Expression of Long-chain Fatty Acyl-CoA Synthetase 4 in Breast and Prostate Cancers Is Associated with Sex Steroid Hormone Receptor Negativity

Abstract

Previous studies have shown that key enzymes involved in lipid metabolic pathways are differentially expressed in normal compared with tumor tissues. However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be established. Fatty acid synthase is overexpressed in a variety of cancers, including breast and prostate. The purpose of the present study was to examine the expression patterns of additional lipid metabolic enzymes in human breast and prostate cancers. This was accomplished by analysis of published expression databases, with confirmation by immunoblot assays. Our results indicate that the fatty acid-activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), is differentially expressed in human breast cancer as a function of estrogen receptor alpha (ER) status. In 10 separate studies, ACSL4 messenger RNA (mRNA) was overexpressed in ER-negative breast tumors. Of 50 breast cancer cell lines examined, 17 (89%) of 19 ER-positive lines were negative for ACSL4 mRNA expression and 20 (65%) of 31 ER-negative lines expressed ACSL4 mRNA. The inverse relationship between ER expression and ACSL4 expression was also observed for androgen receptor status in both breast and prostate cancers. Furthermore, loss of steroid hormone sensitivity, such as that observed in Raf1-transfected MCF-7 cells and LNCaP-AI cells, was associated with induction of ACSL4 expression. Ablation of ACSL4 expression inMDA-MB-231 breast cancer cells had no effect on cell proliferation; however, sensitivity to the cytotoxic effects of triacsin C was increased three-fold in the cells lacking ACSL4.

Figure 1

Expression of ACSL4 mRNA in human breast tumor samples. (A) Comparison of ASCL4 mRNA levels in ER-negative (solid bars) versus ER-positive human (open bars) breast tumors in ten independent gene expression profile data sets (denoted by study first author). Box-and-whisker plots indicate median, lower, and upper quartiles, and the smallest and largest values. Differences between ER-positive and ER-negative values are significant with P < .001 for all studies represented. Data provided by Oncomine as normalized expression units. (B) Relationship between ACSL4 expression and ESR1 (ER) expression in the study by Miller et al. [11]. The samples evaluated comprised 34 ER-negative and 213 ER-positive tumors. Data provided by Oncomine as normalized expression units.

Figure 2

Effect of ER status on expression of ACSL4 in human breast cancer cell lines. Data provided by Array Express represents the log to the base 2 of the original data. (A) Expression levels of ACSL mRNA in human breast cancer cells. The expression data for 19 ER-positive (open bars) and 31 ER-negative (solid bars) breast cancer cell lines was compared for the five known isoforms of mammalian ACSL (1, 3, 4, 5, and 6). Values shown are the mean ± SD. (B) Individual ACSL4 mRNA expression values for each cell line. The horizontal line indicates the cutoff, as determined by immunoblot, of ACSL4 positivity. (C) ACSL4 and ACSL1 protein expression in selected breast cancer cell lines. Immunoblot analyses were performed as described in Materials and Methods.

Figure 3

Effect of AR status on expression of ACSL4. (A) Relationship between AR and ACSL4 mRNA expression in 77 ER-negative breast tumor samples as reported in a study by Wang et al. [14]. Data provided by Oncomine. (B) Expression of ACSL4 protein in prostate cancer cell lines. Immunoblot analyses were performed as described in Materials and Methods. (C) Relationship between AR and ACSL4 mRNA levels in 98 human prostate tumors as reported by Holzbeierlein et al. [27]. Data provided by Gene Expression Omnibus.

Figure 4

Induction of ACSL4 expression in MCF-7 cells constitutively expressing Raf-1. Data provided by Gene Expression Omnibus. C indicates control; EI, long-term E₂-independent growth; erbB-2, cells overexpressing constitutively active c-erbB-2; MEK, cells overexpressing constitutively active MEK; Raf-1, cells overexpressing constitutively active Raf-1; EGFR, cells overexpressing ligand-activatable EGFR. Results shown are derived from the means of three separate determinations.

Figure 5

Effect of ablation of ACSL4 expression on proliferation of MDA-MB-231 cells. Cells were treated for 48 hours with either a control or ACSL4-specific siRNA as described in the text. Cells were then harvested and replated in multiwell plastic dishes and treated as described. (A) Immunoblot of cellular protein after a 48-hour transfection with no addition (-), control siRNA (C), or ACSL4-siRNA (A). Methods were as described in the text. (B) Growth curve of cells after 48 hours of treatment with control or ACSL4-siRNA. (C) Effects of triacsin C on relative cell number after 48 hours of treatment with control or ACSL4-siRNA. Relative cell numbers were assayed as described in the text. Values shown are means ± SD.

Figure 6

Correlation of ACSL4 mRNA levels with time to distant metastasis in ER-negative tumors. Kaplan-Meier analysis of 77 ER-negative tumors from node-negative patients who did not receive systematic adjuvant therapy. The log-rank test evaluates whether there are significant differences between tumors harboring high versus low ACSL4 levels. The univariate Cox test evaluates the association of ACSL4 with patient outcome, when the expression values are treated as a continuous variable (i.e., without making a cut). Data provided by Oncomine from a study by Wang et al. [14].