GLYCOSPHINGOLIPID ENRICHED MICRODOMAINS IN CANCER CELL INVASION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 鞘糖脂富集微区与癌细胞侵袭 理解和对抗癌症进展的最终目标是研究最终导致侵袭性和转移性细胞表型的机制。癌细胞侵袭是一个高度复杂的多步骤过程，其特征在于细胞粘附分子的表达水平改变和蛋白水解酶的分泌，沿着多种细胞蛋白在多个分支信号通路中的表达或活性的变化。此外，质膜含有富含肿瘤相关鞘糖脂（GSL）-抗原的膜微区，其作为细胞粘附的介质，并在信号传导分子中，为细胞信号传导提供重要的位置。最近的研究表明，肿瘤细胞的侵袭往往是由于GSL和/或膜蛋白的差异组织和聚集以及它们与信号分子的组装所引发的异常信号机制。合成的醚脂质类似物ET-18-OMe（1-O-十八烷基-2-O-甲基-甘油-3-磷酸胆碱）先前显示出影响人乳腺癌和结肠癌细胞的侵袭，并用作诱导细胞侵袭的分子探针，以探索关键分子的组成、易位和组织的变化以及膜微区的可能参与。 我们的研究结果表明，重组，集群和特定的膜蛋白和信号转导与鞘糖脂在膜微区的组装负责细胞-细胞粘附的损失，并随后导致MCF-7乳腺癌细胞的侵袭力增加。细胞-细胞粘附的丧失是由于定位于鞘糖脂MSGb 5簇中并与其相关的表唾液酸对E-钙粘蛋白的空间位阻，随后的侵袭是由于MSGb 5相关的FAK/cSrc信号复合物和下游ERK的激活，导致MMP的表达和激活增加。相反，HCT-8结肠癌细胞的侵袭行为是通过整合素α 1亚基的聚集和相关FAK/cSrc复合物的激活而启动的。然而，随后的下游途径似乎是p130 Cas-JNK途径，也导致MMPs表达增加，并且不涉及与鞘糖脂的特异性关联。后者的结果指出了“经典的”整合素依赖性的癌细胞侵袭，通过整合素的聚集启动。相反，朝向侵袭的信号传导也可以通过GSL发生，由鞘糖脂的糖部分介导的与周围基质的粘附导致GSL依赖性粘附，然后可以诱导信号传导事件以改变细胞表型。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，我们选择使用LNCaP/C4-2/C4-2B进展模型继续我们的研究，该模型与人前列腺癌的临床进展非常相似。该实验模型使得能够研究转移性前列腺癌细胞与其亲本细胞相比的机制改变。我们的初步数据表明，FAK和src，在侵袭中起着至关重要的作用，在转移性亚系C4-2B中被激活，并参与增强基质金属蛋白酶的表达和活性。此外，可能触发这些细胞事件的可能上游调节因子的细胞表面组织的改变已得到证实，目前正在研究以进一步阐明其组织的相关性，这可能将其与下游信号传导途径的激活联系起来，从而改变表型。未来的工作包括在临床相关标本中进行评估，并与UNM动物研究机构新墨西哥州大学医学院的Marco Bisoffi博士合作，使用小鼠异种移植模型在体内验证我们的发现。

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.