Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.
Institute of Pathology, University Medicine Greifswald, Friedrich-Loeffler-Straße 23e, 17475, Greifswald, Germany.
Cell Commun Signal. 2019 Nov 29;17(1):159. doi: 10.1186/s12964-019-0456-x.
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.
核孔蛋白家族的成员作为核输入受体/衔接蛋白，为核质和细胞质之间的大分子交换提供了途径。有新的证据表明，核孔蛋白家族的一部分在肝癌发生中失调，包括核输入蛋白-α2（KPNA2）。然而，KPNA2 在肝癌中的功能和调节作用仍不完全清楚。
使用定量蛋白质组学（LC-MS/MS，总共约 1750 种蛋白质）研究了 HCC 细胞中 siRNA 介导的 KPNA2 敲低后全局蛋白质丰度的变化。功能和机制分析包括集落形成和 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。体外结果与来自小鼠 HCC 模型和 HCC 患者样本（3 个队列，总共超过 600 例）的数据相关。
蛋白质组学方法揭示了在 HCC 细胞中 KPNA2 耗竭后最下调的蛋白质之一是促肿瘤微管（MT）相互作用蛋白 stathmin（STMN1）。我们进一步观察到，KPNA2 敲低导致 HCC 细胞迁移和集落形成减少，而直接敲低 stathmin 可模拟这种现象。作为潜在的调节机制，我们发现 E2F1 和 TFDP1 是 KPNA2 的转运底物，在 KPNA2 缺失后被保留在细胞质中，从而导致 STMN1 表达减少。最后，鼠和人 HCC 数据表明，STMN1 的表达与 E2F1/TFPD1 以及 KPNA2 的表达显著相关，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，从而为 HCC 中核转运和 MT 相互作用蛋白之间提供了新的联系，具有功能和预后意义。
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