Growth Of Uveal Melanoma Cells Research Articles

Quercetin Impairs the Growth of Uveal Melanoma Cells by Interfering with Glucose Uptake and Metabolism.

Monosomy 3 in uveal melanoma (UM) increases the risk of lethal metastases, mainly in the liver, which serves as the major site for the storage of excessive glucose and the metabolization of the dietary flavonoid quercetin. Although primary UMs with monosomy 3 exhibit a higher potential for basal glucose uptake, it remains unknown as to whether glycolytic capacity is altered in such tumors. Herein, we initially analyzed the expression of n = 151 genes involved in glycolysis and its interconnected branch, the "pentose phosphate pathway (PPP)", in the UM cohort of The Cancer Genome Atlas Study and validated the differentially expressed genes in two independent cohorts. We also evaluated the effects of quercetin on the growth, survival, and glucose metabolism of the UM cell line 92.1. The rate-limiting glycolytic enzyme PFKP was overexpressed whereas the ZBTB20 gene (locus: 3q13.31) was downregulated in the patients with metastases in all cohorts. Quercetin was able to impair proliferation, viability, glucose uptake, glycolysis, ATP synthesis, and PPP rate-limiting enzyme activity while increasing oxidative stress. UMs with monosomy 3 display a stronger potential to utilize glucose for the generation of energy and biomass. Quercetin can prevent the growth of UM cells by interfering with glucose metabolism.

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.

CEP-1347 Dually Targets MDM4 and PKC to Activate p53 and Inhibit the Growth of Uveal Melanoma Cells.

Uveal melanoma (UM) is among the most common primary intraocular neoplasms in adults, with limited therapeutic options for advanced/metastatic disease. Since UM is characterized by infrequent p53 mutation coupled with the overexpression of MDM4, a major negative regulator of p53, we aimed to investigate in this study the effects on UM cells of CEP-1347, a novel MDM4 inhibitor with a known safety profile in humans. We also examined the impact of CEP-1347 on the protein kinase C (PKC) pathway, known to play a pivotal role in UM cell growth. High-grade UM cell lines were used to analyze the effects of genetic and pharmacological inhibition of MDM4 and PKC, respectively, as well as those of CEP-1347 treatment, on p53 expression and cell viability. The results showed that, at its clinically relevant concentrations, CEP-1347 reduced not only MDM4 expression but also PKC activity, activated the p53 pathway, and effectively inhibited the growth of UM cells. Importantly, whereas inhibition of either MDM4 expression or PKC activity alone failed to efficiently activate p53 and inhibit cell growth, inhibition of both resulted in effective activation of p53 and inhibition of cell growth. These data suggest that there exists a hitherto unrecognized interaction between MDM4 and PKC to inactivate the p53-dependent growth control in UM cells. CEP-1347, which dually targets MDM4 and PKC, could therefore be a promising therapeutic candidate in the treatment of UM.

Kinome profiling identifies MARK3 and STK10 as potential therapeutic targets in uveal melanoma

Most uveal melanoma cases harbor activating mutations in either GNAQ or GNA11. Despite activation of the mitogen-activated protein kinase (MAPK) signaling pathway downstream of Gαq/11, there are no effective targeted kinase therapies for metastatic uveal melanoma. The human genome encodes numerous understudied kinases, also called the “dark kinome”. Identifying additional kinases regulated by Gαq/11 may uncover novel therapeutic targets for uveal melanoma. In this study, we treated GNAQ-mutant uveal melanoma cell lines with a Gαq/11 inhibitor, YM-254890, and conducted a kinase signaling proteomic screen using multiplexed-kinase inhibitors followed by mass spectrometry. We observed downregulated expression and/or activity of 22 kinases. A custom siRNA screen targeting these kinases demonstrated that knockdown of microtubule affinity regulating kinase 3 (MARK3) and serine/threonine kinase 10 (STK10) significantly reduced uveal melanoma cell growth and decreased expression of cell cycle proteins. Additionally, knockdown of MARK3 but not STK10 decreased ERK1/2 phosphorylation. Analysis of RNA-sequencing and proteomic data showed that Gαq signaling regulates STK10 expression and MARK3 activity. Our findings suggest an involvement of STK10 and MARK3 in the Gαq/11 oncogenic pathway and prompt further investigation into the specific roles and targeting potential of these kinases in uveal melanoma.

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.

Regulator of G protein signaling 2 inhibits Gαq-dependent uveal melanoma cell growth

Activating mutations in Gαq/11 are a major driver of uveal melanoma (UM), the most common intraocular cancer in adults. While progress has recently been made in targeting Gαq/11 for UM therapy, the crucial role for these proteins in normal physiology and their high structural similarity with many other important GTPase proteins renders this approach challenging. The aim of the current study was to validate whether a key regulator of Gq signaling, regulator of G protein signaling 2 (RGS2), can inhibit Gαq-mediated UM cell growth. We used two UM cell lines, 92.1 and Mel-202, which both contain the most common activating mutation GαqQ209L and developed stable cell lines with doxycycline-inducible RGS2 protein expression. Using cell viability assays, we showed that RGS2 could inhibit cell growth in both of these UM cell lines. We also found that this effect was independent of the canonical GTPase-activating protein activity of RGS2 but was dependent on the association between RGS2 and Gαq. Furthermore, RGS2 induction resulted in only partial reduction in cell growth as compared to siRNA-mediated Gαq knockdown, perhaps because RGS2 was only able to reduce mitogen-activated protein kinase signaling downstream of phospholipase Cβ, while leaving activation of the Hippo signaling mediators yes-associated protein 1/TAZ, the other major pathway downstream of Gαq, unaffected. Taken together, our data indicate that RGS2 can inhibit UM cancer cell growth by associating with GαqQ209L as a partial effector antagonist.

Abstract 6406: FAK and MEK co-targeting: A new multimodal precision therapy for GNAQ-driven uveal melanoma

Abstract Uveal melanoma (UM) is characterized by gain-of-function mutations in GNAQ or GNA11, encoding Gα proteins from the Gq/11 family. UM is the most common eye malignancy in adults. Approximately 50% of UM patients develop liver metastasis (mUM) within 5-10 years after diagnosis, independently of the successful treatment of the primary lesions. mUM is refractory to cytotoxic, targeted, and immunotherapies, with most mUM patients dying within a year. Recent information suggests that GNAQ-oncogenic signaling involves a non-canonical pathway distinct from the activation of PLCβ and PKC-MEK-ERK, which may explain the failure of MEK inhibitors (MEKi) in increasing mUM patient survival. Instead, we found that GNAQ promotes the activation of YAP1, a key oncogenic driver, by a mechanism involving the activation of RhoA by the direct association of Gαq to TRIO, a Rho-GEF (Cancer Cell, 2014). In turn, YAP1 is essential for uveal melanoma cell growth, however no effective and safe YAP1 inhibitors are currently available. Using a novel bioinformatics pipeline, we recently found that PTK2, encoding Focal Adhesion Kinase (FAK), is a synthetic lethal gene with GNAQ activation, and uncovered that GNAQ controls YAP1 through FAK (Cancer Cell, 2019). This study identified FAK as a druggable signaling hub downstream from GNAQ in UM. However, activation of compensatory pathways often results in resistance to targeted agents. Here, we combined the use of CRISPR-Cas9 sgRNA screens with a recently described Cancer Signaling Toolkit approach to identify synthetic lethal interactions enhancing the response to FAKi and signaling networks mediating drug resistance, respectively. Remarkably, both approaches converged to reveal that co-targeting FAK and the MEK-ERK pathway would be a promising combination for treatment of UM. Indeed, MEK-ERK pathway inhibition by multiple approved MEKis (e.g., trametinib), combined with FAK inhibition (VS-4718 or defactinib), showed remarkable synergistic growth inhibitory effects in UM cells. Additionally, the novel RAF/MEK inhibitor RO5126766 also showed synergistic anti-proliferative effects with defactinib. Accordingly, FAKi combination with MEKi exerted cytotoxic effects (apoptotic death) leading to rapid tumor shrinkage in UM xenografts, whereas single drugs were primarily cytostatic. Furthermore, the FAKi/MEKi combination was successful at reducing tumor burden in recently developed liver metastasis UM models. By coupling the unique genetic landscape of UM with the power of unbiased computational pipelines and systems biology genetic screens, our studies revealed that FAK and MEK-ERK co-targeting may provide a new network-based precision therapeutic strategy for mUM treatment. Indeed, the combination of defactinib and RO5126766 is currently being evaluated in patients with various solid tumors (NCT03875820), and could be explored in mUM based on these preclinical findings. Citation Format: Justine S. Paradis, Monica Acosta, Nadia Arang, Robert Saddawi-Konefka, Ayush Kishore, Takahito Sugase, Xiaodong Feng, Kris C. Wood, Silvia Coma, Mizue Terai, Takami Sato, Jonathan A. Pachter, J. Silvio Gutkind. FAK and MEK co-targeting: A new multimodal precision therapy for GNAQ-driven uveal melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6406.

Abstract IA08: Regulation of YAP by tyrosine phosphorylation of core Hippo pathway components: Lessons learned from the GNAQ oncogene and FAK

Abstract Dysregulation of the Hippo signaling pathway and the consequent YAP1 activation is a frequent event in human malignancies, yet the underlying molecular mechanisms are still poorly understood. In prior studies we have focused on uveal melanoma, the most frequent eye malignancy in adults that is caused by constitutively active mutations in the GNAQ oncogenes, which encode Gαq and Gα11 heterotrimeric G protein α subunits. We found that GNAQ promotes the activation of YAP1 by a mechanism involving the activation of RhoA by the direct association of Gαq to TRIO, a Rho-GEF, and the consequent stimulation of acting polymerization and the release of YAP from its complex with AMOT. We also showed that YAP1 is essential for uveal melanoma cell growth, thus representing a potential therapeutic target for this malignancy. However, no effective and safe YAP1 inhibitors are currently available. Using a novel integrated bioinformatics pipeline, we recently found that PTK2, encoding Focal Adhesion Kinase (FAK), represents a candidate synthetic lethal gene with GNAQ activation (Feng et al., Cancer Cell 2019). We found that Gαq activates FAK through the TRIO-RhoA noncanonical Gαq-signaling, and that genetic ablation or pharmacologic inhibition of FAK inhibits uveal melanoma growth. Surprisingly, analysis of the FAK-regulated transcriptome indicated that GNAQ stimulates YAP1 through FAK. Dissection of the underlying mechanism revealed that FAK regulates YAP1 by tyrosine phosphorylation of MOB1, thereby disrupting MOB1/LATS association and core Hippo pathway signaling. By coupling the power of an unbiased computational pipeline to the unique genetic landscape of uveal melanoma, we uncovered a molecular framework regulating YAP1 and identified FAK as a druggable signaling hub downstream from GNAQ in uveal melanoma. Our findings established FAK as a potential therapeutic target for uveal melanoma and other diseases that may involve FAK activation and unrestrained YAP1 function. Further studies on how multiple growth promoting and oncogenic tyrosine kinase signaling networks may converge to phosphorylate core Hippo pathway components to activate YAP1 in cancer will be also discussed. Citation Format: J. Silvio Gutkind. Regulation of YAP by tyrosine phosphorylation of core Hippo pathway components: Lessons learned from the GNAQ oncogene and FAK [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA08.

Arylsulfonamide 64B Inhibits Hypoxia/HIF-Induced Expression of c-Met and CXCR4 and Reduces Primary Tumor Growth and Metastasis of Uveal Melanoma.

Uveal melanoma (UM) is the most prevalent and lethal intraocular malignancy in adults. Here, we examined the importance of hypoxia in UM growth and tested the antitumor effects of arylsulfonamide 64B, an inhibitor of the hypoxia-induced factor (HIF) pathway in animal models of UM and investigated the related mechanisms. UM cells were implanted in the uvea of mice eyes and mice systemically treated with 64B. Drug effect on primary eye tumor growth, circulating tumor cells, metastasis formation in liver, and survival were examined. 64B effects on UM cell growth, invasion and hypoxia-induced expression of C-X-C chemokine receptor type 4 (CXCR4) and mesenchymal-epithelial transition factor (c-Met) were measured. Luciferase reporter assays, chromatin immunoprecipitation, co-immunoprecipitation, and cellular thermal shift assays were used to determine how 64B interferes with the HIF transcriptional complex. Systemic administration of 64B had potent antitumor effects against UM in several orthotopic mouse models, suppressing UM growth in the eye (∼70% reduction) and spontaneous liver metastasis (∼50% reduction), and extending mice survival (P < 0.001) while being well tolerated. 64B inhibited hypoxia-induced expression of CXCR4 and c-Met, 2 key drivers of tumor invasion and metastasis. 64B disrupted the HIF-1 complex by interfering with HIF-1α binding to p300/CBP co-factors, thus reducing p300 recruitment to the MET and CXCR4 gene promoters. 64B could thermostabilize p300, supporting direct 64B binding to p300. Our preclinical efficacy studies support the further optimization of the 64B chemical scaffold toward a clinical candidate for the treatment of UM.

The bidirectional crosstalk between metastatic uveal melanoma cells and hepatic stellate cells engenders an inflammatory microenvironment

Uveal melanoma is the most common primary ocular neoplasm in adults. It is peculiar for its hematogenous dissemination and its high propensity to spread to the liver. Current treatments rarely prolong patient survival. We hypothesized that metastatic uveal melanoma cells modulate the function of surrounding hepatic stellate cells to facilitate their own growth and survival. This study was conducted to investigate the role of the hepatic microenvironment on uveal melanoma aggressiveness. We demonstrated that the paracrine signaling of surrounding hepatic stellate cells have more transcriptional impact on metastatic uveal melanoma cells. Upregulated transcripts were linked to inflammation and included several interleukins. The uveal melanoma-stellate cell crosstalk induced as well the expression of transmembrane integrins. In addition, the interleukin-6 receptor inhibitor Tocilizumab did not reduce the growth of uveal melanoma cells. Our results provide evidence that inflammatory mediators are key players in the homing of uveal melanoma cells to the liver. The bidirectional crosstalk between uveal melanoma cells and hepatic stellate cells involved pro-fibrogenic interleukins. The inflammatory characteristics of the metastatic microenvironment might offer relevant therapeutic opportunities in uveal melanoma.

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

Growing evidences indicated that Long noncoding RNAs (lncRNAs) played important roles in tumor initiation and progression. However, the function and mechnism of lncRNA ferritin heavy chain 1 pseudogene 3 (FTH1P3) remain unknown in uveal melanoma. We showed that the expression level of FTH1P3 was upregulated in uveal melanoma cell lines and tissues. Elevated expression of FTH1P3 promoted uveal melanoma cell proliferation, cell cycle and migration. Moreover, we found that FTH1P3 was a direct target gene of miR-224-5p in uveal melanoma cell. Overexpression of FTH1P3 suppressed miR-224-5p expression and promoted the expression of Rac1 and Fizzled 5, which were the direct target genes of miR-224-5p. Furthermore, we showed that miR-224-5p expression level was downregulated in uveal melanoma cell lines and tissues. FTH1P3 expression was inversely correlated with the miR-224-5p expression in uveal melanoma tissues. Ectopic expression of miR-224-5p decreased uveal melanoma cell proliferation, cell cycle and migration. Elevated expression of FTH1P3 enhanced uveal melanoma cell proliferation and migration by inhibiting miR-224-5p expression. These results suggest that lncRNA FTH1P3 plays a crucial role in uveal melanoma. Investigation of the underlying mechanism may be a target for the treatment of uveal melanoma.

MiR-367 promotes uveal melanoma cell proliferation and migration by regulating PTEN.

We aimed to investigate the biological role of miR-367 in uveal melanoma cell growth and migration, and the underlying mechanism responsible. Quantitative real-time polymerase chain reaction was performed to evaluate miR-367 expression in uveal melanoma tissue samples and cell lines. A miR-367 mimic, miR-367 inhibitor, and negative control oligonucleotide were transfected into these cells to investigate the function of this microRNA. In addition, the role of PTEN in miR-367-mediated uveal melanoma cell growth and migration was evaluated. miR-367 was significantly upregulated in uveal melanoma cells and tissue samples (both P < 0.01). Its inhibition suppressed the proliferation, cell cycle transition, and migration of such cells, and increased levels had the opposite effect. PTEN was confirmed to be a target gene of miR-367. More importantly, co-transfection with a PTEN construct lacking the 3'-untranslated region mitigated miR-367 mimic-induced promotion of uveal melanoma cell proliferation and migration. In summary, miR-367 was found to be upregulated in this malignancy, and may promote uveal melanoma cell proliferation and migration, at least in part by regulating PTEN.

Hippo-Independent Activation of YAP by the GNAQ Uveal Melanoma Oncogene through a Trio-Regulated Rho GTPase Signaling Circuitry

Mutually exclusive activating mutations in the GNAQ and GNA11 oncogenes, encoding heterotrimeric Gαq family members, have been identified in ∼ 83% and ∼ 6% of uveal and skin melanomas, respectively. However, the molecular events underlying these GNAQ-driven malignancies are not yet defined, thus limiting the ability to develop cancer-targeted therapies. Here, we focused on the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway that controls organ size. We found that Gαq stimulates YAP through a Trio-Rho/Rac signaling circuitry promoting actin polymerization, independently of phospholipase Cβ and the canonical Hippo pathway. Furthermore, we show that Gαq promotes the YAP-dependent growth of uveal melanoma cells, thereby identifying YAP as a suitable therapeutic target in uveal melanoma, a GNAQ/GNA11-initiated human malignancy.

Uveal melanoma cell growth is inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP-dependent kinase.

To evaluate the effects and mechanism of aminoimidazole carboxamide ribonucleotide (AICAR), an AMP-dependent kinase (AMPK) activator, on the growth of uveal melanoma cell lines. Four different cell lines were treated with AICAR (1-4 mM). Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Cell cycle analysis was conducted by flow cytometry; additionally, expression of cell-cycle control proteins, cell growth transcription factors, and downstream effectors of AMPK were determined by RT-PCR and Western blot. Aminoimidazole carboxamide ribonucleotide inhibited cell growth, induced S-phase arrest, and led to AMPK activation. Aminoimidazole carboxamide ribonucleotide treatment was associated with inhibition of eukaryotic translation initiation factor 4E-BP1 phosphorylation, a marker of mammalian target of rapamycin (mTOR) pathway activity. Aminoimidazole carboxamide ribonucleotide treatment was also associated with downregulation of cyclins A and D, but had minimal effects on the phosphorylation of ribosomal protein S6 or levels of the macroautophagy marker LC3B. The effects of AICAR were abolished by treatment with dipyridamole, an adenosine transporter inhibitor that blocks the entry of AICAR into cells. Treatment with adenosine kinase inhibitor 5-iodotubericidin, which inhibits the conversion of AICAR to its 5'-phosphorylated ribotide 5-aminoimidazole-4-carboxamide-1-D-ribofuranosyl-5'-monophosphate (ZMP; the direct activator of AMPK), reversed most of the growth-inhibitory effects, indicating that some of AICAR's antiproliferative effects are mediated at least partially through AMPK activation. Aminoimidazole carboxamide ribonucleotide inhibited uveal melanoma cell proliferation partially through activation of the AMPK pathway and downregulation of cyclins A1 and D1.

MicroRNA 145 may play an important role in uveal melanoma cell growth by potentially targeting insulin receptor substrate-1

Background MicroRNAs (miRNAs) contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. In this study, we investigated the role of miR-145 in the pathogenesis of uveal melanoma. Methods Expression profiles of miRNAs in uveal melanoma were performed using Agilent miRNA array. Quantitative real-time polymerase chain reaction was used to screen the expression levels of miR-145 in normal uveal tissue, uveal melanoma tissue, and uveal melanoma cell lines. Lenti-virus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145. Cell proliferation, cell cycle, and cell apoptosis of these miR-145 overexpression cell lines were examined by MTT assay and flow cytometry respectively. The target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase reporter assay. The expression of insulin-like growth factor-1 receptor (IGF-1R), insulin receptor substrate-1 (IRS-1) proteins was determined by Western blotting analysis. IRS-1 was knocked down in OCM-1 cells. TUNEL, BrdU, and flow cytometry assay were performed in IRS-1 knocked down OCM-1 cell lines to analyze its function. Results Forty-seven miRNAs were up regulated in uveal melanoma and 61 were down regulated. miR-145 expression was significantly lower in uveal melanoma sample and the cell lines were compared with normal uveal sample. Overexpression of miR-145 suppressed cell proliferation by blocking the G1 phase entering S phase in uveal melanoma cells, and promoted uveal melanoma cell apoptosis. IRS-1 was identified as a potential target of miR-145 by dual luciferase reporter assay. Knocking down of IRS-1 had similar effect as overexpression of miR-145. Conclusion miR-145 might act as a tumor suppressor in uveal melanoma, and downregulation of the target IRS-1 might be a potential mechanism.

Simultaneous Inhibition of the HGF/MET and Erk1/2 Pathways Affect Uveal Melanoma Cell Growth and Migration

PurposeNearly all primary uveal melanoma (UM) that metastasize involve the liver. Hepatocyte growth factor (HGF) is proposed to be an important microenvironmental element in attracting/supporting UM metastasis through activation of MET. The majority (>85%) of UM express mutations in the G-alpha proteins, that drive the MEK-ERK1/2 pathway. Thus, we proposed that the combination of MET and MEK inhibition would inhibit the growth and migration of G-alpha protein mutant versus non-mutant UM cells.MethodsWestern-blots demonstrated the relative protein levels of ERK1/2 and MET in UM cells. Cells were treated with the small molecule inhibitors AZD6244 (MEKi) and/or MK-8033 (METi) and downstream markers evaluated. Further studies determined the effect of combination MEKi and METi treatment on cell growth, apoptosis and migration.ResultsAll G-alpha protein mutant UM cell lines express MET mRNA and protein. The level of mRNA expression correlates with protein expression. MEKi, but not METi treatment results in markedly reduced ERK1/2 phosphorylation. Either MEKi or METi treatment alone results in reduced cell proliferation, but only modest induction of apoptosis. The combination MEKi+METi results in significant reduction of proliferation in G-alpha protein mutant cells. UM cell migration was blocked by METi, but not MEKi treatment.ConclusionsMET protein expression showed no correlation with G-alpha protein mutation status. Combining MEKi with METi treatment has added benefit to either treatment alone in reducing G-alpha protein mutant UM cell growth. Combining METi with MEKi treatment adds the effect of limiting uveal melanoma cell migration.

Tumor-targeting TRAIL expression mediated by miRNA response elements suppressed growth of uveal melanoma cells

Malignant uveal melanoma severely damages eye function and is prone to metastasize to other organs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent to treat uveal melanoma because of its induction of apoptosis in cancer cells both at primary and metastatic sites. However, TRAIL therapy lacks tumor specificity in the current delivery systems for uveal melanoma treatment, thereby causing cytotoxiciy to normal tissues. To improve uveal melanoma specificity of adenovirus-based TRAIL introduction, we used miRNA response elements (MREs) of miR-34a, miR-137 and miR-182, which have been shown to have reduced expression in uveal melanoma cells, to regulate its expression. miR-34a, miR-137 and miR-182 all had lower expression levels in uveal melanoma cell lines, compared with normal cells. MREs-regulated luciferase activity was reduced in normal cell lines, but not significantly attenuated in uveal melanoma cells. The infection of MRE-regulated TRAIL-expressing adenoviral vector (Ad-TRAIL-3MREs) led to high level of TRAIL expression in uveal melanoma cell lines, but not in normal cells. Strong expression of TRAIL had a high anti-tumor capacity by inducing apoptosis in uveal melanoma cells. In contrast, Ad-TRAIL-3MREs had no cytotoxicity to normal cell lines. Animal experiments further confirmed tumor-suppressing effect of Ad-TRAIL-3MREs on uveal melanoma xenografts and its biosafety to hepatic tissues. Collectively, we constructed an MRE-directed TRAIL-expressing adenoviral vector and provided evidence that this vector possessed high anti-tumor activity and uveal melanoma specificity.

The Pharmacological NF-κB Inhibitor BAY11-7082 Induces Cell Apoptosis and Inhibits the Migration of Human Uveal Melanoma Cells

Uveal melanomas are highly metastatic and have high rate of recurrence due to the lack of effective systemic therapy. The identification of important survival pathways in uveal melanomas provides novel therapeutic targets for effective treatment. In the present study, we found that the NF-κB signaling pathway was constitutively and highly activated in uveal melanoma cells. Treatment with the pharmacological NF-κB specific inhibitor BAY11-7082 markedly decreased the nuclear translocation of NF-κB. In a dose-dependent setting, BAY11-7082 inhibited the proliferation and growth of uveal melanoma cells by inducing apoptosis without effect on cell cycle. The migration capacity of uveal melanoma cells was also significantly suppressed by BAY11-7082 treatment. Mechanistically, BAY11-7082 increased the activity of caspase 3 and reduced the expression of anti-apoptotic protein Bcl-2, but did not influence the expression of pro-apoptotic protein Bax. Furthermore, BAY11-7082 induced uveal melanoma cell apoptosis and inhibited xenograft tumor growth in vivo. Collectively, the present study identified NF-κB as an important survival signal for uveal melanoma cells and suggested that administration of specific NF-κB inhibitor BAY11-7082 could serve as an effective treatment for patients with uveal melanoma.

Protein Kinase C Inhibitor AEB071 Targets Ocular Melanoma Harboring GNAQ Mutations via Effects on the PKC/Erk1/2 and PKC/NF-κB Pathways

Somatic GNAQ mutations at codon 209 have been identified in approximately 50% of uveal melanomas and have been reported to be oncogenic through activating PLCβ/PKC/Erk1/2 pathways. We hypothesized that protein kinase C (PKC) may provide new opportunities for therapeutic targeting of uveal melanoma carrying GNAQ mutations. To test this hypothesis, uveal melanoma cells harboring wild-type or mutant GNAQ were treated with the PKC inhibitor AEB071 (sotrastaurin) or infected with lentivirus-expressing short hairpin RNAs (shRNA) targeting PKC isoforms. Notably, AEB071 at low micromolar concentrations significantly inhibited the growth of uveal melanoma cells harboring GNAQ mutations through induction of G(1) arrest and apoptosis. However, AEB071 had little effect on uveal melanoma cells carrying wild-type GNAQ. AEB071-mediated cell inhibition in the GNAQ-mutated uveal melanoma was accompanied by inhibition of extracellular signal-regulated kinase (Erk)1/2 phosphorylation, NF-κB, decreased expression of cyclin D1, survivin, Bcl-xL, and XIAP, and increased expression of cyclin-dependent kinase inhibitor p27(Kip1). AEB071 suppressed the expression of PKC α, β, δ, ε, and θ in GNAQ-mutated uveal melanoma cells. Our findings from shRNA-mediated knockdown studies revealed that these PKC isoforms are functionally important for uveal melanoma cells harboring GNAQ mutations. Furthermore, inhibitors of Erk1/2 and NF-κB pathways reduced viability of uveal melanoma cells. Together, our findings show that AEB071 exerts antitumor action on uveal melanoma cells carrying GNAQ mutations via targeting PKC/Erk1/2 and PKC/NF-κB pathways. Targeted PKC inhibition with drugs such as AEB071 offers novel therapeutic potential for uveal melanoma harboring GNAQ mutations.

Role of MicroRNA-182 in Posterior Uveal Melanoma: Regulation of Tumor Development through MITF, BCL2 and Cyclin D2

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play central roles in diverse pathological processes. In this study, we investigated the effect of microRNA-182 (miR-182) on the development of posterior uveal melanomas. Initially, we demonstrated that miR-182 expression was dependent on p53 induction in uveal melanoma cells. Interestingly, transient transfection of miR-182 into cultured uveal melanoma cells led to a significant decrease in cell growth, migration, and invasiveness. Cells transfected with miR-182 demonstrated cell cycle G1 arrest and increased apoptotic activity. Using bioinformatics, we identified three potential targets of miR-182, namely MITF, BCL2 and cyclin D2. miR-182 was shown to have activity on mRNA expression by targeting the 3′ untranslated region of MITF, BCL2 and cyclin D2. Subsequent Western blot analysis confirmed the downregulation of MITF, BCL2 and cyclin D2 protein expression. The expression of oncogene c-Met and its downstream Akt and ERK1/2 pathways was also downregulated by miR-182. Concordant with the findings that miR-182 was decreased in uveal melanoma tissue samples, overexpression of miR-182 also suppressed the in vivo growth of uveal melanoma cells. Our results demonstrated that miR-182, a p53 dependent miRNA, suppressed the expression of MITF, BCL2, cyclin D2 and functioned as a potent tumor suppressor in uveal melanoma cells.