Delta-6 desaturase FADS2 is a tumor-promoting factor in cholangiocarcinoma

Abstract

Cholangiocarcinoma is a fatal disease with limited therapeutic options. We screened genes required for cholangiocarcinoma tumorigenicity and identified FADS2, a delta-6 desaturase. FADS2 depletion reduced in vivo tumorigenicity and cell proliferation. In clinical samples, FADS2 was expressed in cancer cells but not in stromal cells. FADS2 inhibition also reduced the migration and sphere-forming ability of cells and increased apoptotic cell death and ferroptosis markers. Lipidome assay revealed that triglyceride and cholesterol ester levels were decreased in FADS2-knockdown cells. The oxygen consumption ratio was also decreased in FADS2-depleted cells. These data indicate that FADS2 depletion causes a reduction in lipid levels, resulting in decrease of energy production and attenuation of cancer cell malignancy.

Keywords: FADS2; cholangiocarcinoma; cholesterol esters; delta‐6 desaturase; triglyceride.

FIGURE 1

Fatty acid desaturase 2 (FADS2) is enriched in serially transplanted xenograft cancer cells (A) Schematic outline of the serially transplanted xenograft model. (B) FADS2 mRNA at each passage point was determined by microarray analysis. (C) FADS2 was knocked down by siRNA, and FADS2 mRNA expression was measured by real‐time PCR. **p < 0.01, ***p < 0.001. (D, E) FADS2 knockdown and control cells were transplanted into NOG mice, and the tumor volumes were measured weekly. (D) CHOL1 cells; (E) TFK‐1 cells. p‐Values were calculated by comparing the nonlinear regression curves of the two groups. (F) Cell proliferation was measured after treatment with siFADS2 using an Incucyte system. Arrowheads indicate the treatment of siRNA. (G) Representative images of FADS2 mRNA expression in cholangiocarcinoma tissues based on RNAscope assay. FADS2 positive appeared as brown spots. Bar (low‐magnification images), 20 μm. Bar (high‐magnification images), 5 μm.

FIGURE 2

Fatty acid desaturase 2 (FADS2) depletion reduces cell proliferation activity. (A) Cell proliferation was measured after treatment with FADS2 inhibitor SC26196 using MTT assay. (B) Western blotting. CHOL1 and TFK‐1 cells were harvested at 9 and 4 days after SC26196 addition, respectively. The values indicate the band densities quantified using ImageJ software. (C) Sphere formation assay with SC26196 in CHOL1 and TFK‐1 cells using MTT assay. (D) Scratch‐wound assay. The migrated areas were measured after 24 h with or without SC26196 treatment. The wound‐healing size was quantified using ImageJ software and is shown in the bar graphs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3

Fatty acid desaturase 2 (FADS2) inhibitor alters characteristics of cancer cells. (A) Apoptotic cells were counted by flow cytometry. Apoptotic cells were detected by propidium iodide and/or annexin V‐positive staining. (B) Apoptosis assay. Caspase 3/7 activity was measured with a luminometer. (C) Western blotting. Cells were treated with FADS2 inhibitor or erastin (a ferroptosis inducer) for 3 days, and GPX4 protein expression was determined by Western blotting. The values indicate the band densities quantified using ImageJ software. (D) The GSH/GSSG ratio was measured. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4

Triglyceride is altered in fatty acid desaturase 2 (FADS2)‐depleted cells. (A) Lipidomics in FADS2 knockdown CHOL1 cells. Lipids were quantitatively identified by supercritical fluid chromatography–tandem mass spectrometry (SFC‐MS/MS) spectrometry and the relative expression of siFADS2s (n = 9) to siControl (n = 3) was calculated. TG, triglycerides; CE, cholesterol esters; FA, free fatty acids; MG, monoglycerides; DG, diglycerides; LPC, lysophosphatidylcholine; PG, phosphatidylglycerol; PC, phosphatidylcholines; SM, sphingomyelin; PE; phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; LPE, lysophophatidylethanolamine; HexCer, hexosylceramine; Cer, ceramide. (B) Volcano plot. Fold change was calculated based on the values of siFADS2 to siControl samples. Triglycerides are marked with red circles, and the others are gray. (C) CHOL1 and TFK‐1 cells were stained by Lipi‐Green, and the intensity was measured by flow cytometry. Representative images of CHOL1 cells are shown (scale bar = 10 μm). (D) The oxygen consumption ratio was measured with a flux analyzer. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.