GLUCOSYLCERAMIDE SYNTHASE IN A NOVEL TARGET FOR CANCER TREATMENT

葡萄糖神经酰胺合成酶作为癌症治疗的新靶点

• 批准号: 8168132
• 负责人: Yong-Yu Liu
• 金额: $ 10.98万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168132
• 关键词: Aftercare; Apoptosis; Base Pairing; Cell Line; Cells; Ceramide glucosyltransferase; Ceramides; Computer Retrieval of Information on Scientific Projects Database; Drug resistance; Exons; Failure; Funding; Genes; Glycosphingolipids; Goals; Grant; Institution; MCF7 cell; Mixed-Backbone Oligonucleotide; Mus; Neoplasm Metastasis; Oligonucleotides; Pharmaceutical Preparations; Phenotype; RNA Splicing; Regulation; Relapse; Research; Research Personnel; Resources; Sorting - Cell Movement; Source; Specificity; Steam; Stem cells; TP53 gene; United States National Institutes of Health; Work; base; cancer cell; cancer stem cell; cancer therapy; chemotherapy; design; enzyme activity; improved; in vivo; malignant breast neoplasm; mutant; neoplastic cell; new therapeutic target; novel; overexpression; prevent; tumor

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 耐药性是化学疗法衰竭的细胞基础。识别耐药性的新靶标对于改善癌症治疗至关重要。该项目的总体目标是检查葡萄糖基酶合酶（GCS）是否是一种新型的治疗靶标，并且对GCS的体内有效寡核苷酸是耐药性的逆转剂。具体目的包括设计混合背骨寡核苷酸（MBO-ASGC）并检查其在细胞和肿瘤小鼠中的特异性和功效，并表征其化学敏感性机制。先前的工作表明，MBO-ASGC逆转了耐药性。为了表征其机制，我们假设沉默的GC可以恢复p53依赖性凋亡并破坏癌症干细胞。我们发现，沉默的GC恢复了p53突变癌细胞（NCI/ADR-RES，在外显子5中删除21个碱基对）中的依赖性凋亡。 MBO-ASGC提高了野生型p53，p21和Bax（p53的下降效应子）的表达水平，并在细胞和肿瘤中造成药物诱导的凋亡。 GCS基因的引入降低了p53多肌凋亡。 MBO-ASGC通过神经酰胺调制的RNA剪接恢复p53。我们发现GCS过表达与乳腺癌干细胞（BCSC）的增加相关。与MCF-7细胞系相比，具有CD44+/CD24-/ESA+表型的BCSC增加了5倍，在药物耐药的MCF-7/DOX和NCI-ADR/RES细胞系中增加了3倍。在BCSC中，GCS酶活性比其他排序的非茎细胞大2倍。通过使用MBO-ASGC对GC进行沉默，可显着减少MCF-7/DOX细胞中BCSC的数量。在接种富含BCSC的NCI/ADR-RES和NCI/ADR-RE/GCS细胞的所有小鼠中都发现了侵略性肿瘤；但是，在注射NCI-ADR/ASGCS细胞的小鼠中未观察到肿瘤或转移。 MBO-ASGC显着减少了治疗后NCI-ADR/RES肿瘤分离的BCSC数量。这些结果表明，糖脂脂参与癌症干细胞的调节。沉默的GC消除了可以逆转耐药性并防止肿瘤复发的BCSC。通过这些发现，我们的进一步研究将回答神经酰胺如何恢复野生型p53表达以及GSL如何调节癌症干细胞的形成。