The NF2 Tumor Suppressor Gene Product, Merlin, Inhibits Cell Proliferation and Cell Cycle Progression by Repressing Cyclin D1 Expression

Abstract

Inactivation of the NF2 tumor suppressor gene has been observed in certain benign and malignant tumors. Recent studies have demonstrated that merlin, the product of the NF2 gene, is regulated by Rac/PAK signaling. However, the mechanism by which merlin acts as a tumor suppressor has remained obscure. In this report, we show that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibits cell proliferation and arrests cells at G₁ phase, concomitant with decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of pRB. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene results in upregulation of cyclin D1 and S-phase entry. Furthermore, PAK1-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and PAK activity was inhibited by expression of merlin. Interestingly, the S518A mutant form of merlin, which is refractory to phosphorylation by PAK, was more efficient than the wild-type protein in inhibiting cell cycle progression and in repressing cyclin D1 promoter activity. Collectively, our data indicate that merlin exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression, suggesting a unifying mechanism by which merlin inactivation might contribute to the overgrowth seen in both noninvasive and malignant tumors.

ACKNOWLEDGMENTS

We thank Peter Adams for helpful suggestions regarding some of the experiments conducted in this study and Warren Kruger and Davide Ruggero for critical review of the manuscript. We also thank Bert Vogelstein for providing the pAdEasy system to generate the NF2 adenoviral vectors.

This work was supported by National Institutes of Health grants CA-45745 (to J.R.T.), training grant T32-16850, ACS institutional grant 38102 (to G.H.X.), and CA-06927 (to Fox Chase Cancer Center), by an appropriation from the Commonwealth of Pennsylvania, and by a gift from the Local No. 14 Mesothelioma Fund of the International Association of Heat and Frost Insulators and Asbestos Workers in memory of Hank Vaughan and Alice Haas. R.G.P is supported by National Institutes of Health grants CA-70896, CA-75503, CA-86072, and CA-93596. The following Fox Chase Cancer Center shared facilities were used in the course of this work: Cell Culture Facility, DNA Sequencing Facility, DNA Synthesis Facility, DNA Microarray Facility, and Flow Cytometry and Cell Sorting Facility.