Role of the released forms of L1-CAM in breast cancer cell motility
Date
2010
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
L1-CAM (L1) is a neural cell adhesion protein which plays a major role in neural development. Recently, L1 is found to be abnormally expressed in several cancers and has been shown to be a biomarker indicating poor outcome and bad prognosis of the cancer. L1 is a transmembrane protein which is abnormally shed in cancer cells to release the cleaved ectodomain. The L1 ectodomain stimulates cell motility and migratory ability of ovarian, colon and other cancerous cells. L1 is also released by the cancer cells in the form of exosomes. Expression of L1 was found in few breast cancer cell lines and was also associated with aggressive nature of the breast cancer disease. However, the molecular mechanisms of L1 in breast cancer are not known. Breast cancer metastasis to the brain leads to very quick death. The extracellular environment in the brain contains L1 protein and the dissemination and metastasis of breast cancer cells in the brain could depend on the L1 expressed by the breast cancer cells. In order to understand the role of L1, I have characterized the presence of L1 in three breast cancer cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-
468. The amount of L1 expressed by the cell lines was correlated with their known metastatic potential. Having found L1 in breast cancer cells, I have performed experiments to find the role of the two released forms of L1, the soluble L1 ectodomain and the L1 released in the form of exosomes. Overexpression of L1 ectodomain caused the MDA-MB-468 cells to flatten out more and they developed long cytoplasmic extensions. L1 ectodomain overexpression also led to an increase in the motility of both MDA-MB-468 and the MDA-MB-231 cells. Random motility analysis of MDA-MB-468 cells overexpressing L1 ectodomain showed a statistically significant increase in the average velocity of about 45%, and MDA-MB-231 cells overexpressing L1 ectodomain showed a significant increase in the average velocity of about 18%. As MDA-MB-468 cells have very less expression of L1 protein, they have shown a greater change in cell motility due to overexpression of L1. However, scratch assay of MDA-MB-468 cells with L1 ectodomain overexpression showed an increase of only about 10% and there seemed to be a subpopulation of cells which were highly motile compared to the other cells along the scratch. These results show that L1 ectodomain can stimulate breast cancer cell motility. Attenuation of L1 expression using short hairpin RNA in MDA-MB-231 cells reduced the cell motility and random motility analysis showed a statistically significant decrease of 25% in the average cell velocity. Exosomes were isolated from the breast cancer cells and they were confirmed as exosomes based on the presence of the exosomal markers TSG101 or CD9. To establish the role of exosomes in cell motility, exosomes from 231shL1 cells containing little L1 or exosomes from 231Ctrl cells were incubated with 231shL1 cells. Random motility analysis of 231shL1 showed a very high increase of about 80-100% increase in cell velocity with exosomes from 231Ctrl cells compared to the exosomes from 231shL1 cells containing lesser L1. This shows the importance of L1 in exosomes in stimulating breast cancer cell motility. Overall, this study shows the importance of the two released forms of L1, soluble L1 ectodomain and the exosomal L1, in stimulating cell motility of breast cancer cells. It also unravels the importance of intact exosomes containing L1 in cancer cell motility, which has not yet been demonstrated in any of the other types of cancerous cells releasing L1 in the form of exosomes. Therapeutic antibodies to L1 have already been tested in in vivo models showing reduced tumor growth in the case of ovarian cancer. With my current study of L1 in breast cancer, understanding the role of soluble L1 ectodomain and the exosomal L1 in breast cancer would take us a step further to possibly developing therapeutics antibodies to L1 to prevent breast cancer metastasis, in the near future.