GLYCOSPHINGOLIPID ENRICHED MICRODOMAINS IN CANCER CELL INVASION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 鞘糖脂富集型微域在癌细胞侵袭中的作用 了解和抗击癌症进展的最终目标是研究最终导致侵袭和转移细胞表型的机制。癌细胞的侵袭是一个高度复杂和多步骤的过程，其特征是细胞黏附分子的表达水平和蛋白水解酶的分泌水平发生变化，同时多种细胞蛋白在多个分支信号通路中的表达或活性发生变化。此外，质膜含有富含肿瘤相关糖脂(GSL)的膜微区，GSL抗原起到细胞黏附的中介作用，并在信号分子中为细胞信号提供重要位置。最近的研究表明，肿瘤细胞的侵袭往往是由GSLs和/或膜蛋白的差异组织和聚集及其与信号分子的组装所启动的异常信号机制所致。合成的乙醚脂质类似物ET-18-OME(1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine)先前被证明影响人乳腺癌细胞和结肠癌细胞的侵袭，并被用作诱导细胞侵袭的分子探针，以探索关键分子的组成、移位和组织的变化以及膜微区的可能参与。 我们的结果表明，特定的膜蛋白和信号转导分子与膜微区的糖鞘糖脂重组、聚集和组装是导致细胞与细胞之间黏附丧失的原因，从而导致MCF-7乳腺癌细胞侵袭力的增加。细胞-细胞间黏附的丧失是由于定位于糖鞘糖脂MSGb5簇并与之相关的表皮生长因子对E-钙粘蛋白的立体阻碍，随后的侵袭是由于MSGb5相关的FAK/CSRC信号复合体和ERK下游的激活，导致MMPs表达和激活的增加。相反，HCT-8结肠癌细胞的侵袭行为是通过整合素α1亚单位的聚集和相关的FAK/CSRC复合体的激活而启动的。然而，下游的通路似乎是p130Cas-JNK通路，也导致MMPs表达增加，而不涉及与神经鞘糖脂的特异性联系。后者的研究结果指出，癌细胞的侵袭依赖于整合素，这种侵袭是通过整合素的聚集性开始的。相反，侵袭的信号也可能通过GSLS发生，糖鞘脂糖部分介导的与周围基质的黏附导致GSL依赖的黏附，然后可能诱导信号事件改变细胞表型。这些结果表明，癌症恶性可能是由于现有成分的重组导致细胞内信号事件的变化。因此，我们选择用LNCaP/C4-2/C4-2B进展模型继续我们的研究，该模型与人类前列腺癌的临床进展非常相似。这一实验模型能够研究转移性前列腺癌细胞与其亲代细胞相比的机制变化。我们的初步数据表明，在侵袭过程中起关键作用的FAK和src在转移的C4-2B亚系中被激活，并参与了基质金属蛋白酶的表达和活性的增强。此外，可能触发这些细胞事件的上游调控因子的细胞表面组织的变化已经被证实，目前正在进行研究，以进一步阐明它们的组织结构可能与下游信号通路的激活，从而改变表型的相关性。未来的工作包括与新墨西哥大学医学院的Marco Bisoffi博士合作，在临床相关标本中进行评估，并在体内使用异种移植模型验证我们的发现。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glycosphingolipid Enriched Microdomains in Cancer Cell Invasion The ultimate goal for understanding and combating cancer progression is to investigate mechanisms culminating in an invasive and metastatic cell phenotype. Cancer cell invasion is a highly complex and multistep process, that is characterized by altered expression levels of cell adhesion molecules and secretion of proteolytic enzymes, along with changes in expression or activities of a variety of cellular proteins in multiple branching signaling pathways. In addition, the plasma membrane contains membrane microdomains enriched in tumor-associated glycosphingolipid (GSL)-antigens that function as mediators of cell adhesion, and in signaling molecules, providing important locations for cell signaling. Recent studies showed that tumor cell invasion often results from aberrant signaling mechanisms initiated by differential organization and clustering of GSLs and/or membrane proteins, and their assembly with signaling molecules. The synthetic ether lipid analog ET-18-OMe (1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine) was previously shown to influence invasion of human breast and colon cancer cells and is used as a molecular probe to induce cellular invasion in order to explore changes in composition, translocation and organization of crucial molecules and the possible involvement of membrane microdomains. Our results show that reorganization, clustering and assembly of specific membrane proteins and signal transducers with glycosphingolipids in membrane microdomains are responsible for loss of cell-cell adhesion and subsequent result in increased invasiveness of MCF-7 breast cancer cells. Where loss of cell-cell adhesion was due to sterical hindrance of E-cadherin by episialin both localized in and associated with clusters of the glycosphingolipid, MSGb5, subsequent invasion resulted from activation of MSGb5-associated FAK/cSrc signaling complexes and downstream ERK, leading to increased expression and activation of MMPs. In contrast, the invasive behavior of HCT-8 colon cancer cells was initiated through clustering of integrin alpha1 subunits and the activation of associated FAK/cSrc complexes. The followed path downstream, however, appeared to be the p130Cas-JNK pathway also leading to increased expression of MMPs and did not involve specific association with glycosphingolipids. The latter results point out to the 'classic' integrin-dependent invasion of cancer cells, initiated through clustering of integrins. In contrast, signaling towards invasion may also occur via GSLs, adhesion to the surrounding matrix mediated by the sugar moieties of glycosphingolipids resulting in GSL-dependent adhesion may then induce signaling events to alter cellular phenotype. These results suggested that cancer malignancy may result from reorganization of existing components that lead to changes in intracellular signaling events. Therefore, we choose to continue our study with the LNCaP/C4-2/C4-2B progression model, which closely resembles the clinical progression of human prostate cancer. This experimental model enables the study of mechanistic alterations of metastatic prostate cancer cells compared to their parental cells. Our preliminary data indicate that FAK and src, that play a crucial role in invasion, are activated in the metastatic subline C4-2B, and are involved in enhanced expression and activity of matrix metalloproteinases. In addition, alterations in cell surface organization of possible upstream regulators that may trigger these cellular events were demonstrated and are currently investigated to further elucidate the relevance of their organization which may link them to activation of downstream signaling pathways and thus alteration of phenotype. Future work includes assessment in clinically relevant specimen and validation of our findings in vivo using xenograft models in mice in collaboration with Dr. Marco Bisoffi from the University of New Mexico, School of Medicine at the UNM Animal Research Facility.