Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer drug resistance through cSrc and β-catenin signaling

Background: Drug resistance is the outcome of multiple-gene interactions in cancer cells under stress of anticancer agents. MDR1 overexpression is most commonly detected in drug-resistant cancers and accompanied with other gene alterations including enhanced glucosylceramide synthase (GCS). MDR1 encodes for P-glycoprotein that extrudes anticancer drugs. Polymorphisms of MDR1 disrupt the effects of P-glycoprotein antagonists and limit the success of drug resistance reversal in clinical trials. GCS converts ceramide to glucosylceramide, reducing the impact of ceramide-induced apoptosis and increasing glycosphingolipid (GSL) synthesis. Understanding the molecular mechanisms underlying MDR1 overexpression and how it interacts with GCS may find effective approaches to reverse drug resistance. Results: MDR1 and GCS were coincidently overexpressed in drug-resistant breast, ovary, cervical and colon cancer cells; silencing GCS using a novel mixed-backbone oligonucleotide (MBO-asGCS) sensitized these four drug-resistant cell lines to doxorubicin. This sensitization was correlated with the decreased MDR1 expression and the increased doxorubicin accumulation. Doxorubicin treatment induced GCS and MDR1 expression in tumors, but MBO-asGCS treatment eliminated "in-vivo" growth of drug-resistant tumor (NCI/ADR-RES). MBO-asGCS suppressed the expression of MDR1 with GCS and sensitized NCI/ADR-RES tumor to doxorubicin. The expression of P-glycoprotein and the function of its drug efflux of tumors were decreased by 4 and 8 times after MBO-asGCS treatment, even though this treatment did not have a significant effect on P-glycoprotein in normal small intestine. GCS transient transfection induced MDR1 overexpression and increased P-glycoprotein efflux in dose-dependent fashion in OVCAR-8 cancer cells. GSL profiling, silencing of globotriaosylceramide synthase and assessment of signaling pathway indicated that GCS transfection significantly increased globo series GSLs (globotriaosylceramide Gb3, globotetraosylceramide Gb4) on GSL-enriched microdomain (GEM), activated cSrc kinase, decreased beta-catenin phosphorylation, and increased nuclear beta-catenin. These consequently increased MDR1 promoter activation and its expression. Conversely, MBO-asGCS treatments decreased globo series GSLs (Gb3, Gb4), cSrc kinase and nuclear beta-catenin, and suppressed MDR-1 expression in dose-dependent pattern. Conclusion: This study demonstrates, for the first time, that GCS upregulates MDR1 expression modulating drug resistance of cancer. GSLs, in particular globo series GSLs mediate gene expression of MDR1 through cSrc and beta-catenin signaling pathway.