Inhibited Melanoma Cell Proliferation Research Articles

Repurposing of Antidiarrheal Loperamide for Treating Melanoma by Inducing Cell Apoptosis and Cell Metastasis Suppression In vitro and In vivo.

Melanoma is the most common skin tumor worldwide and still lacks effective therapeutic agents in clinical practice. Repurposing of existing drugs for clinical tumor treatment is an attractive and effective strategy. Loperamide is a commonly used anti-diarrheal drug with excellent safety profiles. However, the affection and mechanism of loperamide in melanoma remain unknown. Herein, the potential anti-melanoma effects and mechanism of loperamide were investigated in vitro and in vivo. In the present study, we demonstrated that loperamide possessed a strong inhibition in cell viability and proliferation in melanoma using MTT, colony formation and EUD incorporation assays. Meanwhile, xenograft tumor models were established to investigate the anti-melanoma activity of loperamide in vivo. Moreover, the effects of loperamide on apoptosis in melanoma cells and potential mechanisms were explored by Annexin V-FITC apoptosis detection, cell cycle, mitochondrial membrane potential assay, reactive oxygen species level detection, and apoptosis-correlation proteins analysis. Furthermore, loperamide-suppressed melanoma metastasis was studied by migration and invasion assays. What's more, immunohistochemical and immunofluorescence staining assays were applied to demonstrate the mechanism of loperamide against melanoma in vivo. Finally, we performed the analysis of routine blood and blood biochemical, as well as hematoxylin- eosin (H&E) staining, in order to investigate the safety properties of loperamide. Loperamide could observably inhibit melanoma cell proliferation in vitro and in vivo. Meanwhile, loperamide induced melanoma cell apoptosis by accumulation of the sub-G1 cells population, enhancement of reactive oxygen species level, depletion of mitochondrial membrane potential, and apoptosis-related protein activation in vitro. Of note, apoptosis-inducing effects were also observed in vivo. Subsequently, loperamide markedly restrained melanoma cell migration and invasion in vitro and in vivo. Ultimately, loperamide was witnessed to have an amicable safety profile. These findings suggested that repurposing of loperamide might have great potential as a novel and safe alternative strategy to cure melanoma via inhibiting proliferation, inducing apoptosis and cell cycle arrest, and suppressing migration and invasion.

Artesunate induces melanoma cell ferroptosis and augments antitumor immunity through targeting Ido1

Artesunate (ART), a natural product isolated from traditional Chinese plant Artemisia annua, has not been extensively explored for its anti-melanoma properties. In our study, we found that ART inhibited melanoma cell proliferation and induced melanoma cell ferroptosis. Mechanistic study revealed that ART directly targets Ido1, thereby suppressing Hic1-mediated transcription suppression of Hmox1, resulting in melanoma cell ferroptosis. In CD8+ T cells, ART does not cause cell ferroptosis due to the low expression of Hmox1. It also targets Ido1, elevating tryptophan levels, which inhibits NFATc1-mediated PD1 transcription, consequently activating CD8+ T cells. Our study uncovered a potent and synergistic anti-melanoma efficacy arising from ART-induced melanoma cell ferroptosis and concurrently enhancing CD8+ T cell-mediated immune response both in vivo and in vitro through directly targeting Ido1. Our study provides a novel mechanistic basis for the utilization of ART as an Ido1 inhibitor and application in clinical melanoma treatment.

Tumor eradication by triplet therapy with BRAF inhibitor, TLR 7 agonist, and PD-1 antibody for BRAF-mutated melanoma.

Programmed death 1 (PD-1)/programmed death-ligand 1 inhibitors are commonly used to treat various cancers, including melanoma. However, their efficacy as monotherapy is limited, and combination immunotherapies are being explored to improve outcomes. In this study, we investigated a combination immunotherapy involving an anti-PD-1 antibody that blocks the major adaptive immune-resistant mechanisms, a BRAF inhibitor that inhibits melanoma cell proliferation, and multiple primary immune-resistant mechanisms, such as cancer cell-derived immunosuppressive cytokines, and a Toll-like receptor 7 agonist that enhances innate immune responses that promote antitumor T-cell induction and functions. Using a xenogeneic nude mouse model implanted with human BRAF-mutated melanoma, a BRAF inhibitor vemurafenib was found to restore T-cell-stimulatory activity in conventional dendritic cells by reducing immunosuppressive cytokines, including interleukin 6, produced by human melanoma. Additionally, intravenous administration of the Toll-like receptor 7 agonist DSR6434 enhanced tumor growth inhibition by vemurafenib through stimulating the plasmacytoid dendritic cells/interferon-α/natural killer cell pathways and augmenting the T-cell-stimulatory activity of conventional dendritic cells. In a syngeneic mouse model implanted with murine BRAF-mutated melanoma, the vemurafenib and DSR6434 combination synergistically augmented the induction of melanoma antigen gp100-specific T cells and inhibited tumor growth. Notably, only triplet therapy with vemurafenib, DSR6434, and the anti-PD-1 antibody resulted in complete regression of SIY antigen-transduced BRAF-mutated melanoma in a CD8 T-cell-dependent manner. These findings indicate that a triple-combination strategy targeting adaptive and primary resistant mechanisms while enhancing innate immune responses that promote tumor-specific T cells may be crucial for effective tumor eradication.

Salidroside induces mitochondrial dysfunction and ferroptosis to inhibit melanoma progression through reactive oxygen species production

Reactive oxygen species (ROS) induces necroptotic and ferroptosis in melanoma cells. Salidroside (SAL) regulates ROS in normal cells and inhibits melanoma cell proliferation. This study used human malignant melanoma cells treated with SAL either alone or in combination with ROS scavenger (NAC) or ferroptosis inducer (Erastin). Through cell viability, wound healing assays, and a Seahorse analyze found that SAL inhibited cell proliferation, migration, extracellular acidification rate, and oxygen consumption rate. Metabolic flux analysis, complexes I, II, III, and IV activity of the mitochondrial respiratory chain assays, mitochondrial membrane potential assay, mitochondrial ROS, and transmission electron microscope revealed that SAL induced mitochondrial dysfunction and ultrastructural damage. Assessment of malondialdehyde, lipid ROS, iron content measurement, and Western blot analysis showed that SAL activated lipid peroxidation and promoted ferroptosis in A-375 cells. These effects were abolished after NAC treatment. Additionally, SAL and Erastin both inhibited cell proliferation and promoted cell death; SAL increased the Erastin sensitivity of cells while NAC antagonized it. In xenograft mice, SAL inhibited melanoma growth and promoted ROS-dependent ferroptosis. SAL induced mitochondrial dysfunction and ferroptosis to block melanoma progression through ROS production, which offers a scientific foundation for conducting SAL pharmacological research in the management of melanoma.

Development of nanoemulgel of 5-Fluorouracil for skin melanoma using glycyrrhizin as a penetration enhancer

The purpose of this study was to enhance the topical delivery of 5-Fluorouracil (5-FU), a cancer treatment, by developing a nanoemulgel formulation. Glycyrrhizin (GLY), a natural penetration enhancer has been investigated to exhibit synergistic effects with 5-FU in inhibiting melanoma cell proliferation and inducing apoptosis, Hence, GLY, along with suitable lipids was utilized to create an optimized nanoemulsion (NE) based gel. Solubility studies and ternary phase diagram revealed isopropyl myristate (IPM), Span 80, Tween 80 as Smix and Transcutol P as co-surfactant. IPM demonstrates excellent solubilizing properties facilitates higher drug loading, ensuring efficient delivery to the target site.,The optimized formulation consisting of 40 % IPM, 30 % of mixture of Tween80: Span80 (Smix) and 15 % Transcutol P provides with a nanometric size of 64.1 ± 5.13 nm and drug loading of 97.3 ± 5.83 %. The optimized formulation observed with no creaming and breakeing of NE and found thermodynamically stable during different stress conditions (temperatures of 4.0 °C and 45.0 °C) and physical thawing (−21.0 ± 0.50 °C to 20.0 ± 0.50 °C). The NE was then transformed into a nanoemulgel (NEG) using 1.5 % w/w Carbopol base and 0.1 % w/w glycyrrhizin. The ex vivo permeability studies showed significant enhancements in drug permeability with the GLY-based 5-FU-NEG formulation compared to pure 5-FU gel in excised pig skin upto1440 min in PBS 7.4 as receptor media. The IC50 values for Plain 5-FU gel, 5-FU-NEG, and GLY-based 5-FU-NEG were found to be 20 µg/mL, 1.1 µg/mL, and 0.1 µg/mL, respectively in B16F10 cell lines. The percentage intracellular uptake of GLY-5-FU-NEG and 5-FU-NEG was found to be 44.3 % and 53.6 %, respectively. GLY-based 5-FU-NEG formulation showed alterations in cell cycle distribution, in compared to 5-FU-NE gel. The overall findings suggest that the GLY-based 5-FU-NEG holds promise for improving anti-melanoma activity.

Inhibition of melanoma cell proliferation by strobilurins isolated from mushrooms and their synthetic analogues.

Strobilurins A and X, isolated from Mucidula venosolamellata culture extracts, demonstrated potent inhibition of human melanoma G-361 cell proliferation. Strobilurin X exhibited milder inhibitory effects on human fibroblast cells (NB1RGB) compared to strobilurin A. Additional strobilurin-related compounds were isolated from the other mushroom species. Oudemansins A and B displayed weaker activities on G-361 cells than strobilurins A and B, respectively, emphasizing the importance of a conjugated double-bond structure. Among isolated compounds, strobilurin G showed the lowest IC50 value for G-361 cells. Additional strobilurins bearing various substituents on the benzene ring were synthesized. Synthetic intermediates lacking the methyl β-methoxyacrylate group and a strobilurin analogue bearing modified β-methoxyacrylate moiety showed almost no inhibitory activity against G-361 cells. The introduction of long or bulky substituents at the 4' position of the benzene ring of strobilurins enhanced the activity and selectivity, suggesting differential recognition of the benzene ring by G-361 and NB1RGB cells.

Screening of anti-melanoma compounds from Morus alba L.: Sanggenon C promotes melanoma cell apoptosis by disrupting intracellular Ca2+ homeostasis

Ethnopharmacological relevanceMorus alba L. is a widespread plant that has long been considered to have remarkable medical values, including anti-inflammation in Traditional Chinese Medicine (TCM). The components of Morus Alba L. constituents have been extensively studied and have been shown to have high prospects for cancer therapy. However, limited investigations have been done on the bioactive compounds in Morus alba L. Aim of the studyThis study aimed to systematically examine the anticancer properties of 28 commercially available compounds from Morus alba L. against melanoma cells in vitro. Additionally, the anticancer mechanisms of the bioactive compound exhibiting the most significant potential were further studied. Materials and methodsThe anti-proliferative effects of Morus alba L.-derived compounds on melanoma cells were determined by colony formation assays. Their effects on cell viability and apoptosis were determined using the CCK8 assay and flow cytometry, respectively. The binding affinity of identified Morus alba L. compounds with anticancer activities towards melanoma targets was analyzed via molecular docking. The molecular mechanism of Sanggenon C was explored using soft agar assays, EdU incorporation assays, flow cytometry, western blotting, transcriptome analysis, and xenograft assays. ResultsBased on colony formation assays, 11 compounds at 20 μM significantly inhibited colony growth on a panel of melanoma cells. These compounds displayed IC50 values (half maximal inhibitory concentrations) ranging from 5 μM to 30 μM. Importantly, six compounds were identified as novel anti-melanoma agents, including Sanggenon C, 3′-Geranyl-3-prenyl-2′,4′,5,7-tetrahydroxyflavone, Moracin P, Moracin O, Kuwanon A, and Kuwanon E. Among them, Sanggenon C showed the most potent effects, with an IC50 of about 5 μM, significantly reducing proliferation and inducing apoptosis in melanoma cells. Based on the xenograft model assay, Sanggenon C significantly inhibited melanoma cell proliferation in vivo. Sanggenon C triggered ER stress in a dose-dependent manner, which further disrupted cellular calcium ion (Ca2+) homeostasis. The Ca2+ chelator BAPTA partially restored cell apoptosis induced by Sanggenon C, confirming that Ca2+ signaling contributed to the anticancer activity of Sanggenon C against melanoma. ConclusionsIn our study, 11 compounds demonstrated anti-melanoma properties. Notably, Sanggenon C was found to promote apoptosis by disrupting the intracellular calcium homeostasis in melanoma cells. This study provides valuable information for the future development of novel cancer therapeutic agents from Morus alba L.

Lnc-PKNOX1-1 inhibits tumor progression in cutaneous malignant melanoma by regulating NF-κB/IL-8 axis.

Cutaneous malignant melanoma is one of the most lethal cutaneous malignancies. Accumulating evidence has demonstrated the potential influence of long non-coding RNAs (lncRNAs) in biological behaviors of melanoma. Herein, we reported a novel lncRNA, lnc-PKNOX1-1 and systematically studied its functions and possible molecular mechanisms in melanoma. Reverse transcription-quantitative PCR assay showed that lnc-PKNOX1-1 was significantly decreased in melanoma cells and tissues. Low lnc-PKNOX1-1 expression was significantly correlated with invasive pathological type and Breslow thickness of melanoma. In vitro and in vivo experiments showed lnc-PKNOX1-1 dramatically inhibited melanoma cell proliferation, migration and invasion. Mechanically, protein microarray analysis suggested that interleukin-8 (IL-8) was negatively regulated by lnc-PKNOX1-1 in melanoma, which was confirmed by western blot and ELISA. Western blot analysis also showed that lnc-PKNOX1-1 could promote p65 phosphorylation at Ser536 in melanoma. Subsequent rescue assays proved IL-8 overexpression could partly reverse the tumor-suppressing function of lnc-PKNOX1-1 overexpression in melanoma cells, indicating that lnc-PKNOX1-1 suppressed the development of melanoma by regulating IL-8. Taken together, our study demonstrated the tumor-suppressing ability of lnc-PKNOX1-1 in melanoma, suggesting its potential as a novel diagnostic biomarker and therapeutic target for melanoma.

Downregulated BIRC5 inhibits proliferation and metastasis of melanoma through the β-catenin/HIF-1α/VEGF/MMPs pathway.

Melanoma is a malignant skin tumor caused by melanocytes and associated with high mortality rates. This study aims to investigate the specific mechanism of ZWZ-3 in melanoma proliferation and metastasis. RNA sequencing was performed to identify the effect of ZWZ-3 on gene expression. siRNA was used to inhibit BIRC5 gene expression in the B16F10 cell line. A zebrafish tumor model was used to assess the therapeutic effect of ZWZ-3 in vivo. Mechanistic insights into the inhibition of tumor metastasis by ZWZ-3 were obtained through analysis of tumor tissue sections in mice. Our findings demonstrated that ZWZ-3 suppressed melanoma cell proliferation and migration. We performed RNA sequencing in melanoma cells after the treatment with ZWZ-3 and found that Birc5, which is closely associated with tumor metastasis, was significantly down-regulated. Bioinformatics analysis and the immuno-histochemical results of tissue chips for melanoma further confirmed the high expression of BIRC5 in melanoma and its effect on disease progression. Moreover, Birc5 knock-down significantly inhibited melanoma cell proliferation and metastasis, which was correlated with the β-catenin/HIF-1α/VEGF/MMPs pathway. Additionally, ZWZ-3 significantly inhibited tumor growth in the zebrafish tumor model without any evident side effects. Histological and immuno-histochemical analyses revealed that ZWZ-3 inhibited tumor cell metastasis by down-regulating HIF-1α, VEGF, and MMP9. Our findings revealed that ZWZ-3 could downregulate BIRC5 and inhibit melanoma proliferation and metastasis through the β-catenin/HIF-1α/VEGF/MMPs pathway. Therefore, BIRC5 represents a promising therapeutic target for the treatment of melanoma.

Vitamin D3 regulates NSUN2 expression and inhibits melanoma cell proliferation and migration.

The activated form of vitamin D3 [1,25-dihydroxyvitamin D3; 1,25(OH)2D3] is important for various physiological processes, such as bone mineralization and calcium metabolism, and plays an anticancer role in numerous cancers as well. Its role in melanoma cells has yet to be proven. NOP2/Sun RNA methyltransferase 2 (NSUN2) is a typical RNA methyltransferase and is highly expressed in a variety of cancer cells. However, the molecular mechanisms underlying the role of 1,25(OH)2D3 and NSUN2 in melanoma cells remain largely unknown. The current study showed that 1,25(OH)2D3 could significantly and specifically inhibit the proliferation and migration of melanoma B16 cells. 1,25(OH)2D3 enhances vitamin D receptor expression while simultaneously reducing NSUN2 expression in melanoma cells. Subsequently, knockdown of NSUN2 suppressed B16 cell proliferation and migration. RNA-Seq results illuminated that DNA replication, cell proliferation and cell cycle pathways were enriched, and genes promoting these pathways were reduced after knocking down Nsun2. Dual-luciferase reporter assays showed that 1,25(OH)2D3 downregulated reporter gene expression was controlled by the Nsun2 promoter. The results suggest that 1,25(OH)2D3 binds to the vitamin D response element located upstream of the Nsun2 promoter to downregulate Nsun2 transcription activity and then affects the gene expression pattern related to cell proliferation and the cell cycle, thereby restraining B16 cell proliferation and migration.

MiR-5195-3p targets the PCBP2/PI3K/AKT pathway to inhibit melanoma cell proliferation and migration

Although miR-5195-3p has been acknowledged for its tumor suppressor role in diverse cancer categories, its precise functions and mechanisms concerning melanoma have not been comprehensively elucidated. In this study, we employed quantitative reverse transcription PCR, Western blot analysis, and immunohistochemistry staining to investigate the expression patterns of miR-5195-3p and poly (rC) binding protein 2 (PCBP2) in melanoma tissues compared to adjacent tissues. Our findings revealed downregulation of miR-5195-3p and upregulation of PCBP2 in melanoma tissues. Through the implementation of a luciferase reporter assay, we successfully identified PCBP2 as a newly discovered target of miR-5195-3p in melanoma cells. Enforced expression of miR-5195-3p via mimics inhibited cell proliferation and migration in A375 and A2058 cells, as demonstrated by CCK-8 and transwell migration assays. In melanoma cells, reintroduction of PCBP2 partially reversed the inhibitory effects of miR-5195-3p overexpression. Treatment with LY294002, an inhibitor of the PI3K/AKT signaling pathway, also reversed the effects of PCBP2 in melanoma cells. Furthermore, our results suggest that miR-5195-3p inhibits the activation of the PI3K/AKT signaling pathway in melanoma by inhibiting PCBP2. In conclusion, our research has identified the miR-5195-3p targeting of the PCBP2-mediated PI3K/AKT signaling pathway as a potential therapeutic target for melanoma treatment.

The anti-cancer effect and mechanism of animal scale-derived extract on malignant melanoma cells

Melanoma is a type of cancer with abnormal proliferation of melanocytes and is one of the most diagnosed cancer types. In traditional Chinese medicine, pangolin scales have been used to treat various diseases, including human cancers. However, its efficacy has not been scientifically proven. Here we studied the anticancer effect and mechanism of pangolin scale extract (PSE) on melanoma cell lines using scientific approaches. Our cell viability assay shows that PSE exhibits up to approximately 50–80% inhibition on SK-MEL-103 and A375 melanoma cell lines. Mechanically, PSE inhibits melanoma cell proliferation, migration, and causes changes in cell morphology. The apoptosis assay showed a significant chromosomal condensation inside the PSE-treated melanoma cells. The sequencing and analysis of A375 melanoma cell transcriptomes revealed 3077 differentially expressed genes in the 6 h treatment group and 8027 differentially expressed genes in the 72 h treatment group. Transcriptome analysis suggests that PSE may cause cell cycle arrest in melanoma cells and promote apoptosis mainly by up-regulating the p53 signaling pathway and down-regulating the PI3K-Akt signaling pathway. In this study, the anticancer effect of PSE was demonstrated by molecular biological means. PSE shows a significant inhibition effect on melanoma cell proliferation and cell migration in vitro, causes cell cycle arrest and promotes apoptosis through p53 and PI3K-AKT pathways. This study provides better insights into the anti-cancer efficacy and underlying mechanism of PSE and a theoretical basis for mining anticancer compounds or the development of new treatments for melanoma in the future. It is worth noting that this study does not advocate the use of the pangolin scale for disease treatment, but only to confirm its usefulness from a scientific research perspective and to encourage subsequent research around the development of active compounds to replace pangolin scales to achieve the conservation of this endangered species.

A novel mitochondria-targeting compound exerts therapeutic effects against melanoma by inducing mitochondria-mediated apoptosis and autophagy in vitro and in vivo.

Melanoma is the most invasive skin cancer, with a high mortality rate. However, existing therapeutic drugs have side effects, low reactivity, and lead to drug resistance. As the power source in cells, mitochondria play an important role in the survival of cancer cells and are an important target for tumor therapy. This study aimed to develop a new anti-melanoma compound that targets mitochondria, evaluate its effect on the proliferation and metastasis of melanoma cells, and explore its mechanism of action. The novel mitochondria-targeting compound, SCZ0148, was synthesized by modifying the structure of cyanine. Then, A375 and B16 cells were incubated with different concentrations of SCZ0148, and different doses of SCZ0148 were administered to A375 and B16 xenograft zebrafish. The results showed that SCZ0148 targeted mitochondria, had dose- and time-dependent effects on the proliferation of melanoma cell lines, and had no obvious side effects on normal cells. In addition, SCZ0148 induced melanoma cell apoptosis through the reactive oxygen species-mediated mitochondrial pathway of apoptosis and promoted autophagy. SCZ0148 significantly inhibited the migration of melanoma cells via a matrix metalloprotein 9-mediated pathway. Similarly, SCZ0148 inhibited melanoma cell proliferation in a concentration-dependent manner in vivo. In summary, SCZ0148 may be a novel anti-melanoma compound that targets mitochondria.

Combination of niclosamide and quinacrine inactivates Akt/HK2/Cyclin D1 axis mediated by glucose deprivation towards the inhibition of melanoma cell proliferation

Malignant melanoma is one of the most aggressive and lethal skin cancer. At present, the treatment methods for melanoma have shortcomings. Glucose is the primary energy source of cancer cells. However, it is unclear whether glucose deprivation can be used to treat melanoma. Herein, we first found glucose played an essential role in melanoma proliferation. We then further found a drug combination of niclosamide and quinacrine could inhibit melanoma proliferation and glucose intake. Thirdly, we revealed the mechanism of anti-melanoma effect of the drug combination, which suppressed the Akt pathway. In addition, the first-rate limiting enzyme HK2 of glucose metabolism was inhibited. This work also disclosed that the decrease of HK2 inhibited cyclin D1 by reducing the activity of transcription factor E2F3, which further suppressed the proliferation of melanoma cells. The drug combination treatment also resulted in significant tumor regression in the absence of obvious morphologic changes in primary organ in vivo. In summary, our study demonstrated that the drug combination treatment created glucose deprivation to inactive the Akt/HK2/cyclin D1 axis, thereby inhibited the proliferation of melanoma cells, providing a potential anti-melanoma strategy.

CDCA2 promotes melanoma progression by inhibiting ubiquitin-mediated degradation of Aurora kinase A

BackgroundMalignant melanoma is one of the most aggressive types of malignant skin cancer. CDCA2 is of great significance in many tumours, but its role in melanoma is unclear. MethodsCDCA2 expression in melanoma samples and benign melanocytic naevus tissues was detected by GeneChip and bioinformatics analysis as well as immunohistochemistry. The gene expression in melanoma cells was detected by quantitative PCR detecting system and Western blot. Melanoma models with gene knockdown or overexpression were constructed in vitro, and the effects of gene knockdown or overexpression on melanoma cell phenotype and tumour growth were evaluated by celigo cell counting, transwell, wound healing, flow cytometry and subcutaneous nude mouse tumour models. GeneChip primeview, Ingenuity pathway analysis and bioinformatics analysis combined with co-immunoprecipitation, protein stability experiments and ubiquitination analysis were performed to demonstrate the downstream genes and regulatory mechanism of CDCA2. ResultsCDCA2 was highly expressed in melanoma tissues, and CDCA2 level was positively correlated with tumour stage and poor prognosis. CDCA2 downregulation significantly reduced cell migration and proliferation by inducing G1/S phase arrest and apoptosis. CDCA2 knockdown suppressed tumour growth and Ki67 expression in vivo. Mechanistically, CDCA2 inhibited ubiquitin-dependent Aurora kinase A (AURKA) protein degradation by acting on SMAD specific E3 ubiquitin protein ligase 1. AURKA downregulation inhibited melanoma cell proliferation and migration and promoted apoptosis. High expression of AURKA implied poor survival in melanoma patients. Moreover, AURKA knockdown constricted CDCA2 overexpression-induced proliferation and migration. ConclusionCDCA2, which was upregulated in melanoma, enhanced AURKA protein stability by inhibiting SMAD specific E3 ubiquitin protein ligase 1-mediated AURKA ubiquitination, thus playing a carcinogenic role in melanoma progression.

Abstract LB013: Integrated in vivo functional screens and multi-omics analyses identify alpha-2,3-sialylation as essential for melanoma survival

Abstract Glycosylation is a hallmark of cancer biology, and altered glycosylation influences multiple facets of melanoma growth and progression. To identify glycosyltransferases, glycans, and glycoproteins essential for melanoma survival, we conducted an in vivo growth screen with a pooled shRNA library of glycosyltransferases, lectin microarray profiling of benign nevi and melanoma patient samples, and mass spectrometry-based glycoproteomics. We found that alpha-2,3 sialyltransferases ST3GAL1 and ST3GAL2 and corresponding alpha-2,3-linked sialosides are upregulated in melanoma compared to nevi and are essential for melanoma growth in vivo and in vitro. Glycoproteomics revealed that glycoprotein targets of ST3GAL1 and ST3GAL2 are enriched in transmembrane proteins involved in growth signaling, including the amino acid transporter Solute Carrier Family 3 Member 2 (SLC3A2/CD98hc). CD98hc suppression mimicked the effect of ST3GAL1 and ST3GAL2 silencing, inhibiting melanoma cell proliferation. We found that both CD98hc protein stability and its pro-survival effect in melanoma are dependent upon alpha–2,3 sialylation mediated by ST3GAL1 and ST3GAL2. In summary, our studies reveal that alpha-2,3-sialosides functionally contribute to melanoma maintenance, supporting ST3GAL1 and ST3GAL2 as novel therapeutic targets in these tumors. Citation Format: Praveen Agrawal. Integrated in vivo functional screens and multi-omics analyses identify alpha-2,3-sialylation as essential for melanoma survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB013.

HOXB9 a miR-122–5p regulated gene, suppressed the anticancer effects of brusatol by upregulating SCD1 expression in melanoma

Brusatol (Bru), a Chinese medicine Brucea javanica extract, has a variety of antitumour effects. However, its role and underlying mechanism in melanoma have not been fully elucidated. In this study, we found that brusatol inhibited melanoma cell proliferation and migration and promoted cell apoptosis in vitro, in addition to suppressing melanoma cell tumorigenesis in vivo. Further studies on the mechanism revealed that brusatol significantly downregulated the expression of stearoyl-CoA desaturase 1 (SCD1). Increased SCD1 expression could impair the antitumour effects of brusatol on melanoma cells. Subsequently, we found that HOXB9, an important transcription factor, was directly bound to the promoter of SCD1, facilitating its transcription. Overexpression of HOXB9 inhibited brusatol-induced SCD1 reduction and promoted cell survival. Furthermore, our results revealed that miR-122–5p was significantly increased in response to brusatol treatment and led to a decrease in HOXB9 in melanoma. Collectively, our data suggested that the miR-122–5p/HOXB9/SCD1 axis might play an important role in the antitumour effects of brusatol and that brusatol might have potential clinical implications in melanoma therapy.

MAGOH and MAGOHB Knockdown in Melanoma Cells Decreases Nonsense-Mediated Decay Activity and Promotes Apoptosis via Upregulation of GADD45A

Cutaneous malignant melanoma is a highly proliferative and aggressive skin cancer with a steadily increasing incidence and a low long-term survival rate after metastatic progression. The protein MAGOH and its highly identical homologue MAGOHB are core components of the exon junction complex (EJC), which regulates splicing, stability and translation of mRNAs. The EJC, and especially MAGOH, has been shown to be involved in the development and progression of several cancers. In melanoma, the expression and function of both homologues remain essentially unexplored. This study identifies high MAGOH and MAGOHB protein expression in cutaneous melanoma cell lines and patient derived tissue samples. An siRNA-mediated knockdown of MAGOH significantly inhibits melanoma cell proliferation. The loss of MAGOH does not affect cell cycle progression, but induces apoptosis, an effect that is enhanced by a simultaneous knockdown of MAGOH and MAGOHB. MAGOH and MAGOHB do not influence the expression of the pro-apoptotic protein Bcl-XS or exon skipping. However, the knockdown of MAGOH and MAGOHB strongly decreases nonsense-mediated decay (NMD) activity, leading to an upregulation of the pro-apoptotic protein GADD45A. In conclusion, simultaneous inhibition of MAGOH and MAGOHB expression substantially affects cell survival, indicating both MAGOH homologues as promising new targets for the treatment of melanoma.

KCNQ1OT1 sponges miR-34a to promote malignant progression of malignant melanoma via upregulation of the STAT3/PD-L1 axis.

Malignant melanoma is a leading cause of skin cancer-related death. In over 30% of cases, the melanoma is invasive and has a metastatic phenotype. KCNQ1 overlapping transcript 1 (KCNQ1OT1) was previously identified as an oncogenic long noncoding RNA (lncRNA). Our study intends to uncover the mechanism of KCNQ1OT1 functioning in melanoma. qRT-PCR, immunohistochemical analysis, and Western blotting were used to investigate mechanisms of the lncRNA KCNQ1OT1, on its downstream genes in melanoma tissues, cells as well as the impact on CD8+ T cells. Proliferation, apoptosis, and migration/invasion were assessed in melanoma cells to evaluate the effects of KCNQ1OT1, miR-34a, and signal transducer and activator of transcription 3 (STAT3). The RNA interactions were determined by dual-luciferase reporter, and melanoma cells were co-cultured with CD8+ T cells to study immune evasion. A lactate dehydrogenase (LDH) cytotoxicity assay was used to investigate the cytotoxicity of CD8+ T cells toward melanoma cells. The in vivo tumorigenic potential of KCNQ1OT1 was defined using xenograft models. KCNQ1OT1 was upregulated in melanoma tissues leading to a poor prognosis, and knocking down it inhibited melanoma cell proliferation, migration, and invasion. KCNQ1OT1 regulated the progression of the melanoma via its action as a miR-34a sponge. STAT3 was found to be a downstream target of miR-34a, resulting in transcriptional regulation of Programmed cell death 1 ligand 1 (PD-L1). KCNQ1OT1 regulated STAT3 by targeting miR-34a. Knockdown of KCNQ1OT1 reduced PD-L1 level, enhanced CD8+ T cell cytotoxicity, and proliferation and inhibited apoptosis of CD8+ T cells. Melanoma cells overexpressed KCNQ1OT1, which influenced the miR-34a/STAT3 axis, to promote proliferation, migration, and invasion of melanoma cells. In addition, KCNQ1OT1 inhibited CD8+ T cell function, also via the miR-34a/STAT3/PD-L1 axis, thus promoting immune evasion of melanoma cells. The current findings expose a potential therapeutic target of melanoma.

Upregulation LncRNA MEG3 expression suppresses proliferation and metastasis in melanoma via miR-208/SOX4.

Long non-coding RNAs (lncRNAs) regulate gene expression and play a significant role in cancer progression. Previously, downregulation of lncRNA MEG3 was shown to associate with poor clinical outcomes in melanoma patients. The basis for this association has not been described and the aims of this study were to identify a role for lncRNA MEG3 in melanoma and to describe its regulatory mechanism of action. RT-qPCR was used to detect lncRNA MEG3 expression in melanoma cells and tissues. Luciferase reporter assays were used to identify lncRNA MEG3 downstream targets. Melanoma cells were transfected with various expression vectors and these transfected cells were assessed for; migration, colony formation, proliferation, in vivo tumorigenesis, and metastatic potential. Melanoma cell lines were found to be sensitive to lncRNA MEG3 expression levels and overexpression was found to inhibit melanoma cell proliferation and invasion, both in vitro and in vivo. Luciferase reporter assays identified miR-208 and SOX4 as downstream targets of lncRNA MEG3. Overexpression of miR-208 and silencing of SOX4 rescued invasion and proliferation by cells that overexpressed lncRNA MEG3. Moreover, lncRNA MEG3 inhibited cancer stem cell differentiation and suppressed melanoma progression and metastasis through inhibition of miR-208 by SOX4.