Malignant Transformation Of Melanocytes Research Articles

SIRT7 promotes mitochondrial biogenesis to render the adaptive resistance to MAPK inhibition in melanoma

Melanoma, arising from the malignant transformation of melanocytes, stands as the most lethal type of skin cancer. While significant strides have been made in targeted therapy and immunotherapy, substantially enhancing therapeutic efficacy, the prognosis for melanoma patients remains unoptimistic. SIRT7, a nuclear-localized deacetylase, plays a pivotal role in maintaining cellular homeostasis and adapting to external stressors in melanoma, with its activity closely tied to intracellular nicotinamide adenine dinucleotide (NAD+). However, its involvement in adaptive resistance to targeted therapy remains unclear. Herein, we unveil that up-regulated SIRT7 promotes mitochondrial biogenesis to render the adaptive resistance to MAPK inhibition in melanoma. Initially, we observed a significant increase of SIRT7 expression in publicly available datasets following targeted therapy within a short duration. In consistent, we found elevated SIRT7 expression in melanoma cells subjected to BRAF or MEK inhibitors in vitro. The up-regulation of SIRT7 expression was also confirmed in xenograft tumors in mice after targeted therapy in vivo. Furthermore, we proved that SIRT7 deficiency led to decreased cell viability upon prolonged exposure to BRAF or MEK inhibitors, accompanied by an increase in cell apoptosis. Mechanistically, SIRT7 deficiency restrained the upregulation of genes associated with mitochondrial biogenesis and intracellular ATP levels in response to targeted therapy treatment in melanoma cells. Ultimately, we proved that SIRT7 deficieny could sensitize BRAF-mutant melanoma cells to MAPK inhibition targeted therapy in vivo. In conclusion, our findings underscore the role of SIRT7 in fostering adaptive resistance to targeted therapy through the facilitation of mitochondrial biogenesis. Targeting SIRT7 emerges as a promising strategy to overcome MAPK inhibitor adaptive resistance in melanoma.

Melanoma-associated melanocortin 1 receptor variants confer redox signaling-dependent protection against oxidative DNA damage

Cutaneous melanoma, a lethal skin cancer, arises from malignant transformation of melanocytes. Solar ultraviolet radiation (UVR) is a major environmental risk factor for melanoma since its interaction with the skin generates DNA damage, either directly or indirectly via oxidative stress. Pheomelanin pigments exacerbate oxidative stress in melanocytes by UVR-dependent and independent mechanisms. Thus, oxidative stress is considered to contribute to melanomagenesis, particularly in people with pheomelanic pigmentation. The melanocortin 1 receptor gene (MC1R) is a major melanoma susceptibility gene. Frequent MC1R variants (varMC1R) associated with fair skin and red or yellow hair color display hypomorphic signaling to the cAMP pathway and are associated with higher melanoma risk. This association is thought to be due to production of photosensitizing pheomelanins as well as deficient induction of DNA damage repair downstream of varMC1R. However, the data on modulation of oxidative DNA damage repair by MC1R remain scarce. We recently demonstrated that varMC1R accelerates clearance of reactive oxygen species (ROS)-induced DNA strand breaks in an AKT-dependent manner. Here we show that varMC1R also protects against ROS-dependent formation of 8-oxodG, the most frequent oxidative DNA lesion. Since the base excision repair (BER) pathway mediates clearance of these DNA lesions, we analyzed induction of BER enzymes in human melanoma cells of varMC1R genotype. Agonist-mediated activation of both wildtype (wtMC1R) and varMC1R significantly induced OGG and APE-1/Ref1, the rate-limiting BER enzymes responsible for repair of 8-oxodG. Moreover, we found that NADPH oxidase (NOX)-dependent generation of ROS was responsible for AKT activation and oxidative DNA damage repair downstream of varMC1R. These observations provide a better understanding of the functional properties of melanoma-associated MC1R alleles and may be useful for the rational development of strategies to correct defective varMC1R responses for efficient photoprotection and melanoma prevention in fair-skinned individuals.

Comparative Evaluation of Anticancer Activity of Natural Methoxylated Flavones Xanthomicrol and Eupatilin in A375 Skin Melanoma Cells.

Melanoma is a skin cancer caused by the malignant transformation of melanocytes and cutaneous melanoma represents the most aggressive and deadliest type of skin cancer with an increasing incidence worldwide. The main purpose of the present research was to evaluate the anticancer effects of the natural bioactive compounds xanthomicrol (XAN) and eupatilin (EUP) in human A375 malignant skin melanoma cells, a cell line widely used as an in vitro model of cutaneous melanoma. XAN and EUP are lipophilic methoxylated flavones with antioxidant, anti-inflammatory, and antitumor properties. The effects of XAN and EUP on cell viability, morphology, lipid profile, oxidative status, apoptosis, and mitochondrial membrane polarization were determined and compared in A375 cells. At 24 h-incubation (MTT assay), XAN significantly reduced viability at the dose range of 2.5-200 μM, while EUP showed a significant cytotoxicity from 25 μM. Moreover, both methoxylated flavones induced (at 10 and 25 μM, 24 h-incubation) marked cell morphological alterations (presence of rounded and multi-nucleated cells), signs of apoptosis (NucView 488 assay), and a noteworthy mitochondrial membrane depolarization (MitoView 633 assay), coupled to a marked lipid profile modulation, including variations in the ratio of phospholipid/cholesterol and a decrease in the oleic, palmitic, and palmitoleic acid amounts. Moreover, a remarkable time-dependent ROS generation (2',7'-dichlorodihydrofluorescein diacetate assay) was observed during 3 h-incubation of A375 cancer cells in the presence of XAN and EUP (10 and 25 μM). Our results confirm the potential antitumor effect of natural EUP and XAN in cutaneous melanoma by the activation of multiple anticancer mechanisms.

Active natural compounds perturb the melanoma risk-gene network.

Cutaneous melanoma is an aggressive type of skin cancer with a complex genetic landscape caused by the malignant transformation of melanocytes. This study aimed at providing an in silico network model based on the systematic profiling of the melanoma-associated genes considering germline mutations, somatic mutations, and genome-wide association study signals accounting for a total of 232 unique melanoma risk genes. A protein-protein interaction network was constructed using the melanoma risk genes as seeds and evaluated to describe the functional landscape in which the melanoma genes operate within the cellular milieu. Not only were the majority of the melanoma risk genes able to interact with each other at the protein level within the core of the network, but this showed significant enrichment for genes whose expression is altered in human melanoma specimens. Functional annotation showed the melanoma risk network to be significantly associated with processes related to DNA metabolism and telomeres, DNA damage and repair, cellular ageing, and response to radiation. We further explored whether the melanoma risk network could be used as an in silico tool to predict the efficacy of anti-melanoma phytochemicals, that are considered active molecules with potentially less systemic toxicity than classical cytotoxic drugs. A significant portion of the melanoma risk network showed differential expression when SK-MEL-28 human melanoma cells were exposed to the phytochemicals harmine and berberine chloride. This reinforced our hypothesis that the network modeling approach not only provides an alternative way to identify molecular pathways relevant to disease but it may also represent an alternative screening approach to prioritize potentially active compounds.

Piezo1 Activation Prevents Spheroid Formation by Malignant Melanoma SK-MEL-2 Cells.

Melanoma is a highly aggressive type of skin cancer produced through the malignant transformation of melanocytes, and it is usually associated with a poor prognosis. Clinically, melanoma has several stages associated with migration and invasion of the cells through the skin's layers, the rapid spreading of cells and the formation of tumors in multiple organs. The main problem is the emergence of resistance in melanoma to the applied methods of treatment; thus, it is of primary importance to find more crucial signaling pathways that control the progression of this type of cancer and could be targeted to prevent melanoma spreading. Here, we uncover novel aspects of the role of the mechanosensitive ion channel Piezo1 in melanoma tumor formation. Using a combinative approach, we showed the functional expression of mechanosensitive Piezo1 channels in the aggressive human melanoma SK-MEL-2 cell line. We found that chemical activation of Piezo1 by its agonist, Yoda1, prevents melanoma spheroid formation; thus, Piezo1 could be a potential target for selective modulation aimed at the prevention of melanoma development.

Abstract 27 Secretome of Umbilical Cord Mesenchymal Stem Cells: Potential Effects on Melanoma

Abstract Introduction Melanoma, a tumor resulting from the malignant transformation of melanocytes, is characterized by its aggressive nature and propensity to metastasize. Current treatment options for advanced melanoma are limited and mostly ineffective, highlighting the need for novel therapeutic approaches. Mesenchymal stem cells (MSCs) have increased considerable attention due to their anti-cancer and immunomodulatory properties. In particular, human umbilical cord MSCs (hUCMSCs) have shown promise in various therapeutic applications. Objectives The objective of this research was to assess and understand the potential therapeutic effects of conditioned media (CM) derived from hUCMSCs on melanoma cells in vitro. Methods hUCMSC, isolated from the umbilical cord of seven healthy neonates by enzymatic digestion followed by direct plastic adherence method, were cultivated and their CM stored for subsequent assays. Then, malignant melanoma B16F10 cells were treated using a final concentration of 100% of hUCMSC-CM for 24 hours. Afterward, their viability, adhesion, and motility were accessed by MTT, crystal violet, and scratch wound healing assays, respectively. Results The MTT assay showed the inhibition of B16F10 cell viability in all treated groups compared to the control group (32%; p<0,05 vs control, n=7). Moreover, crystal violet staining revealed that hUCMSC-CM enhanced the adhesion capacity of melanoma cells by 16% (p<0,05 vs control, n=7). In terms of motility, the hUCMSC-CM reduced migration by 24% (p<0,05 vs control, n=7) after 24 hours. Discussion Our results suggest that hUCMSCs may exhibit dual effects on melanoma cells. On one hand, certain factors produced by hUCMSCs have shown potential in reducing the viability and motility of B16F10 melanoma cells, indicating a potential therapeutic benefit. On the other hand, it has also been observed that CM of hUCMSCs can increase the adhesion capacity of melanoma cells. While hUCMSC-released factors may exhibit beneficial effects on melanoma cells, further research and clinical trials are needed to establish their safety, efficacy, and optimal therapeutic protocols for malignant melanoma.

Use of immunomodulation in cancer wound care: a case study

Skin cancer is characterized by an abnormal proliferation of cells and is considered the most malignant cutaneous tumor, originating from the malignant transformation of melanocytes. The objective of this research was to assess the progression of oncological wounds after six months of specific supplementation aimed at accelerating wound healing. A descriptive analysis of the data obtained during the intervention was conducted, utilizing graphs, tables, and Excel 365 spreadsheets. The immunomodulatory nutritional therapy demonstrated to be of utmost importance, yielding a positive and substantiated enhancement in the wound healing process, providing excellent aesthetic and functional outcomes, and positively impacting the quality of life of the patients.

BRAF Mutations in Melanoma: Biological Aspects, Therapeutic Implications, and Circulating Biomarkers.

Melanoma is an aggressive form of skin cancer resulting from the malignant transformation of melanocytes. Recent therapeutic approaches, including targeted therapy and immunotherapy, have improved the prognosis and outcome of melanoma patients. BRAF is one of the most frequently mutated oncogenes recognised in melanoma. The most frequent oncogenic BRAF mutations consist of a single point mutation at codon 600 (mostly V600E) that leads to constitutive activation of the BRAF/MEK/ERK (MAPK) signalling pathway. Therefore, mutated BRAF has become a useful target for molecular therapy and the use of BRAF kinase inhibitors has shown promising results. However, several resistance mechanisms invariably develop leading to therapeutic failure. The aim of this manuscript is to review the role of BRAF mutational status in the pathogenesis of melanoma and its impact on differentiation and inflammation. Moreover, this review focuses on the mechanisms responsible for resistance to targeted therapies in BRAF-mutated melanoma and provides an overview of circulating biomarkers including circulating tumour cells, circulating tumour DNA, and non-coding RNAs.

Assessment of tumoral and peritumoral inflammatory reaction in cutaneous malignant melanomas.

Skin cancer is one of the most common types of cancer, with an increasing worldwide incidence in recent decades. The main risk factor for increasing the skin cancer incidence is ultraviolet (UV) radiation. Of the two major forms of skin cancer (melanomas and non-melanotic cancers), the cutaneous melanoma (CM) is the most aggressive form, causing about 80% of the deaths resulted from this type of tumor. Malignant melanoma develops through malignant transformation of melanocytes in the skin because of prolonged exposure to solar or artificial UV. The malignant transformation of the melanocytes in the skin is accompanied by the presence of a local inflammatory reaction that, in the initial stages of carcinogenesis, would oppose to tumor development. Chronic exposure to UV or other etiopathogenic factors induces chronic inflammation, which, by producing inflammatory molecules (cytokines, chemokines, prostaglandins), constitutes a tumoral microenvironment that favors carcinogenesis, tumor invasion, metastasis, and the presence of neoplastic "mutant cells" that avoid the protective action of the immune system. Using immunohistochemistry techniques, we assessed the intra- and peritumoral inflammatory infiltrate cells in CM. The chronic inflammatory infiltrate presented more intense in the peritumoral stroma compared to the intratumoral one, heterogenous, more intensely composed of lymphocytes, plasma cells, macrophages, and mast cells (MCs), the most numerous cells in the inflammatory infiltrate being T-lymphocytes, plasma cells and macrophages; B-lymphocytes and MCs were in a small number, especially intratumorally. Inflammatory cells had a direct contact with tumor cells, blood vessels, connective matrix, suggesting that the inflammatory microenvironment plays an important role in carcinogenesis, tumor invasion, local angiogenesis, and tumor metastasis.

Abstract 3755: Dlx4os: a lncRNA associated with malignant melanocyte transformation

Abstract It is estimated that more than 90% of the human genome is transcribed into non-coding RNAs (ncRNA). It has been demonstrated that ncRNAs have an important role in biological processes such as proliferation, differentiation, and cell migration. Changes in their expression have been associated with several diseases, including cancer. Melanoma is one of the most aggressive types of cancer, with high probability of metastasis and an unfavorable response to therapies. Our lab has developed an in vitro melanoma progression model, which was established by exposing nontumorigenic melanocytes (melan-a) to sustained stress conditions (cycles of adhesion blockage), sequentially generating different cell lines (i.e., 4C, premalignant melanocytes; 4C11-, non-metastatic melanoma cells; and 4C11+, metastatic melanoma cells), in which each cell line represents a distinct stage of tumor progression. Previous RNA sequencing data comparing these 4 cell lines identified a total of 3404 long non-coding transcripts which displayed differential expression (DE) levels when comparing two or more cell lines. We have clustered 531 of them into three distinct signatures: malignancy, epithelial-to-mesenchymal transition (EMT), or metastasis, according to their expression profiles. We also analyzed the neighboring genes of those lncRNAs, as it is known that lncRNAs might regulate gene expression in cis. We identified the lncRNA Dlx4os, highly expressed in undifferentiated and mesenchymal-like 4C and 4C11- cells, and 6 neighboring genes whose expression correlate to the EMT signature and overall survival in human melanoma patients. Among these genes we found Dlx4, which shares the same promoter site with Dlx4os and whose expression correlates to a poor prognosis. We silenced Dlx4os in the 4C11- cells through interference RNA technology. The downregulation of Dlx4os led to expression changes in EMT-related genes, such as Snail1, Sox10 and Mitf, indicating that it might play a role in EMT. Dlx4os downregulation also inhibited cell migration in vitro and tumorigenesis in vivo. These results overall indicate a role of Dlx4os in melanocyte malignant transformation. Citation Format: Ana Luísa P. Ayub, Hatylas Azevedo, Diogo Pessoa, Leinal Sejour, Ioannis Vlachos, Eduardo M. Reis, Frank J. Slack, Miriam G. Jasiulionis. Dlx4os: a lncRNA associated with malignant melanocyte transformation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3755.

A feasibility study comparing car-t cell therapy and separate limb end perfusion therapy in the treatment of melanoma

There are several potential advancements within cancer immunotherapy, but cells of chimeric antigen receptor-T stand out. Recently, two CAR-T cell designs targeting CD19 have been approved for use in the US and the European Union due to their promising results in the treatment of hematologic malignancies. Currently, researchers are focusing on evaluating the efficientness of CAR-T cell treatment for a variety of cancer of the solid tissues. Melanoma is caused by the malignant transformation of melanocytes. In addition to the skin, melanomas can also develop in other locations where neural crest cells migrate, such as the digestive system or the brain. Melanocytes are formed in the neural crest. Since transformed cells must overcome extra obstacles to survive, the treatment of solid cancers with cells that produce CAR-T has been less successful than the treatment of hematologic malignancies with cells that produce CAR-T. A solid cancer's immunosuppressive microenvironment and the inability to migrate cells that produce CAR-T to the site of the cancer are two significant barriers. In addition, finding the optimal target antigens to avoid on-target toxicity and non-cancer toxicity is a challenge. To lessen the harmful effects of systemic chemotherapy, Creech & Krementz developed isolated limb perfusion in 1956 to create high levels of chemotherapy in limbs affected by unresectable cancers, particularly soft tissue sarcomas and melanomas. By using these targets, it is possible to remove the wounded limb's circulation from the body's circulation and connect it to an extracorporeal system. When the patient becomes excessively hot, chemotherapeutic medications, primarily melphalan & cancer necrosis factor, are administered via a perfusion circuit. The objective of this article is to provide a summary of the benefits and drawbacks of employing car-t-cell therapy to cure solid cancers, particularly melanoma. It also investigates the possibilities of curing melanoma using car-T treatment as well as isolated limb perfusion.

Extracellular Vesicles in Cancer Drug Resistance: Implications on Melanoma Therapy.

Extracellular vesicles (EVs) are involved in the pathogenesis of neoplastic diseases. Their role in mediating drug resistance has been widely described in several types of cancers, including melanoma. EVs can mediate drug resistance through several different mechanisms, such as drug-sequestration, transfer of pro-survival proteins and RNA, induction of cancer stem cell-like features and interaction with cells of the tumor microenvironment and immune-system. Melanoma is a highly immunogenic tumor originating from the malignant transformation of melanocytes. Several therapeutic strategies currently used in the treatment of melanoma and the combination of BRAF and MEK-inhibitors, as well as immune check-point inhibitors (ICI), have consistently improved the overall survival time of melanoma patients. However, the development of resistance is one of the biggest problems leading to a poor clinical outcome, and EVs can contribute to this. EVs isolated from melanoma cells can contain "sequestered" chemotherapeutic drugs in order to eliminate them, or bioactive molecules (such as miRNA or proteins) that have been proven to play a crucial role in the transmission of resistance to sensitive neoplastic cells. This leads to the hypothesis that EVs could be considered as resistance-mediators in sensitive melanoma cells. These findings are a pivotal starting point for further investigations to better understand EVs' role in drug resistance mechanisms and how to target them. The purpose of this review is to summarize knowledge about EVs in order to develop a deeper understanding of their underlying mechanisms. This could lead to the development of new therapeutic strategies able to bypass EV-mediated drug-resistance in melanoma, such as by the use of combination therapy, including EV release inhibitors.

The dual role of Nrf2 in melanoma: a systematic review

Melanoma is the most lethal type of skin cancer that originates from the malignant transformation of melanocytes. Although novel treatments have improved patient survival in melanoma, the overall prognosis remains poor. To improve current therapies and patients outcome, it is necessary to identify the influential elements in the development and progression of melanoma.Due to UV exposure and melanin synthesis, the melanocytic lineage seems to have a higher rate of ROS (reactive oxygen species) formation. Melanoma has been linked to an increased oxidative state, and all facets of melanoma pathophysiology rely on redox biology. Several redox-modulating pathways have arisen to resist oxidative stress. One of which, the Nrf2 (nuclear factor erythroid 2-related factor 2), has been recognized as a master regulator of cellular response to oxidative or electrophilic challenges. The activation of Nrf2 signaling causes a wide range of antioxidant and detoxification enzyme genes to be expressed. As a result, this transcription factor has lately received a lot of interest as a possible cancer treatment target.On the other hand, Nrf2 has been found to have a variety of activities in addition to its antioxidant abilities, constant Nrf2 activation in malignant cells may accelerate metastasis and chemoresistance. Hence, based on the cell type and context, Nrf2 has different roles in either preventing or promoting cancer. In this study, we aimed to systematically review all the studies discussing the function of Nrf2 in melanoma and the factors determining its alteration.

Metastatic malignant melanoma mimicking urinary bladder mass: A rare presentation

Introduction. Melanoma is a solid aggressive tumor characterized by the malignant transformation of melanocytes. To date, only about 35 primary and about 30 metastatic malignant melanomas of the bladder have been reported. Our objective is to report a rare case of secondary tumor of urinary bladder. Case outline. A 57-year-old man presented to the Urologic Clinic due to lower urinary tract symptoms. The urologist indicated transurethral resection (TUR). His medical history was significant for cutaneous malignant melanoma resected 3 years prior, which were localized at scapular region on the left side. Microscopic examination of the TUR specimen showed several fragments of ureter mucosa with presence of tumor and focally with normal urothel. Tumor cells were markedly atypical and polygonal in a solid pattern. The nuclei were large with variation in size and prominent eosinophilic nucleoli. Also, there were present areas with abundant brown pigment. Immunohistochemical analysis of tumor cells showed positivity for Melan A and HMB45 and negativity for GATA3. Molecular analysis showed that BRAF was mutated. Conclusion. The incidence of malignant melanoma is high and increasing, but the urinary bladder is a rare location of metastasis. However, both primary and metastatic melanomas can occur in the bladder, so the urologist and the pathologist have to consider it when it is the primary site of onset, or when it represents the first symptomatic metastasis.

YTHDF3 promotes melanoma metastasis by reading N6‐methyladenosine

YTHDF3 promotes melanoma metastasis by reading N⁶‐methyladenosine

RAI14 Promotes Melanoma Progression by Regulating the FBXO32/c-MYC Pathway.

Melanoma originates from the malignant transformation of melanocytes. Compared with other skin cancers, melanoma has a higher fatality rate. The 5-year survival rate of patients with early-stage primary melanoma through surgical resection can reach more than 90%. However, the 5-year survival rate of patients with metastatic melanoma is only 25%. Therefore, accurate assessment of melanoma progression is critical. Previous studies have found that Retinoic Acid Induced 14(RAI14) is critical in tumorigenesis. However, the biological function of RAI14 for the development of melanoma is unclear. In this study, RAI14 is highly expressed in melanoma and correlated with prognosis. The expression of RAI14 can affect the proliferation, migration and invasion of melanoma cells. F-Box Protein 32(FBXO32) is an E3 ubiquitin ligase of c-MYC. We found that RAI14 affects the transcriptional expression of FBXO32 and regulates the stability of c-MYC. These results suggest that RAI14 play an important role in the growth of melanoma and is expected to be a therapeutic target for melanoma.

Metabolic rewiring directs melanoma immunology.

Melanoma results from the malignant transformation of melanocytes and accounts for the most lethal type of skin cancers. In the pathogenesis of melanoma, disordered metabolism is a hallmark characteristic with multiple metabolic paradigms involved in, e.g., glycolysis, lipid metabolism, amino acid metabolism, oxidative phosphorylation, and autophagy. Under the driving forces of oncogenic mutations, melanoma metabolism is rewired to provide not only building bricks for macromolecule synthesis and sufficient energy for rapid proliferation and metastasis but also various metabolic intermediates for signal pathway transduction. Of note, metabolic alterations in tumor orchestrate tumor immunology by affecting the functions of surrounding immune cells, thereby interfering with their antitumor capacity, in addition to the direct influence on tumor cell intrinsic biological activities. In this review, we first introduced the epidemiology, clinical characteristics, and treatment proceedings of melanoma. Then, the components of the tumor microenvironment, especially different populations of immune cells and their roles in antitumor immunity, were reviewed. Sequentially, how metabolic rewiring contributes to tumor cell malignant behaviors in melanoma pathogenesis was discussed. Following this, the proceedings of metabolism- and metabolic intermediate-regulated tumor immunology were comprehensively dissertated. Finally, we summarized currently available drugs that can be employed to target metabolism to intervene tumor immunology and modulate immunotherapy.

080 Current treatment options for patients with unresectable cutaneous melanoma

Cutaneous Melanoma (CM) remains the leading cause of skin-related mortality in the U.S. The U.S. Surveillance, Epidemiology and End Results database reports 5.6% of all new cancer cases in 2021 attributed to CM, with incidences rising annually since 2009. Several subtypes of CM are thought to arise from early driver mutations in BRAF, RAS, and NF1 genes resulting in unopposed activation of MAPK and PI3K signaling pathways, unregulated growth and malignant transformation of melanocytes. Due to the metastatic potential of CM, emphasis on systemic treatment has been an area of interest when surgical resection is insufficient for curative prognosis.

Abstract 112: Epac signaling facilitates malignant transformation of melanocytes

Abstract Cyclic AMP (cAMP) signaling plays a critical role in melanocyte proliferation and differentiation. However, the role of cAMP signaling in malignant transformation of human melanocytes and initiation of melanoma is not fully understood. Previously, we demonstrated that cAMP signaling plays opposite roles in primary and metastatic melanoma cells, i.e., pro- and anti-proliferative roles, respectively. This switch in cAMP action is mediated by the alternative cAMP signaling axis involving EPAC-RAP1 (exchange protein directly activated by cyclic AMP-Ras-related protein 1). In this study, we tested the hypothesis that activation of EPAC signaling is an early event in malignant transformation of melanocytes and is required for survival and proliferation of malignant melanocytes. Here, we show that shRNA-mediated knockdown of PTEN alone or PTEN-KD with lentiviral transduction with BRAF(V600E) results in upregulation of EPAC expression and activation of RAP1 (i.e., RAP1-GTP) in human primary melanocytes. Interestingly, activation of phosphoinositide 3-kinase (PI3K) signaling alone is sufficient for stimulation of maximal EPAC expression as well as proliferation suggesting that EPAC activation is an early event associated with loss of PTEN and melanoma tumor initiation. Inhibition of EPAC activity by either EPAC 1/2 or EPAC2-selective inhibitors suppressed the growth of transformed melanocytes by inhibiting cell cycle progression as evident by downregulation of cyclins E, -A2 and -B1. Inhibition of EPAC signaling also increased cells expressing senescence-associate beta galactosidase activity suggesting that EPAC may contribute to the senescence escape mechanisms in BRAF(V600E) melanocytes. Our data suggest that activation of PI3K pathway alone or in combination with mitogen-activated protein kinase pathway activates EPAC signaling in melanocytes. Since primary but not metastatic melanoma cells remain dependent on EPAC signaling, understanding the mechanisms of action EPAC can open new avenues to prevent melanomagenesis and inhibit melanoma tumor progression. Citation Format: Mithalesh Kumar Singh, Sarah Altameemi, Marcos Lares, Shreyans Sadangi, Vijayasaradhi Setaluri. Epac signaling facilitates malignant transformation of melanocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 112.

Abstract 907: CD133 stimulates cell proliferationviaupregulation of amphiregulin in melanoma

Abstract Melanoma, a malignant transformation of melanocytes, accounts for 90% of skin cancer deaths due to its ability to efficiently metastasize to other organs. CD133, also known as Prominin1, is a pentaspan glycosylated transmembrane protein that is overexpressed in cancer stem cells from various tumors, including melanoma, and has been associated with tumor recurrence, chemoresistance, and metastasis. To characterize and target CD133-positive cancer stem cells, patient-derived melanoma cell lines BAKP and POT, harboring BRAFWT/NRASQ61K and BRAFWT/NRASQ61R driver mutations, respectively, were co-transduced with a Tet activator (rtTA3) and a Tet-on vector expressing CD133 (TRE3G-CD133) to produce stable doxycycline (Dox)-inducible cell lines. These cell lines were exposed to Dox (1µg/mL) to induce CD133 expression for 24 h; mRNA was extracted and subjected to RNA-seq analysis, followed by bioinformatic analyses, which revealed genes that are significantly up- or down-regulated by Dox-induced CD133 expression. Among the most significantly altered genes was amphiregulin (AREG), which increased 7-fold with Dox induction. These results were validated in both BAKP and POT cell lines by qPCR and immunoblot analysis. AREG binds EGFR and activates RAS/RAF/MEK/ERK and PI3K/AKT signaling pathways, leading to cell proliferation, survival, and migration. Consistently, cell cycle and cell growth analyses revealed that Dox-induced CD133 expression increases the percent of cells in S-phase of the cell cycle, leading to increased cell growth in BAKP cells. The role of CD133 in cell proliferation via AREG upregulation was further studied with immunofluorescent and flow cytometry analysis of proliferation markers PCNA, Ki67, as well as BrdU incorporation into newly synthesized DNA in Dox-induced BAKP cells, with or without siRNA knockdown of AREG. AREG may play an integral role in melanoma progression due to its vital function in cell proliferation, serving as an attractive target for novel therapeutics in melanoma and cancers exhibiting overexpression of both CD133 and AREG. Further studies using specific AREG inhibitors or RNAi knockdown in melanoma cell lines is currently ongoing, and can further elucidate molecular mechanisms highlighting the role(s) of CD133 and AREG in melanoma cancer stem cells. Citation Format: Cynthia M. Simbulan-Rosenthal, Nusrat Islam, Yoga Haribabu, Ryyan Alobaidi, Li-Wei Kuo, Azadeh Shalamzari, Adam Leibowitz, Joanna Moulton, Dean S. Rosenthal. CD133 stimulates cell proliferationviaupregulation of amphiregulin in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 907.