Ceramide glycosylation determines the stemness of cancer stem cells

描述（由申请人提供）：神经酰胺糖基化决定了癌症干细胞的干细胞性（stemness）。具有播散性转移的耐药性是癌症死亡的一个原因;在乳腺癌中，每年最终导致约40，500名美国妇女死亡。一旦治疗失败，肿瘤通常迅速进展，并且这些肿瘤与癌症干细胞的富集通常与患者预后不良相关。因此，启动肿瘤发生并驱动肿瘤进展的癌症干细胞可能是化疗失败的根本原因。肿瘤干细胞与正常成体干细胞的区别在于它们的肿瘤行为。众所周知，抗癌药物可以杀死正常的干细胞，如骨髓干细胞，这是严重副作用的来源;然而，相反，一些证据表明，抗癌药物可能会扩大癌症干细胞的分数或数量。我们的长期目标是了解如何特异性地操纵癌症干细胞用于预防和治疗目的。本文提出的研究的目的是确定在化疗过程中选择的抗癌药物如何影响乳腺癌干细胞（BCSC）群体。据报道，葡萄糖神经酰胺合酶（GCS）在转移性乳腺癌中过表达，是预测化疗反应不良的危险因素。胚胎干细胞至关重要地需要由GCS催化的神经酰胺糖基化以保持其多能性。然而，关于GCS可能参与调节癌症干细胞的情况知之甚少。我们的假设是，通过GCS进行的神经酰胺糖基化是使肿瘤富集BCSC的关键步骤，并且一旦化疗失败，癌症干细胞的富集驱动肿瘤快速进展和转移。本申请是根据我们自己的初步数据和先前发表的工作制定的，其中发现GCS在转移性乳腺癌中过表达，并且GCS的沉默能够逆转耐药性以及防止BCSC富集。我们将：（1）确定抗癌药物对BCSC富集和正常干细胞凋亡的影响;（2）确定GCS催化的神经酰胺糖基化是控制BCSC积累和正常干细胞凋亡的关键过程;如果是这样，（3）确定神经酰胺糖基化促进BCSC形成的信号通路。体外研究的结果将在体内进一步探索，在暴露于抗癌药物的原位肿瘤中，采用基因沉默和过表达策略，并重点关注GCS在增加BCSC形成方面的拟议作用，对比骨髓干细胞和正常乳腺上皮干细胞的凋亡作用。结果将通过对接受化疗患者的肿瘤和骨髓样本的研究进一步证实。最后，将检查通过反义寡核苷酸或小分子破坏神经酰胺糖基化以实现原位肿瘤中BCSC的减少的治疗功效，其中成功将为这种通过缺失BCSC选择性消除癌症干细胞的独特方法提供有效性。 关键鞘糖脂。完成拟议的研究将向参与的学生介绍癌症干细胞和药物发现的新概念和先进技术，从而大大提高他们的生物医学科学教育。

DESCRIPTION (provided by applicant): Ceramide glycosylation determines the stemness of cancer stem cells Drug resistance with disseminated metastases is a cause of cancer death; in breast cancer, it ultimately claims approximately 40,500 American women per year. Tumors often progress rapidly once treatments fail, and enrichment of these tumors with cancer stem cells generally correlates with poor patient prognosis. Thus, cancer stem cells that initiate tumorigenesis and drive tumor progression might be a root cause of chemotherapy failure. Cancer stem cells are distinguished from normal adult stem cells by their tumorous behaviors. It is well known that anticancer drugs can kill normal stem cells, such as bone marrow stem cells-a source of severe side-effects; in contrast, however, some evidence suggests that anticancer drugs might act to expand cancer stem cell fractions or numbers. Our long-term goal is to understand how cancer stem cells can be specifically manipulated for preventive and therapeutic purposes. The objective of the research proposed herein is to determine how selected anticancer drugs affect breast cancer stem cell (BCSC) populations during the course of chemotherapy. It has been reported that glucosylceramide synthase (GCS) is overexpressed in metastatic breast cancers, and is a risk factor predicting poor chemotherapy response. Embryonic stem cells crucially require ceramide glycosylation catalyzed by GCS in order to retain their pluripotency. Little is known, though, concerning the possible involvement of GCS in regulating cancer stem cells. Our hypothesis is that ceramide glycosylation by GCS is a key step enabling tumor enrichment with BCSCs, and that enrichment with cancer stem cells drives rapid tumor progression and metastasis once chemotherapy fails. This application is formulated from our own preliminary data and previously published work, in which it was found that GCS is over-expressed in metastatic breast cancer, and that silencing of GCS was able to reverse drug resistance as well as prevent BCSC enrichment. We will: (1) identify the effects of anticancer drugs on enrichment of BCSCs and apoptosis of normal stem cells; (2) determine GCS-catalyzed ceramide glycosylation is a key process governing the accumulation of BCSCs and apoptosis of normal stem cells; and if so, (3) determine the signaling pathway(s) by which ceramide glycosylation promotes BCSC formation. Findings from in-vitro studies will be further explored in vivo, in orthotopic tumors exposed to anticancer drugs, employing gene silencing and overexpression strategies, and focusing on proposed role(s) of GCS in increasing the formation of BCSCs vs. contrasting apoptotic effects on bone marrow stem cells and normal mammary epithelial stem cells. Results will be further corroborated via studies of tumor and bone marrow samples from patients undergoing chemotherapy. Lastly, the therapeutic efficacy of disrupting ceramide glycosylation by an antisense oligonucleotide or a small molecule in order to effect reductions in BCSCs in orthotopic tumors will be examined, wherein success will lend validity to this unique approach for selectively eliminating cancer stem cells via deletion of key glycosphingolipids. Completion of the proposed studies will introduce new concepts and advanced technologies in cancer stem cells and drug discovery to students who participate, thus greatly enhancing their biomedical sciences education.