Ceramide glycosylation by glucosylceramide synthase (GCS) and breast cancer stem cells.

摘要

Cancer stem cells (CSCs) initiate tumorigenesis and are the betacauses of metastasis and relapse. CSCs, rather than other differentiated cells in tumors display resistance to cytotoxicity of anticancer drugs, but little is known whether by-product of chemotherapy can induce cancer stem cells leading to chemotherapy failure. Glucosylceramide synthase (GCS) is a limiting enzyme regulating the synthesis of glycosphingolipids that play an essential role in the maintenance of embryonic stem cells. The purpose of this study was to identify and characterize the effect of ceramide glycosylation in the formation and maintenance of breast cancer stem cells (BCSCs). It was found that GCS overexpression was interrelated to the increment BCSCs and drug resistance in human breast cancer MCF-7 cell lines after doxorubicin induction. In MCF-7/Dox (doxorubicin-resistant) cells, GCS protein was increased by 8-fold accompanied with enhanced enzyme activity, as compared to wild-type MCF-7 cells. The BCSCs with CD44+/CD24-/ESA+ phenotype were increased by 5-fold in MCF-7/Dox cells as compared to MCF-7 cells. Silencing GCS by using mixed-backbone oligonucleotide (MBO-asGCS) significantly decreased the numbers of BCSCs more than 2-fold in MCF-7/Dox cells. In the soft agar colony formation assay, MCF-7/Dox cells showed significantly higher colonies formed as compare to the MCF-7 cells. In addition, MCF-7/Dox tumors grew aggressively in athymic nude mice compared to MCF-7 or MCF-7/Dox cells treated with MBO-asGCS (4 mg/kg, intraperitoneal injection). MBO-asGCS treatment significantly decreased the numbers of BCSCs in MCF-7/Dox cells as compared with control groups. BCSCs sorted from MCF-7/Dox cells displayed 3-fold higher GCS enzyme activity, and formed 2-fold more colonies, as compare with other non-stem cell subsets. The BCSCs cells had significantly higher tumorigenicity and metastases as compared to non-stem cells (CD44-/CD24-) in athymic nude mice. The western blot results indicated that the BCSCs highly expressed stem cells mediators such as FGF-2, OCT-4 and CD44, compared to the CD44-/CD24 - cells. More interestingly, doxorubicin treatment (1 mg/kg, once a week for 40 days) considerably increased BCSCs, by 2-fold in tumors; MBO-asGCS treatment eliminated the tumor growth and reduced metastasis due to reduction of BCSCs in vivo. Comparison of glycosphingolipids and gene profile of stem cells indicated GCS or globo-series glycosphingolipids upregulated FGF1, FGFR1, ALDH2, ISL1, MSX1, SIGMARI, PPARD, CDH2 and CCND2, while it downregulated SOX-2 and JAG1 to accumulate BCSCs during the course of chemotherapy. Further assessment of signaling pathway indicated that formation and maintenance of BCSCs relied on activated cSrc kinase and beta-catenin. This consequently led to increment of CD44, OCT-4 and FGF-2 expression, which involved in the maintenance of BCSCs. These results, for the first time, demonstrate that ceramide glycosylation by GCS accumulates BCSCs formation and causes tumor progression. Silencing of GCS that eliminates BCSCs is a new approach to prevent and treat drug-resistant tumors and relapse.