Fatty acid synthase, a key enzyme of de novo lipogenesis, is an attractive therapeutic target in cancer. The novel fatty acid synthase inhibitor, TVB-3664, shows anti-cancer activity in multiple cancers including colorectal cancer; however, it is unclear whether uptake of exogeneous fatty acids can compensate for the effect of fatty acid synthase inhibition. This study demonstrates that inhibition of fatty acid synthase selectively upregulates fatty acid translocase (CD36), a fatty acid transporter, in multiple colorectal cancer models including colorectal cancer cells with shRNA mediated knockdown of fatty acid synthase and genetically modified mouse tissues with heterozygous and homozygous deletion of fatty acid synthase. Furthermore, human colorectal cancer tissues treated with TVB-3664 show a significant and selective upregulation of CD36 mRNA. shRNA-mediated knockdown of CD36 and inhibition of CD36 via sulfosuccinimidyl oleate, a chemical inhibitor of CD36, decreased cell proliferation in vitro and reduced tumor growth in subcutaneous xenograft models. Isogenic cell populations established from patient derived xenografts and expressing high levels of CD36 show a significantly increased ability to grow tumors in vivo. The tumor-promoting effect of CD36 is associated with an increase in the levels of pAkt and survivin. Importantly, combinatorial treatment of primary and established colorectal cancer cells with TVB-3664 and sulfosuccinimidyl oleate shows a synergistic effect on cell proliferation. In summary, our study demonstrates that upregulation of CD36 expression is a potential compensatory mechanism for fatty acid synthase inhibition and that inhibition of CD36 can improve the efficacy of fatty acid synthase-targeted therapy.

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

Altered fatty acid metabolism continues to be an attractive target for therapeutic intervention in cancer. We previously found that colorectal cancer (CRC) cells with a higher metastatic potential express a higher level of fatty acid translocase (CD36). However, the role of CD36 in CRC metastasis has not been studied. Here, we demonstrate that high expression of CD36 promotes invasion of CRC cells. Consistently, CD36 promoted lung metastasis in the tail vein model and GI metastasis in the cecum injection model. RNA-Seq analysis of CRC cells with altered expression of CD36 revealed an association between high expression of CD36 and upregulation of MMP28, a novel member of the metallopeptidase family of proteins. Using shRNA-mediated knockdown and overexpression of CD36, we confirmed that CD36 regulates MMP28 expression in CRC cells. siRNA-mediated knockdown of MMP28 decreases invasion of CRC cells, suggesting that MMP28 regulates the metastatic properties of cells downstream of CD36. Importantly, high expression of MMP28 leads to a significant decrease in active E-cadherin and an increase in the products of E-cadherin cleavage, CTF1 and CTF2. In summary, upregulation of CD36 expression promotes the metastatic properties of CRC via upregulation of MMP28 and an increase in E-cadherin cleavage, suggesting that targeting the CD36–MMP28 axis may be an effective therapeutic strategy for CRC metastasis.

Altered lipid metabolism is a potential target for therapeutic intervention in cancer. Overexpression of Fatty Acid Synthase (FASN) correlates with poor prognosis in colorectal cancer (CRC). While multiple studies show that upregulation of lipogenesis is critically important for CRC progression, the contribution of FASN to CRC initiation is poorly understood. We utilize a C57BL/6-Apc/Villin-Cre mouse model with knockout of FASN in intestinal epithelial cells to show that the heterozygous deletion of FASN increases mouse survival and decreases the number of intestinal adenomas. Using RNA-Seq and gene set enrichment analysis, we demonstrate that a decrease in FASN expression is associated with inhibition of pathways involved in cellular proliferation, energy production, and CRC progression. Metabolic and reverse phase protein array analyses demonstrate consistent changes in alteration of metabolic pathways involved in both anabolism and energy production. Downregulation of FASN expression reduces the levels of metabolites within glycolysis and tricarboxylic acid cycle with the most significant reduction in the level of citrate, a master metabolite, which enhances ATP production and fuels anabolic pathways. In summary, we demonstrate the critical importance of FASN during CRC initiation. These findings suggest that targeting FASN is a potential therapeutic approach for early stages of CRC or as a preventive strategy for this disease.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the USA. Development of metastasis is the most common cause of death in these patients. Fatty acid synthase (FASN) and Sphingosine Kinases 1 and 2 (SPHK1 and 2) are overexpressed in many cancers, including CRC. However, the contribution of FASN-mediated upregulation of sphingolipid metabolism to CRC metastasis and potential of these pathways as targets for therapeutic intervention remains unknown. The purpose of this study was to determine (i) expression of FASN, SPHK1 and SPHK2 in human CRC tissues, (ii) the effect of upregulation of FASN on sphingolipid metabolism and (iii) functional significance of the FASN/SPHK axis in advanced CRC. Methods: Expression of FASN, SPHK1 and SPHK2 was assessed in a CRC tumor microarray (matched normal colon and tumor; 56 cases) by immunohistochemistry. Sphingolipids were measured by mass spectrometry. Primary CRC cells were established from CRC patient-derived xenograft (PDX) tumors and treated with TVB-3664, a novel FASN inhibitor, or FTY-720, an S1P mimetic that inhibits SPHKs and S1P receptors. Cellular proliferation was measured using a cell counter. Migration capabilities of cells were assessed by live cell imaging using Nikon BioStation. Invadopodia and focal adhesions were assessed by total internal reflection fluorescence microscopy. Tumor tissues were implanted into 6 to 8-week-old NOD scid gamma mice to establish PDX models. Expression of proteins involved in adhesion, migration and invasion were assessed by western blot. Results: SPHK1 and SPHK2 were overexpressed in CRC as compared to normal mucosa and expression of FASN correlates with expression of SPHK2 (Spearman's r=0.27894, p=0.0374). Furthermore, FASN and SPHKs co-localized within invadopodia of primary CRC cells. Moreover, FASN inhibition decreased expression of SPHK2 and the level of Sphingosine-1-phosphate (S1P) in primary and established CRC cells. Inhibition of de novo lipogenesis using TVB-3664 or FTY-720 significantly inhibited proliferation, migration, focal adhesion formation and gelatin degradation ability of primary CRC cells. Inhibition of the FASN/SPHK/S1P axis was accompanied by a decrease in activation of p-MET, p-FAK, and p-Paxilin in vitro and in vivo. S1P treatment rescued FASN-mediated inhibition of these proteins suggesting that FASN promotes metastatic properties of CRC cells, in part, through an increase in sphingolipid metabolism. Conclusion: Upregulation of the FASN/SPHK/S1P axis promotes CRC progression by enhancing cellular proliferation, adhesion and migration. Therefore, this study provides a strong rationale for further investigation of the interconnection of de novo lipogenesis and sphingolipid metabolism that would potentially lead to identification of new therapeutic targets and strategies for CRC. Citation Format: Naser Jafari, James Drury, Andrew J. Morris, Fredrick O. Onono, Payton D. Stevens, Tianyan Gao, Eun Y. Lee, Heidi L. Weiss, B Mark Evers, Yekaterina Zaytseva. De novo fatty acid synthesis-driven sphingolipid metabolism promotes metastatic potential of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1437.