Oncogene (2020) 39:2212–2223 https://doi.org/10.1038/s41388-019-1137-3 BRIEF COMMUNICATION Disease-specific alteration of karyopherin-α subtype establishes feed-forward oncogenic signaling in head and neck squamous cell carcinoma 1,2,3 1 3 4 2 2 Masaharu Hazawa ● Kie Sakai ● Akiko Kobayashi ● Hironori Yoshino ● Yoshihiro Iga ● Yuki Iwashima ● 3 5 6 1,7 6 Kee Sing Lim ● Dominic Chih-Cheng Voon ● Yan-Yi Jiang ● Shin-ichi Horike ● De-Chen Lin ● Richard W. Wong 1,2,3 Received: 29 March 2019 / Revised: 25 November 2019 / Accepted: 27 November 2019 / Published online: 10 December 2019 © The Author(s) 2019. This article is published with open access Abstract Nuclear import, mediated in part by karyopherin-α (KPNA)/importin-α subtypes, regulates transcription factor access to the genome and determines cell fate. However, the cancer-specific changes of KPNA subtypes and the relevancy in cancer biology remain largely unknown. Here, we report that KPNA4, encoding karyopherin-α4 (KPNA4), is exclusively amplified and overexpressed in head and neck of squamous cell carcinoma (HNSCC). Depletion of KPNA4 attenuated nuclear 1234567890();,: 1234567890();,: localization signal-dependent transport activity and suppressed malignant phenotypes and induced epidermal differentiation. Mechanistically, KPNA4-mediated nuclear transport of Ras-responsive element-binding protein (RREB1), which sustains Ras/ERK pathway signaling through repressing miR-143/145 expression. Notably, MAPK signaling enhanced trafficking activity of KPNA4 via phosphorylation of KPNA4 at Ser60. These data reveal that KPNA4 establishes a feed-forward cascade that potentiates Ras/ERK signaling in HNSCC. Introduction Supplementary information The online version of this article (https:// doi.org/10.1038/s41388-019-1137-3) contains supplementary material, which is available to authorized users. Head and neck squamous cell carcinoma (HNSCC) is lethal malignancies arising from oral squamous epithelium. * Masaharu Hazawa HNSCC is characterized by aggressive behavior such as [email protected] metastasis and recurrence. Previous studies highlighted a * Richard W. Wong key subset of oncogenic transcription factors (TFs) and gene [email protected] targets in human SCCs including HNSCC [1–7]. Current gene-expression network approaches commonly focus on 1 Cell-Bionomics Research Unit, Institute for Frontier Science TFs, biasing network-based discovery efforts away from Initiative, Kanazawa University, Kanazawa, Ishikawa, Japan potentially important non-TF proteins. 2 Laboratory of Molecular Cell Biology, School of Natural System, The nuclear pore complex (NPC) is constructed by 30 Institute of Science and Engineering, Kanazawa University, Kanazawa, Ishikawa, Japan nucleoporin (NUP) proteins and establishes the sole gate- way between the nucleus and the cytoplasm. Systematic 3 WPI Nano Life Science Institute, Kanazawa University, Kanazawa, Ishikawa, Japan nucleocytoplasmic transport occurs through NPCs on the 4 nuclear envelope [8–11]. Accumulating evidence has sug- Department of Radiation Science, Hirosaki University Graduate fi School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036- gested that tissue/disease-speci c NUP arrangements 8564, Japan determine cell fate [12–18]. We recently reported that 5 Cancer Model Research Innovative Unit, Institute for Frontier expression levels of NUP62, which regulates nuclear Science Initiative, Kanazawa University, Kanazawa, Japan transport of the oncogenic TF p63, are elevated in SCCs 6 Department of Medicine, Cedars-Sinai Medical Center, [16]. Later, Roriguez-Bravo et al. further showed that Los Angeles, CA 90048, USA overexpressed POM121, a transmembrane NPC compo- 7 Advanced Science Research Center, Institute for Gene Research, nent, drives nuclear import of oncogenic TFs and promotes Kanazawa University, Kanazawa, Ishikawa, Japan lethal prostate cancer [17]. Together these findings suggest Disease-specific alteration of karyopherin-α subtype establishes feed-forward oncogenic. 2213 that cancer-specific alterations of nuclear transport factors Results may facilitate the transduction of oncogenic signaling. Nuclear transport receptors (NTRs), such as karyopherin- Identification of KPNA4 as a HNSCC-specifically α (KPNA) and karyopherin-β (KPNB) (also known as altered gene importins) family members, selectively aid the shuttle of karyophilic cargo proteins, which harbor nuclear localiza- To comprehensively profile the genetic abnormalities tion signals (NLSs), through NPCs [19–21]. The human affecting KPNA, the Cancer Genome Atlas (TCGA) was re KPNA family consists of 7 subtypes and each KPNA has analyzed. The results showed that KPNA4 was the most cargo specificity [21]. Although the significance of KPNA amplified in HNSCC (Fig. 1a). Analysis of KPNA4 subtypes during differentiation and development has been amplification across multiple cancers revealed that HNSCC established [22, 23]. Onset NTRs abnormality of human showed remarkable amplification (Fig. 1b), which was also HNSCC and its significance remain unknown. observed other types of SCCs arising from lung and cervix Here, we performed genomic profiling of KPNA family (data not shown). We next analyzed TCGA RNA-seq data members in HNSCC and identified KPNA4, encoding kar- to examine mRNA levels of each KPNA subtype. The yopherin-α4 (KPNA4), as a specifically amplified and mRNA expression level of KPNA4 was markedly higher overexpressed KPNA subtype in HNSCC. KPNA4 is than other KPNA subtypes (Fig. 1c). Next, we compared required for prevention of epidermal differentiation and the KPNA4 transcript amounts between 433 cases of activation of the Ras/MAPK pathway through mediating HNSCC patients with various pathological stages and 43 nuclear transport of Ras-responsive element-binding protein cases of normal subjects via Cancer RNA-Seq Nexus (See (RREB1). Further, MAPK-dependent phosphorylation of “URLs”). The analysis revealed that KPNA4 is significantly importin-β binding (IBB) domain of KPNA4 enhances its elevated in HNSCC patients (Fig. 1d). Notably, KPNA4 traffic activity, resulting in feed-forward loop of MAPK mRNA levels were especially higher in HNSCC based on pathway in HNSCC. Cancer Cell Line Encyclopedia datasets (Fig. 1e). Fig. 1 Profiling of KPNA alteration in HNSCC. a Analysis of KPNA expression of KPNA4 in nontumor tissue, and HNSCC samples from copy number alteration (CNA) in HNSCC from TCGA (http://ca Cancer RNA-Seq Nexus (http://syslab4.nchu.edu.tw/). SI, SII, SIII, ncergenome.nih.gov/). b Summary of KPNA4 amplification across SIV denote stages I, II, III and IV. e KPNA4 mRNA expression across different tumor types from TCGA. SA stomach adenocarcinoma, LA different types of cancer cells from CCLE (http://portals.broadinstitute. lung adenocarcinoma, CA colon adenocarcinoma, BIA breast invasive org/ccle/). f High KPNA4 expression (mRNA expression z-Scores adenocarcinoma, LHC liver hepatocellular carcinoma, GM glio- (RNA-Seq V2 RSEM) >mean +2.0SD) was associated with poor blastoma multiform, AML acute myeloid leukemia, SC sarcoma. c The disease-free survival of HNSCC patients in the TCGA cohorts. expression of KPNA family transcripts in HNSCC from TCGA. d The 2214 M. Hazawa et al. Kaplan–Meier analysis on the TCGA cohorts further dependent nuclear transport in those cells since NLS- revealed that the upregulation of KPNA4 was significantly dependent nuclear import is dominantly regulated by Kar- correlated with poorer outcome of HNSCC patients (Fig. 1f). yopheryin family [24]. To investigate the role of KPNA4 on Collectively, these findings suggested that, within KPNA nuclear import ability, GFP harboring SV40-derived NLS family, KPNA4 is uniquely upregulated in HNSCC. (GFPNLS) was generated (Supplementary Fig. 1) and expressed to HNSCC cells. We found that KPNA4 is required for cellular malignancy and KPNA4 silencing caused reduction of nuclear GFPNLS regulates global gene-expression network of HNSCC amounts while cytoplasmic GFPNLS levels was increased cells upon KPNA4 knockdown (Fig. 2b, c), suggesting KPNA4 mediates NLS-dependent nuclear imports [24]. To study the biological role of KPNA4 in HNSCC, we Next, we investigated whether KPNA4 is functionally silenced KPNA4 in several HNSCC cell lines by shRNA- involved in cancer cell biology. Silencing of KPNA4 mediated knockdown (Fig. 2a). First, we investigated NLS- resulted in a significant reduction in short-term cell Fig. 2 KPNA4 mediates cellular malignant phenotype and affects 0.05. d Short-term proliferation was analyzed MTT assay Data gene expression in HNSCC. a Western blot analysis of each KPNA4 represent means ± SD (n = 3). e The heat map from microarray data as well as KPNB1 in various HNSCC cell lines expressing shRNA for showing upregulated and downregulated genes after knockdown of KPNA4. b Western blot analysis of GFPNLS in both cytoplasmic and KPNA4. f GO analysis of the upregulated genes upon KPNA4 nucleus fraction. c Quantification of the protein levels were performed. depletion. g Pathway analysis of the downregulated genes after Data show mean ± SD from three independent experiments (n = 3). KPNA4 knockdown. One sample t test was performed using GraphPad QuickCalcs. *p < Disease-specific alteration of karyopherin-α subtype establishes feed-forward oncogenic. 2215 proliferation, foci formation, migration ability and resis- analysis indicated NF-κB signaling, Ras signaling and MAPK tance to radiation in HNSCC cells (Fig. 2d and Supple- signaling were affected by silencing of KPNA4 (Fig. 2g). mentary Fig. 2). Importazol,