Metastasis Of Melanoma Cells Research Articles

Mast cells impair melanoma cell homing and metastasis by inhibiting HMGA1 secretion.

Metastatic disease is the major cause of death from cancer. From the primary tumour, cells remotely prepare the environment of the future metastatic sites by secreted factors and extracellular vesicles. During this process, known as pre-metastatic niche formation, immune cells play a crucial role. Mast cells are haematopoietic bone marrow-derived innate immune cells whose function in lung immune response to invading tumours remains to be defined. We found reduced melanoma lung metastasis in mast cell-deficient mouse models (Wsh and MCTP5-Cre-RDTR), supporting a pro-metastatic role for mast cells in vivo. However, due to evidence pointing to their antitumorigenic role, we studied the impact of mast cells in melanoma cell function in vitro. Surprisingly, in vitro co-culture of bone-marrow-derived mast cells with melanoma cells showed that they have an intrinsic anti-metastatic activity. Mass spectrometry analysis of melanoma-mast cell co-cultures secretome showed that HMGA1 secretion by melanoma cells was significantly impaired. Consistently, HMGA1 knockdown in B16-F10 cells reduced their metastatic capacity in vivo. Importantly, analysis of HMGA1 expression in human melanoma tumours showed that metastatic tumours with high HMGA1 expression are associated with reduced overall and disease-free survival. Moreover, we show that HMGA1 is reduced in the nuclei and enriched in the cytoplasm of melanoma metastatic lesions when compared to primary tumours. These data suggest that high HMGA1 expression and secretion from melanoma cells promote metastatic behaviour. Targeting HMGA1 expression intrinsically or extrinsically by mast cells actions reduce melanoma metastasis. Our results pave the way to the use of HMGA1 as anti-metastatic target in melanoma as previously suggested in other cancer types.

Dioscin induces M1 macrophage polarization through Connexin-43 Channels in Tumor-associated-macrophages-mediated melanoma metastasis

BackgroundTumor-associated macrophages (TAMs) are important constituent parts of tumor microenvironment that connected with tumor metastasis in melanoma. Connexin 43 (Cx43) was expressed in all the immune cells which modulated different aspects of immune response. However, the concrete molecular mechanism maintains unclear. PurposeThe study aimed to find a natural drug monomer effectively reversed the polarity of tumor-associated macrophages inhibiting melanoma metastasis and improving survival time. MethodsFlow cytometry was used to determine the effects of dioscin on the macrophage phenotype. Western bolt and ELISA were performed to explore the underlying mechanism of dioscin and a co-culture experiment in vitro was applied to assess the role of dioscin on TAMs-mediated melanoma proliferation, invasion and migration. Moreover, in vivo melanoma metastasis models were established for examining effects of dioscin on TAMs-mediated melanoma metastasis. ResultsDioscin repolarized macrophages from M2 towards M1-like phenotype. Dioscin suppressed M2-like phenotype macrophages through enhanced the expression and transport function of Cx43. Furthermore, the stimulation IFN-γ/STAT1 pathway and suppression IL-4/JAK2/STAT3 pathway were major mechanism of dioscin. Importantly, dioscin suppressed Cx43G21R mutation TAMs induced proliferation, invasion, migration and metastasis of melanoma cells. It worthily noting that dioscin ameliorated tumor-associated-macrophages-mediated melanoma metastasis in vitro and vivo. ConclusionDioscin re-polarized macrophages from M2 to M1 phenotype through activation of Cx43-gap-junction-intercellular-communications (Cx43-GJs)/IFN-γ/STAT1 pathway and inhibition of Cx43-GJs/IL-4/JAK2/STAT3 suppressing migration, invasion and metastasis of melanoma, which provided a theoretical and experimental basis for treating melanoma metastasis.

A circulating subset of iNKT cells mediates antitumor and antiviral immunity.

Invariant natural killer T (iNKT) cells are a group of innate-like T lymphocytes that recognize lipid antigens. They are supposed to be tissue resident and important for systemic and local immune regulation. To investigate the heterogeneity of iNKT cells, we recharacterized iNKT cells in the thymus and peripheral tissues. iNKT cells in the thymus were divided into three subpopulations by the expression of the natural killer cell receptor CD244 and the chemokine receptor CXCR6 and designated as C0 (CD244-CXCR6-), C1 (CD244-CXCR6+), or C2 (CD244+CXCR6+) iNKT cells. The development and maturation of C2 iNKT cells from C0 iNKT cells strictly depended on IL-15 produced by thymic epithelial cells. C2 iNKT cells expressed high levels of IFN-γ and granzymes and exhibited more NK cell-like features, whereas C1 iNKT cells showed more T cell-like characteristics. C2 iNKT cells were influenced by the microbiome and aging and suppressed the expression of the autoimmune regulator AIRE in the thymus. In peripheral tissues, C2 iNKT cells were circulating that were distinct from conventional tissue-resident C1 iNKT cells. Functionally, C2 iNKT cells protected mice from the tumor metastasis of melanoma cells by enhancing antitumor immunity and promoted antiviral immune responses against influenza virus infection. Furthermore, we identified human CD244+CXCR6+ iNKT cells with high cytotoxic properties as a counterpart of mouse C2 iNKT cells. Thus, this study reveals a circulating subset of iNKT cells with NK cell-like properties distinct from conventional tissue-resident iNKT cells.

Claudin-3 inhibits tumor-induced lymphangiogenesis via regulating the PI3K signaling pathway in lymphatic endothelial cells

Claudin-3 is a tight junction protein that has often been associated with the progression and metastasis of various tumors. Here, the role of claudin-3 in tumor-induced lymphangiogenesis is investigated. We found an increased lymphangiogenesis in the B16F10 tumor in claudin-3 knockout mice, accompanied by augmented melanoma cell metastasis into sentinel lymph nodes. In vitro, the overexpression of claudin-3 on lymphatic endothelial cells inhibited tube formation by suppressing cell migration, resulting in restricted lymphangiogenesis. Further experiments showed that claudin-3 inhibited lymphatic endothelial cell migration by regulating the PI3K signaling pathway. Interestingly, the expression of claudin-3 in lymphatic endothelial cells is down-regulated by vascular endothelial growth factor C that is often present in the tumor microenvironment. This study indicates that claudin-3 plays an important role as a signaling molecule in lymphatic endothelial cell activity associated with tumor lymphangiogenesis, which may further contribute to melanoma metastasis.

Terminalia catappa leaf extracts inhibited metastasis of A2058 and A375 melanoma cells via downregulating p-Src and β-catenin pathway in vitro.

Background: Melanoma is a highly aggressive, lethal, and malignant cancer. Once diagnosed early, it can be easily removed and cured with satisfaction. Although many methods such as surgery, chemotherapy, radiotherapy, and immunotherapy have been used to treat this disease at an advanced stage, the outcomes are poor. Terminalia catappa leaves have been shown to have various biological benefits, including antitumor activity. The specific effects and molecular mechanisms of Terminalia catappa leaf in treating A2058 and A375 melanoma cells in vitro need to be clarified. Methods: The A2058 and A375 melanoma cancer cells were treated with Terminalia catappa leaf extracts, and then the effect of Terminalia catappa leaf extracts on migration and invasion was examined. The cell migration/invasion capacities of A2058 and A375 cells were investigated by a modified Boyden chamber assay. Zymography was used to clarify the activities of matrix metalloproteinases-2 and urinary type plasminogen activator. We performed a Western blot to verify the related expression of phospho-Src (Tyr416), phospho-Focal adhesion kinase (Tyr397), Vimentin, and β-catenin. Results: Modified Boyden chamber assays demonstrated that treatment of Terminalia catappa leaf extracts significantly inhibited A2058 and A375 cell migration/invasion capacities. In the zymography results, we showed that Terminalia catappa leaf extracts negatively modulated the activities of matrix metalloproteinases-2 and urinary type plasminogen activator. Western blot indicated that Terminalia catappa leaf extracts reduced the expression of phospho-Src (Tyr416), phospho-Focal adhesion kinase (Tyr397), Vimentin, and β-catenin. Conclusion: Terminalia catappa leaf extracts affected the antimetastasis of the A2058 and A375 melanoma cell lines by inhibiting the Focal adhesion kinase/Src interaction and Wingless-int1/β-catenin pathways in vitro. Terminalia catappa leaf extracts may serve as an effective chemopreventive agent against metastasis of melanoma cancer.

Follistatin-like 1 mitigates intermittent hypoxia-induced melanoma lung metastasis in mice.

Intermittent hypoxia (IH) mimicking obstructive sleep apnea (OSA) has been confirmed to induce tumor lung metastasis via oxidative stress and inflammation responses. Follistatin-like 1 (Fstl1), as a matricellular protein, plays critical roles in inflammatory diseases and cancer. This study aimed to investigate the effect and mechanism of Fstl1 on OSA-IH-induced tumor lung metastasis. Fstl1+/+ or Fstl1+/- mice inoculated with B16F10 melanoma cells were exposed to OSA-IH. The number and area of mouse lung metastatic colonies were assessed. Markers for tumor metastasis, oxidative stress, and inflammation in lung melanoma tissue or B16F10 melanoma cells were quantified by western blotting, qRT-PCR, and immunohistochemistry. The migration of B16F10 cells was examined by wound healing assay. Fstl1 levels are decreased in lung tissues from OSA-IH injured mice inoculated with melanoma cells. Fstl1-deficient mice were highly susceptible to the OSA-IH model of melanoma lung metastasis, as assessed by increased number and area of lung metastatic colonies, and by the elevated levels of HIF-1α, Vegf, N-cadherin, and E-cadherin. Lung melanoma tissue in Fstl1+/- mice provided evidence of increased oxidative stress, as determined by increased levels of NRF2 and P22phox and decreased level of Sod2, as well as increased inflammatory response, as determined by elevated levels of NF-κB P65, Tnf-α and Il-6. Conversely, stable overexpression of Fstl1 in B16F10 cells under OSA-IH exposure attenuated the migration of B16F10 cells and levels of tumor-related markers, as well as decreased oxidative stress and inflammatory responses. These results suggest that Fstl1 may protect against OSA-IH-induced tumor lung metastasis through oxidative stress and inflammatory responses. Fstl1 may serve as a promising target for OSA-related cancer.

Kaempferol impairs aerobic glycolysis against melanoma metastasis via inhibiting the mitochondrial binding of HK2 and VDAC1

Metastasis is the leading cause of death in melanoma patients. Aerobic glycolysis is a common metabolic feature in tumor and is closely related to cell growth and metastasis. Kaempferol (KAM) is one of the active ingredients in the total flavonoids of Chinese traditional medicine Sparganii Rhizoma. Studies have shown that it interferes with the cell cycle, apoptosis, angiogenesis and metastasis of tumor cells, but whether it can affect the aerobic glycolysis of melanoma is still unclear. Here, we explored the effects and mechanisms of KAM on melanoma metastasis and aerobic glycolysis. KAM inhibited the migration and invasion of A375 and B16F10 cells, and reduced the lung metastasis of melanoma cells. Extracellular acidification rates (ECAR) and glucose consumption were obviously suppressed by KAM, as well as the production of ATP, pyruvate and lactate. Mechanistically, the activity of hexokinase (HK), the first key kinase of aerobic glycolysis, was significantly inhibited by KAM. Although the total protein expression of HK2 was not significantly changed, the binding of HK2 and voltage-dependent anion channel 1 (VDAC1) on mitochondria was inhibited by KAM through AKT/GSK-3β signal pathway. In conclusion, KAM inhibits melanoma metastasis via blocking aerobic glycolysis of melanoma cells, in which the binding of HK2 and VDAC1 on mitochondria was broken.

Pharmacological attenuation of melanoma by tryptanthrin pertains to the suppression of MITF-M through MEK/ERK signaling axis.

Melanoma is the most aggressive among all types of skin cancers. The current strategies against melanoma utilize BRAFV600E, as a focal point for targeted therapy. However, therapy resistance developed in melanoma patients against the conventional anti-melanoma drugs hinders the ultimate benefits of targeted therapies. A major mechanism by which melanoma cells attain therapy resistance is via the activation of microphthalmia-associated transcription factor-M (MITF-M), the key transcription factor and oncogene aiding the survival of melanoma cells. We demonstrate that tryptanthrin (Tpn), an indole quinazoline alkaloid, which we isolated and characterized from Wrightia tinctoria, exhibits remarkable anti-tumor activity towards human melanoma through the down-regulation of MITF-M. Microarray analysis of Tpn-treated melanoma cells followed by a STRING protein association network analysis revealed that differential expression of genes in melanoma converges at MITF-M. Furthermore, in vitro and in vivo studies conducted using melanoma cells with differential MITF-M expression status, endogenously or ectopically, demonstrated that the anti-melanoma activity of Tpn is decisively contingent on its efficacy in down-regulating MITF-M expression. Tpn potentiates the degradation of MITF-M via the modulation of MEK1/2-ERK1/2-MITF-M signaling cascades. Murine models demonstrate the efficacy of Tpn in attenuating the migration and metastasis of melanoma cells, while remaining pharmacologically safe. In addition, Tpn suppresses the expression of mutated BRAFV600E and inhibits Casein Kinase 2α, a pro-survival enzyme that regulates ERK1/2 homeostasis in many tumor types, including melanoma. Together, we point to a promising anti-melanoma drug in Tpn, by virtue of its attributes to impede melanoma invasion and metastasis by attenuating MITF-M.

Tumor-promoting properties of karyopherin β1 in melanoma by stabilizing Ras-GTPase-activating protein SH3 domain-binding protein 1

The nuclear import receptor karyopherin β1 (KPNB1), a member of the Karyopherin protein family, is reported to be overexpressed in various cancers and promote carcinogenesis. By analyzing the correlation between the expression of KPNB1 and the overall survival rate of melanoma patients, we found that melanoma patients with higher expression of KPNB1 had worse survival. Furthermore, the database analyzed that the KPNB1 mRNA level was higher in melanoma samples than that in skin nevus tissues. We thus proposed that KPNB1 played a role in promoting melanoma development, and conducted gain-of- and loss-of-function experiments to test our hypothesis. We found that KPNB1 knockdown significantly retarded the growth and metastasis of melanoma cells in vitro and in vivo, and increased their sensitivity towards the anti-tumor drug cisplatin. KPNB1 overexpression had opposite effects. Notably, in melanoma cells, KPNB1 overexpression significantly decreased Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) protein level, which was also overexpressed in melanoma samples and enhanced malignant behaviors of melanoma cells. We further demonstrated that KPNB1 overexpression induced deubiquitination of G3BP1, and prevented its degradation. However, KPNB1 overexpression hardly affected the nuclear translocation of G3BP1. Additionally, alterations induced by KPNB1 overexpression were partly reversed by G3BP1 inhibition. Therefore, the results suggest that KPNB1 may promote melanoma progression by stabilizing the G3BP1 protein. KPNB1-G3BP1 axis represents a potential therapeutic targetable node for melanoma.

Abstract 3640: Adipocyte age dependent metabolic programming dictates melanoma metastatic burden and tropism

Abstract The aged skin microenvironment and aged fibroblasts contribute to melanoma progression. A key hallmark of skin ageing is the loss of subcutaneous fat. Because previous work has established that lipids contribute to melanoma invasion and proliferation, we studied whether aged adipocytes contribute to the poor outcome of aged patients. We compared the distinct lipid secretory profile of young and aged human subcutaneous adipocytes, and discovered young and aged adipocytes contribute different lipids to the melanoma microenvironment, activating aged-dependent signaling pathways and driving distinct metabolic programms in melanoma cells. To test how aged and young adipocytes affect melanoma, we obtained human preadipocytes from donors of different ages and differentiated them to functional adipocytes in vitro. We studied the morphological, transcriptomic, proteomic, and lipid profile of adipocytes by age, then exposed melanoma cells to the young or aged adipocyte secretome. We observed melanoma cells exposed to young adipocyte secretome increased the rate of proliferation, invasion, and migration compared to melanoma cells exposed to aged adipocyte secretomes. This effect was linked to a greater amount of total lipids secreted by young adipocytes, as well as a unique ratio of lipids by age, which were taken up by melanoma cells. Critically, lipid transfer led to distinct signaling pathway activation and metabolic reprogramming. In vivo, these differences led to a greater rate of metastasis and distinct tropism of melanoma cells exposed to aged or young adipocyte secretomes. Finally, we systematically tested the contribution of differentially secreted lipids by age and identified the specific lipids that function as signaling molecules to switch metabolic dependencies and fuel proliferation, invasion, and migration of melanoma cells Citation Format: Shilpa Gurung. Adipocyte age dependent metabolic programming dictates melanoma metastatic burden and tropism [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 3640.

Loss of YY1, a Regulator of Metabolism in Melanoma, Drives Melanoma Cell Invasiveness and Metastasis Formation.

Deregulation of cellular metabolism through metabolic rewiring and translational reprogramming are considered hallmark traits of tumor development and malignant progression. The transcription factor YY1 is a master regulator of metabolism that we have previously shown to orchestrate a metabolic program required for melanoma formation. In this study, we demonstrate that YY1, while being essential for primary melanoma formation, suppresses metastatic spreading. Its downregulation or loss resulted in the induction of an invasiveness gene program and sensitized melanoma cells for pro-invasive signaling molecules, such as TGF-β. In addition, NGFR, a key effector in melanoma invasion and phenotype switching, was among the most upregulated genes after YY1 knockdown. High levels of NGFR were also associated with other metabolic stress inducers, further indicating that YY1 knockdown mimics a metabolic stress program associated with an increased invasion potential in melanoma. Accordingly, while counteracting tumor growth, loss of YY1 strongly promoted melanoma cell invasiveness in vitro and metastasis formation in melanoma mouse models in vivo. Thus, our findings show that the metabolic regulator YY1 controls phenotype switching in melanoma.

FMRP ligand circZNF609 destabilizes RAC1 mRNA to reduce metastasis in acral melanoma and cutaneous melanoma

BackgroundMelanoma is a type of malignant tumor with high aggressiveness and poor prognosis. At present, metastasis of melanoma is still an important cause of death in melanoma patients. However, the potential functions and molecular mechanisms of most circular RNAs (circRNAs) in melanoma metastasis remain unknown.MethodscircRNAs dysregulated in melanoma cell subgroups with different metastatic abilities according to a screening model based on repeated Transwell assays were identified with a circRNA array. The expression and prognostic significance of circZNF609 in skin cutaneous melanoma and acral melanoma cells and tissues were determined by qRT–PCR, nucleoplasmic separation assays and fluorescence in situ hybridization. In vitro wound healing, Transwell and 3D invasion assays were used to analyse melanoma cell metastasis ability. Tail vein injection and intrasplenic injection were used to study in vivo lung metastasis and liver metastasis, respectively. The mechanism of circZNF609 was further evaluated via RNA immunoprecipitation, RNA pull-down, silver staining, and immunofluorescence colocalization assays.ResultscircZNF609 was stably expressed at low levels in melanoma tissues and cells and was negatively correlated with Breslow depth, clinical stage and prognosis of melanoma patients. circZNF609 inhibited metastasis of acral and cutaneous melanoma in vivo and in vitro. Mechanistically, circZNF609 promoted the binding of FMRP protein and RAC1 mRNA, thereby enhancing the inhibitory effect of FMRP protein on the stability of RAC1 mRNA and ultimately inhibiting melanoma metastasis.ConclusionsOur findings revealed that circZNF609 plays a vital role in the metastasis of acral and cutaneous melanoma through the circRNF609-FMRP-RAC1 axis and indicated that circZNF609 regulates the stability of RAC1 mRNA by combining with FMRP, which might provide insight into melanoma pathogenesis and a new potential target for treatment of melanoma.

Ablation of Proton/Glucose Exporter SLC45A2 Enhances Melanosomal Glycolysis to Inhibit Melanin Biosynthesis and Promote Melanoma Metastasis

Sequence variation in SLC45A2 are responsible for oculocutaneous albinism type 4 in many species and are associated with melanoma susceptibility, but the molecular mechanism is unclear. In this study, we used Slc45a2-deficient melanocyte and mouse models to elucidate the roles of SLC45A2 in melanogenesis and melanoma metastasis. We found that the acidified cellular environment impairs the activity of key melanogenic enzyme tyrosinase in Slc45a2-deficient melanocytes. SLC45A2 is identified as a proton/glucose exporter in melanosomes, and its ablation increases the acidification of melanosomal pH through enhanced glycolysis. Intriguingly, 13C-glucose-labeled metabolic flux and biochemical assays show that melanosomes are active glucose-metabolizing organelles, indicating that elevated glycolysis mainly occurs in melanosomes owing to Slc45a2 deficiency. Moreover, Slc45a2 deficiency significantly upregulates the activities of glycolytic enzymes and phosphatidylinositol 3-kinase/protein kinase B signaling to promote glycolysis-dependent survival and metastasis of melanoma cells. Collectively, our study reveals that the proton/glucose exporter SLC45A2 mediates melanin synthesis and melanoma metastasis primarily by modulating melanosomal glucose metabolism.

Molecular docking study of vemurafenib derivatives on melanoma inhibitory activity (MIA) as anti-melanoma

Background: Melanoma is one of the cancers with high mortality rates in Indonesia. The newest potential anti-melanoma target is the Melanoma Inhibitory Activity (MIA/5IXB) macromolecule, but the location of the binding pocket interaction is unknown. Vemurafenib is known to be active in vivo as against melanoma. Thus, it can be used as a lead compound. This study aimed to examine the location of the binding pocket and the interactions that occur between macromolecules and the test compounds. Methods: It was carried out using the blind docking method between vemurafenib and an MIA macromolecule. Results: The results showed that the best molecular docking was obtained in the distribution of the grid box area in the third area with a ∆G value of -7.80 kcal/mol. Docking validation results showed the value of ∆G 11.06 kcal/mol with an average value of RMSD 1.788 Å, and the amino acid residues that played a role in the interaction were MET31, TYR30, and PRO33. Targeted docking to the binding pocket results on 45 test compounds showed that the most potent compound was Compound-17 with a ∆G value of 11.31 kcal/mol with hydrophobic bond interactions on amino acid residues TYR30. This hydrophobic bond is responsible for the MIA mechanism as an inhibitor of melanoma cell metastasis in the body. It can be concluded that the binding pocket of the MIA(5IXB) macromolecule with the vemurafenib compound is at the amino acid residues MET31, TYR30, and PRO33 with the interaction of hydrogen bonds and hydrophobic bonds. Also, it was observed that T.C-17 is the most potential anti-melanoma test compound.

Antitumor activity and molecular mechanism of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase family of proteins that activate other proteins in cells. Functionally, PCSK9 binds to the receptor of low-density lipoprotein particles (LDL) to regulate cholesterol metabolism and lipoprotein homeostasis in human body. PCSK9 inhibition is a novel pharmacological strategy to control hypercholesterolemia and cardiovascular diseases. Recently accumulating evidence realizes that PCSK9 possesses other roles in cells, such as regulation of tissue inflammatory response, intratumoral immune cell infiltration, and tumor progression. This review discussed the advancement of PCSK9 research on its role and underlying mechanisms in tumor development and progression. For example, PCSK9 inhibition could attenuate progression of breast cancer, glioma, colon tumor, hepatocellular cancer, prostate cancer, and lung adenocarcinoma and promote apoptosis of glioma, prostate cancer, and hepatocellular cancer cells. PCSK9 deficiency could reduce liver metastasis of B16F1 melanoma cells by lowering the circulating cholesterol levels. PCSK9 gene knockdown substantially attenuated mouse tumor growth in vivo by activation of cytotoxic T cells, although PCSK9 knockdown had no effect on morphology and growth rate of different mouse cancer cell lines in vitro. PCSK9 inhibition thus can be used to control human cancers. Future preclinical and clinical studies are warranted to define anti-tumor activity of PCSK9 inhibition.

CircITCH suppresses cell proliferation and metastasis through miR-660/TFCP2 pathway in melanoma.

BackgroundMelanoma is an aggressive disease that is rising in incidence. Advanced melanoma is still a life‐threatening disease. CircRNAs are documented to be involved in melanoma progression. But circITCH role in melanoma remains unclear.Methods and ResultsTo explore the functions of circITCH in melanoma, levels of circITCH in melanoma tissues and paracarcinoma normal tissues were detected. To study the roles of circITCH in melanoma in terms of cell proliferation and migration, in vitro and in vivo experiments were performed. Mechanism study was designed to investigate the potential regulatory effect of circITCH in melanoma. Results revealed that circITCH expression was repressed in melanoma versus adjacent normal tissues. Function study showed that circITCH suppressed melanoma cell proliferation and metastasis. The mechanism study showed that circITCH‐sponged miR‐660 to upregulate TFCP2 and suppress melanoma progression.ConclusionsThe circITCH/miR‐660/TFCP2 axis is involved in melanoma progression hence circITCH can be a diagnostic biomarker as well as a target for treating melanoma.

Icaritin ameliorates extracellular microparticles-induced inflammatory pre-metastatic niche via modulating the cGAS-STING signaling.

The concept of the inflammatory pre-metastatic niche (PMN) provides a new and promising direction for the prevention and treatment of metastasis. The excessive activation of the GAS-STING signaling leads to augmented metastasis by promoting the formation of the inflammatory PMN. In this study, tumor-derived microparticles (MP) were used to establish the PMN model both in vitro and in vivo, and pro-inflammatory mediators were also employed to evaluate the effects of Icaritin (ICT). It was demonstrated that ICT could inhibit the pulmonary metastasis of B16BL6 melanoma cells in mice via interfering with PMN. The phosphorylation and dimerization of STING and its downstream signaling TBK1-IFNβ were proved to be diminished in the presence of ICT. Furthermore, we revealed that ICT suppressed the generation of pro-inflammatory PMN through conferring the inactivation of the STING signaling pathway. CETSA and DARTS assay also confirmed that STING tended to be a target for the action of ICT. Collectively, our findings highlight a new binding mechanism between STING and ICT for the inhibition of transduction of the STING signaling pathway, suggesting that pharmacological or therapeutic intervention of the STING-TBK1-IFNβ singling axis may serve as an effective strategy to prevent the progression of inflammatory PMN and lung metastasis.

Synthesis of selective PAK4 inhibitors for lung metastasis of lung cancer and melanoma cells

The p21 activated kinase 4 (PAK4) is serine/threonine protein kinase that is critical for cancer progression. Guided by X-ray crystallography and structure-based optimization, we report a novel subseries of C-3-substituted 6-ethynyl-1H-indole derivatives that display high potential and specificity towards group II PAKs. Among these inhibitors, compound 55 exhibited excellent inhibitory activity and kinase selectivity, displayed superior anti-migratory and anti-invasive properties against the lung cancer cell line A549 and the melanoma cell line B16. Compound 55 exhibited potent in vivo antitumor metastatic efficacy, with over 80% and 90% inhibition of lung metastasis in A549 or B16-BL6 lung metastasis models, respectively. Further mechanistic studies demonstrated that compound 55 mitigated TGF-β1-induced epithelial-mesenchymal transition (EMT).

PLP2-derived peptide Rb4 triggers PARP-1-mediated necrotic death in murine melanoma cells

Malignant melanoma is the main cause of death in patients with skin cancer. Overexpression of Proteolipid protein 2 (PLP2) increased tumor metastasis and the knockdown of PLP2 inhibited the growth and metastasis of melanoma cells. In the present work, we studied the antitumor activity of peptide Rb4 derived from protein PLP2. In vitro, Rb4 induced F-actin polymerization, prevented F-actin depolymerization and increased the ER-derived cytosolic calcium. Such effects were associated with necrosis of murine melanoma B16F10-Nex2 cells and with inhibition of the viability of human cancer cell lines. Loss of plasma membrane integrity, dilation of mitochondria, cytoplasm vacuolation and absence of chromatin condensation characterized tumor cell necrosis. Cleavage of PARP-1 and inhibition of RIP1 expression were also observed. In vivo, peptide Rb4 reduced the lung metastasis of tumor cells and delayed the subcutaneous melanoma growth in a syngeneic model. Rb4 induced the expression of two DAMPs molecules, HMGB1 and calreticulin, in B16F10-Nex2. Our results suggest that peptide Rb4 acts directly on tumor cells inducing the expression of DAMPs, which trigger the immunoprotective effect in vivo against melanoma cells. We suggest that peptide Rb4 is a promising compound to be developed as an anticancer drug.

Disruption of LTBP4 Inhibition-Induced TGFβ1 Activation Promoted Cell Proliferation and Metastasis in Skin Melanoma by Inhibiting the Activation of the Hippo-YAP1 Signaling Pathway.

Melanoma is a malignant tumor derived from melanocytes, which is the most fatal skin cancer. The present study aimed to explore and elucidate the candidate genes in melanoma and its underlying molecular mechanism. A total of 1,156 differentially expressed genes were obtained from the GSE46517 dataset of Gene Expression Omnibus database using the package “limma” in R. Based on two algorithms (LASSO and SVM-RFE), we obtained three candidate DEGs (LTBP4, CDHR1, and MARCKSL1). Among them, LTBP4 was identified as a diagnostic marker of melanoma (AUC = 0.985). Down-regulation of LTBP4 expression was identified in melanoma tissues and cells, which predicted poor prognosis of patients with melanoma. Cox analysis results discovered that LTBP4 with low expression was an independent prognostic factor for overall survival in patients with melanoma. LTBP4 inhibition reduced cell apoptosis and promoted cell proliferation and metastasis. These changes were correlated with the expression levels of caspase-3, Ki67 and E-cadherin. Further, as indicated by tumor formation study of nude mice, LTBP4 silencing improved the tumorigenic ability of melanoma cells. Knockdown of LTBP4 increased the percentage of active TGFβ1 secreted by melanoma cells. CTGF, Gyr61, and Birc5 expression levels were reduced, YAP1 phosphorylation was inhibited, and YAP1 was translocated from the cytoplasm to the nucleus in melanoma cells treated with TGF-β1. These effects were reversed by LTBP4 overexpression. As evidenced by immunofluorescent staining, Western blotting and luciferase reporter assay, LTBP4 overexpression activated the Hippo signaling pathway, which was characterized by the increased nuclear-cytoplasmic translocation of YAP1 and the enhanced phosphorylation of YAP1, MST1, and MOB1. In addition, the effects of LTBP4 overexpression on inhibiting CTGF, Cyr61 and Birc5 expression, promoting the apoptosis, and inhibiting the metastasis and proliferation of melanoma cells were reversed by the overexpression of YAP1 or MST1. In conclusion, the LTBP4-TGFβ1-Hippo-YAP1 axis is a critical pathway for the progression of skin melanoma.