The Role of EpCAM Glycosylation in Breast Cancer Metastasis

项目总结 乳腺癌是一种广泛传播的疾病，仅在2022年就会夺走超过4万名美国女性的生命， 这些死亡中的大多数是由IV期疾病造成的，在这种疾病中，癌症已经转移到远处， 因为转移性疾病很难治疗，而且几乎没有有效的治疗选择。乳腺癌细胞的转移 通过侵入基底膜，侵入血管，循环到远处组织，留下血液 血管，并定植该组织。上皮细胞黏附分子(EpCAM)在细胞黏附中发挥作用， 迁移和侵袭，并与几种上皮性癌症有关，包括乳腺癌、前列腺癌和 结直肠癌。尽管它有可能促进转移性级联反应的几个步骤以及它与上皮细胞的联系 在癌症中，EpCAM在转移中的具体作用尚不清楚。在他对正常组织的研究中，初级 17名转移性乳腺癌患者的肿瘤和转移结节，我们的合作者Pedram Argani博士 确定在转移性结节中EpCAM蛋白表达增加，而没有相应的 EPCAM基因表达增加。因此，我们假设EpCAM的翻译后修饰， 它具有三个已知的N-糖基化位点和未知的O-糖基化位点，稳定并增加 转移癌组织中EpCAM的表达丰富。我们最近对这些样本的研究发现，丰度 转移性组织中N-糖链的含量显著高于原发性肿瘤和正常组织 确定了25个差异显著丰富的N-糖链。我们的研究还发现，EpCAM蛋白 与原发肿瘤相比，转移瘤组织中的表达显著增加，并显著 统计上与7种N-糖链的表达相关。综上所述，这些数据表明EpCAM N-糖基化在肿瘤转移过程中可能起一定作用。这个项目的中心假设是增加了 N-糖基化的变化稳定了EpCAM，并使其促进了转移。我提议测试一下这一点 通过以下具体目的提出假说：目的1：阐明EPCAM N-糖基化对细胞外信号转导的影响 EpCAM在人乳腺癌细胞中的稳定性和粘附性；目标2：评估EpCAM N- 糖基化对小鼠乳腺癌模型转移的影响。这些目标将通过一个 癌症生物学、生化和质谱学方法在人类三阴性乳房中的联合应用 癌细胞系和乳腺癌异种移植模型。这项工作意义重大，因为它将阐明 转移性乳腺癌中EpCAM的N-糖基化，确定EpCAM的特异性N-糖基化图谱 在转移的乳腺癌细胞，并可能随后发现新的治疗靶点和战略 转移性乳腺癌。

PROJECT SUMMARY Breast cancer is a widespread disease that will claim the lives of over 40,000 US women in 2022 alone, with the majority of these deaths resulting from stage IV disease, in which cancer has metastasized to distant regions, as metastatic disease is difficult to treat and has few effective treatment options. Breast cancer cells metastasize by invading the basement membrane, invading into blood vessels, circulating to distant tissues, leaving the blood vessel, and colonizing that tissue. The epithelial cell adhesion molecule (EpCAM) plays a role in cell adhesion, migration, and invasion, and has been linked to several epithelial cancers, including breast, prostate, and colorectal cancer. Despite its potential to facilitate several steps of the metastatic cascade and its link to epithelial cancers, the specific role of EpCAM in metastasis remains unknown. In his study of normal tissues, primary tumors, and metastatic nodules of 17 metastatic breast cancer patients, our collaborator Dr. Pedram Argani determined that EpCAM protein expression was increased in metastatic nodules, without a corresponding increase in EpCAM mRNA expression. We therefore hypothesize that post-translational modification of EpCAM, which has three known N-glycosylation sites and no known O-glycosylation sites, stabilizes and increases the abundance of EpCAM in metastatic tissues. Our recent study of these same samples found that the abundance of N-glycans was significantly increased in metastatic tissues versus primary tumors versus normal tissues and identified 25 significantly differentially abundant N-glycans of interest. Our study also found that EpCAM protein expression was significantly increased in metastatic tissues compared to primary tumors and was significantly statistically correlated with the expression of seven N-glycans. Taken together, these data suggest that EpCAM N-glycosylation may play a role in metastatic progression. The central hypothesis of this project is that increases and changes in N-glycosylation stabilize EpCAM and allow it to facilitate metastasis. I propose to test this hypothesis through the following specific aims: Aim 1: To elucidate the impact of EpCAM N-glycosylation on EpCAM stability and adhesion in human breast cancer cells; Aim 2: To assess the impact of EpCAM N- glycosylation on metastasis in mouse models of breast cancer. These aims will be achieved through a combination of cancer biology, biochemistry, and mass spectrometry approaches in human triple-negative breast cancer cell lines and breast cancer xenograft models. This work is significant because it will elucidate the role of N-glycosylation of EpCAM in metastatic breast cancer, determine the specific N-glycosylation profile of EpCAM in metastasizing breast cancer cells, and may subsequently uncover novel therapeutic targets and strategies in metastatic breast cancer.