Nuclear Localization of BRAF(V600E) Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells

SIMPLE SUMMARY: Despite some successes of selective anti-BRAF(V600E) inhibitors, resistance remains a major challenge. The aim of our study is to determine the role of nuclear BRAF(V600E) and its newly identified partner, HMOX1, in melanoma aggressiveness and drug resistance. We identified the mechanism by which drug resistance is developed via the nuclear localization of BRAF(V600E) and its partner HMOX1 in melanoma tissues and cell lines. According to our studies, the outcomes of our manuscript have a direct clinical impact on establishing novel prognostic markers and therapeutic intervention strategies in metastatic melanoma. This study provides new information on the ability to selectively classify patients with cytosolic BRAF for selective BRAF inhibitors and offers an alternative treatment to patients with nuclear BRAF(V600E) and high HMOX1 expressions. ABSTRACT: Background: Previously, we have demonstrated that nuclear BRAF(V600E) is associated with melanoma aggressiveness and vemurafenib resistance. However, the underlying mechanisms of how nuclear localization of BRAF(V600E) 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 BRAF(V600E) in promoting melanoma aggressiveness. Methods: Proteomics analysis was performed to identify the interacting partner(s) of nuclear BRAF(V600E). Immunohistochemistry was applied to evaluate the levels of HMOX-1 and nuclear BRAF(V600E) expression in melanoma and adjacent healthy tissues. Immunofluorescence assessed the nuclear localization of BRAF(V600E) in vemurafenib-resistant A375R melanoma cells. Further study of HMOX-1 knockdown or BRAF(V600E) 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. Results: 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 BRAF(V600E) 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 BRAF(V600E) localization, thus promoting melanoma proliferation via a persistent activation of the AKT pathway. Conclusions: Our results highlight a previously unknown mechanism in which the nuclear BRAF(V600E)/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.