REVIEW ARTICLE

Journal of

Fatty Acids and Breast Cancer: Make Them on Site or Have Them Delivered

Cellular Physiology

WILLIAM B. KINLAW,1* PAUL W. BAURES,2 LESLIE E. LUPIEN,3,4 WILSON L. DAVIS,1 5,6 AND NANCY B. KUEMMERLE 1

Division of Endocrinology and Metabolism, Department of Medicine, The Geisel School of Medicine at Dartmouth, Norris Cotton Cancer Center, Lebanon, New Hampshire

2

Department of Chemistry, Keene State University, Keene, New Hampshire

3

The Geisel School of Medicine at Dartmouth, Program in Experimental and Molecular Medicine, Lebanon, New Hampshire

4

Division of Oncology, Department of Medicine, The Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire

5

The Geisel School of Medicine at Dartmouth, Norris Cotton Cancer Center, Lebanon, New Hampshire

6

Division of Hematology/Oncology, Department of Medicine, White River Junction VAMC, White River Junction, Vermont

Brisk fatty acid (FA) production by cancer cells is accommodated by the Warburg effect. Most breast and other cancer cell types are addicted to fatty acids (FA), which they require for membrane phospholipid synthesis, signaling purposes, and energy production. Expression of the enzymes required for FA synthesis is closely linked to each of the major classes of signaling molecules that stimulate BC cell proliferation. This review focuses on the regulation of FA synthesis in BC cells, and the impact of FA, or the lack thereof, on the tumor cell phenotype. Given growing awareness of the impact of dietary fat and obesity on BC biology, we will also examine the less-frequently considered notion that, in addition to de novo FA synthesis, the lipolytic uptake of preformed FA may also be an important mechanism of lipid acquisition. Indeed, it appears that cancer cells may exist at different points along a “lipogenic-lipolytic axis,” and FA uptake could thwart attempts to exploit the strict requirement for FA focused solely on inhibition of de novo FA synthesis. Strategies for clinically targeting FA metabolism will be discussed, and the current status of the medicinal chemistry in this area will be assessed. J. Cell. Physiol. 231: 2128–2141, 2016. ß 2016 Wiley Periodicals, Inc.

Oncogenic antigen 519

In 1989, Kuhajda et al. (1989) demonstrated that overexpression of a protein, which they termed “haptaglobinrelated protein” (Hpr), was associated with a poor prognosis in breast cancer (BC). Hpr was subsequently referred to as “oncogenic antigen 519” (OA-519) until peptide sequencing revealed it to be the cytosolic enzyme fatty acid synthase (FASN) (Kuhajda et al., 1994). In the intervening years there has been intense interest in the significance of fatty acid (FA) metabolism in general, and FASN in particular, to cancer

Abbreviations: ACACA/ACACB, acetyl-CoA carboxylase a/b; ACLY, ATP citrate lyase; ADSF, adipocyte-derived fibroblast; ALA, a-linolenic acid; ATGL, adipose triglyceride lipase; BC, breast cancer; CCND1, cyclin D1; ChREBP, carbohydrate response element-binding protein; CLA, conjugated linoleic acid; CLL, chronic lymphocytic leukemia; CPT1, carnitine palmitoyltransferase 1; DH, dehydratase domain of FASN; EGCG, (
)-Epigallocatechin gallate; ER, b-enoyl reductase domain of FASN; FA, fatty acid; FASN, fatty acid synthase; FFA, free fatty acid; GPIHBP1, glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1; HSPG, heparan sulfate proteoglycan; KR, bketoacyl reductase domain of FASN; KS, b-ketoacyl synthase domain of FASN; LPL, lipoprotein lipase; MAGL, monoglyceride lipase; MAT, malonyl-CoA/acetyl-CoA ACP transacylase domain of FASN; MFD, milk fat depression; mTOR, mammalian target of rapamycin; MUFA, monounsaturated fatty acid; OA-519, oncogenic antigen 519; PL, phospholipid; PUFA, polyunsaturated fatty acid; S14, spot 14; THRSP; SCD, stearoyl-CoA desaturase; SREBP, sterol response element binding protein; TCA, tricarboxylic acid; TE,

© 2 0 1 6 W I L E Y P E R I O D I C A L S , I N C .

biology. Gene products related to FA metabolism have been identified as both prognostic biomarkers and therapeutic targets. Investigative interest in the nexus between FA metabolism and cancer has been further spurred by the recent recognition that the obesity epidemic in westernized countries is accompanied by an upsurge in the incidence of certain tumor types, including BC (Eheman et al., 2012). In addition to increased risk, the presence of obesity at the time of diagnosis also confers a worse outcome for BC patients (Potani et al., 2010). This review will focus on the dependence of most BC, as well as other tumor types, on an ongoing supply of fatty acids to

thioesterase domain of FASN; THRSP, thyroid hormone responsive spot protein; TG, triglyceride; VLDL, very low density lipoprotein. Contract Contract Contract Fund; Contract

grant sponsor: NIH/NCI; grant number: R01CA126618. grant sponsor: Norris Cotton Cancer Center Prouty grant number: NIH NH-INBRE 5 P20 GM103506.

*Correspondence to: William B. Kinlaw, Division of Endocrinology and Metabolism, Department of Medicine, The Geisel School of Medicine at Dartmouth, Norris Cotton Cancer Center, 606 Rubin Building, Medical Center Drive, Lebanon, NH 03756. E-mail: [email protected] Manuscript Received: 1 February 2016 Manuscript Accepted: 2 February 2016 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 4 February 2016. DOI: 10.1002/jcp.25332

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maintain proliferation and prevent programmed cell death, and on the potential to clinically target this facet of tumor metabolism. Despite the overwhelming focus of investigative attention on de novo FA synthesis as the mechanism for tumor cells to satisfy their strict metabolic requirement, we will also examine the potential importance of cellular uptake of preformed FA by tumor cells as an alternative source of supply. Mammalian FA synthesis

Palmitic acid (C16:0) is the primary product of de novo mammalian FA synthesis. This saturated FA may be subsequently monodesaturated and/or elongated, but mammalian cells do not produce polyunsaturated FA (PUFA). The carbon used to synthesize palmitate is derived primarily from pyruvate, the end-product of glycolysis, and glutamine (DeBerardinis et al., 2007). Glutamine is particularly important in cancer cells, in which the entry of pyruvate into the mitochondrion may be curtailed as a manifestation of the hypoxia-like glucose metabolism of the Warburg effect (Warburg, 1956), where pyruvate dehydrogenase, the rate-limiting enzyme for entry of pyruvate into mitochondria, is deactivated (Kim et al., 2006). Indeed, the growth of cultured BC cells and xenograft tumors in immunodeficient mice is significantly slowed by inhibition of the enzyme aspartate aminotransferase, which converts glutamine to the tricarboxylic acid cycle intermediate aketoglutarate in these cells (Thornburg et al., 2008). It is important to note that a-ketoglutarate is downstream of citrate in the tricarboxylic acid (TCA) cycle, which is the precursor for FA synthesis. Wise et al. (2008) demonstrated that glutaminolysis in tumor cells is driven by the MYC oncogene. Amazingly they also found that the cells may actually reverse the flow of metabolites in the TCA cycle to accommodate the synthesis of citrate from aketoglutarate (Wise et al., 2008). The initial step in FA synthesis is the export of citrate from the mitochondrion to the cytosol. Three cytosolic enzymes then act sequentially to produce palmitic acid. ATP citrate lyase (ACLY) cleaves citrate to yield acetyl-CoA and oxaloacetate, which is transported back into the mitochondrion. Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme that carboxylates the 2-carbon acetyl-CoA substrate to yield the 3carbon product, malonyl-CoA, which forms the nidus for subsequent elongation by fatty acid synthase (FASN). Carboxylation of acetyl-CoA is the pace-setting step in long chain FA synthesis, and ACC is regulated at the transcriptional level as well as by allosteric feed-forward activation by citrate and phosphorylation/dephosphorylation (reviewed in Brownsey et al. (2006)). There are two ACC isoforms, and both are found in BC cells (Witters et al., 1994). The aisoenzyme (ACACA) is involved primarily in de novo FA synthesis, whereas the b form (ACACB) is implicated in the regulation of FA b-oxidation in the mitochondrion. This regulation is accomplished by modulation by ACACB of the concentration of its product, malonyl-CoA. Malonyl-CoA is a potent inhibitor of carnitine palmitoyltransferase I (CPT1), the rate-determining enzyme of mitochondrial FA b-oxidation. Malonyl-CoA is the substrate for the multifunctional enzyme FASN, which repetitively adds two carbon units (from acetylCoA) to ultimately yield palmitic acid. The FA chain is bound to the acyl carrier moiety of FASN during elongation, and the FFA is finally released by thioesterase, the seventh, and final activity present in FASN (Smith, 1994). Excess saturated fatty acids are cytotoxic, and thus stearoyl-CoA desaturase (Scd1), which introduces a single double bond, performs this important “post-lipogenic” enzymatic modification of palmitate. Regulation of FA synthesis in BC

Physiological FA synthesis occurs in a wide variety of tissues, but at low levels except in adipose cells, liver, and lactating JOURNAL OF CELLULAR PHYSIOLOGY

mammary gland (Weiss et al., 1986). Coordinate transcriptional regulation of each of the three enzymes of FA synthesis is mediated primarily by two major transcription factors: sterol response element-binding protein 1c (SREBF, most commonly known as SREBP1c) and carbohydrate response element binding protein (MLXIPL, but often referred to as CHREBP). These transcription factors enter the cell nucleus in response to insulin and carbohydrate metabolisminitiated signals, respectively. Swinnen et al. (1997) demonstrated that SREBP-1c mediates the induction of FASN gene transcription in response to both androgens and cell surface-acting growth factors (Swinnen et al., 2000) in prostate cancer cells. Analogous circuitry was subsequently demonstrated in BC. In BC cells that express the appropriate receptors, progestins and androgens induce the enzymes of the FA synthetic pathway (Chalbos et al., 1987) in a SREBP-1c dependent manner (Martel et al., 2005). Growth factormediated FASN induction involves the MAP kinase and PI 3kinase signaling cascades in BC cells (Yang et al., 2003). Furthermore, transformation of a non-tumorigenic mammary epithelial cell line induced FASN gene expression using those same signaling mechanisms (Yang et al., 2002). The ubiquitous transcription factor Sp1 is required for sex steroid-mediated proliferation and increased FA synthesis in BC cells, where it participates in SREBP1 gene activation (Lu and Archer, 2010). Unlike many cancer-promoting gene networks, the evidence indicates that variants or mutations of the FASN, SREBP1, or CHREBP genes do not seem to be important contributors to BC risk (Campa et al., 2009), although FASN polymorphisms have been linked to both obesity and prostate cancer risk (Nguyen et al., 2010). It appears that SREBP-1c is the proximal mediator of the induction of lipogenesis-related genes in cancer cells, but its activation is generally driven by growth-promoting signals, rather than metabolism-related hormones and substrate availability. Lipogenesis is thus a key facet of the anabolic program of BC cells, and it is driven by each of the major classes of pro-proliferative molecules. FASN is a prominent target for Her2/neu-initiated signaling in BC cells, and its induction is inhibited by trastuzumab (Kumar-Sinha et al., 2003). Transcriptome analysis of nonHer2/neu expressing BC cells after enforced Her2/neu expression showed the FASN gene to be a major responder. Furthermore, Her2/neu positive BC cells are particularly sensitive to apoptosis resulting from inhibition of FASN enzyme activity. A surprising explanation for these findings was revealed by Lupu and coworkers. They demonstrated a bidirectional crosstalk, where Her2/neu signaling activated transcription of the FASN gene, and, inhibition of FASN enzyme activity suppressed expression of the Her2/neu gene (Menendez et al., 2004). Moreover, enforced expression of FASN in non-transformed mammary epithelial cell lines resulted in phosphorylation-induced activation of both Her1 and Her2/neu, and elicited a phenotype resembling that of transformed cells (Vazquez-Martin et al., 2008). The primacy of FASN as a Her2/neu target gene was underscored by the observation that the development of Her2/neu-independent FASN expression contributes substantially to the aggressive phenotype of trastuzumab-resistant cells (Yun et al., 2014). The bidirectional crosstalk between Her2/neu signaling and fatty acid synthesis predicted the observed therapeutic synergy between agents targeting these two pathways. Interestingly, a high fat diet promoted the development of an increased number of tumors in a Her2/neu-driven mouse model of BC, suggesting that exogenous FA may substitute for endogenously formed FA in this circuitry (Khalid et al., 2010). The effects of exogeneous FAs have yet to be fully examined. One exception is a-linolenic acid (ALA), an essential FA found in flax seed. ALA actually suppressed Her2/neu gene expression and slowed BC cell growth (Menendez et al., 2006). Overall, activation of the

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FASN gene appears to be a consistent element of malignant transformation. The finding of FASN overexpression in ductal carcinoma in situ (DCIS), the earliest stage of BC progression, underscores the notion that enhanced lipogenesis is an early event in mammary cell transformation (Milgraum et al., 1997). The intensity of FASN expression in metastatic deposits was found to be site-dependent, with brain showing the highest, and liver the least signal on immunohistochemistry (Jung et al., 2015). As expected, a recent immunohistochemical study by Kim et al. (2015b) showed that FASN expression was highest in Her2/neu-expressing breast tumors, and lowest in triple negative disease (i.e., tumors lacking receptors for sex steroids or trastuzumab). Mechanisms other than transcriptional activation may also contribute to Her2/neu effects on FASN expression in BC cells. Yoon et al. (2007) showed that, in contrast to Her2/neu nonexpressing cells, Her2/neu over-expressing BC cells achieved increased ACACA and FASN enzyme content without an increase in nuclear translocation of the active fragment of SREBP-1. Rather, lipogenic enzyme induction was attributable to increased translational efficiency of the respective mRNAs mediated by the mTOR signaling pathway. Using non-cancer cell models, however, both Porstmann and Duvel and their respective coworkers have convincingly shown that Aktmediated cell growth and enhanced lipogenesis does result from downstream activation of SREBP-1c through the action of mTOR complex 1 (mTORC1), and that this is accompanied by nuclear translocation of the active SREBP1c fragment and the expected induction of mRNAs coding the lipogenic enzymes (Porstmann et al., 2008; Duvel et al., 2010). As anticipated from this signaling cascade, simultaneous inhibition of FASN with Cerulenin and mTOR signaling with rapamycin exerted a synergistic anticancer effect in Her2/neu-expressing BC cells (Yan et al., 2014). Much less attention has focused on the role of ChREBP, as compared to SREBP-1c, in BC or other cancer biology thus far. Tong et al. (2009) reported that ChREBP knockdown in nonBC cancer cells inhibited growth and caused a shift toward aerobic metabolism. This has been attributed to inhibition of lipogenesis, resulting in reduced formation of malonyl-CoA and subsequent derepression of mitochondrial FA b–oxidation. Cytosolic ChREBP is dephosphorylated in response to brisk glycolysis, and this promotes its entry into the nucleus. The glycolytic signal that triggers ChREBP activation was initially believed to be the intracellular content of xylulose-5phosphate, an obscure metabolite of the pentose phosphate shunt. The range of candidates has now been expanded, however, and other glycolytic metabolites, including glucose-6phosphate, are now favored (reviewed in Filhouland et al. (2013)). Results from investigation of the relationship of ChREBP expression in BC tissue and clinical outcome revealed a different picture from that seen in cultured cells. Airley and coworkers found an inverse relationship between expression of ChREBP mRNA and protein and clinical BC virulence (Airley et al., 2014). They concluded that reduced ChREBP activation reflected an aerobic metabolic phenotype (i.e., reduced glycolytic flux), inconsistent with hypoxia- or pseudohypoxiainduced signaling. Indeed, the intensity of a hypoxic gene expression signature was inversely correlated with ChREBP expression. Thus, evidence of a shift away from anaerobic glycolysis was observed in both the cells and tumor tissue, but the net effect on indices of tumor virulence differed between the cell-based and clinical studies. The role of ChREBP in cancer biology therefore remains unsettled. Thyroid hormone responsive spot protein (THRSP; S14) is a nuclear protein that has been strongly linked to FA synthesis in nonmalignant lipogenic tissues, such as adipose and liver, and also in BC cells and tissue (reviewed in Kinlaw et al. (2006)). Progestins and androgens induce S14 gene expression in JOURNAL OF CELLULAR PHYSIOLOGY

receptor-positive BC cells (Heemers et al., 2000), and this is mediated by SREBP-1c and stimulates proliferation (Martel et al., 2005). The S14 gene arose by duplication of the ancestral MID1IP1 gene (also known as MIG12) around the time that mammals evolved (Zhan et al., 2006), and appears to be specifically dedicated to the regulation of FA synthesis in the mammary gland. In contrast to other lipogenic tissues, mammary epithelial cells do not express MIG12 that could functionally substitute for S14, as is possible in the liver. Thus, the S14 knockout mouse shows deficient mammary lipogenesis (Zhu et al., 2005). Brisk S14 expression in primary BC tissue is strongly predictive of recurrence (Wells et al., 2006). S14 was also found to be a component of a succinct metastasis gene expression signature in mouse BC models, consistent with the findings in human disease (Yang et al., 2005). The S14 gene is found, in the company of the mammary oncogene cyclin D1 (CCND1), on the BC amplicon on chromosome 11q13, providing a genetic mechanism for enhanced S14 expression in about 20% of BC cases (Moncur et al., 1998). The THRSP and CCND1 genes are located on opposite ends of the 11q13 amplicon, separated by a large, gene-poor intervening sequence. It is tempting to speculate that they exert complementary tumor-promoting influences: CCND1 drives cell cycle progression, while S14 promotes metabolic adjustments to accommodate the proliferation. The precise function(s) of S14 remains elusive. On the one hand, S14 has been shown to be a required intermediary for the induction of lipogenesis-related genes in hepatocytes and BC cells (Kinlaw et al., 1995; Martel et al., 2005). Alternatively, S14 has also been shown to heterodimerize with MIG12, and thus attenuate the ability of MIG12 homodimers to activate cytosolic ACC enzyme activity (Colbert et al., 2010). Such deployment of S14, however, appears to be at odds with its localization to the cell nucleus (Kinlaw et al., 1992; Wells et al., 2006), the aforementioned mammary metabolic phenotype of the S14 knockout mouse (Zhu et al., 2005), its strong association with enhanced lipogenesis in multiple tissues (Jump and Oppenheimer, 1985), and its correlation with a phospholipidomic pattern characteristic of aggressive BC (Hilvo et al., 2011). On the other hand, the S14 knockout mouse unexpectedly demonstrated enhanced hepatic lipogenesis (Zhu et al., 2001). It appears possible that MIG12 homodimers stimulate ACC enzyme activity more efficiently than do S14 homodimers or MIG12-S14 heterodimers. Thus, subtraction of S14 from the liver would result in enhanced lipogenesis, while the opposite effect would occur in the lactating mammary gland. These considerations aside, S14 is clearly a regulator of FA synthesis in BC cells, and a biomarker for enhanced FA synthesis and poor prognosis in clinical BC. As previously mentioned, monounsaturation of newly synthesized saturated fatty acids is an important metabolic step, both for avoiding the cytotoxicity associated with excess saturated FA, and for achieving the requisite repertoire of acyl group species to be esterified into membrane phospholipids (PL). In cancer cells monounsaturation is performed primarily by stearoyl CoA desaturase (SCD), which introduces a single double bond into palmitoyl- or stearoyl-CoA to produce the monounsaturated fatty acids (MUFA) palmitoleoyl- or oleoylCoA. This is critical because membrane PL synthesis requires substantial quantities of MUFA, and because excess saturated fatty acid content induces apoptosis of BC cells (Hardy et al., 2003). As is the case for the lipogenic enzymes, overexpression of SCD in primary BC tissue is associated with a poor clinical outcome (Holder et al., 2013). The SCD gene is also a target of the Her2/neu oncogene (Kumar-Sinha et al., 2003). Like the lipogenic enzymes, SCD is induced by mTOR signaling (Luyimbazi et al., 2010) and estrogen in estrogen receptorexpressing BC cells but not in non-transformed mammary epithelial cells (Belkaid et al., 2015). Interestingly, stimulation of

FATTY ACIDS AND BREAST CANCER

renal cell carcinoma cell growth by unsaturated FA, including the MUFA oleate, has been demonstrated to result from stabilization of b-catenin, and concentrations of unsaturated FA and b-catenin were correlated in clinical tumor tissues (Kim et al., 2015a). This circuitry, which did not involve ubiquitination of b-catenin, has yet to be studied in BC. Qiang et al. (2015) observed a link between the histone deacetylase sirtuin 1 (SirT1) and Scd1 in a prostate cancer model. They ablated the deleted-in-breast-cancer-1 (Dbc1) gene, and found that the SirT1-dependent induction of Scd1 was derepressed. This led to a more aggressive tumor phenotype in a mouse model, and this was ameliorated by a chemical Scd1 inhibitor. Moreover, the Dbc1 knockout also engendered features of metabolic syndrome, providing a potential mechanistic link between obesity, insulin resistance, and cancer. Given the dependence of lipogenesis-related genes in cancer cells on oncogenic signaling pathways that are generally considered to be constitutively activated, one would not expect that the metabolism of these cells would be responsive to nutrient availability, as occurs in normal tissues. This, however, does not always appear to be the case. Daniels et al. (2014) have shown that various cancer cells will indeed increase the expression of genes required for FA synthesis in response to lipid deprivation. Fate and functional roles of FA in BC cells

Excess FFA, particularly if they are saturated, are cytotoxic to BC cells (Hardy et al., 2003). As discussed previously, stearoylCoA desaturase is a major cellular buffer against such toxicity. Another buffer is rapid esterification of FA after cellular FA production or uptake. In normal cells the liberation of FFA from intracellular triglyceride (TG) stores is accomplished by a series of intracellular lipases. In adipocytes, intracellular lipolysis is initiated by adipose triglyceride lipase (ATGL), which releases FA and diacylglycerol, followed by hormone sensitive lipase (HSL), which produces FA and monoacylglycerol, and finally monoglyceride lipase (MGLL, or MAGL), which produces FA and glycerol. In cancer cells, investigative attention thus far has focused primarily on MAGL. Nomura et al. (2010) demonstrated that MAGL was markedly overexpressed in aggressive cell variants derived from melanoma, ovarian cancer, and BC, as compared to their less aggressive counterparts. They showed that the increased MAGL activity was associated with raised cellular FFA content, and also with several important features of tumor cell virulence, including enhanced cellular migration, invasion, and survival as well as tumor growth in in vivo xenografts. Similar results were observed in a colorectal cancer model (Ye et al., 2011). It thus appears that MAGL promotes an aggressive malignant phenotype by providing a steady, but not excessive, stream of intracellular FFA. Subsequent work by Nomura et al. (2011) using prostate cancer cells showed that this stream includes two general structural and functional FFA species. On the one hand, FFA were produced for generation of structural components, such as PL, while free endocannabinoids, which are regulatory FA, were also hydrolytically released. The full malignant phenotype was recapitulated in MAGL-knockdown cells only after adding back both classes of FFA. An early study of BC tissue compared the membrane lipid composition of BC tumor tissue with that of adjacent normal breast (Chajes et al., 1995). Tumor membranes contained more monounsaturated and less essential acyl groups than did the control mammary tissue, consistent with a greater reliance on FA synthesis, as opposed to exogenous lipid uptake, including diet-derived PUFA. This is not surprising because normal mammary tissue is essentially a fat pad, and adipose cells are designed to take up exogenous FA. Swinnen et al. (2003) JOURNAL OF CELLULAR PHYSIOLOGY

studied the disposition of newly synthesized FA in prostate cancer cells, and found that 81% is used for PL synthesis. The most prominent PL class was phosphatidylcholine, although the synthesis of other PL types was also increased. Importantly, these PL were largely deployed in plasma membrane lipid rafts (Swinnen et al., 2003). Lipid rafts are the locus for signaling systems, including transmembrane growth receptors that drive important aspects of tumor cell biology. It seems likely that altered membrane fluidity associated with the degree of plasma membrane PL saturation could substantially modulate such signaling. Subsequent phospholipidomic analyses of matched BC tissue and normal mammary gland revealed a pattern similar to that of the prostate cancers. Hilvo et al. (2011) assessed the membrane lipid composition of BC tissue, and found increased content of palmitate-containing phosphatidylcholine species in BC, as opposed to normal adjacent tissue. This trend was accentuated with BC progression, predicted reduced survival, and was more pronounced in estrogen receptor negative and high histological grade tumors. Thus, the phospholipidomic signature compared favorably with a classic gene expression survey as a prognostic biomarker. Cifcova et al. (2015) found increased content of all PL classes, with phosphatidyl-inositol, -ethanolamine, and -choline being the largest, in BC of the luminal A and B subtypes. Analysis of the acyl groups found in these PL revealed a major increase in C16:0 and C18:1 FA, again consistent with enhanced de novo synthesis as their source. The degree of saturation of the acyl groups incorporated into membranes also appears to be important for cancer cell survival. Rysman et al. (2010) examined several cancer-derived cell lines, including BC, and found that the plasma membrane saturation index of lipogenic cancer cells was markedly reduced upon inhibition of de novo lipogenesis. Unsaturated acyl groups in membranes are more susceptible to peroxidation, so this alteration was accompanied by enhanced susceptibility to lipid peroxidative damage and oxidative stressinduced apoptosis. Moreover, the fluidity of saturated membranes was affected in a manner that caused reduced uptake of the cytotoxic chemotherapeutic agent doxorubicin. In line with this observation, a lipogenic proteomics signature was associated with cisplatin resistance in a mouse BC model (Warmoes et al., 2013). Sounni et al. (2014) noted that enhanced glycolysis and lipogenesis occurred in resurgent tumors following the withdrawal of anti-angiogenic therapy in mouse cancer models, including BC. Importantly, the recurrences were blocked by administration of Orlistat. The effect was attributed to inhibition of FASN, but since Orlistat inhibits both LPL and FASN enzyme activities, the interpretation is not entirely clear (Kridel et al., 2004). These findings emphasize that cancer cell lipogenesis yields qualitative, as well as quantitative, alterations in the cellular plasma membrane phenotype. It is well recognized that cancer cells may use FA for structural, signaling, and energy-producing purposes. Recent work by Schoors et al. (2015) in vascular endothelial cells surprisingly revealed that these cells, as well as fibroblasts, dedicate FA-derived carbon to deoxyribonucleotide synthesis to support angiogenesis. Although not directly pertinent to tumor cells per se, this unexpected requirement for FAderived carbon is of obvious potential relevance to the biology of tumor angiogenesis. Moreover, perusal of their data derived from a screen of a large panel of cancer-derived cell types reveals that BC cells use relatively more FA-derived carbon for DNA synthesis that any other non-vascular/fibroblast cell type examined. The previously cited work of Sounni et al. (2014) also demonstrated increased lactate production by vascular endothelial cells in the face of anti-angiogenic drug treatment, suggesting a Warburg-like effect. DNA and FA synthesis were not examined in these cells, so a linkage of endothelial cell

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metabolism to DNA synthesis could not be assessed. It will be of interest to see subsequent studies in this area focused on the nexus between lipogenesis and DNA synthesis in BC. FA uptake: An underappreciated pathway in cancer

Since the identification of OA-519 as FASN the vast bulk of investigative effort on FA and breast and other cancer types has focused on the cellular machinery involved in de novo FA synthesis. Several observations, however, suggested that alternate mechanisms for FA acquisition are also important. First was the increased tumorigenesis and accelerated tumor growth seen in a variety of rodent BC models when the animals were fed a high saturated fat diet (Freedman et al., 1990). Second was the observation that BC and other cancer cell types could be rescued from the pro-apoptotic effect of FASN inhibition by the provision of exogenous palmitate (Kuhajda et al., 1994; Olsen et al., 2010). This indicated that cancer cells can internalize exogenous FFA, and that these FA may subsume the function of those synthesized on site. Last, a recent prospective study of a large cohort of women (n ¼ 337,327, 10,062 cases of BC) spanning 11.5 years of follow-up revealed increased risk of ER þ BC in subjects consuming high total and saturated dietary fat (hazard ratio 1.2) (Sieri et al., 2014). This obviously did not address mechanism, although it was speculated that high fat intake may have been associated with higher levels of estrogen. Cellular acquisition of FFA requires a source of extracellular FFA, which arise from hydrolysis of TG, and CD36, the cell surface channel for cellular FA uptake (aka fatty acid translocase) (Goldberg et al., 2009). Circulating FFA may arise from the intracellular hydrolysis and release of adipose TG stores, or from intravascular release of FFA by extracellular hydrolysis of TG transported in chylomicrons or very low density lipoproteins (VLDL). Extracellular TG hydrolysis is achieved primarily by lipoprotein lipase (LPL), a secreted enzyme that is affixed to the inner surface of the capillaries serving adipose, striated muscle, and lung tissue. CD36 is a multifunctional transmembrane protein that serves as a channel for long chain fatty acid uptake. The CD36 knockout mouse exhibits raised circulating levels of FFA, impaired FFA uptake by adipocytes, and reduced mitochondrial b-oxidation of FA (Febbrario et al., 1999). In adipose tissue, insulin promotes translocation of CD36 to the plasma membrane (Stahl et al., 2002), analogous to its effect on the trafficking of glucose transporters. Kuemmerle et al. (2011) analyzed breast tumors for CD36 expression by immunohistochemistry, and observed that the majority of cases express abundant cell surface CD36. Interestingly there was a statistically significant trend for estrogen receptor negative tumors to exhibit CD36 immunoreactivity in the cytoplasm, but not at the cell surface, suggesting a role for sex steroids in CD36 trafficking to the cell surface in BC. Expression of the CD36 gene is down-regulated by estrogen in BC cells (Uray et al., 2004). LPL is viewed as a secreted enzyme produced primarily by adipocytes and striated muscle cells, especially cardiomyocytes (reviewed in Goldberg et al. (2009)). The enzyme decorates the inner surface of capillary beds that serve the LPL-secreting tissues, and thus exhibits tissue specificity. The association of LPL with the surface of vascular endothelial cells has long been known to be non-covalent, and to be disruptable by heparin, suggesting binding to a heparan sulfate motif displayed on a cell surface heparan sulfate proteoglycan (HSPG) core protein. Recent studies using physiological, noncancer models by Young’s group, however, have modified the traditional model of physiological LPL deployment (Goulbourne et al., 2014). They have implicated a protein found on the surface of vascular epithelial cells, glycosylphosphatidylinositol high density JOURNAL OF CELLULAR PHYSIOLOGY

lipoprotein binding protein 1 (GPIHBP1), rather than a heparan sulfate moiety, as the LPL binding site. GPIHBP1 acts to chaperone secreted LPL from the outer to the luminal surface of the capillary endothelial cell by transcytosis, and then serves as a platform for intravascular lipolysis (Beigneux et al., 2007). Importantly, the interaction between GPIHBP1 and LPL is disrupted by heparin. LPL binds to GPIHBP1 as a homodimer, and in this configuration can hydrolyze TG within circulating TG-rich lipoproteins (chylomicrons, very low density lipoproteins [VLDL]) to release glycerol and FFA. These FFA are thus available to local cells that express cell surface CD36. The GPIHBP1-LPL interaction has yet to be studied in cancer. In addition to its metabolic role, older studies implicated LPL in the adhesion of monocytes to vascular endothelial cells in a HSPG-dependent fashion (Mamputu et al., 1997). The role of LPL in tumor cell binding to VEC and subsequent extravasation could be relevant to metastasis, but it has not been studied. Although the importance of LPL in the realms of dyslipidemia and cardiovascular disease is well established, its potential role in cancer biology has been recognized only recently, initially in a series of studies of chronic lymphocytic leukemia (CLL) by several groups. CLL patients may exhibit a long, indolent course, or succumb quickly to rapidly advancing disease. Surprisingly, unbiased assessment of gene expression profiles of CLL cells obtained at the time of diagnosis revealed LPL as the single mRNA that best predicts a virulent course (Heintel et al., 2005; Oppezzo et al., 2005; Nuckel et al., 2006; van’t Veer et al., 2006). Two groups have examined the functional significance of LPL in CLL, as opposed to its role as a biomarker. This work showed that inhibition of LPL enzyme activity in CLL cells with the obesity drug Orlistat caused apoptosis (Pallasch et al., 2008). The result, however, is difficult to interpret because Orlistat is an inhibitor of both LPL and FASN enzyme activities (Menendez et al., 2005). The second study revealed apoptosis of CLL cells in response to LPL knockdown. The data also indicated that LPL overexpression in these cells is driven by the transcription factor STAT3 (Rozovski et al., 2015). STAT3 exerts antiapoptotic effects in BC cells, but its involvement in LPL gene expression has not been assessed in these cells. Kuemmerle et al. (2011) examined BC cells and tumors for expression of LPL mRNA, protein, and enzyme activity. A small minority of BC cell lines expressed the enzyme, while nearly all tumors (including breast, prostate, and liposarcoma) surprisingly showed brisk expression, comparable to that seen in the lactating mammary gland. Addition of exogenous LPL to tissue culture media of BC cells that do not express the enzyme stimulated cell growth, but only in the presence of TG-rich VLDL. This suggests the novel possibility that LPL secreted by nearby nonmalignant cells, such as adipocytes, could be utilized to support the growth of cancer cells. This could be of particular importance for primary BC arising in the mammary fat pad, or in selected fatty metastatic sites, such as bone marrow. Conversely, LPL knockdown in LPL-expressing HeLa cells impaired cell growth. Some BC cell lines secrete lipase activity into tissue culture medium (Kuemmerle et al., 2011), but obviously there are no vascular endothelial cells for it to bind to, and the panel of BC cells that have been examined express a very small quantity of GPIHBP1 compared to that observed in heart (L. Lupien, W. Davis, W. Kinlaw: unpublished data). The GPIHBP1 expression status of human BC tumor tissue is unknown. The precise deployment of the LPL expressed by BC is not settled. Heparin displacement studies using BC tumor homogenate or intact cells, however, has provided a clue regarding this issue: in each case 30% of the LPL was displaced. In the case of tumor, this could represent displacement from GPIHBP1 present on fragments of capillaries in the homogenate. In the case of the cultured BC

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cells however, this is not possible, and the data indicate the novel possibility of cell surface binding of LPL to a HSPG that displays a heparin-like motif. Overall, these findings indicate that BC may employ LPL and CD36 to access the rich source of energy and building blocks for plasma membrane synthesis found in TG-rich lipoproteins, and also provide a link between dietary fat, obesity, and BC biology. Furthermore, the ability of cancer cells to acquire exogenous FA could thwart therapeutic attempts based solely on inhibition of de novo FA synthesis. Representative sections of liposarcoma, prostate cancer, and BC immunohistochemically stained for components of the lipogenic pathway (FASN, S14) and the FA uptake apparatus (LPL, CD36) are depicted in Figure 1. It was previously mentioned that the intracellular lipase MAGL releases a stream of FFA from intracellular stores for both structural and signaling purposes (Nomura et al., 2011). It

was also shown that the reduced virulence of tumor xenografts formed from MAGL-knockdown cells was restored by provision of a high fat diet (Nomura et al., 2010). In this regard, it is notable that LPL-mediated extracellular lipolysis of esterified FA transported in VLDL, as opposed to other lipoprotein classes, generated ligands for peroxisome proliferated receptor-a (Ziouzenkova et al., 2003). Thus, it appears that both extra- and intra-cellular lipolysis may feed FA into signaling as well as structural functions. A meta-analysis of numerous rodent BC models indicated that high fat feeding accelerated tumorigenesis and tumor growth (Freedman et al., 1990), and it is now clear that obesity increases the risk of BC (Eheman et al., 2012) and worsens the prognosis as well (Potani et al., 2010). Fuentes-Mattei et al. (2014) compared global gene expression patterns in a panel of estrogen receptor positive BC tissue from obese and non-

Fig. 1. Representative sections of breast cancer, liposarcoma, and prostate cancer tissue immunostained for (A) FASN, (B) S14, (C) LPL, or (D) CD36. Slides were counterstained with hematoxylin (blue). FASN is cytosolic, while S14 shows nuclear localization. LPL exhibits a perinuclear pattern consistent with localization in the Golgi apparatus (insets “C”). CD 36 exhibits cell surface and cytoplasmaic signals except in an example of estrogen receptor negative breast cancer, where only cytoplasmic staining is seen (upper “D”). The image was originally published in Kuemmerle et al. (2011).

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obese patients, and found numerous body weight-related differences in gene expression signatures. Major findings included enrichment of AKT target genes and genes associated with epithelial to mesenchymal transition in the obese cases. They also demonstrated that tumor tissue from the obese patients expressed more LPL and less FASN mRNAs compared to tumors from non-obese subjects. This suggests that tumors of obese patients may differ with respect to markers of tumor virulence as well as metabolically in that they may rely more on lipid uptake than on de novo FA synthesis. This could represent exploitation of the elevated circulating FFA concentrations observed in obese subjects. It is clear that various BC cells may occupy different positions along a continuum of relying primarily on de novo lipogenesis or on lipid uptake (Kuemmerle et al., 2011). Optimal clinical targeting of the dependence of BC cells on FA will require phenotyping to determine the balance between lipogenesis and lipolysis in individual cases. Fuentes-Mattei et al. (2014) performed co-culture of BC cells and adipocytes, and found that cytokines produced by the fat cells augmented BC cell growth, and the cytokine production and growth acceleration were inhibited by Metformin. Bochet et al. (2013) demonstrated bidirectional communication between BC cells and adipocytes in co-culture experiments. The BC cells promote transformation of adipocytes to adipocyte-derived fibroblasts (ADFs) through Wnt/b-catenin signaling, and the ADFs promote a more aggressive BC phenotype (Bochet et al., 2013). Additional coculture studies by Lee et al. (2015) confirmed the impact of adipocytes on BC cells, and further demonstrated the induction of epithelial-mesenchymal transition markers in the BC cells. In the case of ovarian cancer, which is known to preferentially metastasize to omental fat, elegant studies by Nieman et al. (2011) showed that the ovarian cancer cells actually induce mobilization of adipose TG stores to produce FFA for use by the tumor cells. Such exchange of FA between BC and adipose cells has not been reported. Conversely, however, Kleinfeld and Okada (2005) showed that FFA released by BC cells participate in suppression of host anti-tumor immunity by inhibiting the function of killer T-cells. Conjugated linoleic acid and BC

Conjugated linoleic acids (CLA) comprise a group of octadecadieneoate isomers containing two double bonds in apposition. CLA, particularly the 10t, 12c- and 9c, 11t- isomers, have long been known to exert anticancer effects (Belury, 2002). CLA cannot be synthesized by mammalian cells, but is produced by specific bacteria in the rumen of cows, sheep, and goats, and it is present in their meat and milk. Numerous hypotheses have been advanced to explain the mechanism of the anticancer effects of CLA. Surprisingly, a firm linkage to metabolism was established in the course of studies focused a problem affecting dairy cattle. Harvatine and Bauman (2006) demonstrated that milk fat depression (MFD), a condition in which the fat content of cow’s milk suddenly plummets, is caused by systemic absorption of CLA derived from bacterial metabolism of oleic acid found in certain forage, from the rumen. Comparison of cDNA microarray data from bovine mammary gland before and after infusion of CLA and recapitulation of MFD revealed that one major alteration in gene expression accompanying MFD was diminished expression of the THRSP gene, strongly suggesting that diminished de novo lipogenesis was the cause of the lowered milk fat content. Donnelly et al. (2009) thus addressed the idea that the anticancer effects of CLA might be mediated by its ability to inhibit lipogenesis. Indeed, 9c, 11t-, and particularly 10t, 12cCLA killed BC and liposarcoma cells, and the cells were JOURNAL OF CELLULAR PHYSIOLOGY

rescued by provision of exogenous palmitic acid. Her2/neuexpressing BC cells were particularly sensitive to the cytotoxic effect, as has been shown for the inhibition of FASN enzyme activity by Cerulenin or siRNA (Kumar-Sinha et al., 2003). The reduced cellular content of mRNA coding either THRSP or FASN was attributable to reduced gene transcription in response to CLA treatment, but the precise molecular biology of the modulation of gene expression by CLA remains unknown. There has been some controversy regarding the impact of CLA on mammary tumorigenesis. As mentioned previously, numerous studies have shown chemopreventive, therapeutic, and anti-metastasis effects of CLA in various rodent models of BC (Ip et al., 1991, 1994; Hubbard et al., 2000, 2003). A human study using serum CLA concentrations as a biomarker for dietary CLA intake also found a reduced circulating CLA concentrations in BC patients compared to controls (Aro et al., 2000). Two studies have appeared, however, that seemed to contradict these results. Ip et al. (2007) reported that CLA caused increased tumorigenesis in a mouse model of BC driven by Her2/neu over-expression, and another group observed similar results in a mouse BC model driven by the polyomavirus middle T-antigen (Flowers et al., 2010). In both cases there was a background of mammary epithelial cell hyperplasia in the CLA-treated animals. Bauman’s group appears to have resolved these discrepant results by performing a study of the effects of graded doses of CLA on mouse mammary gland development and metabolism (Foote et al., 2010). This showed that high dose CLA elicited mammary epithelial hyperplasia and enhanced tumorigenesis, whereas low dose CLA caused full inhibition of lipogenesis without causing undesirable proliferative and tumorigenic effects. It thus appears that unnecessarily high doses of CLA elicit an increased number of mammary epithelial cells, resulting in more tumors in mice. CLA is widely used as a nutritional supplement by people seeking weight loss and a leaner body composition. A 2 year trial of 7.5 g CLA/day in healthy subjects resulted in mildly reduced body weight and no detectable toxicity (Gaullier et al., 2005). Based on the safety profile, McGowan and coworkers undertook a proof-of-principle trial of 7.5 g CLA/day taken orally during the 12 day interval between BC diagnosis and surgery in a group of 24 patients. No adverse effects were observed. The brief treatment resulted in significantly reduced expression of S14 and the proliferation marker Ki67 in the tumors, as assessed by immunohistochemistry. Expression of FASN was not impaired, and an apoptosis marker was unaffected. This was the first clinical trial designed to target lipid metabolism in human tumors, and the results appear encouraging. Targeting dependence on FA: Inhibiting ACLY and ACC

Inhibiting any of the enzymes known to have a role in the de novo lipogenesis pathway can reduce or block production of palmitate by FASN. ACLY uses citrate to produce acetyl-CoA and that is subsequently used by FASN as well as for the synthesis of malonyl-CoA by ACC. Thus, this enzyme links glycolysis with lipogenesis, and expression levels of ACLY have been correlated to the pathology of diseases like non-alcoholic fatty liver disease (Wang et al., 2009; Tallino et al., 2015), type 2 diabetes (Guay et al., 2007; MacDonald et al., 2009), obesity, and to many cancers, including that of the breast (Szutowicz et al., 1979). Likewise, ACC is required for palmitate synthesis due to the use of malonyl-CoA by FASN, in addition to the important regulatory functions of malonyl-CoA in the cell, as previously mentioned. In addition to inhibition of ACLY and ACC to prevent de novo lipogenesis, knocking down ACLY expression using RNAi reduces the conversion of glucose to lipid and as a consequence impairs cell growth (Bauer et al.,

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2005). The suppression of tumor growth has also been demonstrated with a small molecule inhibitor of ACLY (Hatsivassiliou et al., 2005). Today, ACLY is recognized as an upregulated enzyme in many cancers (Chypre et al., 2012; Zaidi et al., 2012) and inhibiting ACLY using small molecules to potentially treat cancer is an active area of research (Zu et al., 2012). There are three categories of inhibitors of ACLY: those with a structural relationship to citric acid, a class of benzenesulfonamides, and selected natural product macrolides. The citric acid derivatives include (þ) and (
)-2,2difluorocitrate (Saxty et al., 1991) and (
)-hydroxycitrate (Watson et al., 1969). These polar compounds are useful for studying the role of ACLY inhibition in vitro and in vivo, but are limited from potential clinical use by poor pharmacokinetic properties (Zu et al., 2012). A class of substituted butanedioic acid compounds that contained a hydrophobic 2,4dichlorophenyl group and the citric acid based head group was developed as alternative to the early citric acid derivatives, and these compounds proved to be potent ACLY inhibitors (Gribble et al., 1996). Yet, like previous citric acid derivatives, these compounds did not enter cells efficiently. Prodrugs of this class of compounds incorporate lactones that mask an alcohol and carboxylic acid functionality; this modification is reported to improve cell penetration as compared with the parent structures and was useful in demonstrating hypolipidemic effects in animals resulting from ACLY inhibition (Pearce et al., 1998). The benzenesulfonamide class of inhibitors was discovered by a high throughput screen that followed the reduction of lipid synthesis in HepG2 cells, and was undertaken to identify an orally active compound for investigating the potential of ACLY inhibition in an animal model of hypercholesterolemia (Li et al., 2007). The identified compound was also reported to inhibit both isoforms of ACC, making it impossible to rule out the role of off-target effects on the in vivo experimental outcomes. Interestingly, a competitive inhibitor of the mitochondrial citrate transport protein (CTP) fits this same structural class (Aluvila et al., 2010), and it was recently reported that this small molecule reduces cytoplasmic citrate levels in MCF-7 and MDA-MB-231 BC cells, reducing cell viability by 38.3% and 36.4%, respectively (Ozkaya et al., 2015). Radicicol (Kido and Spyhalski, 1950) and antimycin A (Dunshee et al., 1949) are natural product macrolides first known to be antibacterials. Both compounds were later discovered to inhibit ACLY, albeit non-selectively. Radicicol is a noncompetitive inhibitor of ACLY (Ki et al., 2000) whereas the inhibitory behavior of the antimycins is not reported (Barrow et al., 1997). The most well defined activity of radicicol is as a Hsp90 inhibitor (Khandelwal et al., 2016) and the antimycins are now recognized as mitochondrial toxins that operate by increasing reactive oxygen species (Park and You, 2016). There has been no published work following up on the activity of these macrolide natural products on ACLY, likely because unwanted off target effects make them poor candidates for drug development. The development of small molecule therapeutics generally benefits from structural information regarding the binding between the protein target and the inhibitor class. The protein crystal structure of ACLY has been recently determined with citrate bound or with tartrate bound (Sun et al., 2010), as well as with both ADP and tartrate bound (Sun et al., 2011). Yet, no structures with any compounds from the known classes of inhibitors bound to the enzyme have been published to date. The development of ACLY inhibitors to target BC will also benefit from greater understanding of the mechanism regarding apoptosis following treatment. Zaidi et al. (2012) have shown ACLY inhibition in PC3M, HOP62, and HepG2 JOURNAL OF CELLULAR PHYSIOLOGY

cells is most effective when they are grown in lipid deprived conditions, and that addition of oleic acid could rescue HepG2 cells from ACLY silencing. There was only a slight rescue of HepG2 cells when cholesterol was added. In contrast, the viability of PC3M and HOP62 cells was greater with cholesterol added rather than oleic acid. It was also demonstrated in this work that acyl-CoA synthetase short-chain family member 2 (ACSS2) was up regulated following ACLY silencing, allowing rescue of these cells with acetate. As previously mentioned, there are two isoforms of ACAC. The a-isozyme (ACACA) is found in liver and adipose tissue, and is the form predominantly involved in de novo FA synthesis. Similar to ACLY, the enzyme ACACA is up regulated in BC (Milgraum et al., 1997) and is required for BC cell survival (Chajes et al., 2006). ACACA associates with the tumor suppressor protein, BRCA1 (Magnard et al., 2002; Sinilnikova et al., 2004; Brunet et al., 2008), which keeps ACACA in the inactive phosphorylated form. Mutant BRCA1 proteins that fail to interact with ACACA allow dephosphorylation of the enzyme, and thereby increase de novo lipogenesis (Moreau et al., 2006). Identification of small molecules that inhibit ACC (selectively or non-selectively for the two isoforms) and could be used to treat a range of metabolic disorders, including diabetes, obesity, and cancer, has been an active area of research (Tong and Harwood, 2006; Bourbeau and Bartberger, 2015). This multifunctional enzyme has both biotin carboxylase and carboxyl transferase activities. Inhibitors have been developed for each of these catalytic sites, and a large number of crystal structures are available where the individual domains are bound to inhibitors (reviewed in Bourbeau and Bartberger (2015)). There are also ACCs in plants, fungi, and bacteria, making these important targets for the development of small molecule inhibitors to be used as herbicides and antimicrobials, respectively (Tong and Harwood, 2006). The medicinal chemistry of ACC inhibitors is far more advanced in comparison with ACLY, and today there are many different scaffold classes that have been developed, leading to the discovery of potent inhibitors (Bourbeau and Bartberger, 2015) The majority of the compounds across the different scaffolds display non-selective inhibition of the two isoforms. It is still unclear whether a non-selective ACC inhibitor is a disadvantage overall, but the Acaca knockout mouse was embryonically lethal, and this has rightly raised concerns about inhibiting this isoform (Abu-Elheiga et al., 2005). There are now inhibitors developed with significant specificity for ACACB (Gu et al., 2006), which is the isoform found in the mitochondrial membrane of oxidative tissues such as heart and muscle. The current generation of inhibitors fits Lipinski’s Rules for molecular weight, lipophilicity, and hydrogen bonding characteristics, consistent with the majority of marketed, orally administered drugs, and rendering these inhibitors useful in studying the roles of ACAC in cells and animals. The main impetus driving the development of ACC inhibitors is to treat metabolic diseases like obesity and diabetes, although some research is published where cancerrelated endpoints were measured. Inhibition of ACACA expectedly leads to a reduction in de novo FA synthesis (Thupari et al., 2001), with a corresponding inhibition of growth and cytotoxicity in LNCaP, PC-3M, and BPH-1 prostate cancer cells (Beckers et al., 2007), NCI-H460, HCT-8, HCT-15 cells (Wang et al., 2009), and HepG2 cells (Sugimoto et al., 2007). Soraphen A is a natural product polyketide that potently inhibits ACAC (Vahlensieck et al., 1994; Bourbeau and Bartberger, 2015). This compound has been used to demonstrate that ACACA inhibition in cancer stem cells inhibited FA synthesis and resulted in cell death (CorominasFaja et al., 2014). In HepG2 and LnCaP cells, both malonyl-CoA and FA levels decreased following ACACA inhibition by

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Soraphen A (Jump et al., 2011). Reduced invasive behavior of MDA-MB-231 BC and other cancer cell lines, following ACACA inhibition has also been shown to correlate with the inhibition of de novo FA synthesis (Scott et al., 2012). Paradoxically, it was recently reported that screening of lung cancer cell gene expression under hypoxic conditions disclosed how concurrent inhibition of ACLY and ACACA protected cancer cells from hypoxia-induced apoptosis. The enzyme inhibition resulted in elevated a-ketoglutarate levels that caused reduced activity of the oncogenic transcription factor ETV4 (Keenan et al., 2015). This phenomenology was observed in a variety of cancer cells types, including MDA-MD-231 BC cells, and injects a note of caution regarding the concurrent use of ACLY and ACACA inhibitors to treat cancer. What is not clear is how the cells survived FA synthesis inhibition, as the provision of exogenous FA other than that presumably found in fetal calf serum, is not mentioned in the paper. Targeting dependence on FA: The Medicinal Chemistry of FASN inhibitors

The upregulation of FASN in cancer tissue, including that of the breast, has been well established (Menendez et al., 2004; Menendez and Lupu, 2007; Mashima et al., 2009; Baumann et al., 2013; Zaidi et al., 2013; Jones and Infante, 2015). Both ACLY and ACACA inhibition can decrease de novo FA synthesis as previously described, although the direct inhibition of FASN as a means to treat FA-dependent cancers is the most researched target in this pathway (Lupu and Menendez, 2006a,b; Flavin et al., 2010; Olsen et al., 2010; Wang et al., 2010; Lamaziere et al., 2014). Early investigation suggested that the increased malonylCoA mediated the cytotoxicity following FASN inhibition (Pizer et al., 2000; Thupari et al., 2001). A knockdown of FASN in MDA-MB-435 cells using siRNA revealed widespread changes in the expression of genes regulating cell proliferation, DNA replication, transcription, and apoptosis (Knowles et al., 2008). Continued investigation demonstrated apoptosis of BC cells following FASN inhibition to be a caspase-8-mediated event along with increased sensitivity to the apoptotic protein TRAIL, all induced by the downregulation of mTOR (Knowles et al., 2008). FASN is a multidomain enzyme that synthesizes palmitate from acetyl-CoA and malonyl-CoA using a catalytic cycle involving a bketoacyl reductase domain (KR), a dehydratase domain (DH), a benoyl reductase domain (ER), and a b-ketoacyl synthase domain (KS). Entry to the cycle involves a malonyl-CoA/Acetyl-CoA-ACP transacylase domain (MAT), and exit from the cycle is done through a thioesterase domain (TE) that recognizes the appropriate hydrocarbon chain length. Protein crystallography has shed light on the organization and cooperativity of these domains (Maier et al., 2006, 2008). Each of these domains represents a target for medicinal chemistry efforts, though the KR, ER, KS, and TE domains have been exploited the most for drug discovery thus far (Wang et al., 2012). There are three well-known FASN inhibitors, cerulenin, Orlistat, and C75, that have been the most widely used to inhibit FASN and thereby investigate cytotoxicity, animal models of obesity, cancer, or diabetes (Lupu and Menendez, 2006a; Flavin et al., 2010; Olsen et al., 2010; Wang et al., 2012; Lamaziere et al., 2014). Two of these compounds, Cerulenin and Orlistat, have reactive functional groups that lead to the irreversible modification of FASN. Cerulenin was first isolated from Cephalosprium caerulens in the 1960’s, and its ability to inhibit FASN was discovered in the early 1970’s (Vance et al., 1972). Cerulenin was shown to inhibit the KS domain (D’Agnolo et al., 1973) and quickly proved to be a useful compound to investigate the role of FASN in cells (Omura, 1976). JOURNAL OF CELLULAR PHYSIOLOGY

Orlistat was discovered in the 1980’s as a gastric and pancreatic lipase inhibitor (Hogan et al., 1987; Hadvary et al., 1988), and only later was it shown to irreversibly inhibit the TE domain of FASN (Kridel et al., 2004). A crystal structure of Orlistat bound to the TE domain of FASN is known (Pemble et al., 2007), providing valuable clues to the binding interactions for possible optimization in specificity. A structure-activity relationship study was conducted in an effort to increase the activity (Richardson et al., 2008). Orlistat is used clinically as an anti-obesity drug, and is also available over-the-counter despite noxious side effects (Filippatos et al., 2008). Since Orlistat inhibits both FASN and lipases, it has been difficult to interpret experiments using this compound in cells or animals. C75 was designed as a FASN inhibitor based on the mechanism of action of cerulenin and its hypothesized interaction with the FASN KS domain (Kuhajda et al., 2000). C75 was very effective at inhibiting FASN, but it was later discovered that one of the stereoisomers also inhibits carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme of mitochondrial FA b-oxidation, and was responsible for the unwanted anorexic effect observed in animal models of cancer (Makowski et al., 2013). The above three compounds are the most widely used for investigating the role of FASN inhibition in cancer and other diseases. Yet, there are efforts to synthesize or discover new and improved FASN inhibitors. A variety of natural FASN inhibitors have been reported recently. (
)-Epigallocatechin gallate (EGCG) is a polyphenol found in tea with demonstrated potency against the KR domain of FASN, and is perhaps the most noteworthy of the natural flavonols reported with FASN inhibitory activity (Wang et al., 2001). More recently, ginkgolic acids (Oh et al., 2013) and a variety of natural products from Chinese medicine (Cheng et al., 2014) and spices (Jiang et al., 2015) have been shown to inhibit FASN. Additional natural product FASN inhibitors with cytotoxic effects on BC cell lines include tannic acid (Nie et al., 2015), sea buckthorn procyanidins (Wang et al., 2014), a-Mangostin (Li et al., 2014a), stilbene glycosides from rhubarb (Li et al., 2014b), and amentoflavone (Lee et al., 2009). Interestingly, the polarity of these compounds contrasts with conjugated linoleic acid, which was also shown to be an inhibitor of FASN (Oku et al., 2003). The divergent physical properties of these inhibitors could be viewed in two ways by a medicinal chemist. On the one hand, they may represent an optimistic opportunity to employ a wide variety of scaffolds and to fine-tune the pharmacokinetic properties of the initial hits to make better drug candidates. On the other hand, a pessimistic viewpoint perceives FASN as a target that is very promiscuous in its binding to various compounds that is difficult to inhibit with both potency and specificity. The diverse range of scaffolds reported in the literature and patents (Pandey et al., 2012; Wang et al., 2012) seems to favor the optimistic viewpoint. The polyphenolic natural product EGCG inspired the design, synthesis, and identification of additional FASN inhibitors, using the cytotoxicity of the synthesized compounds against FASNdependent BC cell lines as the assay (Puig et al., 2009; Oliveras et al., 2010; Turrado et al., 2012). The FASN inhibitory activity of the most potent compounds was confirmed as part of these efforts. Triclosan is found in many antibacterial products, including hand soaps, toothpastes, and deodorants, following longstanding use in hospital settings (Yueh and Tukey, 2016). In 2002 triclosan was reported to inhibit the ER domain of FASN and as a result, demonstrated cytotoxicity in BC cell lines (Liu et al., 2002). A recent protein crystal structure revealed that inhibition of FASN by triclosan occurs by allosteric binding at a protein-protein interface of the ER domain, rather than binding at the active site (Sippel et al., 2014).

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A startup company, 3-V Biosciences (Menlo Park, CA), is targeting FASN as a means of treating cancer (http:// www.3vbio.com). They have described a series of heterocyclic imidazopyridine-based FASN inhibitors and a refined compound with an IC50 of 17 nM against FASN (Oslob et al., 2013). This compound underwent additional improvement resulting in a new lead compound and inhibitor of the KR domain of FASN, TVB-3166, which was used to extensively detail cellular effects of FASN inhibition (Ventura et al., 2015). A Phase I clinical study is underway according to the company website and clinicaltrials.gov, but it appears to be with a different compound (TVB-2640). The FASN inhibitors cerulenin, Orlistat, and C75 have been used to investigate their effects in cancer cell lines and animal models, and the results of many of the experiments can be found highlighted in several reviews (Menendez and Lupu, 2004; Menendez and Lupu, 2007; Mashima et al., 2009; Baumann et al., 2013; Zaidi et al., 2013; Jones and Infante, 2015). Cerulenin is the most widely used, as the non-specificity of Orlistat and racemic C75 would make in vivo experimental outcomes with those two compounds challenging to interpret. Pizer et al. (1996) established the cytotoxic effect of cerulenin on ZR-75-1 BC cells in 1996, and this was subsequently demonstrated in MCF-7 (Pizer et al., 2000) and SK-BR-3 (Kuhajda et al., 2000) BC cells. Today, measuring the cytotoxicity of FASN inhibitors to BC cells lines is a common experimental approach to use when investigating potential new inhibitors (Puig et al., 2009). There remains a need for potent and selective FASN inhibitors to further define the mechanisms of cytotoxicity, and to investigate the potential of FASN as a target in BC and other tumors. To this end, there are promising advances to report. A probe compound, ML356, has been developed and characterized against the TE domain of FASN as part of the NIH Molecular Libraries Program (Ardecky et al., 2010). This compound is reported to be potent, selective against other thioesterases, and to have drug-like properties. This hit is undergoing further medicinal chemistry development in an effort to improve the pharmacokinetics and thereby enable animal studies. The class of compounds under development by 3-V Biosciences, as previously mentioned, is additionally helping to delineate the cytotoxic mechanisms of FASN inhibition and the susceptibility of certain cancer cells. A FASN inhibitor, TVB-3567, was used to show cytotoxicity that was not predicted by FASN expression levels or de novo palmitate synthesis, but instead by the fractional incorporation of glucose into four specific complex lipids, lysophosphatidic acid, phosphatidic acid, diacylglycerol, and phosphatidylcholine (Benjamin et al., 2015). This result is consistent with the previously cited phospholipidomic analyses of Swinnen and coworkers (Swinnen et al., 2003). The authors indicate that these results may not only be of value as a biomarker for patient responsiveness to FASN inhibition, but also an indicator of possible synergistic chemotherapeutic combinations. Targeting dependence on FA: Targeting expression of genes related to FA metabolism

Inhibiting the expression of genes that play key roles in FA synthesis and its regulation is an alternative strategy to exploit the dependence of tumor cells on FA. One example of this approach is the use of CLA, which was discussed previously in this review. Another class of compounds that has received considerable attention is the triterpenoids. The synthetic triterpenoid CDDO-imidazolide (CDDO-Im) has been reported to promote the differentiation of preadipocytes into mature adipocytes (Suh et al., 1999). Based on those findings, Hughes et al. (2007) tested the capacity of CDDO-Im to differentiate liposarcoma cells. In contrast to expectations, JOURNAL OF CELLULAR PHYSIOLOGY

CCDO-Im induced apoptotic demise of the cells, and this was attributed to inhibition of expression of several lipogenesisrelated genes, including FASN and THRSP. Subsequently, betulin, a similar triterpenoid derived from the bark of birch trees, was identified as an inhibitor of proteolytic processing of SREBP (Tang et al., 2011). Betulin acts to stabilize the interaction of SCAP and INSIG, thus detaining SREBP in the endoplasmic reticulum and preventing its access to the proteases of the Golgi apparatus that produce the active form of the transcription factor. As predicted by the primacy of SREBP1 as a driver of lipogenesis-related gene expression, numerous publications now support the potential of betulin as a small molecule inhibitor that causes apoptosis of lipogenic cancer cells (Li et al., 2010). Flaveny et al. (2015) identified SR9243, a small molecule that comprehensively targets lipogenesis and the Warburg effect. SR9243 binds to the nuclear liver X receptor (LXR), and induces a conformational change that results in the recruitment of corepressors. This inhibited the expression of a panel of target genes involved in glycolysis and lipogenesis, and exerted anticancer effects in a number of non-BC cancer models. It will be of interest to see results using SR9243 in BC models, and to assess its functional interaction with dietary fat.

Conclusion

Lipogenesis, fueled by pyruvate and glutamine, is a major facet of the cancer cell’s anabolic program that is facilitated by the Warburg effect. It is evident from the work reviewed here that the strict requirement of BC and other tumor types for FA is eminently targetable, and the low level of FA synthesis in most nonmalignant tissues predicts acceptable toxicity from this approach. Moreover, for many of the components of the lipogenic apparatus, such as ACLY and FASN, there is no “back up” gene product to replace the function of the targeted molecule. These features of the de novo lipogenic pathway are the impetus for widespread efforts to develop highly specific small molecule inhibitors that could potentially act at several levels, including that of gene expression. The optimal deployment of such inhibitors in the clinic remains to be discovered. In addition to pairing antimetabolic therapeutics with traditional cytotoxic chemotherapy or targeted agents, novel strategies may prove to be useful. One example would be the co-administration of a metabolic inhibitor and a growthpromoting agent to foster an unsupportable conflation of growth promotion and substrate deficiency, with subsequent metabolic catastrophe. There are two major areas in the nexus between BC and fatty acids that are relatively underexplored, and may significantly condition the clinical use of FA synthesis inhibition. First is the issue of BC cell uptake of preformed, exogenous FA. This process is now known to be supported by the expression of LPL and CD36 by BC cells. Several major questions remain unanswered, in particular the cancer cell’s deployment of LPL, and whether FFA or triglyceriderich lipoproteins are the preferred substrates. The possibility that the impact of FA synthesis inhibition could be negated by FA uptake needs to be considered. It appears that the position of a given tumor along the “lipogenic-lipolytic axis” would need to be assessed to guide therapy. Second is the likely related issue of the association of obesity with BC risk and outcome. Several proposed mechanisms, such as enhanced estrogen production, do not appear to be applicable to all of the tumor types that are associated with obesity. This association may be multifactorial and tumortype specific, as the current literature suggests, but it appears that cancer cell FA metabolism will likely to be shown to be a major and nearly generic contributor.

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Acknowledgments

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