ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

Abstract

Background To comprehensively understand the molecular mechanism of tamoxifen resistance (TamR) acquisition by epigenetically regulated genes, it is essential to identify pivotal genes by genome-wide methylation analysis and verify their function in xenograft animal model and cancer patients. Methods The MCF-7/TamR breast cancer cell line was developed and a genome-wide methylation array was performed. The methylation and expression of ELOVL2 was validated in cultured cells, xenografted tumor tissue, and breast cancer patients by methylation-specific PCR, qRT-PCR, Western blot analysis, and immunohistochemistry. Deregulation of ELOVL2 and THEM4 was achieved using siRNA or generating stable transfectants. Tam sensitivity, cell growth, and apoptosis were monitored by colorimetric and colony formation assay and flow cytometric analysis. Pathway analysis was performed to generate networks for the differentially methylated genes in the MCF-7/TamR cells and for the differentially expressed genes in the ELOVL2-overexpressing cells. Results Genome-wide methylation analysis in the MCF-7/TamR cells identified elongation of very-long chain fatty acid protein 2 (ELOVL2) to be significantly hypermethylated and downregulated, which was further verified in the tumor tissues from TamR breast cancer patients (n = 28) compared with those from Tam-sensitive (TamS) patients (n = 33) (P < 0.001). Immunohistochemical analysis of tissues from cancer patients showed lower expression of ELOVL2 in the TamR than TamS tissues. Growth of the MCF-7/TamR cells overexpressing ELOVL2 was retarded in cell culture and also in xenograft tumor tissue. Strikingly, ELOVL2 attenuated resistance to Tam up to 70% judged by the colorimetric and colony formation assay and xenograft mouse model. ELOVL2 contributed to the recovery of Tam sensitivity by regulating a group of genes in the AKT and ERα signaling pathways, e.g., THEM4, which plays crucial roles in drug resistance. Conclusions ELOVL2 was hypermethylated and downregulated in TamR breast cancer patients compared with TamS patients. ELOVL2 is responsible for the recovery of Tam sensitivity. AKT- and ERα-hubbed networks are pivotal in ELOVL2 signaling, where THEM4 contributes to the relaying ELOVL2 signaling. This study implies that deregulation of a gene in fatty acid metabolism can lead to drug resistance, giving insight into the development of a new therapeutic strategy for drug-resistant breast cancer.

Matrix Metalloproteinase-1 (MMP1) Upregulation through Promoter Hypomethylation Enhances Tamoxifen Resistance in Breast Cancer

“…In our previous study, genome-wide methylation was analyzed in MCF-7/tamR cells to identify epigenetically regulated genes responsible for drug resistance [ 29 ]. In the current study, we focused on hypomethylated genes, as numerous oncogenes have been reported to be upregulated by hypomethylation.…”

“…The aforementioned in vitro observations were tested in vivo by evaluating whether MMP1 can drive tumor cell growth and drug resistance in a xenograft mouse model. First, MMP1 expression was examined in the tumor tissues obtained after xenografting MCF-7 and MCF-7/tamR cells into nude mice, as described in our previous study [ 29 ]. In situ immunohistochemistry of tumor sections validated the elevated MMP1 expression in MCF-7/tamR cells compared to that in parental MCF-7 cells ( Figure 4 A,B).…”

“…Human breast cancer MCF-7 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Tamoxifen-resistant MCF-7 (MCF-7/tamR) cells were developed as described in our previous study [ 29 ] and further used for in vitro cell assays as well as in vivo xenografts. All cells were cultured in RPMI 1640 medium (Gibco BRL, Carlsbad, CA, USA) supplemented with 2% penicillin/streptomycin (Capricorn, Ebsdorfergrund, Germany) and 10% fetal bovine serum (Capricorn) at 37 °C in 5% CO 2 .…”

“…All tissues were acquired from patients operated on between 2012 and 2013. The clinical information of the patients has been previously described [ 29 ].…”

Multi-Omic Approaches to Breast Cancer Metabolic Phenotyping: Applications in Diagnosis, Prognosis, and the Development of Novel Treatments

“…The authors examined the relationship between ELOVL2 expression and metastatic relapse-free in a BC cohort with a follow-up of ten years, resulting in the discovery of an association between lower ELOVL2 expression levels and shorter metastasis-free survival and higher tumor grade [116]. A recent study investigating the molecular mechanisms of tamoxifen resistance in BC confirmed these findings and described lower ELOVL2 expression in tamoxifenresistant models and ELOVL2 downregulation in patients with tamoxifen resistance [135].…”