ELOVL5 and IGFBP6 genes modulate sensitivity of breast cancer cells to ferroptosis.

Introduction: Relapse of breast cancer is one of the key obstacles to successful treatment. Previously we have shown that low expression of ELOVL5 and IGFBP6 genes in breast cancer tissue corresponded to poor prognosis. ELOVL5 participates directly in the elongation of polyunsaturated fatty acids (PUFAs) that are considered to play an important role in cancer cell metabolism. Thus, in this work we studied the changes in lipid metabolism in breast cancer cells with reduced expression of either ELOVL5 or IGFBP6 gene. Methods: MDA-MB-231 cells with a stable knockdown of either ELOVL5 or IGFBP6 gene were used in this study. Transcriptomic and proteomic analysis as well as RT-PCR were utilized to assess gene expression. Content of individual fatty acids in the cells was measured with HPLC-MS. HPLC was used for analysis of the kinetics of PUFAs uptake. Cell viability was measured with MTS assay. Flow cytometry was used to measure activation of apoptosis. Fluorescent microscopy was utilized to assess accumulation of ROS and formation of lipid droplets. Glutathione peroxidase activity was measured with a colorimetric assay. Results: We found that the knockdown of IGFBP6 gene led to significant changes in the profile of fatty acids in the cells and in the expression of many genes associated with lipid metabolism. As some PUFAs are known to inhibit proliferation and cause death of cancer cells, we also tested the response of the cells to single PUFAs and to combinations of docosahexaenoic acid (DHA, a n-3 PUFA) with standard chemotherapeutic drugs. Our data suggest that external PUFAs cause cell death by activation of ferroptosis, an iron-dependent mechanism of cell death with excessive lipid peroxidation. Moreover, both knockdowns increased cells' sensitivity to ferroptosis, probably due to a significant decrease in the activity of the antioxidant enzyme GPX4. Addition of DHA to commonly used chemotherapeutic drugs enhanced their effect significantly, especially for the cells with low expression of IGFBP6 gene. Discussion: The results of this study suggest that addition of PUFAs to the treatment regimen for the patients with low expression of IGFBP6 and ELOVL5 genes can be potentially beneficial and is worth testing in a clinically relevant setting.