Modulation of MUC1 Mucin Trafficking

描述(申请人的描述)：MUC1是一个很好的特征标记 许多起源于上皮的癌症也直接导致了 肿瘤侵袭性转移表型。来自研究的累积数据 这些癌症患者表明，特别是高水平的 细胞内MUC1在肿瘤和血清中与强烈的 有可能发生转移，患者预后不佳。这个 这种通常顶端的跨膜粘蛋白的抗粘连性能(40-90 20个氨基酸的高度0-糖基化串联重复序列)破坏细胞-细胞和 非极化肿瘤细胞中的细胞-基质相互作用，从而增强 转移。患者血清和腹水中的可溶性MUC1也有助于 作为肿瘤诱饵的癌症侵袭性表型 与抗体的免疫复合物，通过结合T细胞并阻断其 增殖，并通过与肿瘤重新结合并启动信号转导 小路。由于MUC1在许多肿瘤中含有更少和更小的0-糖链， 初步研究旨在了解这如何有助于 MUC1在细胞内的积累和释放，因为 事件未知。利用正常和糖基化缺陷的CHO细胞，代谢 标记和生物素化，我们发现重组人的细胞表面表达 人的MUC1依赖于0-糖基化。令人惊讶的是，我们发现这一点 巨大的分子通过网状蛋白介导的内吞作用内化，这表明 MUCI的40-90串联重复免疫优势表位是理想的 免疫毒素治疗的靶点。因此，重要的是要了解 调节MUC1内吞和脱落以更好地靶向治疗 MUC1阳性肿瘤，避免血清/腹水中出现可溶性MUCI。我们发现MUC1 内吞作用被双棕榈酰化潜在位点的突变所阻断 并被较小的0-葡聚糖增强，表明它的贩运可能是 受同型聚集性和与脂质微区的关联调节 参与信号转导。因为我们还发现Muci的脱落是 依赖于内吞作用，我们的特定目标是为了了解粘液是如何 转运和脱落受其0-葡聚糖结构的调节， 棕榈酰化和磷酸化。初步实验将利用 未极化的CHO细胞，而后来的实验发现了新的糖基化缺陷 MDCK细胞将定义MUCI运输的尖端特异性调节。

DESCRIPTION (applicant's description): MUC1 is a well characterized marker for many cancers of epithelial origin that also directly contributes to the aggressive metastatic phenotype of tumors. Cumulative data from studies of these cancer patients indicates that high levels of particularly intracellularMUC1 in the tumor and in serum correlates with a strong potential for metastasis and a poor prognosis for the patient. The anti-adhesive property of this normally apical, transmembrane mucin (with 40-90 heavily 0-glycosylated tandem repeats of 20 aa) disrupts both cell-cell and cell-matrix interactions in non-polarized tumor cells, thus enhancing metastasis. The soluble MUC1 in patient serum and ascites also contributes to the aggressive phenotype of the cancer by acting as a tumor decoy by forming immunocomplexes with antibodies, by binding T-cells and blocking their proliferation, and by rebinding to the tumor and initiating signal transduction pathways. Since MUC1 has fewer and smaller 0-glycans in many tumors, preliminary studies were designed to understand how this contributes to the intracellular accumulation and shedding of MUC1 since the mechanisms for either event is unknown. Using normal and glycosylation-defective CHO cells, metabolic labeling and biotinylation, we find that cell surface expression of recombinant human MUC1 is dependent on 0-glycosylation. Surprisingly, we find that this enormous molecule is internalized by clathrin-mediated endocytosis indicating that the 40-90 tandem-repeat immunodominanat epitopes of MUCI are an ideal target for immunotoxin therapy. Thus, it is essential to understand what regulates MUC1 endocytosis and shedding to better target treatments to MUC1-positive tumors and avoid soluble MUCI in serum/ascites. We find that MUC1 endocytosis is blocked by mutation of a potential site for dual palmitoylation and enhanced by smaller 0-glycans, indicating that its trafficking may be modulated by homotypic clustering and association with lipid microdomains involved in signal transduction. Since we also find that MUCI shedding is endocytosis-dependent, our specific aims are designed to understand how MUCI trafficking and shedding is modulated by its 0-glycan structure, palmitoylation, and phosphorylation. Initial experiments will utilize non-polarized CHO cells while later experiments in new glycosylation-defective MDCK cells will define apical-specific modulation of MUCI trafficking.