5.2.1 Merlin

In the 1990s, early characterizations of Merlin described a novel moesin-, ezrin-, radixin-like protein, taking its name from its structure.s. Literatur ^,​ s. Literatur Merlin is a 595-amino acid protein composed of an N-terminal domain, a coil–coil domain, and a carboxyl terminal domain. It can be regarded as a scaffold protein most commonly found at the plasma membrane. Merlin functions as a tumor suppressor that also plays an important role in schwannoma cell motility, cell adhesion, and cell proliferation.s. Literatur ^,​ s. Literatur

Merlin has structural similarity to the protein 4.1 superfamily known as the “ERM family,” and functions in linking plasma membrane receptors to cytoskeletal components.s. Literatur Relative conservation of the N-terminus domain known as the FERM region (F for 4.1 protein, E for ezrin, R for radixin, and M for moesin) characterizes the gene family, and sequence alignment demonstrates 64% similarity for Merlin at the conserved FERM domain.s. Literatur The FERM domain in Merlin differs from the ERM proteins in that Merlin possesses a unique actin-binding motif at the N-terminal domain, in lieu of the conventional C-terminal actin binding site.s. Literatur ^,​ s. Literatur Moreover, while all ERM proteins form inter- and intramolecular associations, Merlin’s associations are relatively weaker and more dynamic.s. Literatur Nevertheless, these interactions appear critical for tumor-suppressor function.

Merlin’s activity is controlled via phosphorylation. The process of dephosphorylation has been thought to activate Merlin by changing its structure from an unfolded, open state to its folded, active form as a tumor suppressor.s. Literatur ^,​ s. Literatur Recent work has suggested that while phosphorylation inactivates Merlin’s role as a tumor suppressor, the protein remains in the closed configuration, without significant conformation change (Fig. 5‑1 ).s. Literatur Activated Merlin appears to play critical roles in multiple molecular pathways that regulate cell cycle, which will be described later in this chapter.

5.2.2 NF2/Merlin Inactivation in Tumor Biology

Inactivation of both alleles of the NF2 tumor-suppressor gene is necessary for the development of both spontaneous and NF2-associated VS.s. Literatur ^,​ s. Literatur Patients with inherited NF2 syndrome obtain one mutated copy of the NF2 gene through autosomal dominant transmission. But unlike most autosomal dominant conditions, in which one copy of the altered gene is sufficient to result in phenotypic disease, both copies of the NF2 gene must be altered to result in tumor formation. Somatic mutations in the NF2 gene then occur during the patient’s life, resulting in schwannomas or other tumors of the nervous system. Given the high probability of intergenerational transmission, selected at-risk populations should pursue genetic testing for the NF2 mutation.s. Literatur

In sporadic VS, spontaneous mutations of NF2 within Schwann cells of the vestibular nerve are thought to be responsible for tumor formation. Isolated, unilateral VSs frequently harbor identifiable mutations in both copies of the NF2 gene.s. Literatur ^,​ s. Literatur ^,​ s. Literatur

5.2.3 NF2 Mutation Type May Predict Severity of Disease

While mutations in the NF2 gene are responsible for both sporadic and NF2-associated VS, there is significant variability in clinical presentation of both sets of patients.s. Literatur ^,​ s. Literatur Differences in disease severity may be related to the mutation type, as a wide range of mutations have been found in genetic studies of NF2 patients.s. Literatur Approximately 65% of mutations seen in NF2 patients result in a truncated gene product (nonsense or frameshift mutations).s. Literatur Studies evaluating genotype–phenotype relationships have determined that truncated NF2 products lead to more severe clinical disease. Patients with milder disease tend to harbor missense mutations or DNA alterations that are not found by conventional mutation detection strategies.s. Literatur Merlin proteins containing missense mutations may be stably expressed but defective in their ability to suppress cell division.s. Literatur ^,​ s. Literatur ^,​ s. Literatur Today, it is known that one risk for a poorer prognosis in NF2 patients is related to the type of mutation—specifically the presence of a truncating mutation.

Patients with sporadic VS tend to have different types of NF2 mutations than patients with NF2 syndrome.s. Literatur Between 76 and 89% of sporadic VS cases have been found to harbor small deletions,s. Literatur ^,​ s. Literatur ^,​ s. Literatur whereas only 42% of NF2-associated tumors harbor small deletions.s. Literatur More common in NF2-associated tumors were single base pair point mutations.s. Literatur Differences in mutation types likely result in varied presence and composition of the defective Merlin protein. However, the downstream effects on intracellular signaling pathways may represent an additional source of differential tumor growth patterns between patients.

5.3.1 Contact Inhibition/Suppression of Cell Proliferation

Merlin is activated in conditions of increased cell density, and the resultant CD4-mediated interactions suppress cell proliferation. Merlin has been found to co-localize with the plasma membrane protein CD44.s. Literatur Merlin and CD44 form a “molecular switch” whereby at low cell density, Merlin exists in a phosphorylated complex with ezrin, moesin, and CD44 that is growth permissive; at high cell density, Merlin becomes hypophosphorylated and inhibits cell growth through signals from the extracellular matrix (ECM; Fig. 5‑2 ).s. Literatur Cell–cell contact, in particular, plays a role in Merlin activity; loss of cell adhesion results a near-complete dephosphorylation of Merlin, while re-plating cells reverses this process.s. Literatur In a murine model, Bai et al showed that increased expression of wild-type Merlin in Tr6BC1 schwannoma cells inhibits hyaluronic acid binding to CD44, supporting the idea that the interaction between CD44 and Merlin is at least partially responsible for tumor growth suppression.s. Literatur

5.3.2 RAC11-Mediated Cytoskeletal Pathway

Merlin localizes predominantly to the membrane–cytoskeleton interface. Similar to the ERM proteins, Merlin most likely acts as a linker between plasma membrane molecules and the cytoskeleton, functioning in membrane organization.s. Literatur Loss of Merlin results in deregulation of the actin cytoskeleton. Acting via CD44, Merlin triggers RAC1-dependent cytoskeleton-associated processes that modulate growth factor and ECM signals (Fig. 5‑2 ).s. Literatur Expression of the activated GTPase RAC1 decreases the association of Merlin with the cytoskeleton via dephosphorylation. Furthermore, increased expression of Merlin inhibits RAC-induced signaling via phosphorylation.s. Literatur Merlin inhibits the activation of the kinase Pak1, preventing cell cycle progression. Studies have shown that p21 (RAC1) activated kinase 1 (PAK1) substrates as well as the associated LIM domain kinases 1 and 2 (LIMK1, LIMK2) modulate actin dynamics and translocate into the nucleus to regulate cell cycle progression.s. Literatur

5.3.3 The Mammalian Target of Rapamycin Pathway

Merlin has also been found to serve a tumor-suppressor function via the pro-proliferative mTOR pathway. Constitutive activation of this signaling pathway has been demonstrated in NF2-schwannomas and meningiomas.s. Literatur The loss of Merlin has been shown to activate PI3K, which starts a signaling cascade resulting in activation of mTOR and cell proliferation (Fig. 5‑3 ). Inhibitors of PI3K have been tested in an in vivo Merlin-deficient mouse, with resultant strong antiproliferative activity.s. Literatur Further downstream, PI3K activates AKT via phosphorylation in a manner that is regulated by two histone deacetylases (HDAC) and protein phosphatase 1 (PP1) (Fig. 5‑3 ). HDAC may provide another potential target for therapy along the mTOR pathway as HDAC inhibitors have been shown to decrease human schwannoma cell proliferation in an allograft model.s. Literatur ^,​ s. Literatur Finally, new studies relate the potential for Merlin to directly inhibit mTOR signaling, independent of PI3K or AKT activation, although the particular mechanism of action remains unclear.s. Literatur These findings reiterate the potential need for multiple, simultaneous therapeutic approaches.