Nuclear Localization of BRAFV600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells.

Mourad Zerfaoui,Moroz Krzysztof,Eman Toraih,Emmanuelle Ruiz,Youssef Errami,Emad Kandil,Zakaria Y Abd Elmageed,Abdallah S Attia

doi:10.3390/cancers14020311

Mourad Zerfaoui, Moroz Krzysztof + Show 6 more

Open Access

https://doi.org/10.3390/cancers14020311

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Abstract

Previously, we have demonstrated that nuclear BRAFV600E is associated with melanoma aggressiveness and vemurafenib resistance. However, the underlying mechanisms of how nuclear localization of BRAFV600E promotes cell aggressiveness have not yet been investigated. Despite therapeutic advancements targeting cutaneous melanoma, unknown cellular processes prevent effective treatment for this malignancy, prompting an urgent need to identify new biological targets. This study aims to explore the association of inducible heme oxygenase 1 (HMOX-1) with nuclear BRAFV600E in promoting melanoma aggressiveness. Proteomics analysis was performed to identify the interacting partner(s) of nuclear BRAFV600E. Immunohistochemistry was applied to evaluate the levels of HMOX-1 and nuclear BRAFV600E expression in melanoma and adjacent healthy tissues. Immunofluorescence assessed the nuclear localization of BRAFV600E in vemurafenib-resistant A375R melanoma cells. Further study of HMOX-1 knockdown or BRAFV600E overexpression in melanoma cells suggested a role for HMOX-1 in the regulation of cell proliferation in vivo and in vitro. Finally, Western blot analysis was performed to confirm the pathway by which HMOX-1 mediates Akt signaling. Proteomics results showed that HMOX-1 protein expression was 10-fold higher in resistant A375R cells compared to parental counterpart cells. In vitro and in vivo results illustrate that nuclear BRAFV600E promotes HMOX-1 overexpression, whereas HMOX-1 reduction represses melanoma cell proliferation and tumor growth. Mechanistic studies revealed that HMOX-1 was associated with nuclear BRAFV600E localization, thus promoting melanoma proliferation via a persistent activation of the AKT pathway. Our results highlight a previously unknown mechanism in which the nuclear BRAFV600E/HMOX-1/AKT axis plays an essential role in melanoma cell proliferation. Targeting HMOX-1 could be a novel method for treating melanoma patients who develop BRAF inhibitor resistance.

Highlights

Melanoma is an aggressive solid neoplasm with a highly critical prognosis
We demonstrated that the conventional nuclear BRAFV600E localization was associated with melanoma aggressiveness and vemurafenib resistance [24]
In order to explore whether BRAFV600E has a nuclear localization in A375R cells, we monitored the cellular localization after 24 h of cell synchronization followed by serum stimulation

Summary

Introduction

Melanoma is an aggressive solid neoplasm with a highly critical prognosis. Surgery and combinational chemotherapies have been shown to confer only modest survival benefits in advanced melanoma, with a five-year survival rate below 16% in patients with metastatic melanoma [1]. There are many unanswered questions related to advanced-stage BRAFV600E melanomas, the major indecision in current practice is the choice between BRAF/MEK inhibitors or immunotherapy for metastatic or high-risk disease patients [4]. NRF2 (nuclear factor (erythroid-derived 2)-like 2) is a transcription factor that regulates the antioxidative response as a result of ROS and protects cells against oxidative damage. This metabolic adaptation allows BRAF inhibitor-resistant melanoma cells to survive under oxidative stress [8]. We have demonstrated that nuclear BRAFV600E is associated with melanoma aggressiveness and vemurafenib resistance. The underlying mechanisms of how nuclear localization of BRAFV600E promotes cell aggressiveness have not yet been investigated. Staining intensity was scored in a blinded manner on a scale of 0 to 3, with 0 representing no staining, 1 low staining,

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