GLUCOSYLCERAMIDE SYNTHASE IN A NOVEL TARGET FOR CANCER TREATMENT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 耐药是化疗失败的细胞学基础。识别耐药的新靶点对于改善癌症治疗至关重要。该项目的总体目标是研究葡萄糖神经酰胺合成酶(GCS)是否是一个新的治疗靶点，以及体内有效的针对GCS的寡核苷酸是否是耐药的逆转剂。具体目的包括设计一种混合骨架寡核苷酸(MBO-asGCS)，检测其在细胞和荷瘤小鼠中的特异性和有效性，并表征其化疗敏感性机制。以往的研究表明，MBO-asGCS逆转耐药。为了描述其机制，我们假设沉默GCS可以恢复P53依赖的细胞凋亡并破坏肿瘤干细胞。我们发现沉默GCS恢复了p53突变癌细胞的P53依赖性凋亡(NCI/ADR-RES，缺失外显子-5的21个碱基对)。MBO-asGCS可上调野生型P53、p21和Bax(P53的下游效应因子)的表达水平，并可诱导细胞和肿瘤细胞的凋亡。GCS基因的导入减少了P53依赖的细胞凋亡。MBO-asGCS通过神经酰胺调节的RNA剪接恢复P53。我们发现GCS的过度表达与乳腺癌干细胞(BCSCs)的增加密切相关。耐药细胞株MCF-7/Dox和NCI-ADR/RES细胞中CD44+/CD24-/ESA+表型的BCSCs分别是MCF-7细胞株的5倍和3倍。在BCSCs中，GCS酶活性是其他分选的非干细胞的2倍。应用MBO-asGCS沉默GCS可显著减少MCF-7/Dox细胞中BCSCs的数量。接种富含BCSCs的NCI/ADR-RES和NCI/ADR-RE/GCS细胞的小鼠均出现侵袭性肿瘤，而注射NCI-ADR/asGCS细胞的小鼠未见肿瘤或转移。MBO-asGCS治疗后可显著减少NCI-ADR/RES肿瘤的BCSCs数量。这些结果表明，鞘糖脂参与了对肿瘤干细胞的调控。沉默GCS可以消除BCSCs，后者可以逆转耐药性，防止肿瘤复发。有了这些发现，我们的进一步研究将回答神经酰胺如何恢复野生型P53的表达，以及GSLS如何调节癌症干细胞的形成。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Drug resistance is the cellular basis of chemotherapy failure. Identifying novel targets in drug resistance is essential for improving cancer treatment. The overall goals of this project are to examine whether glucosylceramide synthase (GCS) is a novel therapeutic target, and an in vivo efficient oligonucleotide against GCS is a reversal agent for drug resistance. The specific aims include to design a mixed-backbone oligonucleotide (MBO-asGCS) and examine its specificity and efficacy in cells and in tumor-bearing mice, and to characterize its chemo-sensitivity mechanisms. Previous works have showed that MBO-asGCS reverses drug resistance. To characterize its mechanisms, we hypothesize that silencing GCS can restore p53-dependent apoptosis and disrupt cancer stem cells. We found that silencing GCS restored p53 dependent-apoptosis in p53 mutant cancer cells (NCI/ADR-RES, deleted 21 base pairs in exon-5). MBO-asGCS increased the expression levels of wild-type p53, p21 and Bax (down-steam effectors of p53), and drug-induced apoptosis in cells and tumors. Introduction of GCS gene reduced p53-depedent apoptosis. MBO-asGCS restores p53 through ceramide modulated-RNA splicing. We found that GCS overexpression was interrelated to the increase of breast cancer stem cells (BCSCs). The BCSCs with CD44+/CD24-/ESA+ phenotype were increased by 5-fold, and 3-fold in drug-resistant MCF-7/Dox and NCI-ADR/RES cell lines, as compared to MCF-7 cell lines, respectively. In BCSCs, GCS enzyme activity was 2-fold greater than other sorted non-stem cells. Silencing GCS by using MBO-asGCS significantly decreased the numbers of BCSCs in MCF-7/Dox cells. Aggressive tumors were found in all mice inoculated with NCI/ADR-RES and NCI/ADR-RE/GCS cells that were BCSCs-enriched; however, no tumor or metastasis was observed in mice injected with NCI-ADR/asGCS cells. MBO-asGCS significantly decreased the numbers of BCSCs isolated from tumors of NCI-ADR/RES after treatment. These results demonstrate that glycosphingolipids are involved in the regulation of cancer stem cell. Silencing GCS eliminates BCSCs that can reverse drug resistance as well as prevent tumor relapse. With these findings, our further studies will answer how ceramide restores wild-type p53 expression and how GSLs modulate the formation of cancer stem cells.