Abstract A09: ACSL4 is a target of the 17beta-estradiol/estrogen-receptor alpha pathway in mammary carcinoma cell lines
Authors: Belkaid Anissa , Ouellette Rodney J. , Surette Marc E. 
Published: May 2016

Abstract
Polyunsaturated fatty acids (PUFA) are important building blocks for cellular membranes and are incorporated into membrane glycerophospholipids to sustain many cellular functions. Newly absorbed PUFA like arachidonic acid (AA) and eicosapentaenoic acid (EPA) are rapidly transformed to acyl-CoAs by members of the acyl-CoA synthetase family (ACSL1, 3, 4, 5 and 6) prior to their incorporation into membrane phospholipids. However, there is little information on the impact of estrogen on PUFA uptake and ACSL expression in estrogen receptor-positive (ER+ve) breast cancer cells. In the current study, the effect of 17 beta-estradiol on PUFA uptake and ACSL expression was measured in (ER+ve) mammary carcinomas cell lines. Stimulation of estrogen-starved ER+ve MCF-7 and T47D mammary carcinomas cells with 17 beta-estradiol resulted in a significant increase in the cellular uptake of exogenous AA and EPA. This was accompanied by a three-fold increase in the expression the ACSL4 isoform measured by western blot in both MCF-7 and T47D cells, but not in immortalized MCF-10A mammary epithelial cells. There was no measured change in the expression of the other ACSL isoforms. ER alpha silencing with siRNA reversed the 17 beta-estradiol-induced ACSL4 expression in both cells lines showing for the first time that hormonal stimulation of ER+ve breast cancer cells is a driver for ACSL4 expression. However, ACSL4 mRNA levels measured by qPCR were unchanged by 17 beta-estradiol treatment, and no change in mRNA stability was measured with 17beta-estradiol treatment as assessed in actinomycin D-treated cells. Conversely, the half-life of ACSL4 protein after treatment with 17 beta-estradiol was extended from 8±0.5 hours in untreated cells to 26±2 hours in 17 beta-estradiol treated cells as assessed following incubation in the absence or presence of cycloheximide. ER alpha silencing with specific siRNA reversed this half-life extension. SHP2 is a tyrosine phosphatase that regulates ACSL4 expression, however the investigation of possible SHP2-mediated changes in ACSL4 in 17 beta-estradiol-treated cells incubated or not with the SHP2 inhibitor NSC87877 showed no difference in ACSL4 expression. Importantly, ACSL4 silencing with siRNAs reversed the 17 beta-estradiol-induced uptake of AA and EPA in both MCF-7 and T47D cells, indicating that ACSL4 was responsible for the enhanced uptake of these PUFA in ER+ve breast cancer cells. Moreover, ACSL4 silencing completely reversed 17 beta-estradiol-induced cell migration as assessed by an in vitro wound healing assay. Overall, these results demonstrate that 17 beta-estradiol enhances the ability of ER+ve mammary carcinoma cells to incorporate PUFA that are important membrane building blocks, via the induction of ACSL4 protein stability. The requirement of enhanced ASCL4 expression for 17 beta-estradiol-induced cell migration suggests that it is a potential therapeutic target in ER+ve mammary carcinoma cells.
This work was supported by the Canadian Breast Cancer Foundation, the Canadian Institutes of Health Research and the Canada Research Chairs Program.
Citation Format: Anissa Belkaid, Rodney J. Ouellette, Marc E. Surette. ACSL4 is a target of the 17beta-estradiol/estrogen-receptor alpha pathway in mammary carcinoma cell lines. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A09.