Chemical genetics in tumor lipogenesis

Accepted Manuscript Chemical genetics in tumor lipogenesis

Simone Braig PII: DOI: Reference:

S0734-9750(18)30023-5 doi:10.1016/j.biotechadv.2018.02.007 JBA 7217

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Biotechnology Advances

Received date: Revised date: Accepted date:

30 October 2017 6 February 2018 11 February 2018

Please cite this article as: Simone Braig , Chemical genetics in tumor lipogenesis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jba(2018), doi:10.1016/j.biotechadv.2018.02.007

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ACCEPTED MANUSCRIPT Chemical genetics in tumor lipogenesis.

Authors: Simone Braig

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Affiliations:

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Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich, Munich, Germany.

Corresponding author: Dr. Simone Braig, Department of Pharmacy, Butenandtstr.

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5-13, 81377 Munich, Germany, Phone: 49-89-2180-77189, Fax: + 49-89-2180-

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77170, Email: [email protected]

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Abstract Since cancer cells depend on de novo lipogenesis for energy supply, highly active membrane biosynthesis and signaling, enhanced fatty acid synthesis is a crucial characteristic of cancer cells. Hence, targeting lipogenic enzymes and signaling

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cascades is a very promising approach in developing innovative therapeutic agents

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for the fight against cancer. This review summarizes main aspects of altered fatty acid synthesis in cancer cells and emphasizes the power of chemical genetic approaches in identifying and analyzing novel anti-cancer drug candidates interfering

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with lipid metabolism.

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Keywords

Lipogenesis, chemical genetics, cancer therapy, fatty acid synthase, acetyl-CoA

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carboxylase, lipid metabolism

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Introduction

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In order to proliferate, replicate and survive in unfavorable environments, cancer cells

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exhibit an extraordinary need of nucleic acids, proteins and lipids. Therefore, rewiring the metabolism is crucial for malignant cells. As stated by Hanahan and Weinberg, deregulated energy metabolism is an emerging hallmark of cancer (Hanahan and Weinberg 2011). One of the most prominent examples of metabolic reprogramming in cancer cells is aerobic glycolysis (also known as Warburg effect), which is characterized as an increased oxygen independent glucose uptake and fermentation to lactate even in presence of intact mitochondria (Warburg et al. 1927). It could be shown in the early 1950s that fatty acid synthesis is dramatically enhanced in cancer

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ACCEPTED MANUSCRIPT tissues compared to their healthy counterparts (Medes et al. 1953). Interestingly, in contrast to normal cells being able to take up fatty acids from the environment, cancer cells synthesize the required lipids and fatty acids on their own and thus, strongly rely on de novo lipogenesis (Swinnen et al. 2006). Regarding the role of fatty acids in a myriad of cellular processes, the feasibility of targeting lipogenesis in

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treatment of cancer became quite evident: By forming building blocks for

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membranes, acting as second messenger and hormones, serving as energy storage and generating lipid rafts, alterations in lipid metabolism impact proliferation, motility, angiogenesis, oxidative stress resistance, cell growth, differentiation and survival

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(Rohrig and Schulze 2016).

Next to altered fatty acid synthesis, also uptake and degradation of lipids is severely

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impaired in cancer cells. For example, a recent study indicates that high cholesterol and HDL (high density lipoprotein) levels in men are associated with high-grade

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prostate cancer (Jamnagerwalla et al. 2017). In addition, inhibiting the uptake of fatty acids by targeting the fatty acid receptor CD36 impairs metastasis in oral cancer

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(Pascual et al. 2017). Since fatty acids are stored as triacylglycerides in lipid droplets,

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blocking the lipolysis and thus their release as free fatty acids imbalances the energy demand and hence proliferation and growth of cancer cells, as shown by the group of

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Teodoro (Zagani et al. 2015). In line with that, cancer cells display a higher number of lipid droplets, which correlates which cancer aggressiveness (Nieva et al. 2012). Importantly, as lipogenesis and lipolysis processes interact with each other to dynamically adjust the high energy demand in cancer cells, targeting lipid metabolism and its associated genes is an encouraging strategy in current cancer research. While there are several excellent reviews referring to cancer lipid metabolism in general (Wenk 2005, Wymann and Schneiter 2008, Santos and Schulze 2012, Rohrig and Schulze 2016, Liu et al. 2017), this review summarizes current 3

ACCEPTED MANUSCRIPT knowledge on altered lipogenesis in cancer, its impact on cancer progression and highlights

the strength of chemical genetics approaches in identifying new

pharmacological addressable targets and potential anticancer drugs in the field of fatty acid synthesis. Thereby, we are focusing on cancer-relevant enzymes and proteins in the lipogenesis pathway which exhibit high potential to serve as lead

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1. Main pathways of lipid biosynthesis

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compounds for antineoplastic intervention.

The main metabolic intermediate providing the substrate for fatty acid synthesis is acetyl-CoA. Under normoxic conditions, pyruvate, the end product of glycolysis is

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converted to acetyl-CoA by pyruvate dehydrogenase (PDH) in the mitochondria

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(Fig.1). Acetyl-CoA feeds the mitochondrial tricarboxylic acid cycle resulting in production of citrate. Citrate is transported to the cytosol, where ATP-citrate lyase

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(ACLY) in turn cleaves citrate into oxaloacetate and acetyl-CoA. The rate limiting step of fatty acid synthesis is the ATP-dependent carboxylation of acetyl-CoA to

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malonyl-CoA by acetyl-CoA carboxylase (ACC). Malonyl-CoA and acetyl-CoA are further processed by fatty acid synthase (FASN) to the 16-carbon saturated fatty acid

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palmitic acid. These cyclic condensation reactions require NADPH as a reducing

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agent. Elongation and desaturation processes by elongases such as ELOVL1-7 (elongation of very long chain fatty acid proteins) and SCD (stearoyl CoA desaturase) take part at the cytosolic part of the ER, forming a wide variety of very long fatty acids of various degrees of saturation, which are used for synthesis of triacylglyerides, cholesterolester, phospholipids, sphingolipids and acylated proteins. Cholesterol, an important component of cellular membranes controlling membrane dynamics and functions, is synthesized from acetyl-CoA via the mevalonate pathway involving the

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ACCEPTED MANUSCRIPT main enzymes, HMG-CoA synthase (3-hydroxy-3-methyl glutaryl CoA synthase) and HMG-CoA reductase. Most enzymes involved in fatty acid synthesis, including ACLY, ACC and FASN are regulated by transcription factors of the helix-loop-helix leucine zipper family, namely the SREBPs (sterol regulatory element binding proteins) (Fig. 2A). Intracellular

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sterols in turn control the activity of SREBPs, allowing binding of SREBP to the

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promotor of its target genes when sterol levels are low and vice versa (Brown and Goldstein 1997). Next to transcriptional regulation and allosteric inhibition, the activity of lipogenic enzymes is influenced by phosphorylation and dephosphorylation at

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various residues. It could be shown that AMPK (AMP-dependent protein kinase) mediated phosphorylation of ACC1, the key enzyme in fatty acid synthesis,

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inactivated the enzyme, whereas dephosphorylation by PP2A (protein phosphatase 2A) results in activation of ACC (Fig. 2B). AMPK itself is activated by AMP and

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inhibited by ATP, thus acting as an energy sensor of the cell resulting in stimulation of AMPK activity and inhibition of fatty acid synthesis when cellular ATP levels are

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low (Hardie and Carling 1997).

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2. Lipogenesis in cancer

Due to the reliance of cancer cells to de novo lipogenesis in order to satisfy the increased need for energy, lipid signaling molecules and membrane building blocks, a plethora of proteins involved in lipid biosynthesis is overexpressed in fast proliferating tumor cells compared to their healthy counterparts, even when high levels of extracellular lipids prevail. In normal cells, lipogenesis is tightly regulated since accumulation of palmitate, the product of de novo fatty acid biosynthesis, induces cellular apoptosis (Hardy et al. 2000). By activation of growth factors, cancer 5

ACCEPTED MANUSCRIPT cells are able to circumvent these cytotoxic effects and benefit from enhanced lipogenesis (Menendez et al. 2004, Baumann et al. 2016). Of note, newly synthesized fatty acids of cancer cells are in most cases saturated, whereas dietderived fatty acids are polyunsaturated. This has strong implications on cell membrane characteristics, modulating their physical and chemical properties.

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Rysman and colleagues demonstrated that the shift towards fatty acid saturation

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renders cancer cells unsusceptible towards chemotherapy and reactive oxygen species (Rysman et al. 2010). Moreover, modulated lipogenesis affects the composition of lipid rafts, which are main platforms for the association of signaling

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molecules and involved in membrane protein trafficking. Thus, signal transduction, intracellular trafficking, migration and invasion is severely impaired in cancer cells

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(Patra 2008, Murai 2012).

ATP-citrate lyase (ACLY) which converts mitochondria-derived citrate to oxaloacetate

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and acetyl-CoA was shown to be upregulated in various malignancies, including lung, prostate, ovarian, breast and colorectal cancer (Zaidi et al. 2012). By inhibiting the

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expression of ACLY, de novo lipogenesis as well as cancer cell growth was inhibited

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both in vitro as well as in vivo (Migita et al. 2008, Khwairakpam et al. 2015). Carboxylation of acetyl-CoA to form malonyl-CoA is catalyzed by acetyl-CoA

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carboxylase (ACC). This enzyme exists in two isoforms, ACC1 and ACC2. Although both isoforms convert acetyl-CoA to malonyl-CoA, knockout of ACC1

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embryonically lethal in mutant mice, whereas ACC2 knockout mice have a normal life span and accumulate less fat compared to controls (Abu-Elheiga et al. 2003, AbuElheiga et al. 2005). Malonyl-CoA produced by cytosolic ACC1 is used by FASN for the generation of fatty acids, while ACC2 is localized to the outer mitochondrial membrane to abrogate fatty acid oxidation via malonyl-CoA mediated inhibition of carnitine palmitoyltransferase 1 (CPT1). Although both isoforms are reported to be 6

ACCEPTED MANUSCRIPT upregulated in several types of cancer, current focus is set on the role of ACC1 in cancer progression (Wang et al. 2015). Silencing of ACC1 in cancer cells leads to induction of apoptosis, impaired migration and reduced proliferation capacity of different cell lines (Brusselmans et al. 2005, Chajes et al. 2006). Acetyl-CoA and malonyl-CoA are further processed by fatty acid synthase (FASN), one of the most

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studied lipogenic enzymes in the field of cancer. Comparable to the role of ACC in

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cancer, overexpression of FASN is involved in proliferation, apoptosis and resistance towards DNA-damaging drugs (Menendez and Lupu 2007, Wu et al. 2014). Regarding the mechanisms underlying overexpression of lipogenic enzymes, studies

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have concentrated on factors promoting gene transcription. Various growth factors, including EGF (Swinnen et al. 2000), HER2 (Kumar-Sinha et al. 2003) and KGF

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(Chang et al. 2005), stimulate gene expression by activation of the transcription factor SREBP via ERK/MAPK, JNK and PI3K pathways (Yang et al. 2002,

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Porstmann et al. 2005). Next to activation by growth factors, SREBP mediated gene expression is induced by steroids such as androgens, progestogens and estrogens

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(Shimano and Sato 2017).

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3. Chemical genetics to identify new drugs and targets in fatty acid synthesis pathways

Since

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the activity of lipogenic enzymes has antiproliferative and

antimetastatic effects on cancer cells, targeting de novo lipogenesis is a highly promising option in cancer treatment and development of small molecule inhibitors to pharmacologically address lipogenesis is crucial to bring this therapeutic strategy from bench to bedside.

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ACCEPTED MANUSCRIPT Chemical genetics describes an emerging field in drug discovery, which utilizes small molecules to study biological systems by modulating the function of distinct proteins in a rapid, time conditional and reversible manner (Spring 2005). Thereby, small molecule libraries are screened for compounds inducing a phenotype of interest (socalled forward chemical genetics or phenotype-based approach), or the function of a

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distinct protein is characterized by targeting them with specific compounds (reverse

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or hypothesis-based approach) (Fig.3). Within the course of phenotype based screening, compounds mediating the desired phenotype are selected and their cellular targets are determined, thus offering the potential not only to discover highly

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effective anticancer agents but also to identify innovative cellular targets being implicated in cancer progression. Although target identification is still a major

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challenge, this approach paves the way to gain advanced knowledge of the functions of unknown and even familiar proteins in a so far unexplored context. By using

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chemical compounds instead of genetic interference methodologies, which are out to eliminate the expression of a given gene, small molecules can perturb the formation

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and disassembly of multifunctional enzyme complexes, influence posttranslational

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modifications or impede protein-protein interactions. The concept of chemical genetics has been applied for centuries especially in the field of natural products.

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Prominent examples of natural products are statins, which were isolated in the 1970’s from Penicillium and Aspergillus fungi (Endo et al. 1976, Alberts et al. 1980). Statins are very potent inhibitors of HMG-CoA reductase, the rate-limiting enzyme in the mevalonate pathway. Their high effectiveness to abrogate cholesterol synthesis led to the development of extremely successful anti-hypercholesterolemia drugs, which nowadays are used around the world. 4. Selected drugs targeting fatty acid synthesis

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ACCEPTED MANUSCRIPT Due to the importance of fatty acid synthesis in cancer progression, numerous chemical compounds inhibiting lipogenesis have been described so far (see Fig. 4 for exemplary chemical structures). FASN, converting malonyl-CoA and acetyl-CoA to palmitate, was the one of the first enzymes of the fatty acid synthesis pathway which was targeted by small molecules. Cerulenin, an antifungal antibiotic, and its synthetic

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analog C75 inhibit FASN activity and thus proliferation of cancer cells in vitro and in

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vivo (Kuhajda 2000). In addition, a pancreatic lipase inhibitor named orlistat, a polyketide-like β-lactone, is approved by the FDA for obesity management. Interestingly, by performing a forward chemical genetics approach to discover novel

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FASN inhibitors, the group of Jeffrey Smith identified orlistat as a quite potent and selective inhibitor of FASN in tumor cells (Kridel et al. 2004). They unraveled that

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orlistat interferes with fatty acid synthesis, abrogates cell proliferation and inhibits the growth of prostate tumors in vivo (Kridel et al. 2004). This study highlights the

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strength of chemical genetics as a valuable approach to identify additional and novel targets for known compounds and drugs.

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In 2016, Harriman and colleagues identified a series of potent allosteric inhibitors of

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ACC (Harriman et al. 2016). They followed a structure-based drug design approach to discover compounds that prevent the dimerization of the ACC subunits and thus

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their enzymatic activity. By virtual screening of chemical structures potentially binding within the dimerization site of ACC and subsequent in vitro verification of their binding mode, ND-022 was determined as lead structure for further chemical modifications of allosteric ACC1/2 inhibitors (Harriman et al. 2016). In their following study, a derivative of ND-022, namely ND-646, was extensively studied in various preclinical models of non-small-cell-lung cancer. Chronic treatment with the ACC inhibitor results in a strong inhibition of fatty acid synthesis in vitro and in vivo and consequently, in vivo lung tumor growth is markedly diminished upon single 9

ACCEPTED MANUSCRIPT administration and in combination with carboplatin (Svensson et al. 2016). Hence, by introducing a ACC inhibitor that exhibits favorable drug-like properties and shows potent anticancer effects, the key enzyme of the fatty acid synthesis pathway could set on stage as a pharmacologically addressable therapeutic target in cancer drug development.

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Soraphen A is a myxobacterial compound, which inhibits the fatty acid synthesis

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pathway also by impeding the activity of ACC. A direct impact of soraphen A on the membrane physiology of cancer cells and the subsequent consequences on membrane-associated key players of cancer-relevant signaling cascades was

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evaluated by Stoiber et al quite recently (Stoiber et al. 2018). Inhibition of fatty acid synthesis by soraphen A results in changed phospholipid composition of cellular

deformation and

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membranes, which affects main tasks of cell mechanics, including membrane rigidity, fluidity (Braig et al. 2015). Interestingly, these modulated

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membrane properties impair the activity of growth-factor related signaling cascades by impeding dimerization, localisation and recycling of transmembrane receptors

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(Stoiber et al. 2017, in press). Hence, main functional aspects of cancer progression,

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namely tumor growth and metastasis are significantly inhibited upon treatment with soraphen A. By using soraphen A as a chemical tool to unravel the molecular

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consequences of impaired lipogenesis in cancer cells, the critical role of membrane characteristics in progression and metastasis of cancer was clarified and ACC inhibitors could be identified as promising candidates for development of new anticancer strategies. Next to targeting enzymes being directly involved in lipogenesis, several studies concentrated on manipulating transcriptional regulators of fatty acid synthesis. As mentioned above, the transcription factor SREBP activates gene expression of ACLY, ACC, FASN and SCD-1, thus pharmacologically targeting this master 10

ACCEPTED MANUSCRIPT regulator of lipogenesis might be an efficient approach to suppress cancer progression. Indeed, Fatostatin, which inhibits the ER-Golgi translocation and thus activation of SREBP, shows antitumoral effects in different cancer cells lines alone and in combination with chemotherapeutics (Kamisuki et al. 2009, Li et al. 2014, Siqingaowa et al. 2017). However, the group of Jorge Torres demonstrated that next

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to targeting SREBP, fatostatin inhibits tubulin polymerization and disturbs mitotic

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microtubule spindle apparatus, which is crucial for cell division (Gholkar et al. 2016). As stated by the authors, inhibiting lipogenesis and cell division is especially beneficial for tumors characterized by an enhanced metabolic activity and rapid

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proliferation, such as glioblastoma cells. Hence, multi-targeting chemical compounds offer the potential to address several cancer relevant pathways in parallel, which

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emphasizes the power of small molecules in treatment of aggressive cancer. Interestingly, current research is out to investigate a potential repurposing of already

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established drugs that target lipid metabolism, like statins or metformin, for anticancer therapy. By inhibiting the HMG-CoA-reductase, statins impede cholesterol

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biosynthesis and are thus used to prevent cardiovascular diseases since decades.

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However, numerous case control studies indicate that statins treatment reduces the risk of cancer development (reviewed in (Ahmadi et al. 2017) and inhibit cancer cell

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proliferation (Kubatka et al. 2014). However, large prospective clinical trials in order to evaluate the potential usage of statins as anti-cancer drugs either as single treatment or in combination with chemotherapeutics (Corcos and Le Jossic-Corcos 2013, May and Glode 2016) are still ongoing. Metformin is an antidiabetic drug which decreases gluconeogenesis in the liver and enhances insulin activated glucose uptake in muscle cells. The anti-tumoral and chemosensitizing activities of metformin have been demonstrated in various preclinical studies (reviewed in (HeckmanStoddard et al. 2017). Next to its AMPK-independent effects, metformin targets 11

ACCEPTED MANUSCRIPT AMPK, which results in inhibition of lipogenesis via abrogating the expression and activity of lipogenic markers, such as ACC and FASN (Loubiere et al. 2015, Ikhlas and Ahmad 2017). Still, since most of the clinical data have been derived from retrospective cohort studies, prospective and randomized controlled trials in nondiabetic patients are needed to substantiate the anticancer potential of this worldwide

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used antidiabetic drug (Wang et al. 2017).

5. Conclusion

Numerous studies throughout the last years have shown that cancer cells reprogram

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their lipid metabolism to enable proliferation und metastasis. Hence, targeting key

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enzymes in fatty acid synthesis is a very promising approach in cancer therapy. However, up to date only one compound (TVB-2640, a FASN inhibitor developed by

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3-V Biosciences) has entered clinical trials. One main drawback in targeting lipogenesis might be the issue of selectively targeting cancer cells without causing

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severe systemic effects. In addition, due to the wide variety of lipid species and the very dynamic routes of synthesis, remodeling and breakdown, understanding and

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targeting lipid metabolism is quite challenging.

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Chemical genetic approaches offer the opportunity to gain in depth knowledge of biological processes and their role in distinct diseases. By using selective and cellpermeable small molecules, the function of enzymes and proteins can be inhibited in a reversible and conditional manner. This enables studying highly dynamic processes such as lipogenesis and further metabolic pathways, which are hardly addressable by classical genetics methodologies like RNA interference. Of note, next to new and unexplored compounds, already known and established small molecules, such as natural products and derivatives thereof, can be investigated in these

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ACCEPTED MANUSCRIPT approaches to learn more on their mode of action in a different context which finally might lead to the development of anti-cancer drugs. In addition to identify and characterize novel targets for anticancer treatment, the discovery of small molecules as potential drug candidates highlights the strength of chemical genetics as a tremendously powerful approach towards new therapeutic

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options in anticancer treatment.

Acknowledgements

The valuable help of Prof. Angelika Vollmar for writing assistance and proof-reading

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of the manuscript is highly acknowledged.

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ACCEPTED MANUSCRIPT FIGURE LEGENDS

Figure 1: Overview of fatty acid biosynthesis. For details see text. ACC: acetylCoA Carboxylase, ACYL: ATP-citrate lyase, ELOV: elongation of very long chain fatty acid proteins, FASN: fatty acid synthase, HMGCR: HMG-CoA reductase,

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HMGCS: HMG-CoA synthase, PDH: pyruvate dehydrogenase, SCD: stearoyl CoA

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desaturase, TCA: tricarboxylic acid cycle.

Figure 2: Regulation of fatty acid synthesis. (A) Upon activation by growth factors

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or sterols, the transcription factor SREBP (sterol regulatory element binding protein) binds to SRE promotor regions and induces the expression of lipogenic genes, such

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as ACC (acetyl-CoA carboxylase), FASN (fatty acid synthase), ACYL (ATP-citrate lyase) and SCD-1 (stearoyl CoA desaturase). (B) Phosphorylation of ACC by AMPK

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(AMP-dependent protein kinase) led to inactivation of ACC and reduced fatty acid synthesis, whereas PP2A (protein phosphatase 2A) mediated dephosphorylation of

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ACC1 activates ACC. AMPK in turn is regulated by ATP and AMP, respectively.

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Figure 3: Chemical genetic approaches. For details see text.

Figure 4: Chemical structures of selected FASN, ACC and SREB inhibitors.

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