Retinoid X Receptor Alpha and Melanocytes: A Translational Perspective

Retinoid receptors comprise a large family, including retinoic acid receptors (RAR) and retinoid X receptors (RXR) alpha, beta, gamma, and retinoid orphan receptors. Previous experimental studies have suggested that ablation of RXR alfa can lead to larger, thicker, and more vascularized melanomas [1]. Other studies have shown constitutive expression of RXR alfa in melanoma cells [2] and reported an anti-proliferative effect in melanoma cells exposed to retinoid agonists in vitro [2]. Chakravarti et al. conducted an immunohistochemistry assay utilizing a polyclonal antibody containing RXR alpha. They found increased RXR alfa positivity in melanomas exhibiting vertical growth and correlation between loss of cytoplasmic RXR alfa and worse prognosis [3]. Despite this, most evidence supporting RXR alfa's role in regulating melanoma cell proliferation is based on experimental rather than clinical data. Contrary to Chakravarti's findings, another study examining melanocytic lesions (intradermal nevi, dysplastic nevi, and melanomas) using a monoclonal antibody, which is more specific than polyclonal ones, observed RXR alfa loss in all lesions compared to normal skin [4]. Given these findings, we designed the present study to evaluate RXR alfa expression specifically in melanocytes from normal skin samples. We conducted a retrospective, cross-sectional study using paraffin-embedded skin samples collected from surgical excisions between 2012 and 2018. All samples were derived from melanoma margin amplification procedures. Cases were excluded if they contained residual or concurrent lesions (e.g., nevi) or if keratinocytes (used as internal positive controls) were negative. Samples were subjected to immunohistochemical analysis using a monoclonal anti-RXR alfa antibody/NR2B1 antibody (Clone X52773, R&D Systems®). According to the antibody datasheet, it specifically recognizes human RXRα and does not cross-react with human RXRβ or RXRγ. Double counterstaining with Melan A and Magenta (both from Dako®) was performed using the Autostainer Link 48 platform (Dako®). Slide evaluation was performed by one certified dermatopathologist and one dermatologist. Positivity was assessed based on two criteria: (1) the percentage of positively stained melanocytes and (2) staining intensity (0 = none, weak, moderate, or strong). The chosen antibody dilution (1:200) produced a strong, diffuse signal in external (mucosal epithelium and lymph node tissue) and internal (keratinocytes and fibroblasts) controls visible at 40× magnification without background staining. Double staining ensured accurate identification of melanocytes. Of the 68 patients, 34 were men and 34 were women. The median age was 50 years (SD: 16.73 years). Regarding the sample sites, 58 (85.3%) were from the trunk, 5 (7.3%) from the head and neck, and 5 (7.3%) from the limbs. All melanocytes in the 68 samples were negative for RXR alfa (0% positive cells, 0 intensity), while keratinocytes consistently showed positive staining, as demonstrated in Figure 1. In recent years, PReferentially expressed Antigen in Melanoma (PRAME) has emerged as a potential biomarker for malignant melanocytic tumors. In the presence of RA, PRAME likely binds to RARs and prevents transcriptional activation, acting as an epigenetic repressor of RAR signaling [5]. There is evidence that the link between RAR and PRAME arises from a direct and ligand-dependent physical interaction [6]. PRAME expression likely represents a poorly understood mechanism that enables tumor cells to evade RAR-mediated tumor suppression, while also potentially affecting the activity of other nuclear receptors [6]. Considering the current findings and prior study from our group [4], we propose that RXR alfa is not involved in the pathogenesis of melanocytic neoplasms. Methylation of the PRAME promoter region is one possible explanation for the lack of PRAME expression in normal melanocytes [7]; however, the absence of RAR expression may also contribute to PRAME negativity in these cells. Previous reports suggested that PRAME is expressed in melanoma cells but absent in normal and benign melanocytes [8], however it is actually well established that its assessment has several limitations [9]. The current WHO classification of skin tumors categorizes melanomas according to distinct genetic pathways, highlighting their biological heterogeneity [10]. Therefore, it might be reasonable to infer that PRAME may play a role in certain tumors within this group and/or specific stages of tumorigenesis, potentially in association with RAR receptors, but not in all cases. Considering the negative findings for RXRα in normal melanocytes, along with our previous immunohistochemical [4] and transcriptomic [11] analyses demonstrating RXRα loss in melanomas and intradermal nevi, and the evidence that PRAME can interact with RXRβ and RXRγ [6], it appears unlikely that targeting RXRα with specific agonists would yield meaningful therapeutic benefits in melanocytic tumors from a translational perspective. Discrepancies in the literature may be explained by differences in antibody clones and experimental methodologies (e.g., in vitro studies vs. human tissue analyses, and immunohistochemistry vs. molecular approaches). Future research should focus on identifying which members of the retinoid receptor family are implicated in melanocytic transformation. Our data indicate that RXR alfa is absent in normal melanocytes. This result should be interpreted with caution due to the antibody used, which differs from those applied in previous studies. Additionally, although the influence of the tumor microenvironment related to the sample origin cannot be fully assessed, it is likely minimal because the specimens were obtained from surgical re-excisions of previously margin-free tumors. As a result, we can hypothesize RXR alfa-targeting therapies are unlikely to be effective in patients with melanocytic neoplasms or to prevent them. Further studies are needed to determine (1) which retinoid receptor, if any, plays a pivotal role in melanocyte malignancy (2) the RXR alpha potential role during melanoma malignant transformation or progression (3) the specific regulatory network between PRAME and RAR signaling. ChatGPT was used for language review. Financial support—Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). The funder had no role in the design, data collection, data analysis, and reporting of this study. This study was performed in accordance with the Declaration of Helsinki. It was approved by the Research Ethics Committee (protocol 03058918.4.0000.5411). The authors declare no conflicts of interest. The data that support the findings of this study are not publicly available due to privacy reasons, but are available from the corresponding author upon reasonable request.