Figures and data
Ectopic expression of Snail confers epithelial cells nitidine chloride resistance via induction of ABCA1 expression
(A) Enhancement of growth inhibitory effect of nitidine chloride (NC) on human renal carcinoma cells by co-treatment with an ABCA1 inhibitor cyclosporin A (CsA). Cells were grown with or without 20 μM NC and 10 μM CsA for 24 h, and cell number was determined with CCK-8. Fold change in cell number by NC was shown as relative to controls without NC. Results are shown as mean of at least four independent experiments ± SD (Tukey-Kramer’s multiple comparison test: **, p<0.01; ***, p<0.001; ****, p<0.0001).
(B) Immunoblot analysis of whole cell lysate of human renal carcinoma cell lines.
(C) Comparison of ABCA1 expression between normal tissue and primary tumor of indicated subtypes of renal cancers analyzed using UCSC Xena. Bars indicate means (Welch’s t-test: ***, p<0.001; ****, p<0.0001).
(D) Immunohistochemistry of a surgically extracted renal tissue from a patient with Fuhrman grade 3 primary ccRCC, indicating upregulation of ABCA1 in the lesion site. The ABCA1 staining was verified by the accumulation of known signals in renal tubules and glomeruli in normal tissue (Yang et al, 2010). Scale bars, 250 μm (left), 50 μm (right).
(E) Phase contrast images of EpH4 wild-type cells (top) and Snail-overexpression cells (bottom, EpH4-Snail). Scale bar, 50 μm.
(F) Immunofluorescence images of EpH4 and EpH4-Snail cells. Scale bars, 20 μm.
(G) Acquisition of NC resistance by exogenous expression of Snail in EpH4 cells and enhancement of NC effect on EpH4-Snail cells by co-treatment with CsA. Shown are means of three independent experiments ± SD (Tukey-Kramer’s multiple comparison test: *, p<0.05; ****, p<0.0001).
(H-J) Immunoblot analyses of whole cell lysates of (H) EpH4, EpH4-Snail, E-cadherin KO and α-catenin KO EpH4 cells, (I) MDCK II cells expressing KRAS G12V treated with 5 μg/ml TGFβ for 0, 3 and 8 days and (J) human esophageal carcinoma cell lines TE-15 and TE-8. Phase contrast images are also shown on top (J). Scale bars, 50 μm.
Alteration of cellular cholesterol distribution induces ABCA1 expression in hybrid E/M cells
(A) Immunofluorescence images of cells stained with the LXRα+β antibody. Scale bars, 10 μm.
(B) Immunoblot analyses of whole cell lysates of EpH4-Snail cells treated with 1 μM GSK-2033 for 24 h.
(C) Immunoblot analyses of whole cell lysates of EpH4-Snail cells treated with indicated concentration of simvastatin or 0.1% DMSO (‘0’ μM) in DMEM supplemented with 10% normal or lipid-free fetal bovine serum (FBS) for 48 h.
(D) Subcellular distribution of Chol stained with filipin. Scale bar, 10 μm.
(E) Live cell images of lipid droplets (LDs) visualized by Lipi-Green dye.
(F) Quantification of LD signal per cell. The Lipi-Green signals of all optical sections were summed and divided by the number of cells counted using DAPI images. Shown are means of three different fields of view ±SD.
(G) Comparison of cholesterol (Chol) content relative to phospholipid concentration.
Shown are means of three biological replicates ± SDs. (F,G) p-value of Student’s t-test are shown.
Effects of Snail-induced EMT on sphingomyelin profile
(A) Comparison of sphingomyelin (SM) content relative to phospholipid content.
(B) Comparison of cholesterol (Chol)/SM ratio between EpH4 and EpH4-Snail cells.
(C) Fatty acid composition of SM determined using LC-MS. All peaks corresponding to d18:1-even fatty acid-SMs were included in the analysis and each peak value is expressed as a percentage. n.d., peak not detected.
(D) Pie charts of SM chain length profile from c. SMs were classified into long-chain fatty acid (LCFA, orange) (≤20) and very long-chain fatty acid (VLCFA, purple) (>20) SMs.
(E) Chain length specificity of Elovls and CerSs responsible for fatty acid profile of sphingolipids reported (Levy & Futerman, 2010; Sassa & Kihara, 2014). Elovls catalyze a rate-limiting step of fatty acid elongation cycle, and CerSs catalyze N-acylation of sphingoid bases to produce ceramides.
(F) Comparison of transcription levels of ELOVLs and CERSs. Notable decreases in expressions of ELOVL7 and CERS3 in EpH4-Snail cells are indicated by asterisks.
(G) Immunoblot analyses of whole cell lysates of mouse epithelial (MTD-1A, CSG) and fibroblast (L-929, NIH3T3) cells.
(H) Comparison of Chol/SM ratio of EpH4, EpH4-Snail and normal fibroblasts.
(I) Comparison of Chol/SM ratio between TE-15 and TE-8.
(J) Scatter plot of Chol content against SM content used in B, H and I. Dashed lines indicate Chol/SM = 1.0 (blue) and 1.5 (red). The region of Chol/SM > 1.5 is shown with red background.
(K) Possible mechanism of LD enlargement induction through decrease in SM. In EpH4, a certain amount of Chol is sequestered through interaction with SM. In EpH4-Snail, SM-unbound form of Chol is increased, detected by cellular cholesterol sensors, internalized and stored in LDs. In A, B, C, H and I, data are means of three independent experiments ± SDs (Student’s t-test (A,B,C,I) or Dunnett’s test (H): n.s., p ≥ 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001).
Selective growth inhibition of Snail-positive cells by an ACAT inhibitor TMP-153
(A) Schematic image of molecular mechanisms for handling excess cholesterol to avoid cell death. Cells eliminate excess cholesterol through efflux mediated by transporters including ABCA1 or isolation into lipid droplets (LD) via esterification of cholesterol by acyl-CoA: cholesterol acyltransferases (ACATs) to avoid cell death. Cyclosporin A (CsA) and TMP-153 inhibits ABCA1 and ACATs, respectively.
(B-E), Effects of treatments with cyclosporin A (B,D) or TMP-153 (C,E) to cellular growth of EpH4 and EpH4-Snail (B,C) or human kidney cancer cell lines (D,E). Cells were treated with drugs for 24 h and relative cell number to 0.1% DMSO control was determined by CCK-8. Results are shown as mean of at least three independent experiments ± SD.
(F-H) TMP-153 inhibits the growth of tumor xenografts composed of Snail-positive renal cancer 786-O cells. Experiments were performed as described in Materials and Methods. In brief, 50 mg/kg TMP-153 was administered intraperitoneally to nude mice bearing 786-O tumor xenografts. After 21 days of the first administration, the overall appearance (F) and the excised tumors (G) of control and treated mice were compared. (H) Tumor growth curve in the xenograft model. Results are shown as mean of four biological replicates ± SD (Student’s t-test: *, p < 0.05; **, p < 0.01).
Cancer types that exhibit tumor specific upregulation of ABCA1 transcription
(A-C) Comparison of mRNA transcription of ABCAI (A,B) and Snail (C) in different cancer types. Expression data from GDC TCGA database were analyzed using USCS Xena platform. Significant differences (***,p < 0.001) by Welch’s t-tests are shown.
Comparison of ABCA1 expression in different progression grades of kidney cancer cases
Immunohistochemistry of surgically extracted renal tissues from patients with Fuhrman grade 13 primary ccRCC, indicating elevation of ABCAI signal at the lesion site of higher-grade ccRCC. All samples were collected from independent patients. The ABCAI staining was verified by the accumulation of known signals in renal tubules and glomeruli in normal tissues (32). Scale bars, 100 µm.
Contribution of transcription factors involved in ABCA1 transcription in epithelial cells
(A) Immunoblot analysis of whole cell lysates of EpH4-Snail (without treatment) and EpH4 cells treated with inhibitors of PBK-Akt pathway (5 µM GSK2334470: PDKI, 50 µM LY294002: PBK, or I µM Wortmannin: PBK for 24 h).
Comparison of SM and cholesterol content
(A,B) Comparison among EpH4, EpH4-Snail, and fibroblasts.
(C,D) Comparison between esophageal cancer cell lines TE-15 (Snail-negative) and TE-8 (Snail-positive). N = 3, ** p < 0.0l, * p < 0.05, n.s.,p 2: 0.05 by Tukey’s multiple comparison test (A,B) and Student’s t-test
Selective growth inhibition of a Snail positive esophageal cancer cell line by combined treatment of cyclosporin A and TMP-153
(A-C) Effects of treatments with cyclosporin A (a), TMP-153 (b) or combination of 50 µM cyclosporin A and indicated concentration of TMP-153 (c) to cellular growth of human esophageal cancer cell lines TE-15 and TE-8. Cells were treated with drugs for 24 hand relative cell number to 0.1% DMSO control was determined by CCK-8. n 2: 3, error bars, SDs. Significant differences between TE-15 and TE-8 by unpaired one-sided Student’s t-test are shown (*,p<0.05).
(D-F), Combined treatment of cyclosporin and TMP-153 inhibits the growth of TE-8 tumor xenografts. Experiments were performed as described in Materials and Methods. In brief, IO mg/kg cyclosporin A and I 0 mg/kg TMP-153 were simultaneously administered intraperitoneally to nude mice bearing TE-8 tumor xenografts. After 36 days of the first administration, the overall appearance (D), the excised tumors (E), and the tumor size (F) of control and treated mice were compared. n = 5, error bars, SDs. Significant differences by unpaired one-sided Student’s t-test are shown(*, p < 0.05).
