An Untested Approach: Biomarkers for Colon Cancer

描述（由申请人提供）：癌症研究中最苛刻的领域之一是寻找早期检测癌症的生物标志物。已知癌症与包括糖脂和糖蛋白的糖缀合物的糖基化模式的改变相关。基于这一观察结果，本项目的目标是确定新的粘液生物标志物，用于结肠癌的早期检测和预后评估。我们的方法不同于传统的方法，确定癌症相关的聚糖，因为我们检查细胞糖基化的水平，并使用此信息沿着我们的知识聚糖生物合成途径，以预测可能的候选生物标志物。该研究的总体假设是，“癌症相关聚糖结构的变化是由参与其生物合成的糖基转移酶的细胞表达的根本变化驱动的。癌细胞表达不同的糖基转移酶，导致不同的聚糖特征。“为了支持上述概念，我们进行了研究，以测定不同结肠癌细胞系以及正常和癌性结肠组织中岩藻糖基-、β-N-乙酰半乳糖胺基-、唾液酸基-和磺基转移酶的活性。这些研究集中在产生聚糖中寡糖链外端的酶。在这些研究中，我们一致地观察到癌组织中两种不同的Gal：3-O-磺基转移酶水平升高。这些酶中的一种介导SE 3-Ga（31）-> 3GalNAc-型结构的形成，而另一种介导粘蛋白核心2四糖Gal（1，4）GlcNAc（1，6）GalNAc-中SE 3-Gal（1，3）->4GlcNAc末端单元的形成。这些组织还显示出明显水平的（1，3）岩藻酰基转移酶和（2，3）唾液酸转移酶。基于这些来自癌细胞系和组织的研究，我们选择了可能存在于结肠癌而不是正常细胞上的独特表达的新型硫酸化碳水化合物表位。我们认为这些硫酸化聚糖为结肠癌的治疗和检测提供了新的靶点。为了证实这种可能性，我们建议合成选定的硫酸化碳水化合物抗原，并将它们连接到KLH。将针对这些靶结构开发单克隆抗体。最后，将这些抗体应用于结肠细胞系和临床标本，以评估其在鉴定新型结肠相关抗原方面的功效。我们的合成能力还提供了一系列寡糖来确定新抗体试剂的特异性，因此这些试剂可能不仅适用于结肠癌的研究，而且适用于癌症和血管生物学领域的其他问题。结肠癌细胞硫酸化聚糖特异性单克隆抗体的产生可能导致新的诊断策略。目前还没有结肠癌的血清标志物，因此我们的项目还将研究结肠癌期间血清中的硫酸化聚糖是否升高。该计划的成功结果将引导我们开展未来的结肠癌计划，其中包括使用癌细胞上表达的聚糖作为癌症治疗的靶点。

DESCRIPTION (provided by applicant): One of the most demanding areas of cancer research is finding biomarkers for the early detection of cancer. It is known that cancer is associated with alterations in the glycosylation patterns of glycoconjugates including glycolipids and glycoproteins. Based on this observation, the objective of this project is to identify novel mucinous biomarkers for the early detection and prognostic evaluation of colon cancer. Our approach differs from conventional methods of identifying cancer-associated glycans in that we examine the level of cellular glycosylation and use this information along with our knowledge of glycan biosynthetic pathways to predict likely candidate biomarkers. The overall hypothesis of the study is that "Changes in the structures of cancer-associated glycans are driven by fundamental changes in the cellular expression of the glycosyltransferases involved in their biosynthesis. Cancer cells express different glycosyltransferases leading to distinct glycan signatures." In support of the above concept, we performed studies to assay the activity of fucosyl-, (3-N- acetylgalactosaminyl-, sialyl- and sulfotransferases in different colon cancer cell lines, and normal and cancerous colon tissue. These studies focused on those enzymes that generate the outer ends of the oligosaccharide chains in glycans. In these studies, we consistently observed elevated levels of two distinct Gal:3-O-sulfotransferases in cancerous tisue. One of these enzymes mediated formation of SE3-Ga(31->3GalNAca- type structure and the other mediated formation of SE3-Gal(->4GlcNAc terminal units in the mucin core 2 tetrasaccharide Gal(,4GlcNAc(1,6 (Gal(1,3)GalNAca-. These tissues also exhibited appreciable levels of ((1,3)fucoyltranferase and ((2,3)sialyltranferase. Based on these studies from cancer cell lines and tissues, we have selected uniquely expressed novel sulfated carbohydrate epitopes that are likely present on colon cancer but not normal cells. We propose that these sulfated glycans provide novel targets for treatment and detection of colon cancer. In order to confirm this possibility, we propose to synthesize selected sulfated carbohydrate antigens and link them to KLH. Monoclonal antibodies will be developed against these target structures. Finally, these antibodies will be applied to colon cell lines and clinical specimens to assess their efficacy in identifying novel colon associated antigens. Our synthetic capability also provides a series of oligosaccharides to determine the specificity of the new antibody reagents, and thus these reagents are likely to be useful not only for studies of colon cancer but also other problems in the fields of cancer and vascular biology. Generation of monoclonal antibodies unique to sulfated glycans of colon cancer cells can lead to new diagnosis strategies. There are currently no serum markers of colon cancer, and thus our project will also study if sulfated glycans in serum are elevated during colon cancer. The successful outcome of this program will lead us to a future program on colon cancer that includes the use of glycans expressed on cancer cells as targets for cancer therapy.