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Published in final edited form as: Cell Chem Biol. 2016 Jun 2;23(6):678–688. doi: 10.1016/j.chembiol.2016.04.011

Fasnall, a Selective FASN Inhibitor, Shows Potent Anti-tumor Activity in the MMTV-Neu Model of HER2+ Breast Cancer

Yazan Alwarawrah 1, Philip Hughes 1, David Loiselle 1, David A Carlson 1, David B Darr 2, Jamie L Jordan 2, Jessie Xiong 2, Lucas M Hunter 2, Laura G Dubois 1, J Will Thompson 1, Manjusha M Kulkarni 3, Annette N Ratcliff 3, Jesse J Kwiek 3,4,*, Timothy AJ Haystead 1,4,*
PMCID: PMC6443244  NIHMSID: NIHMS996490  PMID: 27265747

SUMMARY

Many tumors are dependent on de novo fatty acid synthesis to maintain cell growth. Fatty acid synthase (FASN) catalyzes the final synthetic step of this pathway, and its upregulation is correlated with tumor aggressiveness. The consequences and adaptive responses of acute or chronic inhibition of essential enzymes such as FASN are not fully understood. Herein we identify Fasnall, a thiophenopyrimidine selectively targeting FASN through its co-factor binding sites. Global lipidomics studies with Fasnall showed profound changes in cellular lipid profiles, sharply increasing ceramides, diacylglycerols, and unsaturated fatty acids as well as increasing exogenous palmitate uptake that is deviated more into neutral lipid formation rather than phospholipids. We also showed that the increase in ceramide levels contributes to some extent in the mediation of apoptosis. Consistent with this mechanism of action, Fasnall showed potent anti-tumor activity in the MMTV-Neu model of HER2+ breast cancer, particularly when combined with carboplatin.

Graphical Abstract

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In Brief

Many tumors are dependent on de novo fatty acid synthesis. Using a chemoproteomic screen, Alwarawrah et al. identified Fasnall, a thiophenopyrimidine fatty acid synthase inhibitor that displays anti-neoplastic activity against breast cancer in vitro and in vivo.

INTRODUCTION

In humans, de novo fatty acid synthesis is active in a limited number of tissues such as liver, adipose, cycling endometrium, and lactating mammary gland. This contrasts with the other bodily tissues, which largely meet their fatty acid requirements from dietary sources (Brusselmans and Swinnen, 2009) (Iwanaga et al., 2009) (Swinnen et al., 2006). However, some pathological conditions promote cells to become dependent on de novo fatty acid synthesis including solid tumors, leukemic cells, and host cells of certain viruses (Ameer et al., 2014). Fatty acid synthase I (FASN) catalyzes the final steps leading to the synthesis of long-chain fatty acids in vivo. The active form of FASN is composed of a homodimer where each monomer has seven different catalytic domains. These domains include the acyl carrier protein, which is responsible for substrate channeling from one domain to another; the ketoacyl synthetase domain, which catalyzes the condensation step; ketoacyl reductase and enoyl reductase, which both are responsible for saturating the acyl chain; the dehydratase domain, responsible for removing a water molecule from the acyl chain between the two reduction steps; malonylacetyl transferase, which catalyzes the transfer of both malonylcoenzyme A (CoA) and acetyl-CoA; and the thioesterase domain, which clips the palmitate off the enzyme after reaching the desired acyl-chain length (Maier et al., 2008).

In breast cancer, the level of FASN expression is correlated with tumor progression, where high FASN expression leads to more tumor aggressiveness and poor prognostic outcome (Alo et al., 1996). Inhibiting FASN activity in vitro by pharmacological means or its message levels by small interfering RNA (siRNA) has been shown to stop cancer cell growth and induce apoptosis. As a consequence, many research groups have tried to exploit FASN as a target for cancer by developing inhibitors including C75, C93, EGCG (epigallocatechin gallate), G28UCM, orlistat, GSK2194069, and GSK837149A (Hardwicke et al., 2014; Kuhajda et al., 2000; Landis-Piwowar et al., 2007; McFadden et al., 2005; Oliveras et al., 2010; Orita et al., 2007; Puig et al., 2009, 2011; Thupari et al., 2002; Tian, 2006; Turrado et al., 2012; Ueda et al., 2009; Vazquez et al., 2008; Wang and Tian, 2001; Zhou et al., 2007). Despite these efforts, however, the majority of FASN inhibitors have failed to even advance in translational studies largely due selectivity issues in vivo resulting in unexpected toxicities. The only FASN inhibitor advanced to clinical trial for the treatment of advanced solid tumors to date is the FASN inhibitor TVB-2640. This molecule is based on a potent imidazopyridine scaffold and also has anti-hepatitis C virus activity (Oslob et al., 2013).

One of the common themes among current FASN inhibitors is a mechanism of action favoring competition with substrate intermediates over co-factor binding. Even in the case of GSK2194069, despite acting on the β-ketoacyl reductase step, the triazolone is only competitive with trans-1-decalone binding and is uncompetitive with NADPH (Hardwicke et al., 2014). Inhibitors targeting the FASN co-factor domain therefore remain largely unexplored. Targeting of the substrate domains may in part explain the toxicities and lack of efficacy in vivo of the majority of FASN inhibitors, since in order to act competitively the molecules are lipid like in nature. A second concern relates to the broader physiological consequences of selectively inhibiting FASN in vivo, either acutely or chronically. The de novo fatty acid synthesis pathway is highly regulated at several steps and is therefore highly prone to compensatory adaptive responses that would potentially mitigate the efficacy of any selective FASN inhibitor in vivo. Likely compensations could include increased overexpression of FASN itself, increased uptake of exogenous dietary lipids, alteration in expression of enzymes regulating malonyl-CoA levels, such as acetyl-CoA carboxylase or malonyl-CoA decarboxylase, or even switching of the cell to a glycolytic phenotype. In this paper, we have set out to define new scaffolds specifically targeting the largely unexplored sites of purine interaction within FASN. Three of the FASN enzymatic activities (ketoacyl reductase, enoyl reductase, and malonyl/acetyltransferase) use purine-containing co-factors in the form of NADPH, acetyl-CoA, and malonyl-CoA. Importantly, inhibitors targeting purine-utilizing enzymes are generally not lipophilic and have formed the basis of many drugs in clinical use from reverse transcriptase inhibitors to the newer cutting-edge inhibitors targeting protein kinases or heat-shock proteins (Felder et al., 2012; Haystead, 2006; Knapp et al., 2006; Murray and Bussiere, 2009). We now report the identification of a thiophenopyrimidine-based FASN inhibitor (Fasnall) that was discovered with the native enzyme using the chemoproteomic platform FLECS (fluorescence-linked enzyme chemoproteomic strategy; Carlson et al., 2013). Fasnall has potent broad anti-tumor activity against various breast cancer cell lines and in detailed lipidomic analysis produced a profound change in the global cellular lipid profiles remarkably consistent with selective inhibition of FASN. Based on these studies we evaluated Fasnall for safety, its pharmacokinetic properties in normal mice, and efficacy in the mouse mammary tumor virus (MMTV)-Neu mouse model of human epidermal growth factor receptor 2-positive (HER2+) breast cancer. We show that Fasnall is well tolerated, exhibits bioavailability in vivo, and acts a potent inhibitor of HER2+ breast tumor growth.

RESULTS

Identification of Fasnall, a New Scaffold Targeting Fatty Acid Synthase

To specifically identify inhibitors of FASN targeting its nucleotide binding pockets, we utilized Cibacron blue Sepharose. This medium has been used previously to purify NAD and NADP binding proteins from crude tissue extracts (Miyaguchi et al., 2011; Muratsubaki et al., 1994). FASN-enriched extract from lactating pig mammary gland was bound to the resin and labeled with cysteine reactive fluorescein. Having established that labeled FASN could be competitively released from the resin with adenine-containing nucleotides (Figure S2), we subsequently screened the bound enzyme against a single concentration of an in-house small-molecule library comprising compounds with structural similarity to any purine or known purine analog scaffold (Carlson et al., 2013). Of the 3,379 compounds screened, 247 were found to yield a fluorescent signal at 488ex/522em nm (Figure 1). One hundred fifty-five of the molecules selectively eluted FASN from the resin, and 20 potential lead compounds progressed according to their FASN selectivity (assessed with SDS-PAGE, silver staining, and mass spectrometry [MS]). These 20 compounds were reduced to 13 based on the absence of any obvious chemical liabilities. Next, the molecules were tested for their ability to inhibit FASN activity in a HepG2 cell-based assay that measured the incorporation of [3H]glucose into lipids (Figures 1B and S1).

Figure 1. Discovery of the FASN Inhibitor Fasnall.

Figure 1.

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(A) Cibacron blue Sepharose was incubated with porcine lactating mammary gland extracts (a rich source of FASN), washed, and the bound proteins labeled with fluorescein. A small-molecule library of druggable molecules with structural similarity to any purine or known purine analog scaffold was assembled and tested for the ability of each molecule to compete fluorescein-labeled proteins off Cibacron blue resin, proteins from the eluents that had high fluorescence intensity were separated by SDS-PAGE and silver stained, then proteins were identified by MS.

(B) The screen of 3,379 purine-based compounds identified 247 hits with high fluorescent signal. Of the 247 hits, 155 were selected by virtue of both a high FASN intensity and a low number of non-FASN protein bands. The 20 most selective compounds were tested for anti-FASN activity in a [3H]glucose incorporation assay (Figure S1) and the molecule with the highest activity (Fasnall) was selected as a lead molecule.

Of the 13 molecules tested, Fasnall was the most potent inhibitor. In more detailed cell-based assays, Fasnall potently blocked both acetate and glucose incorporation into total lipids, with IC50 values of 147 and 213 nM, respectively, in HepG2 cells (Figure 2A). Subsequently, we confirmed direct inhibition of FASN using the purified human enzyme isolated from the BT474 cell line (IC50 = 3.71 μM, Figure 2B). We also synthesized the enantiomers of Fasnall (HS-79 and HS-80) and a truncated version of Fasnall (HS-102) as a negative control (Figure 2C). The molecules were tested in the acetate incorporation assay in BT474 cells. Both of the Fasnall enantiomers were able to inhibit the incorporation of tritiated acetate (IC50 5.84 μM, 1.57 μM, and 7.13 μM for Fasnall, HS-79, and HS-80, respectively) into lipids. The truncated molecule HS-102 had no effect on acetate incorporation. The molecules were also tested in the blue Sepharose elution assay (Figure S2) and there was no significant difference between them except for HS-102, which had no activity. Finally, as an additional testament to the selectivity of Fasnall, we surveyed data derived from prior screens against our in-house library (Carlson et al., 2013) (Figure 3). None of the previously screened proteins (ACC [acetyl-CoA carboxylase], Hsp90, Hsp70, TRAP-1, DAP kinase 3, IRAK 2, AMPK α and γ subunits, NEK9, dengue nonstructural protein 5, malarial kinase PfPK9, and HSF-1) were targeted by Fasnall. In addition, we tested the ability of Fasnall to eluted proteins of ATP Sepharose loaded with BT474 lysate and found that Fasnall does not elute any proteins over that observed with the vehicle DMSO (Figure S3D), further confirming selectivity.

Figure 2. In Vitro Activity of Fasnall and Its Enantiomers.

Figure 2.

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(A) HS molecules inhibit both tritiated acetate (IC50 147 nM) and glucose (IC50 213 nM) incorporation into lipids.

(B) Fasnall inhibits the human purified FASN activity of [14C]malonyl-CoA incorporation into lipids with an IC50 of 3.71 μM.

(C) Structures of Fasnall enantiomers and the truncated molecule HS-102.

(D) Both Fasnall enantiomers were able to inhibit the incorporation of tritiated acetate (IC50 5.84 μM,1.57 μM, and 7.13 μM for Fasnall, HS-79, and HS-80, respectively) into lipids in BT474 cells while the truncated molecule HS-102 had no significant effect on acetate incorporation into lipids (mean ± SEM).

Figure 3. Selectivity of Fasnall.

Figure 3.

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Individual compounds were assayed for their ability to elute proteins from Cibacron blue resin. Blue-red color spectrum indicates protein concentration as measured by fluorescence (see FLECS Methods). SDS-PAGE and MS analysis showed that Fasnall selectively elutes FASN compared with strong (HS-206160) and weak (HS-202889) hits. Bottom (red graph): compound library was screened for inhibitory activity against the following enzymes: ACC, ZipK, AMPKα, AMPKγ, TRAP1, HSP70, NS5, and IRAK2; Fasnall was a potent inhibitor of FASN (only).

Fasnall Inhibits Proliferation in Breast Cancer Cell Lines

To evaluate the potential of Fasnall in breast cancer, we first tested its effects on proliferation across a panel of non-tumorigenic (MCF10A) and aggressive tumor-forming breast cancer cell lines including estrogen receptor positive (ER+) (MCF7), triple negative (MDA-MB-468), and HER2+ (BT474 and SKBR3). Fasnall inhibited the proliferation of aggressive cell lines with potency similar to that of C75, but showed lower activity in the non-tumorigenic cell line MCF10A (Figures 4A–4E). The weaker effects of Fasnall in MCF10A cells correlated with low expression of FASN in this cell line relative to the more aggressive lines, suggesting that the former cells are less dependent on FASN for growth (Figure 4G). Fasnall treatment of BT474 cells did not induce cell-cycle arrest except for an increase in the Sub 2N cell population (Figure 4F).

Figure 4. Anti-proliferative Activity of Fasnall.

Figure 4.

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(A–E) Based on the DNA content measured by staining with Hoechst, treating various types of breast cancer cell lines with one dose of 50 μM Fasnall (closed circles) was able to inhibit cell proliferation with potency similar to 50 μM C75 (open triangles) except for the non-tumorigenic cell line MCF10A when compared with control (closed squares).

(F) Cell-cycle analysis for BT474 cells treated with different concentrations of Fasnall for 24 hr shows an increase in the Sub 2N population (mean ± SEM).

(G) Treating breast cancer cell lines with 10 μM Fasnall for 24 hr did not have any effect on the expression of FASN.

Fasnall Alters the Global Cellular Lipid Profile of BT474 Cells, Consistent with Selective FASN Inhibition

To determine the effects of Fasnall on the whole-cell lipid profile, we carried out lipidomic analysis by liquid chromatography-tandem MS (LC-MS/MS) following 2 hr of exposure to 10 μM Fasnall in BT474 cells (Figure 5A). Using electrospray ionization (ESI+ and ESI−) profiling, more than 3,000 lipid features can be simultaneously quantified, and our analysis showed that Fasnall induced more than 2-fold change in abundance of 167 specific molecules (p < 0.01 relative to vehicle). Many fatty acids were found to increase over the control, most significantly the polyunsaturated lipids (Figure 5B), suggesting a compensatory effect as a result of their uptake from the media. This was confirmed by a [14C]palmitate uptake assay whereby Fasnall treatment increased 14C labeling of free fatty acids (Figure 5C). Other lipids of particular note that increased many fold with Fasnall are ceramides, which are considered as pro-apoptotic lipids. The increase of ceramides would be expected due to malonyl-CoA (the direct substrate of FASN) accumulation and its effects on CPT-1 inhibition (Bandyopadhyay et al., 2006). As a consequence, any free fatty acids (derived primarily from the extracellular media) are likely to be condensed to 3-keto dihydrosphingosine and on through a series of reduction and acylation steps to various ceramides such as dihydroceramide and ceramide. Diacylglycerols were also found to increase significantly, which can indicate an overall increase in the lipolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) or an increase in de novo synthesis of diacylglycerols. Increase in diacylglycerol accumulation would be expected as a consequence of FASN inhibition, since this would be predicted to promote accumulation of glycerol, a precursor of triglyceride and diacylglycerols. This is because flux of carbons normally supplied by glycolysis for de novo fatty acid is now blocked at the level of FASN itself, causing accumulation of all upstream intermediates (Haystead et al., 1989). We were able to confirm the accumulation of neutral lipids by performing an oil red O stain for lipid droplets under different serum conditions, showing an increase in lipid droplet formation when BT474 are exposed to Fasnall in full serum (Figure 5D).

Figure 5. Fasnall Effects on the Lipidome.

Figure 5.

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BT474 cells were treated with 10 μM Fasnall for 2 hr, then lipids were extracted and subjected to LC-MS. More than 3,000 lipid features were quantified using both ESI+ and ESI− analyses.(A) Each point represents one of the lipid molecules that were quantified, aggregated for ESI+ and ESI−. The color of each dot represents extent of significant difference in the abundance between the control and treatment according to the scale shown on the right.

(B) Of the lipids that were identified, many were diacylglycerols. Ceramides and fatty acids were found to increase over the control (*p < 0.01, #p < 0.05, n = 5) (mean ± SEM).

(C) BT474 cells were treated with different concentrations of Fasnall for 1 hr and lipids were separated by aminopropyl cartridges after incubating the cells with [3H]acetate for 2 hr. Fasnall was able to inhibit the incorporation of acetate into the different types of lipids, especially the more abundant phospholipids. A similar experiment was done with [14C]palmitate, which showed a dose-dependent increase in palmitate sequestering into free fatty acids and reduction in its incorporation into phospholipids. Similar to free fatty acids, neutral lipids increased except for at 50 μM Fasnall, when they decreased (mean ± SEM).

(D) Treatment of BT474 cells with different concentrations of Fasnall under 10% FBS conditions induce the formation of lipid droplets as shown by oil red O staining, indicating an increase in neutral lipids formation when compared with 1% FBS.

Anti-proliferative Activity of Fasnall Is Due to the Induction of Apoptosis

Inhibition of FASN in rapidly proliferating tumorigenic cells would be predicted to have two major effects; first, limiting the oxidative capacity of the mitochondria through increasing malonyl-CoA levels; second, triggering program cell death pathways mostly via accumulation of ceramide. To investigate the latter mechanism, we examined caspase-3 and −7 activation in response to Fasnall and C75 (Figure 6A). Consistent with their tumorigenic capacities, SKBR3 and BT474 cells had 2- to 10-fold, respectively, higher caspase activity than MCF10A cells in response to Fasnall or C75 treatment. The ability of Fasnall to induce apoptosis was also confirmed by detecting the presence of phosphatidylserine and phosphatidylcholine on the outer leaflet of the plasma membrane using fluorescently labeled annexin V and flow cytometry (Figure S5A). To validate the on-target effect of Fasnall, we performed an siRNA knockdown of FASN (Figure S4A) and assessed its effect on Fasnall induction of apoptosis and reduction of viability and proliferation. FASN siRNA pretreatment was able to reduce Fasnall-induced toxicity in BT474 cells, suggesting that Fasnall-induced toxicity is due to FASN inhibition (Figures S4C–S4E). To further confirm that Fasnall induction of apoptosis is directly related to the inhibition of FASN, we tried to rescue the cells by pretreating them with different combinations of palmitate (the end product of FASN) and the ACC inhibitor 5-tetradecyloxy-2-furoic acid (TOFA) to prevent malonyl-CoA accumulation (Figure 6C). However, in our hands TOFA treatment was able to completely reverse the effect of Fasnall only in BT474 cells, which was not due to a general anti-apoptotic activity of TOFA (Figure S5B), while palmitate, or the combination of both palmitate and TOFA, did not fully reverse the effect of the inhibitor. In SKBR3 cells, TOFA, palmitate, and the combination of both was able to partially reverse the effect of Fasnall. We tried further verify whether ceramide accumulation is the main cause of apoptosis by treating the cells with different ceramide synthesis inhibitors targeting various enzymes in both the de novo and salvage ceramide synthesis pathway (Figure S5C). Only myriocin, a serine palmitoyl transferase inhibitor, was able to partially reverse the Fasnall-induced apoptosis (Figure S5D), indicating that ceramide accumulation is not the only cause of apoptosis.

Figure 6. Fasnall Induces Apoptosis in HER2+ Breast Cancer Cells.

Figure 6.

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(A) The indicated cells were treated with different concentrations of Fasnall or C75 for 24 hr, then caspase-3/−7 activity was assayed using the fluorogenic substrate (DEVD)2-r110.

(B) The same experiment was repeated for longer treatment time (48 hr), further showing that Fasnall has more selective induction of apoptosis in the HER2+ cell lines when compared with C75.

(C) Cells were pretreated for 1 hr with 100 μM of a mixture of 1:2 palmitate/oleate in complex with 0.1% BSA (FA) or 15 μM TOFA, or both (TOFA + FA). All the treatments contained the exact amount of BSA and DMSO. Cells were then treated with different concentrations of Fasnall or C75 for 24 hr, and caspase-3/−7 activity was assayed (mean ± SEM).

Toxicity and Pharmacokinetic Studies in Mice

In an acute toxicity study, FVB/J mice received 5, 20, or 80 mg/kg Fasnall via intraperitoneal injection on days 1 and 3, and blood was collected on day 4. Fasnall was toxic at 80 mg/kg, but at 5–20 mg/kg was well tolerated with no adverse effects on white blood cell counts, hemoglobin levels, kidney, or liver functions (Table S2). To test for the long-term effects of Fasnall, we administered biweekly intraperitoneal injections of 5, 10, or 15 mg/kg Fasnall to mice for 8 weeks. None of these doses induced any signs of toxicity, stress, or significant change in mouse weight (Figure 7A). Next, we carried out pharmacokinetic studies to determine the uptake and biodistribution of Fasnall in MMTV-Neu mice by LC-MS (Figure S6). These studies showed that Fasnall appears rapidly in the plasma within 5 min of the injection and is cleared rapidly (T1/2 = 9.81 ± 0.02 min, n = 3). Similar uptake and clearance was also observed in liver and kidney (liver T1/2 = 9.84 ± 0.09 min, n = 3; kidney T1/2 = 9.90 ± 0.01 min, n = 3). Although our MS analysis focused primarily on the parent compound (amu 339Da), preliminary examination of the entire LC profile following drug extraction of the tissues did not reveal any obvious Fasnall metabolites (data not shown). These findings suggest that Fasnall is rapidly cleared through the kidney and liver in its parent ion state.

Figure 7. Fasnall Activity in MMTV-Neu Mice.

Figure 7.

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(A) Fasnall does not induce weight change. Mice were assessed weekly, treated twice weekly with an intraperitoneal injection of different concentrations of Fasnall made in DMSO/saline (1:1) (mean ± SEM).

(B and C) Combination of Fasnall and carboplatin (Carbo) significantly reduces tumor volume (t test, p = 0.014).

(D) Fasnall increases the median survival of mice from 29 to 63 days, with a log-rank p value of 0.049.

biw, twice weekly; qw, weekly.

MMTV-Neu Mouse Survival Increases upon Treatment with Fasnall

Having determined that Fasnall was well tolerated in mice, we next tested its efficacy on tumor progression in the MMTV-Neu model of HER2+ breast cancer (Muller et al., 1988) (Jackson Laboratory strain 002376). Cohorts of MMTV-Neu mice were treated with a biweekly intraperitoneal injection of 15 mg/kg Fasnall (Figures 7B and 7C). When given alone, Fasnall reduced tumor volume compared with vehicle-treated animals (day 21, Fasnall treatment volume 436 ± 218 [SD of mean] mm, n = 7, control volume 628 ± 381 mm, n = 9; p = 0.85). Significantly, Fasnall also increased the median survival of the MMTV-Neu mice to 63 days (p = 0.049) compared with animals treated with vehicle alone (Figure 7D). Importantly, MS analysis of tumor tissue verified Fasnall uptake and also showed a significantly longer elimination time (T1/2 = 65.71 ± 0.32 min, n = 3) than all other tissues tested. The long duration of treatment in our studies suggest that the dosing frequency of Fasnall can be greatly increased to achieve greater effects on survival and tumor volume. These findings are consistent with effects of Fasnall as an anti-proliferative agent in tumors.

More dramatic acute tumor responses were observed when Fasnall was combined with 50 mg/kg of the platinum-based chemotherapeutic agent carboplatin administered weekly. Here, 88% of tumors achieved an objective response rate of stable disease or better compared with carboplatin only at 25% (Fisher’s exact test, p = 0.01). This response was not durable, however, as there was no long-term benefit of the combination therapy at this dosing regimen (Figure 7D). As often seen in the clinic, tumors that are responsive initially will develop resistance, which is likely the case here. These findings are consistent with the actions of two compounds acting independently of one another whereby one anti-neoplastic develops resistance while the other maybe unaffected. Importantly, carboplatin is a front-line chemotherapeutic agent for the treatment of breast cancer. Similar in action to cisplatin (Knox et al., 1986), carboplatin stops tumor progression by binding to DNA and inducing a DNA-damage response that leads to a halt in proliferation and activation of apoptosis (Chu, 1994). Although carboplatin is less toxic than cisplatin (Harland et al., 1984), toxicity is still a major issue where determination of the drug dose is based on the target area under the curve and the evaluated drug clearance (Etienne et al., 2003), and in most cases is administered once every 4 weeks (Martin et al., 1992). Due to the efficacy of Fasnall when combined with carboplatin in the MMTV-Neu model, we tested a similar combination in the C3Tag mouse model of triple-negative breast cancer (TNBC). Unlike HER2+ or ER+ breast cancers, there are no molecularly targeted drugs for TNBC and platinum-based compounds are the most commonly used chemotherapeutics for treatment. The combination of Fasnall and carboplatin was able to significantly reduce tumor volume in the C3Tag model (Figure S7). These data, combined with our studies in the Neu model, indicate a strong well-tolerated synergism between a fatty acid synthase inhibitor and a front-line chemotherapeutic agent that is extensively used for breast cancer therapy.

DISCUSSION

Here we report the discovery of a thiophenopyrimidine, Fasnall, that selectively targets FASN both in cells and in animal tumor models of human breast cancer. Fasnall can be readily synthesized in one efficient step from the chloropyrimidine and amine starting materials. The synthetic route to Fasnall is readily adaptable for the preparation of analogs that are similar in structure, suggesting that the pharmacological properties of Fasnall can be improved. In liver and breast tumor cells, Fasnall was able to inhibit both acetate and glucose incorporation into lipids. Also, Fasnall showed anti-proliferative activity in triple-negative, ER+, and HER2+ breast tumor cell lines. In contrast, anti-proliferative activity of Fasnall was lower in the non-tumorigenic cell line MCF10A, which has lower dependence on FASN activity (Yang et al., 2002). As we show that the anti-proliferative activity is due to the induction of apoptosis, we tested whether lowering FASN expression in BT474 would reduce the efficacy of Fasnall in a way similar to what we see in the MCF10A cells. FASN siRNA treatment was able to significantly reduce Fasnall-induced toxicity but due to the inability to achieve 100% transfection efficacy, the siRNA was not able to completely block Fasnall toxicity because of the residual FASN expression.

Blocking malonyl-CoA accumulation by inhibiting ACC activity was able to reverse Fasnall-induced apoptosis. This indicates that Fasnall-induced apoptosis is due to malonyl-CoA accumulation, which is known to inhibit CPT-1 activity (McGarry et al., 1983), which connects FASN inhibition to apoptosis through the increase in ceramide concentration (Bandyopadhyay et al., 2006). The accumulation of ceramides was confirmed by our lipidomics analysis, which may indicate, in addition to the inhibition of CPT-1, induction of sphingomyelinase activity. To investigate which pathway is responsible for the increase in ceramide concentration, we used different inhibitors of ceramide accumulation to block Fasnall-induced apoptosis. The inability of the ceramide salvage pathway inhibitors to rescue the Fasnall-induced apoptosis indicates that ceramide accumulation is unlikely to be due to an increase in sphingomyelinase activity. In addition, the partial rescue by myriocin suggests that there is another signal that contributes to apoptosis downstream of the blockage of fatty acid oxidation.

Fasnall treatment was also found to induce an increase in diacylglycerols and unsaturated fatty acid abundance. There are two main pathways by which diacylglycerols can be formed; by de novo synthesis from glycerol and fatty acids, which increases when there are large quantities of these precursors; and from the lipolysis of PIP2. Based on our findings with Fasnall, the former pathway would be favored since inhibition of the fatty acid synthesis pathway in general leads to the glucose being diverted to the synthesis of glycerol (Haystead et al., 1989). When combined with the uptake of fatty acids from the media and the inhibition of CPT-1, these conditions will favor an increase in diacylglycerol abundance. The palmitate uptake experiment and the lipid droplet staining at different serum concentrations confirms the previous finding by showing the partitioning of palmi-tate into neutral lipids instead of phospholipids. This explains the inability of palmitate to completely rescue Fasnall-induced apoptosis. Collectively, these data therefore reveal why FASN inhibition would induce anti-proliferative activity in vivo despite the provision of fatty acids from the circulation.

Combining the accumulation of ceramides and neutral lipids with the inhibition of phospholipid formation by blocking de novo fatty acid synthesis could induce changes in the plasma membrane composition. The uptake of exogenous unsaturated fatty acids while de novo fatty acid synthesis is inhibited would limit the cells’ ability to produce phospholipids with saturated acyl chains, which are important for membrane functions (Rysman et al., 2010), while ceramides are known to displace cholesterol from lipid rafts, which may affect lipid raft structure and function (Megha and London, 2004). These potential changes in lipid raft structures are noteworthy due to their effect on the localization of growth factor receptors to the cell surface, especially HER2 in the case of BT474 cells.

In contrast to most FASN inhibitors, Fasnall is well tolerated in mice and does not induce any overt weight loss or change in feeding behavior. Even on a conservative twice-weekly dosing regimen, Fasnall reduced tumor size in both the MMTV-Neu and C3Tag models, and had a profound effect on median survival. Moreover, combining Fasnall with carboplatin synergistically reduced tumor volumes and affected survival over the first 40 days of combination treatment. Although overall survival was not extended beyond that with Fasnall alone, the dramatic early response to the combination has clinical relevance. Normally, carboplatin treatment is restricted to 21 days in patients due to its toxicity and tendency to develop resistant tumors when used over the longer term. Our pharmacokinetic study shows that there is room to increase Fasnall dosing to improve its performance in vivo. The compound is rapidly cleared from plasma and tissues, indicating that it is possible to increase the dosing schedule from twice weekly to at least a daily regimen. Fasnall may therefore enable significant reduction of the carboplatin dose, which may increase the combined drugs’ efficacy while reducing the toxicity of the latter compound. In addition, our findings suggest that the Fasnall scaffold can be developed further to optimize its pharmacological properties as an inhibitor of FASN in vivo.

SIGNIFICANCE

FASN is considered an attractive target for cancer therapy due to the selective dependence of many tumors on de novo fatty acid synthesis. Many trials for the development of FASN inhibitors had previously failed to advance to translational studies, and only one FASN inhibitor was able to progress forward in clinical trials. Herein, we present a novel and selective FASN inhibitor (Fasnall) that has an in vivo anti-neoplastic activity in the clinically relevant HER2+ breast cancer mouse model MMTV-Neu. Fasnall shows the ability to selectively inhibit proliferation and induce apoptosis in HER2+ breast cancer cell lines that is coupled with profound changes in the lipidome. The anti-neoplastic activity of Fasnall was due to the induction of apoptosis resulting from CPT-1 inhibition, ceramide accumulation, and changes in lipids raft composition, all of which can be rescued by ACC inhibition rather than exogenous palmitate supplementation, which is diverted into neutral lipids instead of phospholipids. In addition to its in vitro and in vivo anti-neoplastic activity, Fasnall is chemically tractable and can be modified for further optimization.

EXPERIMENTAL PROCEDURES

ATP, NAD, NADPH, acetyl-CoA, malonyl-CoA, propidium iodide, Hoechst 33258, RNase A, and Cibacron blue Sepharose were obtained from Sigma-Aldrich. [3H]Acetate, 3-D-[3H]glucose, and 2-[14C]malonyl-CoA were purchased from PerkinElmer. Fluorescein-5-maleimide, Alexa Fluor 488, annexin V, and Sytox red were purchased from Invitrogen. Humulin R insulin was purchased from Lilly. Cells were obtained from the American Type Culture Collection.

FLECS Screen

Porcine mammary glands were collected from lactating pigs as previously described (Hughes et al., 2012). Tissues were homogenized in lysis buffer A (100 mM sodium fluoride, 5 mM EDTA, 1 mM DTT, and 5% glycerol made in 10 mM sodium phosphate buffer [pH 7.5]) in a ratio of 3 ml of buffer per each gram of tissue. After removing cell debris by centrifugation at 142,000 × g for 45 min and filtering through glass wool, the homogenate was applied to Cibacron blue Sepharose pre-equilibrated with buffer B (100 mM sodium fluoride, 5 mM EDTA, 1 mM DTT, and 50 mM sodium citrate made in 10 mM sodium phosphate buffer [pH 7.5]) in a ratio of 4.5 g of tissue to each 1 ml of settled resin. For removal of dehydrogenases and reduction of the amount of ATP binding proteins bound to resin, the resin was washed with ten bed volumes of buffer B and then one bed volume of 5 mM NAD made in buffer B followed by one bed volume of buffer B. Thereafter, the resin was washed with one bed volume of 10 mM ATP. For labeling of FASN attached to the resin, to each 1 ml of resin, 1 ml of 10 mM sodium phosphate buffer (pH 7.5) containing 50 μg of fluorescein-5-maleimide (pre-dissolved in DMF) was added to the resin and incubated overnight at 4°C with slow rotation. The resin was then washed with 20 bed volumes of buffer B to remove any excess fluorescein. The resin was suspended in buffer B (1:1, v/v) and distributed in 96-well filter plates (50 μl/well). Fluorescein-labeled proteins were eluted from the resin by an in-house library of 3,379 purine-based compounds. For each well, 50 μl of each compound was added (1 mM made in buffer B with 10% DMSO). Different concentrations of ATP were used as a control. The eluents were collected in 96-well black plates by centrifugation at 1,260 × g for 5 min. Fluorescence in each well was measured at excitation/emission 485/535 nm. Eluents with the highest fluorescent intensity were run on SDS-PAGE. After silver staining, proteins in each band were identified by MALDITOF/TOF MS.

[3H]Glucose and [3H]Acetate Incorporation into Lipids

Incorporation of radiolabeled glucose or acetate into total lipids was measured according to published methods (Haystead and Hardie, 1986). HepG2 cells (80% confluent in 12-well plates) were starved overnight in MEM-α. The medium was then changed with DMEM medium containing 0.1 g/l glucose, 10% fetal bovine serum (FBS), 5 μM insulin, and 1 μCi of D-[3H]glucose or 1 μCi of [3H]acetate in addition to different concentrations of each compound. After incubation for 1 hr at 37°C and 5% CO2, the cells were washed with ice-cold PBS and detached by treating with 100 μl of trypsin-EDTA for 10 min, followed by adding 1 ml of ice-cold PBS. From each well, 1 ml of cell lysate was added to a 4-ml scintillation vial, and 2 ml of toluene containing 25 g/l butyl PBD was added. The vials were mixed thoroughly and centrifuged for 30 min at 1,600 × g, and the 3H radioactive counts were measured by liquid scintillation counting.

FASN Activity Assay

Human FASN activity was measured by monitoring the incorporation of 2-[14C]malonyl-CoA into fatty acids using liquid scintillation counting by a method similar to the one described by Richardson and Smith (2007). FASN (10 μg/ml in PBS containing 1 mM DTT and 1 mM EDTA) was pre-incubated with different concentrations of Fasnall (final DMSO concentration 1%) at 37°C for 30 min, then substrates were added (20 μM acetyl-CoA and 200 μM NADPH) in a total reaction volume of 100 μl. The reaction was initiated by adding 10 μl of 50 μM malonyl-CoA spiked with 0.05 μCi of 2-[14C]malonyl-CoA. After incubation for 30 min at 37°C, lipids were extracted three times with 150 μl of chloroform/methanol (2:1, v/v). Then, to the pooled organic phases 1 ml of toluene containing 25 g/l butyl PBD was added and radioactivity was measured by liquid scintillation counting.

Proliferation Assay

MCF10A (5,000 cells/well), MCF7 (7,500 cells/well), MDA-MB-468 (5,000 cells/well), BT474 (7,500 cells/well), and SKBR3 (5,000 cells/well) were seeded in 96-well plates with 10% FBS, 4 g/l glucose DMEM medium except for MCF10A which was in DMEM/F12 medium. After 24 hr, cells were treated with different concentrations of Fasnall or C75. Every 24 hr for 5 days, medium from one of the plates was removed and the plate was frozen to −80°C. After collecting all the time points, to each well 100 μl of ddH2O was add and the plates were frozen again. Then 100 μl of Hoechst 33258 solution made in TNE buffer (1 μl from Hoechst stock [1 mg/ml in 1:4 DMSO/H2O] in 1 ml of TNE buffer [which contains 10 mM Tris, 2 M NaCl, and 1 mM Na2EDTA]) was added to each well and fluorescence was measured at excitation/emission 355/460 nm.

Cell-Cycle Analysis

After treating BT474 cells with different concentrations of Fasnall for 24 hr, cells were collected and fixed with 70% ethanol, washed with PBS, then treated with 20 mg/ml RNase A. The cells were then stained with 50 μg/ml propidium iodide and DNA content for each cell was quantified using a BD Accuri C6 flow cytometer (Becton Dickinson), and the data were analyzed using CFlow Plus software (BD).

Western Blot Analysis

Cell lysate from cells treated for 24 hr with 10 mM Fasnall or DMSO were loaded (28 μg/well) and run on Criterion XT Tris-HCl gel (4%–15% gradient) (Bio-Rad) according to manufacturer’s instructions, and the proteins were transferred to a polyvinylidene fluoride membrane overnight using 25 V at 4°C. Thereafter, membranes were blocked and blotted for FASN (Cell Signaling Technology, antibody number 3180) and GAPDH (Cell Signaling, antibody number 5174).

Caspase-3/−7 Activity Assay

The assay was performed using a similar protocol to the one described by Fritz et al. (2001). Cells were seeded at a density of 10,000 cells/well and treated with different concentrations of Fasnall or C75. After 24 hr, to each well 50 μl of caspase assay/lysis buffer (50 mM HEPES [pH 7.5], 100 mM KCl, 5 mM EDTA, 10 mM MgCl2, 10 mM CHAPS, 20% sucrose, 10 mM DTT, 10 μM (Z-DEVD)2-Rh110 [Santa Cruz Biotechnology], and complete protease inhibitor [Roche]) was added. After 6 hr of incubation at 37°C, fluorescence was measured at excitation/emission 485/535 nm.

Lipidomics Sample Preparation

BT474 cells were seeded in 15-cm dishes at density of 200,000 cells/ml in DMEM and 10% FBS. After overnight incubation, the cells were treated with 10 mM Fasnall for 2 hr, washed with ice-cold PBS, and flash-frozen. Cell pellets (five vehicles and five treated with 10 μM Fasnall) were separately thawed on ice, and 100 μl of ammonium bicarbonate (pH 8) was added to each. Pellets were then probe sonicated at power level 3 for three bursts of 5 s each burst, cooling on ice between bursts. A Bradford assay was performed on each solubilized pellet using 10× diluted material. 1 mg from each was taken out and normalized to 137 μl in total with AmBic in a 96-well plate. To each sample well, 200 μl of methanol was added followed by the addition of 600 μl of methyl tert-butyl ether (MTBE). The plate was capped and mixed at 800 rpm at room temperature for 1 hr. The plate was then centrifuged at 2,000 rpm at room temperature for 10 min, and 400 μl of the MTBE/MeOH layer was pipetted out and transferred to another plate. The extract was dried under nitrogen gas and samples were reconstituted in 100 μl of isopropanol/acetonitrile/H2O (2:1:1). A pool was made by taking an equal volume from all ten samples.

Mass Spectrometry Lipid Profiling

Each sample was analyzed twice using ultra performance liquid chromatography (UPLC)/ESI/MS/MS in positive ion mode (3 μl) and negative ion mode (10 μl). UPLC separation was performed using a binary gradient separation on an Acquity UPLC (Waters) using an Acquity 2.1 × 10-mm, 1.7-μm CSH C18 column. Mobile phase A contained 60:40:0.1 (v/v/v) MeCN/water/formic acid with 10 mM ammonium formate, and mobile phase B contained 90:10:0.1 (v/v/v) isopropanol/MeCN/formic acid. Lipid separation was performed at 0.6 ml/min and 60°C column temperature, using a complex gradient program as follows: initial conditions 40% B, ramp to 43% B at 1.3 min, ramp to 50% B at 1.4 min, ramp to 54% B at 8 min, ramp to 70% B at 8.2 min, ramp to 99% B at 12.2 min, ramp to initial condition 40% B at 12.3 min, then hold at 40% B for re-equilibration until 14 min. Via ESI, the LC eluent was introduced into a G2 Synapt (Waters), and data collected between 50 and 1,200 m/z in 0.3 s; MS/MS was collected at a scan rate of 0.2 s for peaks above a threshold of 3,000 intensity/s for positive ion and 1,000 intensity/s for negative ion. Source parameters are as follows: capillary at 2.7 kV/2.3 kV for positive/negative ion, respectively; cone voltage of 30 V; 500°C desolvation temperature; 700 l/hr desolvation gas; 150 l/hr cone gas; and source temperature of 100°C. Lockmass calibration was performed every 30 s using a solution of 500 fmol/μl leucine-enkephalin in positive (556.2771 m/z) or negative mode (554.2615 m/z).

Quantitative data were analyzed in Progenesis QI (Nonlinear Dynamics/Waters). Quantitative data including accurate mass, charge state, retention time, and intensity were exported for additional statistical analysis and have been made available at https://discovery.genome.duke.edu/express/resources/3745/3745_IDandStats_HvsD_ProgenesisQI_062514.xlsx. Putative identifications were made by searching against compiled LipidMaps databases with theoretical fragmentation where available, using 10 ppm precursor and fragment ion tolerance. Putative identifications were confirmed based on accurate mass and retention time using standards for fatty acids, ceramides, and diacylglycerols where standards were available. Data for the fatty acid standards, ceramides, and diacylglycerols are provided in Table S1. The Skyline software package (https://skyline.gs.washington.edu/) was additionally utilized to verify accurate mass, isotope distribution, and quantitative measurements performed by Progenesis QI. Skyline documents for fatty acid verification may be downloaded at https://discovery.genome.duke.edu/express/resources/3745/Skyline_FattyAcid_Verification.sky.zip. Skyline document for diacylglycerol verification may be downloaded at https://discovery.genome.duke.edu/express/resources/3745/Skyline_DAG_verification.sky.zip. The Skyline document for ceramide verification may be downloaded at https://discovery.genome.duke.edu/express/resources/3745/Skyline_Cer_verification.sky.zip.

Determination of Acetate and Palmitate Incorporation into the Main Lipid Classes

BT474 cells were seeded in six-well plates in 10% FBS and 4.5 g/l glucose DMEM at a density of 400,000 cells/well. After 24 hr the medium was changed with 0.1 g/l glucose DMEM containing different concentrations of Fasnall. After 1 hr, to each well, 10 μCi of [3H]acetate or 0. 5μCi of [14C]palmitate (in complex with BSA) was added and incubated for 1 hr. Cells were then treated with 500 μl of trypsin per well for 5 min and subsequently 500 μl of ice-cold PBS was added to each well. Lipids were separated as previously described (Kaluzny et al., 1985). In brief, lipids were extracted three times with 700 μl of chloroform and injected into Sep-PaK aminopropyl cartridges containing 360 mg of resin (Waters) pre-equilibrated with 10 ml of chloroform. The cartridges were then injected with 5 ml of chloroform/isopropanol (2:1), 2% acetic acid in ether and methanol to elute neutral lipids, free fatty acids, and phospholipids, respectively. To each fraction, 1 ml of 25 g/l butyl PBD dissolved in toluene was added and the radioactivity measured by scintillation counting.

Lipid Droplet Staining

BT474 cells were treated with different concentrations of Fasnall under two serum conditions (1% and 10%). After 72 hr, the cells were washed with PBS and fixed for 1 hr in 10% formalin, then incubated in 60% isopropanol for 5 min after being washed with water. Subsequently, the cells were incubated for 5 min in oil red O solution (0.3% oil red O in isopropanol diluted 3:2 in water), then washed and stained with hematoxylin.

Determination of Fasnall Efficacy In Vivo

Single-time parous female MMTV-NEU mice (Jackson Laboratory Strain 002376) were used to test the efficacy of Fasnall (15 mg/kg, intraperitoneally, twice weekly) alone and in combination with carboplatin (50 mg/kg, intraperitoneally, once weekly). Mice were monitored for tumor development by palpating them weekly as per UNC Lineberger Mouse Phase 1 Unit protocol. Once tumors were observed, the mice were placed on treatment. The tumor-bearing mice were injected weekly with Fasnall and/or carboplatin. The solvent for Fasnall consists of 50% DMSO and 50% saline (0.9% sodium chloride solution). Clinical-grade carboplatin was purchased from the UNC Hospital pharmacy. Tumor volume was measured at the time of injection by calipers, and width (short diameter) and length (long diameter) in millimeters were recorded. The volume was calculated using the formula length × width2 × 0.5. At the time of injection, body composition was assessed and weight measurements (in grams) were recorded and used to determine toxicity. After 3 weeks, tumor progression was calculated using the formula (21-day volume minus initial volume)/initial volume × 100. This percent change in tumor volume, at 21 days, was used to assess the objective response rate of the therapies. Mice were treated and monitored until euthanized according to predetermined humane endpoints per UNC IACUC protocol 13–190. Overall survival was calculated by time from initial treatment date to date of necropsy. The same protocol was used for the assessment of Fasnall and Fasnall-carboplatin combo in the TNBC C3Tag genetically engineered mouse model approved by UNC-CH IACUC.

Supplementary Material

1
S1
S2

Highlights.

  • Fasnall is a thiophenopyrimidine targeting fatty acid synthase

  • Fasnall has anti-proliferative activity and induces apoptosis in breast cancer cells

  • Fasnall promotes fatty acid uptake, ceramide and diacylglycerol accumulation

  • Fasnall is well tolerated and shows efficacy against HER2+ breast tumors in vivo

ACKNOWLEDGMENTS

This work was funded by NIH grants 1R01-AI089526–04 and 1R01AI090644–04 to J.J.K. and T.A.J.H. A.N.R. was supported by a Pelotonia Fellowship. There is a patent-pending application by Y.A., P.F.H., J.J.K., and T.A.J.H. for several of the small-molecule inhibitors listed in this work. Fasnall is available upon request from T.A.J.H. We would like to acknowledge Dr. Donald P. McDonnell and Dr. Rachid Safi (Duke University, Department of Pharmacology and Cancer Biology) for allowing us to use the flow machine from their laboratory and sharing their protocols and reagents. We also thank Dr. Lauretta A. Rund (University of Illinois, Department of Animal Sciences) for providing the pig mammary gland tissues.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures, seven figures, and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.chembiol.2016.04.011.

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Associated Data

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Supplementary Materials

1
NIHMS996490-supplement-1.pdf (1.8MB, pdf)
S1
NIHMS996490-supplement-S1.pdf (48.7KB, pdf)
S2
NIHMS996490-supplement-S2.pdf (86.3KB, pdf)

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