian cancer cells is through LPA2-Gq-PLC-AMPK signaling pathway. Many small molecule inhibitors for ACL and ACC have been developed as potential therapeutic agents for cancer [133, 139].
