Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2025;15(2):4952 1 Melanoma on Chronically Sun-Damaged Skin: Deciphering Gene Expression Signatures Alejandra Avila1*, Varsha Thakur1,2*, Natalie Vincent1, Pilar Valencia1, Mecker G. Möller1,3, Rimpi Khurana2, Guo Yan2, Jennifer C. Tang1,2, Barbara Bedogni1,2, Natalia Jaimes1,2 1 Dr. Phillip Frost Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA 2 Sylvester Comprehensive Cancer Center, University of Miami Health System, Miami, Florida, USA 3 Pritzker School of Medicine, University of Chicago, Illinois, USA *Equal contribution Key words: Melanoma, Gene expression, Ultraviolet radiation, Sequence analysis RNA, Inflammation, sun-damaged skin Citation: Avila A, Thakur V, Vincent N, et al. Melanoma on Chronically Sun-Damaged Skin: Deciphering Gene Expression Signatures. Dermatol Pract Concept. 2025;15(2):4952. DOI: https://doi.org/10.5826/dpc.1502a4952 Accepted: January 7, 2025; Published: April 2025 Copyright: ©2025 Avila et al. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original authors and source are credited. Funding: None. Competing Interests: None. Authorship: All authors have contributed significantly to this publication. Acknowledgments: Valeria De Bedout, MD for her support with sample collection. Corresponding Author: Natalia Jaimes, MD, Dr. Phillip Frost Department of Dermatology & Cutaneous Surgery. University of Miami Miller School of Medicinen and Sylvester Comprehensive Cancer Center, University of Miami Health System, Miami, FL. ORCID ID: 0000-0002-3651-1019, E-mail: nmw50@med.miami.edu Introduction: Melanoma of the skin is responsible for most skin cancer-related deaths. It is well known that exposure to ultraviolet radiation is the most common and modifiable risk factor for melanoma. Melanomas arising on chronically sun-damaged skin (CSDS) have shown a higher mutational burden. Objectives: To analyze skin samples of patients with melanoma on CSDS to identify possible gene expression signatures that may contribute to melanomagenesis. Methods: This experimental observational analysis, conducted at the Dermatology Melanoma and Pigmented Lesion Clinic at University of Miami Hospitals/Sylvester Comprehensive Cancer Center, Miami, Florida, included a total of 10 patients over 18 years of age with a recent diagnosis of mel- anoma on CSDS. For each patient, two skin samples were obtained using a 2-mm punch (one from CSDS within 2 cm of the primary melanoma, another from sun-protected skin). Skin samples were sent to the Sylvester Onco-genomics Shared Resource (OGSR) for library preparation and RNA se- quencing. Main outcome was the identification of differentially expressed genes between CSDS and non-CSDS of patients with a recent diagnosis of melanoma. Results: A total of four skin samples met the necessary quality standards for molecular analyses. Sig- nificant differences were observed between the CSDS and non-CSDS samples. Pathways involved in inflammation (e.g., IL-17 signaling), immune responses (e.g., ABC transporters), and oxidative phos- phorylation were overexpressed in CSDS. ABSTRACT 2 Original Article | Dermatol Pract Concept. 2025;15(2):4952 Introduction It is well known that exposure to ultraviolet radiation (UVR) is the most common, modifiable, environmental risk factor for melanoma. UVR exposure causes oxidative stress, direct and indirect damage to the DNA, and alteration to DNA- repair proteins [1]. From a biological and genetic stand- point, melanomas arising on chronically sun-damaged skin (CSDS) demonstrate a higher mutational burden [2], includ- ing increased frequency of mutations such as BRAF-V600K, NF1, TP53, and KIT as well as increased levels of PD-L. [3] Studies show that the skin surrounding a melanoma on CSDS can harbor an increased mutational load (similar to that seen within the melanoma), suggesting that the high number of oncogenic mutations within the melanoma may be acquired at an earlier stage, and therefore additional mutations may not be required for its progression [3]. Objectives To identify possible gene expression signatures in the skin of patients with melanoma on CSDS that may contribute to melanomagenesis. Methods This observational experimental research study included a total of 10 patients over 18 years of age with a recent diag- nosis of melanoma on CSDS (i.e., diagnosis no more than 6 months before the day of enrollment). CSDS was defined by the clinical presence of multiple solar lentigines involv- ing at least three anatomical areas, and pigmentary changes including dyschromia, hypo- and/or hyperpigmentation. For each patient, two skin samples were obtained using a 2-mm punch biopsy, and specifically, one sample from CSDS within 2 cm of the primary melanoma and a second sample from sun-protected skin (i.e, retroauricular, inner buttock/gluteal fold). Each skin sample was embedded in RNA-later buffer. RNA was isolated utilizing the RNA extraction kit (Qiagen Cat#74134) and sent to the Sylvester Onco-genomics Shared Resource for library preparation and RNA sequencing. This study was approved by the Institutional Review Board at the University of Miami. The RNA-seq raw expression data was processed in the R-programming environment to perform statistical calculations and differential expression analysis. To evaluate the different signaling pathways between CSDS and non-CSDS, the gene set enrichment analysis (GSEA) was conducted using the cluster Profiler R-package [4], and Mul- tiRankSeq [5] was used to identify differentially expressed genes (DEG). Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analysis were performed to understand the gene functional annota- tion and functional enrichment for DEG, respectively. False discovery rate P< 0.05 was set as the significance criterion. Conclusions Over the course of one year, a total of 10 patients met the inclusion criteria. Due to the small skin sample size (2 mm punch) and fragile nature of RNA, which requires optimal processing to prevent degradation, only the skin samples from four patients met the necessary quality standards for molecular analyses. The patients’ average age at diagnosis was 47.7 years; 75% (3/4) were females. Two patients had an invasive melanoma (average thickness 0.65 mm; range 0.3–1 mm), and two, in-situ disease. Superficial spreading mela- noma (50%) and lentigo maligna (50%) were the histolog- ical subtypes. Tumors were found on the scalp (50%, N=2), neck and chest. Patients’ average body mass index was 32.35 (obesity range) (Figure 1). To screen out the hub CSDS- related genes that may have contributed to the development of mel- anoma on CSDS, the principal component analysis (PCA) was performed to classify the relationship between the eight samples. The PCA showed that three pairs of the analyzed samples clustered together. Significant differences were ob- served between the CSDS and non-CSDS samples (Figure 2). Applying the defined cutoff values (log2FoldChange >2.0 and p.Adj 0.05), six genes of interest were identified and as- sociated with CSDS compared to non-CSDS, including four upregulated (i.e., SERPINB4, RHCG, CHST2, KRT16) and one downregulated gene on CSDS (i.e., CACNA1H). To in- vestigate the potential regulatory mechanism of the DEGs in CSDS, we performed gene ontology and KEGG enrich- ment analysis. The biological functions of these skin samples were detected by KEGG pathways analysis, which revealed that the main pathways were IL-17 signaling pathway, ABC transporters, and oxidative phosphorylation (activated path- ways), proliferator-activated receptor (PPAR) as well as Conclusions: CSDS can be an adequate milieu for the development and progression of melanoma. CSDS reveals overexpression of pathways involved in inflammation, immune responses, and oxidative phosphorylation, all of which may facilitate interactions between the skin microenvironment and me- lanocytes/melanoma cells, predisposing to melanoma development and progression. Original Article | Dermatol Pract Concept. 2025;15(2):4952 3 endocytosis and Notch signaling pathway (suppressed path- ways). The significantly enriched pathways in the two groups were selected by adjusted p-value <0.05. We found that ke- ratinization, epidermal cell differentiation, and keratinocyte differentiation genes were significantly enriched in the CSDS group (Figure 3). Melanoma development and progression is a complex and dynamic process involving melanoma cells and their interaction with the skin microenvironment. Evi- dence demonstrates the role of the microenvironment in the malignant transformation of melanocytes, with some niches (e.g., hair follicle bulge) protecting against their malignant transformation and tumor initiation and other interactions supporting proliferation, angiogenesis, and metastasis. [6]. Depending on specific factors, the skin can be a favorable microenvironment for tumor growth, hosting initial muta- tions that may maintain their normal function until further mutations lead to melanoma formation. Moreover, depend- ing on specific genetic drivers as well as internal and/or ex- ternal stimuli (e.g., UVR), some microenvironments may or may not be permissive to melanocyte transformation. For Figure 1. Primary cutaneous melanoma of two patients. A-B: Clinical and dermoscopic images of lentigo maligna on the frontal scalp. C-D: Clinical and dermoscopic images of superficial spreading melanoma 0.7mm on the chest. Figure 2. Principal component analysis of the gene expression profiles of skin samples from chron- ically sun-damaged skin (CSDS) and non-CSDS of patients with melanoma on CSDS. Each dot represents an RNA-Seq sample, with red dots indicating CSDS and blue dots indicating non-CSDS. Samples with similar gene expression profiles are clustered together. 4 Original Article | Dermatol Pract Concept. 2025;15(2):4952 receptors [8]. On the other hand, the Notch signaling path- way, which plays a role in cell renewal, differentiation, ho- meostasis, and repair, was found to be suppressed in CSDS, suggesting an imbalance between pro-inflammatory and re- pair responses. This pathway has anti-apoptotic functions in keratinocytes UVB-response through down-modulation of FoxO3a expression [10]. The limitations of our study include the limited number of cases and their heterogeneity. Our findings support the pivotal role of the skin micro- environment, which under the influence of UVR facilitates inflammatory and immune responses that can lead to mel- anomagenesis. CSDS reveals overexpression of pathways involved in inflammation, immune response, and oxidative phosphorylation, which allow interactions between the skin microenvironment and melanocytes/melanoma cells, predis- posing to melanoma development/progression. These find- ings may serve as a valuable baseline for future studies and therapeutic strategies targeting the microenvironmental fac- tors that may contribute to melanoma progression. References 1. Pecorelli A, Valacchi G. Oxidative-stress-sensitive microRNAs in UV-promoted development of melanoma. Cancers (Basel). 2022;14(13):3224. DOI: 10.3390/cancers14133224. 2. Millán-Esteban D, Peña-Chilet M, Garcia-Casado Z, et al. Mu- tational characterization of cutaneous melanoma supports di- vergent pathways model for melanoma development. Cancers (Basel). 2021;13(20):5219. DOI: 10.3390/cancers13205219. example, it is suggested that melanocyte stem cells, which are usually quiescent on the hair follicle bulge, can only proliferate and migrate to the epidermis in response to UVR- induced inflammation or by paracrine signals secreted by the epidermal microenvironment [7]. Indeed, it is well known that UVR promotes skin inflammation, decreases immune surveillance, induces cellular stress (e.g., oxidative stress, accumulation of reactive oxygen species), and induces changes in gene expression and protein production as well as nuclear and mitochondrial DNA damage. In our study, we found pathways and genes that were overexpressed on CSDS but not on sun-protected skin of patients with mela- noma on CSDS. Interestingly, these overexpressed pathways (i.e., IL-17, ABC transporters, oxidative phosphorylation, PPAR signaling pathway) are all involved in inflammatory conditions. For example, the oxidative phosphorylation pathway, which is crucial to cell energy homeostasis and metabolic demands, may induce a hypoxic, glucose-defi- cient, and acidic tumor microenvironment that may inhibit the function of immune cells [8]. Furthermore, the IL-17 and ABC transporters are involved in immune cell responses. 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