Review

Interacting Cancer Machineries: CellSignaling, Lipid Metabolism, and Epigenetics

Thomas W. Grunt1,2,3,*

Cancer-specific perturbations of signaling, metabolism, and epigenetics canbe a cause and/or consequence of malignant transformation. Evidence indi-cates that these regulatory systems interact with each other to form highlyflexible and robust cybernetic networks that promote malignant growth andconfer treatment resistance. Deciphering these plexuses using holisticapproaches known from systems biology can be instructive for the futuredesign of novel anticancer strategies. In this review, I discuss novel findingselucidating the multiple molecular interdependence among cancer-specificsignaling, cell metabolism, and epigenetics to provide an insightful understand-ing of how major cancer machineries interact with each other during cancerdevelopment and progression, and how this knowledge may be used for futureco-targeting strategies.

From Linear Pathways to Crosslinked NetworksTraditional cancer research has provided deep insight into the molecular aberrations occurringin cancer cells. After the identification and characterization of numerous oncogenic driverpathways, research has been increasingly focused on the molecular mechanisms of inter-actions between individual pathways. Accordingly, it has become evident that cancer cells arecontrolled by complex, crosslinked regulatory networks rather than by 1D cascades ofreactions. This enables neoplastic cells to quickly respond to unfavorable and harsh conditionstypically associated with excessive tumor expansion, metastatic spread, and cytotoxic drugtreatments, assuring cell survival in a hostile environment. Herein, I delineate the molecularrelationships between three crucial cancer machineries: cell signaling, lipid metabolism, andepigenetics. Understanding the mechanisms of multilateral crosstalk provides great oppor-tunities for novel anticancer treatment approaches to optimize therapeutic efficacy and preventthe occurrence of drug resistance.

Oncogenic SignalingCells are required to react properly to their environment to enable their survival, maintenance,and growth. Extracellular messenger molecules bind to cognate cell receptors, which conveyexternal stimuli to the cell interior. Receptor activation triggers a series of downstream signalingevents that cause altered gene expression. This transduction of extracellular signals to the cellnucleus is deregulated in cancer cells. Despite the overwhelming number of molecular alter-ations occurring in malignant cells, a recent high-throughput screen of a cDNA library forneoplastic drivers identified a set of only 17 core pro-oncogenic signaling pathways that areoften activated in cancer [1]. Among them, phosphatidylinositol-3-kinase (PI3K)-AKT-mecha-nistic target of rapamycin (mTOR) complex 1 (mTORC1) and RAF-MEK-extracellular signal-regulated kinases (ERK) were the most common. PI3K-AKT-mTORC1, in particular, stimulatesthe proliferation, growth, and survival of cancer cells. It also controls anabolic and catabolic

TrendsDevelopment of resistance to antion-cogenic drugs is caused by the highdegree of regulatory plasticity of malig-nant cells. This plasticity is supportedby the intricate architecture of net-works that comprise crucial pathways,including mitogenic signaling, meta-bolic homeostasis, and epigeneticcontrol of gene expression.

Important functional interactions havebeen identified between growth signal-ing and lipid metabolism, growth sig-naling and epigenetics, as well as lipidmetabolism and epigenetics.

Elucidation of the molecular mechan-isms of these relationships is requiredfor understanding the promotive func-tion of metabolism and its disorders (e.g., hyperphagia, diabetes, or meta-bolic syndrome) for the developmentof cancer and progression to resistantdisease.

1Signaling Networks Program, Divisionof Oncology, Department of MedicineI, Medical University of Vienna,Vienna, Austria2Comprehensive Cancer Center,Medical University of Vienna, Vienna,Austria3Ludwig Boltzmann Cluster Oncology,Medical University of Vienna, Vienna,Austria

*Correspondence:thomas.grunt@meduniwien.ac.at(T.W. Grunt).

86 Trends in Endocrinology & Metabolism, February 2018, Vol. 29, No. 2 https://doi.org/10.1016/j.tem.2017.11.003

© 2017 Elsevier Ltd. All rights reserved.

GlossaryAcetyl-coenzyme A (acetyl-CoA):participates in carbohydrate, lipid,and protein metabolism; can bederived from carbohydrates(glycolysis), FAs (b-oxidation), aminoacids, or free acetate; delivers acetylto the citric acid (Krebs) cycle forenergy production, for the synthesisof FAs for cell growth, to the MVApathway for steroid synthesis, for theproduction of amino acids andketone bodies, or for the post-translational modification of lysineresidues of histone proteins forepigenetic regulation. It is a crucialbuilding block for FA production andhistone acetylation.Acetyl-CoA carboxylase (ACC):the rate-limiting enzyme in FAbiosynthesis. It irreversiblycarboxylates acetyl-CoA to malonyl-CoA. Malonyl-CoA also inhibitscarnitine palmitoyltransferase 1(CPT1) and, thus, blocks FAb-oxidation.ATP-citrate lyase (ACLY): incarbohydrate catabolism, ACLYconverts Krebs cycle-derived citrateto acetyl-CoA.Cancer stem cells (CSCs): afraction of undifferentiated cancercells; have characteristics associatedwith normal stem cells and acapacity for self-renewal growth andfor the production of descendantcells showing variable degrees ofdifferentiation. CSCs give rise to allother cell types in a cancer sample,are tumorigenic, and can causerelapse and/or metastasis.Carnitine palmitoyltransferase 1(CPT1): located in the outermitochondrial membrane. It transfersacyl groups of long-chain FAs fromthe cytosol into the mitochondrialintermembrane space. CPT1represents the first step duringdegradation of FAs by b-oxidation. Itis inhibited by malonyl-CoA.Diacylglycerol (DAG): a signalinglipid that stimulates protein kinase C.Endoplasmic reticulum (ER)stress: arising upon accumulation ofunfolded and/or misfolded proteins inthe ER in response to various stimuli.The ensuing unfolded proteinresponse (UPR) lowers the stresscondition for the ER and enables cellmaintenance and survival.Fatty acid (FA): a carboxylic acidwith a usually unbranched aliphatic

processes, and promotes aerobic glycolysis, a hallmark of neoplastic cells (also known as theWarburg effect), as well as lipid biosynthesis [2]. In this review, I specifically focus on the functionof PI3K-AKT-mTORC1 in the regulation of cancer cell physiology. More-detailed analyses of themolecular architecture of the PI3K-AKT-mTORC1 pathway are provided elsewhere [3,4].

Lipid MetabolismLipid BiosynthesisAn overwhelming amount of preclinical and clinical data collected over the years demonstratesthat malignant cells depend on hyperactive lipogenesis. Fatty acid synthase (FASN; seeGlossary) provides new fatty acids (FAs) that are required for the build-up of plasma mem-branes during cell multiplication. FASN catalyzes the condensation of one molecule of acetyl-coenzyme A (acetyl-CoA) with seven molecules of malonyl-CoA to give rise to the 16-carbonsaturated FA palmitate. Apart from its main localization in the cytosol, FASN can also integrateinto membrane compartments and can be subject to activating phosphorylation by membranereceptor tyrosine kinases (RTKs), such as ERBB2, or by the serine/threonine kinasemTOR, which is a constituent of mTORC1 (Figure 1A) [5–7]. Moreover, FASN has been shownto interact with caveolin-1, a palmitoylated lipid raft protein. Overall, data indicate that FASN isrequired not only for supplying additional lipids, but also for conditioning the membranestructure and function for growth and survival signaling (e.g., by providing second messengersignaling lipids), and for epigenetic reprogramming of gene expression (see below) [7–10].FASN reveals typical features of an anticancer drug target: it is overexpressed in cancer; it isrequired for neoplastic growth; and it is readily accessible by small-molecule inhibitors. Yet,most of the available FASN-targeting drugs are not suitable for clinical use due to unfavorablepharmacokinetics, poor bioavailability, and substantial adverse effects [11]. Nevertheless, anovel promising FASN-antagonistic drug (TVB-2640) recently entered Phase I and II clinicaltrials [12]. Enrollment has already been completed and a first-outcome evaluation is expectedsooni.

By sharp contrast, the enzymes of a related lipogenic pathway, the mevalonate (MVA)pathway, provide several well-proven oncology drug targets, including 3-hydroxy 3-methyl-glutaryl-CoA reductase, isopentenyl-PP isomerase, and farnesyl-PP synthase. These enzymescan be antagonized by statins (e.g., simvastatin) or bisphosphonates (e.g., zoledronate). Bothtypes of drug have already been used for many years in the clinic, albeit not against cancer cells:statins for lowering cholesterol levels and bisphosphonates to diminish osteoclast-mediatedbone loss. Therefore, drug safety is no longer an issue for these compounds [13]. Generally,MVA pathway enzymes are positively regulated by various oncogenic signaling pathways. Theyuse acetyl-CoA as a starting material to produce sterols and isoprenoids. These isoprene-likeprecursor molecules provide functional groups for post-translational modification of oncopro-teins and are crucial for carcinogenesis and neoplastic progression. Therefore, the MVApathway enzymes and oncogenic signal effectors are stimulated by mutually regulatory cross-talk loops.

Lipid DegradationIn contrast to FA synthesis, which yields building blocks for cell growth, oxidative degradation ofFAs (b-oxidation) provides ATP for energy balance, NADPH for reduction of oxidative stress,and acetyl-CoA for (histone) protein acetylation. For a long time, lipogenesis and lipolysis wereconsidered incompatible and not to proceed simultaneously. However, recent evidencerevealed that the master regulator of FA metabolism, acetyl-CoA carboxylase (ACC), whichconverts acetyl-CoA to malonyl-CoA, comes in two flavors: ACC1 is found in the cytoplasm andpromotes the production of FAs, whereas ACC2 is localized to the outer mitochondrial

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membrane and prevents FA oxidation. Both have a role in cancer cell maintenance. Thus, it isconceivable that they occur simultaneously and are balanced by prevailing intra- and extracel-lular conditions, such as the availability of nutrients and ATP. When cellular energy is low, thelevel of AMP increases relative to ATP and AMP-activated protein kinase (AMPK), the keykinase regulator of ACC, phosphorylates and inactivates both ACC1 and ACC2 [14]. Recently,cancer cell phenotypes have been identified that depend on either FA synthesis or FA oxidation.The former are characterized by overexpression of ACC1, whereas the latter contain an inactivenonhydroxylated form of ACC2 and lack prolyl hydroxylase domain-containing protein 3(PHD3), which is known to hydroxylate ACC2 [15]. Under low energy conditions, ACC1and ACC2 are negatively regulated by phosphorylation through AMPK, which promotes thedegradation rather than production of FA. However, under nutrient abundance, AMPK isdownregulated, abolishing its repressive function on ACC1 and ACC2. Moreover, ACC2becomes further activated by PHD3 and can efficiently suppress FA oxidation. Intriguingly,it has been proposed that the function of quiescent cancer stem cells (CSCs) benefits fromactive FA oxidation [16], while the bulk of proliferating progeny cells require de novo synthesis ofFAs for growth [17]. Thus, concepts of discrete or combined targeting of anabolic and catabolicFA metabolism are novel and have high therapeutic relevance. The corresponding pathwayscontrol the availability not only of lipids, but also of metabolic intermediates that regulate geneexpression.

Epigenetic Gene RegulationMalignant transformation of cells is characterized by typical alterations in the nucleotidesequence of the DNA (mutations) (Box 1). Gain-of-function mutations turn growth-promotinggenes into oncogenes expressing either large amounts of corresponding wild-type proteins orregular levels of hyperactive proteins. Growth-inhibiting (tumor-suppressor) genes usuallybecome disabled in cancer. This can occur either by an inactivating mutation leading to lossof function or loss of protein, or by epigenetic silencing, which also results in protein down-regulation (Box 2). Intriguingly, although epigenetic patterns are inherited in regeneratingsomatic cells, these patterns, unlike genetic blueprints, are potentially reversible. This nurtureshope that epigenetic modifications occurring in cancer may provide additional drug targets forimproved therapy [18,19].

Interaction between Oncogenic Signaling and Lipid MetabolismOncogenic Signaling Regulates Lipid MetabolismIn prostate, breast, and ovarian cancer, ligand stimulation or overexpression of members of theepidermal growth factor receptor (EGFR) family (EGFR and ERBB2) or the fibroblast growthfactor receptor (FGFR) family correlates with high FASN levels, whereas blockade of theseRTKs causes FASN downregulation. Notably, FASN is a genuine substrate of ERBB2, which isactivated by ERBB2-mediated phosphorylation. However, the specific phosphorylation siteswithin FASN remain elusive [20–26]. Inhibition of EGFR, ERBB2, or downstream PI3K-AKT-mTORC1 by kinase blockers or small interfering (si)RNAs has been found to downregulateFASN, whereas inhibition of RAF-MEK-ERK signaling was ineffective. Moreover, phosphataseand tensin homolog deleted on chromosome 10 (PTEN), which counteracts PI3K function,is inversely correlated with FASN [27,28]. Collectively, the data suggest that FASN is regulatedby the RTK-PI3K-AKT-mTORC1 but not by the RTK-RAS-RAF-MEK-ERK route in prostate,breast, and ovarian cancer (Figure 1A) [20–28]. By contrast, in bronchial [29] and pancreaticductal adenocarcinoma [30], FASN appears to be modulated primarily by RTK-RAS-RAF-MEK-ERK signaling.

chain containing an even number (4–28) of carbons.Fatty acid synthase (FASN): keyenzyme of de novo lipid biosynthesisproviding new FAs.Lipid raft: specialized cellmembrane microdomain containingglycosphingolipids and receptorproteins that form glycolipoproteinstructures. Lipid rafts serve as anorganizing center for the assembly ofsignaling molecules, includingreceptor and adaptor proteins.Lipidation: attachment of lipids toproteins.Mevalonate (MVA) pathway:begins with acetyl-CoA andproduces isoprenoids, a diverseclass of over 30 000 biomolecules,including cholesterol and steroidhormones.Noncoding RNAs (ncRNAs): RNAmolecules that are not translated intoproteins; includes tRNAs, rRNAs,small nucleolar (sno)RNAs, miRNAs,siRNAs, small nuclear (sn)RNAs,extracellular RNA (ex)RNAs, Piwi-interacting (pi)RNAs, small Cajalbody-specific (sca)RNAs, and longncRNAs. Their precise functions arenot yet fully identified.Phosphatase and tensin homologdeleted on chromosome 10(PTEN): an endogenousphosphatidylinositol-3-kinase (PI3K)inhibitory phosphatase thatdephosphorylates PIP3.Phosphatidylinositol 3,4,5-trisphosphate (PIP3): a signalinglipid produced by PI3K.Receptor tyrosine kinases(RTKs): transmembrane proteinswith an extracellular polypeptide-binding domain, a transmembranelinker domain, and a cytoplasmictyrosine kinase domainphosphorylating substrate proteins attyrosine.Serine/threonine kinases:cytoplasmic enzymes thatphosphorylate substrate proteins atserine and/or threonine.Sterol regulatory element-bindingprotein-1c (SREBP-1c):transcription factor regulating genesthat harbor sterol regulatory element-1 DNA motifs in their promoter.Unfolded protein response (UPR):a response related to ER stress;activated upon accumulation ofunfolded and/or misfolded proteins inthe ER lumen to halt protein

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translation, degrade misfoldedproteins, and activate production ofchaperones for cell survival.RTK

GF

Glucose-6-P

Glucose

GCK

Pyruvate

PI3K

AKT

PIP3

mTORC1

SREBP1c

Krebs cycle

DNMT1

p300/CBP

Histoneacetyla on

Citrate

Oxalo-acetate

Acetyl-CoA

Acetyl-CoA

GlutamineMevalonate

pathway

ACLYPalmitoyl-CoAMalonyl-CoA

FASN

HIF-1αAMPKα

REDD1TSC2TSC1

RHEB

ACC

Glycolysis

β-oxida on

Fa y acid synthesisCPT1

Oncogenic signalingDNAmethyla on

(A)

Histoneacetyla on

RAS

Protein synthesis,inhibi on of autophagy,

signal protein stabiliza on

REDD1

PI3Kp110p85

PTEN

PDK1AKT

PIP3PIP2DAG + PI(P) pool

TSC2

TSC1

Rheb

mTORC1

S6K1

S6 eIF4E

4EBP1

HIF-1α

AMPK

Step 5

Step 6

GRB2Lipid ra s

RTK

Step 1

Step 7

Step 4

Step 2

Step 3

(B)

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In any case, RTK downstream signaling leads to the upregulation and/or activation of sterolregulatory element-binding protein-1c (SREBP-1c). This transcription factor controls theexpression of many lipogenic and glycolytic genes, such as those encoding FASN, ATP-citrate lyase (ACLY), and glucokinase [23,31,32]. Moreover, AKT-mTORC1-SREBP-1c sig-naling not only upregulates ACLY transcription, but also directly activates its catalytic functionby phosphorylation at Ser455 (Box 3). ACLY produces acetyl-CoA, which is carboxylated byACC to malonyl-CoA and used for lipogenesis and/or for inhibition of carnitine palmitoyl-transferase 1 (CPT1), the rate-limiting enzyme in FA oxidation. This indicates that oncogenicRTK signaling causes the activation of lipogenic and inhibition of lipolytic genes and unveils vitalmolecular links between oncogenic signaling and lipid metabolism (Figure 1A).

(C)

FASNACCACLY

SAHMethylSAM

Histoneacetyla on

Histonemethyla on

PEmethyla on

DNAmethyla on

PP2Amethyla on

Acetyl-CoA Malonyl-CoA Palmitoyl-CoA

Acetate

Citrate

ACSS

Krebs cycle

β-oxida on

Fa y acid synthesis

DNMT1

Figure 1. Interaction between Oncogenic Signaling, Lipid Metabolism, and Epigenetic Regulation. (A) Growthfactors (GF) activate receptor tyrosine kinases (RTK), which signal to phosphatidylinositol-3-kinase (PI3K). PI3K producesphosphatidylinositol 3,4,5-trisphosphate (PIP3) and recruits the serine/threonine kinase AKT. AKT activates mechanistictarget of rapamycin complex 1 (mTORC1) by blocking tuberous sclerosis complexes-1 (TSC1-hamartin) and -2 (TSC2-tuberin), which are repressors of the mTORC1-activator GTPase RHEB. mTORC1 activates sterol regulatory element-binding protein-1c (SREBP-1c), which upregulates fatty acid synthase (FASN) and glucokinase (GCK), linking oncogenicsignaling with lipogenesis and glycolysis. FASN upregulates RTK and PIP3, blocks AMP-activated protein kinase a

(AMPKa) and hypoxia-inducible factor-1a (HIF-1a), which otherwise would activate TSC1/TSC2 via regulated in devel-opment and DNA damage response 1 (REDD1). RTK and mTORC1 phosphorylate FASN. AKT and SREBP-1c stimulatethe lipogenic enzyme ATP-citrate lyase (ACLY), whereas AMPKa impedes acetyl-CoA carboxylase (ACC)-mediatedproduction of malonyl-CoA. Malonyl-CoA is used for FA synthesis and inhibits carnitine palmitoyltransferase 1 (CPT1)-dependent shuttling of FAs into mitochondria for b-oxidation. AKT interacts with histone acetyltransferase p300/CREB-binding protein (CBP) and DNA methyltransferase DNMT1, which is controlled by ACLY. Therefore, Acetyl-CoA connectsglycolysis, Krebs cycle, b-oxidation, glutamine, lipogenesis, mevalonate pathway, and histone acetylation. (B) Lipogenicpathways stimulate RTK-PI3K-AKT-mTORC1. Step 1: facilitating lipid raft formation. Step 2: membrane RTK upregulation.Step 3: RTK-GRB2 recruitment. Step 4: restocking the pools of signaling lipids [diacylglycerol (DAG), phosphatidylinositol(PI), and phosphatidylinositol phosphate (PIP)]. Step 5: RAS lipidation/palmitoylation. Step 6: HIF-1a blockade. Step 7:AMPK inhibition. (C) Acetyl-CoA and methyl interconnect metabolic [FA synthesis, b-oxidation, phosphatidylethanolamine(PE)-membrane lipid methylation, Krebs cycle], epigenetic (histone-acetylation and/or methylation and DNA methylation)and oncogenic pathways (phosphatase PP2A-methylation). Abbreviations: ACSS, acetyl-CoA synthetase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.

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Lipid Metabolism Regulates Oncogenic SignalingIntriguingly, evidence indicates that cancer signaling controls lipid biosynthesis and, vice versa,that lipogenesis regulates oncogenic signaling. Overall, an intricate crosstalk coordinates cellcycle, DNA replication, and anabolic processes to provide building blocks for cell proliferationand growth.

Most of the FASN-generated newly synthesized FAs are integrated into phospho- and gly-colipids, which are major constituents of cell membranes [33–35]. FASN has been found tosupport the formation of membrane lipid rafts (Figure 1B, Step 1) facilitating the clustering ofsignal protein complexes in these functionally important membrane microdomains. Moreover,FASN promotes the expression of membrane RTKs, such as EGFR, ERBB2, and hepatocytegrowth factor receptor/C-MET, in breast, ovarian, and prostate cancer, and non-Hodgkinlymphoma [21,22,36,37] (Figure 1B, Step 2), and stimulates the recruitment of adaptorproteins, such as GRB2, to EGF-activated EGFR [8] (Figure 1B, Step 3). Consequently,expression of EGFR and ERBB2, and clustering of macromolecular signaling complexes atthe membrane, are impaired upon exposure to FASN inhibitory drugs [22]. Overall, FASN isrequired for the production and/or formation of signaling complexes in the membrane [38].Another way by which FASN can promote signal transduction is by providing signaling lipids,such as diacylglycerol, phosphatidylinositol, or phosphatidylinositol 3,4,5-trisphosphate(PIP3) (Figure 1B, Step 4) [8,9,39]. These second messengers exert crucial functions duringmitogenic and prosurvival signaling. In addition, FASN-produced lipids can be used for post-translational lipidation of proteins, a maturation process required for correct intracellularlocalization and function of many signaling proteins (Figure 1B, Step 5) (Box 4). Moreover,lipidomic analyses have shown that hyperactive FASN elevates the proportion of saturated FAsrelative to unsaturated FAs in the membrane of neoplastic cells, thereby inducing a ‘saturated’lipid phenotype, which affects signaling processes and confers resistance to oxidative stress

Box 1. The Relationship between Genetics and Epigenetics in Cancer

Epigenetics is defined as ‘the study of changes in gene function that are mitotically and/or meiotically heritable and thatdo not involve a change in the sequence of DNA’ [90]. Today, neoplastic growth is considered to result from aberrationsat both the genetic and the epigenetic level [91]. These alterations occur early during carcinogenesis and increase duringprogression. Notably, epigenetic changes in turn are triggered by mutational events in genes that control theepigenome. These epigenetic genes encode epigenetic-modifying enzymes that modulate the activity of regulatoryDNA sequences or chromatin histone proteins, or they produce epigenetic-related noncoding RNAs (ncRNA). Thus,cancer is still exclusively a genetic disease, although both genetic and epigenetic aberrations can be identified inmalignant cells.

Box 2. Mechanisms of Epigenetic Gene Regulation

A main mechanism of epigenetic gene regulation routinely seen in cancer is hypermethylation of cytosines in CpG-richpromoter regions (CpG islands). This process is mediated by DNA methyltransferases (DNMTs). It impedes transcriptionof the respective gene, which typically encodes a tumor-suppressor protein.

Another mechanism of epigenetic tumor-suppressor gene silencing is through condensation of the nucleosomalchromatin in the respective region of the chromosome. Histone deacetylases (the antagonists of histone acetyltrans-ferases) remove acetyl groups from acetylated lysines at the N terminus of histone core proteins and, thus, transformloose, transcriptionally permissive euchromatin into compact, transcriptionally nonpermissive heterochromatin. Like-wise, enzymatic addition of methyl groups, ubiquitin, or phosphate to amino acids at the N terminus of histone proteinsalso affects the surface charge of the histones and contributes to the structural balance between eu- and hetero-chromatin [91].

This delicate balance is indirectly controlled by epigenetic-related ncRNAs, which regulate the expression of epigenetic-modifying enzymes at the transcriptional and/or post-transcriptional level [92,93].

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and cytotoxic drugs [8,40]. These mechanisms of signal generation, propagation, and trans-mission at the cell membrane, and along downstream signaling cascades become graduallyimpaired, because the supply of de novo synthesized lipids ceases upon blockade or down-regulation of FASN [4,8,41].

In contrast to this delayed response to abrogation of FASN, a rapid inhibitor-mediatedupregulation of hypoxia-inducible factor (HIF) and its downstream effector REDD1 can bediscerned even before silencing of membrane-signaling proteins occurs [8]. Therefore, itappears that active lipogenesis can repress HIF pathways (Figure 1B, Step 6). Both HIF-1aand HIF-2a are upregulated upon endoplasmic reticulum (ER) stress [42,43]. The latter hasbeen found to promote the formation of lipid droplets to alleviate cytotoxic ER stress responses[44]. Altogether, the data are consistent with the notion that FASN blockade or silencing rapidlyelicits an ER stress response [45,46]. The ER membrane is generally sensitive to perturbation ofphosphatidylcholine, its main constituent. This causes ER stress and the unfolded proteinresponse (UPR) [45]. Several studies have demonstrated that SREBP ablation or FASNblockade causes depletion of functionally important ER membrane lipids followed by ER stressand UPR, which is characterized by the upregulation of HIF-1a and its downstream effectorREDD1 [8,42–47]. REDD1 blocks mTORC1 downstream signaling [4]. Upon continued inhibi-tion of FASN, HIF-1a and REDD1 levels gradually decrease, and a lack of ATP and bioenergyoccurs. In this situation, AMPK, another mTORC1 repressor, becomes activated and takesover mTORC1 inhibition [8]. Thus, the synthesis of lipids occurs only when energy is available,keeping stress stimuli and AMPK activity low (Figure 1B, Step 7). Altogether, blockade of FASNcauses the depletion of lipids, resulting in derangement of membrane lipid rafts and disinte-gration of raft-anchored signal protein complexes. It also elicits ER stress and UPR, leading tothe upregulation of stress response proteins, such as HIF, REDD1, and AMPK, which silenceRTK-PI3K-AKT-mTORC1 signal transduction by repressing mTORC1 function. Moreover,abrogation of FASN causes the ubiquitination and degradation of PI3K-AKT-mTORC1

Box 3. Oncogenic Signaling Promotes Catabolic Production of Acetyl-CoA

Growth signaling and the epigenome are deregulated in cancer. Recent research uncovered the mechanisms of howoncogenic signaling controls epigenetic gene regulation. Growth-promoting ligands interact with cognate RTKs andstimulate RAS, which activates effector pathways, including PI3K-AKT-mTORC1. PI3K activity stimulates cell meta-bolism through multiple mechanisms. AKT, for example, promotes glycolysis by upregulation of the glucose transporter1 (GLUT1) at the membrane, by activation of phosphofructokinase, and by mitochondrial association of hexokinases 1and 2. In addition, AKT stimulates transcription of the acetyl-CoA-producing enzyme ACLY via the mTORC1-SREBP-1caxis [94] and directly phosphorylates it, which activates its catalytic function [95].

In summary, AKT signaling spurs glucose consumption and glycolytic catabolism, providing high amounts of citric acid(Krebs) cycle-derived citrate. It also upregulates ACLY expression and activates ACLY-mediated cleavage of citrate toacetyl-CoA. Thus, AKT promotes the cytosolic production of acetyl-CoA, which is a substrate necessary for histonelysine acetyl transferases during acetylation of nucleosomal core histone proteins and for FASN during lipidbiosynthesis.

Box 4. Various Forms of Post-Translational Lipidation of Signal Proteins

Palmitate, the product of FASN, can be attached to G proteins, membrane receptors, or RAS and RAS-homologous(RHO) GTPases at C-terminal CAAX-boxes and directs their intracellular localization and function [96–98]. Products of arelated lipogenic pathway (the mevalonate pathway) can be used for other forms of lipid-dependent post-translationalmodification of proteins, including prenylation or N-glycosylation. Prenylated variants of RAS and RHO GTPases and N-glycosylated forms of insulin-like growth factor receptor have been observed [99]. These types of lipid-mediatedstructural ‘maturation’ of proteins alter their physicochemical properties and regulate cell growth, cell cycle, carcino-genesis, neoplastic progression, and drug-mediated apoptosis [100,101].

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signaling proteins via the autophagy pathway, thereby reducing the steady-state levels of theseeffector proteins [8,41].

Interactions between Oncogenic Signaling and Epigenetic Gene RegulationRecent experimental evidence indicates that oncogenic signaling controls several key pro-cesses of epigenetic gene regulation. In particular, a close link between PI3K-AKT-mTORC1and the catabolic production of acetyl-CoA, the key substrate for histone acetylation, has beenobserved (Box 3). AKT downstream signaling has been found to facilitate continuous allocationof acetyl-CoA, even when the substrate, citrate, is limited [48]. The level of lysine-specificacetylation of histone proteins, a key determinant of epigenetic regulation of gene expression, issensitive to the availability of acetyl-CoA and is balanced by histone lysine acetyl transferases(HATs or KATs), such as GCN5, and by histone deacetylases (HDACs) [48,49–54]. Thus, RTK-PI3K-AKT-mTORC1 signal transduction controls histone acetylation through modulation ofACLY and the availability of acetyl-CoA (Figure 1A). Moreover, evidence indicates that PI3K-AKT may directly phosphorylate epigenetic-modifying enzymes to promote transcriptionalcompetence. For instance, PI3K-AKT stimulates the HAT p300 and its paralog CREB-bindingprotein (CBP) by phosphorylation. Phosphorylation of the histone H3 methyltransferase EZH2by AKT reduces repressive methylation of H3, and AKT-mediated phosphorylation of thehistone lysine demethylase KDM5A causes its nuclear exit and prevents its inhibitory action onH3. DNMT1 phosphorylated by AKT at S143 is unable to associate with, and subsequentlymethylate, DNA (Figure 1A) [55]. Therefore, PI3K-AKT can stabilize the transcriptionally com-petent conformation of euchromatin and enables locus-specific hypomethylation of DNA.

Interactions between Lipid Metabolism and Epigenetic Gene RegulationIt is now well established that cell metabolism and epigenetic gene regulation interact with eachother and that cancer cells exploit this molecular link [56]. For instance, ACC was recently foundto modulate global histone acetylation, thus providing a direct link between anabolic metabo-lism and epigenetic gene regulation (Figure 1A,C) [57]. Moreover, free acetate, which isconverted to acetyl-CoA by acetyl-CoA synthetase (ACSS), was found to act as an epigeneticmetabolite and to promote lipid synthesis under hypoxic conditions [58]. As shown above,ACLY is critical for histone acetylation [50]. Lipids can directly function as a carbon source andcan generate acetyl-CoA to be used for histone acetylation [59]. In addition, experimentalevidence indicates that ACLY regulates DNMT1 (Figure 1A,C) and maintains DNA methylationpatterns in adipocytes [60]. DNA methylation is dependent on the availability of methyl groupdonors. These are provided by the enzyme methionine adenosyltransferase, which metabolizesand converts the essential amino acid methionine to S-adenosylmethionine (SAM). All cells cansynthesize SAM. It is required as a general precursor for the methylation of a variety ofacceptors (DNA, RNA, proteins, lipids, and many other small molecules) [61,62] and yieldsthe demethylated derivative S-adenosylhomocysteine (SAH), which drives cellular sulfurmetabolism. In particular, SAM is a co-substrate involved in methyl group transfers by DNMTsfor the methylation of CpG islands in the promoter regions of genes. Apart from this keyepigenetic regulatory process, mono-, di- and tri-methylation of specific lysines in nucleosomalcore histone proteins H3 and H4 have been observed. These methylated lysines can eitheractivate or inhibit gene transcription depending on the number of methyl groups added and onthe overall structural conditions surrounding the methylated lysine in the histone protein [63,64].Interestingly, an unexpected relationship between the SAM:SAH ratio and the methylation ofeither the membrane phospholipid phosphatidylethanolamine (PE) or the histone H3 and thephosphatase PP2A has been observed (Figure 1C). Available evidence indicates that PE canact as a SAM-consuming sink for cellular methyl and provides SAH that can be used for trans-

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sulfuration of methionine to cysteine. However, in nonhepatic cells, methyl becomes absorbedby histone H3 and the phosphatase PP2A rather than by PE [65]. These data suggest aninverse interaction between the methylation of membrane lipids, the epigenetic regulation ofhistone proteins, and phosphorylation-mediated cellular signaling (Figure 1C). Altogether, it isnow widely accepted that cancer epigenetics, cancer cell metabolism, and oncogenic signalingare connected via a multidirectional, cross-connected regulatory network [66].

Nutrition and Cancer RiskUptake of sugar elevates insulin production [67]. Insulin cooperates with insulin-like growthfactor-I (IGF-I) to modulate the sensitivity of cells to glucose [68]. Insulin and IGF-I stimulate thecellular uptake of glucose by increasing membrane expression of glucose transporters(GLUTs). Insulin and IGF-I bind to their cognate membrane RTKs (insulin receptor and IGF-1 receptor, respectively) and activate downstream RAF-MEK-ERK and PI3K-AKT-mTORC1pathways that support proliferation and survival. Moreover, mTORC1 acts as a biochemicalrelay. It promotes anabolic processes, including the biosynthesis of proteins, lipids, andorganelles, and restricts catabolic processes, such as autophagy [69]. mTORC1 also receivesa variety of different inputs from hormones, growth factors, and intracellular sensors of ATP (e.g., AMPK), oxygen (e.g., HIF-1a), and amino acids (e.g., RAS-related GTPase, RAG). Recently,functional links between a high-fat diet, activation of peroxisome proliferator-activated receptordelta (PPAR-d) and the mTORC1 constituent mTOR have been established that enhance therisk for colorectal and mammary carcinogenesis [70,71]. Thus, mTORC1 integrates multiplecellular inputs to ensure homeostatic metabolic balance [72]. Therefore, excessive consump-tion of carbohydrates and fats fuels cell growth, which favors cancer development. By contrast,mild calorie restriction leads to a decrease in circulating growth factors, anabolic hormones,inflammatory cytokines, and oxidative stress markers associated with cancer [73]. Dataindicate that hyperactivation of cell anabolism stimulates mitogenic signaling, which in turnspurs epigenetic programs enabling cell growth and mitosis. These conditions provide amechanistic explanation for the well-known links between a physically inactive life-style,malnutrition, hyperphagia, obesity, metabolic syndrome, diabetes, and increased cancer risk(Figure 2).

Epigene�c generegula�on

Cancer

Cellsignaling

Lipidmetabolism

CancerMetabolicdisorders

Figure 2. The Functional Interactions between Cell Signaling, Lipid Metabolism, and Epigenetic GeneRegulation and their Links to the Development of Metabolic Disorders and Cancer.

94 Trends in Endocrinology & Metabolism, February 2018, Vol. 29, No. 2

Exploiting Cancer Lipid Metabolism for Diagnosis, Prognosis, and TreatmentExperimental evidence gives reason for hope that characterization of global lipid profiles thatreflect tumor classification and correlate with malignant progression will provide a basis forenhanced cancer diagnosis and improved cancer targeting [33]. For instance, increased lipiddesaturation was recently observed in an actively growing subpopulation of ovarian cancercells with stem cell-like properties. The whole gene set regulating lipid synthesis and maturationis activated in proliferating ovarian CSCs, disclosing a potential vulnerability in an otherwiserecalcitrant progenitor cell population [74–76]. Accordingly, high levels of FASN associate withunfavorable prognosis and poor survival, whereas inhibition of FASN results in cancer cell deathand reduction in tumor volume [77–80], and in resensitization of chemoresistant cancer cells,including proliferating CSCs [81–83]. Similarly, as stated, blockade of the MVA synthesispathway also blocks cancer cell growth and induces apoptosis [84–87]. By contrast, quiescentCSCs have been found to use FA b-oxidation to provide ATP and NADPH for maintenance andsurvival. Accordingly, blockade of b-oxidation resulted in the elimination of this crucial cancercell pool [16,17]. Thus, targeting lipid metabolism may become a useful strategy to eradicateCSCs and to overcome resistance of cancer cells to standard chemotherapeutics [88].

Concluding Remarks and Future Prospects to Overcome Drug ResistanceToday, cancer therapy involves complicated schedules of the simultaneous, sequential, orintermittent application of multiple anticancer drugs to minimize the risk of drug resistance and/or to maximize therapeutic efficacy, which ideally results in synthetic cell lethality. Thesestrategies of combination treatment are motivated by the type of evidence presented here,which elucidates how regulatory pathways, such as proliferative signaling, lipid metabolism,and epigenetic gene regulation, interact with each other to enable drug-stressed cancer cells topersevere. The interdependence can be illustrated by the following setting: blockade ofangiogenic kinase pathways by antiangiogenic drugs induces metabolic rewiring in the neo-plastic cells. Establishing autonomous metabolic loops, such as autophagy and FA synthesis,renders these cells inured to nutrient deficiency and hypoxia. Thus, the targeted impact on suchreactive salvage mechanisms may improve the outcome of antiangiogenic therapies. There-fore, approaches combining angiogenesis inhibitors with antimetabolic drugs are under intensepreclinical and clinical investigation [89]. Moreover, evidence indicates that oncogenic signalingmodulates the epigenetic control of metabolic genes. Nevertheless, a series of burningquestions remains (see Outstanding Questions). Solving these issues will be helpful in under-standing the characteristic perturbations that occur in the cybernetic networks of cells andtissues during cancer development. In the years to come, we expect to see a variety of noveldrugs that specifically interfere with these regulatory systems being applied in combination witheach other and/or with conventional chemotherapeutics for the benefit of patients.

AcknowledgmentsThe author apologizes to colleagues whose important contributions to the field could not be cited because of space

limitations. Research described in the article was funded, in part, by grants from the Medical Scientific Fund of the Mayor of

the City of Vienna, by the ‘Initiative Krebsforschung’ of the Medical University of Vienna, and by the Herzfelder

Familienstiftung, Vienna, Austria.

Resourcesihttps://clinicaltrials.gov/ct2/show/record/NCT02223247?term=TVB-2640&rank=1

Supplemental InformationSupplemental information associated with this article can be found, in the online version, at https://doi.org/10.1016/j.tem.

2017.11.003.

Outstanding QuestionsWhat are the chances to develop anti-metabolic drugs that specifically targetcancer without harming the metabolicand/or energetic condition of unrelatedtissues and/or of the whole organism?

What contribution can metabolomicsand lipidomics make to biomarkerdevelopment in oncology? Is it feasibleto establish reliable and invariantmetabolomic and/or lipidomic profilesof cancer tissues that could serve asbiomarker for cancer classification,treatment, and/or prognosis? Howwould these marker patterns relateto signalomic and epigenomicprofiles?

Heterogeneity within cancer tissuesleads to the coexistence of functionallydistinct cancer cell subpopulations. DoCSCs reveal specific metabolomic, lip-idomic, and/or epigenomic patterns?Is there a typical CSC niche that wouldbe characterized by specific microen-vironmental conditions enabling CSCmaintenance?

Are the processes of FA synthesis andFA oxidation compatible with eachother? Can both pathways activelyproceed simultaneously in a singlecell? Which cancer cell phenotypeswould be sensitive to blockade of FAsynthesis and which would be suscep-tible to inhibition of FA oxidation?

What is the contribution of noncoding(nc)RNA to overall rewiring in cancercells, and what is the contribution ofmutated ncRNA genes to cancer?

Trends in Endocrinology & Metabolism, February 2018, Vol. 29, No. 2 95

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1. Acetyl-CoA is crucial for (choose two): a. Synthesis of nucleotides b. Post-translational modification of histones c. Methylation of CpG islands d. Unfolded protein response e. Lipogenesis

2. The mevalonate pathway (choose two): a. Produces isoprenoids b. Produces steroids c. Produces biogenic amines d. Is induced when ATP levels are getting low

3. Fatty acid synthase is located in (choose one): a. The intermembrane space of mitochondria b. The cytosol c. The karyoplasm d. The Golgi

4. Lipidation of RAS is a mechanism for (choose one): a. Epigenetic silencing of oncogene expression b. Degradation of fatty acids c. Facilitation of signal transduction d. Termination of signal transduction

5. Activation of oncogenic signaling via PI3K and/or MAPK/ERK a. Inhibits b. Induces

expression and/or function of SREBP-1c (choose one)

6. FASN inhibitors downregulate (choose one) a. PIP3 b. PTEN c. HIF-1α

7. ATP-citrate lyase is directly inhibited by AMPK-α (choose one) a. Correct b. False c. Depends on cell type

8. SAM is produced by the enzyme (choose one) a. DNA methyltransferase 1 b. Methionine adenosyltransferase c. Acetyl-CoA synthetase

9. FASN inhibitors cause (choose one) a. Increase of prostaglandins b. Increase of malonyl-CoA c. Increased level of saturated fatty acids d. Resistance against chemotherapeutic drugs

10. Which type of biomolecules is not directly involved in epigenetic regulation of gene expression (choose one)

a. DNA methyltransferases

b. Histone acetyltransferases c. Histone deacetylases d. 3-Hydroxy 3-methylglutaryl-CoA reductase e. Histone methyltransferase EZH2