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. Author manuscript; available in PMC: 2016 Aug 31.
Published in final edited form as: Cell Rep. 2016 Jul 28;16(6):1614–1628. doi: 10.1016/j.celrep.2016.07.009

Fatty Acid Oxidation Mediated by Acyl-CoA Synthetase Long Chain 3 Is Required for Mutant KRAS Lung Tumorigenesis

Mahesh S Padanad 1,2,12, Georgia Konstantinidou 1,2,12,13, Niranjan Venkateswaran 1,2, Margherita Melegari 2, Smita Rindhe 1,2, Matthew Mitsche 3,4, Chendong Yang 5, Kimberly Batten 6, Kenneth E Huffman 7, Jingwen Liu 8, Ximing Tang 9, Jaime Rodriguez-Canales 9, Neda Kalhor 9, Jerry W Shay 6, John D Minna 1,7, Jeffrey McDonald 4, Ignacio I Wistuba 10,11, Ralph J DeBerardinis 3,5, Pier Paolo Scaglioni 1,2,*
PMCID: PMC4981512  NIHMSID: NIHMS806820  PMID: 27477280

SUMMARY

KRAS is one of the most commonly mutated oncogenes in human cancer. Mutant KRAS aberrantly regulates metabolic networks. However, the contribution of cellular metabolism to mutant KRAS tumorigenesis is not completely understood. We report that mutant KRAS regulates intracellular fatty acid metabolism through Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrates for lipid synthesis and β-oxidation. ACSL3 suppression is associated with depletion of cellular ATP and causes the death of lung cancer cells. Furthermore, mutant KRAS promotes the cellular uptake, retention, accumulation, and β-oxidation of fatty acids in lung cancer cells in an ACSL3-dependent manner. Finally, ACSL3 is essential for mutant KRAS lung cancer tumorigenesis in vivo and is highly expressed in human lung cancer. Our data demonstrate that mutant KRAS reprograms lipid homeostasis, establishing a metabolic requirement that could be exploited for therapeutic gain.

Graphical abstract

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INTRODUCTION

Lung cancer remains one of the leading causes of cancer-related death. Activating mutations of the proto-oncogene KRAS (mutant KRAS) occur in ~30% of the cases of non-small cell lung cancer (NSCLC) (Cancer Genome Atlas Research Network, 2014; Pylayeva-Gupta et al., 2011). Continuous expression of mutant KRAS is required for the survival of NSCLC both in mouse cancer models and human-derived NSCLC cells (Fisher et al., 2001; Singh et al., 2009). Mutant KRAS promotes tumorigenesis by regulating several oncogenic networks, for instance, the RAF/MEK/ERK, PI3K/AKT/mTOR, and RHOA-focal adhesion kinase. These observations establish mutant KRAS as a therapeutic target. However, there are currently no approved therapies that target tumors that harbor mutant KRAS (Gysin et al., 2011; Konstantinidou et al., 2013; Pylayeva-Gupta et al., 2011).

Cancer cells undergo oncogene-directed metabolic reprogramming to support cell growth and survival. For instance, cancer cells harboring mutant KRAS display a high level of carbon flux through aerobic glycolysis and activation of glucose-dependent biosynthetic pathways, including the synthesis of hexosamines and nucleotides (Boroughs and DeBerardinis, 2015; Hu et al., 2012; Ying et al., 2012). Therefore, mutant KRAS drives both the acquisition of nutrients and the orchestration of cellular metabolism to convert carbon sources into biomass. However, the relevance of metabolic reprogramming in tumorigenesis is not completely understood.

The metabolism of fatty acids (FAs) is emerging as a mechanism to cope with oncogenic stress. For instance, mutant KRAS stimulates the cellular uptake of lysophospholipids, and cancer cells with deregulated mTORC1 are dependent on unsaturated FAs in hypoxic conditions (Kamphorst et al., 2013; Young et al., 2013). Autophagy is also emerging as a mechanism to maintain functional mitochondria, which are important for lipid metabolism (Guo et al., 2013). However, the mechanistic details of the regulation and the biological significance of the cellular metabolism of FAs in cancer cells are not completely understood.

Fatty acids are fundamental cellular components that may be used as building blocks for cellular membranes, as moieties for post-translational protein modification, and as substrates for energy generation through β-oxidation. De novo FA synthesis involves several key enzymes: ATP citrate lyase (ACL) generates acetyl-coenzyme A (CoA) from citrate, which is typically produced in the mitochondrial tricarboxylic acid cycle (TCA) cycle; acetyl-CoA carboxylase (ACC) catalyzes the irreversible carboxylation of acetyl-CoA to form malonyl-CoA, the committed metabolite in FA synthesis; and fatty acid synthase (FASN) then sequentially adds 2-carbon units until a long-chain FA is produced.

In most tissues, FASN expression is low; thus, most non-transformed cells preferentially use dietary (exogenous) lipids for energy generation and membrane maintenance (Menendez and Lupu, 2007). However, proliferating cells avidly take up free FAs from the environment and use them to generate phospholipids, which constitute a substantial fraction of the dry weight of mammalian cells (Deberardinis et al., 2006; Spector, 1967). Furthermore, overexpression of FASN occurs in several human cancers, suggesting that some cancer cells and tumors endogenously synthesize FAs (Furuta et al., 2008; Menendez and Lupu, 2007).

Acyl-CoA synthetases (ACSLs) are a family of enzymes (i.e., ACSL1, 3, 4, 5, and 6) that convert free long-chain FAs into fatty acyl-CoA esters, which then serve as a substrate for both lipid synthesis and β-oxidation (Coleman et al., 2002). FAs are not only de novo synthesized but also are taken from exogenous sources. Once FAs are in the cell, ACSLs promote their retention by converting them into hydrophilic fatty acyl-CoA esters that cannot exit cells (Kamp and Hamilton, 2006).

ACSL enzymes utilize a wide range of saturated and unsaturated FAs with a preference for FAs of chain lengths of 8–22; however, they vary greatly in substrate specificity: for instance, ACSL1 and ACSL5 prefer oleate, ACSL3 palmitate, and ACSL4 arachidonic acid (Grevengoed et al., 2014; Soupene and Kuypers, 2008).

ACSL enzymes are ubiquitously expressed, even though individual genes are differentially expressed in individual tissues and differ in subcellular localization. For instance, ACSL3 is mainly expressed in the endoplasmic reticulum (ER) and lipid droplets and ACSL4 in peroxisomes and ER, whereas ACSL1, ACSL5, and ACSL6 are expressed in mitochondria, plasma membrane, and cytoplasm (Grevengoed et al., 2014; Soupene and Kuypers, 2008). Moreover, ACSL enzymes are expressed in pneumocytes, where they participate in the synthesis of surfactant (Coleman et al., 2002; Schiller and Bensch, 1971).

ACSL enzymes also participate in the metabolic reprogramming of cancer cells. For instance, pharmacologic inhibition of ACSLs results in apoptosis in a subset of TP53-deficient cancer cells (Mashima et al., 2005; Yamashita et al., 2000). Nevertheless, the biological significance of ACSL enzymes in promoting tumorigenesis is still largely unknown. For instance, it remains to be determined whether they play a role in the maintenance of cancers expressing mutant KRAS. Furthermore, it is not known whether glucose or fatty-acid-dependent cellular metabolic processes are worthy of therapeutic targeting in KRAS tumors.

In this manuscript, we show that ACSL3 is essential for the oncogenic capacity of mutant KRAS in lung cancer. Our data provide the rationale for the development of inhibitors that specifically target ACSL3 as anticancer drugs.

RESULTS

Mutant KRAS Regulates Glycolysis and Lipid Biosynthetic Processes In Vivo

To gain insight into mutant KRAS-regulated cellular networks that are required for tumor maintenance, we employed a transgenic mouse expressing a doxycycline (doxy)-inducible mutant Kras transgene in the respiratory epithelium. For this purpose, we crossed tetracycline operator-regulated KrasG12D (tet-op-KrasG12D) mice with Clara cell secretory protein-tet activator (CCSP-rtTA) mice. When fed with doxy, CCSP-rtTA/tet-op-KrasG12D mice invariably develop lung tumors, which are dependent on continuous expression of KrasG12D (Fisher et al., 2001).

To obtain well-established lung tumors, we fed CCSP-rtTA/tet-op-KrasG12D mice with doxy for 11 weeks. In agreement with previous findings, withdrawal of doxy from the diet caused KrasG12D extinction (Figure 1A; Fisher et al., 2001). Accordingly, total RAS activity and activated PDK1, a well-known downstream target of KRAS, were also downregulated (Figures 1B and 1C). Lung tumors completely regressed 6 days after doxy withdrawal (Figure 1D).

Figure 1. Mutant KRAS Regulates ACSL3 and ACSL4 in Lung Cancer Cells Both In Vitro and In Vivo.

Figure 1

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(A) Transgenic (KrasG12D) and endogenous (KrasWT) mRNA was quantified in micro-dissected lung tumors of CC10-rtTA/Tet-op-KrasG12D mice by real-time PCR at the indicated time points after doxy withdrawal.

(B) Pan-Ras activity was measured with an ELISA assay in micro-dissected lung tumors at the indicated time points after doxy withdrawal in CC10-rtTA/Tet-o-KrasG12D mice; ***p < 0.0003.

(C) Immunoblot detection of P-Pdk1 in lysates of micro-dissected lung tumors at the indicated time points after KrasG12D extinction.

(D) H&E-stained lung tissue sections demonstrating lung tumor regression at the indicated time points after KrasG12D extinction; the scale bar represents 1 mm.

(E) Heatmaps of the genes found enriched in a genome-wide expression profiling, illustrating the changes in gene expression of the indicated metabolic pathways 24 hr upon doxy withdrawal. Expression level shown is representative of log2 values of each replicate from micro-dissected lung tumors (n = 4). Red signal denotes higher expression relative to the mean expression level within the group, and blue signal denotes lower expression relative to the mean expression level within the group.

(F) GSEA plot of glycolytic processes based on the 24-hr off-doxy versus on-doxy gene expression profiles. NES, normalized enrichment score; p, nominal value.

(G) GSEA plot of and lipid metabolic processes based on the 24-hr off-doxy versus on-doxy gene expression profiles.

(H) Real-time PCR showing mRNA levels of Hk2 and Ldh-A in lung tumors of KrasG12D mice before or 24 hr after KrasG12D extinction.

(I) Real-time PCR showing mRNA levels of Acsl family members in lung tumors of KrasG12D mice before or 24–48 hr after KrasG12D extinction. *p < 0.02; **p < 0.004.

(J) ACSL enzymatic activity in healthy lung parenchyma or in micro-dissected lung tumors after 11 weeks of KrasG12D induction or after the indicated length of KrasG12D extinction. **p < 0.003; ***p < 0.001.

(K) ACSL enzymatic activity measured in BK fibroblasts treated with doxy for 24 hr.

(L) Real-time PCR showing mRNA levels of Acsl family members in BK fibroblasts treated with doxy for 24 hr.

(M) ACSL enzymatic activity in A549 NSCLC cells after transfection with the indicated siRNAs.

See also Figure S1.

To identify proximal molecular changes linked to KrasG12D extinction, we performed whole-genome gene expression analysis of micro-dissected lung tumors 24 or 48 hr after doxy withdrawal. Database for Annotation, Visualization and Integrated Discovery-based (DAVID) functional enrichment analysis and gene set enrichment analysis (GSEA) between experimental groups revealed a significant downregulation of genes that regulate glycolysis and lipid metabolic processes in mice undergoing KrasG12D withdrawal (Figures 1E–1G; data not shown).

Mutant KrasG12D extinction significantly downregulates the expression of the glycolytic enzymes hexokinase 2 (Hk2) and lactate dehydrogenase-A (Ldh-A) in lung tumors in vivo (Figure 1H). This observation is consistent with the fact that mutant KRAS has been associated with increased lactate production in cancer cells in vitro (Yun et al., 2009). Furthermore, Hk2 and Ldh-A play an important role in mutant Kras-driven lung tumorigenesis (Patra et al., 2013; Xie et al., 2014). However, we found that silencing of glycolytic enzymes decreases cell proliferation, glucose consumption, and lactate production without causing overall cell loss in vitro (Figures S1A–S1F).

Mutant KRAS Upregulates ACSL3 and ACSL4

We found that the lipid metabolic enzymes Acsl3 and Acsl4 were significantly downregulated in mouse lung tumors undergoing KrasG12D extinction, whereas we did not detect significant changes in mRNA levels of their family members Acsl1, Acsl5, and Acsl6 (Figure 1I). Furthermore, KrasG12D lung tumors display a striking upregulation of ACSL enzymatic activity compared to lung tissue of non-transgenic littermates (Figure 1J). Conversely, ACSL activity is downregulated by about 50% in lung tumors as early as 24 hr after KrasG12D extinction (Figure 1J). These findings suggest that ACSL enzymatic activity plays a significant role in the maintenance of mutant KRAS lung cancer.

We tested whether Acsl3 and Acsl4 are dependent on KrasG12D in cell culture with several complementary approaches. We found that KrasG12D induction increases ACSL activity and selectively upregulates Acsl3 and Acsl4 mRNA levels (Figures 1K, 1L, and S1G) in mouse embryonic fibroblasts derived from tetracycline operator-regulated Tet-op-KrasG12D;p53−/− transgenic mice (hereafter BK cells). These cells stably express rtTA, allowing tight doxy-dependent expression of KrasG12D (Pao et al., 2003).

Next, to demonstrate that KrasG12D upregulates Acsl3, we performed transactivation assays in BK cells. We observed that KrasG12D increases approximately by 3-fold the activity of an ACSL3 promoter reporter (pGL3-ACSL3luc; Figure S1H; Cao et al., 2010). Furthermore, treatment with rapamycin, a well-known inhibitor of mTORC1, inhibits the ability of KRAS to upregulate the ACSL3 promoter reporter (pGL3-ACSL3luc; Figure S1I) and also Acsl3 mRNA (Figure S1J). These findings suggest that mutant KRAS regulates ACSL3 expression through the mTOR signaling pathway. In addition, KRAS silencing reduces ACSL activity by about 50% in several NSCLC cells (Figure S1K and S1L). Finally, silencing of KRAS with a siRNA pool significantly reduced ACSL3 mRNA levels (Figure S1M).

We silenced each of the ACSL family members (i.e., ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6) to determine their relative contribution to ACSL cellular activity in mutant KRAS NSCLC cells: knockdown of ACSL3 leads to the largest reduction of total ACSL activity followed by the knockdown of ACSL4. However, knockdown of ACSL1, ACSL5, and ACSL6 had modest to no effect on total ACSL activity (Figures 1M and S1N).

Taken together, these results indicate that mutant KRAS regulates ACSL activity both in vivo and in vitro and that ACSL3 is the functionally predominant ACSL gene in mutant KRAS NSCLC.

ACSL3 Is Required for the Survival of Mutant KRAS In Vitro

In order to investigate whether ACSL3 promotes the survival of lung cancer cells expressing mutant KRAS, we silenced ACSL3 in a panel of human NSCLC cells, which have been extensively characterized with respect to the presence of oncogenic mutations (Sanger Institute Catalogue of Somatic Mutations in Cancer and the Cancer Genome Atlas; Table S1). ACSL3 silencing with two non-overlapping short hairpin RNAs (shRNAs) striking reduced the proliferation of ten mutant KRAS NSCLC cell lines and less-pronounced but still statistically significant inhibitory effects in the mutant KRAS HCC4017 and HCC461 cells (Figures 2A, 2B, and S2A). These NSCLC cell lines, with the exception of HCC461, carry also mutations inactivating the TP53 and/or INK4A/ARF tumor suppressors. Notably, ACSL3 silencing did not cause significant antiproliferative effects in immortalized human bronchial epithelial cells (HBEC3KT cells) that ectopically express mutant KRAS (Ramirez et al., 2004). In contrast, the effect of ACSL3 silencing on cell viability was not consistent in lung cancer cells carrying wild-type KRAS: we did not detect significant adverse effects in six lung cancer cells (H522, H596, H838, H1437, H2023, and HCC15) that harbor wild-type KRAS but are mutant for either TP53 or INK4A/ARF (Figures 2A and 2B). However, ACSL3 knockdown leads to inhibitory effects in H125 (wild-type KRAS; TP53;INK4A/ARF mutant), H661 (wild-type KRAS; TP53;INK4A/ARF mutant), and HCC95 (wild-type KRAS; TP53 mutant) lung cancer lines (Figure S2B). In addition, there was no correlation between dependency on ACSL3 and the proliferation rates of these NSCLC cells (Figure 2A; Table S1). Among the cell lines that were used for cell proliferation assays, with exception for H1264, there was a positive correlation between ACSL3 abundance and sensitivity to ACSL3 knockdown (Figures 2B and S2C).

Figure 2. ACSL3 Is Required for the Proliferation of NSCLC Cells Expressing Mutant KRAS.

Figure 2

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(A) Growth curves of NSCLC cells upon transduction with the indicated shRNAs. KRAS status for each cell line is indicated. Doubling time is mentioned in hours in parentheses.

(B) The histogram shows cell viability of the indicated NSCLC cells 96 hr after transfection with ACSL3 or control shRNAs.

(C) Schematic representation of the ACSL3 rescue cDNA mutants. shRNA recognition site and sequence of shRNA mutant 1 and 2 are indicated. Red letters indicate mutated nucleotides.

(D) The histogram shows H460 (mutant KRAS) cell viability of lung cancer cell line transduced with retroviruses expressing the indicated shRNA and ACSL3 cDNAs.

See also Figure S2 and Table S1.

To assess off-target effects, we performed rescue experiments with retroviruses expressing ACSL3 cDNAs harboring mutations ablating binding sites of either shRNA ACSL3 (A) or shRNA ACSL3 (B), named ACSL3 mutant 1 and ACSL3 mutant 2, respectively (Figure 2C). The antiproliferative effect caused by shRNA ACSL3 (A) was rescued by ACSL3 mutant 1, but not by mutant 2 (Figure 2D). Conversely, the phenotype of shRNA ASCL3 (B) was rescued by ACSL3 mutant 2, but not by mutant 1 (Figure 2D). As expected, cells expressing the shRNA-resistant ACSL3 cDNAs had increased levels of ACSL3 as compared to cells expressing wild-type ACSL3 cDNA (Figure S2D). Thus, we conclude that the antiproliferative effects are specific to ACSL3 knockdown and not due to off-target effects.

Next, we tested the effect of knocking down ACSL4, which is also downregulated by mutant Kras extinction in lung tumors (Figure 1I). However, knocking down ACSL4 both in mutant and wild-type KRAS cells did not cause any significant effect on cell proliferation (Figure S2E).

To begin assessing the mechanism underlying the dependency on ACLS3, we assessed the effect of its silencing on ACSL activity. We found that knockdown of ACSL3 reduced ACSL activity by 55%–65% in mutant KRAS NSCLC cells but only by 30%–35% in wild-type KRAS cells (Figure S2F). In addition, ACSL3 knockdown leads to the cell-line-dependent upregulation of ACSL1, ACSL4, and ACSL6 in wild-type KRAS lung cancer cells that are resistant to ACSL3 knockdown (Figures S2G and S2H). In contrast, knockdown of ACSL4 reduced ACSL activity only by 20%–30% in representative mutant KRAS and wild-type KRAS NSCLC cells (Figures S2I and S2J). Finally, we found that ACSL3 knockdown did not affect glucose or glutamine consumption neither in mutant nor in wild-type KRAS lung cancer cells (Figures S2K and S2L; data not shown). Taken together, these results indicate that (1) mutant KRAS regulates ACSL activity both in vivo and in vitro, (2) ACSL3 is the functionally predominant ACSL isoenzyme in mutant KRAS lung cancer cells, (3) upregulation of other ACSL family members and not glycolysis or glutaminolysis is likely to mediate the resistance to ACSL3 knockdown in wild-type KRAS lung cancer cells, and (4) the decrease of ACSL activity caused by ACSL4 knockdown is below the threshold needed to cause detrimental effects on cell viability.

Characterization of the Role of ACSL3 in Mutant KRAS Lung Cancer Cells

ACSL3 silencing was accompanied by induction of apoptosis, as shown by an increase in the percentage of the SubG0 population of cells, cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase 3 in H460, H1264, and HCC44 cells, as representative examples of ACSL3-dependent lung cancer cells (Figures 3A, S3A, and S3B). These changes were not evident in H522 NSCLC cells, which are representative of the cells refractory to ACSL3 silencing (Figure 3A). In addition, we measured mitochondrial membrane potential using JC-1 (5, 5′, 6, 6′-tetracholoro-1, 1′, 3, 3′-tetraethylbenzimi-dazolylcarbocyanine iodide), a cytofluorimetric lipophilic cationic dye. Silencing of ACSL3 leads to significant decrease in membrane potential in H460 mutant KRAS cells (Figure S3C), which is consistent with induction of apoptosis. Next, we assessed the effect of silencing ACSL3 on mitochondrial respiratory activity. We found that ACSL3 silencing significantly reduces basal oxygen consumption rate (OCR) in real time in H460 mutant KRAS cells (Figure S3D). These results lend further support to the notion that lung cancer cells that express mutant KRAS are vulnerable to ACSL3 depletion.

Figure 3. ACSL3 Is Required for the Survival and Anchorage-Independent Growth of NSCLC Cells Expressing Mutant KRAS.

Figure 3

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(A) Flow cytometry showing the effect of shRNA-mediated ACSL3 knockdown on cell cycle of the indicated NSCLC lines. The percentages of cells in specific phases of the cell cycle are indicated. Note the presence of cells in the sub-G0 phase of the cell cycle in the ACSL3-dependent H460 and H1264 cells, but not in the ACSL3-independent H522 cells.

(B) Representative images of tissue culture plates showing soft agar colony formation assay in A549 and H460 (mutant KRAS) cells treated as indicated.

(C) Histograms showing quantification of colonies of A549 and H460 cells grown in soft agar upon transduction with the indicated shRNAs; ***p < 0.001.

See also Figure S3.

To interrogate the functional significance of ACSL3 in KRAS lung tumorigenesis, we performed colony formation assays in soft agar and found that shRNA-mediated ACSL3 silencing results in more than 90% inhibition of colony formation in A549, H460, and H1155, mutant KRAS NSCLC cells, but caused no significant inhibition of colony formation in HCC15 and H2023 wild-type KRAS NSCLC cells (Figures 3B, 3C, S3E, and S3F). These results indicate that ACSL3 is critical for maintaining the ability of mutant KRAS NSCLC cells to grow in an anchorage-independent fashion, which is a hallmark of cancer cells (Hanahan and Weinberg, 2011).

Taken together, these results support several conclusions: (1) ACSL3 plays a critical role in promoting the survival of mutant KRAS NSCLC cells; (2) silencing of ACSL3 does not lead to generalized antiproliferative or proapoptotic effects, as demonstrated by its inability to impair H522, H596, H838, H1437, H2023, HCC15, and HBEC3KT cells; and (3) ACSL3 may exert a prosurvival function in NSCLC cells carrying oncogenotypes other than mutant KRAS, as demonstrated by the inhibitory effects we observed upon ACSL3 knockdown in H125, H661, and HCC95 cells.

ACSL3 Promotes Fatty Acid Uptake, Accumulation of Neutral Lipids, and β-Oxidation in Mutant KRAS NSCLC Cells

Although mutant KRAS cancer cells display both de novo FA synthesis and lipid uptake from extracellular sources, it is not known which pathways predominate. It is also unknown whether mutant KRAS cells predominantly utilize these FAs as a substrate for the synthesis of structural or storage lipids, for instance, phospholipids or triacylglycerides (TAGs), and/or for energy production by β-oxidation (Boroughs and DeBerardinis, 2015).

We observed that KrasG12D extinction in vivo leads to a time-dependent (24–48 hr) downregulation of a set of genes known to control FA entry into the mitochondria/peroxisomes or ER. In particular, we detected genes responsible for long-chain FA transport and FA oxidation (Figures 4A–4C, S4A, and S4B). These observations suggest that KRAS enhances the expression of genes involved in β-oxidation.

Figure 4. Suppression of Fatty Acids Uptake and β-Oxidation upon Mutant KRAS Suppression.

Figure 4

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(A) Heatmaps illustrating the changes in expression of FA metabolism/β-oxidation genes 24 hr upon doxy withdrawal in mutant Kras lung cancer tumors in transgenic mice. Expression level shown is representative of log2 values of each replicate from micro-dissected lung tumors. Red signal, higher expression; blue signal, lower expression relative to the mean expression level within the group.

(B) GSEA plot of FA metabolism/β-oxidation genes based on the 24-hr off-doxy versus on-doxy gene expression profiles.

(C) Real-time PCR showing the mRNA level of the indicated genes upon KrasG12D extinction in vivo; genes responsible for long-chain FA transport carnitine O-octanoyltransferase (Crot) and fatty acid transporter-member 2 (Fatp2/Slc27a2) and 4 (Fatp4/Slc27a4); and genes that control FA β-oxidation, such as enoyl coenzyme A hydratase (Echs1) and carnitine O-acetyltransferase (Crat); ns, statistically non-significant; *p < 0.01; **p < 0.003; ***p < 0.001.

(D) BODIPY-FA uptake in BK cells treated with doxy as indicated. Red and white arrows indicate positive and negative cells, respectively (cells with both cytoplasmic and membrane staining were considered positive); the scale bar represents 50 μm. Note that mutant KRAS induces the uptake/retention of BODIPY-FA.

(E) Fluorescence-activated cell sorting analysis of BK cells after a 30-min pulse labeling with BODIPY-FA. Note uptake/retention of BODIPY-FA in BK cells expressing mutant KRAS.

(F) BODIPY-FA retention in mutant KRAS HCC44 NSCLC cells expressing the indicated shRNAs. Red and white arrows indicate representative positive and negative cells, respectively; the scale bar represents 50 μm.

(G) The histogram shows the percentage of BODIPY-FA-positive HCC44 and H460 cells transduced with indicated ACSL3 shRNA (100 cells were counted in three separate areas).

(H) Composition of lysophosphatidylcholine (LPC) and triacylglycerides (TAGs) in BK cells with and without doxy treatment (*p < 0.05; **p < 0.001).

(I) Composition of LPC and TAGs in H460 NSCLC cells treated with the indicated ACSL3 shRNA (*p < 0.05).

(J) β-oxidation assay in H460 NSCLC cells treated with the indicated ACSL3 shRNAs or etomoxir; ***p < 0.001.

(K) Measurement of ATP in H460 NSCLC cells treated with the indicated ACSL3 shRNAs or etomoxir.

(L) The histogram shows the viability of the indicated mutant KRAS NSCLC cells 72 hr after the indicated treatments; *p < 0.01; **p < 0.004.

(M) The histogram shows the viability of the indicated mutant KRAS NSCLC cells 72 hr after the treatment with 4-bromocrotonic acid; **p < 0.004.

See also Figure S4.

In order to gain insight into the impact of mutant KRAS on FA uptake, synthesis of storage/structural lipids, and β-oxidation, we employed isogenic BK cells. We found that mutant KRAS increases the uptake of FAs from an exogenous source in BK cells pulse labeled with the fluorescent FA BODIPY 500/510 C4,9, which mimics a long-chain FA (BODIPY-FA) (Figures 4D and 4E; Kasurinen, 1992; Nchoutmboube et al., 2013). Conversely, ACSL3 silencing significantly decreases the uptake and cellular retention of fluorescent BODIPY-FA in NSCLC cells (Figures 4F, 4G, and S4C).

To better understand the role of mutant KRAS in cellular lipid metabolism, we used a mass spectrometry method to measure a lipidomic profile, including hundreds of TAGs, 250 phospholipids, cholesteryl esters, diacylglycerides, and free FAs (Fahy et al., 2009; Mitsche et al., 2015). We found that induction of mutant KRAS for 24 hr in BK cells increased TAGs and caused an equivalent downregulation of lysophosphatidylcholine (LPC) (Figures 4H and S4D; Table 1). This finding is consistent with the observation that BODIPY-FA accumulates in structures that resemble lipid droplets, the storage site for neutral lipids (Figures 4D–4F and S4C).

Table 1.

Cellular Lipids Composition in BK Cells with KRAS Induction

Lipid Classes BK Cells (%) BK Cells + Dox (%)
Endocyne   0.02 ± 0.0   0.02 ± 0.0
Lysophospholipids   7.48 ± 1.6   3.44 ± 1.5*
Neutral lipids   8.43 ± 1.1 11.38 ± 1.9**
Phospholipids 76.86 ± 6.3 78.24 ± 2.1
Polar lipids   7.20 ± 1.32   6.92 ± 1.5
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Data shown are the mean ± SD of percentage of total lipids at a steady state. Mutant KRAS was induced in BK cells by doxycycline (Dox) treatment for 24 hr.

*

p < 0.05;

**

p < 0.001.

Direct quantification of lipid classes indicates that the increase in neutral lipids is accounted by an increase in diacylglycerides (DAGs) and TAGs lipid species, which is consistent with the hypothesis that LPCs are consumed in the synthesis of DAGs and TAGs (Figures 4H and S4E). Conversely, ACSL3 knockdown decreases the intracellular content of TAGs (Figures 4I, S4E, and S4F; Table 2). Figures S4G and S4H show a summary of the composition of TAGs, phosphatidylcholines, and lysophosphatidylcholines with respect to their number of carbons and double bonds in BK cells and in H460 prior and after doxy treatment or ACSL3 knockdown, respectively.

Table 2.

Cellular Lipids Composition in H460 NSCLC Cells with shRNA against ACSL3

Lipid Classes Scrambled shRNA (%) shRNA ACSL3(A) (%) shRNA ACSL3(B) (%)
Endocyne   0.02 ± 0.0   0.02 ± 0.0   0.02 ± 0.0
Lysophospholipids   6.76 ± 1.6   6.95 ± 0.8   7.46 ± 0.9
Neutral lipids   7.45 ± 1.1   5.58 ± 0.9*   6.00 ± 0.5*
Phospholipids 78.81 ± 6.3 81.08 ± 0.5 79.82 ± 0.9
Polar lipids   6.96 ± 1.3   6.36 ± 0.5   6.69 ± 0.5
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Data shown are the mean ± SD of percentage of total lipids at a steady state.

*

p < 0.05.

In addition, knockdown of ACSL3 inhibits β-oxidation to a degree similar to etomoxir, a known inhibitor of carnitine palmitoyltransferase, the rate-limiting enzyme that allows entry of acyl-CoA FAs into the mitochondrion (Figure 4J; Pike et al., 2011). Notably, knockdown of ACSL3 inhibits ATP production to a degree similar to that of cells treated with etomoxir (Figure 4K). Importantly, etomoxir impairs cell viability in this context (Figure 4L). Similarly, 4-bromocrotonic acid, an inhibitor of mitochondrial thiolase, a component of β-oxidation, reduces the cell viability (Figure 4M).

These findings support the conclusion that ACSL3 in mutant KRAS NSCLC critically regulates the uptake and cellular retention of long-chain fatty acyl-CoAs and their channeling to β-oxidation for energy generation. Taken together, these data suggest that inhibition of ACSL3 in vivo would have potent anti-tumor effects.

ACSL3 Suppression Impairs Tumor Growth In Vivo

To gain insight into the significance of ACSL3 inhibition in vivo, we performed xenograft experiments using A549 and H460 NSCLC cells, which are representative of our in vitro studies. Silencing ACSL3 with shRNA(A), which we previously validated to be specific, resulted in a remarkable suppression of tumor formation with 3.5-fold and 10-fold difference compared to the control A549 and H460 xenografts, respectively (Figures 5A and 5B). Significantly, mice carrying xenografts with silenced ACSL3 had also a dramatically increased survival (Figures 5C and 5D). These results indicate that ACSL3 is required for the tumor growth in vivo.

Figure 5. ACSL3 Suppression Impairs Tumorigenesis In Vivo.

Figure 5

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(A and B) Tumor volume of xenografts of A549 and H460 NSCLC cells upon transduction with the indicated shRNAs.

(C and D) Kaplan-Meier curves of nude mice carrying xenografts of A549 and H460 NSCLC cells expressing the indicated shRNA upon transduction with lentiviruses (n = 6/group).

Loss of Acsl3 Results in Defective Tumorigenesis in Mutant Kras Lung Cancer in Mice

We obtained mutant mice carrying a gene trap disrupting Acsl3 upstream of exon 1. Acsl3−/− mice were characterized as null based on the absence of transcripts by real-time PCR (LEXKO 011 mouse strain; European Mutant Mouse Archive; Figure S5A). We confirmed the genotype of these mice with a genotyping protocol that distinguishes between wild-type, heterozygous, and homozygous littermates (Figure S5B).

We found that Acsl3−/− mice are born according to the expected Mendelian ratio, without obvious macroscopic defects during development or adult life. Histologically, lung tissue has no morphological differences between the wild-type and Acsl3−/− littermates (Figure S5C). Considering that type II pneumocytes and Clara cells are the probable precursor for lung tumors (Sutherland et al., 2014; Xu et al., 2012), it is noteworthy that there are no significant morphological differences between wild-type and Acsl3−/− littermates as confirmed with SP-C (surfactant-associated protein-C) and CCSP staining (Figure S5D). Furthermore, RAS is present at comparable levels in the lungs of wild-type and mutant littermates (Figure S5E). Nevertheless, the lungs of Acsl3−/− mice show about 40% decrease in ACSL enzymatic activity as compared to their wild-type littermates (Figure S5F). We conclude that, even though the loss of Acsl3 significantly decreases the ACSL activity in the lungs, the overall morphology of the lungs is not affected.

To assess the role of Acsl3 in mutant Kras lung tumors in vivo, we generated compound mutant mice by crossing LSL-KrasG12D mice (LSL-Kras mice) with Acsl3 mutant mice, which allows conditional expression of KrasG12D upon exposure to Cre recombinase in the respiratory epithelium (Jackson et al., 2001). We assessed lung tumorigenesis 8 weeks after intratracheal delivery of an adenovirus that expresses Cre recombinase (adeno-Cre). As expected, ACSL3 is undetectable in the lungs of Ascl3−/− mice and a pan-RAS antibody did not detect differences between KrasG12D;Acsl3 wild-type and KrasG12D;Acsl3−/− mice (Figure S5G). We assessed tumor number and grade according to well-established diagnostic criteria developed for Kras-driven lung cancer mouse models (DuPage et al., 2009; Jackson et al., 2001). The overall tumor burden was significantly decreased in LSL-KrasG12D;Acsl3−/− mice as compared to their LSL-KrasG12D;Acsl3+/+ littermates. As expected, in LSL-KrasG12D;Acsl3+/+ mice, we detected adenomas with uniform nuclei (A), atypical adenomatous hyperplasia (AAH), and bronchial hyperplasia (BH) (Figures 6A and 6B). In contrast, there were no adenomas in the LSL-KrasG12D;Acsl3−/− mice and all lesions present were small areas of AAH or BH (Figures 6A and 6B). We confirmed that the adenomas and AAH originated from type II alveolar cells with SP-C staining (Figure 6C). In both genotypes, there was equivalent activation of phospho-S6 (P-S6), which is one of the downstream targets of KRAS (Figure 6D). However, AAH lesions of KrasG12D;Acsl3−/− mice had decreased phospho-histone 3 (p-H3) as compared to the ones of LSL-KrasG12D;Acsl3+/+ mice (Figures 6D and 6E). These findings suggest that, even though Acsl3 loss does not affect mutant KRAS signaling, it significantly impairs the capacity of KrasG12D to promote cell proliferation and tumor initiation in vivo.

Figure 6. ACSL3 Is Required for the Initiation of KRAS-Driven NSCLC in Mice and Is Expressed in Patients.

Figure 6

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(A) Images of KrasG12D lung cancers stained with H&E after 8 weeks of adeno-Cre infection. Lung nodules are indicated as A: adenoma, AAH: atypical adenomatous hyperplasia, and BH: bronchial hyperplasia (magnification: upper panels 12.5×; bottom panels 100×).

(B) Histogram of tumor type distribution in mice with the indicated genotypes (*p < 0.05; ***p < 0.001; n, number of mice samples per genotype). Tumor types are indicated.

(C) Images of murine lung tumors stained with SP-C antibody. Positive staining confirms their origin from type II alveolar cells (magnification 400×). Arrows indicate the location of the inlets.

(D) Images of murine lung tumors stained with indicated antibodies. Antibody staining is in brown; nuclear counter staining is in blue (magnification 400×). Arrows indicate the location of inlets.

(E) The histogram shows the percentage of P-H3-positive cells in lung tumors of KrasG12D; Acsl3+/+ and KrasG12D; Acsl−/− mice (100 cells were counted in three separate areas).

(F) Representative microphotographs of ACSL3 in the indicated representative human samples (respective H-score for tumor samples are indicated). Black arrowhead indicates ACSL3 in type II pneumocytes. Arrows indicate the location of inlet (the scale bar represents 200 μm).

(G) Histogram showing H-score for ACSL3 staining intensity in tumor samples according to their pathologic stages (*p < 0.05; ns, statistically insignificant; n, number of patient samples).

See also Figure S5.

ACSL3 in Human NSCLC

To determine whether ACSL3 has any significance in patients, we assessed ACSL3 by immunohistochemistry (IHC) on a tissue microarray (TMA) comprising 182 NSCLC patient samples. We found low-level ACSL3 staining in type II pneumocytes of normal lungs, whereas it is readily detectable in NSCLC specimens (Figure 6F). We used the Aperio Image Toolbox (Leica Biosystems) to quantify ACSL3 staining in lung cancer specimens with a scoring scale, which takes into consideration the staining intensity in conjunction with the percentage of positive cells (H-score; ranging from 0 to 300). We considered an H-score of less than 100, from 100 to 200, and above 201 having a low, intermediate, or high staining intensity, respectively. Overall, there was a higher percentage of samples with the higher H-score of >201 (Figure S5H). However, there was no significant difference between the H-score of wild-type and mutant KRAS NSCLC samples (Figure S5I). Notably, a significantly higher percentage of surgical stage I (early stage) specimens displayed a higher H-score as compared to stage III specimens (late stage; Figures 6G and S5J). We conclude that ACSL3 is highly expressed in lung cancer. These results also suggest that ACSL3 may play an important role in the early stages of tumorigenesis, a notion that is consistent with our findings that ACSL3 is required for tumor initiation in the LSL-KrasG12D mouse model of lung tumorigenesis (Figures 6A and 6B).

DISCUSSION

The observation that extinction of mutant Kras in vivo causes a prominent perturbation of the glycolytic and lipid metabolic pathways in lung cancer cells suggests that these networks are involved in lung cancer maintenance.

Glucose metabolism has been intensely studied in cancer cell lines and tumors. Mutant KRAS induces aerobic glycolysis in vitro and in vivo in mouse models of pancreatic and lung cancer (Racker et al., 1985; Ying et al., 2012). Furthermore, abrogation of the glycolytic enzymes Hk2 and Ldh-A impairs without abolishing the formation of mutant Kras lung tumors in mice (Patra et al., 2013; Xie et al., 2014). Our finding that suppression of glycolytic enzymes reduces the proliferation of mutant KRAS NSCLC cells without causing cell death in cell culture is consistent with these recent reports and suggests that alternative bioenergetic pathways, particularly those that oxidize FAs, glutamine, or other nutrients, may support the survival of mutant KRAS lung cancer cells, providing mechanisms that bypass the inhibition of HK2, LHD-A, or other glycolytic enzymes (Boroughs and DeBerardinis, 2015).

We obtained several lines of evidence that ACSL3 is the critical ACSL enzyme required not only for the viability of mutant KRAS NSCLC cells in culture but also for the ability of mutant Kras to promote tumor initiation and progression in vivo. The observation that, in human NSCLC specimens, ACSL3 is readily detected adds support to the notion that ACSL3 plays a role in NSCLC tumorigenesis. Longer follow-up will be needed to fully characterize the phenotype of Acsl3−/− lung tumors, but taken together, our data demonstrate that ASCL3 is a critical requirement for mutant KRAS lung tumorigenesis.

Cancer cells display the ability to both synthetize FAs de novo and to uptake them from extracellular sources (Boroughs and DeBerardinis, 2015). We reason that the dependency on ACSL3 is due to several properties of this enzyme. ACSL3 is placed at a critical metabolic node because ACSL enzymes are essential for both β-oxidation and lipid biosynthesis by activating long-chain FAs derived from either an exogenous or endogenous source. Our observation that suppression of ACSL3 leads to significant inhibition of β-oxidation with depletion of cellular ATP, loss of mitochondrial function, and impaired cell viability underscores the importance of this enzyme in mutant KRAS lung cancer cells. Furthermore, we demonstrated that mutant KRAS promotes the cellular accumulation of neutral lipids (which are mostly by TAGs) in an ACSL3-dependent manner. We speculate that lipid droplets, the organelles that store neutral lipids, may provide substrates that support β-oxidation in conditions of energetic stress, particularly in vivo. Therefore, we conclude that ACSL3 is a critical non-redundant therapeutic target for cancer types that rely on lipid metabolic processes.

From a therapeutic point of view, it is also of interest that even a partial decrease in ACSL enzymatic activity is sufficient to achieve a critical therapeutic threshold without causing generalized toxicity in cultured cells or major phenotypes in Acsl3-null mice. These observations suggest that ACSL3 is limiting in conditions of oncogenic stress. Accordingly, inhibition of ACSL3 is likely to have a therapeutic window in vivo.

Moreover, ACSL3 is distal to FASN, where compensatory mechanisms are less likely to occur (Coleman et al., 2002; Menendez and Lupu, 2007). For instance, others have reported that ACSL short-chain family member 2 (ACAS2) and FASN, which are upstream of ACSL in de novo FA synthesis, may represent therapeutic targets (Hatzivassiliou et al., 2005; Menendez and Lupu, 2007). However ACL and ACAS2 may compensate for each other, whereas the requirement for FASN may be bypassed by the uptake of exogenous lipids. In these regards, our findings also provide a molecular explanation to the recent observation that mutant KRAS promotes the scavenging of longer-chain unsaturated FAs from lipids in the extracellular space. In addition, these serum lipids are shown to promote both proliferation and survival of Ras-driven human cancer cells (Kamphorst et al., 2013; Salloum et al., 2014).

Our results suggest that the catalytic activity is required for the vulnerability to ACSL3 depletion in mutant KRAS NSCLC. However, we were unable to rescue the viability defect caused by ACSL3 suppression with short-chain FAs that enter the mitochondrion in a CPT1A-independent manner. We reason that, in addition to defective β-oxidation, several other processes could contribute to the dependency of mutant KRAS lung cancer cells on ACSL3. For instance, ACSL enzymes also provide substrates for protein lipidation, such as farnesylation and myristoylation, which are required for the activity of several oncogenes, including KRAS (Gysin et al., 2011). It is also possible that these important long-chain acyl-CoAs cannot be replenished by supplementing medium-chain free FAs. Contrary to our expectation, we did not detect changes in phospholipid content upon ACSL3 silencing; thus, we reason that it is not likely that a defect in the synthesis of cellular membranes causes the loss of viability of NSCLC cells, at least during the time frame of our experiments (Yao and Ye, 2008). In principle, inhibition of ACSL3 could also lead to the accumulation of cytotoxic free FAs; however, our mass spectrometry data do not provide support to this hypothesis. Future studies will be necessary to understand in greater detail the mechanisms responsible for the dependency of mutant KRAS lung cancer cells on ACSL3.

The results of our transactivation assays are consistent with the notion that mutant KRAS regulates the ACSL3 promoter through mTORC1, which is a well-known regulator of the SREBPs prolipogenic transcription factors, which in turn may promote aberrant proliferation in cancer cells (Laplante and Sabatini, 2012). Furthermore, SREBPs are required for aberrant proliferation and survival of breast cancer cells (Ricoult et al., 2016). It is noteworthy that the ACSL3 and ACSL4, but not the ACSL1 and ACSL6, promoters contain SREBP-binding sites. Thus, it is likely that mutant KRAS regulates the ACSL3 promoter through the mTORC1/SREBP pathway. It is also of interest that wild-type KRAS NSCLC cells bypass the deleterious effects of ACSL3 knockdown by upregulating other ACSL3 family members. Thus, it will be of interest in the future to define in greater detail the mechanisms that regulate the expression of ACSL family members.

Whereas we focused on NSCLCs with KRAS mutations, it is likely that the detrimental effects on cell viability of ACSL3 knockdown are not restricted only to mutant KRAS lung cancer cells, as demonstrated by the observation that H661, HCC95, and H125 NSCLC cell lines with wild-type KRAS are also sensitive to ACSL3 knockdown. The observation that a significant percentage of lung tumors with high intensity of ACSL3 staining does not carry mutant KRAS suggests that other oncogenic mutations may upregulate ACSL3. Therefore, future studies will be needed to determine the spectrum of lung cancer oncogenotypes vulnerable to ACSL3 depletion.

Our report provides the important demonstration that mutant KRAS positively regulates lipid metabolism, establishing a requirement that can be exploited for therapeutic gain. We propose that ACSL3 is a target for the development of targeted therapies against mutant KRAS lung cancer.

EXPERIMENTAL PROCEDURES

All reagents and detailed methods are described in the Supplemental Information.

Cell Lines, Reagents, and Plasmids

Human NSCLC cell lines and immortalized human bronchial epithelial HBEC3KT cells (which ectopically express Cdk4 and hTERT) were from the Hamon Center cell line repository (UT Southwestern Medical Center; Gazdar et al., 2010; Ramirez et al., 2004). All cell lines were DNA fingerprinted for provenance (PowerPlex 1.2 Kit; Promega) and were mycoplasma free (e-Myco Kit; Boca Scientific). Fluorescent BODIPY-FA was from Molecular Probes, pBabePuro and pBabePuro-KRAS-G12V were from Addgene, the ACSL3 cDNA was from Dr. Takahiro Fujino (Department of Bioscience, Ehime University), and pGL3-ACSL3luc was from Dr. Jingwen Liu (Department of Veterans Affairs Palo Alto Health Care System). We performed site-directed mutagenesis using the Quickchange kit (Agilent). See also Supplemental Information for tissue culture and assay conditions.

Mouse Studies

CCSP-rtTA/Tet-op-Kras (FVB/SV129 mixed background) mice were described previously (Fisher et al., 2001). Acsl3−/− mice (LEXKO-011; Lexicon Genetics) were obtained from the European Mouse Mutant Archive. We generated LSL-KRasG12D;Ascl3−/− mice with standard breeding techniques. We performed xenograft studies as previously described (Konstantinidou et al., 2013). For additional details, refer to the Supplemental Information.

Expression Profiling

Gene expression profiles were obtained from micro-dissected lung tumors of 19-week-old mice (Tusher et al., 2001). The data have been deposited in NCBI’s GEO and are accessible through GEO: GSE40606. See also Supplemental Information.

RNAi

We obtained ACSL3 and ACSL4 or non-targeting small interfering RNAs (siRNAs) (siGenome) and pLKO lentiviral vectors encoding shRNAs from Thermo Scientific. siRNAs were transfected with DharmaFECT 4 Transfection Reagent (Thermo Scientific). See also Supplemental Information.

Quantitative Real-Time PCR

We extracted RNA with a RNA extraction kit (QIAGEN) and used Superscript III (Invitrogen) to generate cDNA and SYBR Green PCR master mix (Applied Biosystems) for quantitative real-time PCR (qRT-PCR) analysis. See also Supplemental Information.

Immunoblotting and Antibodies

We performed immunoblots according to standard procedures (Konstantinidou et al., 2009). A list of the antibodies is provided in the Supplemental Information.

Measurement of ACSL Activity and β-Oxidation

We measured ACSL activity in cell lysates by detecting incorporation of radiolabeled palmitate into palmitoyl-CoA as previously described (Zhou et al., 2007). We measured β-oxidation as described previously (Deberardinis et al., 2006). See also Supplemental Information for details.

Flow Cytometry

We performed flow cytometry to detect the phases of the cell-cycle or BODIPY-FA uptake according to standard procedures (Konstantinidou et al., 2009; Nchoutmboube et al., 2013).

Lactate and Glucose Flux Measurements

Cell culture media was analyzed for glucose and lactate concentration using a NOVA Biomedical Bio-Profile Basic 4 Analyzer, as described previously (Yang et al., 2009).

Microscopy

For BODIPY-FA, immunofluorescent microscopy cells were fixed with 4% formaldehyde in PBS for 20 min. We used Image J software to evaluate fluorescence patterns and intensity (Schneider et al., 2012). For quantification, we analyzed at least 100 cells in three independent fields. Cells positive for BODIPY-FA staining both in the cytoplasm and cell membrane were considered positive.

Statistical Analysis

All data presented are the mean ± SEM or SD of experiments repeated three or more times. We determined significance using two-tailed unpaired Student’s t test or the one-way ANOVA test.

Supplementary Material

1
2

Highlights.

  • ACSL3 expression and activity in lung cancer cells is upregulated by mutant KRAS

  • ACSL3 suppression decreases β-oxidation and ATP levels in mutant KRAS lung cancer cells

  • ACSL3 is required for survival and oncogenic capacity of mutant KRAS lung cancer cells

  • ACSL3 is highly expressed in human lung cancer specimens and promotes cancer initiation

Acknowledgments

We thank Dr. Harold E. Varmus for providing CCSP-rtTA/Tet-op-Kras mice and BK cells and Dr. Luc Girard and Dr. Helen Hobbs (University of Texas Southwestern Medical Center) for sharing bioinformatics data and expertise in lipid analysis, respectively. We also thank Dr. Andrea Rabellino and Dr. Edward Motea for assistance with transactivation assays and flow cytometer, respectively. The following financial support is acknowledged: American Cancer Society Scholar Award 13-068-01-TBG, CDMRP LCRP grant LC110229, CPRIT RP140672, NCI 5P50 CA70907-15, UT Southwestern Friends of the Comprehensive Cancer Center, the Gibson Foundation, and Texas 4000 (to P.P.S.); CPRIT RP140110 (to M.S.P.); CPRIT RP101496 (to G.K.); NCI R01 CA157996-01 and Welch Foundation research grant I-1733 (to R.J.D.); Lung Cancer SPORE (P50CA70907; to J.D.M., P.P.S., J.W.S., K.E.H., and I.I.W.); CPRIT RP120732 and RP110709 (to J.D.M. and K.E.H.); NIH K01GM109317 (to M. Mitsche); and 1P30 CA 142543-01, the Harold C. Simmons Comprehensive Cancer Center through NCI Cancer Center support grant and 2P30CA016672, MD Anderson’s Institutional Tissue Bank, and NIH National Cancer Institute (to I.I.W.).

Footnotes

ACCESSION NUMBERS

The accession number for the KRAS tumor microarray reported in this paper is GEO: GSE40606.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures, five figures, and one table and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2016.07.009.

AUTHOR CONTRIBUTIONS

M.S.P., G.K., and P.P.S. designed the experimental plan. M.S.P., G.K., N.V., M. Melegari, S.R., C.Y., and M. Mitsche performed experiments. M.S.P., G.K., N.V., M. Mitsche, J.M., R.J.D., and P.P.S. analyzed the data. K.E.H. and J.D.M. contributed NSCLC cell lines. K.B. and J.W.S. analyzed microarray data from mouse lung tumors. J.L. contributed to the ACSL3 antibody. X.T., J.R.-C., N.K., and I.I.W. performed and analyzed the data of immunohistochemistry on patient specimens. M.S.P., G.K., and P.P.S. wrote the paper. All authors read and approved the final manuscript.

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Supplementary Materials

1
NIHMS806820-supplement-1.pdf (2.2MB, pdf)
2
NIHMS806820-supplement-2.xlsx (67.7KB, xlsx)

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