Background & Aims

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 MAN2A1−FER in 4 cell lines led to epidermal growth factor receptor activation of BRAF, MEK, and AKT; HUH7 cells with MAN2A1−FER knockout had significant decreases in phosphorylation of these proteins. Cell lines that expressed MAN2A1−FER 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 MAN2A1−FER 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 MAN2A1−FER 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 MAN2A1−FER fusion, which increases proliferation and invasiveness of cancer cell lines and has liver oncogenic activity in mice.

中文翻译：

---

MAN2A1–FER 融合基因由人类肝脏和其他肿瘤类型表达，并在小鼠中具有致癌活性

 背景与目标

人类肿瘤和肝癌细胞系表达甘露糖苷酶 α 2A 类成员 1 基因 ( MAN2A1 ) 的前 13 个外显子与 FER 酪氨酸激酶基因 ( FER ) 的后 6 个外显子之间的融合产物，称为MAN2A1−FER 。我们研究了MAN2A1−FER是否由人类肝脏肿瘤表达及其在肝癌发生中的作用。

 方法

我们对 102 个非小细胞肺癌、61 个卵巢肿瘤、70 个肝肿瘤、156 个多形性胶质母细胞瘤样本、27 个食管腺癌和 269 个前列腺癌样本以及 10 个非肿瘤肝组织和 20 个非肿瘤样本进行了逆转录聚合酶链反应分析。前列腺组织，收集于匹兹堡大学。我们还测量了 15 种人类癌细胞系的表达。我们在 NIH3T3 和 HEP3B（肝癌）细胞中表达了MAN2A1−FER的标记形式；分离高尔基体进行分析。 MAN2A1−FER在 PC3 或 DU145（前列腺癌）、NIH3T3（成纤维细胞）、H23（肺癌）和 A-172（多形性胶质母细胞瘤）细胞系中也过表达，并在 HUH7（肝癌）细胞中被敲除。对细胞进行增殖和侵袭分析分析，和/或将其注射到严重联合免疫缺陷小鼠的胁腹中；评估异种移植肿瘤的生长和转移。对肝脏 PTEN 缺失的小鼠进行尾静脉注射MAN2A1−FER 。

 结果

我们在肝细胞癌细胞系 HUH7 以及肝肿瘤、食管腺癌、多形性胶质母细胞瘤、前列腺肿瘤、非小细胞肺肿瘤和卵巢肿瘤中检测到MAN2A1−FER信使 RNA 和融合蛋白 (114 kD)，但是不是非肿瘤前列腺或肝组织。 MAN2A1−FER 蛋白保留了 MAN2A1 的高尔基体定位信号肽，并在癌细胞系中从细胞质易位至高尔基体。 MAN2A1−FER 的酪氨酸激酶活性几乎是野生型 FER 的 4 倍，并且在其 N 末端的酪氨酸 88 处磷酸化表皮生长因子受体。 MAN2A1 - FER在 4 个细胞系中的表达导致 BRAF、MEK 和 AKT 的表皮生长因子受体激活； MAN2A1 - FER敲除的 HUH7 细胞这些蛋白质的磷酸化显着降低。与不表达融合蛋白的细胞相比，表达MAN2A1 - FER 的细胞系的增殖、集落形成和侵袭性增加，并在小鼠中形成更大（>2 倍）的异种移植肿瘤，具有更多的转移灶。与对照 HUH7 细胞相比， MAN2A1 - FER敲除的 HUH7 细胞形成更小的异种移植肿瘤，转移更少。表达 MAN2A1−FER 的 HUH7、A-172 和 PC3 细胞对 FER 激酶抑制剂克唑替尼和表皮生长因子受体激酶抑制剂卡纳替尼的敏感性高出约 2 倍；这些药物减缓了 MAN2A1−FER 细胞异种移植肿瘤的生长，并防止其在小鼠体内的转移。 MAN2A1 - FER的水动力尾静脉注射导致Pten肝损伤的小鼠迅速发展为肝癌。

 结论

许多人类肿瘤类型和癌细胞系表达MAN2A1 - FER融合蛋白，该融合蛋白会增加癌细胞系的增殖和侵袭性，并在小鼠中具有肝癌活性。