Overcoming Anoikis Resistance in Melanoma Treatment: Combined Therapy Targeting BRAF Inhibitors and Nitric Oxide Synthases

• Grant number: 25/10560-0
• Support Opportunities: Regular Research Grants
• Start date: November 01, 2025
• End date: October 31, 2028
• Field of knowledge: Health Sciences - Pharmacy
• Principal Investigator: Hugo Pequeno Monteiro
• Grantee: Hugo Pequeno Monteiro
• Host Institution: Centro de Terapia Celular e Molecular. Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated researchers: Daniela De Zio ; Luiz Sidney Longo Junior

Abstract

Cancer is the second leading cause of death worldwide and represents a major public health challenge. It is a group of diseases caused by mutations in genes responsible for regulating cell proliferation, differentiation and cell death, and can also be caused by non-mutational epigenetic reprogramming. In Brazil, 704,000 new cases of cancer are estimated for the 2023-2025 triennium, with emphasis on non-melanoma skin cancer, female breast cancer, prostate cancer, colorectal cancer, lung cancer, stomach cancer and cervical cancer. Although melanoma has a lower incidence, its mortality rate is high, especially in metastatic cases, with a five-year survival rate of less than 5%. One of the major problems associated with the treatment of melanoma is the development of resistance to systemic therapy and targeted therapy. In addition, melanoma and other cancers use the high production of reactive oxygen species and nitric oxide (NO) to develop, survive the different treatment strategies used against them and metastasize. In turn, successful metastasis is linked to the resistance of tumor cells to death by loss of adhesion (anoikis). In previously published studies by our group, we showed that resistance to anoikis in murine melanoma B16F10/Nex8H cells was upregulated by high concentrations of NO, which in turn promoted S-nitrosylation and activation of the protein tyrosine kinase Src (1). Recently we showed that the protein tyrosine kinase FAK, the structural proteins ¿-actin, ¿-tubulin, caveolin-1, and the enzyme NO synthase 3 (NOS3) promoted resistance to anoikis in A375 human melanoma cells maintained in suspension (2,3). These cells present the BRAFV600E mutation. An in silico study based on this set of observations led us to propose the existence of a protein network consisting of the proteins FAK, Src, ¿-actin, ¿-tubulin, Caveolin-1 and NOS3, emphasizing the role of NO as a positive regulator of resistance to death by anoikis. In turn, it is well documented that NO activates the MAPK signaling pathway (4) of which the protein kinases BRAF and MEK are part. These proteins accumulate important mutations associated with their activation and resistance to anoikis. These are the premises for the development of this project, which initially aims to characterize at the molecular level the protein network described above and, based on this characterization, use the proteins that constitute the network as possible molecular targets in A375 and SK-MEL-28 cells that present the BRAFV600E mutation, maintained in suspension for prolonged periods, thus seeking to eliminate the resistance to death by anoikis in these cells. In parallel, we will synthesize a series of imidazothiazoles that are potential inhibitors of all RAF isoforms (pan-RAF), using multicomponent reactions to generate structural diversity and explore potential structure-activity relationships. With these new inhibitors, the chemotherapeutic agents that inhibit BRAF and MEK (Vemurafenib and Trametinib), the chemotherapeutic agent that inhibits microtubule formation (Paclitaxel), the Src kinase inhibitor (PP1) and the general NOS inhibitor (L-NAME), we will develop combined strategies for using these drugs to overcome anoikis resistance in A375 and SK-MEL-28 cells maintained in suspension for prolonged periods. With the development of this project, we will contribute to the development of new therapeutic approaches for the treatment of melanoma in advanced stages.(1)da COSTA et al., Free Radic Res. 2018 May;52(5):592-604.(2)Igor R. Nascimento 2022 - Dissertação de Mestrado.(3)Igor R. Nascimento 2024 - Manuscrito submetido à publicação.(4)Liu X, et al,. Biomolecules. 2021 Jul 10;11(7):1009.Keywords: melanoma; Nitric Oxide; Anoikis; Anoikis resistance; RAF Inhibitors; Imidazothiazols (AU)