Cluster of differentiation 44 promotes osteosarcoma progression in mice lacking the tumor suppressor Merlin.

Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin-CD44 complex through loss of Merlin, unleashes putative tumor- or metastasis-promoting functions of CD44. To evaluate the relevance of the Merlin-CD44 interaction in vivo, we compared tumor growth and progression in Cd44-positive and Cd44-negative Nf2-mutant mice. Heterozygous Nf2-mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2-mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4ß1 (very late antigen-4, VLA-4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA-4 ß1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis-promoting role in the absence of Merlin.

CPI-17 drives oncogenic Ras signaling in human melanomas via Ezrin-Radixin-Moesin family proteins.

Hyperactive Ras signaling has strong oncogenic effects causing several different forms of cancer. Hyperactivity is frequently induced by mutations within Ras itself, which account for up to 30% of all human cancers. In addition, hyperactive Ras signaling can also be triggered independent of Ras by either mutation or by misexpression of various upstream regulators and immediate downstream effectors. We have previously reported that C-kinase potentiated protein phosphatase-1 inhibitor of 17 kDa (CPI-17) can drive Ras activity and promote tumorigenic transformation by inhibition of the tumor suppressor Merlin. We now describe an additional element of this oncogenic mechanism in the form of the ezrin-radixin-moesin (ERM) protein family, which exhibits opposing roles in Ras activity control. Thus, CPI-17 drives Ras activity and tumorigenesis in a two-fold way; inactivation of the tumor suppressor merlin and activation of the growth promoting ERM family. The in vivo significance of this oncogenic switch is highlighted by demonstrating CPI-17's involvement in human melanoma pathogenesis.

Merlin Isoforms 1 and 2 Both Act as Tumour Suppressors and Are Required for Optimal Sperm Maturation.

The tumour suppressor Merlin, encoded by the gene NF2, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, NF2 is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, NF2 encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used Nf2 isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new Nf2-dependent process. Additionally, we provide for the first time in vivo evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2.

Merlin's wizardry guides cohesive migration.

Cells often migrate in tightly connected groups with coordinated movement and polarity. The collective migration of epithelial cell sheets is now shown to be mediated by a signalling axis that involves the merlin tumour-suppressor protein, the tight-junction-associated angiomotin-Rich1 complex and the Rac1 small GTPase.

A neuronal function of the tumor suppressor protein merlin.

Mutagenic loss of the NF2 tumor suppressor gene encoded protein merlin is known to provoke the hereditary neoplasia syndrome, Neurofibromatosis type 2 (NF2). In addition to glial cell-derived tumors in the PNS and CNS, disease-related lesions also affect the skin and the eyes. Furthermore, 60% of NF2 patients suffer from peripheral nerve damage, clinically referred to as peripheral neuropathy. Strikingly, NF2-associated neuropathy often occurs in the absence of nerve damaging tumors, suggesting tumor-independent events. Recent findings indicate an important role of merlin in neuronal cell types concerning neuromorphogenesis, axon structure maintenance and communication between axons and Schwann cells. In this review, we compile clinical and experimental evidences for the underestimated role of the tumor suppressor merlin in the neuronal compartment.

Neuronal merlin influences ERBB2 receptor expression on Schwann cells through neuregulin 1 type III signalling.

Axonal surface proteins encompass a group of heterogeneous molecules, which exert a variety of different functions in the highly interdependent relationship between axons and Schwann cells. We recently revealed that the tumour suppressor protein merlin, mutated in the hereditary tumour syndrome neurofibromatosis type 2, impacts significantly on axon structure maintenance in the peripheral nervous system. We now report on a role of neuronal merlin in the regulation of the axonal surface protein neuregulin 1 important for modulating Schwann cell differentiation and myelination. Specifically, neuregulin 1 type III expression is reduced in sciatic nerve tissue of neuron-specific knockout animals as well as in biopsies from seven patients with neurofibromatosis type 2. In vitro experiments performed on both the P19 neuronal cell line and primary dorsal root ganglion cells demonstrate the influence of merlin on neuregulin 1 type III expression. Moreover, expression of ERBB2, a Schwann cell receptor for neuregulin 1 ligands is increased in nerve tissue of both neuron-specific merlin knockout animals and patients with neurofibromatosis type 2, demonstrating for the first time that axonal merlin indirectly regulates Schwann cell behaviour. Collectively, we have identified that neuronally expressed merlin can influence Schwann cell activity in a cell-extrinsic manner.

Merlin isoform 2 in neurofibromatosis type 2-associated polyneuropathy.

The autosomal dominant disorder neurofibromatosis type 2 (NF2) is a hereditary tumor syndrome caused by inactivation of the NF2 tumor suppressor gene, encoding merlin. Apart from tumors affecting the peripheral and central nervous systems, most NF2 patients develop peripheral neuropathies. This peripheral nerve disease can occur in the absence of nerve-damaging tumors, suggesting an etiology that is independent of gross tumor burden. We discovered that merlin isoform 2 (merlin-iso2) has a specific function in maintaining axonal integrity and propose that reduced axonal NF2 gene dosage leads to NF2-associated polyneuropathy. We identified a merlin-iso2-dependent complex that promotes activation of the GTPase RhoA, enabling downstream Rho-associated kinase to promote neurofilament heavy chain phosphorylation. Merlin-iso2-deficient mice exhibited impaired locomotor capacities, delayed sensory reactions and electrophysiological signs of axonal neuropathy. Sciatic nerves from these mice and sural nerve biopsies from NF2 patients revealed reduced phosphorylation of the neurofilament H subunit, decreased interfilament spacings and irregularly shaped axons.

Transduction of peptides and proteins into live cells by cell penetrating peptides.

Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines.