A novel, transferable sialylation-mediated mechanism of chemoradioresistance in GI cancer

摘要-联合放化疗用于治疗多种胃肠道（GI）癌症，包括 直肠癌直肠癌在美国每年影响40，000人。大约85%的患者有 对治疗的反应不完全或不佳，增加了复发的风险。我们发现反应差的人 含有对治疗更具抗性的亚克隆，并且酶ST 6 Gal-1在这些亚克隆中富集， 克隆ST 6 Gal-1是一种高尔基体糖基转移酶，它将带负电荷的糖，唾液酸（SA）， 特定的蛋白质被分配到细胞表面。SA可以对细胞的结构和功能产生深远的影响， proteins. ST 6 Gal-1是癌细胞中最普遍上调的糖基转移酶之一。公司简介 已经显示通过唾液酸化特异性地促进肿瘤细胞存活和抗性。此外，ST 6 Gal-1 已经在癌细胞制造的细胞外囊泡（ECV）中发现。ECV是具有脂质膜的颗粒 含有RNA和蛋白质货物;因此，它们是可转移抗性的潜在介质， 癌症亚克隆尚未研究ST 6 Gal-1和ECV在放化疗抗性中的作用。 本申请的总体目标是确定ST 6 Gal-1在先天和可转移抗性中的作用 到直肠癌的放化疗基于我们的初步数据，我们假设ST 6 Gal-1介导了 直肠癌个体亚克隆对放化疗的抗性，这种抗性被转移到 通过ECV扩展抗性将其传递给其它亚克隆，并且该抗性由ST 6 Gal-1调节 BACE 1的裂解。我们已经发现，在直肠癌模型中，在用抗肿瘤药物治疗后，ST 6 Gal-1增加。 放化疗我们将以3个目的研究我们的假设：目的1 -确定ST 6 Gal-1在肿瘤细胞中的作用。 人直肠癌的放化疗抵抗性。我们假设ST 6 Gal-1导致治疗 通过抑制细胞凋亡来提高放化疗后的抵抗力。我们将采用细胞分选、测序和shRNA技术， 接近。我们还将进行研究，以调查其在患者样本中的功能。AIM 2 -确定是否 携带ST 6 Gal-1的ECV在直肠癌的亚克隆之间转移对放化疗的抗性。我们 假设ECV作为载体从亚克隆到亚克隆传递对放化疗抗性 通过运输ST 6 Gal-1，因此，糖蛋白唾液酸化，在直肠癌中引起细胞凋亡减少， 受体亚克隆。目的3 -确定BACE 1是否促进直肠癌的放化疗敏感性， 部分与ST 6 Gal-1裂解有关。我们发现，BACE 1 mRNA在肿瘤患者的肿瘤中增加， 对放化疗完全有效已知BACE 1切割ST 6 Gal-1，我们发现通过抑制剂 研究表明，由于BACE 1切割ST 6 Gal-1，BACE 1似乎调节SA。这项研究将评估 一种以前未知的直肠癌放化疗耐药机制， 开发新的治疗方法，可以靶向多个GI中的多个耐药亚克隆 腺癌，其中护理标准是术前放化疗治疗。

ABSTRACT — Combination chemoradiation is utilized to treat multiple gastrointestinal (GI) cancers including rectal cancer. Rectal cancer affects 40,000 people per year in the US. Approximately 85% of patients have an incomplete or poor response to treatment increasing their risk of recurrence. We have found that poor responders harbor sub-clones that are more resistant to treatment, and that the enzyme ST6Gal-1 is enriched in these sub- clones. ST6Gal-1 is a Golgi glycosyltransferase that adds the negatively-charged sugar, sialic acid (SA), to specific proteins destined for the cell surface. SA can have profound effects on the structure and function of proteins. ST6Gal-1 is one of the most pervasively upregulated glycosyltransferases in cancer cells. ST6Gal-1 has been shown to specifically promote tumor cell survival and resistance via sialylation. In addition, ST6Gal-1 has been found in extracellular vesicles (ECVs) made by cancer cells. ECVs are particles with a lipid membrane that contains RNA and protein cargo; thus, they are potential mediators of transferable resistance between cancer sub-clones. The role of ST6Gal-1 and ECVs in resistance to chemoradiation has not been investigated. The overall objective of this application is to ascertain the role of ST6Gal-1 in innate and transferable resistance to chemoradiotherapy in rectal cancer. Based on our preliminary data, we hypothesize that ST6Gal-1 mediates resistance to chemoradiation in individual sub-clones in rectal cancer, that this resistance is transferred to other sub-clones via ECVs spreading resistance, and that this resistance is regulated by ST6Gal-1 cleavage by BACE1. We have found that ST6Gal-1 is increased in rectal cancer models after treatment with chemoradiation. We will investigate our hypothesis with 3 aims: AIM 1 — Determine the role of ST6Gal-1 in chemoradiation resistance in human rectal cancer. We hypothesize that ST6Gal-1 causes treatment resistance after chemoradiation by inhibiting apoptosis. We will employ cell sorting, sequencing, and shRNA approaches. We will also conduct studies to investigate its function in patient samples. AIM 2 — Determine if ECVs carrying ST6Gal-1 transfer resistance to chemoradiation between sub-clones in rectal cancer. We hypothesize that ECVs act as vectors that impart resistance to chemoradiotherapy from sub-clone to sub-clone by trafficking ST6Gal-1, and thus, glycoprotein sialylation, in rectal cancer causing decreased apoptosis in the recipient sub-clones. AIM 3 — Determine if BACE1 promotes chemoradiosensitivity in rectal cancer due, in part, to ST6Gal-1 cleavage. We show that BACE1 mRNA is increased in tumors from patients who completely respond to chemoradiotherapy. BACE1 is known to cleave ST6Gal-1, and we found through inhibitor studies that BACE1 appear to regulate SA due to cleavage of ST6Gal-1 by BACE1. This research will evaluate a previously unknown mechanism of resistance to chemoradiotherapy in rectal cancer, with future potential for development of novel therapeutics that could target multiple resistant sub-clones across multiple GI adenocarcinomas, where the standard of care is pre-operative chemoradiation treatment.