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Karyopherin Α2-Dependent Import of E2F1 and TFDP1 Maintains

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Karyopherin Α2-Dependent Import of E2F1 and TFDP1 Maintains Drucker et al. Cell Communication and Signaling (2019) 17:159 https://doi.org/10.1186/s12964-019-0456-x RESEARCH Open Access Karyopherin α2-dependent import of E2F1 and TFDP1 maintains protumorigenic stathmin expression in liver cancer Elisabeth Drucker1,2, Kerstin Holzer2, Stefan Pusch3,4, Juliane Winkler5, Diego F. Calvisi6, Eva Eiteneuer1, Esther Herpel1, Benjamin Goeppert1, Stephanie Roessler1, Alessandro Ori7,8, Peter Schirmacher1, Kai Breuhahn1 and Stephan Singer1,2* Abstract Background: 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. Methods: 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). Results: 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. Conclusion: 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. Keywords: Karyopherin, Stathmin, HCC, E2F1, TFDP1, Nuclear transport Background Sorafenib for systemic treatment of advanced disease stage Hepatocellular carcinoma (HCC) ranks fifth among the [2]. A more detailed understanding in particular of those most common malignancies worldwide and second among molecular mechanisms that have not yet been in the pri- leading causes of cancer-related death [1]. The prognosis of mary focus of liver cancer related research such as alter- HCC is poor and therapeutic options are limited including ations of the nuclear transport system (NTS), holds great partial hepatectomy, liver transplantation, radio frequency potential for improved therapeutic approaches [3]. ablation, transarterial chemoembolization (TACE) and The NTS is essential for the exchange of macromolecules between the nucleus and the cytoplasm [3, 4]. The NTS in- * Correspondence: [email protected] cludes importins and exportins mostly belonging to the kar- 1Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany yopherin superfamily and components of the nuclear pore 2Institute of Pathology, University Medicine Greifswald, complex (NPC), termed Nucleoporins (Nups) [3]. The clas- Friedrich-Loeffler-Straße 23e, 17475 Greifswald, Germany sical protein import pathway involves binding of cargo Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Drucker et al. Cell Communication and Signaling (2019) 17:159 Page 2 of 14 proteins containing a nuclear localization signal (NLS) to siRNA-transfections adaptor proteins of the karyopherin-α family which in turn Small interfering RNAs (siRNAs) KPNA2#1 (5′-AAUCUU interact with importin-β1[4, 5]. This heterotrimeric ACCUGGACACUUU-3′) and KPNA2#2 (5′-UUCGUU complex enters the nucleus through the NPC and dissoci- AAGCUUAAUUGAGAA-3′), STMN1#1 (5′-AGGCAA ates in a RanGTP-dependent fashion releasing its transport UAGAAGAGAACAA-3′) and STMN1#2 (5′-AAGAGA substrate. Karyopherin-α is then re-shuttled by exportin 2/ AACUGACCCACAA-3′), E2F1#1 (5′-AACUCCUCGC Cellular apoptosis susceptibility (XPO2/CAS) to the cyto- AGAUCGUCAUC-3′) and E2F1#2 (5′-CAGAUCUCCC plasm [6] while importin-β1 is exported by binding to UUAAGAGCAAA-3′), TFDP1#1 (5′-CAGAACCTTA RanGDP [3, 4]. GTCCCGGGAAA-3′) and TFDP1#2 (5′-CACATTTGAA Altered nuclear transport factors in cancer have been ATCCACGATGA-3′), c-JUN#1 (5′-AAGAACGTGA primarily studied in the context of Nup-containing fusion CAGATGAGCAG-3′)andc-JUN#2(5′-CCCGAGCTGG proteins [7, 8], but are also observed in a variety of solid AGCGCCTGATA-3′) were purchased from Eurofins tumors including liver cancer [3, 9, 10]. Karyopherin-α2 MWG Operon (Ebersberg, Germany). As negative control (KPNA2; =importin alpha 1) is among the strongest over- siRNA for all knockdown experiments the QIAGEN All- expressed karyopherins in HCC, as previously described Stars duplex (Hilden, Germany) was used. The transfections [9]. However, the functional aspects of KPNA2 in HCC were performed according to the manufacturer’sinstruc- and the underlying mechanisms by which KPNA2 sup- tions using Oligofectamine (Invitrogen, Karlsruhe, Germany) ports tumorigenesis are poorly understood. with a final siRNA concentration of 50 nM. For siRNA Highly dynamic turnover of the microtubule (MT) net- pools, the two respective siRNAs were combined at a con- work is essential to tumor cell growth, migration, invasion centration of 25 nM each to reach a final concentration of and dissemination. MTs consist of α-tubulin and β- 50 nM. tubulin heterodimers and are characterized by a perman- ent transition (dynamic instability) between phases of Immunoblotting depolymerization (catastrophe) and polymerization (res- Immunoblotting was performed as previously described cue) [11]. MT-interacting proteins modulate the dynamic [9]. In brief, whole protein lysates were separated by instability of MTs either by executing MT-stabilizing or SDS/PAGE and transferred to nitrocellulose membranes -destabilizing functions. Stathmin (oncoprotein 18/OP18, (Whatman, Dassel, Germany). Membranes were incu- STMN1) represents the prototype member of a MT- bated with the following primary antibodies diluted in destabilizing phosphoprotein family that encompasses also 5% Milk/TBST-containing blocking solution overnight: stathmin-like 2 (superior cervical ganglion 10; SCG10, anti-KPNA2 (rabbit polyclonal, 1:2000; abcam, Cam- STMN2), stathmin-like 3 (SCG10-like protein; SCLIP, bridge, UK), anti-stathmin (rabbit monoclonal, 1:1000; STMN3), and stathmin-like 4 (RB3, STMN4)[12]. Stath- abcam), anti-E2F1 (rabbit polyclonal, 1:200; Santa Cruz, min is the best characterized member of this protein fam- Heidelberg, Germany), anti-TFDP1 (rabbit polyclonal, 1: ily in the context of cancer biology [13] and has been 500; abcam), anti-ATF-2 (rabbit polyclonal, 1:200; Santa described to facilitate tumor cell migration, invasion and Cruz), anti-FBP-1/2 (goat polyclonal, 1:200; Santa Cruz), colony formation in many cancer types [14–16] including anti-c-JUN (rabbit monoclonal, 1:2000; Cell Signaling HCC [17]. Technology, Frankfurt, Germany), anti-HMOX1 (rabbit Here, we identified by proteome-wide analysis that monoclonal, 1:10,000; abcam), anti-GTSF1 (goat poly- KPNA2 is required for maintaining stathmin overex- clonal, 1:200; Santa Cruz), anti-PARP (rabbit polyclonal, pression in liver cancer cells and dissected the 1:500; Cell Signaling Technology), anti-β-tubulin (mouse underlying regulatory mechanism involving the nu- monoclonal, 1:1000; Becton, Dickinson and Company, clear import of the transcription factors E2F1 and Franklin Lakes, USA) and anti-β-actin (mouse monoclo- TFDP1. nal, 1:10,000; MP Biomedicals, Illkirch, France). Blots were incubated with fluorescence-conjugated secondary antibodies (LI-COR Bioscience, Bad Homburg, Methods Germany) for one hour and detection was performed Cell culture using the Odysee Sa Infrared Imaging System (LI-COR HLE and HLF cells (JCRB0404 and JCRB0405, both ob- Bioscience). tained from JCRB Cell Bank, Osaka, Japan) were cultured in Dulbecco’sModifiedEagle’sMedium(DMEM,ob- Subcellular fractionation tained from Sigma-Aldrich, Taufkirchen, Germany) sup- NE-PER Nuclear and Cytoplasmic Extraction Reagents plemented with 10% fetal calf serum and 1% penicillin/ (Thermo Scientific, Offenbach, Germany) were used ac- streptomycin (Life Technologies, Darmstadt, Germany) in cording to the manufacturer’s protocol with an add- an atmosphere containing 5% CO2. itional washing step after isolation of the cytoplasmic
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