The Neurofibromatosis Type 2 Gene Product, merlin, Reverses the F-Actin Cytoskeletal Defects in Primary Human Schwannoma Cells

Abstract

Schwannoma tumors, which occur sporadically and in patients with neurofibromatosis, account for 8% of intracranial tumors and can only be treated by surgical removal. Most schwannomas have biallelic mutations in the NF2 tumor suppressor gene. We previously showed that schwannoma-derived Schwann cells exhibit membrane ruffling and aberrant cell spreading when plated onto laminin, indicative of fundamental F-actin cytoskeletal defects. Here we expand these observations to a large group of sporadic and NF2-related tumors and extend them to schwannomatosis-derived tumors. Mutation at NF2 correlated with F-actin abnormalities, but the extent of morphological change did not correlate with the type of NF2 mutation. We used a recently described molecular strategy, TAT-mediated protein transfer, to acutely introduce the NF2 protein, merlin, into primary human schwannoma cells in an attempt to reverse the cytoskeletal phenotype. Abnormal ruffling and cell spreading by cells with identified NF2 mutations were rapidly reversed by introduction of TAT-merlin. The effect is specific to TAT-merlin isoform 1, the growth-suppressive isoform of merlin. TAT-merlin isoform 2, a TAT-merlin mutant (L64P), and merlin lacking TAT were ineffective in reversing the cytoskeletal phenotype. Results show that merlin isoform 1 is sufficient to restore normal actin organization in NF2-deficient human tumor cells, demonstrating a key role for merlin in the NF2 phenotype. These results lay the foundation for epigenetic complementation studies in NF2 mouse models and possibly for experiments to evaluate the utility of merlin transduction into patients as protein therapy.

We thank S. Dowdy and coworkers (Washington University, St. Louis, Mo.) for constructs and advice concerning the TAT system; Patrick Wood, Les Olsen, and the University of Miami Organ Procurement Team for providing human nerves; and Mark Marchionni (Cambridge Neuroscience) for rhGGF2. University of Cincinnati GCRC MO1RR 08084 and the Midwest Human Tissue Acquisition Network (Columbus, Ohio) provided some specimens. The IRBs of the University of Cincinnati and Massachusetts General Hospital approved this work. We thank all patients and families who generously donated tissue.

This work was supported by NIH CA 78524 to W.I. and N.R., NS35878 to M.M., and FS CA 80385 to A.-M.B.