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发现癌症的生物标志物。众所周知，癌症与糖缀合物（包括糖脂和糖蛋白）的糖基化模式的改变有关。基于这一观察结果，本项目的目的是确定新的黏液生物标志物，用于结肠癌的早期检测和预后评估。我们的方法与识别癌症相关聚糖的传统方法不同，因为我们检查细胞糖基化水平，并利用这些信息以及我们对聚糖生物合成途径的了解来预测可能的候选生物标志物。该研究的总体假设是“癌症相关聚糖的结构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、正常和癌变结肠组织中focusyl-、(3-N-乙酰半乳糖胺基-、唾液酰-和亚砜转移酶的活性。这些研究集中在那些在聚糖中产生低聚糖链外端的酶上。在这些研究中，我们一致观察到癌组织中两种不同的Gal:3- o -硫转移酶水平升高。其中一种酶介导了SE3-Ga（31- bbb3galnaca -）型结构的形成，另一种酶介导了粘蛋白核心2四糖Gal(,4GlcNAc（1,6)Gal (1,3)GalNAca-）末端单元的形成。这些组织也表现出明显的（1,3）岩藻糖基转移酶和（2,3）唾液基转移酶水平。基于这些来自癌细胞系和组织的研究，我们选择了独特表达的新型硫酸碳水化合物表位，这些表位可能存在于结肠癌中，而不存在于正常细胞中。我们认为这些磺化聚糖为结肠癌的治疗和检测提供了新的靶点。为了证实这一可能性，我们建议合成选择性的硫酸碳水化合物抗原，并将其与KLH连接。针对这些目标结构将开发单克隆抗体。最后，这些抗体将应用于结肠细胞系和临床标本，以评估它们在识别新的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的低聚糖来确定新的抗体试剂的特异性，因此这些试剂不仅可能用于结肠癌的研究，而且可能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的单克隆抗体的产生可以带来新的诊断策略。目前还没有结肠癌的血清标志物，因此我们的项目也将研究血清中硫酸甘聚糖在结肠癌期间是否升高。这个项目的成功结果将引导我们在结肠癌的未来项目，包括使用在癌细胞上表达的聚糖作为癌症治疗的目标。

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.