Searching for molecular mechanisms sustaining tumor formation and progression in Neurofibromatosis type 1

SUMMARY Neurofibromatosis type 1 (NF1, OMIM # 162200), also known as von Recklinghausen, is an autosomal dominant disease caused by mutations of the NF1 gene coding a 2818 amino acid protein, neurofibromin (Nf1). More than 900 different mutations in the NF1 gene have been identified (HGMD, Human Genetic Mutation Database). Mutations of NF1 gene cause a variety of clinical manifestations such as the optic gliomas, neoplasms of the haematopoietic system and learning disabilities. However, the hallmark of NF1 is the development of multiple benign peripheral nerve sheath tumors called neurofibromas. Neurofibromas are complex tumors arising from peripheral nerve sheaths and mainly composed of Schwann Cells (SCs) homozygous for mutated NF1, mast cells (MCs) and fibroblasts (FBRs) both heterozygous for the same mutation. The plexiform variety can progress to highly malignant sarcomas termed Malignant Peripheral Nerve Sheath Tumors (MPNSTs), which are almost invariably lethal. Up to now, any effective therapy able to either reduces neurofibroma size and its incidence or to counteract its formation, has not been developed yet. The main feature of neurofibroma is a rigid structure due to massive deposition of collagen of different types by activated FBRs. These cells, named myofibroblasts (mFBRs), are massively stimulated by mast cell-secreting Transcription Growth Factor-Beta (TGF-Beta) to produce growth factors such as Platelet Growth Factor (PDGF), Fibroblast Growth Factor (FGF) and collagen. This leads to both potent SCs proliferation and deposition of rigid extracellular matrix (ECM). Cells’ haploinsufficient for Nf1 display hyper-activation of Rat Sarcoma (Ras), which further increases when Loss of Heterozigozyty (LOH) of NF1 occurs. Thus, the activation of Ras/ Rapidly Accelerated Fibrosarcoma (Raf) /Extracellular signal-regulated Kinase (ERK) signaling in SCs is sufficient to make them more susceptible to proliferative signals provided by a NF1+/- niche. However, the physiological response to Ras hyper-activation is cell-cycle arrest and/or senescence rather than transformation. Ras-mediated transformation of SCs probably relies on a step-wise process that integrates circuits of amplification signals from the local niche. A major component of the niche is the ECM, a complex network of macromolecules whose the elasticity (ranging from soft to stiff and rigid),contributes to development and cancer. ECM elasticity determines how a cell senses and perceives external forces and thus provides a major environmental cue that determines cell behavior. Indeed, the focal adhesion complex, which consists of integrins, multicomplex of adaptors and signaling proteins, can be viewed as a mechanosensor linking the actomyosin cytoskeleton with the ECM. How lack of Nf1 may impact on the complexity of ECM-cell dynamic and how the great rigidity of the ECM in neurofibromas influences SCs’ behavior, is still unknown. Among the three functional domains described in the Nf1 protein, a Focal Adhesion Kinas (FAK) binding domain has been identified and Nf1 has been shown to interact with FAK, paving the way for the enunciation of new hypothesis aimed to explain the route of SCs transformation toward cancer. Rational: as in other tumors {Lu, 2012 #289}, {Yu, 2011 #292}), also in the plexiform neurofibromas, the tumorigenic phenotype of SCs is fostered by the amplification of integrated signaling pathways triggered by loss of Nf1, Ras hyper activation and deregulated ECM. Changes of the mechanical properties of ECM due to increased collagen secretion by mFBRs might actively contribute to tumor progression by influencing gene expression profile of the cells through the enhancement of Ras signaling pathway triggered by FAK. The deep investigation of these biological changes triggered in SCs by ECM formation is the goal of the present project. Project Goals: To shed light into this issue, we intend 1) to generate a novel three-dimensional experimental model in vitro reproducing the multicellular complexity of neurofibromas with primary cells. Immortalized cells, indeed, are not suitable for our aims since the molecular oncologists consider the immortalization process as the first hit leading to tumorigenic phenotype, because of the changes which made for cell cycle control in gene expression 2) to assess the requirement ECM for SCs transformation in this new in vitro system identifying the proper ECM composition and stiffness in matrigel (structural and non structural components) required for neurofibroma’s formation. Results: Isolation of primary SCs and FBRs from Neurofibromas and their biological characterization: 1) We have already isolated and cultured in 2D our SCs and FBRs NF1+/- according to Serra methodology {Serra, 2000 #210}. These cells have been isolated from plexiform neurofibroma biopsies after informed consent of patients by our Milan and Rome University collaborators. To get two populations of SCs and FBRs we have cultured cells in selective Medium (according to {Serra, 2000 #210}, and our new unpublished protocol) and characterized them biochemically by: S100B {Tucker, 2011 #321} and p75 markers specifically recognizing SCs and collagen I secretion, alpha smooth muscle actin (α-SMA) expression, Smad2/3 activation, Abl kinase activation characterizing mFBR activity {Kojima, 2010 #150}. 2) We have already obtained colonies of SCs growing in 3D in vitro system as described in step 1 and 2 (in transwell-like chambers to permit autocrine stimulation between mFBRs and SCs). Our preliminary data show that primary SCs generate colonies only when plated in an ECM/reconstituted basement membrane Collagen I-Matrigel of at list 3 mg/ml. However, we have still to set up the culture conditions to keep cells in highly proliferating state. Preliminary indications in immortalized Mouse Embrionic Fibroblasts (MEFs) In other cellular models as in mouse FBRs NF1-/-, we have found that the absence of Neurofibromin correlates with deregulation of FAK Y397 and Y925 phosphorylation both in absence of integrin clustering and after ligand stimulation. Further, the tumorigenesis assay showed that MEFNF1-/-ability to form colonies is affected by both MECK inhibitor and FAK inhibitor (Y15) indicating the cooperative role of FAK and PDGFBB growth factor in the tumorigenesis process mediated by Nf1. Consistently, immunoprecipitation experiments showed that in Nf1 null cells, Growth factor receptor-bound protein2 (Grb-2), the RAS pathway initiator, interacts with FAK also in absence of collagen in a PDGFBB ligand-dependent way, thus suggesting that FAK and growth factor receptors can cooperate to increase the Ras activity to a threshold required to induce tumorigenesis.