Melanoma Cell Proliferation Research Articles

In vitro study of cold atmospheric plasma-activated liquids inhibits malignant melanoma by affecting macrophage polarization through the ROS/JAK2/STAT1 pathway

Melanoma is a prevalent malignant skin tumor known for its high invasive ability and a high rate of metastasis, making clinical treatment exceptionally challenging. Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment and play a crucial role in tumor survival and development. Cold atmospheric plasma (CAP) is an emerging tool for tumor treatment that has garnered attention from scholars due to its interaction with non-tumor cells in the tumor microenvironment. Here, we used the macrophage lines THP-1 and RAW264.7, as well as the melanoma cell lines A375 and MV3, as research subjects to investigate the effect of plasma-activated liquid (PAL) on macrophage differentiation and its inhibitory effect on melanoma cell proliferation. We confirmed that the killing effect of PAL on melanoma cells was selective. Using flow cytometry and PCR, we discovered that PAL can influence macrophage differentiation. Through in vitro cell coculture, we demonstrated that PAL-treated macrophages can significantly impede tumor cell development and progression, and the effect is more potent than that of PAL directly targeting tumor cells. Furthermore, we have proposed the hypothesis that PAL promotes the differentiation of macrophages into the M1 type through the ROS/JAK2/STAT1 pathway. To test the hypothesis, we employed catalase and fludarabine to block different sites of the pathway. The results were then validated through Western Blot, qPCR and ELISA. This study illustrates that PAL therapy is an effective tumor immunotherapy and expands the scope of tumor immunotherapy. Furthermore, these findings establish a theoretical foundation for potential clinical applications of PAL.

Major Facilitator Superfamily Domain Containing 12 Is Overexpressed in Lung Cancer and Exhibits an Oncogenic Role in Lung Adenocarcinoma Cells.

Major facilitator superfamily domain containing 12 (MFSD12) regulates lysosomal cysteine import and promotes the proliferation and survival of melanoma cells. However, the expression and function of MFSD12 in other cancers, particularly in lung cancer, remain unclear. The expression of MFSD12 across various types of cancers and corresponding control tissues was examined using TIMER. MFSD12 expression in lung adenocarcinoma (LUAD) and its correlation with distinct clinicopathological features of LUAD patients were analyzed with UALCAN. The correlation between MFSD12 expression and survival of LUAD patients was assessed using the R package, survival, and the relationship between MFSD12 expression and immune infiltration status in LUAD was investigated using CIBERSORT. In addition, MFSD12 expression was knocked down in PC9 LUAD cells and their proliferation, capacity for expansion, cell cycle, apoptosis, and migration/invasion were evaluated through CCK-8 assays, colony formation assays, 7-AAD staining, Annexin V/PI staining, and Transwell assays, respectively. The stemness of these PC9 cells was determined through Western blotting, flow cytometry, and tumor sphere formation assays. MFSD12 mRNA levels were significantly elevated in multiple types of cancers, including LUAD. MFSD12 expression was also positively correlated with cancer stage, nodal metastasis, and infiltration of various immune cells in LUAD, and high MFSD12 levels predicted poor survival among LUAD patients. Knockdown of MFSD12 in PC9 cells resulted in decreased proliferation, attenuated colony formation capacity, cell cycle arrest, elevated apoptosis, impaired migration/invasion, and reduced stemness in PC9 cells. MFSD12 is an oncogene in LUAD.

The involvement of RIPK4 in TNF-α-stimulated IL-6 and IL-8 production by melanoma cells

PurposeThe receptor-interacting protein kinase (RIPK4) has an oncogenic function in melanoma, regulates NF-κB and Wnt/β-catenin pathways, and is sensitive to the BRAF inhibitors: vemurafenib and dabrafenib which lead to its decreased level. As its role in melanoma remains not fully understood, we examined the effects of its downregulation on the transcriptomic profile of melanoma.MethodsApplying RNA-seq, we revealed global alterations in the transcriptome of WM266.4 cells with RIPK4 silencing. Functional partners of RIPK4 were evaluated using STRING and GeneMANIA databases. Cells with transient knockdown (via siRNA) and stable knockout (via CRISPR/Cas9) of RIPK4 were stimulated with TNF-α. The expression levels of selected proteins were assessed using Western blot, ELISA, and qPCR.ResultsGlobal analysis of gene expression changes indicates a complex role for RIPK4 in regulating adhesion, migration, proliferation, and inflammatory processes in melanoma cells. Our study highlights potential functional partners of RIPK4 such as BIRC3, TNF-α receptors, and MAP2K6. Data from RIPK4 knockout cells suggest a putative role for RIPK4 in modulating TNF-α-induced production of IL-8 and IL-6 through two distinct signaling pathways—BIRC3/NF-κB and p38/MAPK. Furthermore, increased serum TNF-α levels and the correlation of RIPK4 with NF-κB were revealed in melanoma patients.ConclusionThese data reveal a complex role for RIPK4 in regulating the immune signaling network in melanoma cells and suggest that this kinase may represent an alternative target for melanoma-targeted adjuvant therapy.

Iodinated 4,4'-Bipyridines with Antiproliferative Activity Against Melanoma Cell Lines.

In the last decade, biological processes involving halogen bond (HaB) as a leading interaction attracted great interest. However, although bound iodine atoms are considered powerful HaB donors, few iodinated new drugs were reported so far. Recently, iodinated 4,4'-bipyridines showed interesting properties as HaB donors in solution and in the solid state. In this paper, a study on the inhibition activity of seven halogenated 4,4'-bipyridines against malignant melanoma (MM) cell proliferation is described. Explorative dose/response proliferation assays were first performed with three 4,4'-bipyridines by using four MM cell lines and the normal BJ fibroblast cell line as control. Among them, the A375 MM cell line was the most sensitive, as determined by MTT assays, which was selected to evaluate the antiproliferative activity of all 4,4'-bipyridines. Significantly, the presence of an electrophilic iodine impacted the biological activity of the corresponding compounds. The 3,3',5,5'-tetrachloro-2-iodo-4,4'-bipyridine showed significant antiproliferation activity against the A375 cell line, and lower toxicity on BJ fibroblasts. Through in silico studies, the stereoelectronic features of possible sites determining the bioactivity were explored. These results pave the way for the utilization of iodinated 4,4'-bipyridines as templates to design new promising HaB-enabled inhibitors of MM cell proliferation.

Nebivolol, an antihypertensive agent, has new application in inhibiting melanoma.

Repurposing existing drugs for cancer therapy has become an important strategy because of its advantages, such as cost reduction, effect and safety. The present study was designed to investigate the antimelanoma effect and possible mechanisms of action of nebivolol, which is an approved and widely prescribed antihypertensive agent. In this study, we explored the effect of nebivolol on cell proliferation and cell activity in melanoma in vitro and the potential antimelanoma mechanism of nebivolol through a series of experiments, including the analysis of the effects with regard to cell apoptosis and metastasis. Furthermore, we evaluated the antimelanoma effect on xenograft tumor models and inspected the antimelanoma mechanism of nebivolol in vivo using immunohistochemical and immunofluorescence staining assays. As results in this work, in vitro , nebivolol possessed a strong activity for suppression proliferation and cell cycle arrest on melanoma. Moreover, nebivolol significantly induced cell apoptosis in melanoma through a mitochondrial-mediated endogenous apoptosis pathway. Additionally, nebivolol inhibited melanoma cell metastasis. More importantly, nebivolol exhibited significantly effective melanoma xenograft models in vivo , which related to the mechanism of apoptosis induction, proliferation inhibition, metastasis blocking and angiogenesis arrest. Overall, the data of the present study recommend that nebivolol holds great potential in application as a novel agent for the treatment of melanoma.

Retracted] MicroRNA‑338‑3p suppresses cell proliferation, migration and invasion in human malignant melanoma by targeting MACC1.

[This retracts the article DOI: 10.3892/etm.2019.7644.].

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.

FOXO3 Inhibits the Cisplatin Resistance and Progression of Melanoma Cells by Promoting CDKN1C Transcription.

Forkhead box O3 (FOXO3) and cyclin dependent kinase inhibitor 1C Gene (CDKN1C) have been shown to be involved in the melanoma process, but their roles in the cisplatin (DDP) resistance of melanoma remain unclear. The mRNA levels of CDKN1C and FOXO3 were measured using quantitative real-time PCR. The protein levels of CDKN1C, FOXO3 and mitochondrial oxidative phosphorylation (mtOXPHOS)-related markers were determinant by western blot analysis. The DDP resistance, proliferation, and apoptosis of melanoma cells were assessed by cell counting kit 8 assay, colony formation assay and flow cytometry. Glucose consumption, lactate production and ATP level were detected to assess glycolysis. The regulation of FOXO3 on CDKN1C was confirmed by ChIP assay and dual-luciferase reporter assay. In vivo experiments were performed to evaluate the effect of FOXO3 on DDP sensitivity in melanoma tumor tissues. CDKN1C and FOXO3 were downregulated in chemoresistant melanoma tissues, and their low expression levels were related to the poor prognosis of melanoma patients. Overexpression of CDKN1C and FOXO3 repressed DDP resistance, proliferation, and glycolysis, while promoted apoptosis and mtOXPHOS in DDP-resistant melanoma cells. Further analysis suggested that FOXO3 could bind to CDKN1C promoter region to enhance its transcription. Besides, CDKN1C knockdown reversed the regulation of FOXO3 on melanoma cell DDP resistance and progression. Moreover, FOXO3 overexpression enhanced the DDP sensitivity of melanoma tumor tissues in vivo. FOXO3 promoted the transcription of CDKN1C, thereby inhibiting the DDP resistance and progression of melanoma cells.

H2A.Z chaperones converge on E2F target genes for melanoma cell proliferation.

High levels of H2A.Z promote melanoma cell proliferation and correlate with poor prognosis. However, the role of the two distinct H2A.Z histone chaperone complexes SRCAP and P400-TIP60 in melanoma remains unclear. Here, we show that individual subunit depletion of SRCAP, P400, and VPS72 (YL1) results in not only the loss of H2A.Z deposition into chromatin but also a reduction of H4 acetylation in melanoma cells. This loss of H4 acetylation is particularly found at the promoters of cell cycle genes directly bound by H2A.Z and its chaperones, suggesting a coordinated regulation between H2A.Z deposition and H4 acetylation to promote their expression. Knockdown of each of the three subunits downregulates E2F1 and its targets, resulting in a cell cycle arrest akin to H2A.Z depletion. However, unlike H2A.Z deficiency, loss of the shared H2A.Z chaperone subunit YL1 induces apoptosis. Furthermore, YL1 is overexpressed in melanoma tissues, and its upregulation is associated with poor patient outcome. Together, these findings provide a rationale for future targeting of H2A.Z chaperones as an epigenetic strategy for melanoma treatment.

A novel long non-coding RNA SLNCR1 promotes proliferation, migration, and invasion of melanoma via transcriptionally regulating SOX5

Steroid receptor RNA activator (SRA)-like non-coding RNA (SLNCR1) has been implicated in various tumorigenic processes, but the precise regulatory role in melanoma progression remains uncertain. We performed a comprehensive analysis to investigate the prognostic value of SLNCR1 expression in patients with melanoma by TCGA database and melanoma tissue samples via the Kaplan–Meier method. Subsequently, we conducted qRT-PCR and Fluorescence in Situ Hybridization (FISH) assays to identify SLNCR1 expression levels and localization in tissues and cells, respectively. Loss-of-function assays utilizing shRNAs vectors were used to investigate the potential impact of SLNCR1. Our data showed that SLNCR1 is significantly up-regulated in human malignant melanoma tissues and cell lines and functions as an oncogene. Silencing of SLNCR1 suppressed melanoma cell proliferation, migration, invasion, and inhibited tumorigenesis in a mouse xenograft model. Additionally, we employed bioinformatic predictive analysis, combined with dual-luciferase reporter analysis and functional rescue assays, to elucidate the mechanistic target of the SLNCR1/SOX5 axis in melanoma. Mechanistically, we discovered that SLNCR1 promotes EMT of human melanoma by targeting SOX5, as downregulation of SLNCR1 expression leads to a decrease in SOX5 protein levels and inhibits melanoma tumorigenesis. Our research offers promising insights for more precise diagnosis and treatment of human melanoma.

Abstract 671: G-protein coupled receptor (GPCR) screening library identifies sertindole, an antipsychotic, as potential therapeutic option to target GNAQ mutation Q209L in uveal melanoma

Abstract Background: Uveal melanoma is a rare but aggressive intraocular malignancy with limited therapeutic options. A hallmark genetic alteration associated with uveal melanoma is the mutation of the GNAQ gene, particularly the Q209L substitution in the Gαq protein, which contributes to oncogenic signaling and tumor progression. The GNAQ gene encodes for a Gαq protein that plays a critical role in signal transduction pathways involving G protein-coupled receptors (GPCRs). The Q209L substitution results in a gain-of-function mutation, leading to the chronic activation of downstream signaling pathways. This persistent signaling activity contributes to the uncontrolled growth of uveal melanoma cells. As G protein-coupled receptors (GPCRs) play a crucial role in this process, screening libraries of GPCR inhibitors has been explored as a potential therapeutic avenue. These libraries comprise a vast array of compounds with the potential to block or modulate GPCR activity. Identifying specific inhibitors that can effectively interfere with the oncogenic signaling cascades triggered by the mutated Gαq protein is a critical step in developing targeted therapies for uveal melanoma. Methods: GPCR library high throughput screening employed isogenic cell lines expressing wild type or mutant (Q209L) GNAQ. This process utilized IP-One assays to identify compounds affecting Gq-coupled receptors by detecting inositol monophosphate accumulation. Subsequently, promising hits were evaluated for their impact on inhibiting the growth and proliferation of uveal melanoma cells. Further analysis involved Western blotting to assess their potential in promoting autophagy and apoptosis, inhibiting downstream signaling pathways, and a migration assay. Finally, ROS generation was assessed using flow cytometry. Results: Using isogenic cell lines expressing GNAQ gene wild type and the Q209L mutant, a high-throughput screening of GPCR libraries identified sertindole among 5 hits. Sertindole showed a dose-dependent inhibition of Q209L-mutated uveal melanoma cells, significantly reducing cell viability, proliferation, and migration. It also suppressed key signaling pathways, including the critical MAPK pathway, and activated autophagy and apoptosis, likely via ROS generation. These findings suggest sertindole's multifaceted approach in suppressing cancer in uveal melanoma. Conclusion: Our study highlights the potential therapeutic value of sertindole in GNAQ Q209L-mutated uveal melanoma. Further research is needed to evaluate the efficacy of sertindole in preclinical mouse models of uveal melanoma. Citation Format: Shelby Rheinschmidt, Taylor Bargenquast, Sydney Adamson, Trason Thode, Alexis Weston, Tithi Ghosh Halder, Serina Ng, Harini Tulasi, Brian Durbin, Justin Moser, Mohan Kaadige, Raffaella Soldi, Sunil Sharma. G-protein coupled receptor (GPCR) screening library identifies sertindole, an antipsychotic, as potential therapeutic option to target GNAQ mutation Q209L in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 671.

Abstract 4648: Metabolic vulnerabilities of drug-resistant melanoma with therapeutic potential

Abstract Melanoma has been at the center of revolutions in both targeted BRAF therapy and immune therapy. However, the resistance to current therapeutic drugs has been a formidable obstacle to continuing benefit to patients. Overcoming resistance and new therapeutic agents and strategies are the primary goals of current cancer research. We currently found that metabolic pathways altered significantly in BRAFi-resistant melanoma. We hypothesized that BRAFi-resistant melanoma rewires its metabolic routes to reach its needs in energy and biosynthetic precursors, both essential for escaping the BRAFi attack. We found that five amino acids transporters and metabolic enzymes (ASNS, slc7a11, slc7a5, slc3a2, slc4a7) were the significant increase in resistant melanoma. Knockdown by shRNA or knockout by crispr/cas9 of these amino acids transporters or/and metabolic enzymes enhanced the sensitivity of BRAFi resistant melanoma to BRAFi. In contrast, overexpression of these amino acid transporters or/and metabolic enzymes promoted the ability of resistance of BRAFi in parent melanoma cells. Moreover, the inhibition of these amino acid transporters and metabolic enzymes by small molecular inhibitors significantly attenuated the resistance of the BRAFi-resistant melanoma whereas less affected the parent melanoma. Notably, we confirmed that blocking these amino acid transporters and metabolic enzymes by shRNA and/or small molecular inhibitors could rescue the sensitivity of resistant melanoma cells and inhibit resistant-melanoma cell growth in the preclinical mouse model in vivo. Importantly, we found that overexpression of slc7a11, slc7a5 and slc3a2 correlated with worse survival in melanoma in SK-TCGA data. Our findings uncovered the mechanism of how metabolic reprogramming fueled melanoma cell growth and proliferation for escaping drug attacks and offered a rational strategy to guide clinical treatment. The identified novel molecular proteins represented a promising therapeutic target for BRAF mutant melanoma patients with resistance to current therapy. Citation Format: Yanlin Yu, Lei Huang, Weiping Chen, Glenn Merlino. Metabolic vulnerabilities of drug-resistant melanoma with therapeutic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4648.

Abstract 3020: The tumor suppressive role of methylthioadenosine phosphorylase in melanoma

Abstract Methylthioadenosine phosphorylase (MTAP) is an enzyme that is expressed in virtually all tissues but lost in many cancers. MTAP deficiency can be due to either deletion of the MTAP gene or methylation of the MTAP promoter. In normal cells, MTAP catalyzes the conversion of methylthioadenosine (MTA), produced during polyamine biosynthesis, to adenine and 5-methylthioribose-1-phosphate, which is subsequently converted to methionine. MTAP deficiency results in intracellular accumulation of MTA. Although recent genome wide association studies (GWAS) have associated the MTAP locus with melanoma risk, the molecular mechanisms linking MTAP loss to increased tumorigenesis are not yet fully understood. In this study, we hypothesized that loss of MTAP and the resulting accumulation of MTA would dysregulate microphthalmia transcription factor (MITF), a master transcriptional regulator of melanocytes that is also an amplified or mutated oncogene in melanoma. We identify a novel signaling mechanism in which MTA accumulation from MTAP deficiency aberrantly induces MITF expression via inhibition of the cAMP phosphodiesterase PDE4D3 in melanocytes. Inhibition of PKA abolishes this MITF induction, suggesting that the PKA pathway is hyperactivated upon MTAP deficiency. Downregulation of MTAP leads to increased proliferation of melanoma cells in vitro and increased melanoma tumor growth in vivo. We also show that MTAP deficient melanoma cells are more resistant to the BRAF inhibitor vemurafenib and observe MTAP downregulation in BRAF inhibitor resistant melanoma clinical specimens. Furthermore, MTAP downregulation via decreased expression and/or genomic loss corresponds to significantly worse outcomes in early-stage cutaneous melanomas from multiple datasets, thereby identifying a clinically important sub-category of “thin-lethal” tumors. Citation Format: Jennifer J. Hsiao, Pedro Andreu-Perez, Lajos V. Kemény, Miroslav Hejna, Jérémie Nsengimana, Jennifer Allouche, Abdullah Al Emran, D. Timothy Bishop, Göran B. Jönsson, Julia Newton-Bishop, Jun Song, David E. Fisher. The tumor suppressive role of methylthioadenosine phosphorylase in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3020.

Abstract 3258: A potential oncogenic role and immunoregulatory mechanisms of MZB1 in melanoma

Abstract While immune checkpoint blockade (ICB) therapies have drastically improved melanoma treatment, a majority of patients fail to achieve a durable response. Thus, there is a critical need to advance our understanding of melanoma immune evasion mechanisms and identify novel targets capable of enhancing ICB responses. MZB1 (Marginal Zone B and B1 Cell Specific Protein), an immune regulator gene, is differentially expressed in metastatic melanoma compared to primary melanoma or normal skin. In a limited number of cancers, MZB1 has been shown to function as either an oncogene or a tumor suppressor. However, its role and significance in melanoma is not known. In this study, we determined the functional significance and immunoregulatory mechanisms of MZB1 in human melanoma cell lines using MZB1 overexpression (OE) (in SK-MEL-2, and SK-MEL-28) and CRISPR knockout (KO) (in A375 and Hs294T) lines. MZB1 OE significantly increased melanoma cell proliferation and expression of the proliferation markers Ki67 and PCNA, while MZB1 KO yielded the opposite effect. To identify immunoregulatory mechanisms associated with MZB1, we performed differential gene expression analysis of 770 immune-related genes using NanoString nCounter technology. nSolver analysis identified 153 differentially expressed genes (DEGs) as a result of MZB1 knockout in Hs294T cells (adjusted p <0.05, 1.5-fold change cutoff). MZB1 KO strongly reduced the expression of the HLA class-II family genes and altered the expression of various cytokines, suggesting an immunoregulatory role of MZB1. Further, MZB1 KO significantly downregulated genes related to cancer progression and metastasis promotion. Additionally, MZB1 KO reduced expression of common “cancer stem cell” genes, such as RXRG, ALDH1A1, AQP1, NFATC2, LPAR1, indicating MZB1 expression in melanoma may promote a dedifferentiated or neural crest-like state. In addition, an Ingenuity Pathway Analysis (IPA) of the DEGs revealed many cancer-related functions altered upon MZB1 KO (p adjusted < 0.05) and displayed a negative enrichment for cancer-promoting pathways including tumor growth, metastasis, and proliferation. IPA regulatory analysis predicted MZB1 KO to decrease the activation of oncogenic signaling components including PI3K, MEK, and ERK1/2. Further, IPA predicted that MZB1 inhibition could positively sensitize melanoma cells towards higher immune responses by activating mechanisms associated with T cell proliferation and activation, leukocyte migration, and macrophage activation. Overall, our study suggests MZB1 promotes oncogenic signaling in melanoma to drive proliferation and dedifferentiation, as well as regulates a wide range of immune-related genes that may be responsible for immune evasion in melanoma cells. Thus, MZB1 merits further investigation to determine if the immune consequences yielded by targeting MZB1 can complement ICB approaches in the management of melanoma. Citation Format: Gagan Chhabra, Carl A. Shirley, Julia M. Krien, Mary A. Ndiaye, Nihal Ahmad. A potential oncogenic role and immunoregulatory mechanisms of MZB1 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3258.

Abstract 6963: Cysteinyl leukotriene receptors promote melanoma progression and metastasis

Abstract Background: Cysteinyl leukotrienes (cys-LTs; LTC4, LTD4, LTE4) are pro-inflammatory mediators mainly produced by hematopoietic cells, which enhance inflammation through their receptors, CysLT1R and CysLT2R. They play a pivotal role in the development of chronic asthma in humans. Recent association of inflammation with cancer has led to immense interest on the role of cysteinyl leukotrienes in cancer progression and metastasis. However, the exact molecular mechanisms regarding how these inflammatory mediators regulate the tumor proliferation and metastasis remain unexplored. A thorough investigation of these precise molecular mechanisms and identification of the specific receptor/s responsible can aid in understanding the therapeutic potential of these receptors in various cancers like melanoma. Since the inhibitors of these receptors are already FDA approved for the treatment of asthma, our goal is to repurpose these drugs to treat melanoma progression. Objective: To determine the mechanistic aspects of how CysLTRs regulate melanoma tumor initiation, progression, and metastasis. Methods: Protein expression by western blotting and ELISA, transcript expression by qPCR, viability, and proliferation by XTT and BrDU, migration using trans-well assay, tumor growth and metastasis were confirmed through in vivo experiments. Results & Conclusion: B16F10 melanoma cells express high CysLT1R compared to CysLT2R and produce endogenous cys-LTs that function in an autocrine signaling pattern. Further, cys-LTs mediated the activation of several signaling proteins such as ERK, p38, AKT, and CREB that are important for melanoma survival and proliferation. Moreover, treatment with CysLTR antagonists significantly reduced melanoma cell proliferation, survival, and migration. Accordingly, we observed a significant reduction in the melanoma tumor volume in vivo in both Cysltr1−/- and Cysltr2−/− mice compared to the WT mice. Interestingly, angiogenesis was significantly reduced in Cysltr2−/- mice but not in Cysltr1−/−.Therefore, we speculate that while both receptors play a crucial role in tumor proliferation in vivo, CysLT2R is the main driver of angiogenesis and metastasis. Therefore, targeting both these receptors using their specific antagonists can offer potential therapy for melanoma progression and metastasis. Citation Format: Emma Elizabeth Sabu Kattuman, Lakshminarayan Reddy Teegala, Somayeh Darzi, Sailaja Paruchuri. Cysteinyl leukotriene receptors promote melanoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6963.

Abstract 296: BRAFi-induced ROCK-mediated non-canonical nuclear β-catenin shuttling drives a phenotypic switch in cancer-associated fibroblasts

Abstract The discovery of BRAF mutations in around 60% of melanoma patients and the subsequent development of targeted inhibitors to counter the hyperactive BRAF-driven pro-tumorigenic signaling pathway have marked a significant milestone in melanoma therapy. However, despite this progress, the long-term outcomes remain unsatisfactory for many patients due to the occurrence of drug resistance. We previously reported that cancer-associated fibroblasts (CAFs) are stimulated by BRAF inhibitors (BRAFi) to drive matrix remodeling and therapeutic escape pathways in melanoma cells. However, the underlying mechanism by which CAFs are reprogrammed by BRAFi to obtain enhanced phenotypes remains elusive. Here, we present compelling evidence showing that BRAFi (PLX4032), but not CRAF inhibitor (GW5074) or pan-RAF inhibitor (RAF709), induces the accumulation of nuclear β-catenin in CAFs harboring wild-type BRAF, a process mediated by BRAF and CRAF but not ARAF isoform. BRAFi binding to BRAF and CRAF leads to robust RAF kinase dimerization, including BRAF and CRAF homodimer and BRAF: CRAF heterodimer. RAF dimers are subsequently recruited to the plasma membrane, where BRAF and CRAF are phosphorylated on residues Thr599 and Ser338, respectively, and activated in a RAS-GTP-dependent manner. RAF activation stimulates the downstream Rho kinase (ROCK) and MEK/ERK signaling pathways simultaneously. Notably, ablating RAS and RAF isoforms effectively suppressed BRAFi-induced activation of ROCK1/2 signaling in CAFs, further confirming that the ROCK pathway is a downstream effector of BRAFi-driven RAF activation and is intricately linked to increased nuclear β-catenin in CAFs. The pharmacological blockade of ROCK activity, using HA-1077, diminished BRAFi-induced nuclear β-catenin accumulation in CAFs, suggesting BRAFi-driven cytoskeletal remodeling plays a pivotal role in the nuclear shuttling of β-catenin. Using a mouse model that mimics human BRAF-mutant melanoma, we found that elevated level of nuclear β-catenin drives the conversion of fibroblasts into α-SMA-positive CAFs, leading to elevated collagen deposition and contributing to melanoma progression in vivo. In summary, BRAFi reprograms the transcriptional activity in CAFs by driving increased nuclear β-catenin to increase their ability to remodel the tumor ECM and promote melanoma cell proliferation. Collectively, the data offer new insights into the molecular mechanisms that underlie the paradoxical reprogramming of CAFs by BRAFi in melanoma therapy through the non-canonical β-catenin pathway. Citation Format: Bruna da Silva Soley, Radhika Athalye, Ryan Zhang, Thomas Andl, Yuhang Zhang. BRAFi-induced ROCK-mediated non-canonical nuclear β-catenin shuttling drives a phenotypic switch in cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 296.

The MITF/mir-579-3p regulatory axis dictates BRAF-mutated melanoma cell fate in response to MAPK inhibitors

Therapy of melanoma has improved dramatically over the last years thanks to the development of targeted therapies (MAPKi) and immunotherapies. However, drug resistance continues to limit the efficacy of these therapies. Our research group has provided robust evidence as to the involvement of a set of microRNAs in the development of resistance to target therapy in BRAF-mutated melanomas. Among them, a pivotal role is played by the oncosuppressor miR-579-3p. Here we show that miR-579-3p and the microphthalmia-associated transcription factor (MITF) influence reciprocally their expression through positive feedback regulatory loops. In particular we show that miR-579-3p is specifically deregulated in BRAF-mutant melanomas and that its expression levels mirror those of MITF. Luciferase and ChIP studies show that MITF is a positive regulator of miR-579-3p, which is located in the intron 11 of the human gene ZFR (Zink-finger recombinase) and is co-transcribed with its host gene. Moreover, miR-579-3p, by targeting BRAF, is able to stabilize MITF protein thus inducing its own transcription. From biological points of view, early exposure to MAPKi or, alternatively miR-579-3p transfection, induce block of proliferation and trigger senescence programs in BRAF-mutant melanoma cells. Finally, the long-term development of resistance to MAPKi is able to select cells characterized by the loss of both miR-579-3p and MITF and the same down-regulation is also present in patients relapsing after treatments. Altogether these findings suggest that miR-579-3p/MITF interplay potentially governs the balance between proliferation, senescence and resistance to therapies in BRAF-mutant melanomas.

Cryptotanshinone inhibits PFK-mediated aerobic glycolysis by activating AMPK pathway leading to blockade of cutaneous melanoma

BackgroundCutaneous melanoma is a kind of skin malignancy with low morbidity but high mortality. Cryptotanshinone (CPT), an important component of salvia miltiorrhiza has potent anti-tumor activity and also indicates therapeutic effect on dermatosis. So we thought that CPT maybe a potential agent for therapy of cutaneous melanoma.MethodsB16F10 and A375 melanoma cells were used for in vitro assay. Tumor graft models were made in C57BL/6N and BALB/c nude mice for in vivo assay. Seahorse XF Glycolysis Stress Test Kit was used to detect extracellular acidification rate and oxygen consumption rate. Si-RNAs were used for knocking down adenosine monophosphate-activated protein kinase (AMPK) expression in melanoma cells.ResultsCPT could inhibit the proliferation of melanoma cells. Meanwhile, CPT changed the glucose metabolism and inhibited phosphofructokinase (PFK)-mediated glycolysis in melanoma cells to a certain extent. Importantly, CPT activated AMPK and inhibited the expression of hypoxia inducible factor 1α (HIF-1α). Both AMPK inhibitor and silencing AMPK could partially reverse CPT’s effect on cell proliferation, cell apoptosis and glycolysis. Finally, in vivo experimental data demonstrated that CPT blocked the growth of melanoma, in which was dependent on the glycolysis-mediated cell proliferation.ConclusionsCPT activated AMPK and then inhibited PFK-mediated aerobic glycolysis leading to inhibition of growth of cutaneous melanoma. CPT should be a promising anti-melanoma agent for clinical melanoma therapy.

Progerin Inhibits the Proliferation and Migration of Melanoma Cells by Regulating the Expression of Paxillin.

Progerin, the underlying cause of Hutchinson-Gilford Progeria Syndrome (HGPS), has been extensively studied for its impact on normal cells and premature aging patients. However, there is a lack of research on its specific effects on tumor cells. Melanoma is one of the most common malignant tumors with high morbidity and mortality. This study aimed to elucidate the potential therapeutic role of progerin in melanoma. We constructed the melanoma A375 cell line and M14 cell line with stable expression of progerin. The expression of progerin, paxillin, and epithelial-mesenchymal transition (EMT) marker proteins in each cell group was measured using Western blot. The migration, proliferation, and cell cycle of cancer cells were assessed using the transwell assay, wound healing assay, colony formation assay, CCK 8 assay, and flow cytometry. RT-qPCR technology was used to examine the impact of progerin overexpression on microRNA expression. Finally, we transfected paxillin into the progerin overexpression cell group to verify whether progerin regulates the phenotype of tumor cells through paxillin. Our study demonstrated that overexpression of progerin leads to decreased expression of paxillin and inhibits cancer cell migration, proliferation, EMT process and cell cycle progression. Additionally, rescue experiments revealed that the migration, proliferation ability, and EMT marker protein expression in progerin overexpressing cancer cells could be partially restored by transfecting a plasmid containing the paxillin gene. Mechanistic investigations further revealed that progerin achieves this inhibition of paxillin expression by upregulating miR-212. This study reveals that progerin may inhibit the migration and proliferation of melanoma cells through the miR-212/paxillin axis, which provides a new approach for the future treatment of this disease.

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.