The Neurofibromatosis Type 1 Gene Product Neurofibromin Enhances Cell Motility by Regulating Actin Filament Dynamics via the Rho-ROCK-LIMK2-Cofilin Pathway

Abstract

Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients.Neurofibromatosis type 1 (NF1), 2 also called von Recklinghausen disease, is autosomal dominant and one of the most common inherited disorders, affecting 1 in 3500 individuals (1). The phenotype of NF1 is highly variable, with several organ systems being affected, including bones, skin, irises, and central and peripheral nervous systems. The disease commonly manifests with "café au lait" macules in the skin, iris Lisch nodules, and learning disability (2, 3). The hallmark of NF1 is benign tumors that develop in the peripheral nervous system accompanied by an increased risk of malignancies.The NF1 gene lies on chromosome 17q11.2 and encodes a large 2818-amino acid protein, termed neurofibromin. Sequence analysis of neurofibromin revealed that a region centered around the 360 amino acids encoded by the NF1 gene shows significant homology to the known catalytic domains of mammalian Ras GTPase-activating protein (p120GAP), which interacts with Ras and promotes hydrolysis of Rasbound GTP (active form) to GDP (inactive form), resulting in inactivation of the Ras protein. Accordingly, loss and/or mutations of neurofibromin elevate Ras activity and are followed by activation of various Ras effectors. Ras activation has been considered to be the causative event for tumor formation and other clinical manifestations in NF1 patients (2, 3).Recent studies have suggested that neurofibromin has additional functions besides regulating cell proliferation ...

The Ras Inhibitor Farnesylthiosalicylic Acid as a Potential Therapy for Neurofibromatosis Type 1

“…This phenomenon seemed to be associated with increased amounts of Ras-GTP in the NF1 cells because FTS induced the complete disappearance of actin stress fibers in these cells. Consistently with these observations, recent studies showed that loss of neurofibromin induced excessive formation of actin stress fibers in HT1080 and HeLa cells (40). In those cell lines, which are derived from tumors not associated with the NF1 phenotype, this effect required Ras activation and was reversed by transfection with the NF1 GAP-related domain.…”

“…A recent study showed that loss of neurofibromin induces excessive formation of actin stress fibers in HT1080 and HeLa cells (40) We therefore examined whether stress fiber formation related to neurofibromin deficiency is detectable in the NF1 MPNST cells and, if so, whether this phenotype can be reversed by FTS. We carried out a comparative analysis of cytoskeleton reorganization in the NF1 MPNST cells and in non-NF1 STS26T MPNST cells.…”

Gene expression profiling of metastatic brain cancer

“…Gene sequence analysis has revealed homology of NF1 with Ras GTPase-activating protein, and as such, the clinical manifestations of neurofibromatosis type 1 have been linked to loss of the GTPase activity, or accordingly, increased Ras activity (19,20). NF1 regulates cell motility and invasion via activation of the Rho-ROCK-LIMK2 pathway, and subsequent phosphorylation of cofilin associated with actin stress fiber formation (21). However, our study showed increased expression of NF1 in metastatic brain tumors, which may indicate its interactions with other oncogenic molecules, a possible result of depletion of the NF1 gene product.…”

Αναστολή Της PKC-ε Και Μηχανισμοί Ογκογένεσης Στο ΚΝΣ

“…Η αλληλεπίδραση της Νευροϊνιδίνης με τον κυτταροσκελετό της ακτίνης είναι ιδιαίτερα σημαντική, καθώς στην ίδια μελέτη οι συγγραφείς κατάφεραν, μετά από μείωση της δραστικότητας της PKCα (24 ώρες έκθεση των κυττάρων σε ΤΡΑ), να αναστρέψουν το μη μιτωτικό φαινότυπο των C62B κυττάρων γλοιώματος σε μιτωτικό μετά από επίδραση με EGF [83]. Επιπλέον, έχει δειχθεί ότι η μείωση της νευροϊνιδίνης, ενεργοποιεί το σηματοδοτικό μονοπάτι Rho-ROCK-LIMK2, το οποίο έχει ως αποτέλεσμα την εξαρτώμενη από φωσφορυλίωση απενεργοποίηση της cofilin, τροποποιώντας με αυτόν το τρόπο τον κυτταροσκελετό των ινιδίων ακτίνης προς ιδιαίτερα διηθητικό φαινότυπο σε κύτταρα HeLa και HT1080 [298]. Τα αποτελέσματα αυτά, κατέδειξαν ότι η νευροϊνιδίνη σχετίζεται τόσο με τον πολλαπλασιασμό των κυττάρων, ως RasGAP, όσο και με την κυτταρική μετανάστευση, κομβικές διαδικασίες που διαταράσσονται κατά τη δημιουργία νεοπλασίας [298].…”

“…Επιπλέον, έχει δειχθεί ότι η μείωση της νευροϊνιδίνης, ενεργοποιεί το σηματοδοτικό μονοπάτι Rho-ROCK-LIMK2, το οποίο έχει ως αποτέλεσμα την εξαρτώμενη από φωσφορυλίωση απενεργοποίηση της cofilin, τροποποιώντας με αυτόν το τρόπο τον κυτταροσκελετό των ινιδίων ακτίνης προς ιδιαίτερα διηθητικό φαινότυπο σε κύτταρα HeLa και HT1080 [298]. Τα αποτελέσματα αυτά, κατέδειξαν ότι η νευροϊνιδίνη σχετίζεται τόσο με τον πολλαπλασιασμό των κυττάρων, ως RasGAP, όσο και με την κυτταρική μετανάστευση, κομβικές διαδικασίες που διαταράσσονται κατά τη δημιουργία νεοπλασίας [298].…”