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In-depth proteomic profiling of the uveal melanoma secretome.

ABSTRACT: Uveal melanoma (UM), the most common primary intraocular tumour in adults, is characterised by a high frequency of metastases to the liver, typically with a fatal outcome. Proteins secreted from cancer cells ('secretome') are biologically important molecules thought to contribute to tumour progression. We examined the UM secretome by applying a label-free nanoLCMS/MS proteomic approach to profile proteins secreted into culture media by primary UM tumours with a high- (HR; n = 11) or low- (LR; n = 4) metastatic risk, compared to normal choroidal melanocytes (NCM) from unaffected post-mortem eyes. Across the three groups, 1843 proteins were identified at a 1% false discovery rate; 758 of these by at least 3 unique peptides, and quantified. The majority (539/758, 71%) of proteins were classified as secreted either by classical (144, 19%), non-classical (43, 6%) or exosomal (352, 46%) mechanisms. Bioinformatic analyzes showed that the secretome composition reflects biological differences and similarities of the samples. Ingenuity® pathway analysis of the secreted protein dataset identified abundant proteins involved in cell proliferation-, growth- and movement. Hepatic fibrosis/hepatic stellate cell activation and the mTORC1-S6K signalling axis were among the most differentially regulated biological processes in UM as compared with NCM. Further analysis of proteins upregulated ? 2 in HR-UM only, identified exosomal proteins involved in extracellular matrix remodelling and cancer cell migration/invasion; as well as classically secreted proteins, possibly representing novel biomarkers of metastatic disease. In conclusion, UM secretome analysis identifies novel proteins and pathways that may contribute to metastatic development at distant sites, particularly in the liver.

SUBMITTER: Angi M

PROVIDER: S-EPMC5226534 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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In-depth proteomic profiling of the uveal melanoma secretome.

Angi Martina M Kalirai Helen H Prendergast Samuel S Simpson Deborah D Hammond Dean E DE Madigan Michele C MC Beynon Robert J RJ Coupland Sarah E SE

Oncotarget 20160801 31

Uveal melanoma (UM), the most common primary intraocular tumour in adults, is characterised by a high frequency of metastases to the liver, typically with a fatal outcome. Proteins secreted from cancer cells ('secretome') are biologically important molecules thought to contribute to tumour progression. We examined the UM secretome by applying a label-free nanoLCMS/MS proteomic approach to profile proteins secreted into culture media by primary UM tumours with a high- (HR; n = 11) or low- (LR; n ...[more]

PMID: 27391064

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Project description:BackgroundUveal melanoma is the most common malignancy of the adult eye. The overall mortality rate is high because this aggressive cancer often metastasizes before ophthalmic diagnosis. Quantitative proteomic analysis of primary metastasizing and non-metastasizing tumors was pursued for insights into mechanisms and biomarkers of uveal melanoma metastasis.MethodsEight metastatic and 7 non-metastatic human primary uveal melanoma tumors were analyzed by LC MS/MS iTRAQ technology with Bruch's membrane/choroid complex from normal postmortem eyes as control tissue. Tryptic peptides from tumor and control proteins were labeled with iTRAQ tags, fractionated by cation exchange chromatography, and analyzed by LC MS/MS. Protein identification utilized the Mascot search engine and the human Uni-Prot/Swiss-Protein database with false discovery ≤ 1%; protein quantitation utilized the Mascot weighted average method. Proteins designated differentially expressed exhibited quantitative differences (p ≤ 0.05, t-test) in a training set of five metastatic and five non-metastatic tumors. Logistic regression models developed from the training set were used to classify the metastatic status of five independent tumors.ResultsOf 1644 proteins identified and quantified in 5 metastatic and 5 non-metastatic tumors, 12 proteins were found uniquely in ≥ 3 metastatic tumors, 28 were found significantly elevated and 30 significantly decreased only in metastatic tumors, and 31 were designated differentially expressed between metastatic and non-metastatic tumors. Logistic regression modeling of differentially expressed collagen alpha-3(VI) and heat shock protein beta-1 allowed correct prediction of metastasis status for each of five independent tumor specimens.ConclusionsThe present data provide new clues to molecular differences in metastatic and non-metastatic uveal melanoma tumors. While sample size is limited and validation required, the results support collagen alpha-3(VI) and heat shock protein beta-1 as candidate biomarkers of uveal melanoma metastasis and establish a quantitative proteomic database for uveal melanoma primary tumors.

Project description:Metastatic uveal melanoma (UM) is a rare, but often lethal, form of ocular cancer arising from melanocytes within the uveal tract. UM has a high propensity to spread hematogenously to the liver, with up to 50% of patients developing liver metastases. Unfortunately, once liver metastasis occurs, patient prognosis is extremely poor with as few as 8% of patients surviving beyond two years. There are no standard-of-care therapies available for the treatment of metastatic UM, hence it is a clinical area of urgent unmet need. Here, the clinical relevance and therapeutic potential of cysteinyl leukotriene receptors (CysLT1 and CysLT2) in UM was evaluated. High expression of CYSLTR1 or CYSLTR2 transcripts is significantly associated with poor disease-free survival and poor overall survival in UM patients. Digital pathology analysis identified that high expression of CysLT1 in primary UM is associated with reduced disease-specific survival (p = 0.012; HR 2.76; 95% CI 1.21-6.3) and overall survival (p = 0.011; HR 1.46; 95% CI 0.67-3.17). High CysLT1 expression shows a statistically significant (p = 0.041) correlation with ciliary body involvement, a poor prognostic indicator in UM. Small molecule drugs targeting CysLT1 were vastly superior at exerting anti-cancer phenotypes in UM cell lines and zebrafish xenografts than drugs targeting CysLT2. Quininib, a selective CysLT1 antagonist, significantly inhibits survival (p < 0.0001), long-term proliferation (p < 0.0001), and oxidative phosphorylation (p < 0.001), but not glycolysis, in primary and metastatic UM cell lines. Quininib exerts opposing effects on the secretion of inflammatory markers in primary versus metastatic UM cell lines. Quininib significantly downregulated IL-2 and IL-6 in Mel285 cells (p < 0.05) but significantly upregulated IL-10, IL-1?, IL-2 (p < 0.0001), IL-13, IL-8 (p < 0.001), IL-12p70 and IL-6 (p < 0.05) in OMM2.5 cells. Finally, quininib significantly inhibits tumour growth in orthotopic zebrafish xenograft models of UM. These preclinical data suggest that antagonism of CysLT1, but not CysLT2, may be of therapeutic interest in the treatment of UM.

Dataset Information

In-depth proteomic profiling of the uveal melanoma secretome.

Publications

In-depth proteomic profiling of the uveal melanoma secretome.

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