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Metabolism

De Novo Fatty Acid Synthesis-Driven Sphingolipid Metabolism Promotes Metastatic Potential of Colorectal Cancer

Naser Jafari, James Drury, Andrew J. Morris, Fredrick O. Onono, Payton D. Stevens, Tianyan Gao, Jinpeng Liu, Chi Wang, Eun Y. Lee, Heidi L. Weiss, B. Mark Evers and Yekaterina Y. Zaytseva
DOI: 10.1158/1541-7786.MCR-18-0199 Published January 2019

Abstract

Metastasis is the most common cause of death in colorectal cancer patients. Fatty acid synthase (FASN) and sphingosine kinase-1 and -2 (SPHK1 and 2) are overexpressed in many cancers, including colorectal cancer. However, the contribution of FASN-mediated upregulation of sphingolipid metabolism to colorectal cancer metastasis and the potential of these pathways as targets for therapeutic intervention remain unknown. This study determined that sphingosine kinases (SPHK) are overexpressed in colorectal cancer as compared with normal mucosa. FASN expression significantly correlated with SPHK2 expression in data sets from The Cancer Genome Atlas (TCGA) and a colorectal cancer tumor microarray. FASN, SPHK1, and SPHK2 colocalized within invadopodia of primary colorectal cancer cells. Moreover, FASN inhibition decreased SPHK2 expression and the levels of dihydrosphingosine 1-phosphate (DH-S1P) and sphingosine 1-phosphate (S1P) in colorectal cancer cells and tumor tissues. Inhibition of FASN using TVB-3664 and sphingolipid metabolism using FTY-720 significantly inhibited the ability of primary colorectal cancer cells to proliferate, migrate, form focal adhesions, and degrade gelatin. Inhibition of the FASN/SPHK/S1P axis was accompanied by decreased activation of p-MET, p-FAK, and p-PAX. S1P treatment rescued FASN-mediated inhibition of these proteins, suggesting that FASN promotes metastatic properties of colorectal cancer cells, in part, through an increased sphingolipid metabolism. These data demonstrate that upregulation of the FASN/SPHK/S1P axis promotes colorectal cancer progression by enhancing proliferation, adhesion, and migration.

Implications: This study provides a strong rationale for further investigation of the interconnection of de novo lipogenesis and sphingolipid metabolism that could potentially lead to the identification of new therapeutic targets and strategies for colorectal cancer.

Introduction

Metabolic alterations are the hallmark of tumor progression and metastasis (1). Most cancers are characterized by increased de novo lipid biosynthesis (2). Abnormally elevated lipid synthesis provides cancer cells with membrane-building blocks, signaling lipid molecules, posttranslational modifications of proteins, and energy supply to support rapid cell proliferation (3). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is overexpressed in many solid cancers, including colorectal cancer (4). Elevated expression of FASN is associated with advanced stages of colorectal cancer and poor prognosis, suggesting that it may play a role in progression of colorectal cancer to metastatic disease (4–6). We previously reported that shRNA-mediated inhibition of FASN in colorectal cancer cells attenuated activation of MET, Akt, FAK, and paxillin, which are known to regulate adhesion, migration, and invasion. Furthermore, we found that inhibition of FASN dramatically reduces lung and hepatic metastases and inhibits tumor angiogenesis in vivo (4, 7).

Potent and selective FASN small-molecule inhibitors (TVB-2640, TVB-3166, TVB-3693, and TVB-3664) have recently been described (8–10). TVBs demonstrate significantly improved potency and selectivity and are typically well tolerated in animals as compared with older compounds such as cerulenin, orlistat, and C-75, which are widely used in preclinical studies (8). The first phase I study (3V2640-CLIN-002) showed that TVB-2640 demonstrates a favorable tolerability profile with no significant adverse events and a biomarker profile demonstrating inhibition of FASN in solid tumor patients (8, 11). TVB-2640 is currently being tested in phase II clinical trials in combination with other therapeutic agents. Its analogues, TVB-3166 and TVB-3664, have shown anticancer activity in lung, prostate, and ovarian cancer models in vitro and in vivo (9, 10). Our laboratory recently reported that oral once-daily TVB-3664 was well tolerated in mice and showed selective antitumor growth properties in colorectal cancer patient-derived xenografts (PDX; ref. 12).

Accumulating evidence suggests that many cancers, including colorectal cancer, have alterations in sphingolipids and their metabolizing enzymes (13). Sphingosine kinase (SPHK) is an evolutionary conserved lipid kinase with two mammalian isoforms, SPHK1 and SPHK2, which catalyze the phosphorylation of sphingosine and dihydrosphingosine to form sphingosine 1-phosphate (S1P) and dihydrosphingosine 1-phosphate (DH-S1P), respectively (14). The dynamic balance between intracellular S1P versus sphingosine and ceramide, and the consequent regulation of opposing signaling pathways, are important factors that determine whether cancer cells survive or die (15, 16).

Condensation of palmitate and serine to form 3-ketosphinganine, a reaction catalyzed by serine palmitoyltransferase, is believed to be the key regulatory and rate-limiting step of de novo ceramide synthesis (17), suggesting that overexpression of FASN and an increase in the flux of de novo palmitate to sphingolipid metabolism may increase the activity of SPHKs and production of DH-S1P and S1P. DH-S1P and S1P have been implicated in the regulation of proliferation, migration, and angiogenesis and strongly correlate with cancer metastasis (18, 19). S1P plays an important role in development of colitis-associated cancer (20); however, its role in colorectal cancer metastasis remains unclear. FTY-720 is an agonist of S1P receptors as well as a substrate for sphingosine kinases that have demonstrated anticancer properties in multiple preclinical studies (21).

Although it is known that upregulation of de novo lipid synthesis is critically important for malignant transformation, tumor growth, and metastasis, the contribution of de novo lipogenesis to colorectal cancer progression via sphingolipid metabolism is not known. Our study is the first to show the crucial role of de novo lipid synthesis in activating metastatic capabilities of colorectal cancer cells via upregulation of sphingolipid metabolism in colorectal cancer. The results of our study demonstrate that upregulation of FASN enhances sphingolipid metabolism and leads to activation of signaling pathways associated with an increase in adhesion, migration, and invasion in colorectal cancer. The results of this study suggest that targeting sphingolipid metabolism should be further explored as a potential target for advanced colorectal cancer.

Materials and Methods

Human primary colorectal cancer cells

Primary colorectal cancer cells in patient (Pt) 93, Pt 130, Pt 2377 primary tumor (PT) and Pt 2377 liver metastasis (LM) were isolated and established from PDX tumors using a previously described protocol of cancer cell isolation from tissues (22). Pt 2377 PT and matched Pt 2377 LM were established from the same patient. Pt 93 was diagnosed with metastatic medullary cancer (T3N1bM1); Pt 130 was diagnosed with metastatic colonic adenocarcinoma (T3N0M1a); and Pt 2377 was diagnosed with metastatic colonic adenocarcinoma (T3N0M1a), and tissues were collected from both PT and liver metastases.

For the present study, cells were maintained as monolayer culture in DMEM supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin–streptomycin. Primary Pt 93 and Pt 130 cells were authenticated using short tandem repeat DNA profiling in March 2016 (Genetica). Furthermore, the mutation profiles of 198 cancer-associated genes were determined using targeted next-generation sequencing and were matched to original patient tissues and PDX models for our ongoing studies (Markey Cancer Center Oncogenomics Shared Resource Facility).

Tissue microarray (TMA)

TMA sections were prepared from 56 tumor tissues and 57 matched normal colon mucosa of patients who were diagnosed with stage I–IV colorectal cancer and had surgery at the University of Kentucky Chandler Medical Center, Lexington, KY (TMA ID BH15991A). Scoring was carried out blindly by a pathologist; immunoreactivity score was determined by multiplication of the values for staining intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and the values for percentage of positive tumor cells (0, no positive cells; 1, 0%–10% positive; 2, 11%–50% positive; 3, 51%–100% positive).

mRNA expression

Microarrays were downloaded from the Oncomine database (PMID:22810696). RNA-seq data were downloaded from Genomic Data Commons (GDC) for TCGA COAD data (accessed March 2018) and normalized to logarithm of transcripts per kilobase million. Gene expressions between tumor and normal samples were compared using linear mixed models. Spearman correlation coefficients were used to quantify correlations between expressions of two genes.

FASN, SPHK1, and/or SPHK2 expression from microarray data (primary solid tumor, n = 215; solid tissue normal, n = 22) as well as RNA-seq data (primary solid tumor, n = 456; solid tissue normal, n = 41) in The Cancer Genome Atlas (TCGA) COAD study was analyzed.

Lipid extraction and liquid chromatography–tandem mass spectrometry

Sphingolipids were extracted and quantified as described previously (23). Extracted lipids were quantified using the Applied Biosystems 4000 Q-Trap mass spectrometer. The measurements used hexadeuterated S1P as an internal standard and were normalized to total lipid phosphorous determined after wet digestion of total lipid extracts in perchloric acid.

Plasmids/siRNAs/antibodies

FASN cDNA (ID6172538; Open Biosystem) was cloned into the pEGFP vector. Stable overexpression was established by transfecting SW480 and Pt 130 cells with pEGFP-FASN vector and gentamicin (Invitrogen) selection. SPHK1 (ID 1181 and s16957) and SPHK2 (ID 1677 and 1587) siRNAs were purchased from Thermo Fisher Scientific. Antibodies for Western blot and immunofluorescent staining were purchased from Cell Signaling Technology: FASN (#3180), p-MET (#3129), MET (#3148), p-paxillin (#2541), paxillin (#2542), p-FAK (#3283), FAK (#3285), p-Akt (#4058L), Akt (#4691L), pERK1/2 (#4695), ERK1/2 (#9107), and RhoA (#2117). Alexa Fluor 568 phalloidin was purchased from Thermo Fisher (cat. #A12380).

Western blot data were analyzed using ImageJ software. The quantification of band intensities was performed using the Gel Analysis method outlined in the ImageJ documentation: http://rsb.info.nih.gov/ij/docs/menus/analyze.html#gels.

Proliferation assay

To determine the IC50, primary colorectal cancer cells were plated in 96-well plates at a density of 3,000 cells per well, cultured overnight, and then treated with TVB-3664 (3-V Biosciences, Inc.; 7 days) and FTY-720 (48 hours) in serial dilutions and analyzed by MTT assay (Promega). For cell count experiments (Beckman-Coulter Vi Cell), cells were seeded in 12-well plates at a confluence of 50,000 cells per well and then treated with TVB-3664 (0.2 μmol/L, 7 days) and 80,000 cells/well and then treated with FTY-720 (2.5 μmol/L, 48 hours).

Cell migration assay

Cells were plated at low densities on glass-bottom dishes (Cellvis) coated with 5 μg/mL fibronectin in DMEM containing 1% FBS and 20 ng/mL HGF, and cultured for 3 hour in a CO2 incubator. Cells were tracked for 8 hours with images acquired every 20 minutes using a Nikon Biostation IMQ (20× objective). All cells that remained entirely within the field of view, did not touch other cells, and did not divide were tracked and analyzed. Cell paths were generated and analyzed using NIS-Elements AR (Nikon).

Focal adhesion staining

Cells were seeded on glass-bottom dishes precoated with 5 μg/mL fibronectin, cultured for 6 hours, then fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% Triton X-100 for 15 minutes, and finally blocked with 5% BSA in PBS for 1 hour. Cells were incubated with p-paxillin (Y118) or p-FAK (Y397) antibodies, washed three times with PBS and incubated with a Dylight 480-labeled goat anti-rabbit secondary antibody and Alexa Fluor 568 phalloidin for 30 minutes. After final washes with PBS, the images were captured with a Nikon Eclipse Ti total internal reflection fluorescence (TIRF) microscope equipped with a 60, 1.45 numerical aperture objective, CoolSNAP HQ2 charge-coupled camera (Roper Scientific). Focal adhesion area distribution was analyzed with NIS-Elements software (Nikon).

Invadopodia formation (gelatin degradation) assay

Glass-bottom dishes were coated with warm Alexa 488–conjugated gelatin (100 μg/mL) in PBS. The coated dishes were dried, fixed with prechilled glutaraldehyde solution (0.3%) for 10 minutes, washed with PBS, and then reduced with 5 mg/mL of sodium borohydride in PBS for 3 minutes. The dishes were washed extensively with PBS and incubated with DMEM containing 10% FBS and antibiotics for 2 hours. Cells were seeded at low confluence in the dishes and cultured for 10 hours, then fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and stained with Alexa 647-Src and 561-phalloidin. Images were captured using a TIRF microscope and analyzed with NIS-Elements software (Nikon).

In Vivo studies

Six- to 8-week-old NSG mice (NOD.Cg-Prkdc Il2rg/SzJ), purchased from The Jackson Laboratory, were used for implantation of tumor tissues and establishment of PDX models. For evaluation of the effect of TVB-3664 on signaling pathways associated with migration, we utilized tissue samples from Pt 2402 PDX model established from a patient diagnosed with metastatic adenocarcinoma (lung) consistent with colon PT (12). Briefly, mice were given a vehicle (30% PEG400) or 6 mg/kg of TVB-3664 by oral gavage daily for 4 weeks. Tissues were lysed and Western blot was performed. To evaluate the effect of FTY-720 on expression of migration/invasion markers, mice were injected with 5 mg/kg of FTY-720 i.p. daily for 5 weeks, and tumor tissues were lysed and analyzed by Western blot.

Results

FASN, SPHK1, and SPHK2 are highly expressed in colorectal cancer and colocalized within invadopodia in primary colorectal cancer cells

Multiple studies, including ours, have reported that FASN is overexpressed in colorectal cancer (4, 7, 24). Consistently, statistical evaluation of immunoreactivity scores showed increased FASN expression in a TMA from 56 patients with stage I–IV colorectal cancer who had surgery at UK Chandler Medical Center. TMA analysis also revealed that sphingosine kinases, SPHK1 and SPHK2, are overexpressed in primary colorectal cancer as compared with normal colon mucosa. SPHK1 expression was observed in cytosol, while SPHK2 expression was detected in both cytosol and nucleus (Fig. 1A). Statistical analysis of correlations among FASN, SPHK1, and SPHK2 expression levels revealed a significant correlation between FASN and SPHK2 (Spearman r = 0.27894, N = 57, P = 0.0374). Spearman analysis showed that expression of SPHK1 is positively correlated with expression of SPHK2 in colorectal cancer tissues (Spearman r = 0.52237, N = 57, P < 0.0001). Interestingly, expression of SPHK1 in normal colon mucosa also showed a strong positive correlation with SPHK2 expression (Spearman r = 0.80422, N = 57, P < 0.0001). The expression levels of FASN, SPHK1, and SPHK2 were also high in liver metastases as compared with matched normal colon tissues (Supplementary Fig. S1A).

Figure 1.
Figure 1.

FASN, SPHK1, and SPHK2 are highly expressed in colorectal cancer and colocalized within invadopodia in primary colorectal cancer cells. A, Immunoreactivity score of FASN, SPHK1, and SPHK2 expression was analyzed in matched normal colon mucosa and tumor tissues (n = 57 normal and 56 tumor tissues; *, P < 0.001 as compared with normal tissue; TMA ID BH15991A). B, Correlation of FASN with either SPHK1 or SPHK2 was determined based on RNA-seq data (primary solid tumor, n = 456; solid tissue normal, n = 41) from The Cancer Genome Atlas. C, Expression of FASN, SPHK1, and SPHK2 in primary Pt 93, Pt 130, and Pt 2377 PT and Pt 2377 LM colorectal cancer cells. D, Colocalization of FASN, SPHK1, and SPHK2 (anti-rabbit 647 labeled purple color secondary antibodies) with cortactin (561 labeled red color) at invadopodia degraded areas in Pt 93, Pt 130, and Pt 2377 LM cells. Background gelatin is stained with Alexa Fluor 488 (green).

To confirm these findings, we analyzed FASN, SPHK1, and SPHK2 mRNA expression using GDC (tumor, n = 456; normal, n = 41) and Oncomine (tumor, n = 215; normal, n = 22) databases. Consistent with the results from the TMA analysis, expression of FASN and SPHK1 mRNA was higher in tumor tissues as compared with normal mucosa. Interestingly, based on both data sets, expression of SPHK2 mRNA was lower in tumor tissues than in normal tissues (Supplementary Fig. S1B–S1C). However, similar to TMA data analysis, we determined significant correlation between FASN and SPHK2 mRNA expression in tumor tissues in both microarray and RNA-seq data sets. No significant correlation was observed between expression of FASN and SPHK1 mRNA (Fig. 1B; Supplementary Fig. S1D).

Pt 93, Pt 130, Pt 2377 PT, and Pt 2377 LM primary cell lines were established from PDX tumors as previously described (12). Pt 2377 PT and Pt 2377 LM cells were established from PT and LM from the same patient, Pt 2377. Western blot analysis revealed that primary colorectal cancer cells have high expression of FASN and SPHK1. Pt 2377 LM cells had the highest level of FASN expression as compared with matched Pt 2377 PT cells and with Pt 93 and Pt 130 cells. The levels of SPHK2 expression varied among primary cell lines. The high level of FASN expression was associated with the high level of SPHK2 in Pt 2377 PT and Pt 2377 LM cells (Fig. 1C).

Invadopodia are actin-rich membrane protrusions through which cancer cells grip the substratum and degrade the extracellular matrix to enable cancer cells to invade and metastasize (25, 26). Using TIRF microscopy, we demonstrated that FASN, SPHK1, and SPHK2 were colocalized within colorectal cancer cell invadopodia with cortactin, a protein associated with increased cancer cell migration and metastatic potential (27). Invadopodia structures of cells were detected as degraded dark spots of background Alexa Fluor 488–labeled gelatin (Fig. 1D). Altogether, these results demonstrate interconnection between de novo lipid synthesis and sphingolipid metabolism in human colorectal cancer and suggest FASN, SPHK1, and SPHK2 might play a role in activating invadopodia, and thus promoting colorectal cancer cell invasion and metastasis.

Inhibition of FASN is associated with a decrease in DH-S1P and S1P in colorectal cancer

Sphingolipids have emerged as bioeffector molecules, controlling various aspects of cell growth, survival, and metastasis in cancer (19, 20, 28). Using a [13C]-sodium acetate tracer, we previously demonstrated that stable inhibition of FASN significantly decreased the level of de novo production of palmitate (4). Because palmitate is a preferred substrate for the first step of sphingolipid synthesis (29), we examined the effect of FASN inhibition on sphingolipid metabolism. Figure 2A–B demonstrates that stable knockdown of FASN in HCT116 cells increases the level of sphingosine and decreases the level of S1P in both normal medium and serum-free conditions, suggesting that overexpression of FASN enhances synthesis of S1P in colorectal cancer.

Figure 2.
Figure 2.

Inhibition of FASN is associated with a decrease in DH-S1P and S1P in colorectal cancer. Total level of (A) sphingosine and (B) S1P in NTC and FASNsh HCT116 colorectal cancer cells as detected by mass spectrometry (NM, normal medium; SFM, serum-free medium; *, P < 0.05 vs. control). C, The effect of TVB-3664 treatment (0.2 μmol/L, 7 days) on the levels of DH-S1P and S1P in cell medium from Pt 93, Pt130, and Pt 2377 PT cells when cultured in NM and SFM conditions. D, The effect of TVB-3664 treatment (4 weeks, 3 mg/kg) on the levels of DH-S1P and S1P in tumor tissues in Pt 2449 PT PDXs (control n = 3; TVB n = 3). C and D, DH-S1P and S1P measurements were performed using HPLC-MS/MS technologies (MUSC Lipidomics Core, SC). Phosphate concentrations were used to normalize samples.

S1P biosynthesis is not directly linked to de novo sphingoid base transformations and is dependent on catabolic generation of sphingosine from complex sphingolipids. On the other hand, DH-S1P is considered the de novo synthesized sphingolipid and SPHKs direct the metabolic flow of newly formed sphingoid bases from ceramide formation toward the synthesis of DH-S1P (30). Therefore, we decided to test whether FASN inhibition affects the level of DH-S1P and S1P in primary colorectal cancer cells and PDX tumors. Figure 2C shows that TVB-3664 treatment of Pt 93, Pt 130, and Pt 2377 PT primary colorectal cancer cell lines significantly decreased the level of DH-S1P in cell medium in both normal and serum-free conditions. Changes in the level of S1P were not consistent among cell lines; however, the average level of S1P was lower in TVB-3664–treated cells as compared with control in both normal and serum-free conditions. Furthermore, analysis of DH-S1P levels in tumor samples from control- and TVB-3664–treated mice PDX Pt 2449 PT from our concurrent studies shows that FASN inhibition is also associated with a decrease in the DH-S1P level. However, this change was not statistically significant. We did not observe changes in the S1P level due to TVB-3664 treatment in this model (Fig. 2D).

Together, these data suggest that overexpression of FASN alters sphingolipid metabolism in both normal and serum-free conditions.

Downregulation of de novo lipid synthesis and sphingolipid metabolism inhibits proliferation in primary colorectal cancer cells

In addition to the synthesis of DNA and proteins, production of lipids is a prerequisite for cell growth and proliferation. To determine whether de novo lipogenesis and sphingolipid metabolism are required for cellular proliferation in colorectal cancer, primary colorectal cancer cells were treated with 0.2 μmol/L TVB-3664, a FASN inhibitor (9), or 2.5 μmol/L FTY-720, a structural analogue of sphingosine that alters de novo sphingolipid synthesis and decreases the level of intracellular S1P (31, 32). As shown in Fig. 3A, FASN inhibition significantly decreased proliferation of Pt 93 and Pt 2377 PT cells. TVB-3664 treatment did not have a significant effect on proliferation of Pt 130 and Pt 2377 LM. In contrast, FTY-720 treatment significantly decreased proliferation in all four colorectal cancer cell lines including Pt 130 and Pt 2377 LM cells (Fig. 3B). Together, these data suggest that sphingolipid metabolism downstream of FASN reinforces proliferative potential of primary colorectal cancer cells.

Figure 3.
Figure 3.

Inhibition of de novo lipid synthesis and sphingolipid metabolism decreases proliferation in primary colorectal cancer cells. Cellular proliferation of primary Pt 93, Pt 130, and Pt 2377 PT and Pt 2377 LM colorectal cancer cells treated with (A) 0.2 μmol/L TVB-3664 for 7 days and (B) 2.5 μmol/L FTY-720 for 48 hours (data shown as mean ± SEM. All experiments were performed three times using multiple replicates; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control).

Inhibition of FASN and sphingolipid metabolism inhibits formation of focal adhesion complexes

Focal adhesion structures are required for cellular interactions with the extracellular matrix and, thus, are crucially important in the migration process (33). We have previously reported that stable knockdown of FASN attenuates the activation of MET, Akt, FAK, and paxillin, which are known to regulate adhesion, migration, and invasion (4). To elucidate the effect of pharmacologic inhibition of de novo lipogenesis and sphingolipid metabolism on focal adhesion formation, we utilized immunofluorescent staining and TIRF microscopy. Our results demonstrate that inhibition of FASN activity decreases the number of focal adhesions in Pt 2377 PT and Pt 2377 LM cells as analyzed by quantifying p-FAK (Y397) staining (Fig. 4A and C). Similarly, FTY-720 treatment of Pt 2377 PT and Pt 2377 LM cells decreased the number of focal adhesions (Fig. 4B and C), suggesting the contribution of both metabolic pathways to focal adhesion dynamics in colorectal cancer cells. These results were confirmed by quantification of p-paxillin (Y118) staining in Pt 2377 PT, Pt 2377 LM, and Pt 130 cell lines when cells were treated with either TVB-3664 or FTY-720 (Supplementary Fig. S2A–C). To demonstrate that the effect of FTY720 on formation of focal adhesions is indeed mediated by SPHKs/S1P, Pt 130 and Pt 93 cells were transfected with SPHK1 and SPHK2 siRNAs and the number of focal adhesions was determined by quantification of p-paxillin (Y118) staining (Supplementary Fig. S3). Both inhibition of SPHK1 and SPHK2 decreased formation of focal adhesions (Supplementary Fig. S3A–S3B). Consistently, we observed attenuated activation of p-MET and p-FAK in these cell lines (Supplementary Fig. S3C).

Figure 4.
Figure 4.

Inhibition of FASN and sphingolipid metabolism diminishes formation of focal adhesion complexes. Primary Pt 2377 PT and Pt 2377 LM colorectal cancer cells were treated with (A) 0.2 μmol/L TVB-3664 for 7 days and (B) 2.5 μmol/L FTY-720 for 48 hours, stained with focal adhesion marker p-FAK (Y397) antibody (green) and phalloidin (red) and images were captured using TIRF microscopy. Representative images are shown. C, Number of focal adhesions in different size classes was quantified using NIS-Elements. D, Primary Pt 2377 PT cells were treated with 0.2 μmol/L TVB-3664 for 7 days. Primary Pt 93 colorectal cancer cells were treated with 2.5 μmol/L FTY-720 for 48 hours. Gelatin degradation ability of cells was assessed. Background gelatin is stained with Alexa Fluor 488 (green). Graphs display quantification of gelatin degradation per cell in both control and treated cells.

Because FASN, SPHK1, and SPHK2 are colocalized in invadopodia structures (Fig. 1D), we tested the effect of FASN and sphingolipid metabolism inhibition on the gelatin degradation capability of colorectal cancer cells. It has been shown that Src contributes to invadopodia formation and extracellular matrix (ECM) degradation in human cancer cells (34). To assess the gelatin degradation capability of control- and TVB-3664–treated primary colorectal cancer cells, staining for Src and Phalloidin was performed. As shown in Fig. 4D, both TVB-3664 and FTY-720 treatments decreased gelatin degradation capability of Pt 2377 PT and Pt 93 cells, suggesting the vital role of the FASN/SPHK/S1P axis in invasion of colorectal cancer cells.

Together, these findings suggest that de novo lipogenesis and sphingolipid metabolism contribute to colorectal cancer cell migration and invasion by activation of focal adhesions and invadopodia.

Inhibition of FASN and sphingolipid metabolism decreases migration of primary colorectal cancer cells

Cancer cell motility is an essential part of the metastatic cascade (35). To determine the functional importance of de novo lipid synthesis and sphingolipid metabolism in cell migration, primary colorectal cancer cells were pretreated with 0.2 μmol/L TVB-3664 (7 days) or 2.5 μmol/L FTY-720 (48 hours) and cell migration was monitored using Live-Cell Imaging for 8 hours. Data analysis demonstrated that inhibition of FASN significantly decreased cell velocity and path length parameters in Pt 93, Pt 2377 PT, and Pt 2377 LM cells and significantly decreased distance from origin in all cell lines. A significant reduction in cell directionality was observed in Pt 130 and Pt 2377 LM cell lines treated with TVB-3664 as compared with control cells (Fig. 5A; Supplementary Fig. S4A). Consistently, stable knockdown of FASN decreased directed migration and Golgi polarization in HCT116 cells (Supplementary Fig. S4C and S4D). FTY-720 treatment had a more prominent effect on inhibition of cell migration as compared with TVB-3664 treatment. FTY-720 significantly decreased all migration parameters in Pt 93, Pt 130, Pt 2377 PT, and Pt 2377 LM cells, with the exception of directionality in Pt 2377 PT, where changes did not reach statistical significance (Fig. 5B; Supplementary Fig. S4B). Inhibition of cell migration by FTY-720 (2.5 μmol/L) was accompanied by a significant decrease in activation of p-FAK, p-MET, and p-PAX in all cell lines (Fig. 5C).

Figure 5.
Figure 5.

Inhibition of FASN and sphingolipid metabolism decreases migration of primary colorectal cancer cells. Primary Pt 93 and Pt 2377 PT colorectal cancer cells were treated with (A) 0.2 μmol/L TVB-3664 for 7 days and (B) 2.5 μmol/L FTY-720 for 48 hours, and cell migration was assessed using Live-Cell Imaging. At least 25 cells from two independent experiments were analyzed. Each line with unique color represents an individual cell's migration path. Migration potential is analyzed in four parameters: velocity (V), path length (PL), distance from origin (DO), and directionality (D). C, The effect of FTY-720 treatment (48 hours) on p-Met, p-Fak, and p-paxillin activation in primary Pt 93, Pt 130, Pt 2377 PT, and Pt 2377 LM colorectal cancer cells. D, The effect of overexpression of FASN in Pt 130 cells on cell migration and activation of signaling pathways associated with cell migration. Equal numbers of cells were cultured for 24 hours prior to cell analysis.

To test whether overexpression of FASN promotes cell migration, Pt 130 and SW480 cells were stably transfected with pEGFP-control or pEGFP-FASN plasmids (7). Overexpression of FASN resulted in a significant increase in all cell migration parameters in Pt 130 and velocity, distance from origin, and path length in SW480 cell lines. Interestingly, FASN overexpression led to an increase in SPHK1 expression in both cell lines and SPHK2 expression in SW480 cells. We also observed that FASN overexpression increased activation of p-PAX and pErk1/2, which was previously reported to activate both SPHK1 and SPHK2 (refs. 36, 37; Fig. 5D; Supplementary Fig. S4E).

To further confirm that FASN-mediated changes in the signaling pathways associated with cell migration occur through upregulation of sphingolipid metabolism, we treated primary colorectal cancer cells with 0.2 μmol/L of TVB-3664 for 7 days and then treated them with 1 μmol/L S1P for 20 minutes. Consistent with the functional data (Figs. 4 and 5), TVB-3664 treatment significantly decreased activation of p-MET, p-FAK, p-PAX, p-Akt, and RhoA in colorectal cancer cells. Furthermore, inhibition of FASN expression led to a decrease in SPHK2 expression in Pt 2377 PT and Pt 2377 LM. S1P treatment rescued the inhibitory effects of TVB-3664 on p-Met, p-FAK, p-PAX, and p-Akt, suggesting that the effect of FASN on activation of these pathways is S1P dependent (Fig. 6). Similar results were observed in Pt 93 and Pt 130 cells (Supplementary Fig. S5). Together, these data suggest that upregulation of the FASN/SPHK/S1P axis promotes migratory capabilities of primary colorectal cancer cells.

Figure 6.
Figure 6.

S1P treatment rescues the effect of TVB-3664 on inhibition of the signaling pathway associated with cell migration and invasion. Activation of p-Met, p-Fak, p-Akt, RhoA, and p-paxillin was assessed in Pt 2377 PT and Pt 2377 LM primary colorectal cancer cells treated with 0.2 μmol/L TVB-3664 (7 days) and 1 μmol/L S1P (20 minutes) alone or in combination.

Inhibition of de novo lipid synthesis and sphingolipid metabolism prevents activation of prometastatic signaling pathways in PDX tumors

To test whether inhibition of the FASN/SPHKs/S1P axis has a similar effect on activation of markers associated with adhesion and migration such as p-MET, p-FAK p-PAX, RhoA, and p-Akt in vivo, we performed Western blot analysis on tumor tissues collected from PDX model Pt 2402, which was established from a patient diagnosed with metastatic colorectal cancer to lung (12). PDXs were treated with either TVB-3664 (6 mg/kg daily by oral gavage for 4 weeks) or FTY-720 (5 mg/kg daily i.p. for 5 weeks). Consistent with our findings in primary colorectal cancer cells, TVB-3664 treatment led to a significant decrease in the expression of SPHK2 and activation of p-FAK and p-paxillin; however, no effect was noted in activation of PARP or Akt and MET pathways (Fig. 7A). FTY-720 treatment did not affect the levels of SPHK1 and SPHK2, but decreased the levels of p-FAK, p-PAX, p-Akt, and RhoA (Fig. 7B). Interestingly, FTY-720 treatment also led to a significant induction of PARP cleavage, which further supports the reported effect of FTY-720 on apoptosis (32). FTY-720 treatment led to approximately 30% reduction in tumor weight (Supplementary Fig. S6).

Figure 7.
Figure 7.

Inhibition of de novo lipid synthesis and sphingolipid metabolism hinders activation of prometastatic signaling pathways in PDX tumors. A, Pt 2402 PDX tumors were treated with TVB-3664 (6 mg/kg for 4 weeks), and tumor tissues were analyzed for expression of SPHK1, SPHK2, and other signaling proteins associated with cell migration and invasion by Western blot. Band intensities were quantified using ImageJ software. B, Pt 2402 PDX tumors were treated with FTY-720 (5 mg/kg daily i.p. for 5 weeks), and tumor tissues were analyzed for expression of SPHK1, SPHK2, and other signaling proteins associated with cell migration and invasion by Western blot. C, Schematic illustration of regulation of cell adhesion, migration, and invasion by the FASN/SPHK/S1P axis. CRC, colorectal cancer.

In summary, our data suggest that de novo lipogenesis and sphingolipid metabolism activate signal transductions involved in cell adhesion, migration, and invasion in vitro and in vivo (Fig. 7C) and warrant further investigation of these metabolic pathways in regulation of adhesion, migration, and invasion in metastatic animal models.

Discussion

In this study, we investigated the role of the FASN/SPHK/S1P axis in regulation of the metastatic potential of primary colorectal cancer cells. Our group and others have reported that upregulation of lipogenic enzymes, including FASN, contributes to cancer cell progression and metastasis (4, 7, 38, 39). However, the mechanisms behind the effect of FASN on colorectal cancer growth and metastasis are not fully understood. Here, we report that alteration of sphingolipid metabolism, including overexpression and activation of SPHKs and an increase in production of DH-S1P and S1P downstream of FASN, plays a vital role in regulation of colorectal cancer cells growth, migration, and invasion.

Altered sphingolipid metabolism has been shown to contribute to cancer progression (40). According to the ceramide–sphingosine–S1P rheostat model, both ceramide and sphingosine promote apoptosis, whereas DH-S1P/S1P enhances cell survival (19, 30). In agreement with this model, we demonstrated an increase in the total sphingosine level in HCT116 cells with knockdown of FASN, while the level of S1P was significantly decreased. Our study also showed that pharmacologic inhibition of FASN is also associated with a decrease in DH-S1P in conditioned medium from primary colorectal cancer cells and in PDX tumors. We did not observe changes in the levels of S1P in PDX tumors, which can be explained by the complexity of S1P signaling in the presence of stroma in PDX tumors (15, 19). Because S1P acts extracellularly in a paracrine and autocrine manner through five specific G protein–coupled receptors S1P1–S1P5 (15, 16), another potential explanation is a change in expression/availability of S1P receptors due to alterations in a cell membrane composition associated with FASN inhibition (10).

In agreement with our presented results, TVB-3166 treatment significantly alters intermediates of de novo sphingolipid metabolism of breast cancer cells (41). Consistently, our recent studies showed that prolonged treatment of colorectal cancer PDXs with TVB-3664 also leads to significant alterations in different sphingolipids (12). Moreover, the important role of de novo lipid synthesis upstream of sphingolipid metabolism was also suggested in a study showing that overexpression of FASN is associated with inhibition of drug-induced ceramide production, thus causing drug resistance in breast cancer cells (42).

An interconnection between FASN and SPHK2 is strongly suggested by the significant correlation between expression of these enzymes in colorectal cancer tissues at mRNA and protein levels. Furthermore, our data show that inhibition of FASN significantly decreases expression of SPHK2 in vitro and in vivo, while overexpression of FASN in Pt 130 and SW480 colorectal cancer cells increases expression of both SPHK1 and SPHK2. The mechanisms of regulation of sphingolipid metabolism downstream of de novo lipid synthesis are currently under investigation in our laboratory.

SPHK1 is located mainly in the cytoplasm and the cell membrane, whereas SPHK2 is located mainly in the mitochondria, nucleus, and the endoplasmic reticulum (43). Surprisingly, we observed localization of FASN and SPHKs in invadopodia, suggesting their role in invadopodia biogenesis and cell migration and invasion. These findings further support our hypothesis that an increase in de novo lipid synthesis alters sphingolipid metabolism to promote metastatic potential of colorectal cancer cells. The role of sphingolipid metabolism in colorectal cancer metastasis is also supported by recent studies showing that upregulation of SPHK1 significantly correlates with parameters such as lymph node metastasis, LM, and advanced TNM stage (44) and SPHK2 is implicated in colorectal cancer cell proliferation and invasion by enhancing MYC expression (45). Interestingly, in agreement with the latter study, our data show that inhibition of sphingolipid metabolism using FTY-720 significantly decreases cellular proliferation in all cell lines tested, and the effect of FTY-720 is more prominent than the effect of FASN inhibition, which seems to be more cell line specific.

Our published studies showed that stable knockdown of FASN inhibits colorectal cancer metastasis by alteration of cell migration, adhesion, and secretion of angiogenic factors (4, 7). Cerulenin, an antifungal antibiotic that inhibits fatty acid and steroid biosynthesis, also suppresses LM of colon cancer in mice (46). In agreement with these studies, our data show that pharmacologic inhibition of FASN using TVB-3664 leads to suppression of colorectal cancer cell growth, migration, focal adhesion, and invadopodia formation.

Our results revealed that FTY-720 treatment is more effective not only in suppressing cellular proliferation, but also in inhibiting migration of colorectal cancer cells. These findings are consistent with another study that reported FTY-720 significantly decreases migration and invasion of glioblastoma cells (47). Our data suggest that, at least in colorectal cancer, activity of SPHKs and production of DH-S1P and S1P are more vital in cell motility than de novo–produced palmitate, which can contribute to diverse cell functions through multiple mechanisms (24). Because sphingolipids are major components of the plasma membrane and lipid raft and also act as signaling molecules, we do not exclude the possibility that intermediates of sphingolipid metabolism, other than DH-S1P and S1P, contribute to the effects of TVB-3664 and FTY-720 on cell migration (40).

Even though the effects of FTY-720 on proliferation and cell motility in cancer cells have been reported (32), its effect on metastatic capabilities of cells and the mechanisms behind it have not yet been elucidated in colorectal cancer. Our data revealed that FTY-720 blocks cell migration and invasion through suppression of focal adhesion formation and abolishment of cells' ability to form invadopodia and digest gelatin. Inhibition of formation of focal adhesions and activation of p-FAK and p-MET was also observed when expression of SPHK1 and SPHK2 is downregulated by siRNAs, suggesting that these effects indeed are mediated by the SPHKs/S1P axis. These findings are consistent with published data from our group showing that FASN upregulates MMP9 in colorectal cancer cell lines (7), and with a study demonstrating that FASN inhibition significantly decreases expression of MMP2 and MMP9 in osteosarcoma cells (48). In agreement with our in vitro studies, we observed that inhibition of FASN and sphingolipid metabolism in PDX models established from a patient who was diagnosed with colorectal cancer metastasized to lung leads to inhibition of p-FAK, p-PAX, RhoA, and p-Akt activation. We are currently exploring anticancer properties of FTY-720 in metastatic colorectal cancer models in vivo.

Our results showing that S1P treatment can rescue the effect of FASN inhibition on activation of p-FAK, p-MET, p-Akt, and p-PAX provide further evidence that the effect of TVB-3664 on the signaling pathways associated with migration and invasion, at least in part, occurs through alteration of S1P synthesis by SPHKs. Even though both S1P and DH-S1P are generated in response to activation of SPHKs, their contribution to the activation of signaling pathways may be different (14). Therefore, further studies are required to better understand the role of FASN overexpression in de novo biosynthesis of sphingolipids and its association with a selective increase in intracellular content of DH-S1P or S1P. Moreover, it is very important to understand the contribution of SPHK1 and SPHK2 to colorectal cancer progression and metastasis using genetic manipulations of these enzymes and to understand how the phosphorylation status of these enzymes contributes to their functions in colorectal cancer. We are currently pursuing these projects in our laboratory.

In recent years, lipogenic enzymes have become an attractive target for therapeutic intervention (8, 32). Our study demonstrates that FASN overexpression alters sphingolipid metabolism, increases DH-S1P and S1P production and, thus, activates signaling pathways associated with cancer cell motility and invasion to promote colorectal cancer metastasis. The compelling data demonstrating the effect of FTY-720 on cellular proliferation and metastatic potential of colorectal cancer cells provide a strong rationale to further explore the role of the FASN/SPHK/S1P axis in metastatic colorectal cancer, which could lead to identification of new therapeutic targets and strategies in colorectal cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: N. Jafari, A.J. Morris, Y.Y. Zaytseva

Development of methodology: N. Jafari, A.J. Morris, F.O. Onono, P.D. Stevens, Y.Y. Zaytseva

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N. Jafari, J. Drury, A.J. Morris, F.O. Onono, P.D. Stevens, E.Y. Lee, B.M. Evers, Y.Y. Zaytseva

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N. Jafari, A.J. Morris, F.O. Onono, J. Liu, C. Wang, E.Y. Lee, H.L. Weiss, Y.Y. Zaytseva

Writing, review, and/or revision of the manuscript: N. Jafari, A.J. Morris, F.O. Onono, B.M. Evers, Y.Y. Zaytseva

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): N. Jafari, A.J. Morris, Y.Y. Zaytseva

Study supervision: A.J. Morris, T. Gao, Y.Y. Zaytseva

Acknowledgments

The University of Kentucky Markey Cancer Center's Research Communications Office and Donna Gilbreath assisted in preparation of this article. The authors thank the Biospecimen and Tissue Procurement Shared Resource Facility (SRF), Redox Metabolism SRF, and Biostatistics and Bioinformatics SRF of the University of Kentucky Markey Cancer Center (supported by NCI grant P30 CA177558).

This work is supported by grants from 3-V Biosciences, Inc. (Y.Y. Zaytseva); ACS IRG 85-001-25 (Y.Y. Zaytseva); and NCI K22 CA197193 (Y.Y. Zaytseva), P20 GM121327 (Y.Y. Zaytseva), R01 CA133429 (T. Gao), and R01 CA208343 (B.M. Evers and T. Gao).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

  • Received February 27, 2018.
  • Revision received June 1, 2018.
  • Accepted August 17, 2018.
  • Published first August 28, 2018.

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De Novo Fatty Acid Synthesis-Driven Sphingolipid Metabolism Promotes Metastatic Potential of Colorectal Cancer
Naser Jafari, James Drury, Andrew J. Morris, Fredrick O. Onono, Payton D. Stevens, Tianyan Gao, Jinpeng Liu, Chi Wang, Eun Y. Lee, Heidi L. Weiss, B. Mark Evers and Yekaterina Y. Zaytseva
Mol Cancer Res January 1 2019 (17) (1) 140-152; DOI: 10.1158/1541-7786.MCR-18-0199
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