| Glucosylceramide synthase (GCS) is a rate-limiting enzyme that regulates glycosphingolipid (GSL) synthesis. Though GCS is a factor responsible for drug resistance, its roles in tumor metastasis, stem cells and cell cycle regulation are still not completely understood. Therefore firstly we investigated its role in cancer metastasis as disseminated metastasis is frequently observed in cancer patients failed in chemotherapy, although it is unclear by which mechanism drug resistance promotes tumor metastasis. Our study reports that GCS potentiates cancer cell metastasis. Drug-resistant MCF-7/Dox cells, which overexpressed GCS, exhibited significantly higher cell migration in wound healing, adhesion to and invasion in extracellular matrix, and expressed 11-fold greater matrix metalloproteinase 9 (MMP-9) than in its drug-sensitive counterpart, human breast cancer MCF-7 cells and other cells. Introduction of GCS gene into MCF-7 cells conferred their metastatic potency, as cell activities in wound healing, matrigel-adhesion and matrigel-invasion increased approximately by 2-fold, compared to mock transfection. Conversely, silencing of GCS expression using mixed-backbone oligonucleotide (MBO-asGCS, 100 nM) substantially diminished the metastatic potency of MCF-7/Dox cells by decreasing their wound healing, matrigel adhesion and invasion. Moreover, MBO-asGCS treatment eliminated lung metastasis of tumors generated from breast cancer stem cells. Further mechanistic studies revealed that enhanced ceramide glycosylation particularly increased globotriaosylceramide (Gb3) in glycosphingolipid-enriched microdomains (GEMs) and upregulated MMP-9, fibroblast growth factor 2 and vascular endothelial growth factor through cSrc/beta-catenin signaling pathway, aiding cell migration and invasion. This clearly demonstrates the role of GCS in potentiating tumor metastasis.;Our second study investigated the probable mechanism which shields the breast cancer stem cells (BCSCs) against doxorubicin but not the bone marrow stem cells (BMSCs), as myelosuppression and drug resistance is often associated with cancer patients undergoing chemotherapy. We report that higher endogenous levels of GCS in BCSCs than in BMSCs is responsible for this shielding effect. It was found that Doxorubicin (Dox) decreased the numbers of BMSCs (ABCG2 +) and the sphere formation in a dose-dependent fashion in isolated bone marrow cells. In tumor-bearing mice, Dox treatments (5 mg/kg, 6 days) decreased the numbers of BMSCs and white blood cells; conversely, those treatments increased the numbers of BCSCs (CD24-/CD44+/ESA +) more than threefold in the same mice. Furthermore, therapeutic-dose of Dox (1 mg/kg/week, 42 days) decreased the numbers of BMSCs while it increased BCSCs in vivo. Breast cancer cells, rather than bone marrow cells, highly expressed GCS, which was induced by Dox and correlated with BCSC pluripotency. These results indicate that Dox may have opposite effects, suppressing BMSCs versus expanding BCSCs and GCS is one of the determinants of the differentiated response of bone marrow and cancer cells.;Several studies report that inhibition of GCS causes cell cycle arrest and leads the cells to apoptosis; however no studies so far have shown how GCS directly regulates the cell cycle. In this study we report that GCS may regulate the cell proliferation through one of its downstream regulators, EST AI798611. EST A1798611 (EST) is an expressed sequence tag that has been isolated from human prostate; however, its functions in cells have never been reported. Based on the expression profiles of microarray in human breast cancer cells, we found the mRNA level of EST was significantly increased with overexpression of GCS, whereas the EST level was decreased with GCS silencing in cells treated with Actinomycin D. Interestingly, it was found that the EST significantly enlarged cell size and promoted tumorsphere formation in EST-overexpressed cells (MCF-7/EST), as compared to mock-transfection control (MCF-7) cells or EST-silenced cells (MCF-7/as-EST). In animal study, tumors were formed in all mice inoculated with both MCF-7 and MCF-7/EST cells, but no tumors were found in the MCF-7/as-EST group. Furthermore, it was found that the EST has less effect on cell responding to doxorubicin that could cause DNA-damage and apoptosis; however, the EST increased the IC50 value for paclitaxel more than 10-fold in the MCF-7/EST cells, as compared to either MCF-7 or MCF-7/as-EST cells. Cell cycle analysis showed that EST overexpression caused 2-fold increase in the number of cells in S-phase thus prolonging it. Tissue-array assay suggested that the protein levels of the EST were increased in some of the tumors. These results thus pinpoint that this EST has a crucial role in modulating cell proliferation. Together our studies show clear implication of GCS in promoting cancer cell metastasis, in assessing bone marrow suppression and in promoting cancer cell proliferation. |
