3 years ago

MAN2A1–FER Fusion Gene Is Expressed by Human Liver and Other Tumor Types and Has Oncogenic Activity in Mice

Human tumors and liver cancer cell lines express the product of a fusion between the first 13 exons in the mannosidase α class 2A member 1 gene (MAN2A1) and the last 6 exons in the FER tyrosine kinase gene (FER), called MAN2A1−FER. We investigated whether MAN2A1−FER is expressed by human liver tumors and its role in liver carcinogenesis. Methods We performed reverse transcription polymerase chain reaction analyses of 102 non−small cell lung tumors, 61 ovarian tumors, 70 liver tumors, 156 glioblastoma multiform samples, 27 esophageal adenocarcinomas, and 269 prostate cancer samples, as well as 10 nontumor liver tissues and 20 nontumor prostate tissues, collected at the University of Pittsburgh. We also measured expression by 15 human cancer cell lines. We expressed a tagged form of MAN2A1−FER in NIH3T3 and HEP3B (liver cancer) cells; Golgi were isolated for analysis. MAN2A1−FER was also overexpressed in PC3 or DU145 (prostate cancer), NIH3T3 (fibroblast), H23 (lung cancer), and A-172 (glioblastoma multiforme) cell lines and knocked out in HUH7 (liver cancer) cells. Cells were analyzed for proliferation and in invasion assays, and/or injected into flanks of severe combined immunodeficient mice; xenograft tumor growth and metastasis were assessed. Mice with hepatic deletion of PTEN were given tail-vein injections of MAN2A1−FER. Results We detected MAN2A1−FER messenger RNA and fusion protein (114 kD) in the hepatocellular carcinoma cell line HUH7, as well as in liver tumors, esophageal adenocarcinoma, glioblastoma multiforme, prostate tumors, non−small cell lung tumors, and ovarian tumors, but not nontumor prostate or liver tissues. MAN2A1−FER protein retained the signal peptide for Golgi localization from MAN2A1 and translocated from the cytoplasm to Golgi in cancer cell lines. MAN2A1−FER had tyrosine kinase activity almost 4-fold higher than that of wild-type FER, and phosphorylated the epidermal growth factor receptor at tyrosine 88 in its N-terminus. Expression of MAN2A1FER in 4 cell lines led to epidermal growth factor receptor activation of BRAF, MEK, and AKT; HUH7 cells with MAN2A1FER knockout had significant decreases in phosphorylation of these proteins. Cell lines that expressed MAN2A1FER had increased proliferation, colony formation, and invasiveness and formed larger (>2-fold) xenograft tumors in mice, with more metastases, than cells not expressing the fusion protein. HUH7 cells with MAN2A1FER knockout formed smaller xenograft tumors, with fewer metastases, than control HUH7 cells. HUH7, A-172, and PC3 cells that expressed MAN2A1−FER were about 2-fold more sensitive to the FER kinase inhibitor crizotinib and the epidermal growth factor receptor kinase inhibitor canertinib; these drugs slowed growth of xenograft tumors from MAN2A1−FER cells and prevented their metastasis in mice. Hydrodynamic tail-vein injection of MAN2A1FER resulted in rapid development of liver cancer in mice with hepatic disruption of Pten. Conclusions Many human tumor types and cancer cell lines express the MAN2A1FER fusion, which increases proliferation and invasiveness of cancer cell lines and has liver oncogenic activity in mice.

Publisher URL: www.sciencedirect.com/science

DOI: S0016508517300045

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