Members of the karyopherin superfamily serve as nuclear transport receptors/adaptor proteins and provide exchange of macromolecules between the nucleo- and cytoplasm. Emerging evidence suggests a subset of karyopherins to be dysregulated in hepatocarcinogenesis including karyopherin-α2 (KPNA2). However, the functional and regulatory role of KPNA2 in liver cancer remains incompletely understood. Quantitative proteomics (LC-MS/MS, ~ 1750 proteins in total) was used to study changes in global protein abundance upon siRNA-mediated KPNA2 knockdown in HCC cells. Functional and mechanistic analyses included colony formation and 2D migration assays, co-immunoprecipitation (CoIP), chromatin immunoprecipitation (ChIP), qRT-PCR, immmunblotting, and subcellular fractionation. In vitro results were correlated with data derived from a murine HCC model and HCC patient samples (3 cohorts, n > 600 in total). The proteomic approach revealed the pro-tumorigenic, microtubule (MT) interacting protein stathmin (STMN1) among the most downregulated proteins upon KPNA2 depletion in HCC cells. We further observed that KPNA2 knockdown leads to reduced tumor cell migration and colony formation of HCC cells, which could be phenocopied by direct knockdown of stathmin. As the underlying regulatory mechanism, we uncovered E2F1 and TFDP1 as transport substrates of KPNA2 being retained in the cytoplasm upon KPNA2 ablation, thereby resulting in reduced STMN1 expression. Finally, murine and human HCC data indicate significant correlations of STMN1 expression with E2F1/TFPD1 and with KPNA2 expression and their association with poor prognosis in HCC patients. Our data suggest that KPNA2 regulates STMN1 by import of E2F1/TFDP1 and thereby provide a novel link between nuclear transport and MT-interacting proteins in HCC with functional and prognostic significance.

中文翻译：

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E2F1和TFDP1依赖核转运蛋白α2的导入维持肝癌中促成瘤性stathmin的表达

核转运蛋白超家族的成员充当核转运受体/适配器蛋白，并在核质和细胞质之间提供大分子的交换。越来越多的证据表明，在肝癌发生过程中，一部分核球蛋白将失调，包括核球蛋白-α2（KPNA2）。但是，KPNA2在肝癌中的功能和调节作用仍未完全了解。定量蛋白质组学（LC-MS / MS，总共约1750种蛋白质）被用于研究HCC细胞中siRNA介导的KPNA2敲低后全局蛋白质丰度的变化。功能和机理分析包括菌落形成和2D迁移测定，共免疫沉淀（CoIP），染色质免疫沉淀（ChIP），qRT-PCR，免疫印迹和亚细胞分级分离。体外结果与来自鼠类HCC模型和HCC患者样品的数据相关（3个队列，总共n> 600）。蛋白质组学方法揭示了HCC细胞中KPNA2耗尽后，促肿瘤原性微管（MT）相互作用蛋白stathmin（STMN1）是最下调的蛋白之一。我们进一步观察到，KPNA2敲低导致肿瘤细胞迁移和HCC细胞集落形成减少，这可以通过直接敲除stathmin来表型复制。作为潜在的调节机制，我们发现E2F1和TFDP1是KPNA2消融后保留在细胞质中的KPNA2的运输底物，从而导致STMN1表达降低。最后，小鼠和人类HCC数据表明STMN1表达与E2F1 / TFPD1和KPNA2表达显着相关，并且与HCC患者的不良预后相关。我们的数据表明，KPNA2通过导入E2F1 / TFDP1来调节STMN1，从而在核转运和肝癌中MT相互作用蛋白之间提供一种新颖的联系，具有功能和预后意义。