Project description:Because it accounts for 70% of all eye cancers, uveal melanoma (UM) is therefore the most common primary ocular malignancy. In this study, we investigated the molecular mechanisms leading to the aberrant expression of the gene encoding the serotonin receptor 2B (HTR2B), one of the most discriminating among the candidates from the class II gene signature, in metastatic and non-metastatic UM cell lines. Transfection analyses revealed that the upstream regulatory region of the HTR2B gene contains a combination of alternative positive and negative regulatory elements functional in HTR2B- but not in HTR23B? UM cells. We demonstrated that both the transcription factors nuclear factor I (NFI) and Runt-related transcription factor I (RUNX1) interact with regulatory elements from the HTR2B gene to either activate (NFI) or repress (RUNX1) HTR2B expression in UM cells. The results of this study will help understand better the molecular mechanisms accounting for the abnormal expression of the HTR2B gene in uveal melanoma.
Project description:MicroRNAs (miRNAs) are a group endogenous small non-coding RNAs that inhibit protein translation through binding to specific target mRNAs. Recent studies have demonstrated that miRNAs are implicated in the development of cancer. However, the role of miR-144 in uveal melanoma metastasis remains largely unknown. MiR-144 was downregulated in both uveal melanoma cells and tissues. Transfection of miR-144 mimic into uveal melanoma cells led to a decrease in cell growth and invasion. After identification of two putative miR-144 binding sites within the 3' UTR of the human c-Met mRNA, miR-144 was proved to inhibit the luciferase activity inMUM-2B cells with a luciferase reporter construct containing the binding sites. In addition, the expression of c-Met protein was inhibited by miR-144. Furthermore, c-Met-mediated cell proliferation and invasion were inhibited by restoration of miR-144 in uveal melanoma cells. In conclusion, miR-144 acts as a tumor suppressor in uveal melanoma, through inhibiting cell proliferation and migration. miR-144 might serve as a potential therapeutic target in uveal melanoma patients.
Project description:The objective of this study was to evaluate the correlation between the expression of p53 gene and the prognosis after local excision in uveal melanoma.Real-time polymerase chain reaction (RT-PCR) test and Western blot were used to detect the expression of p53 in C918, MUM-2B, and D78 cell lines at the levels of messenger RNA (mRNA) and protein. Immunohistochemistry staining was done in the tissues of 68 patients, which were diagnosed with uveal melanoma. Furthermore, the effects of p53 protein on the invasion abilities of both the cell lines were studied by transinfection of p53 small interfering RNA. The clinical and prognostic data regarding the effect of p53 protein on the patient's prognosis were calculated and further analyzed by Kaplan-Meier univariate analysis method.The results of RT-PCR and Western blot revealed that p53 mRNAs were highly expressed in C918 and MUM-2B cells. The high expression rate of p53 among the 88 uveal melanoma tissues was 77.27%. Transinfection of p53 serine could inhibit the expression of p53 in uveal melanoma and the invasion ability of the cells. This study found that the high expression of p53 and the prognosis of uveal melanoma patients were statistically correlated.The expression of p53 protein in uveal melanoma was unusual and was associated with the invasion ability of uveal melanoma. This indicates that the highest expression of p53 protein indicates worse prognosis of uveal melanoma patients.
Project description:Introduction: Uveal melanoma (UM) is the most common type of primary intraocular tumor in the adult population. UM will propagate to the liver as the first metastatic site. Once this organ is invaded, survival becomes a matter of months for the patient as no treatment has proved effectiveness. Among the candidates from the class II gene signature, the serotonin receptor-encoding gene HTR2B appears to be the most discriminating as its expression strongly increases in the tumors of UM patients that will progress toward liver metastases. Our study aims at characterizing the molecular mechanisms that lead to this aberrant expression of HTR2B in metastatic UM cell lines.Materials and Methods: Expression of HTR2B was monitored by microarrays and validated by Western blot in a variety of UM cell lines. A 2 Kbp segment covering both the promoter and 5 '-flanking region of the HTR2B gene was cloned upstream the CAT reporter gene in the plasmid pCATbasic (to yield the -2000/HTR2B construct). Deletion mutants were generated by double digestion of the -2000/HTR2B construct with restriction enzymes and transfected, along with the parental -2000/HTR2B construct, in UM cell lines. The genomic areas of interest were 5’ end-labeled and used as probes in electrophoretic mobility shift assays (EMSAs). DMS methylation footprinting was also used to precisely position the DNA target sites for transcription factors (TFs) that bind the HTR2B regulatory regions.Results and Discussion: Transfection analyses revealed that the upstream regulatory regions of HTR2B promoter is made up of a combination of alternative positive and negative regulatory elements. Repressive regions have an abnormally elevated number of target sites for the TFs NFI. EMSA analyses provided evidence that both the NFI isoforms NFIC and NFIX could interact with the promoter and the upstream negative regulatory element from the HTR2B gene. In addition, the TF RunX1 was shown by DMS methylation interference footprinting to bind a target site from the distal silencer element.Conclusion: This project will help understand better the molecular mechanisms accounting for the abnormal expression of the HTR2B gene in uveal melanoma. In the long term, this study will allow us to identify new potential targets that could help screening patients at risk of progressing to liver metastasis formation. Overall design: Primary culture of humal uveal melanoma cell lines (T97, T98, T108, T111, T128, T131, T132, T142, T143, T151, T157) were analyzed by gene profiling on microarrays at low culture passages (between passage 2 to 7). and their transcriptome analyzed by gen profiling.
Project description:BACKGROUND Dysregulation of the microRNA (miRNA) network is a typical feature of many cancers. However, the key specific miRNAs involved in uveal melanoma carcinogenesis are largely unknown. MATERIAL AND METHODS RT-qPCR was used to detected miR-652 expression in uveal melanoma tissues and cell lines. miR-652 inhibitor was transfected into uveal melanoma cells to decrease miR-652 expression and determine the biological role of miR-652 by CCK-8 and wound healing assays. Bioinformatic analysis and dual luciferase reporter assay were used to predict and validate the target gene of miR-652. HOXA9 siRNA was transfected into cells to confirm that miR-652 relies on regulation of HOXA9 to regulate cell proliferation and migration. RESULTS RT-qPCR showed that miR-652 was overexpressed in uveal melanoma cell lines (MUM-2B, MEL270) compared with melanocyte cells (ARPE-19). Overexpression of miR-652 was also observed in uveal melanoma compared to paired non-tumor tissues. Downregulation of miR-652 inhibited the cell proliferation ability and migration ability of uveal melanoma cells. Using bioinformatic analysis, HOXA9 was found to be a potential target gene of miR-652. The direct regulation of HOXA9 by miR-652 was experimentally validated in uveal melanoma cells by dual luciferase assay and Western blotting. We also observed that miR-652 promoted HIF-1alpha signaling via repression of HOXA9 in uveal melanoma cells. Silencing of HOXA9 attenuated the miR-652 inhibitor decreased cell growth rate and decreased migration ability in uveal melanoma cells. CONCLUSIONS Our data demonstrate an oncogenic role of miR-652 in uveal melanoma, showing that miR-652 may be a useful biomarker for prediction of prognosis for patients with uveal melanoma.
Project description:α9b1 is the most recent addition to the integrin family of membrane-bound receptors and consequently remains the one which is the least characterized. To better understand how transcription of the human gene encoding the α9 subunit is regulated at the molecular level, we cloned the α9 promoter and characterized the regulatory elements that are required to ensure its transcription. Transfection of α9 promoter/CAT recombinant plasmids in primary cultured cells and uveal melanoma cell lines demonstrated the presence of both negative and positive regulatory elements along the α9 promoter and positioned the basal α9 promoter to within 118 bp from the α9 mRNA start site. In vitro DNaseI footprinting and in vivo ChIP analyses demonstrated the binding of the transcription factors Sp1/Sp3, c-Myb and NFI to the most upstream α9 negative regulatory element. The transcription factors Sp1/Sp3 and NFI were found to bind the basal α9 promoter individually but Sp1 binding clearly predominates when both transcription factors are present in the same extract. Most of all, addition of tenascin-C (TNC), the ligand of α9β1, to the tissue culture plates prior to cell seeding increased α9 transcription whereas it simultaneously decreased expression of the α5 integrin subunit gene. This dual regulatory action of TNC on the transcription of the α9 and α5 genes suggests that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing. Primary cultures of human corneal epithelial cells (HCECs; number of replicates: 8), human skin epithelial cells (HSEC; number of replicates: 4), human corneal fibroblast cells (HCFCs; number of replicates: 3), human skin fibroblast cells (HSFCs; number of replicates: 3), human uveal melanocytes (UVM; number of replicates: 3), and various humal uveal melanoma cell lines (T115, T142 and T143; number of replicates: 2-3) were analyzed by gene profiling on microarrays at low culture passages (between passage 2 to 7). HCECs were also cultured on either BSA (number of replicates: 2) or on tenascin-C (number of replicates: 2) and their transcriptome analyzed by gene profiling.
Project description:AIMS:Antimicrobial peptides (AMPs) have been implicated in the pathogenesis of several cancers, although there is also evidence suggesting potential for novel, AMP-based antitumor therapies. Discerning potential roles of AMPs in tumor pathogenesis may provide valuable insight into the mechanisms of novel AMP-based antitumor therapy. METHODS:mRNA expression of the AMPs ? defensin (HNP-1); cathelicidin (LL-37); and ? defensins (hBD-1, hBD-2, hBD-3, hBD-4) in human uveal and cutaneous melanoma cell lines, primary human uveal melanocytes, and primary human uveal melanoma cells was determined by reverse transcriptase polymerase chain reaction. An in vitro scratch assay and custom Matlab analysis were used to determine the AMP effects on melanoma cell migration. Last, the effect of specific AMPs on vasculogenic mimicry was determined by three-dimensional (3D) culture and light and fluorescence microscopy. RESULTS:Low-to-moderate AMP transcript levels were detected, and these varied across the cells tested. Overall, LL-37 expression was increased while hBD-4 was decreased in most melanoma cell lines, compared to primary cultured uveal melanocytes. There was no observable influence of HNP-1 and LL-37 on tumor cell migration. Additionally, aggressive cutaneous melanoma cells grown in 3D cultures exhibited vasculogenic mimicry, although AMP exposure did not alter this process. CONCLUSIONS:Collectively, our data show that although AMP mRNA expression is variable between uveal and cutaneous melanoma cells, these peptides have little influence on major characteristics that contribute to tumor aggressiveness and progression.
Project description:Transcription factors regulating the epithelial-to-mesenchymal transition (EMT) program contribute to carcinogenesis and metastasis in many tumors, including cutaneous melanoma. However, little is known about the role of EMT factors in the growth and metastatic dissemination of uveal melanoma cells. Here, we analyzed the expression and functions of the EMT factors ZEB1, Twist1, and Snail1 in uveal melanoma cell lines and primary tumors.ZEB1, Twist1, and Snail1 mRNA levels were measured using qPCR in five uveal melanoma cell lines and in 30 primary tumors. Gene expression was used to determine class 1 and class 2 signatures in the primary tumors. Short hairpin RNA was used to downregulate the expressions of the EMT factors; then, growth and transwell invasion assays were performed.ZEB1, Twist1, and Snail1 were expressed in all five uveal melanoma lines, with ZEB1 having the highest protein levels. ZEB1 mRNA was significantly elevated in highly metastatic class 2 primary tumors for which survival data were not available, whereas a high gene expression of Twist1 was associated with a worse prognosis in a separate tumor cohort analyzed by expression profiling. The genetic downregulation of ZEB1 in OCM1, OMM1, and 92.1 resulted in a more than 50% reduction in invasion, but only suppressed growth in OMM1 cells. Suppression of Twist1 in Mel290 and OMM1 reduced growth and invasion by more than 50%. The downregulation of Snail1 in the 92.1 cell line reduced invasion by 50%, but did not interfere with growth.The downregulation of ZEB1, Twist1, and Snail1 reduces the invasive properties of uveal melanoma cells, and the elevated mRNA levels of ZEB1 and Twist1 are associated with a more aggressive clinical phenotype in uveal melanoma samples. Therefore, these factors could represent new therapeutic targets in patients with ocular melanoma.
Project description:Metastatic disease from uveal melanoma occurs in almost 50% of patients suffering from this ocular tumour, with median survival from development of symptoms being around 1 year. In contrast to cutaneous melanoma, kinase inhibitors and immune checkpoint inhibitors are usually ineffective in patients with metastatic uveal melanoma. Tebentafusp is a novel form of immunotherapy based on the immune-mobilising monoclonal T cell receptor against cancer (ImmTAC) platform, which comprises a soluble T cell receptor that is fused to an anti-CD3 single-chain variable fragment. The T cell receptor domain of tebentafusp targets cells present a human leukocyte antigen-A*02:01 complexed with a peptide derived from the melanoma-associated antigen gp100, which is expressed strongly by melanoma cells, weakly by normal melanocytes and minimally by other tissues. The anti-CD3 domain recruits CD3+ T cells (and, indirectly, other immune cells), redirecting these to the melanoma cells. The most common adverse events with tebentafusp are manageable and usually transient. Early survival data in patients with metastatic uveal melanoma are promising when considered alongside historical data. Based on these encouraging results, a randomised study comparing tebentafusp to investigator's choice of therapy in metastatic uveal melanoma is ongoing.
Project description:Previous studies of the epithelial specificity of the human papillomavirus type 16 (HPV-16) enhancer pointed out an important role of nuclear factor I (NFI). In epithelial cells, NFI proteins are derived from the NFI-C gene and referred to as NFI/CTF. In contrast, fibroblasts, where the enhancer is inactive, express high levels of NFI from the NFI-X gene. To compare NFI-C and NFI-X derived transcription factors, we cloned and functionally investigated two differentially spliced forms of NFI-X from human fibroblasts. NFI-X1 has 95% homology with a transcript previously identified in hamster liver cells. NFI-X2, a spliced variant, misses 41 amino acids of the proline-rich activation domain. NFI-X expression, examined by Northern blots, shows strong cell-type specific variation in comparison with NFI/CTF. While the transcriptional activation domain of NFI-X2, functionally tested as GAL4-fusion protein in epithelial and fibroblast cells, activates transcription from promoter as well as enhancer position similar to NFI/CTF-1, the activation domain of NFI-X1 fails to activate transcription from enhancer position. In Drosophila cells, void of endogenous NFI proteins, full length NFI/CTF-1 and NFI-X2 activate a reporter construct containing only NFI sites as well as the NFI dependent HPV-16 enhancer. In contrast, NFI-X1 fails to activate the HPV-16 enhancer. Furthermore, overexpression of NFI-X1 in epithelial cells down-regulates the HPV-16 enhancer. Our findings suggest that the family of NFI transcription factors should not be viewed as constitutive activators, but rather, that NFI-C and NFI-X have divergent functions after binding in promoter or enhancer position. This property, combined with the differential expression of NFI-X, can achieve cell-type specificity of NFI dependent promoters and enhancers.