Melanoma Cell Migration Research Articles

Abstract 3186: Keratinocyte desmosomal cadherin Desmoglein 1 as a mediator and target of paracrine signaling in the melanoma niche

Abstract Melanoma is an aggressive cancer arising from the transformation of melanocytes residing in the basal layer of the epidermis, where they are in direct contact with surrounding keratinocytes. The role of keratinocytes in shaping the melanoma tumor microenvironment, however, remains understudied. We previously showed that the keratinocyte-specific desmosomal cadherin, desmoglein 1 (Dsg1) is reduced in response to acute UV exposure. Here, we show that Dsg1 protein is reduced in keratinocytes surrounding melanoma lesions but is unchanged in keratinocytes adjacent to benign nevi. As Dsg1 reduction by UV exposure is transient, we hypothesized that the persistent loss of Dsg1 observed in the melanoma tumor niche is due to melanoma-keratinocyte paracrine crosstalk. To address this idea, keratinocytes were cultured in conditioned media from melanocytes or melanoma cells. Melanoma conditioned media reduced keratinocyte Dsg1 mRNA and protein levels, suggesting that Dsg1 downregulation in keratinocytes is maintained by melanoma cells post-transformation. Keratinocytes in melanoma conditioned media exhibited reduced Grhl1 (Grainyhead like 1), a transcriptional activator of Dsg1, and increased Snai2, a repressor of Grhl1 expression and epidermal differentiation. These data support the idea that increased Snai2 stimulated by factors secreted by melanoma cells reduces keratinocyte Dsg1 mRNA expression by repressing Grhl1. To determine the impact of keratinocyte Dsg1 loss on melanoma cell behavior, melanoma cells were grown in conditioned media from control or Dsg1-deficient keratinocytes. Melanoma cell migration increased in a trans-well assay when melanoma cells were grown in conditioned media from Dsg1-deficient keratinocytes compared to from control keratinocytes. Supporting the in vivo importance of this finding, we found a significant, negative correlation between keratinocyte Dsg1 expression and melanoma cell movement within the melanoma tumor niche in patient samples. Downregulation of keratinocyte Dsg1 by melanoma cell conditioned media increased Erk1/2 signaling upstream of pro-migratory CXCL1 production, while pharmacological inhibition of keratinocyte Erk1/2 abrogated this effect. Furthermore, inhibition of the CXCL1 receptor, CXCR2 on melanoma cells decreased migration in melanoma cells grown in Dsg1-depleted keratinocyte conditioned media. Together, these data support the idea that paracrine crosstalk between melanoma cells and keratinocytes resulting in chronic keratinocyte Dsg1 reduction is important for melanoma progression and melanoma cell movement in the early stages of metastasis. Citation Format: Hope E. Burks, Christopher Arnette, Jennifer Koetsier, Joshua Broussard, Quinn Roth-Carter, Pedram Gerami, Jodi Johnson, Kathleen Green. Keratinocyte desmosomal cadherin Desmoglein 1 as a mediator and target of paracrine signaling in the melanoma niche [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 3186.

Abstract 1868: Targeting adenosine deaminase enhances anticancer effects of 3'-deoxyadenosine in uveal melanoma

Abstract Uveal melanoma, which arises from melanocytes located in the uveal tract, is one of the most common primary intraocular cancers. GNAQ and GNA11 mutations, which are detected most of primary uveal melanoma, activate MAPK signaling pathway, increasing cancer cell proliferation, tumor progression and growth. BAP1 mutation is highly associated with liver metastasis and death. However, there are no good therapeutic compounds for uveal melanoma treatment in clinic. 3’-deoxyadenosine (cordycepin) is an adenosine analog which inhibits growth of several types of cancer. However, its anticancer effects have been showed in limited cancer cell lines. In addition, action mechanism of 3’deoxyadenosin is poorly understood. In this study, we found different adenosine deaminase (ADA) expression or activity affects different anticancer results in uveal melanoma. Inhibition of ADA using either siRNA or pharmacologic approaches sensitized tumors to 3’-deoxyadenosine in vitro and in vivo, resulting in increased apoptosis, reduced clonogenic capacity and slower migration of uveal melanoma cells in vitro. These potent ADA-dependent anti-tumor effects were also seen in various cancer cell lines. Therefore, we suggest the potential of adenosine deaminase (ADA) as both a biomarker predicting response to 3’-deoxyadenosine, and a target for combination therapy. Citation Format: Su-Chan Lee, Lujain Alaali, HyukJean Kwon, Mohammed Rigi, Charles G. Eberhart. Targeting adenosine deaminase enhances anticancer effects of 3'-deoxyadenosine in uveal 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 1868.

Abstract 2443: Knocking out SNCA in SKMEL28 melanoma cells suppresses EMT and prevents invasion

Abstract Malignant melanoma is one of the most aggressive and fifth most prevalent cancer among men and women. Clinical and epidemiological studies shows that Parkinson’s disease (PD) patients have higher risk of developing invasive melanoma, and, reciprocally, patients with invasive melanoma have a higher risk of developing PD. Alpha-synuclein (α-syn; SNCA), an intrinsically disordered protein expressed in neurons, is also highly expressed in malignant melanoma and is a common molecular signature between Parkinson’s disease and melanoma. We have previously reported that SK-Mel-28 SNCA-knockout (KO) cells exhibit suppressed tumor growth and proliferation in vivo and in vitro, and that putting back SNCA via lenti virus restored the phenotype to that of the control cells. The present study is designed to investigate the role of α-syn in melanoma progression in SK-MEL-28 cells in the context of metastasis and the epithelial-to-mesenchymal transition (EMT). The SK-Mel-28 SNCA-KO clones showed a significant decrease in the cell-surface protein L1CAM, a major cell adhesion molecule highly expressed in various melanoma and correlates with melanoma progression, whereas the knock in clones resembled the control. Migration and invasion assay were performed using transwell chambers showed a profound migratory and invasive defect in SNCA-KO clones. To explore further whether this is a proliferative or migratory defect, a single cell phagokinetic motility assay was performed with colloidal gold which showed a marked decrease in cell motility in knockout clones compared to control and knock in clones, which was consistent with our migration and invasion experiments. To determine the proteins involved in the inhibition of invasion mediated by α-syn, we immunoblotted for the mesenchymal markers N-cadherin and vimentin, and these were significantly downregulated in the SNCA-KO clones. These data reinforce that knocking out SNCA impedes melanoma cell migration and invasion in SK-Mel- 28 cell line, implying that α-syn might be a novel therapeutic target for malignant melanoma. Citation Format: Nithya Gajendran, Santhanasabapathy Rajasekaran, Nirjhar M. Aloy, Sahar Shekoohi, Stephan N. Witt. Knocking out SNCA in SKMEL28 melanoma cells suppresses EMT and prevents invasion [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 2443.

MiR-182 Enhanced the Proliferation, Migration and Invasion of Malignant Melanoma Cells by Modulating Zinc Finger Protein 36, C3H Type-Like 1 (ZFP36L1)

Background and Objectives: Malignant melanoma (MM) is the most aggressive skin cancer based on the enhanced proliferative and metastatic ability of melanocytes. Recently, some studies have revealed that higher levels of ZFP36L1 suppressed the proliferation, migration and invasion of multiple types of cancer cells. By searching the database, it was found that miR-182 has the potential to target ZFP36L1 and reduce ZFP36L1 expression. However, whether the possible effects of miR-182 on the development of malignant melanoma and the underlying mechanism remains largely unclear. Materials and Methods: ZFP36L1 and miR-182 expression levels in malignant melanoma cell lines were examined by performing western blotting and RT-qPCR. Then, CCK-8, wound healing and transwell assays were exploited to determine the potential roles of ZFP36L1 and miR-182in MM cells, respectively. Results: In this study, aberrant downregulation of ZFP36L1 was observed in MM cell lines (WM-115, A375). ZFP36L1 overexpression repressed the proliferation, migration and invasion of MM cells. Besides, bioinformatics analysis and luciferase reporter assay confirmed that miR-182 directly target ZFP36L1 and suppressed ZFP36L1 expression. What’s more, miR-182 weakened the inhibitory efficacy of ZFP36L1 on the proliferation, migration and invasion of these two cells. Conclusions: The study validated that miR-182 promoted the proliferation, migration and invasion of malignant melanoma cells through the repression of ZFP36L1.

Inhibition of USP14 enhances anti-tumor effect in vemurafenib-resistant melanoma by regulation of Skp2.

The mutation of BRAF V600E often occurred in melanoma and results in tumorigenesis. BRAF mutation drives hyperactivation of the RAF-MAPK-ERK pathway. The acquired drug resistance upon prolonged use of BRAF inhibitors (such as vemurafenib) still remains the main obstacle. Previously, we have found that E3 ligase Skp2 over-expresses vemurafenib-resistant melanoma cells, and knockdown of Skp2 enhances the anti-tumor effect of vemurafenib. Interestingly, the literature has reported that the selective USP14/UCHL5 inhibitor b-AP15 displays great potential in melanoma therapy; however, the molecular mechanism still remains unknown. In vitro, the effect of the combination regimen of vemurafenib (Vem, PLX4032) and b-AP15 on vem-sensitive and vem-resistant melanoma has been investigated by wound healing, colony formation, transwell invasion assay, flow cytometry, lysosome staining, and ROS detection. In vivo, the combination effect on vem-resistant melanoma has been evaluated with a nude mice xenograft tumor model. GST-pulldown and co-immunoprecipitation (co-IP) assays have been applied to investigate the interactions between USP14, UCHL5, and Skp2. Cycloheximide (CHX) assay and ubiquitination assays have been used to explore the effect of USP14 on Skp2 protein half-life and ubiquitination status. In the present study, we have revealed that repression of USP14 sensitizes vemurafenib resistance in melanoma through a previously unappreciated mechanism that USP14 but not UCHL5 stabilizes Skp2, blocking its ubiquitination. K119 on Skp2 is required for USP14-mediated deubiquitination and stabilization of Skp2. Furthermore, the mutated catalytic activity amino acid cysteine (C) 114 on USP14 abrogates stabilization of Skp2. Stabilization of Skp2 is required for USP14 to negatively regulate autophagy. The combination regimen of Skp2 inhibitor vemurafenib and USP14/UCHL5 inhibitor b-AP15 dramatically inhibits cell viability, migration, invasion, and colony formation in vemurafenib-sensitive and vemurafenib-resistant melanoma. Vemurafenib and b-AP15 hold cells in the S phase thus leading to apoptosis as well as the formation of the autophagic vacuole in vemurafenib-resistant SKMEL28 cells. The enhanced proliferation effect of USP14 and Skp2 is mainly due to a more effective reduction of cell apoptosis and autophagy. Further evaluation of various protein alterations has revealed that the increased expression of cleaved-PARP, LC3, and decreased Ki67 are more obvious in the combination of vemurafenib and b-AP15 treatment than those in single-drug treatment. Moreover, the co-treatment of vemurafenib and b-AP15 dramatically inhibits the growth of vemurafenib-resistant melanoma xenograft in vivo. Collectively, our findings have demonstrated that the combination of Skp2 inhibitor and USP14 inhibitor provides a new solution for the treatment of BRAF inhibitor resistance melanoma.

EIF6 as a Promising Diagnostic and Prognostic Biomarker for Poorer Survival of Cutaneous Melanoma.

BackgroundSkin cutaneous melanoma (SKCM) is the deadliest skin cancer and has the most rapidly increasing incidences among all cancer types. Previous research elucidated that melanoma can only be successfully treated with surgical abscission in the early stage. Therefore, reliable and specific biomarkers are crucial to melanoma diagnosis since it often looks like nevi in the clinical manifestations. Moreover, identifying key genes contributing to melanoma progression is also highly regarded as a potential strategy for melanoma therapy. In this respect, translation initiator eIF6 has been proved as a pro-tumor factor in several cancers. However, the role of eIF6 in the skin cutaneous melanoma progression and its potential as a prognostic marker is still unexplored.MethodsThe immunochemical analysis of clinical specimens were served to assess eIF6 expression levels. Gene Expression Profiling Interactive Analysis (GEPIA) database consultations allowed us to find the survival rates of the eIF6-overexpressed patients. eIF6 cellular effects were evaluated in an eIF6-overexpressed A375 cell line constructed with a lentivirus. The analysis of down-stream effectors or pathways was conducted using C-Bioportal and STRING databases.ResultsOur results revealed that eIF6 was highly over-expressed in melanomas compared to normal skin specimens, and thus the abnormally high level of eIF6 can be a diagnostic marker for melanoma. The in silica analysis indicated that patients with eIF6 over-expression had lower survival rates than that low-expression in SKCM. Meanwhile, similar results also could be found in the other four types of cancers. In vitro, over-expression of eIF6 increased the proliferation and migration of melanoma cells. Correspondingly, pan-cancer clustering analysis indicated the expression level of intermediate filament proteins was correlated with that of eIF6 expression. In our study, all over-expressed keratin proteins, in accordance with over-expressed eIF6, had a negative correlation with melanoma prognosis. Moreover, the decreased methylation level of keratin genes suggested a new potential regulation mode of eIF6.ConclusionsThe up-regulated eIF6 could be a potential diagnostic and prognostic biomarker of melanoma. This study also provides insights into the potential role of eIF6 in pan-cancer epigenetic regulation.

Synthesis, Screening and Characterization of Novel Potent Arp2/3 Inhibitory Compounds Analogous to CK-666

Branched actin networks polymerized by the Actin-related protein 2 and 3 (Arp2/3) complex play key roles in force generation and membrane remodeling. These networks are particularly important for cell migration, where they drive membrane protrusions of lamellipodia. Several Arp2/3 inhibitory compounds have been identified. Among them, the most widely used is CK-666 (2-Fluoro-N-[2-(2-methyl-1H-indol-3-yl)ethyl]-benzamide), whose mode of action is to prevent Arp2/3 from reaching its active conformation. Here 74 compounds structurally related to CK-666 were screened using a variety of assays. The primary screen involved EdU (5-ethynyl-2′-deoxyuridine) incorporation in untransformed MCF10A cells. The resulting nine positive hits were all blocking lamellipodial protrusions and cell migration in B16-F1 melanoma cells in secondary screens, showing that cell cycle progression can be a useful read-out of Arp2/3 activity. Selected compounds were also characterized on sea urchin embryos, where Arp2/3 inhibition yields specific phenotypes such as the lack of triradiate spicules and inhibition of archenteron elongation. Several compounds were filtered out due to their toxicity in cell cultures or on sea urchin development. Two CK-666 analogs, 59 (N-{2-[5-(Benzyloxy)-2-methyl-1H-indol-3-yl] ethyl}-3-bromobenzamide) and 69 (2,4-Dichloro-N-[2-(7-chloro-2-methyl-1H-indol-3-yl) ethyl]-5-[(dimethylamino) sulfonyl] benzamide), were active in all assays and significantly more efficient in vivo than CK-666. These best hits with increased in vivo potency were, however, slightly less efficient in vitro than CK-666 in the classical pyrene-actin assay. Induced-fit docking of selected compounds and their possible metabolites revealed interaction with Arp2/3 that suppresses Arp2/3 activation. The data obtained in our screening validated the applicability of original assays for Arp2/3 activity. Several previously unexplored CK-666 structural analogs were found to suppress Arp2/3 activation, and two of them were identified as Arp2/3 inhibitors with improved in vivo efficiency.

Arginyl-tRNA-protein transferase 1 (ATE1) promotes melanoma cell growth and migration.

Arginyl-tRNA-protein transferase 1 (ATE1) catalyses N-terminal protein arginylation, a post-translational modification implicated in cell migration, invasion and the cellular stress response. Herein, we report that ATE1 is overexpressed in NRAS-mutant melanomas, while it is downregulated in BRAF-mutant melanomas. ATE1 expression was higher in metastatic tumours, compared with primary tumours. Consistent with these findings, ATE1 depletion reduced melanoma cell viability, migration and colony formation. Reduced ATE1 expression also affected cell responses to mTOR and MEK inhibitors and to serum deprivation. Among putative ATE1 substrates is the tumour suppressor AXIN1, pointing to the possibility that ATE1 may fine-tune AXIN1 function in melanoma. Our findings highlight an unexpected role for ATE1 in melanoma cell aggressiveness and suggest that ATE1 constitutes a potential new therapeutic target.

Hsa_circ_0103232 promotes melanoma cells proliferation and invasion via targeting miR-661/RAB3D

ABSTRACT Little is known about the role of hsa_circ_0103232 in melanoma. This study researched the role of hsa_circ_0103232 in melanoma progression. Hsa_circ_0103232 expression in clinical tissues of melanoma patients and melanoma cells was detected by qRT-PCR. Hsa_circ_0103232 localization in melanoma cells was visualized by fluorescence in situ hybridization. Hsa_circ_0103232 effect on melanoma cells viability, proliferation, migration, and invasion was explored by cell counting kit-8 (CCK-8) assay, Edu experiment, wound healing assay, and Transwell experiment. RNA pull-down assay and dual-luciferase reporter gene assay were performed to verify the binding of hsa_circ_0103232 with miR-661, and the binding of miR-661 and RAB3D. Xenograft tumor models were constructed. Western blot and immunohistochemistry were used for protein expression detection. Hsa_circ_0103232 expression was increased in melanoma patients, indicating lower overall survival. Hsa_circ_0103232 was mainly expressed in the cytoplasm of melanoma cells. Silencing hsa_circ_0103232 suppressed melanoma cell viability, proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT) (P < 0.01). Hsa_circ_0103232 functioned as a sponge of miR-661 to increase RAB3D expression. miR-661 overexpression partially reversed hsa_circ_0103232 promoting effect on melanoma cells viability, proliferation, migration, invasion, and EMT (P < 0.01). In melanoma patients, hsa_circ_0103232 expression was negatively correlated with miR-661 and positively correlated with RAB3D. Silencing hsa_circ_0103232 suppressed melanoma cell growth in vivo and Ki67 and RAB3D expression in xenograft tumors (P < 0.01). Hsa_circ_0103232 is a tumor promoter in melanoma to enhance malignant phenotype and growth in vivo via sponging miR-661/RAB3D. Hsa_circ_0103232 may be a novel target for melanoma treatment.

Curcumin Inhibits the Growth and Metastasis of Melanoma via miR-222-3p/SOX10/Notch Axis

Background The aim of this study was to investigate the effect of curcumin on melanoma and its mechanism. Methods Curcumin (0, 0.125, 0.25, or 0.5 mg/ml) was utilized to treat A375 and HT144 cell lines. The MTT analysis was used to confirm the proliferation ability. Wound healing and transwell analysis showed the migration and invasion ability. Immunofluorescence assay was used to demonstrate the effect of curcumin on SOX10 expression. Multiple bioinformatic analysis to confirm the SOX10 associated miRNA. The correlation of miR-222-3p and SOX10 was detected by Luciferase reporter assays. qRT-PCR showed the miR-222-3p level. Western blot analyzed the expression of SOX10, Notch1, and HES1 in melanoma cell treated with or without miR-222-3p inhibitor. Results Curcumin could inhibit the proliferation, migration, and invasion of melanoma cells. Furthermore, curcumin repress the expression of SOX10, Notch1, and HES-1, and increase the expression of miR-222-3p. And the miR-222-3p could directly target to SOX10 mRNA to inhibit its expression. In addition, inhibition of miR-222-3p expression reversed the inhibitory effect of curcumin the growth of melanoma cells. Conclusion Curcumin enhances the miR-222-3p level to reduce SOX10 expression, and ultimately inactivates the Notch pathway in repressing melanoma proliferation, migration, and invasion.

Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure.

Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we identify the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, we show that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, our data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure.

Proliferation and Invasion of Melanoma Are Suppressed by a Plant Protease Inhibitor, Leading to Downregulation of Survival/Death-Related Proteins.

Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death (hallmarks of cancer) in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment.

A novel ribosomal protein S6 kinase 2 inhibitor attenuates the malignant phenotype of cutaneous malignant melanoma cells by inducing cell cycle arrest and apoptosis

ABSTRACT Malignant melanoma (MM) is a highly life-threatening tumor causing the majority of the cutaneous cancer-related deaths. Previously, ribosomal protein S6 kinase 2 (RSK2), the downstream effector of the MAPK pathway, represents a therapeutic target in melanoma. AE007 is discovered as a targeted RSK2 inhibitor, and subsequent results showed that AE007 inhibits RSK2 by directly binding to its protein kinase domain. AE007 causes cell cycle arrest and cellular apoptosis, thereby dramatically inhibiting proliferation, migration, and invasion of melanoma cells. Nevertheless, melanocytes and keratinocytes are not affected by this compound. In addition, suppression of RSK2 abrogates the inhibitory effect of AE007 on melanoma cell proliferation. AE007 treatment significantly inhibits the expression of Cyclin D1, Cyclin B1, CDK2, and Bcl-2, while raises the cleavage of PARP. Moreover, RNA sequencing results show that AE007 treatment can affect the genes expression profile, including the expression of cell cycle and DNA replication genes. In conclusion, AE007 is a promising melanoma therapeutic agent by targeting RSK2.

Statement of Retraction: Overexpressed VEPH1 inhibits epithelial-mesenchymal transition, invasion, and migration of human cutaneous melanoma cells through inactivating the TGF-β signaling pathway

This article refers to:RETRACTED ARTICLE: Overexpressed VEPH1 inhibits epithelial-mesenchymal transition, invasion, and migration of human cutaneous melanoma cells through inactivating the TGF-β signaling pathway

Emerin regulation of nuclear stiffness is required for fast amoeboid migration in confined environments.

When metastasizing, tumor cells must traverse environments with diverse physicochemical properties. Recently, the cell nucleus has emerged as a major regulator of the transition from mesenchymal to fast amoeboid (leader bleb-based) migration. Here, we demonstrate that increasing nuclear stiffness through elevating lamin A, inhibits fast amoeboid migration in melanoma cells. Importantly, nuclei may respond to force through stiffening. A key factor in this process is the inner nuclear membrane (INM) protein emerin. Accordingly, we determined the role of emerin in regulating fast amoeboid migration. Strikingly, we found that both the up- and downregulation of emerin results in an inhibition of fast amoeboid migration. However, when key Src phosphorylation sites were removed, upregulation of emerin no longer inhibited fast amoeboid migration. Interestingly, as measured by using a Src biosensor, activity of Src was low in cells within a confined environment. Thus, the fast amoeboid migration of melanoma cells depends on the precise calibration of emerin activity.

KLF10 upregulates ACSM3 via the PI3K/Akt signaling pathway to inhibit the malignant progression of melanoma.

Malignant melanoma is a type of skin cancer caused by mutations in the DNA of melanocytes. Melanoma is relatively rare compared with other types of skin tumors, but has a highly aggressive biological behavior and consequently, a poorer prognosis. Therefore, the present study aimed to explore the role and mechanism of Kruppel-like factor 10 (KLF10) and acyl-CoA medium-chain synthetase 3 (ACSM3) in melanoma progression. KLF10 expression in melanoma tissues was predicted using Gene Expression Profiling Interactive Analysis (GEPIA). KLF10 expression in healthy and melanoma cells was also detected using reverse transcription-quantitative PCR and western blotting. Cell transfection was performed to overexpress KLF10 or silence ACSM3. Cell viability, proliferation, migration, invasion and apoptosis were detected using Cell Counting Kit-8, colony formation, wound healing, Transwell and TUNEL assays, respectively. The activity of the ACSM3 promoter was detected using a dual-luciferase reporter assay, and the relationship between KLF10 and ACSM3 was detected using the GEPIA database and chromatin immunoprecipitation (ChIP). The results demonstrated that KLF10 expression was significantly downregulated in melanoma cells, especially in A375 cells. Compared with the Ov-NC group, KLF10 overexpression significantly inhibited the proliferation, invasion and migration of melanoma cells and promoted their apoptosis. Similar to KLF10, ACSM3 was also downregulated in A375 cells compared with that in the HEM group, and the GEPIA database analysis and ChIP assay results demonstrated that KLF10 expression was positively associated with ACSM3 expression. Furthermore, silencing ACSM3 significantly reversed the effect of KLF10 overexpression on cell proliferation, invasion and migration, and ACSM3 knockdown increased the levels of phosphorylated (p)-PI3K and p-Akt compared with the levels in the Ov-KLF10 + sh-NC group. Overall, the present study suggested that KLF10 inhibited the proliferation, invasion and migration of melanoma cells by targeting ACSM3 via the PI3K/Akt signaling pathway.

Tepotinib suppresses proliferation, invasion, migration, and promotes apoptosis of melanoma cells via inhibiting MET and PI3K/AKT signaling pathways.

Malignant melanoma seriously threatens public health and lowers the quality of life of the affected subjects. The present study was designed to explore the effects of tepotinib, a selective tyrosine kinase inhibitor of MET proto-oncogene, receptor tyrosine kinase (MET), on the progression of melanoma. Firstly, MTT assays were used to detect the proliferation of tepotinib-treated WM451 cells. The cell invasive and migratory activities were assessed using Transwell and wound healing assays, respectively. In addition, TUNEL staining was employed to determine cell apoptosis. Western blot analysis was utilized for the evaluation of the expression levels of apoptotic and epithelial-mesenchymal transition-related proteins, as well as of proteins involved in the PI3K/AKT signaling pathway. Subsequently, hepatocyte growth factor (HGF), a natural agonist of MET, was administered to WM451 cells to unravel the detailed mechanism of action of tepotinib in melanoma. The results indicated that the proliferation of WM451 cells was significantly decreased by tepotinib treatment. The inhibitory effects of tepotinib on the proliferation of WM451 cells occurred in a concentration-dependent manner. In addition, the migratory and invasive activities of WM451 cells were significantly suppressed following tepotinib treatment. It was also shown that tepotinib exhibited promotive effects on the induction of apoptosis of WM451 cells. Moreover, activation of MET and PI3K/AKT signaling pathways may be blocked by tepotinib treatment, whereas addition of HGF to the cells reversed the effects of tepotinib treatment on the malignant progression of WM451 cells. In conclusion, the data demonstrated that tepotinib suppressed the proliferation, invasion and migration of melanoma cells, whereas it could also induce their apoptosis. This evidence may provide a new perspective for the improvement of malignant melanoma.

LncRNA LNCOC1 is Upregulated in Melanoma and Serves as a Potential Regulatory Target of miR-124 to Suppress Cancer Cell Invasion and Migration

BackgroundA cascade of genes and pathways have been reported in the precise regulation of malignant melanoma (MM). Previous study has demonstrated that lncRNA LNCOC1 is an oncogenic factor in the pathogenesis and development of various cancers. The present study explored the functionalities of LNCOC1 and its interactions with miR-124 in MM.MethodsA total of 65 melanoma patients were enrolled in this study. The expression of LNCOC1 and miR-124 after cell transfection were detected by RT-qPCR. The migration rates of SK-MEL-3 and A375 cells after transient transfection with LNCOC1 expression vector and miR-124 mimic was detected by trans-well assay.ResultsLNCOC1 was accumulated to high levels in melanoma, and it was significantly correlated with the low survival rate of melanoma patients. Our bioinformatics data showed that miR-124 could target LNCOC1. Overexpression of miR-124 could downregulate LNCOC1. However, up-regulated the expression of LNCOC1 did not affect the expression of miR-124. Our correlation analysis also revealed that the expression of LNCOC1 and miR-124 were inversely correlated in both melanoma tissues and non-tumor tissues. The trans-well invasion and migration assays indicated that overexpression of miR-124 inhibited the melanoma cell invasion and migration. However, overexpression of LNCOC1 promoted melanoma cell invasion and migration.ConclusionLNCOC1 is upregulated in melanoma, which can be considered as a potential target of miR-124 in modulating melanoma cell invasion and migration. LNCOC1 may also be an interfering target of MM therapy.

C-phycocyanin decreases proliferation and migration of melanoma cells: In silico and in vitro evidences

The incidence and number of deaths caused by melanoma have been increasing in recent years, and the pigment C-phycocyanin (C-PC) appears as a possible alternative to treat this disease. So, the objective of this study was to combine in silico and in vitro analysis to understand the main anti-melanoma pathways exerted by C-PC. We evaluated the ability of C-PC to bind to the main cellular targets related in the progression of melanoma through molecular docking, and the reflection of this bind in the biological effects in the B16F10 cell line through in vitro analysis. Our results showed that C-PC was able to bind BRAF and MEK, which are related to the signal transduction pathway for proliferation and survival. There was also an interaction between C-PC and cyclin-dependent kinase 4 and 6. In vitro analysis demonstrated that C-PC decreased B16F10 cell proliferation, as observed by cell viability and mitotic index assays. C-PC also interacted with matrix metalloproteinase 2 and 9 and N-cadherin, which may have caused the decrease in cell migration observed in vitro. Besides that, C-PC interacts with VEGF, a factor responsible for regulating the proliferation and cellular invasion pathways. Finally, C-PC did not alter the cell viability of the non-tumoral melanocytes. Therefore, C-PC is a strong anti-tumor candidate for the treatment of melanoma, since it acts in different cellular pathways of melanoma, without causing damage to non-tumoral cells.

Immune Landscape and an RBM38-Associated Immune Prognostic Model with Laboratory Verification in Malignant Melanoma.

Simple SummaryThe primary treatment of malignant melanoma is a classical regimen of surgery combined with chemotherapy, targeted drugs, and immunotherapy. The purpose of this study was to explore the immune response mechanism of RNA binding protein RBM38 in the development of melanoma with the screening of effective immunodiagnostic models and targeted therapy. We found that RBM38, as an oncogene, promotes the proliferation, invasion, and migration of melanoma cells and is associated with immune infiltration and pathways. Our investigation presented the prognostic significance of RBM38-associated immune signature. In addition, this model may provide a potential strategy for improving the survival and immunotherapy of melanoma patients.Background: Current studies have revealed that RNA-binding protein RBM38 is closely related to tumor development, while its role in malignant melanoma remains unclear. Therefore, this research aimed to investigate the function of RBM38 in melanoma and the prognosis of the disease. Methods: Functional experiments (CCK-8 assay, cell colony formation, transwell cell migration/invasion experiment, wound healing assay, nude mouse tumor formation, and immunohistochemical analysis) were applied to evaluate the role of RBM38 in malignant melanoma. Immune-associated differentially expressed genes (DEGs) on RBM38 related immune pathways were comprehensively analyzed based on RNA sequencing results. Results: We found that high expression of RBM38 promoted melanoma cell proliferation, invasion, and migration, and RBM38 was associated with immune infiltration. Then, a five-gene (A2M, NAMPT, LIF, EBI3, and ERAP1) model of RBM38-associated immune DEGs was constructed and validated. Our signature showed superior prognosis capacity compared with other melanoma prognostic signatures. Moreover, the risk score of our signature was connected with the infiltration of immune cells, immune-regulatory proteins, and immunophenoscore in melanoma. Conclusions: We constructed an immune prognosis model using RBM38-related immune DEGs that may help evaluate melanoma patient prognosis and immunotherapy modalities.