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: Esther Lopez-Rivera, Padmini Jayaraman, Falguni Parikh, Michael A Davies, Suhendan Ekmekcioglu, Sudeh Izadmehr, Denái R Milton, Jerry E Chipuk, Elizabeth A Grimm, Yeriel Estrada, Julio Aguirre-Ghiso, Andrew G Sikora
• Journal: Cancer research (Cancer Res) Vol. 74 Issue 4 Pg. 1067-78 (Feb 15 2014) ISSN: 1538-7445 [Electronic] United States
• PMID: 24398473 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: ©2014 AACR.

Chemical References

• RNA, Small Interfering
• TSC2 protein, human
• Tuberous Sclerosis Complex 2 Protein
• Tumor Suppressor Proteins
• Nitric Oxide
• NOS2 protein, human
• Nitric Oxide Synthase Type II
• MTOR protein, human
• TOR Serine-Threonine Kinases

Topics

• Animals
• Cell Proliferation (drug effects)
• Chick Embryo
• Gene Knockdown Techniques
• Humans
• Melanoma (metabolism, pathology)
• Nitric Oxide (metabolism)
• Nitric Oxide Synthase Type II (antagonists & inhibitors, physiology)
• Nitrosation (drug effects, genetics)
• Protein Multimerization
• Protein Processing, Post-Translational (drug effects, genetics)
• RNA, Small Interfering (pharmacology)
• Signal Transduction (drug effects, genetics)
• TOR Serine-Threonine Kinases (metabolism)
• Tuberous Sclerosis Complex 2 Protein
• Tumor Cells, Cultured
• Tumor Suppressor Proteins (metabolism)