Metastatic Melanoma Progression Is Associated with Endothelial Nitric Oxide Synthase Uncoupling Induced by Loss of eNOS:BH4 Stoichiometry.

Fabiana Henriques Machado De Melo,Marcelo Yudi Icimoto,Denise De Castro Fernandes,Miriam Galvonas Jasiulionis,Ricardo Xisto De Sousa,Francisco R M Laurindo,Diego Assis Gonçalves

doi:10.3390/ijms22179556

Abstract

Melanoma is the most aggressive type of skin cancer due to its high capability of developing metastasis and acquiring chemoresistance. Altered redox homeostasis induced by increased reactive oxygen species is associated with melanomagenesis through modulation of redox signaling pathways. Dysfunctional endothelial nitric oxide synthase (eNOS) produces superoxide anion (O2−•) and contributes to the establishment of a pro-oxidant environment in melanoma. Although decreased tetrahydrobiopterin (BH4) bioavailability is associated with eNOS uncoupling in endothelial and human melanoma cells, in the present work we show that eNOS uncoupling in metastatic melanoma cells expressing the genes from de novo biopterin synthesis pathway Gch1, Pts, and Spr, and high BH4 concentration and BH4:BH2 ratio. Western blot analysis showed increased expression of Nos3, altering the stoichiometry balance between eNOS and BH4, contributing to NOS uncoupling. Both treatment with L-sepiapterin and eNOS downregulation induced increased nitric oxide (NO) and decreased O2• levels, triggering NOS coupling and reducing cell growth and resistance to anoikis and dacarbazine chemotherapy. Moreover, restoration of eNOS activity impaired tumor growth in vivo. Finally, NOS3 expression was found to be increased in human metastatic melanoma samples compared with the primary site. eNOS dysfunction may be an important mechanism supporting metastatic melanoma growth and hence a potential target for therapy.

Highlights

Cutaneous melanoma originates from melanocytes, neural-crest-derived pigmentproducing cells located in the epidermis
Intracellular BH4 amount is mainly conamount is mainly controlled by the activity of the de novo pathway comprising the trolled by the activity of the de novo pathway comprising the enzymes guanosine triphosphate (GTP) cyclohydrolase enzymes GTP cyclohydrolase 1 (GTPCH1), the rate‐limiting enzyme in BH4 biosynthesis; 1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis; 6-pyruvoyltetrahydrobiopterin
The results suggested that the imbalance between nitric oxide (NO) and O2 − is caused by the alteration in BH4:endothelial nitric oxide synthase (eNOS) stoichiometry and decreased BH4:BH2 ratio [9]

Summary

Introduction

Cutaneous melanoma originates from melanocytes, neural-crest-derived pigmentproducing cells located in the epidermis. Melanoma occurrence is low compared to other types of cutaneous malignancies, it is responsible for approximately 75% of all skin-cancer-related deaths, which is associated with its chemoresistance to available therapeutic approaches and high ability to metastasize. Data from our group and others have shown that the establishment of a prooxidant intracellular milieu contributes to melanocyte malignant transformation through modulation of signaling pathways that favor proliferation and cell survival, and inhibit apoptosis, as was shown for other skin cancers [5,6,7,8,9]. The contribution of DNA and protein damage in the carcinogenesis induced by UV-derived ROS is well established in non-melanoma skin cancers [10,11]. It was shown that the activation of RAS/RAC1/ERK signaling pathway in melanocytes submitted to a chronic stress condition is associated with oncogenesis, through regulation of global DNA methylation and DNMT1 expression

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Conclusion