Glycan control of stem cell-associated pathways in pancreatic cancer

描述（由申请人提供）：异常表面糖基化是肿瘤细胞的一个定义特征，然而聚糖对致癌作用的功能贡献在很大程度上被忽视。与肿瘤相关的主要变化之一是β 6唾液酸的增加，这是一种通过ST 6 Gal-I糖基转移酶添加到选定受体上的带负电荷的糖。在初步研究中，我们首次发现，ST 6 Gal-I升高， 胰腺癌（PC），并且ST 6 Gal-I上调导致PC细胞获得“癌症干细胞”（CSC）的特征性行为，CSC是一种干细胞样细胞群，其固有地具有转移性，对细胞死亡具有抗性，并且对化疗药物不敏感。我们还做出了里程碑式的发现，即PC细胞中的ST 6 Gal-I上调诱导CSC相关转录因子Sox 9的表达。Sox 9诱导是胰腺导管腺癌的关键起始事件。本申请的目标是确定ST 6 Gal-I在以下方面的作用：（i）由新的ST 6 Gal-I-NFkB-Sox 9信号传导轴指导的CSC样行为;（ii）遗传工程小鼠中的PC发病机制;和（iii）PC细胞对吉西他滨（一线PC治疗）的抗性。为了实现这些目标，我们将采用独特的细胞模型系统、具有强制ST 6 Gal-I过表达或缺失的自发PC鼠模型以及患者来源的原发性肿瘤和肿瘤异种移植物组织。这项研究将扩展糖如何调节肿瘤细胞行为的基础知识，并通过将ST 6 Gal-I鉴定为耐药细胞的标志物和恢复治疗反应的潜在临床靶点来影响患者护理。目的1：ST 6 Gal-I通过Sox 9信号传导促进驱动发病机制的干细胞样PC行为。将使用患者来源的原代PC细胞或具有强制ST 6 Gal-I表达或敲低的PC细胞系来检验以下假设：ST 6 Gal-I活性通过NF kB-Sox 9刺激信号传导以促进CSC行为，包括自我更新;肿瘤球状体生长;和肿瘤引发潜力。将在患者胰腺癌组织中检查ST 6 Gal-I、活化的NFkB和Sox 9的共表达，并且将利用具有胰腺特异性ST 6 Gal-I敲入或敲除的自发性PC鼠模型来建立ST 6 Gal-I在PC发展和进展中的作用。目的2：ST 6 Gal-I对化疗耐药性的贡献和作为治疗靶点的潜力。将用吉西他滨处理具有强制ST 6 Gal-I表达或敲低的患者来源的PC细胞或细胞系，以检验ST 6 Gal-I活性促进化学抗性的假设。为了显示表达ST 6 Gal-I的细胞在体内选择性地在吉西他滨处理中存活，将在从用或不用吉西他滨处理的小鼠收获的患者来源的肿瘤异种移植物中定量ST 6 Gal-I表达。将从吉西他滨处理的肿瘤异种移植物中收获活细胞，以获得体内选择的化疗耐药群体。然后将这些细胞进行ST 6 Gal-I敲低，然后进行吉西他滨处理，以确定是否可以在具有已知稳定抗性的细胞群中恢复吉西他滨敏感性。

DESCRIPTION (provided by applicant): Abnormal surface glycosylation is a defining feature of a tumor cell, however the functional contribution of glycans to carcinogenesis has been largely ignored. One of the predominant tumor-associated changes is an increase in �6 sialic acid, a negatively-charged sugar added to selected receptors by the ST6Gal-I glycosyltransferase. In preliminary studies we find for the first time that ST6Gal-I is elevated in pancreatic cancer (PC), and that ST6Gal-I upregulation causes PC cells to acquire behaviors characteristic of "cancer stem cells" (CSCs), a stem-like cell population inherently metastatic, resistant to cell death, and insensitive to chemotherapeutic drugs. We have also made the landmark discovery that ST6Gal-I upregulation in PC cells induces expression of the CSC-associated transcription factor, Sox9. Sox9 induction is a pivotal initiating event in pancreatic ductal adenocarcinoma. The goals of this application are to establish a role for ST6Gal-I in: (i) CSC-like behaviors directed by a novel ST6Gal-I-NFkB-Sox9 signaling axis; (ii) PC pathogenesis in genetically engineered mice; and (iii) PC cell resistance to gemcitabine, a first-line PC treatment. To achieve these objectives we will employ unique cell model systems, spontaneous PC murine models with forced ST6Gal-I overexpression or deletion, and patient-derived primary tumor and tumor xenograft tissues. This research will extend fundamental knowledge of how sugars regulate tumor cell behavior, and also impact patient care by identifying ST6Gal-I as a marker for resistant cells and potential clinical target for restoring treatment response. AIM 1: ST6Gal-I signaling through Sox9 promotes stem-like PC behaviors that drive pathogenesis. Patient-derived primary PC cells, or PC cell lines with forced ST6Gal-I expression or knockdown, will be used to test the hypothesis that ST6Gal-I activity stimulates signaling through NFkB-Sox9 to promote CSC behaviors including self-renewal; tumor spheroid growth; and tumor-initiating potential. Co-expression of ST6Gal-I, activated NFkB and Sox9 will be examined in patient pancreatic adenocarcinoma tissues, and spontaneous PC murine models with pancreas-specific ST6Gal-I knockin or knockout will be utilized to establish a role for ST6Gal-I in PC development and progression. AIM 2: ST6Gal-I's contribution to chemoresistance and potential as a therapeutic target. Patient-derived PC cells or cell lines with forced ST6Gal-I expression or knockdown will be treated with gemcitabine to test the hypothesis that ST6Gal-I activity promotes chemoresistance. To show that ST6Gal-Iexpressing cells selectively survive gemcitabine treatment in vivo, ST6Gal-I expression will be quantified in patient-derived tumor xenografts harvested from mice treated with or without gemcitabine. Viable cells will be harvested from the gemcitabine-treated tumor xenografts to obtain an in vivo-selected chemoresistant population. These cells will then be subjected to ST6Gal-I knockdown, followed by gemcitabine treatment, to determine whether gemcitabine sensitivity can be restored in a cell population with known stable resistance.