Abstract
The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes.
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Abbreviations
- ADAM10:
-
A disintegrin and metalloprotease 10
- FACS:
-
Flow cytometry
- FAK:
-
Focal adhesion kinase
- FN Repeats:
-
Fibronectin-like repeats
- GBM:
-
Glioblastoma multiforme
- Ig domains:
-
Immunoglobulin-like domains
- RGD:
-
Arg-Gly-Asp
- RIPA buffer:
-
Radioimmunoprecipitation assay buffer
- RT-PCR:
-
Reverse transcription-polymerase chain reaction
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Acknowledgments
This work was funded by grant Number 2 P20 RR016472 under the INBRE program of the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) and by research resources of the Genetically Defined Microbe and Expression Core of the UAB mucosal HIV and Immunology Center (R24DK64400). We thank Dr. Kirk Czymmek at the U.D. Bio-Imaging center for assistance with confocal microscopy, Dr. Ulhas Naik (U.D.) for MDA-MB-231 cells, Dr. Robert Sikes (U.D.) for 293T/17 cells, and Dr. Vance Lemmon (U. of Miami) for the hL1-pcDNA3 vector.
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Yang, M., Li, Y., Chilukuri, K. et al. L1 stimulation of human glioma cell motility correlates with FAK activation. J Neurooncol 105, 27–44 (2011). https://doi.org/10.1007/s11060-011-0557-x
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DOI: https://doi.org/10.1007/s11060-011-0557-x