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.

MMP1 and MMP9 are potential prognostic biomarkers and targets for uveal melanoma.

BACKGROUND: Uveal melanoma (UVM) is the leading cause of eye-related mortality worldwide. This study aimed to explore the expression and prognostic value of matrix metalloproteinases (MMPs) in UVM. METHODS: Gene expression levels were obtained from the Gene Expression Omnibus (GEO) and Oncomine databases. Functional and pathway enrichment analyses were performed using the Metascape database. GeneMANIA was then applied to construct a protein-protein interaction network and identify the hub genes. Moreover, overall survival (OS) and disease-free survival (DFS) analysis for the hub genes was performed using the UALCAN and Gene Expression Profiling Interactive Analysis (GEPIA) online tool. Furthermore, TRRUST was used to predict the targets of the MMPs. RESULTS: Our results revealed that the transcriptional levels of MMP1, MMP9, MMP10, MMP11, MMP13, MMP14, and MMP17 were upregulated in UVM tissues compared to normal tissues. A protein-protein interaction (PPI) network was constructed and the top 50 hub genes were identified. The functions of MMPs and their neighboring proteins are mainly associated with ECM-receptor interaction, proteoglycans in cancer, the IL-17 signaling pathway, and microRNAs in cancer. Among the MMPs, MMP1/2/9/11/14/15/16/17/24 played significant roles in the progression of UVM from stage 3 to stage 4. We also found that the expression of MMP1, MMP2, MMP9, and MMP16 positively correlated with OS and DFS in patients with UVM. Additionally, 18 transcription factors associated with nine MMPs were identified. CONCLUSIONS: The results of this study may provide potential biomarkers and targets for UVM. However, further studies are required to confirm these results.

Albumin-based nanostructures for uveal melanoma treatment.

Uveal melanoma (UM) is an intraocular tumor which is almost lethal at the metastatic stage due to the lack of effective treatments. In this regard, we have developed an albumin-based nanostructure (ABN) containing AZD8055 (ABN-AZD), which is a potent mTOR kinase inhibitor, for its efficient delivery to the tumors. The drug has been conjugated to ABN using tailored linkers that have a disulfide moiety, allowing its release selectively and effectively in the presence of an elevated concentration of glutathione, such as inside the tumoral cells. Our therapeutic approach induced significant cellular toxicity in uveal melanoma cells, but not in non-tumoral keratinocytes, highlighting the excellent selectivity of the system. In addition, these nanostructures showed excellent activity in vivo, decreasing the tumor surface compared to the free AZD8055 in mice models. Remarkably, the results obtained were achieved employing a dose 23 times lower than those used in previous reports.

Tryptophanyl-tRNA synthetase (WARS) expression in uveal melanoma - possible contributor during uveal melanoma progression.

This study aimed to explore the influence of Tryptophanyl-tRNA synthetase (WARS) expression on the proliferation and migration of uveal melanoma (UM) cells, and the potential mechanisms. Bioinformatics analysis based on Gene Expression Omnibus (GEO) database showed that WARS expression in metastatic cancer was significantly higher than that in no-metastatic group. Kaplan-Meier analysis based on The Cancer Genome Atlas (TCGA) database showed that high WARS expression was associated with lower survival. Biological function experiments showed that overexpression of WARS in OCM-1A cells can promote cell proliferation, migration, and invasion, whereas knockdown of WARS in C918 cells showed the opposite effect. Finally, we observed that the up-regulation of WARS induced the activation of phosphatidylinositol 3-kinase/AKT (PI3K/AKT) signaling, whilst depletion of WARS resulted in opponent outcomes. Taken together, our results illustrated that WARS was overexpressed in UM cells and contributed to the viability and motility of UM cells via modulating PI3K/AKT signaling pathway.

Genetic Biomarkers in Melanoma of the Ocular Region: What the Medical Oncologist Should Know.

Melanoma of the ocular region (ocular melanoma) comprises about 5% of all patients with melanoma and covers posterior uveal melanoma, iris melanoma, and conjunctival melanoma. The risk of metastasis is much higher in patients with ocular melanoma compared to a primary melanoma of the skin. The subtypes of ocular melanoma have distinct genetic features, which should be taken into consideration when making clinical decisions. Most relevant for current practice is the absence of BRAF mutations in posterior uveal melanoma, although present in some iris melanomas and conjunctival melanomas. In this review, we discuss the genetic biomarkers of the subtypes of ocular melanoma and their impacts on the clinical care of these patients.

MiR-216a-5p/Hexokinase 2 axis regulates uveal melanoma growth through modulation of Warburg effect.

Hexokinase-2 (HK2), the initial as well as the rate-limiting step in glycolysis, is overexpressed in many human cancers, and correlates with poor clinical outcomes. Aerobic glycolysis is a hallmark of cancer, and drugs targeting its enzymes, including HK2, are being developed. However, the mechanisms of HK2 inhibition and the physiological significance of the HK2 inhibitors in cancer cells are rarely reported. Here, we show that microRNA-216a-5p (miR-216a-5p) inhibits HK2 expression by directly targeting its 3'-UTR in uveal melanoma cells. Through inhibition of HK2, miR-216a-5p dampens glycolysis by reducing HK activity, glucose uptake, lactate production, ATP generation, extracellular acidification rate (ECAR), and increasing oxygen consumption rate (OCR) in uveal melanoma cells. Importantly, glycolysis regulated by miR-216a-5p is critical for its regulating uveal melanoma tumor growth both in vitro and in vivo. miR-216a-5p expression is negatively correlated with HK2 expression and predicts better outcome in uveal melanoma patients. Our findings provide clues regarding the role of miR-216a-5p as a tumor suppressor in uveal melanoma through the inhibition of HK2. Targeting HK2 through miR-216a-5p could be a promising therapeutic strategy in uveal melanoma.

Mutational status of IDH1 in uveal melanoma.

Uveal (intraocular) melanoma is an uncommon malignancy that comprises a small percentage of all melanoma cases. While many uveal melanomas harbor mutations in the BRCA-Associated Protein 1 (BAP1) gene, the genetics of non-BAP1 associated tumors are not completely understood. Recent studies have shown that a small subset of non-uveal melanomas hold mutations in isocitrate dehydrogenase (IDH), but the mutational status of IDH in uveal melanoma is unclear. Mutations in IDH are strongly prognostic and predictive of tumor behavior in other cancers, mainly diffuse gliomas, which commonly contain the IDH1-R132H mutation. For this study, we hypothesized that uveal melanoma may contain the IDH1-R132H mutation, similar to non-uveal melanoma and other cancers. A search of our institutional pathology files identified 50 consecutive cases of uveal melanoma with additional material utilized for retrospective IDH1-R132H immunohistochemical testing. The demographics of these patients included similar ages, gender distributions, and other clinical characteristics as described in previous studies. Similarly, histological subtype distributions and the presence of high risk pathologic features were consistent with other reports. All 50 of the uveal melanoma cases demonstrated negativity for IDH1-R132H by immunohistochemistry. This rate is unlike that of non-uveal melanoma and further supports their distinct molecular oncogenic profile.

Analysis of caveolin-1 and phosphoinositol-3 kinase expression in primary uveal melanomas.

BACKGROUND: To evaluate the regulation of blood supply in primary uveal melanomas through caveolin-1 (Cav-1)/phosphoinositol-3 kinase (PI3K). METHODS: The expression of Cav-1 and PI3K was analysed in 51 paraffin sections of metastatic (n = 30) and non-metastastic uveal melanomas (n = 21). Two trained observers quantified Cav-1 and PI3K immunofluorescensce expression by determining intensity of staining and percentage of positive cells. The expression was correlated with known prognostic factors. Besides angiogenesis by means of endoglin expression, the normal vasculature (von Willebrand Factor expression) was evaluated semi-quantitatively. Vasculogenic mimicry (VM) was analysed by CD31/PAS staining. RESULTS: All examined specimens expressed Cav-1 with a mean of 90.34% Cav-1 positive cells (range, 3.23-100%). Metastatic disease was associated with a higher Cav-1 expression. The correlation of Cav-1 with well-established prognostic factors showed a significant association between Cav-1 expression and largest tumour diameter (P = 0.022), tumour node metastasis classification (P = 0.008) and invasion of optic nerve head (P = 0.048). PI3K was expressed by all uveal melanomas with a mean of 87.28% cells showing PI3K expression. A higher level of PI3K was significantly associated with larger height (P = 0.042) and progressed tumour node metastasis stage (P = 0.016). The percentage of PI3K and Cav-1 positive cells were significantly associated (P = 0.034). For PI3K and Cav-1 expression a non-significant association with VM was shown (P = 0.064 and P = 0.072, respectively). No correlation of PI3K or Cav-1 with angiogenesis or mature vasculature was seen (P > 0.05). CONCLUSIONS: Cav-1 expression may be especially up-regulated in larger uveal melanomas. As it was correlated with PI3K expression and VM in this series of uveal melanoma, Cav-1 might induce the formation of VM via the PI3K-signalling cascade.

Oridonin induces apoptosis in uveal melanoma cells by upregulation of Bim and downregulation of Fatty Acid Synthase.

Oridonin is an orally available drug isolated from Traditional Chinese Medicine. Previous studies with oridonin have demonstrated broad-spectrum anticancer activity in a variety of cancer types. However, the effect of oridonin in uveal melanoma has not been addressed. In this study, we aimed to investigate whether oridonin elicited anticancer activity and its underlying mechanism in human uveal melanoma cells. We demonstrated that oridonin potently reduced cell viability, induced apoptosis and inhibited clonogenic survival and growth with single digit micromolar concentrations in uveal melanoma OCM-1 and MUM2B cell lines. We found that oridonin markedly increased the expression of proapoptotic Bcl-2 family protein Bim in uveal melanoma cells, and knockdown Bim by small interfering RNA significantly attenuated oridonin-induced cell death, indicating an essential role of Bim in oridonin-mediated anticancer activity. Additionally, we observed that oridonin suppressed Fatty Acid Synthase (FAS) expression in uveal melanoma cells, and enforced FAS expression by insulin partially rescued the cells from oridonin-induced apoptosis, showing that inhibition of FAS also contributed to oridonin-mediated apoptosis. Taken together, we reported that oridonin displays potent anticancer effect against uveal melanoma cells through upregulation of Bim and inhibition of FAS. Since oridonin is a popular anticancer agent, our study therefore may have translational implication on the management of patients with uveal melanoma.

Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.

BRAF and NRAS are common targets for somatic mutations in benign and malignant neoplasms that arise from melanocytes situated in epithelial structures, and lead to constitutive activation of the mitogen-activated protein (MAP) kinase pathway. However, BRAF and NRAS mutations are absent in a number of other melanocytic neoplasms in which the equivalent oncogenic events are currently unknown. Here we report frequent somatic mutations in the heterotrimeric G protein alpha-subunit, GNAQ, in blue naevi (83%) and ocular melanoma of the uvea (46%). The mutations occur exclusively in codon 209 in the Ras-like domain and result in constitutive activation, turning GNAQ into a dominant acting oncogene. Our results demonstrate an alternative route to MAP kinase activation in melanocytic neoplasia, providing new opportunities for therapeutic intervention.

Impact of DNA methyltransferases on the epigenetic regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression in malignant melanoma.

Aberrant promoter methylation and resultant silencing of TRAIL decoy receptors were reported in a variety of cancers, but to date little is known about the relevance of this epigenetic modification in melanoma. In this study, we examined the methylation and the expression status of TRAIL receptor genes in cutaneous and uveal melanoma cell lines and specimens and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b. DR4 and DR5 methylation was not frequent in cutaneous melanoma but on the contrary it was very frequent in uveal melanoma. No correlation between methylation status of DR4 and DR5 and gene expression was found. DcR1 and DcR2 were hypermethylated with very high frequency in both cutaneous and uveal melanoma. The concordance between methylation and loss of gene expression ranged from 91% to 97%. Here we showed that DNMT1 was crucial for DcR2 hypermethylation and that DNMT1 and DNMT3a coregulate the methylation status of DcR1. Our work also revealed the critical relevance of DcR1 and DcR2 expression in cell growth and apoptosis either in cutaneous or uveal melanoma. In conclusion, the results presented here claim for a relevant impact of aberrant methylation of decoy receptors in melanoma and allow to understand how the silencing of DcR1 and DcR2 is related to melanomagenesis.

Inhibition of Mn(2+)-induced error-prone DNA synthesis with Cd(2+) and Zn(2+).

Bivalent metal cations are key components in the reaction of DNA synthesis. They are necessary for all DNA polymerases, being involved as cofactors in catalytic mechanisms of nucleotide polymerization. It is also known that in the presence of Mn2+ the accuracy of DNA synthesis is considerably decreased. The findings of this work show that Cd2+ and Zn2+ selectively inhibit the Mn2+-induced error-prone DNA polymerase activity in extracts of cells from human and mouse tissues. Moreover, these cations in low concentrations also can efficiently inhibit the activity of homogeneous preparations of DNA polymerase iota (Pol ι), which is mainly responsible for the Mn2+-induced error-prone DNA polymerase activity in cell extracts. Using a primary culture of granular cells from postnatal rat cerebellum, we show that low concentrations of Cd2+ significantly increase cell survival in the presence of toxic Mn2+ doses. Thus, we have shown that in some cases low concentrations of Cd2+ can display a positive influence on cells, whereas it is widely acknowledged that this metal is not a necessary microelement and is toxic for organisms.

[Effect of Mn(II) on the error-prone DNA polymerase iota activity in extracts from human normal and tumor cells].

The DNA polymerase iota (Pol iota), which has some peculiar features and is characterized by an extremely error-prone DNA synthesis, belongs to the group of enzymes preferentially activated by Mn2+ instead of Mg2+. In this work, the effect of Mn2+ on DNA synthesis in cell extracts from a) normal human and murine tissues, b) human tumor (uveal melanoma), and c) cultured human tumor cell lines SKOV-3 and HL-60 was tested. Each group displayed characteristic features of Mn-dependent DNA synthesis. The changes in the Mn-dependent DNA synthesis caused by malignant transformation of normal tissues are described. It was also shown that the error-prone DNA synthesis catalyzed by Pol iota in extracts of all cell types was efficiently suppressed by an RNA aptamer (IKL5) against Pol iota obtained in our work earlier. The obtained results suggest that IKL5 might be used to suppress the enhanced activity of Pol iota in tumor cells.

Activity of the oral MEK inhibitor trametinib in patients with advanced melanoma: a phase 1 dose-escalation trial.

BACKGROUND: MEK is a member of the MAPK signalling cascade that is commonly activated in melanoma. Direct inhibition of MEK blocks cell proliferation and induces apoptosis. We aimed to analyse safety, efficacy, and genotyping data for the oral, small-molecule MEK inhibitor trametinib in patients with melanoma. METHODS: We undertook a multicentre, phase 1 three-part study (dose escalation, cohort expansion, and pharmacodynamic assessment). The main results of this study are reported elsewhere; here we present data relating to patients with melanoma. We obtained tumour samples to assess BRAF mutational status, and available tissues underwent exploratory genotyping analysis. Disease response was measured by Response Evaluation Criteria in Solid Tumors, and adverse events were defined by common toxicity criteria. This study is registered with ClinicalTrials.gov, number NCT00687622. FINDINGS: 97 patients with melanoma were enrolled, including 81 with cutaneous or unknown primary melanoma (36 BRAF mutant, 39 BRAF wild-type, six BRAF status unknown), and 16 with uveal melanoma. The most common treatment-related adverse events were rash or dermatitis acneiform (n=80; 82%) and diarrhoea (44; 45%), most of which were grade 2 or lower. No cutaneous squamous-cell carcinomas were recorded. Of 36 patients with BRAF mutations, 30 had not received a BRAF inhibitor before; two complete responses (both confirmed) and ten partial responses (eight confirmed) were noted in this subgroup (confirmed response rate, 33%). Median progression-free survival of this subgroup was 5·7 months (95% CI 4·0-7·4). Of the six patients who had received previous BRAF inhibition, one unconfirmed partial response was recorded. Of 39 patients with BRAF wild-type melanoma, four partial responses were confirmed (confirmed response rate, 10%). INTERPRETATION: Our data show substantial clinical activity of trametinib in melanoma and suggest that MEK is a valid therapeutic target. Differences in response rates according to mutations indicate the importance of mutational analyses in the future. FUNDING: GlaxoSmithKline.

The AMP-dependent kinase pathway is upregulated in BAP1 mutant uveal melanoma.

Metastatic uveal melanoma (UM) responds poorly to targeted therapies and immune checkpoint inhibitors. Loss of BRCA1-associated protein 1 (BAP1) via inactivating mutations in the BAP1 gene is associated with UM progression. Thus, molecular alterations caused by BAP1 dysfunction may be novel therapeutic targets for metastatic UM. Here, we found that phosphorylation of AMP-dependent kinase (AMPK) was elevated in BAP1-altered (or mutant) compared to BAP1-unaltered (or wild-type [WT]) UM tumors. As a readout of AMPK pathway activation, phosphorylation of an AMPK downstream effector, acetyl-CoA-carboxylase (ACC), was also elevated. BAP1 re-expression in BAP1-null UM cell lines decreased phospho-AMPK (pAMPK) and phospho-ACC (pACC) levels. AMPK phosphorylation is mediated by calcium/calmodulin dependent protein kinase kinase 2 (CaMKK2) and potentially liver kinase B1 (LKB1) in BAP1 mutant UM cells. Knockdown of AMPKα1/2 reduced the viability of BAP1 mutant UM cells, indicating a survival function of AMPK in BAP1 mutant UM. Our data suggest that the AMPK pathway is an important mechanism mediating the survival of BAP1 mutant UM. Targeting the AMPK pathway may be a novel therapeutic strategy for metastatic UM.

Resistance of uveal melanoma to the interstrand cross-linking agent mitomycin C is associated with reduced expression of CYP450R.

BACKGROUND: Uveal melanoma (UM) is the most common primary intraocular tumour of adults, frequently metastasising to the liver. Hepatic metastases are difficult to treat and are mainly unresponsive to chemotherapy. To investigate why UM are so chemo-resistant we explored the effect of interstrand cross-linking agents mitomycin C (MMC) and cisplatin in comparison with hydroxyurea (HU). METHODS: Sensitivity to MMC, cisplatin and HU was tested in established UM cell lines using clonogenic assays. The response of UM to MMC was confirmed in MTT assays using short-term cultures of primary UM. The expression of cytochrome P450 reductase (CYP450R) was analysed by western blotting, and DNA cross-linking was assessed using COMET analysis supported by γ-H2AX foci formation. RESULTS: Both established cell lines and primary cultures of UM were resistant to the cross-linking agent MMC (in each case P<0.001 in Student's t-test compared with controls). In two established UM cell lines, DNA cross-link damage was not induced by MMC (in both cases P<0.05 in Students's t-test compared with damage induced in controls). In all, 6 out of 6 UMs tested displayed reduced expression of the metabolising enzyme CYP450R and transient expression of CYP450R increased MMC sensitivity of UM. CONCLUSION: We suggest that reduced expression of CYP450R is responsible for MMC resistance of UM, through a lack of bioactivation, which can be reversed by complementing UM cell lines with CYP450R.

Active notch protects MAPK activated melanoma cell lines from MEK inhibitor cobimetinib.

The crosstalk between Notch and MAPK pathway plays a role in MEK inhibitor resistance in BRAFV600E metastatic melanoma (MM) and promotes migration in GNAQQ209L uveal melanoma (UM) cells. We determined the cytotoxicity of combinatorial inhibition of MEK and Notch by cobimetinib and γ-secretase inhibitor (GSI) nirogacestat, in BRAFV600E and BRAF wt MM and GNAQQ209L UM cells displaying different Erk1/2 and Notch activation status, with the aim to elucidate the impact of Notch signaling in the response to MEK inhibitor. Overall the combination was synergic in BRAFV600E MM and GNAQQ209L UM cells and antagonistic in BRAF wt one. Focusing on UM cells, we found that cobimetinib resulted in G0/G1 phase arrest and apoptosis induction, whereas the combination with GSI increased treatment efficacy by inducing a senescent-like state of cells and by blocking migration towards liver cancer cells. Mechanistically, this was reflected in a strong reduction of cyclin D1, in the inactivation of retinoblastoma protein and in the increase of p27KIP1 expression levels. Of note, each drug alone prevented Notch signaling activation resulting in inhibition of c-jun(Ser63) and Hes-1 expression. The combination achieved the strongest inhibition on Notch signaling and on both c-jun(Ser63) and Erk1/2 activation level. In conclusion we unveiled a coordinate action of MAPK and Notch signaling in promoting proliferation of BRAFV600E MM and GNAQQ209L UM cells. Remarkably, the simultaneous inhibition of MEK and Notch signaling highlighted a role for the second pathway in protecting cells against senescence in GNAQQ209L UM cells treated with the MEK inhibitor.

Expression and prognostic significance of iNOS in uveal melanoma.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Disease metastasis occurs in half of the patients and is uniformly fatal despite systemic therapy. Inducible nitric oxide synthase (iNOS) is associated with disease progression in various malignancies including cutaneous melanoma. In this retrospective cohort, we examined the prognostic value of iNOS in UM by performing immunohistochemistry on paraffin-embedded sections of primary tumors (90 patients) and matched primary and metastatic hepatic tumors (19 patients) with complete histopathological and clinical data. We show that iNOS is expressed in UM (57% of the patients) and high iNOS levels significantly (p = 0.04; hazard ratio (HR) = 2.3) predict disease-specific survival (DSS) as assessed by Kaplan-Meier analysis and univariate Cox's proportional hazards regression model. Furthermore, high iNOS expression in the UM primary tissue was significantly associated with metastatic disease and vice versa. Expression of iNOS in hepatic metastases significantly (p = 0.02) predicted a shortened survival as assessed by Kaplan-Meier analysis. However, iNOS did not appear to be a significant (p = 0.16; HR = 1.9) factor in the multivariate Cox's regression analysis performed together with the clinical parameters tumor diameter, tumor cell type, and tumor location in which only tumor diameter predicted DSS. In conclusion, iNOS predicts DSS in UM and may play a role in disease progression but it is not an independent prognostic factor.

Decitabine limits escape from MEK inhibition in uveal melanoma.

MEK inhibitors (MEKi) demonstrate anti-proliferative activity in patients with metastatic uveal melanoma, but responses are short-lived. In the present study, we evaluated the MEKi trametinib alone and in combination with drugs targeting epigenetic regulators, including DOT1L, EZH2, LSD1, DNA methyltransferases, and histone acetyltransferases. The DNA methyltransferase inhibitor (DNMTi) decitabine effectively enhanced the anti-proliferative activity of trametinib in cell viability, colony formation, and 3D organoid assays. RNA-Seq analysis showed the MEKi-DNMTi combination primarily affected the expression of genes involved in G1 and G2/2M checkpoints, cell survival, chromosome segregation and mitotic spindle. The DNMTi-MEKi combination did not appear to induce a DNA damage response (as measured by γH2AX foci) or senescence (as measured by ß-galactosidase staining) compared to either MEKi or DNMTi alone. Instead, the combination increased expression of the CDK inhibitor p21 and the pro-apoptotic protein BIM. In vivo, the DNMTi-MEKi combination was more effective at suppressing growth of MP41 uveal melanoma xenografts than either drug alone. Our studies indicate that DNMTi may enhance the activity of MEKi in uveal melanoma.

P53 apoptosis mediator PERP: localization, function and caspase activation in uveal melanoma.

p53 apoptosis effector related to PMP-22 (PERP) is a transcriptional target gene of p53 tumour suppressor that is specifically induced during apoptosis and not during cell cycle arrest. In primary uveal melanoma (UM), the most common intraocular malignancy in adults that has a reportedly unaffected signalling pathway upstream of and including p53, PERP expression is down-regulated in the metastatic monosomy 3-type tumours, compared with the less aggressive disomy 3-type tumours. Here, we demonstrate experimentally, by the use of full-length PERP-green fluorescent protein (GFP) fusions and real-time confocal microscopy, the intracellular targeting and plasma membrane localization of PERP in living UM cells and show that expression of PERP induces caspase-mediated apoptosis in UM cells. Induction of PERP expression in GFP-PERP-transfected UM cells leads to increased levels of cleaved caspase-8 forms, as well as to reduction of its full-length substrate Bid, but not to detectable processing of caspase-9. The levels of mature caspase-8, -9 and -3 proteins significantly correlate with PERP expression levels in primary UMs. Transcriptional profiling of PERP and caspase-8 in tumour specimens indicates that the positive association of PERP and caspase-8 proteins is a consequence of post-translational processing, most likely at the level of caspase-8 cleavage, and not of increased transcription of pro-caspase-8. We conclude that PERP expression leads to activation of an extrinsic receptor-mediated apoptotic pathway, with a possible subsequent engagement of the intrinsic apoptotic pathway. The findings underline the apoptotic pathway mediated by PERP as a critical mechanism employed by UM tumours to modulate susceptibility to apoptosis.