Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Merlin (NF2)

  • Mateus Mota
  • Rajeev S. Samant
  • Lalita A. Shevde
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101780

Synonyms

Historical Background

Neurofibromatosis type 2 (NF2) is a tumor suppressor gene that when deleted or mutated causes a disorder with the same name. This condition is primarily characterized by benign tumors of the nervous system such as bilateral vestibular schwannomas – originating from Schwann cell – in addition to meningiomas and ependymomas, which arise from cells that make up the membrane surrounding the brain and spinal cord and from the ependyma, a tissue of the central nervous system, respectively (Petrilli and Fernandez-Valle 2016). The incidence ratio of this dominantly inherited disease is 1:25,000, and 50–60% of cases are caused by de novo mutations with somatic mosaicism. NF2 somatic mutations have also been found in patients with other nonnervous system tumors, including mesothelioma, melanoma, glioma, breast, and prostate cancer, suggesting that NF2’s tumor suppressor role is not only restricted to the nervous system but also extends to other tissues (Petrilli and Fernandez-Valle 2016).

The NF2 tumor suppressor gene located on chromosome 22q12 contains 17 exons and encodes a 69 kDa protein named schwannomin or merlin (moesin-ezrin-radixin-like protein). Merlin is a member of the band 4.1 protein superfamily and of the ERM (ezrin, radixin, moesin) protein subfamily, usually referred as the FERM (4.1 protein, ezrin, radixin, moesin) domain family of proteins. Merlin has approximately 64% sequence similarity with the FERM domain of the proteins of this family. As a product of alternative splicing, two major isoforms of merlin are produced. Isoform 1 is predominantly involved in the tumor suppressor activity of merlin. In isoform 1, exon 16 is spliced and exon 17 encodes a long carboxy-terminal end. Isoform 2 contains exon 16, which presents a stop codon and therefore displays a truncated carboxy-terminal end (Cooper and Giancotti 2014; Petrilli and Fernandez-Valle 2016).

The most studied function of FERM domain family of proteins, including merlin, is to serve as a scaffold-like protein that localizes beneath the cell membrane. This cell localization is strategic for merlin’s role as a connector between transmembrane cell receptors, intracellular effectors and F-actin. The downstream cell signaling pathways triggered by the transmembrane cell receptors that merlin associates with, are mostly involved in cell proliferation and survival. Thus, merlin is an important protein that indirectly integrates extracellular information to ultimately modulate cellular behavior. As an example, in isolated cultured mammalian cells, merlin is concentrated in actin-rich structures such as membranes ruffles and, in cells growing near to each other, merlin is situated along cell-cell boundaries. This configuration of localization is coherent with one of merlin’s important roles which is in mediating contact-dependent growth inhibition (McClatchey and Giovannini 2005).

Merlin’s Conformation and Structure

In order to understand merlin’s biology and function, it is important to first address its structure, and this has been aided by its association with the FERM domain family of proteins. The FERM domain is situated at the N-terminal domain, which is involved in binding with transmembrane cell receptors, followed by an α-helix domain and a C-terminal domain (Fig. 1); the latter being associated with the binding to the cortical actin cytoskeleton. However, unlike the other FERM domain proteins, merlin does not have an actin-binding region at the C-terminal domain, instead it may bind actin directly via the N-terminal domain (Cooper and Giancotti 2014; Petrilli and Fernandez-Valle 2016).
Merlin (NF2), Fig. 1

Merlin protein structure. The protein structurally spans three domains, comprising 595 amino acids: the FERM (4.1 protein, ezrin, radixin, moesin) domain is located at the N-terminal domain and contains three subdomains (A, B, and C), followed by the α-helix domain and C-terminal domain, where the phosphorylation site serine 518 (Ser518) is located

Merlin’s conformation and structure are regulated by posttranslational modifications in which phosphorylation seems to be the most important. The most studied phosphorylation site is serine 518 (Ser518) located on the C-terminal domain. In vitro studies show that cell culture conditions such as cell density, cell-substrate attachment, and growth factor availability influence merlin’s function primarily by modulating phosphorylation at Ser518 which ultimately activates or inactivates mechanisms involved in cell proliferation (Shaw et al. 1998).

One intriguing feature about merlin is the relationship between its scaffold-like structure, phosphorylation status at Ser518, and tumor suppressor activity. As a member of the FERM domain protein family, when phosphorylated at Ser518, it is expected of merlin to be an active scaffold-like protein beneath the cell membrane, displaying an open conformation and enabling transmembrane receptor-mediated cell signaling events involved in cell proliferation and survival. Therefore, in such context, the tumor suppressor function of merlin is inactive. However, upon dephosphorylation of Ser518, merlin’s scaffold-like structure closes and poses a hindrance to link receptors on the cell membrane and the cytoskeleton to trigger cell signaling pathways. In such a scenario, the tumor suppressor role of merlin becomes active (Fig. 2a). Although this explanation is very compelling, a recent study based on resonance energy transfer analysis experimentally proposed a new conformational model in which, in solution, both dephosphorylated and phosphorylated forms of merlin are in a closed conformation shape and capable of binding to receptors and cytoskeleton. However, the dephosphorylated, active tumor suppressor conformation is slightly open, while the phosphorylated, inactive tumor suppressor conformation is fully closed (Fig. 2b) (Petrilli and Fernandez-Valle 2016).
Merlin (NF2), Fig. 2

Relationship between the scaffold-like structure and tumor suppressor role of merlin. (a) Based on ERM protein analogy, when phosphorylated at Ser518, merlin is an active scaffold-like protein in a fully open conformation, but it has its tumor suppressor function inactivated; upon dephosphorylation of Ser518, the protein’s structure is fully closed, becoming an inactive scaffold-like protein; in turn, its tumor suppressor function is activated. (b) According to experimental resonance energy transfer analysis data, when phosphorylated at Ser518, merlin acquires a fully closed conformation and has its tumor suppressor function inactivated, whereas upon dephosphorylation at Ser518, merlin’s conformation is slightly open, and its tumor suppressor capabilities are regained

Merlin’s Tumor Suppressor Activation

In low cell density environment and proper supply of growth factors, cells tend to proliferate in order to expand over the space available. In an organism, cells will stop proliferating and undergo cell growth arrest when they make tight cell-cell or cell-extracellular matrix contacts, characterizing a contact inhibition of growth response. In cancer, unresponsiveness to contact inhibition of growth is one of the mechanisms that lead to tumor formation. Merlin, when activated, is one of the proteins critically involved in contact inhibition of growth. The activation or inactivation of merlin’s tumor suppressor activity is directly determined by the dephosphorylation or phosphorylation at Ser518, respectively.

In proliferating cells, receptor tyrosine kinase and integrin activate Cdc42 and Rac proteins, which in turn activate p21-activated kinase (PAK). PAK directly phosphorylates merlin at Ser518, increasing the linking of the FERM domain with the carboxy-terminal domain and fully closing the protein structure. As a result, the tumor suppressor activity of merlin is inactivated. In addition, protein kinase A (PKA) is also involved in merlin’s Ser518 phosphorylation, contributing to the inactivation of its tumor suppressor function (Fig. 3). Other phosphorylation sites of merlin are at threonine 230 and serine 315, which are substrates for protein kinase B (PKB), commonly known as AKT. AKT phosphorylation controls merlin by ubiquitination and degradation via proteasome (Cooper and Giancotti 2014).
Merlin (NF2), Fig. 3

Phosphorylation and inactivation of merlin’s tumor suppressor activity. Integrins and receptor tyrosine kinase (RTK) stimulate Cdc42 and Rac proteins that further activate p21-activated kinase (PAK). PAK directly phosphorylates merlin protein at Ser518 leading to the inactivation of its tumor suppressor activity. Protein kinase A (PKA) is another protein that contributes to merlin’s Ser518 phosphorylation and inactivation of merlin’s tumor suppressor function

When cells are adjacent and make contact with each other, cadherin-dependent cell-cell adhesions are formed, which inhibit PAK consequently leading to decreased phosphorylation of merlin and activation of its tumor suppressor activity. Moreover, merlin regulates contact-dependent growth inhibition through association with the receptor CD44. Hyaluronate (HA), the ligand of CD44, is the major component of extracellular matrix, and as a result of high cell density, there is an increase of HA surrounding the cells. In such a growth inhibitory case, HA binds to CD44, resulting in decreased phosphorylation of merlin and its direct interaction with the ERM-binding domain of the CD44 cytoplasmic tail. Furthermore, myosin phosphatase MYPTI-PP δ (MYPT1) is believed to be the key player in this process since it dephosphorylates Ser518, activates merlin’s tumor suppressor activity, and enables contact inhibition of growth (Fig. 4) (Morrison et al. 2001;Cooper and Giancotti 2014).
Merlin (NF2), Fig. 4

Dephosphorylation and activation of merlin’s tumor suppressor activity. Cadherin-dependent cell-cell adhesions inhibit p21-activated kinase (PAK) causing decreased phosphorylation of merlin and activation of its tumor suppressor activity. Moreover, in high cell density situations, hyaluronate (HA), a ligand of CD44 transmembrane receptor and a major component of extracellular matrix, binds to CD44, also resulting in reduced phosphorylation of merlin. Phosphatases, such as myosin phosphatase MYPTI-PP δ (MYPT1), play a role in the activation process by dephosphorylating merlin at the Ser518 site, ultimately activating its tumor suppressor activity and contact inhibition of growth

Merlin and Its Association with Different Cell Signaling Pathways

PAK

When activated, merlin associates with various cell signaling pathways in order to modulate cellular behavior. Rac, the protein that activates PAK (that directly phosphorylates merlin at Ser518), is a subfamily of the Rho family of small GTPases. One of merlin’s roles in contact inhibition of growth is to stop the recruitment of Rac to matrix adhesion. However, this process is compromised by activated PAK that phosphorylates and inhibits merlin. Experiments with endothelial cells showed that activation of PAK alone is sufficient to enable them to escape contact inhibition of growth restraint. In a low-density cell culture in which proliferation is expected, the culture ceases to expand upon the introduction of a dominant-negative PAK; this is due to lack of phosphorylation and inhibition of merlin, rendering merlin in an active, growth-restrictive form. This suggests that PAK is necessary for normal cell growth. On the other hand, in a high-density, confluent cell culture, cells would normally form cadherin-dependent cell-cell adhesions and undergo contact inhibition of growth. However, stimulation of PAK activation promotes Rac recruitment, in an amplification loop Rac activates PAK, and cells escape cell growth inhibition. Upon introduction of dominant-negative Rac to this circumstance, the cells lose their ability to escape the restraint of contact inhibition provided by PAK activation and halt growth. This suggests that PAK induces the progression of cell cycle by regulating Rac recruitment. As a way to underscore this cross talk between PAK and Rac in relation to merlin’s activation status, it is relevant to note that two distinct loss-of-function mutant forms of merlin showed recruitment of Rac to matrix adhesions and lack of response to contact inhibition. On the other hand, a constitutively unphosphorylated, active mutant form of merlin opposed PAK and turned cells responsive to contact inhibition of growth (Okada et al. 2005). These observations altogether show that Rac is a key GTPase involved in merlin’s activation.

PIKE-L

Another GTPase involved in merlin’s tumor suppressor activity is the long isoform of PI3K enhancer (PIKE-L). PIKE-L is a brain-specific GTPase that binds to PI3K and stimulates its cellular function. The FERM domain of merlin binds to the N-terminal domain of PIKE-L, disrupting PIKE-L binding to PI3K (Rong et al. 2004). Therefore, the loss of merlin in schwannomas and meningiomas leads to the activation of PI3K/AKT pathway and cell proliferation (Petrilli and Fernandez-Valle 2016). In patient-derived mutant L64P merlin cells, there is no interaction between the mutated merlin and PIKE-L, and consequently no negative regulation on PI3K activity is observed. Likewise, a mutation in PIKE-L, P187L, disrupts its interaction with merlin but still allows PIKE-L binding to PI3K and stimulation of activity. These results indicate that the loss of merlin’s ability to bind to PIKE-L, either by a mutation of merlin’s FERM domain or PIKE-L’s N-terminal domain, facilitates continuous activation of PI3K and cell proliferation (Rong et al. 2004).

Hippo

In mammals, the Hippo signaling pathway controls cell growth by phosphorylating and inactivating the transcriptional coactivator YAP. Once inactivated, YAP is degraded in the cytosol and does not translocate to the nucleus for its final association with TEAD transcription factor coactivators. It has been reported that merlin loss in human meningioma tumors and cell lines is associated with an increase in YAP expression and nuclear localization, suggesting merlin as an upstream activator of Hippo signaling pathway (Petrilli and Fernandez-Valle 2016). Furthermore, another study has demonstrated that liver development is regulated by the interrelationship between Hippo and NF2 via YAP. Inactivation of NF2 tumor suppressor caused hyperplasia of hepatocytes and biliary epithelial cells accompanied by hepatocellular carcinoma and bile duct hamartoma. On the other hand, upon inactivation of YAP, the hepatocytes and biliary epithelial cells showed developmental defects. These results suggest that both NF2 and YAP, despite exerting different effects on the cell, are important for liver homeostasis. In addition, loss of NF2 in vivo resulted in a decrease of phosphorylated YAP, which meant reduced YAP inactivation and decreased phosphorylation of Hippo pathway members Lats1/2 that are involved in cell division. Collectively, these are indications that merlin acts as an upstream regulator of the Hippo signaling pathway in mammals (Zhang et al. 2010).

FAK

Focal adhesion kinase (FAK), also known as protein tyrosine kinase 2 (PTK2), is a cytoplasmic protein kinase involved in cell proliferation, adhesion, migration, and invasion by integrating inputs from transmembrane proteins signaling, such as integrins and growth factor receptors. Because of the cellular functions that FAK is associated with, it is not surprising that FAK upregulation has been seen in different cancer types. A study with mesothelioma cells, a malignancy that affects the lining of organs specially the lungs, has reported that an additional mechanism whereby merlin exerts its tumor suppressor activity is by negatively regulating FAK. In order to have its kinase activity activated, FAK must be phosphorylated at the tyrosine 397 (Tyr397) site, which in turn enables its binding to SH2-containing proteins, such as Src family kinases and the p85 subunit of PI3K. This process is crucial because the binding of Src triggers the activation and phosphorylation of downstream effectors of FAK that ultimately potentiate FAK activity, in addition to PI3K activation. It was observed that upon re-expression of merlin in two mesothelioma cells that lacked expression of endogenous NF2, phosphorylation at Tyr397 was significantly decreased. This was accompanied by an interruption in the binding between FAK and Src and p85 and followed by a decrease in cell invasiveness (Poulikakos et al. 2006).

This relationship between merlin and FAK reveals a new path in the pursuit of targeted therapies for cancer. In fact, some studies have reported that merlin-deficient cells are more sensitive to FAK inhibitors. A detailed study reported that among a panel of 47 human cancer cell lines, including mesothelioma, ovarian and breast carcinoma, and melanomas, that were treated with FAK inhibitor VS-4718, the most sensitive cell lines lacked merlin expression. Furthermore, experiments in vivo demonstrated that VS-4718 treatment had an expressively higher antitumor effect, measured by tumor regression, in merlin-lacking human triple-negative breast cancer xenograft mice compared to merlin expressors. In addition, upon VS-4718 treatment, merlin-negative mesothelioma cell lines expressed higher levels of caspase 3/7 and lower levels of Ki-67, markers of apoptosis and cell proliferation, respectively, than merlin-positive mesothelioma cells lines. Therefore, the inhibition of FAK can be particularly beneficial for treatments of malignancies with loss of merlin. Furthermore, in the search for mechanisms behind the higher sensitivity of merlin-deficient cells upon FAK inhibition, it was observed that the blockade of cell-cell contacts by N-cadherin-blocking antibody increased the sensitivity of merlin-positive mesothelioma cells to VS-4718 inhibitor. On the other hand, merlin-negative mesothelioma cells blocked for ß1 integrin exhibited insensitivity to VS-4718 inhibitor. As previously discussed, while cadherin is important for the formation of cell-cell adhesion and is associated with merlin activation, integrins are involved in cell-extracellular matrix interaction and contribute to merlin phosphorylation and inactivation. Therefore, it is proposed that merlin-expressing cells engage in cell-cell junctions as a source of survival and proliferation signal and are independent of FAK and thus insensitive to FAK inhibition. On the contrary, merlin-non-expressing cells are more adherent to extracellular matrix and dependent on FAK for cell survival and consequently are more likely to be affected by FAK inhibition (Shapiro et al. 2014).

Another specific FAK inhibitor that has been studied is the PF-271 ATP-competitive molecule. Given FAK’s involvement in migration and invasion, one of the consequences of FAK inhibition is the impediment of anchorage-independent growth of cancer cells. It was observed that human ovarian cancer cell lines highly sensitive to PF-271 treatment, defined by anchorage-independent cell growth inhibition, had lower levels of merlin in comparison to cells that were insensitive and exhibited high levels of merlin. In vivo experiments using an orthotopic tumor model with high merlin levels have demonstrated that PF-271 treatment had no significant impact on the tumor weight, suggesting lack of sensitivity due to merlin sufficiency. However, at least in this ovarian cancer setting, there was no apparent direct link between high merlin protein expression and FAK inhibition since NF2 knockdown did not confer an increase in PF-271 sensitivity. Therefore, although a causal relationship has not been found, merlin expression levels still seem to be a potential predictive marker for FAK inhibitor response (Shah et al. 2014).

ErbB Family Receptor

The ErbB family receptor activates different signaling pathways involved in the modulation of cell proliferation, survival, motility, and differentiation. The two receptors of the ErbB family more commonly studied and associated with merlin are ErbB1, commonly known as epidermal growth factor receptor (EGFR) and ErbB2 (Ammoun and Hanemann 2011). It was observed that merlin stabilizes the formation of adherens junction and blocks the activation of EGFR by isolating it on the cell membrane, suggesting a new mechanism of tumor suppression. As examples, the levels of active, phosphorylated EGFR were decreased in WT mouse embryo fibroblast (MEF) membrane upon reaching cell confluency; however this effect was not seen in NF2 −/− MEF. Furthermore, NF2 −/− MEFs, primary osteoblasts, and liver-derived epithelial cells were able to escape contact inhibition of growth by failing to downregulate the EGFR receptor. These processes were reverted upon restoration of merlin, suggesting another possible mechanism for merlin in exerting its tumor suppressor role. Upon cell-cell contact, the formation of cadherin-dependent cell-cell adhesion is concordant with the recruitment and activation of merlin to stabilize the association between the cell-cell adhesion setting and the cortical cytoskeleton. Moreover, activated merlin and EGFR are linked by the PDZ domain-containing adaptor NHE-RF1. As a result, upon EGFR ligand binding, EGFR will neither be internalized as part of its activation nor it will interact with its signaling effectors, bringing EGFR signaling pathway to a state of inactivation (Curto et al. 2007). Although ErbB3 and ErbB4 have also been observed to be associated with merlin, ErbB2 is the other receptor of the ErbB family that has more investigations associated with merlin. In membrane lipid rafts of Schwann cells, ErbB2 is activated and triggers downstream signaling pathways, a process inhibited by merlin. Therefore, in schwannoma, a tumor originating from Schwann cells that harbor NF2 mutation(s), ErbB2 is constitutively activated and contributes to cell proliferation (Ammoun and Hanemann 2011). A separate study demonstrated hyperactivation of ErbB2 and increased ErbB2-Src binding in NF2 −/− glia in vitro and in vivo. In this NF2 −/− glia setting, it was seen that Src activation led to the phosphorylation and activation of FAK and paxillin. As discussed before, FAK promotes cell proliferation and survival; paxillin is another protein associated with cell proliferation in NF2 −/− glia in vitro and in vivo. Therefore, it is suggested that in WT cells, merlin competes with Src for binding to ErbB2 cytoplasmic domain; upon merlin binding, Src remains dephosphorylated and inactive, compromising the phosphorylation and activation of its downstream effectors, such as FAK and paxillin. Thus, normal cell growth is protected. However, when merlin is inactivated, Src binds to ErbB2 and undergoes phosphorylation and activation; activated Src phosphorylates and activates FAK and paxillin, resulting in intense cell growth (Houshmandi et al. 2009).

PDGFR

Platelet-derived growth factor receptor (PDGFR) is a receptor tyrosine kinase that exists in two types, PDGFRα and PDGFRβ, in Schwann cells. PDGFR activates signaling pathways involved in cell proliferation and survival. Overexpression of PDGFRβ in human schwannomas and meningiomas increases activity of its downstream effectors, such as ERK1/2 and AKT. It has been reported that merlin associates with and modulates PDGFRβ. Upon indirect interaction, merlin downregulates PDGFRβ signaling. Moreover, the adaptor protein (Na+/H+ exchanger regulating factor 1; ezrin-radixin-moesin (ERM)-binding phosphoprotein) connects merlin to PDGFRβ, causing the internalization of activated PDGFRβ. As such, deficiency of NF2/merlin results in upregulation of PDGFRβ receptor signaling (Ammoun and Hanemann 2011).

mTOR

The other pathway involved with merlin is the Ser/Thr kinase mechanistic target of rapamycin (mTOR); mTOR consists of two complexes, mTORC1, which is sensitive to rapamycin and regulates various cellular functions, such as energy metabolism and autophagy, and mTORC2, which is less sensitive to acute rapamycin treatment and is involved in cytoskeletal regulation and AKT activation. In human schwannomas and meningiomas, constitutive activation of the mTORC1 signaling complex has been reported. Although not yet elucidated, the loss of merlin activated mTORC1 signaling independent of AKT and ERK (James et al. 2009). Another study on mesothelioma cells showed that loss of merlin led to the activation of mTORC1 signaling. As such, these findings suggest that one possible mechanism by which mutations in NF2 lead to merlin malfunction or loss and contribute to tumorigenesis is through the activation of mTORC1 signaling. In addition, as expected, with the loss of merlin and activation of mTORC1, tumor cells become more sensitive to rapamycin. This turns out to be a benefit in the search for treatment for cases of mesothelioma with NF2 mutations since mTORC1 presents as a potential therapeutic target (Lopez-Lago et al. 2009).

DCAF1

Although first described to be localized and exerting its function at the cell membrane, merlin has also been described as a dynamic protein being transported into the nucleus as in the inhibition of CRL4DCAF1. DBB [DNA damage-binding protein 1] and Cul4-associated factor 1 (DCAF1) is a substrate recognition protein for CRL4 (Cullin-Ring ligase 4), a class of ubiquitin E3 ligase that promotes the ubiquitylation and degradation of targets, resulting in cell proliferation, survival, DNA repair, and genomic integrity regulation. Dephosphorylated and activated merlin is guided into the nucleus by its FERM domain; once in the nucleus, merlin binds to the C-terminal domain of DCAF1 and impedes its association with targets for CRL4 ubiquitylation. One suggestive explanation of how merlin would block such DCAF1 activity relies on the fact that one of the subdomains of merlin’s FERM domain has a ubiquitin-like fold; therefore merlin would function as an inhibitory mimic substrate for the CRL4DCAF1 complex. As an illustration, in vitro and in vivo experiments have reported that upon silencing of DCAF1, merlin-deficient tumor cells lost their capacity to grow indicating cell entered growth arrest. Thus, it is obvious that merlin’s function as a tumor suppressor goes beyond its primary location at the cell membrane (Li et al. 2010).

Merlin and Nonnervous System Cancers

NF2 gene mutations and inactivation are predominantly seen in benign nervous system tumors, such as schwannomas, ependymomas, and meningiomas, but recently various studies have reported alterations of NF2 and merlin in other nonnervous system malignancies. For being initially involved in benign tumors, one can speculate that NF2 is associated with early tumorigenetic steps in development of cancer, leading to further disruption of other key signaling pathways (Petrilli and Fernandez-Valle 2016). However, merlin has already been implicated in other aspects of cancer biology and not only in its early establishment. As an example, loss of adherens junction (AJ) functions may be the cellular mechanism, whereby NF2 deficiency leads to tumor metastasis development. Mouse embryo fibroblasts and primary epithelial keratinocytes with NF2 deficiency fail to form cadherin-mediated adhesion between cells. These outcomes suggest that in a normal situation, merlin would associate with AJ and mediate the formation of cadherin junction between adjacent cells. However, this process is compromised in conditions of loss of merlin protein and/or function leading to tumor invasion and metastasis (Lallemand et al. 2003). The types of nonnervous system tumors that NF2/merlin impacts are summarized below:

Mesothelioma – This is a highly aggressive cancer that arises from mesothelial cells of the pleura, peritoneum, and pericardium and is the malignancy that has the highest rate of NF2 aberrations. It has been reported that approximately 30–50% of mesothelioma tumors have mutations in the NF2 coding gene. A report from (Sekido et al. 1995) showed that seven out of 17 cases (representing 41%) of mesotheliomas had genetic alterations in NF2. All of these seven alterations were predicted to generate a truncated or no protein as a consequence of in-frame deletions or premature stop codon. Another study (Bianchi et al. 1995) screened a set of malignant mesothelioma cell lines and matched primary tumors for mutations within the NF2 coding region. Alterations were detected in eight out of 15 cell lines analyzed, and six of these eight altered cells had their NF2 gene transcript alterations confirmed at the genomic level in primary tumors. These studies provide evidence of NF2 alteration in mesothelioma tumorigenesis. Moreover, a recent study speculates that in mesothelioma without detectable NF2 gene mutations, the damage could be at the translational level in which merlin would be inactivated by regulators such as CPI-17, a phosphatase MYPTI-PP δ inhibitor that would impede the dephosphorylation and activation of merlin. In a large panel of clinical samples and primary cultures obtained from mesothelioma patients, merlin was either absent or inactive (phosphorylated on Ser518). Thus, disruption of NF2/merlin is encountered to some extent in all mesothelioma cases (Thurneysen et al. 2009).

Melanoma – This is the most aggressive form of skin cancer where approximately 5% of cases have NF2 mutations. Abrogating endogenous NF2 knockdown in the WM1552C human melanoma cells induced subcutaneous growth in vivo, whereas overexpression of merlin in MeWo melanoma cells caused growth inhibition in vitro. Increased merlin expression activated the MST1/2 kinases, the mammalian homologues of Drosophila Hippo, which are established tumor suppressors and key regulators of cell proliferation and apoptosis, invoking a clear role for merlin in activating Hippo signaling to keep melanoma cell growth in check (Murray et al. 2012).

Glioma – In high-grade human malignant glioma, merlin protein expression is significantly reduced; a clear concordance is seen between the loss of merlin at the transcript levels and the protein. NF2 knockdown promotes glioma growth in vivo; in contrast, restoration of merlin inhibits the growth of human glioma both in vitro and in vivo. Microarray analyses and functional assays revealed that merlin positively regulated Lats2, a kinase in the Hippo signaling cascade, corroborating with the idea of merlin as an activator of Hippo pathway. Merlin was also found to increase the expression of the DKK Wnt inhibitors' simultaneous with a decrease in the Frizzled co-receptor, indicating an inhibition of Wnt signaling (Lau et al. 2008).

Breast cancer – This tumor type was not associated with any significant mutations in NF2 gene; however, merlin protein expression level was significantly reduced as demonstrated by immunohistochemistry staining. The reduced merlin levels in breast cancer were mechanistically determined to be due to its elimination that was initiated by osteopontin-dependent Akt signaling. Phosphorylation of merlin at Ser315 position was critically determinative of its stability. Interestingly enough, introduction of merlin into malignant breast cancer cells attenuated their tumorigenic ability, suggesting that merlin functions in a tumor-suppressive role when restored for its cellular presence (Morrow et al. 2011). In addition, we reported that merlin interacts with β-catenin and assures its localization at the cell membrane of breast cancer cells. This is important because β-catenin maintains cell-cell adhesion by forming a complex with E-cadherin and α-catenin, a process crucial for contact inhibition of growth. Restoration of merlin in metastatic breast cancer cell lines that lack endogenous merlin induced β-catenin proteasomal degradation, via GSK-3β-mediated activated Axin1, and blocked its migration to the nucleus, resulting in downregulation of the expression of the oncoprotein, osteopontin. From the perspective of therapeutic targeting, these findings are very promising since they reveal β-catenin and its destruction complex as potential targets for therapy in merlin-deficient breast tumors. Indeed, it was observed that merlin deficiency sensitizes breast cancer cells to compounds that inhibit the activity of β-catenin (Morrow et al. 2016).

Prostate cancer – Merlin expression was low in four of five prostate cancer cell lines analyzed; DU145 prostate cancer cells had the highest level of phosphorylated merlin (Ser518). The kinase PAK predominantly regulates merlin in the DU145 cells keeping merlin in a constitutively phosphorylated state. Intriguingly, in these cells serum deprivation or increased cell density did not inhibit merlin phosphorylation. Moreover, CD44 binding to hyaluronate or siRNA-mediated inhibition of CD44 expression did not alter merlin phosphorylation, whereas regulation of ERM phosphorylation remained intact. The cumulative observations indicated that in these cells, merlin was characteristically inactivated by PAK (Horiguchi et al. 2008).

Summary

Neurofibromatosis type 2 (NF2) is a tumor suppressor gene that when defective causes a nervous system disorder with the same name, characterized by benign tumors such as schwannomas, meningiomas, and ependymomas. This gene encodes a protein named merlin, which is a member of the FERM domain family of proteins, and connects transmembrane receptors in the cell membrane, cortical actin cytoskeleton, and intracellular effectors in order to regulate cellular functions involved in proliferation and survival. Merlin has a conserved FERM domain in the N-terminus, an α-helix and a C-terminus. The determinant regulatory mechanism of the protein is by phosphorylation, particularly of serine 518 (Ser518), the main phosphorylation site. Upon phosphorylation of Ser518, mediated by p21-activated kinase (PAK) and Cdc42, merlin adopts a fully closed conformation and has its tumor suppressor activity inactivated; upon dephosphorylation of Ser518 by myosin phosphatase MYPTI-PP δ (MYPT1), merlin’s closed conformation opens and has its tumor suppressor activity activated. Merlin interacts with various signaling pathways, such as AKT, mTOR, Rho family of GTPases, Hippo, and E3 ubiquitin ligase CRL4. Besides its association with benign nervous system tumors, NF2/merlin has also been associated with malignant tumors such as mesotheliomas, melanoma, glioma, breast cancer, and prostate cancer. In conclusion, further investigation of merlin biology is encouraged due to its negative regulation of key players of cell proliferation and survival promotion. A better understanding of how merlin activation/inactivation dynamics work and also how the lack of NF2/merlin is responsible for the pathogenesis of neurofibromatosis type 2 disorder and contributes to the development of different malignancies is still evolving. These studies unlock a path to seek potential therapeutic targets of merlin-associated proteins, merlin-regulated proteins, as well as to use merlin expression status as a determinant for patient treatment or survival.

Notes

Acknowledgments

NIH R01CA138850 and Breast Cancer Research Foundation of Alabama (BCRFA) grants to L.A.S.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Mateus Mota
    • 1
  • Rajeev S. Samant
    • 2
    • 3
  • Lalita A. Shevde
    • 1
  1. 1.Division of Molecular and Cellular Pathology, Department of Pathology and Comprehensive Cancer CenterUniversity of Alabama at Birmingham, Wallace Tumor InstituteBirminghamUSA
  2. 2.Division of Molecular and Cellular Pathology, Department of PathologyUniversity of Alabama at Birmingham, Wallace Tumor InstituteBirminghamUSA
  3. 3.Department of Pathology and Comprehensive Cancer CenterThe University of AlabamaBirminghamUSA