title: Inducible nitric oxide synthase drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2.
abstract: Melanoma is one of the cancers of fastest-rising incidence in the world. Inducible nitric oxide synthase (iNOS) is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K-AKT-mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p70 ribosomal S6 kinase (p-P70S6K), p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of tuberous sclerosis complex (TSC) 2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Ras homolog enriched in brain (Rheb), a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of the mTOR pathway members. Exogenously supplied NO was also sufficient to reverse the mTOR pathway inhibition by the B-Raf inhibitor vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers.
authors.affiliation: Authors' Affiliations: Departments of Otolaryngology, Dermatology, Immunology, and Oncological Sciences; Division of Hematology and Oncology, Department of Medicine, Department of Otolaryngology, The Tisch Cancer Institute, Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai; The Tisch Cancer Institute; Department of Genetics and Genomic Sciences, One Gustave L. Levy Place, New York, New York; and Departments of Melanoma Medical Oncology and Biostatistics, University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Esther
authors.initials: E
authors.lastname: Lopez-Rivera
authors.forename: Padmini
authors.initials: P
authors.lastname: Jayaraman
authors.forename: Falguni
authors.initials: F
authors.lastname: Parikh
authors.forename: Michael A
authors.initials: MA
authors.lastname: Davies
authors.forename: Suhendan
authors.initials: S
authors.lastname: Ekmekcioglu
authors.forename: Sudeh
authors.initials: S
authors.lastname: Izadmehr
authors.forename: Denái R
authors.initials: DR
authors.lastname: Milton
authors.forename: Jerry E
authors.initials: JE
authors.lastname: Chipuk
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Grimm
authors.forename: Yeriel
authors.initials: Y
authors.lastname: Estrada
authors.forename: Julio
authors.initials: J
authors.lastname: Aguirre-Ghiso
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Sikora
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002642:Chick Embryo; D055785:Gene Knockdown Techniques; D006801:Humans; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000502:physiology*; D015538:Nitrosation / Q000187:drug effects / Q000235:genetics; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D000077005:Tuberous Sclerosis Complex 2 Protein; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D009569:Nitric Oxide; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases