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Antibody-independent targeted quantification of TMPRSS2-ERG fusion protein products in prostate cancer.

ABSTRACT: Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. Studies of TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies suitable for quantitative studies. Herein, we applied a recently developed PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM-SRM assays provided confident detection of 6 unique ERG peptides in both TMPRSS2-ERG positive cell lines and tissues, but not in cell lines or tissues lacking the TMPRSS2-ERG rearrangement, clearly indicating that ERG protein expression is significantly increased in the presence of the TMPRSS2-ERG gene fusion. Significantly, our results provide evidence that two distinct ERG protein isoforms are simultaneously expressed in TMPRSS2-ERG positive samples as evidenced by the concomitant detection of two mutually exclusive peptides in two patient tumors and in the VCaP prostate cancer cell line. Three peptides, shared across almost all fusion protein products, were determined to be the most abundant peptides, providing "signature" peptides for detection of ERG over-expression resulting from TMPRSS2-ERG gene fusion. The PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products in prostate cancer.

SUBMITTER: He J

PROVIDER: S-EPMC4183720 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The transmembrane protease serine 2:v‑ets erythroblastosis virus E26 oncogene homolog (TMPRSS2:ERG) gene fusion is common in prostate cancer, while its functional role is not fully understood. The present study aimed to investigate the significance of the TMPRSS2:ERG gene fusion in human prostate cancers using bioinformatics tools. Comprehensive alteration analysis of TMPRSS2 and ERG in 148 different human cancer studies was performed by cBioPortal, and the mRNA expression level of the ERG gene was evaluated using Oncomine analysis. Furthermore, lentiviral short hairpin (sh)RNA‑mediated knockdown of TMPRSS2:ERG was performed to study the impact of ERG silencing on cell proliferation and cell cycle distribution in prostate cancer cells. The results demonstrated that the TMPRSS2 and ERG genes were mostly altered in prostate cancer, and the most frequent alteration was gene fusion. Oncomine analysis demonstrated that the ERG gene was significantly upregulated in prostate clinical samples compared with the normal prostate gland in four independent datasets, and a positive association was observed between potassium inwardly‑rectifying channel subfamily J member 15, down syndrome critical region gene 4, potassium inwardly‑rectifying channel subfamily J member 6 and ERG gene expression. There were 272 mutations of the ERG gene identified in the cBioPortal database; among the mutations, 2 missense mutations (R367C and P401H) were regarded as functional mutations (functional impact score >1.938). Furthermore, the present study successfully knocked down ERG gene expression through a lentiviral‑mediated gene silencing approach in VCaP prostate cancer cells. The ERG mRNA and protein expression levels were both suppressed significantly, and a cell‑cycle arrest at G0/G1 phase was observed after ERG gene silencing. In conclusion, these bioinformatics analyses provide novel insights for TMPRSS2:ERG fusion gene study in prostate cancer. Target inhibition of ERG expression could significantly cause cell growth arrest in prostate cancer cells, which could be a potentially valuable target for prostate cancer treatment. However, the precise mechanism of these results remains unclear; therefore, further studies are required.

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Antibody-independent targeted quantification of TMPRSS2-ERG fusion protein products in prostate cancer.

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