Uveal Melanoma Cells Research Articles

Selamectin increases cisplatin sensitivity by inhibiting cisplatin-resistant genes expression and autophagy in uveal melanoma

Cisplatin resistance is the main reason for uveal melanoma (UM) treatment failure. Thus, developing strategy that increasing cisplatin sensitivity is needed. In this study, we performed drug repositioning analysis with the Connectivity Map database using a panel of previously identified cisplatin sensitivity–associated genes and cisplatin resistance–associated genes as the signature and obtained the antiparasitic drug selamectin. We demonstrated that the selamectin and cisplatin combination showed a synergistic effect on inhibiting UM cell growth. Experiments in tumor-bearing nude mice further showed that selamectin and cisplatin have synergistic effects in reducing tumor growth. Previous studies have linked increased autophagy with tumor resistance to chemotherapy. We found that selamectin inhibited the expression of the autophagy-related gene ATG9B, thus reducing autophagy. The cisplatin resistance–associated genes PDGFRB, DUSP1, MAST1 and IL11 were significantly downregulated in UM cells treated with selamectin. In summary, our study shows that selamectin enhanced the sensitivity of UM to cisplatin, through the mechanism of inhibiting cisplatin resistance–associated gene expression and autophagy. These findings may provide a new strategy for the treatment of UM.

The GPER Agonist LNS8801 Induces Mitotic Arrest and Apoptosis in Uveal Melanoma Cells.

Current treatments against metastatic uveal melanoma have shown limited clinical activity and there is an urgent need for effective therapies. Here, we demonstrate that the GPER agonist LNS8801 induced both GPER-dependent and GPER-independent effects and elicited potent anticancer activities in vitro and in vivo. Our results complement and support the ongoing clinical trial of LNS8801 in advanced uveal melanoma.

Abstract 50: Tumorigenic properties of uveal melanoma cancer cells cultured in 3D or on a reconstructed extracellular matrix

Abstract Background: Uveal melanoma (UM) is the most common intraocular tumor in adults. It arises from the transformation of melanocytes, and despite the effective treatment of the ocular tumor, hepatic metastases lead to a poor prognosis. Our hypothesis is that the invasive potential and therapeutic resistance of UM cells (UMCs) depend on interactions with stromal cells and properties of the extracellular matrix (ECM). Our study aims to determine the impact of UMCs in stromal cell activation, and to define the role of the ECM produced by choroidal fibroblasts or hepatic stellate cells (HSteCs) on UM cell proliferation and invasion. Methods: Two types of tumor spheroids were generated i) one corresponding to primary melanoma (UMCs from eye tumor + choroidal fibroblasts and endothelial cells) and ii) the other to liver metastasis (UMCs from liver metastasis + HSteCs and sinusoidal endothelial cells). The organization of these mixed spheroids were studied by confocal microscopy using the following markers: Melan-A (UM cells), alpha-smooth muscle actin (aSMA; activated fibroblasts and stellate cells), CD31 (endothelial cells) and Ki67 (proliferation). The viability and invasion capacity of the mixed spheroids were analyzed using an ATP consumption assay, and the measurement of the invasion area in Matrigel. Next, two types of matrices were produced by tissue engineering using the self-assembly approach with choroidal fibroblasts or HSteCs. These matrices were devitalized and characterized by confocal microscopy (collagens, fibronectin), before seeding UMCs to study their morphology, proliferation and ECM remodeling. Results: A higher concentration of alpha-SMA and proliferative cells were observed in the outer layers of the spheroids by immunofluorescence analyses. Choroidal and hepatic matrices were produced in 21 days. These tissue-engineered matrices contained fibronectin and collagen I. These devitalized matrices were seeded with UMCs in monolayer to study their change of morphology, proliferative state and ECM remodeling. Conclusions: We characterized the viability and cellular organization in both choroidal and hepatic spheroid models, and successfully generated tissue-engineered choroidal and hepatic matrices. With the optimization of these 3D models, we will better understand the steps involved in the progression of ocular melanoma to a metastatic stage. These complex models may replace the 2D model using tumor cells in monolayer for future drug screening, which will consider the impact of the microenvironment on UM therapeutic resistance. Citation Format: Olivier Chancy, Léo Piquet, Kelly Coutant, Andrew Mitchell, Solange Landreville. Tumorigenic properties of uveal melanoma cancer cells cultured in 3D or on a reconstructed extracellular matrix [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 50.

Abstract 1313: Hepatic pre-metastatic niche formation by uveal melanoma extracellular vesicles

Abstract Background: Uveal melanoma (UM) is the most common primary intraocular tumor in adults, and half cases develop incurable liver metastases. As the liver is the first and often the only metastatic site of UM, this suggests the existence of a favorable microenvironment for UM cells. Extracellular vesicles (EVs) are released by cancer cells, which allow oncoproteins or genetic material to be transferred to distant cells in order to modify their microenvironment and promote the spread of cancer. Our hypothesis is that EVs from the ocular tumor precondition the hepatic stroma by activating hepatic stellate cells (HSteCs) to promote the colonization by metastatic cells. Our study aims to determine the role of melanomic EVs in the mechanisms governing the remodeling of the liver microenvironment, including the stromal stiffness. Methods: EVs were isolated from UM cells and choroidal melanocytes by differential centrifugation. The mechanisms of EV internalization in HSteCs were investigated by confocal microscopy using inhibitors of endocytosis pathways. The impact of melanomic EVs on the viability, energy metabolism and gene expression profile of targeted cells was studied using MTS assay, LIVE/DEAD assay, and RNA-Sequencing. In addition, activation of the mechanotransduction signaling pathways were studied by immunofluorescence and Western blotting (FAK phosphorylation and YAP nuclear translocation. HSteCs were seeded on hydrogels of varying stiffness (0.8/2.5/8 kPa; which mimic the stiffness of healthy to highly fibrotic liver), and then exposed to EVs to study their level of activation/contractility by confocal microscopy (⍺SMA+) and traction force microscopy. The preconditioning of the liver by melanomic EVs were studied by in vivo fluorescence imaging in immunodeficient mice. The liver was collected, and the EV-driven activation of HSteCs and their remodeling of the hepatic stroma were analyzed by confocal and polarization microscopy. The number of metastatic lesions and the fibrosis were studied by histological staining using Masson's Trichrome and Hematoxylin & Eosin stains. Results: Melanomic EVs increased the proliferation and energy metabolism of HSteCs, and activated pro-fibrotic signaling pathways. The increased rigidity of hydrogels, which mimic advanced pathological stages, favored the internalization of melanomic EVs by HSteCs. The preconditioning of the liver by melanomic EVs promoted the development of metastases and the production of a stiffer extracellular matrix in the animal model. Conclusions: Melanomic EVs preconditioned the liver by establishing a pre-metastatic niche through the activation of stellate cells. A better understanding of the bidirectional crosstalk between metastatic UM cells and stellate cells at the premetastatic and micro-metastatic stages in the liver will allow to identify new molecular mechanisms or biophysical features that could be therapeutically targeted. Citation Format: Kelly Coutant, Léo Piquet, Enola Bollmann, Andrew Mitchell, François Bordeleau, Solange Landreville. Hepatic pre-metastatic niche formation by uveal melanoma extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1313.

Abstract 2580: Nuclear receptor coactivator 3 (NCOA3) fuels oncogenic pathways in uveal melanoma

Abstract Background: Uveal melanoma (UM) originates from melanocytes residing in the uveal tract of an eye. Despite effective local control, approximately half of patients develop metastatic disease resistant to systemic chemotherapy and presenting poor response to immunotherapy or targeted therapy. UM patients have a median overall survival of 6 to 12 months. The basis of the pathogenesis of UM are mutations in GNAQ and GNA11 genes resulting in constitutive activation of PKC/MAPK signaling. Here, we analyzed the role of NCOA3, a transcription factor coactivator that was previously shown to be modified by PKC in multiple cancer, as a factor potentiating oncogenic outcome of multiple oncogenic pathways activated in UM downstream of GNAQ or GNA11. Methods: We examined the impact of altered NCOA3 mRNA in UM, using publicly available TCGA dataset. Next, we used CRISPR/Cas9 technology to delete NCOA3 protein in MP41 UM cell line. We evaluated proliferation of MP41/NCOA3 KO cell subline and its parental counterpart in vitro by a colony formation assay. Next, we analyzed growth of xenografts of MP41/NCOA3 KO and parental cells in SCID/beige mice. To understand the molecular consequences of NCOA3 activity in UM, we performed a genome wide comparison of gene expression in MP41/NCOA3 KO and parental cells. Functional enrichment analysis was used to identify classes of genes associated with NCOA3 activity and determine similarities with datasets obtained by targeting proteins of GNAQ/GNA11 pathways and inhibiting activity of NCOA3 using a tool small molecule inhibitor SI-2 in UM cell lines. Results: We found that NCOA3 overexpression negatively correlated with UM patients’ progression free and overall survival. Knockout of NCOA3 gene in MP41 UM cells substantially reduced their proliferation in vitro and xenograft growth in mice. Global gene expression analysis revealed that NCOA3 activity is linked with transcriptional signatures associated with cell mitotic activity (cell cycle and DNA replication) and transcriptional machinery binding (especially affecting E2F and myc regulated programs). The MP41 NCOA3 KO normalized enrichment scores (NES) show high concordance with NES obtained after silencing GNAQ or GNA11 in UM cell lines and pharmacological targeting PKC or NCOA3. Conclusions: Inhibition of NCOA3 activity inhibits several oncogenic pathways in a concerted manner, limiting UM cell survival and tumor growth. Our results deliver proof of concept that NCOA3 is a master transcriptional co-regulator of oncogenic signaling in uveal melanoma and a potential therapeutic target. Citation Format: Aleksandra Rusin, Maria E. Ruiz Echartea, Darlene G. Skapura, Karen Berman deRuiz, Christel M. Davis, Eric A. Ehli, Sandra L. Grimm, Christian Coarfa, Salma Kaochar. Nuclear receptor coactivator 3 (NCOA3) fuels oncogenic pathways in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2580.

Abstract 3289: Melanoma patients with high and low target expression can benefit from TCR-CD3 bispecifics through direct and indirect mechanisms of tumor control

Abstract Background: ImmTAC molecules are TCR-CD3 bispecific fusion proteins that can redirect and activate polyclonal T cells to kill tumors. Tebentafusp, which targets gp100, is the first ImmTAC to demonstrate an overall survival benefit and is approved for the treatment of HLA-A*02:01 adult patients with unresectable or metastatic uveal melanoma1. Clinical benefit was observed in patients with both high and low gp100 expression2, suggesting direct and indirect mechanisms of tumor control, which we explored using in vitro models. Method: Uveal and cutaneous melanoma cell lines were pulsed with 0-1 µM peptide concentrations or were treated with supernatants from ImmTAC-activated PBMC, with or without blocking antibodies against IFNβ, IFNγ, TNFα, and IL6. Peptide-HLA (pHLA) molecules on tumor cell surface were quantified by microscopy. ImmTAC-redirected T cell activation and killing were measured by ELISPOT, MSD, and xCelligence assays. Antigen presentation machinery (APM) gene expression was measured by qPCR. Linear regression models were used to assess the influence of variables on ImmTAC-mediated T cell activation. Results: Peptide-pulsing of tumor cells showed a dose response relationship between surface pHLA number and ImmTAC-mediated T cell killing (R2= 0.85), which was confirmed by ImmTAC redirected killing assays using tumor lines with differential endogenous expression of pHLA targets. Higher effector: target (E: T) and higher pHLA number are the key factors, by regression analyses, influencing ImmTAC mediated T cell activation. These account for 53% and 29% variability in IFNγ production (p<0.01), respectively. To assess the indirect impact of ImmTAC-activated T cells on tumor cells, cell lines were pre-treated with supernatants. ImmTAC-activated PBMC supernatants reduced tumor growth (2 to 25-fold) and increased tumor death (20 - 80%) in 5 of 8 cell lines tested. Blocking IFNγ restored tumor growth by over 40%, which was further enhanced by additional blocking of IFNβ, TNFα, and IL6. All cell lines significantly upregulated APM genes and became more susceptible to direct ImmTAC-mediated T cell killing (1.4 to 2-fold increase). Conclusions: In vitro, ImmTAC bispecifics kill cancer cells by both direct and indirect mechanisms. Redirection of polyclonal T cells to kill melanoma cells is greatly influenced by surface pHLA number and E:T ratio. ImmTAC-induced cytokines inhibited tumor growth, increased tumor apoptosis and resulted in higher antigen presentation demonstrating the indirect effect of ImmTAC. These observations highlight how ImmTAC-treated melanoma patients could benefit through direct and indirect mechanisms of tumor control, and in part help to explain how patients with low gp100 expression can derive benefit from tebentafusp.

Abstract 2541: Heterogeneity of VDAC2-Bak/Bax mediated mitochondrial apoptosis can be exploited to develop effective and selective treatment against metastatic uveal melanoma

Abstract Apoptosis, a form of programmed cell death, is an evolutionarily conserved and genetically regulated phenomenon that is pivotal in maintaining tissue homeostasis and is an important player in regulating carcinogenesis. Mitochondria along with its resident proteins are the central player in regulating apoptosis. In recent years, VDAC2, an Outer Mitochondrial Membrane (OMM) protein has been shown to be the key recruitment factor for Bak, a pro-apoptotic protein to the OMM and, therefore is crucial for tBid-induced OMM permeabilization. We have previously found that unexpectedly, hepatocellular carcinoma (HCC) cells are vastly more sensitive to tBid induced apoptosis compared to normal hepatocytes and this difference is owed to high abundance of VDAC2-Bak in HCC. These findings are in line with multiple datasets posted in Oncomine, and protein atlas websites and may have clinical relevance. Combination of S63845, a selective Mcl-1 inhibitor, with TRAIL was effective at reducing tumor growth in vivo, but only in tumors expressing VDAC2. We hypothesized that the liver metastases of some extrahepatic primary tumors might also show enhanced sensitivity to VDAC2-Bak mediated apoptosis as compared to the normal liver tissue. Testing this hypothesis is important for uveal melanoma (UM) that primarily metastasizes in the liver, and these metastases lack an effective treatment. Interestingly, on testing several UM cell lines and patient samples we observed that VDAC2-Bak are upregulated in UM liver metastasis as well as compared to normal liver tissue. As expected, high VDAC2 expressing cell lines were also more sensitive to tBid induced OMM permeabilization. We propose that these differences can be exploited to design an effective therapy for metastasized UM. Citation Format: Piyush Mishra. Heterogeneity of VDAC2-Bak/Bax mediated mitochondrial apoptosis can be exploited to develop effective and selective treatment against metastatic uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2541.

Novel prognostication biomarker adipophilin reveals a metabolic shift in uveal melanoma and new therapeutic opportunities.

Metastatic uveal melanoma remains incurable at present. We previously demonstrated that loss of BAP1 gene expression in tumour cells triggers molecular mechanisms of immunosuppression in the tumour microenvironment (TME) of metastatic uveal melanoma. Adipophilin is a structural protein of lipid droplets involved in fat storage within mammalian cells, and its expression has been identified in uveal melanoma. We comprehensively evaluated adipophilin expression at the RNA (PLIN2) and protein levels of 80 patients of the GDC-TCGA-UM study and in a local cohort of 43 primary uveal melanoma samples respectively. PLIN2 expression is a survival prognosticator biomarker in uveal melanoma. Loss of adipophilin expression is significantly associated with monosomy 3 status and nuclear BAP1 losses in uveal melanoma tumours. Integrative transcriptomic and secretome studies show a relationship between transient loss of adipophilin expression and increased levels of tumour-associated macrophages and hypoxia genes, suggesting PLIN2-dependent changes in oxygen and lipid metabolism in the TME of low and high-metastatic risk uveal melanoma. We designed four adipophilin-based multigene signatures for uveal melanoma prognostication using a transcriptomic and secretome survival-functional network approach. Adipophilin-based multigene signatures were validated in BAP1-positive and BAP1-negative uveal melanoma cell lines using a next-generation RNA sequencing approach. We identified existing small molecules, mostly adrenergic, retinoid, and glucocorticoid receptor agonists, MEK, and RAF inhibitors, with the potential to reverse this multigene signature expression in uveal melanoma. Some of these molecules were able to impact tumour cell viability, and carvedilol, an adrenergic receptor antagonist, restored PLIN2 levels, mimicking the expression of normoxia/lipid storage signatures and reversing the expression of hypoxia/lipolysis signatures in co-cultures of uveal melanoma cells with human macrophages. These findings open up a new research line for understanding the lipid metabolic regulation of immune responses, with implications for therapeutic innovation in uveal melanoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Retraction: Long non-coding RNA FTH1P3 facilitates uveal melanoma cell growth and invasion through miR-224-5p.

Retraction: Long non-coding RNA FTH1P3 facilitates uveal melanoma cell growth and invasion through miR-224-5p.

Identification and validation of a CCL18-related signature for prediction of overall survival in patients with uveal melanoma

Uveal melanoma (UM), the most frequent primary intraocular tumor in adults, has poor prognosis. High C–C motif chemokine ligand 18 (CCL18) has been detected in various tumors and is closely correlated with patients’ clinicopathological characteristics. However, the essential role of CCL18 in UM remains unclear. Therefore, this study aimed to explore the prognostic value of CCL18 in UM. Uveal melanoma cells (M17) were transfected with pcDNA3.1-CCL18 si-RNA using Lipofectamine™ 2000. Cell growth and invasion abilities were measured through Cell Counting Kit-8 assay and invasion assay. RNA expression data and clinical and histopathological details were downloaded from the UM in The Cancer Genome Atlas (TCGA-UM) and GSE22138 datasets, which were defined as the training and validation cohorts, respectively. Univariate and multivariate Cox regression analyses were performed to identify significant prognostic biomarkers. The coefficients of these significant biomarkers generated by multivariate Cox proportional hazard regression analysis were used to establish a risk score formula. Functional enrichment analyses were also carried out. We found that downregulated CCL18 inhibits M17 cell growth and invasion in vitro. CCL18 may affect UM progression by altering C–C motif receptor 8 related pathways. Higher CCL18 expression was associated with worse clinical outcomes and tumor-specific death in the TCGA-UM dataset. Based on the coefficients obtained from the Cox proportional hazard regression analysis, a CCL18-related prognostic signature formula was constructed as follows: risk score = 0.05590 × age +2.43437 × chromosome 3 status +0.39496 × ExpressionCCL18. Notably, in this formula, the normal chromosome 3 was coded as 0, whereas the chromosome 3 loss was coded as 1. Each patient was assigned to either low-risk or high-risk groups using the median cut-off in the training cohort. High-risk patients survived for a shorter time than low-risk patients. The time-dependent and multivariate receiver operating characteristic curves showed promising diagnostic efficacy. Multivariate Cox regression analysis demonstrated the potential of this CCL18-related signature as an independent prognostic indicator. These results were validated using the GSE22138 dataset. In addition, in both TCGA-UM and GSE22138 datasets, stratification of clinical correlations and survival analyses based on this signature indicated the involvement of clinical progression and survival outcome in UM. In the high-risk group, Gene Ontology analyses mainly indicated the enrichment of immune response pathways, such as the T cell activation, response to interferon-gamma, antigen processing and presentation, interferon-gamma-mediated signaling pathway, MHC protein complex, MHC class II protein complex, antigen binding, and cytokine binding. Meanwhile, Kyoto Encyclopedia of Genes and Genomes analyses showed enrichments of pathways in cancer, cell adhesion, cytokine-cytokine receptor interaction, chemokine signaling pathway, Th1 and Th2 cell differentiation, and chemokine signaling pathway. Moreover, single-sample gene set enrichment analysis demonstrated the enrichment of almost all immune cells and immune functions in the high-risk group. In summary, a new prognostic CCL18-related signature was successfully established using the TCGA-UM dataset and validated using the GSE22138 dataset with meaningful predictive and diagnostic efficacies. This signature could serve as an independent and promising prognostic biomarker for patients with UM.

Monoacylglycerol lipase regulates macrophage polarization and cancer progression in uveal melanoma and pan-cancer.

Although lipid metabolism has been proven to play a key role in the development of cancer, its significance in uveal melanoma (UM) has not yet been elucidated in the available literature. To identify the expression patterns of lipid metabolism in 80 UM patients from the TCGA database, 47 genes involved in lipid metabolism were analyzed. Consensus clustering revealed two distinct molecular groups. ESTIMATE, TIMER, and ssGSEA analyses were done to identify the differences between the two subgroups in tumor microenvironment (TME) and immune state. Using Cox regression and Lasso regression analysis, a risk model based on differentially expressed genes (DEGs) was developed. To validate the expression of monoacylglycerol lipase (MGLL) and immune infiltration in diverse malignancies, a pan-cancer cohort from the UCSC database was utilized. Next, a single-cell sequencing analysis on UM patients from the GEO data was used to characterize the lipid metabolism in TME and the role of MGLL in UM. Finally, in vitro investigations were utilized to study the involvement of MGLL in UM. Two molecular subgroups of UM patients have considerably varied survival rates. The majority of DEGs between the two subgroups were associated with immune-related pathways. Low immune scores, high tumor purity, a low number of immune infiltrating cells, and a comparatively low immunological state were associated with a more favorable prognosis. An examination of GO and KEGG data demonstrated that the risk model based on genes involved with lipid metabolism can accurately predict survival in patients with UM. It has been demonstrated that MGLL, a crucial gene in this paradigm, promotes the proliferation, invasion, and migration of UM cells. In addition, we discovered that MGLL is strongly expressed in macrophages, specifically M2 macrophages, which may play a function in the M2 polarization of macrophages and M2 macrophage activation in cancer cells. This study demonstrates that the risk model based on lipid metabolism may be useful for predicting the prognosis of patients with UM. By promoting macrophage M2 polarization, MGLL contributes to the evolution of malignancy in UM, suggesting that it may be a therapeutic target for UM.

Development of Uveal Melanoma-Specific Aptamer for Potential Biomarker Discovery and Targeted Drug Delivery.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. However, challenges in early diagnosis, high risk of liver metastasis, and lack of effective targeted therapy lead to poor prognosis and high mortality of UM. Therefore, generating an effective molecular tool for UM diagnosis and targeted treatment is of great significance. In this study, a UM-specific DNA aptamer, PZ-1, was successfully developed, which could specifically distinguish molecular differences between UM cells and noncancerous cells with nanomolar-range affinity and presented excellent recognition ability for UM in vivo and clinical UM tissues. Subsequently, the binding target of PZ-1 on UM cells was identified as JUP (junction plakoglobin) protein, which held great potential as a biomarker and therapeutic target for UM. Meanwhile, the strong stability and internalization capacity of PZ-1 were also determined, and a UM-specific aptamer-guided "nanoship" was engineered to load and selectively release doxorubicin (Dox) to targeted UM cells, with lower toxicity to nontumor cells. Taken together, the UM-specific aptamer PZ-1 could serve as a molecular tool to discover the potential biomarker for UM and to achieve the targeted therapy of UM.

VE-Cadherin modulates β-catenin/TCF-4 to enhance Vasculogenic Mimicry

Vasculogenic Mimicry (VM) refers to the capacity to form a blood network from aggressive cancer cells in an independent way of endothelial cells, to provide nutrients and oxygen leading to enhanced microenvironment complexity and treatment failure. In a previous study, we demonstrated that VE-Cadherin and its phosphorylation at Y658 modulated kaiso-dependent gene expression (CCND1 and Wnt 11) through a pathway involving Focal Adhesion kinase (FAK). In the present research, using a proteomic approach, we have found that β-catenin/TCF-4 is associated with nuclear VE-cadherin and enhances the capacity of malignant melanoma cells to undergo VM in cooperation with VE-Cadherin; in addition, preventing the phosphorylation of Y658 of VE-cadherin upon FAK disabling resulted in VE-Cadherin/β-catenin complex dissociation, increased β-catenin degradation while reducing TCF-4-dependent genes transcription (C-Myc and Twist-1). Uveal melanoma cells knockout for VE-Cadherin loses β-catenin expression while the rescue of VE-Cadherin (but not of the phosphorylation defective VE-Cadherin Y658F mutant) permits stabilization of β-catenin and tumor growth reduction in vivo experiments. In vivo, the concomitant treatment with the FAK inhibitor PF-271 and the anti-angiogenic agent bevacizumab leads to a strong reduction in tumor growth concerning the single treatment. In conclusion, the anomalous expression of VE-Cadherin in metastatic melanoma cells (from both uveal and cutaneous origins), together with its permanent phosphorylation at Y658, favors the induction of the aggressive VM phenotype through the cooperation of β-catenin with VE-Cadherin and by enhancing TCF-4 genes-dependent transcription.

TAP1, a potential immune-related prognosis biomarker with functional significance in uveal melanoma

BackgroundTAP1 is an immunomodulation-related protein that plays different roles in various malignancies. This study investigated the transcriptional expression profile of TAP1 in uveal melanoma (UVM), revealed its potential biological interaction network, and determined its prognostic value.MethodsCIBERSORT and ESTIMATE bioinformatic methods were used on data sourced from The Cancer Genome Atlas database (TCGA) to determine the correlation between TAP1 expression, UVM prognosis, biological characteristics, and immune infiltration. Gene set enrichment analysis (GSEA) was used to discover the signaling pathways associated with TAP1, while STRING database and CytoHubba were used to construct protein–protein interaction (PPI) and competing endogenous RNA (ceRNA) networks, respectively. An overall survival (OS) prognostic model was constructed to test the predictive efficacy of TAP1, and its effect on the in vitro proliferation activity and metastatic potential of UVM cell line C918 cells was verified by RNA interference.ResultsThere was a clear association between TAP1 expression and UVM patient prognosis. Upregulated TAP1 was strongly associated with a shorter survival time, higher likelihood of metastasis, and higher mortality outcomes. According to GSEA analysis, various immunity-related signaling pathways such as primary immunodeficiency were enriched in the presence of elevated TAP1 expression. A PPI network and a ceRNA network were constructed to show the interactions among mRNAs, miRNAs, and lncRNAs. Furthermore, TAP1 expression showed a significant positive correlation with immunoscore, stromal score, CD8+ T cells, and dendritic cells, whereas the correlation with B cells and neutrophils was negative. The Cox regression model and calibration plots confirmed a strong agreement between the estimated OS and actual observed patient values. In vitro silencing of TAP1 expression in C918 cells significantly inhibited cell proliferation and metastasis.ConclusionsThis study is the first to demonstrate that TAP1 expression is positively correlated with clinicopathological factors and poor prognosis in UVM. In vitro experiments also verified that TAP1 is associated with C918 cell proliferation, apoptosis, and metastasis. These results suggest that TAP1 may function as an oncogene, prognostic marker, and importantly, as a novel therapeutic target in patients with UVM.

Targeting BAP1 with small compound inhibitor for colon cancer treatment

BRCA1-associated protein-1 (BAP1) is a ubiquitin C-terminal hydrolase domain-containing deubiquitinase. The gene encoding BAP1 is mutated in various human cancers, including mesothelioma, uveal melanoma and renal cell carcinoma. BAP1 plays roles in many cancer-related cellular functions, including cell proliferation, cell death, and nuclear processes crucial for genome stability, such as DNA repair and replication. While these findings suggest that BAP1 functions as a tumor suppressor, recent data also suggest that BAP1 might play tumor-promoting roles in certain cancers, such as breast cancer and hematopoietic malignancies. Here, we show that BAP1 is upregulated in colon cancer cells and tissues and that BAP1 depletion reduces colon cancer cell proliferation and tumor growth. BAP1 contributes to colon cancer cell proliferation by accelerating DNA replication and suppressing replication stress and concomitant apoptosis. A recently identified BAP1 inhibitor, TG2-179-1, which seems to covalently bind to the active site of BAP1, exhibits potent cytotoxic activity against colon cancer cells, with half-maximal inhibitory concentrations of less than 10 μM, and inhibits colon tumor growth. TG2-179-1 exerts cytotoxic activity by targeting BAP1, leading to defective replication and increased apoptosis. This work therefore shows that BAP1 acts oncogenically in colon cancer and is a potential therapeutic target for this cancer. Our work also suggests that TG2-179-1 can be developed as a potential therapeutic agent for colon cancer.

MiR-204 suppresses uveal melanoma cell migration and invasion through negative regulation of RAB22A.

Uveal melanoma (UM), a frequently seen adulthood primary ocular malignancy, shows high aggressiveness. Accumulating studies have revealed the crucial effects of microRNAs (miRNAs) on tumorigenesis and development in various human tumors. miR-204, the cancer-associated miRNA, shows dysregulation and is related to several human malignancies, but its effect on UM remains unknown. The present work focused on exploring miR-204's effect on UM and elucidating its possible molecular mechanisms. According to our results, miR-204 expression markedly increased within both UM tissues and cell lines. As revealed by functional analysis, miR-204 suppressed UM cell invasion and migration. Besides, RAB22A expression decreased through directly binding miR-204 into the corresponding 3' untranslated region (3'UTR) in UM cells. Furthermore, the RAB22A mRNA level increased, which was negatively related to the miR-204 level within UM samples. As revealed by mechanical research, miR-204 exerted its inhibition on the invasion and migration of UM cells via RAB22A. Taken together, this study suggested the tumor-suppressing effect of miR-204 on UM through down-regulating RAB22A. Thus, miR-204 may serve as the new anti-UM therapeutic target.

Targeting the cMET pathway to enhance immunotherapeutic approaches for mUM patients.

The liver is the most preferential initial site of metastasis for uveal melanoma (mUM), and this preference is associated with rapid mortality in mUM patients. Despite the significant clinical benefits of Immune checkpoint inhibitors (ICIs) in metastatic cutaneous melanoma patients, ICIs have shown little to no benefit in mUM patients. A potential reason for this inefficiency of ICI could be partly devoted to the involvement of the liver itself, thanks to its rich source of growth factors and immunosuppressive microenvironment. Uveal melanoma cells show increased expression of a transmembrane protein called cMET, which is known as the sole receptor for the Hepatocyte growth factor (HGF). Hyperactivation of cMET by HGF contributes to mUM development, and the liver, being the major source of HGF, may partially explain the metastasis of uveal melanoma cells to the liver. In addition, cMET/HGF signaling has also been shown to mediate resistance to ICI treatment, directly and indirectly, involving tumor and immune cell populations. Therefore, targeting the cMET/HGF interaction may enhance the efficacy of immunotherapeutic regimes for mUM patients. Hence in this minireview, we will discuss the rationale for combining cMET inhibitors/antibodies with leading immune checkpoint inhibitors for treating mUM. We will also briefly highlight the challenges and opportunities in targeting cMET in mUM.

PTK6 inhibits autophagy to promote uveal melanoma tumorigenesis by binding to SOCS3 and regulating mTOR phosphorylation

Autophagy dysfunction is one of the common causes of tumor formation and plays an important role in uveal melanoma (UM). However, little is known about the regulatory mechanisms of autophagy in UM. Here, we show that PTK6 can promote the proliferation, migration, and invasion of UM cells by inhibiting autophagy. SOCS3 can inhibit the proliferation, migration, and invasion of UM cells. Overexpression of SOCS3 can partially rescue the PTK6-induced promotion of UM cell proliferation, migration, and invasion. Mechanistically, PTK6 can bind to SOCS3, and SOCS3 can downregulate the expression of PTK6. Furthermore, PTK6 can upregulate the phosphorylation of mTOR to inhibit autophagy. Taken together, our findings demonstrate the functions of PTK6 and SOCS3 in UM cells and targeting the SOCS3-PTK6 signaling axis might be a novel and promising therapeutic strategy for patients with UM.

LINC01278 Induces Autophagy to Inhibit Tumour Progression by Suppressing the mTOR Signalling Pathway.

Uveal melanoma (UM) is an aggressive intraocular malignant tumour that is closely related to autophagic dysfunction. We aimed to identify autophagy-related long noncoding RNAs (lncRNAs) to elucidate the molecular mechanism of UM. Here, we show that LINC01278 is a new potential biomarker with clinical prognostic value in UM through bioinformatics analysis. Application of an autophagy inhibitor (3-MA) and an autophagy agonist (MG-132) indicated that LINC01278 can inhibit UM cell proliferation, migration, and invasion by inducing autophagy. A xenograft nude mouse model was used to examine the tumorigenesis of UM cells in vivo. Mechanistically, LINC01278 can inhibit the mTOR signalling pathway to activate autophagy, as shown by experiments with an mTOR agonist (MHY1485) and mTOR inhibitor (rapamycin) treatment. Our findings indicate that LINC01278 functions as a tumour suppressor by inhibiting the mTOR signalling pathway to induce autophagy. Targeting the LINC01278-mTOR axis might be a novel and promising therapeutic approach for UM.

Abstract A009: NR2F1 inhibits Gaq/11-YAP-TAZ signaling and limits reactivation of uveal melanoma disseminated cancer cells via induction of dormancy

Abstract Introduction: At the time of diagnosis, less than 5% of uveal melanoma (UM) patients have evidence of metastasis. However, up to 50% of UM patients will succumb to advanced metastatic in liver. UM cancer cells can persist undetected in liver for prolonged periods mainly and often take years-to-decades to grow out and do so without recurrence at the primary site. However, the mechanisms underlying the prolonged dormancy and why the liver is a preferred site for survival and eventual regrowth are a long-standing medical mystery. Results: GNAQ/11 are the main driving mutations in UM that activate downstream oncogenic YAP signaling. Using Gaq/11mut /BAPwt human uveal melanoma lines, we reveal that NR2F1, a neural cell commitment regulator is the main driver of regulating dormancy program. RNA-seq of rare UM disseminated cancer cells (DCCs) in livers identified that dormant DCCs upregulate the expression of neural differentiation genes, including NR2F1, while silencing Gaq downstream YAP/TAZ and cell cycle gene expression compared to proliferative cell clusters. Targeted pharmacological inhibition of Gaq/11mut signaling using YM-254890, an inhibitor of Gaq/11 resulted in NR2F1 upregulation and robust UM growth arrest. In contrast, inhibition of NR2F1 either by siRNA or CRISPR/cas9 upregulated YAP1 and its downstream targets at both transcript and protein levels along with increased colony growth in 3D culture. Importantly, UM solitary DCCs in nude mouse upregulated NR2F1 and in vivo CRISPR KO of NR2F1 results in dormant UM DCC awakening and massive metastatic growth in the murine liver. Immunofluorescence of UM patients’ liver metastatic lesions showed high expression of NR2F1 in solitary DCCs compared to the tumor mass. Chromatin-immunoprecipitation assays revealed that NR2F1 binds to the YAP1 promoter and regulates its gene expression. OMM1.3 CRISPR/Cas9 KO of NR2F1 cells showed increased recruitment of H3K4me3 and H3K27ac histone marks onto the YAP1 promoter while overexpression of NR2F1 decreased the recruitment of histone activating marks. Co-immunoprecipitation assay showed increased YAP1-TEAD1 complex formation in NR2F1 KO OMM1.3 cells while overexpression of NR2F1 reduced the abundance of this complex. Summary: Our work suggests that NR2F1 is required to maintain the single cell state of UM cells in the liver by inhibiting the YAP signally pathway. This study provides insight into the previously unrecognized mechanism of uveal melanoma dormancy opening a new space to design anti-metastatic therapies for UM patients. Citation Format: Rama Kadamb, Melisa Lopez Anton, Vivian Chua, Timothy Purwin, Maria Angelo Nieto Toledano, Andrew Aplin, Julio Aguirre-Ghiso. NR2F1 inhibits Gaq/11-YAP-TAZ signaling and limits reactivation of uveal melanoma disseminated cancer cells via induction of dormancy [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A009.