Department of Medicine/Division of Liver Diseases, Mount Sinai School of Medicine, New York, NY 10029-6574, USA.
Hepatology. 2012 Oct;56(4):1361-70. doi: 10.1002/hep.25810. Epub 2012 Aug 27.
KLF6-SV1 (SV1), the major splice variant of KLF6, antagonizes the KLF6 tumor suppressor by an unknown mechanism. Decreased KLF6 expression in human hepatocellular carcinoma (HCC) correlates with increased mortality, but the contribution of increased SV1 is unknown. We sought to define the impact of SV1 on human outcomes and experimental murine hepatocarcinogenesis and to elucidate its mechanism of action. In hepatitis C virus (HCV)-related HCC, an increased ratio of SV1/KLF6 within the tumor was associated with features of more advanced disease. Six months after a single injection of diethylnitrosamine (DEN), SV1 hepatocyte transgenic mice developed more histologically advanced tumors, whereas Klf6-depleted mice developed bigger tumors compared to the Klf6fl(+/+) control mice. Nine months after DEN, SV1 transgenic mice with Klf6 depletion had the greatest tumor burden. Primary mouse hepatocytes from both the SV1 transgenic animals and those with hepatocyte-specific Klf6 depletion displayed increased DNA synthesis, with an additive effect in hepatocytes harboring both SV1 overexpression and Klf6 depletion. Parallel results were obtained by viral SV1 transduction and depletion of Klf6 through adenovirus-Cre infection of primary Klf6fl(+/+) hepatocytes. Increased DNA synthesis was due to both enhanced cell proliferation and increased ploidy. Coimmunoprecipitation studies in 293T cells uncovered a direct interaction of transfected SV1 with KLF6. Accelerated KLF6 degradation in the presence of SV1 was abrogated by the proteasome inhibitor MG132.
An increased SV1/KLF6 ratio correlates with more aggressive HCC. In mice, an increased SV1/KLF6 ratio, generated either by increasing SV1, decreasing KLF6, or both, accelerates hepatic carcinogenesis. Moreover, SV1 binds directly to KLF6 and accelerates its degradation. These findings represent a novel mechanism underlying the antagonism of tumor suppressor gene function by a splice variant of the same gene.
KLF6-SV1（SV1）是 KLF6 的主要剪接变体，通过未知机制拮抗 KLF6 肿瘤抑制因子。人类肝细胞癌（HCC）中 KLF6 表达的降低与死亡率的增加有关，但增加的 SV1 的贡献尚不清楚。我们试图确定 SV1 对人类结局和实验性小鼠肝癌发生的影响，并阐明其作用机制。在丙型肝炎病毒（HCV）相关的 HCC 中，肿瘤内 SV1/KLF6 的比值增加与更晚期疾病的特征有关。在单次注射二乙基亚硝胺（DEN）后 6 个月，SV1 肝细胞转基因小鼠发展出更具组织学特征的晚期肿瘤，而 Klf6 耗尽的小鼠比 Klf6fl(+/+)对照小鼠发展出更大的肿瘤。在 DEN 后 9 个月，SV1 转基因小鼠的 Klf6 耗竭有最大的肿瘤负担。来自 SV1 转基因动物和具有肝细胞特异性 Klf6 耗竭的原代小鼠肝细胞显示出增加的 DNA 合成，在同时具有 SV1 过表达和 Klf6 耗竭的肝细胞中具有附加效应。通过病毒 SV1 转导和通过腺病毒-Cre 感染原代 Klf6fl(+/+)肝细胞对 Klf6 的耗竭获得了平行结果。增加的 DNA 合成归因于细胞增殖的增强和倍性的增加。在 293T 细胞中的共免疫沉淀研究揭示了转染的 SV1 与 KLF6 的直接相互作用。在存在 SV1 的情况下，KLF6 的降解加速被蛋白酶体抑制剂 MG132 阻断。
增加的 SV1/KLF6 比值与更具侵袭性的 HCC 相关。在小鼠中，无论是通过增加 SV1、减少 KLF6 还是两者兼而有之，增加的 SV1/KLF6 比值都会加速肝肿瘤发生。此外，SV1 直接与 KLF6 结合并加速其降解。这些发现代表了一种新的机制，说明了剪接变体通过相同基因拮抗肿瘤抑制基因功能。
