The Effect Of The Aberrations Of IGF1R And EGFR Signaling Pathways On Malignant Peripheral Nerve Sheath Tumor Cell Invasion And Migration

Objective: Malignant peripheral nerve sheath tumour(MPNST) refers to the sarcomas originating from peripheral nerves or nerve sheath, accounting for about 5-10% of malignant soft tissue sarcomas. MPNST is highly malignant and the 5-year survival rate is about 34-52%. The lesions generally show rapid progress and the prognosis is very poor. The traditional surgery, radiotherapy and chemotherapy is not so effective. In the future it is likely to benefit the MPNST patients by investigating the pathogenesis of the disease and finding new prognostic factores and specific therapeutic targets at the gene and molecular level. We intend to investigate the genetic aberrations of important signaling pathways and its biological significance. We also want to investigate the effect of the berrations of IGF1 R and EGFR signaling pathways on MPNST cell invasion and migration.Methods: By microarray based-comparative genomic hybridization(a CGH), fluorescence in situ hybridization(FISH), bioinformatic analysis, immunohistochemistry(IHC), cell culture, cell biology and molecular biological methods, we intend to investigate the role of IGF1 R and EGFR signaling pathways in progression of MPNST.Results 1. Genomic characterization of 51 MPNST tissue samples collected from Tianjin Medical University Cancer Hospital(26 cases) and MD Anderson Cancer center(25 cases) identified the following resutls.(1)We investigated the genomic differences between tumors from patients with different ethnicity and found that the most significant difference is the increased overall aberration rate in the American patients, yet the overall pattern of aberrations remains similar.(2) several frequently amplified regions harboring 2,599 genes and regions of deletion including 4,901 genes. Frequent deletion inluded 9p21.3(approximately 65% of patients in the region containing the tumor suppressor gene CDKN2 A and CDKN2B), topoisomerase(DNA) II alpha(TOP2A), cyclin-dependent kinase 4(CDK4) and forkhead box M1(FOXM1) genes. Frequent amplifications included epidermal growth factor receptor insulin-like growth factor(EGFR), IGF1 R, cyclin-dependent kinase 6(CDK6), potassium channel, subfamily K member 12(KCNK12), MET proto-oncogene(MET) and platelet-derived growth factor receptor alpha polypeptide(PDGFRA) genes.(3)At the pathway level, there were 11 significant amplicated signaling pathways, including the TFF, ERK, ARF, IGF1 R and EGFR signaling pathways. 2. Intergated characterization of IGF1 R signal pathway identified these following results.(1) We identified a significant enrichment of copy number–altering events in the insulin-like growth factor 1 receptor(IGF1R) pathway, including frequent amplifications of the IGF1 R gene itself. The patients with significant increased genetic aberrations of IGF1 R signal pathway had worse OS.(2) To validate the IGF1 R pathway as a potential target in MPNSTs, we first confirmed that high IGF1 R protein correlated with worse DFS and OS in an independent set of MPNST samples using immunohistochemistry.(3) Two MPNST cell lines(ST88-14 and STS26T) were used to determine the effect of attenuating IGF1 R. Inhibition of IGF1 R in ST88-14 cells using small interfering RNAs led to significant decreases in cell proliferation, invasion, and migration accompanied by attenuation of the PI3K/AKT and MAPK pathways.(4) Inhibition of IGF1 R in ST88-14 cells using IGF1 R inhibitor, MK-0646, led to significant decreases in cell proliferation, invasion, and migration accompanied by attenuation of the PI3K/AKT and MAPK pathways. 3. a CGH and pathway analysis of the 51 MPNSTs identified:(1) there were significant gene amplification events in EGFR pathway, including frequent amplifications of EGFR gene itself, which was subsequently validated by FISH assay.(2) High expression of EGFR protein was associated with poor DFS and OS of human MPNST patients.(3) In human MPNST cell lines ST88-14 and STS26 T, inhibition of EGFR by si RNA led to decreased cell proliferation, migration, and invasion accompanied by attenuation of PI3K/AKT and MAPK pathways.(4) In human MPNST cell lines ST88-14 and STS26 T, inhibition of EGFR by Gefitinib led to decreased cell proliferation, migration, and invasion accompanied by attenuation of PI3K/AKT and MAPK pathways. 4. Attenuation of IGF1 R and EGFR by combined si RNAs in ST88-14 cells significantly decreased cell proliferation without noticeable addictive or combinational synergistic effects. By using EGFR and IGF1 R inhibitors, we noticed that treatment with gefitinib in ST88-14 cells led to a decrease in the activated forms of EGFR as well as a decrease in cell proliferation relative to control without the activation of IGF1 R signal pathways. Most importantly, combined treatment with both inhibitors did not result in a stronger inhibition of cell proliferation。Conclusion: 1. There are lots of genetic aberrations in MPNST, including amplification of 2,599 genes and deletion of 4,901 genes. there were 11 significant amplicated signaling pathways, including the TFF, ERK, ARF, IGF1 R and EGFR signaling pathways. 2. Our integrated genomic and molecular studies provide evidence that the IGF1 R pathways affect MPNST tumor cell invasion and migration. 3. The integrated genomic studies provide evidence that the EGFR pathways affect MPNST tumor cell invasion and migration. 4. Probablely there is no crosstalk of IGF1 R and EGFR sigaling pathways in MPNST.