In this report, we show that adenovirus-mediated expression of wild-type merlin inhibits cell proliferation and blocks cell cycle progression at G1
phase in NF2
-deficient mesothelioma cell lines. These results are in agreement with previous studies implicating merlin as a negative growth regulator in other cell types, including NIH 3T3 fibroblasts (22
), schwannoma cells (25
), and meningioma cells (14
We further demonstrated that merlin regulates cell proliferation by modulating cyclin D1 transcription. Furthermore, our results with human mesothelioma cells parallel the aberrant upregulation of cyclin D1 reported in mouse embryonic fibroblasts from Nf2
null mice (20
). Cyclin D1 (also known as bcl-1 or PRAD1) is a proto-oncogene that encodes a regulatory subunit of the cyclin-dependent kinase holoenzymes. Activation of the holoenzymes leads to phosphorylation and inactivation of the RB tumor suppressor and thereby promotes entry into S phase (32
). Our results demonstrate that expression of merlin induces a decrease in both cyclin D1 RNA and protein levels and CDK4 kinase activity, concomitant with dephosphorylation of pRB and reduced DNA synthesis. We also found that cell cycle arrest at G1
phase caused by AdNF2 or AdNF2S518A can be partially overridden by ectopic expression of a cyclin D1 plasmid, indicating that cyclin D1 is an essential mediator of merlin's observed effect on cell cycle progression. Moreover, our RNA interference experiments point to a physiological role for merlin in the regulation of cyclin D1 expression. Interestingly, Hulit et al. found that even a 50% reduction in cyclin D1 expression is sufficient to reduce tumor incidence and progression in Min
mice harboring a mutant Apc
). This is comparable to the downregulation observed in our studies and emphasizes the importance of cyclin D1 regulation in modulating tumorigenesis.
PAK has been suggested to serve as the key effector for Rac1 activation of cyclin D1 (34
). Recent studies show that PAK signaling mediates cyclin D1 expression in mammary epithelial and cancer cells (2
), and PAK is essential for RAS-induced upregulation of cyclin D1 during the G1
-to-S transition (26
). The data presented here demonstrate that restoration of merlin in NF2
-deficient mesothelioma cells inhibits PAK1 activity and represses PAK1-induced cyclin D1 promoter activity. Our results with human cancer cells are consistent with previous studies, using rodent cells, demonstrating that merlin can inhibit PAK activity (12
). More importantly, our findings indicate that merlin can repress cyclin D1 expression through inhibition of PAK. As noted above, NF2
inactivation has been reported not only in malignant mesotheliomas, but also in benign neurological tumors, particularly schwannomas, seen in patients with NF2 disorder. How a tumor suppressor gene contributes to both malignant mesotheliomas and benign schwannomas may seem somewhat paradoxical, given that Rac-PAK1 signaling can regulate cell motility and invasiveness. Thus, a mechanism other than tumor invasiveness must be invoked to attribute a role for NF2
inactivation in both benign and malignant tumors. Germane to this, Westwick et al. (34
) showed that the Rac interaction with PAK was required for cyclin D1 stimulation but was dispensable for other Rac functions, such as induction of lamellipodia and activation of JNK. Their results therefore support our contention that inhibition of cyclin D1 expression represents perhaps merlin's major function as a tumor suppressor.
It has been shown previously that PAK1 regulates cyclin D1 transcription by means of an NF-κB-dependent pathway (2
), and merlin can block NF-κB activity (16
). Taken together, these data suggest a model in which merlin regulates transcription of cyclin D1 through PAK-NF-κB signaling.
Regulation of cyclin D1 expression by merlin might also involve ERK signaling. It is known that ERK activity is critical in the transcriptional induction of cyclin D1 in several cell types (reviewed in reference 3
). ERK induction of the cyclin D1 promoter activity occurs through sequences targeted by ETS, which is distinct from the Rac/PAK pathway (15
). Because PAK is thought to be required for efficient activation of Raf and MEK/ERK (8
), feedback inhibition of the Rac/PAK axis by merlin could play a role in downregulating ERK activity and therefore repressing cyclin D1 expression. However, neither ERK expression nor phosphorylation was changed in response to AdNF2 expression, indicating that this pathway is not involved in modulating cyclin D1 expression by merlin in our system.
Reexpression of merlin in Meso 17 and Meso 35 cells not only represses cyclin D1 expression but also results in the accumulation of the p27 protein. In addition to phosphorylation of the pRB protein, cyclin D-CDK4 complexes also function to sequester the cell cycle inhibitors p21 and p27, thereby inducing CDK2 activity and promoting cell cycle progression into S phase. Activated CDK2 then phosphorylates and triggers proteolysis of p27 (32
). Thus, by repressing cyclin D-CDK4 activity, it is likely that merlin increases p27 accumulation by inhibiting sequestration and degradation of p27.
Previous work had demonstrated that the phosphorylation status of merlin specifies cell growth arrest or cell proliferation. Phosphorylation of merlin by Rac/PAK signaling inactivates merlin and potentiates Rac/PAK activities (17
). The data presented here further support this feed-forward mechanism. In confluent cell cultures, hypophosphorylated merlin acts as an inhibitor of PAK and blocks Rac/PAK-stimulated cyclin D1 expression. This form of merlin is growth inhibitory and represents the functionally active tumor suppressor. At low cell density, however, merlin is phosphorylated in response to activation of Rac/PAK signaling. Phosphorylation of merlin attenuates its activity as a negative regulator of PAK and potentiates Rac/PAK functions. This form of merlin is growth permissive and therefore represents the functionally inactive tumor suppressor. Collectively, these findings indicate that there is a stimulatory-inhibitory loop between PAK and merlin and that a fine balance must be maintained between these two molecules to regulate normal cellular proliferation.
Merlin is phosphorylated at Ser518 by Rac/PAK signaling (17
). As predicted, McClatchey and colleagues have shown that the A518 phosphorylation-defective form of merlin inhibits Rac-induced activation of AP-1 reporter activity as well as or better than the wild-type protein (30
). In this study, we also examined whether the A518 mutant can act as a constitutively active form of the tumor suppressor in modulating PAK activity and cyclin D1 expression. We found that PAK1-mediated induction of the cyclin D1 promoter was more effectively inhibited by cotransfection with the A518 mutant form of NF2
than with wild-type NF2
. We also observed that AdNF2 A518 arrested more cells at G1
phase than did AdNF2. In addition, Meso 17 cells transfected with a plasmid encoding the A518 mutant showed significantly reduced colony-forming efficiency compared to cells transfected with wild-type NF2
(unpublished observations). Based on these results and the findings by McClatchey and colleagues (30
), we conclude that the phosphorylation-refractory A518 mutant represents an active form of the tumor suppressor.
Collectively, the data presented here suggest that the NF2 tumor suppressor gene exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression. These observations have significant implications for elucidating tumorigenic mechanisms involved in neoplasms associated with merlin inactivation. Our experiments with AdNF2 achieved 100% transduction efficiency in mesothelioma cells, which demonstrates the technical feasibility of using adenovirus-mediated transfer of the NF2 gene as a potential therapeutic strategy for human malignant mesothelioma.