Merlin Phosphorylation by p21-activated Kinase 2 and Effects of Phosphorylation on Merlin Localization

The Nf2 tumor suppressor gene product merlin is related to the membrane-cytoskeleton linker proteins of the band 4.1 superfamily, including ezrin, radixin, and moesin (ERMs). Merlin is regulated by phosphorylation in a Rac/cdc42-dependent fashion. We report that the phosphorylation of merlin at serine 518 is induced by the p21-activated kinase PAK2. This is demonstrated by biochemical fractionation, use of active and dominant-negative mutants of PAK2, and immunodepletion. By using wild-type and mutated forms of merlin and phospho-directed antibodies, we show that phosphorylation of merlin at serine 518 leads to dramatic protein relocalization. Neurofibromatosis type 2 (NF2)1 is an inherited disorder characterized by the development of Schwann cell tumors of the eighth cranial nerve. Mutations and loss of heterozygosity of theNF2 gene have been detected in NF2 patients and in various sporadic tumors, including schwannomas, meningiomas, and ependymomas (1). In further support of a role for NF2 in tumor suppression, mice heterozygous for an Nf2 mutation are predisposed to a wide variety of tumors with high metastatic potential (2). In a separate model in which Nf2 is inactivated specifically in Schwann cells, mice develop schwannomas and Schwann cell hyperplasia (3). The longest and predominant splice form of the Nf2gene codes for a 595-amino acid protein highly similar to the band 4.1 family of proteins. It is most closely related to the ERM proteins,moesin, ezrin, and radixin. The ERM proteins are thought to function as cell membrane-cytoskeleton linkers and are localized to cortical actin structures near the plasma membrane such as microvilli, membrane ruffles, and lamellipodia (4, 5). Likewise, merlin is localized to cortical actin structures, in patterns that partially overlap with the ERMs (1). It has been proposed that intramolecular binding of the N-terminal and C-terminal domains conformationally regulates the ERM proteins by masking binding sites for interacting proteins. The ERMs can also form homodimers and heterodimers, among themselves and with merlin, adding an additional level of complexity to the regulation of these proteins (6). The recently solved crystal structure of the moesin N/C-terminal complex strengthens this model of conformational regulation (7). Given the sequence and, most likely, structural similarities of merlin to the ERM proteins, it is possible that merlin itself could be regulated in a similar fashion. Recent studies (8, 9) have implicated additional factors in the regulation of the ERMs, including phospholipids and phosphorylation. Previous work from our group and others (10, 11) has shown that merlin is differentially phosphorylated as well and that merlin protein levels are affected by growth conditions such as cell confluency, loss of adhesion, or serum deprivation. Merlin is found in an hypophosphorylated form when the combination of cellular and environmental conditions are growth-inhibitory (10). ERMs can be phosphorylated by Rho kinase, and this phosphorylation can affect intramolecular association and cellular localization. Phosphorylation and/or phospholipids may promote the transition of the proteins to an active form by “opening” intra- and intermolecular associations. These active monomers can then bind to other interacting proteins and the actin cytoskeleton and induce actin-rich membrane projections (5,8, 12, 13). The induction of merlin phosphorylation by activated alleles of the Rho family GTPases has also been examined. Interestingly, although activated Rho did not induce noticeable phosphorylation of merlin, activated forms of Rac and cdc42 did. The site of Rac-induced phosphorylation was determined to be a serine at position 518; mutation of serine 518 results in reduced basal phosphorylation and eliminated Rac-induced phosphorylation (11). Although Rac and cdc42 are implicated in the regulation of many pathways, they are most associated with regulation of cytoskeleton reorganization and gene expression (for recent reviews see Refs.14-16). In light of the data demonstrating that activated Rac/cdc42 leads to phosphorylation and possible inactivation of merlin, the elucidation of the responsible effector pathways and their effects on merlin function are of major importance. Understanding this regulation of merlin could lead to a more complete appreciation of the effects of merlin loss in tumors

Crizotinib inhibits NF2-associated schwannoma through inhibition of focal adhesion kinase 1

Neurofibromatosis type 2 (NF2) is a dominantly inherited autosomal disease characterized by schwannomas of the 8th cranial nerve. The NF2 tumor suppressor gene encodes for Merlin, a protein implicated as a suppressor of multiple cellular signaling pathways. To identify potential drug targets in NF2-associated malignancies we assessed the consequences of inhibiting the tyrosine kinase receptor MET. We identified crizotinib, a MET and ALK inhibitor, as a potent inhibitor of NF2-null Schwann cell proliferation in vitro and tumor growth in vivo. To identify the target/s of crizotnib we employed activity-based protein profiling (ABPP), leading to identification of FAK1 (PTK2) as the relevant target of crizotinib inhibition in NF2-null schwannoma cells. Subsequent studies confirm that inhibition of FAK1 is sufficient to suppress tumorigenesis in animal models of NF2 and that crizotinib-resistant forms of FAK1 can rescue the effects of treatment. These studies identify a FDA approved drug as a potential treatment for NF2 and delineate the mechanism of action in NF2-null Schwann cells

Rac1 Targeting Suppresses Human Non-Small Cell Lung Adenocarcinoma Cancer Stem Cell Activity

The cancer stem cell (CSC) theory predicts that a small fraction of cancer cells possess unique self-renewal activity and mediate tumor initiation and propagation. However, the molecular mechanisms involved in CSC regulation remains unclear, impinging on effective targeting of CSCs in cancer therapy. Here we have investigated the hypothesis that Rac1, a Rho GTPase implicated in cancer cell proliferation and invasion, is critical for tumor initiation and metastasis of human non-small cell lung adenocarcinoma (NSCLA). Rac1 knockdown by shRNA suppressed the tumorigenic activities of human NSCLA cell lines and primary patient NSCLA specimens, including effects on invasion, proliferation, anchorage-independent growth, sphere formation and lung colonization. Isolated side population (SP) cells representing putative CSCs from human NSCLA cells contained elevated levels of Rac1-GTP, enhanced in vitro migration, invasion, increased in vivo tumor initiating and lung colonizing activities in xenografted mice. However, CSC activity was also detected within the non-SP population, suggesting the importance of therapeutic targeting of all cells within a tumor. Further, pharmacological or shRNA targeting of Rac1 inhibited the tumorigenic activities of both SP and non-SP NSCLA cells. These studies indicate that Rac1 represents a useful target in NSCLA, and its blockade may have therapeutic value in suppressing CSC proliferation and metastasis

Factors contributing to posttraumatic growth and its buffering effect in adult chidren of cancer patients undergoing treatment

This study examined relationships among demographic, clinical, and psychosocial variables in adult children of cancer patients. Two hundred and fourteen participants completed measures of posttraumatic growth (PTG), distress, posttraumatic stress disorder (PTSD) symptoms, social support, and family functioning. Significant gender differences in all PTG dimensions were found, as well as associations among PTG, gender, parental dependency, distress, PTSD, and family functioning. Social support was not a mediator in the relationship between gender and PTG. Gender, education, disease duration, dependency, distress, and family flexibility predicted PTG. Finally, PTG had amoderating effect in the relationship between distress and PTSD/social support. These results may guide psychosocial interventions in this population.Fundação para a Ciência e Tecnologia (FCT

Non-Small Cell Lung Carcinoma Cell Motility, Rac Activation and Metastatic Dissemination Are Mediated by Protein Kinase C Epsilon

Background: Protein kinase C (PKC) e, a key signaling transducer implicated in mitogenesis, survival, and cancer progression, is overexpressed in human primary non-small cell lung cancer (NSCLC). The role of PKCe in lung cancer metastasis has not yet been established. Principal Findings: Here we show that RNAi-mediated knockdown of PKCe in H358, H1299, H322, and A549 NSCLC impairs activation of the small GTPase Rac1 in response to phorbol 12-myristate 13-acetate (PMA), serum, or epidermal growth factor (EGF). PKCe depletion markedly impaired the ability of NSCLC cells to form membrane ruffles and migrate. Similar results were observed by pharmacological inhibition of PKCe with eV1-2, a specific PKCe inhibitor. PKCe was also required for invasiveness of NSCLC cells and modulated the secretion of extracellular matrix proteases and protease inhibitors. Finally, we found that PKCe-depleted NSCLC cells fail to disseminate to lungs in a mouse model of metastasis. Conclusions: Our results implicate PKCe as a key mediator of Rac signaling and motility of lung cancer cells, highlighting its potential as a therapeutic target

Merlin in organ size control and tumorigenesis: Hippo versus EGFR?

The role of the NF2 gene as a tumor suppressor has been well established. In this issue of Genes & Development, Benhamouche and colleagues (pp. 1718–1730) demonstrate that NF2 is also involved in the regulation of organ size control in mammals. Conditional knockout of Nf2 in the mouse liver results in massive organ enlargement and eventual tumor development, which is attributed to the specific expansion of oval cells. Here we discuss these findings and the proposed molecular mechanisms involved within the context of our current understanding of the pathways regulated by NF2

Cellular transformation by a FERM domain mutant of the Nf2 tumor suppressor gene

Mutations in the Nf2 tumor suppressor gene lead to tumor formation in humans and mice and cellular overproliferation phenotypes in Drosophila. The Nf2 encoded protein, merlin, shares close sequence similarity in its amino terminus to members of the band 4.1 family of membrane-cytoskeletal linkers. Similarities between merlin and this family suggest a role for merlin in regulating cytoskeletal function. However, the mechanism of the tumor suppressing activity of merlin is not yet understood. Mutational analysis of Nf2 in flies has led to the identification of a dominant-negative allele, which harbors mutations in the amino terminus of the protein. Here, we report that expression of a murine analog of this amino-terminal mutant of Nf2 leads to complete transformation of NIH3T3 fibroblasts in culture. Cells that express this Nf2 mutant allele display disruptions of the actin cytoskeleton, lack of contact inhibition of growth, and anchorage-independent growth. Finally, fibroblasts that express this mutant Nf2 allele form tumors when injected into nude mice

WT1: a weak spot in KRAS-induced transformation

Activating mutations in the Ras alleles are found frequently in tumors, making the proteins they encode highly attractive candidate therapeutic targets. However, Ras proteins have proven difficult to target directly. Recent approaches have therefore focused on identifying indirect targets to inhibit Ras-induced oncogenesis. For example, RNAi-based negative selection screens to identify genes that when silenced in concert with activating Ras mutations are incompatible with cellular proliferation, a concept known as synthetic lethality. In this issue of the JCI, Vicent et al. report on the identification of Wilms tumor 1 (Wt1) as a Kras synthetic-lethal gene in a mouse model of lung adenocarcinoma. Silencing of Wt1 in cells expressing an endogenous allele of activated Kras triggers senescence in vitro and has an impact on tumor progression in vivo. These findings are of significant interest given previous studies suggesting that the ability of oncogenic Kras to induce senescence versus proliferation depends on its levels of expression