UGT8/GalCer-dependent resistance of breast cancer cells to drug-induced apoptosis is potentially regulated by the LIM/homeobox protein LHX6

Ceramide galactosyltransferase (UGT8) is overexpressed in basal-like breast cancer (BC) tumors and is associated with an increased risk of lung metastasis. UGT8 synthesizes galactosylceramide (GalCer), an anti-apoptotic molecule that promotes BC cell survival within the tumor microenvironment and enhances resistance to anticancer drugs. In this study, we aimed to elucidate the molecular mechanisms underlying UGT8 overexpression in BC cells. UGT8 promoter constructs and deletion mutants were generated by PCR. Promoter activity was assessed using a dual-luciferase assay, and key regulatory sequences were identified by electrophoretic mobility shift assay (EMSA). Candidate transcription factors were predicted using the JASPAR database and validated by qPCR, Western blotting, and immunohistochemistry. GalCer levels were measured by thin-layer chromatography (TLC) binding assay, and LHX6-DNA interactions were analyzed by surface plasmon resonance. Apoptotic cells were detected using the Thermo Dead Cell Apoptosis Kit. UGT8 promoter activity was significantly higher in UGT8-positive MDA-MB-231 cells than in UGT8-negative T47D and MCF-7 cells, indicating that UGT8 gene expression is regulated at the transcriptional level. Sequential promoter deletions combined with EMSA localized a key regulatory region (- 1132 to - 1618 bp), termed the UGT8 response element (UGT8RE). In silico analysis identified potential transcription factors, among which the LIM/homeobox protein LHX6 was markedly upregulated in MDA-MB-231 cells. Site-directed mutagenesis of two predicted LHX6 binding sites (LHX6BS1 and LHX6BS2) demonstrated that LHX6BS2 is essential for UGT8 promoter activity. RNAi-mediated inhibition of LHX6 reduced UGT8 expression and GalCer synthesis, thereby sensitizing MDA-MB-231 cells to doxorubicin-induced apoptosis. The LIM/homeobox protein LHX6 may regulate the UGT8 expression in BC cells. Targeting LHX6 decreases UGT8 expression and GalCer synthesis, thereby sensitizing MDA-MB-231 cells to doxorubicin. Given UGT8's role in cell survival and drug resistance, inhibition of LHX6 may represent a promising therapeutic strategy for drug-resistant BC.