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CSE1L promotes nuclear accumulation of transcriptional coactivator TAZ and enhances invasiveness of human cancer cells

2021; Elsevier BV; Volume: 297; Issue: 1 Linguagem: Inglês

10.1016/j.jbc.2021.100803

ISSN

1083-351X

Autores

Shunta Nagashima, Junichi Maruyama, Kaori Honda, Yasumitsu Kondoh, Hiroyuki Osada, Makiko Nawa, Ken‐ichi Nakahama, Mari Ishigami‐Yuasa, Hiroyuki Kagechika, Haruhiko Sugimura, Hiroaki Iwasa, Kyoko Arimoto‐Matsuzaki, Hiroshi Nishina, Yutaka Hata,

Tópico(s)

RNA Research and Splicing

Resumo

The transcriptional coactivator with PDZ-binding motif (TAZ) (WWTR1) induces epithelial–mesenchymal transition and enhances drug resistance in multiple cancers. TAZ has been shown to interact with transcription factors in the nucleus, but when phosphorylated, translocates to the cytoplasm and is degraded through proteasomes. Here, we identified a compound TAZ inhibitor 4 (TI-4) that shifted TAZ localization to the cytoplasm independently of its phosphorylation. We used affinity beads to ascertain a putative target of TI-4, chromosomal segregation 1 like (CSE1L), which is known to be involved in the recycling of importin α and as a biomarker of cancer malignancy. We found that TI-4 suppressed TAZ-mediated transcription in a CSE1L-dependent manner. CSE1L overexpression increased nuclear levels of TAZ, whereas CSE1L silencing delayed its nuclear import. We also found via the in vitro coimmunoprecipitation experiments that TI-4 strengthened the interaction between CSE1L and importin α5 and blocked the binding of importin α5 to TAZ. WWTR1 silencing attenuated CSE1L-promoted colony formation, motility, and invasiveness of human lung cancer and glioblastoma cells. Conversely, CSE1L silencing blocked TAZ-promoted colony formation, motility, and invasiveness in human lung cancer and glioblastoma cells. In human cancer tissues, the expression level of CSE1L was found to correlate with nuclear levels of TAZ. These findings support that CSE1L promotes the nuclear accumulation of TAZ and enhances malignancy in cancer cells. The transcriptional coactivator with PDZ-binding motif (TAZ) (WWTR1) induces epithelial–mesenchymal transition and enhances drug resistance in multiple cancers. TAZ has been shown to interact with transcription factors in the nucleus, but when phosphorylated, translocates to the cytoplasm and is degraded through proteasomes. Here, we identified a compound TAZ inhibitor 4 (TI-4) that shifted TAZ localization to the cytoplasm independently of its phosphorylation. We used affinity beads to ascertain a putative target of TI-4, chromosomal segregation 1 like (CSE1L), which is known to be involved in the recycling of importin α and as a biomarker of cancer malignancy. We found that TI-4 suppressed TAZ-mediated transcription in a CSE1L-dependent manner. CSE1L overexpression increased nuclear levels of TAZ, whereas CSE1L silencing delayed its nuclear import. We also found via the in vitro coimmunoprecipitation experiments that TI-4 strengthened the interaction between CSE1L and importin α5 and blocked the binding of importin α5 to TAZ. WWTR1 silencing attenuated CSE1L-promoted colony formation, motility, and invasiveness of human lung cancer and glioblastoma cells. Conversely, CSE1L silencing blocked TAZ-promoted colony formation, motility, and invasiveness in human lung cancer and glioblastoma cells. In human cancer tissues, the expression level of CSE1L was found to correlate with nuclear levels of TAZ. These findings support that CSE1L promotes the nuclear accumulation of TAZ and enhances malignancy in cancer cells. The transcriptional coactivator with PDZ-binding motif (TAZ) (TAZ is widely used but as its official gene symbol is WWTR1 (HUGO Gene Nomenclature Committee [HGNC] ID: HGNC 24042; Entrez Gene 25937), we used TAZ and WWTR1 in this article for the protein [NP001161750.1] and the gene, respectively) was first identified as a protein that binds to 14-3-3 (1Kanai F. Marignani P.A. Sarbassova D. Yagi R. Hall R.A. Donowitz M. Hisaminato A. Fujiwara T. Ito Y. Cantley L.C. Yaffe M.B. TAZ: A novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins.EMBO J. 2000; 19: 6778-6791Crossref PubMed Scopus (528) Google Scholar). The phosphorylation at serine 89 by large tumor suppressor (LATS) kinases triggers the interaction with 14-3-3, resulting in the cytoplasmic segregation of TAZ (2Lei Q.Y. Zhang H. Zhao B. Zha Z.Y. Bai F. Pei X.H. Zhao S. Xiong Y. Guan K.L. TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway.Mol. Cell. Biol. 2008; 28: 2426-2436Crossref PubMed Scopus (636) Google Scholar). Phosphorylation by LATS kinases also causes protein degradation (3Liu C.Y. Zha Z.Y. Zhou X. Zhang H. Huang W. Zhao D. Li T. Chan S.W. Lim C.J. Hong W. Zhao S. Xiong Y. Lei Q.Y. Guan K.L. The hippo tumor pathway promotes TAZ degradation by phosphorylating a phosphodegron and recruiting the SCF{beta}-TrCP E3 ligase.J. Biol. Chem. 2010; 285: 37159-37169Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 4Huang D.C. Adams J.M. Cory S. The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4.EMBO J. 1998; 17: 1029-1039Crossref PubMed Scopus (198) Google Scholar). Thus, TAZ is negatively regulated by LATS kinases, core kinases of the tumor suppressor Hippo pathway. In human cancers, the dysregulation of the Hippo pathway and WWTR1 gene amplification lead to TAZ hyperactivation (5Piccolo S. Cordenonsi M. Dupont S. Molecular pathways: YAP and TAZ take center stage in organ growth and tumorigenesis.Clin. Cancer Res. 2013; 19: 4925-4930Crossref PubMed Scopus (105) Google Scholar, 6Zhang K. Qi H.X. Hu Z.M. Chang Y.N. Shi Z.M. Han X.H. Han Y.W. Zhang R.X. Zhang Z. Chen T. Hong W. YAP and TAZ take center stage in cancer.Biochemistry. 2015; 54: 6555-6566Crossref PubMed Scopus (61) Google Scholar). TAZ activation induces epithelial–mesenchymal transition, enhances drug resistance, confers stemness to cancer cells, and is associated with poor prognosis in cancers. Hence, TAZ is regarded as a potential target for cancer therapy. We screened for TAZ inhibitors by means of a cell-based assay (7Nagashima S. Maruyama J. Kawano S. Iwasa H. Nakagawa K. Ishigami-Yuasa M. Kagechika H. Nishina H. Hata Y. Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.Cancer Sci. 2016; 107: 791-802Crossref PubMed Scopus (3) Google Scholar). We expressed GFP-tagged TAZ (GFP-TAZ) in human osteosarcoma U2OS cells, in which the Hippo pathway is activated and inactivated depending on the cell density. In U2OS cells plated at low density, GFP-TAZ is accumulated in the nucleus. We applied 18,606 small chemical compounds to the cells and obtained as putative TAZ inhibitors compounds that shifted GFP-TAZ to the cytoplasm. We further selected compounds (IBS000540, IBS001594, IBS015181, and IBS015625) that significantly suppressed TAZ/TEAD-driven luciferase reporter activity in HEK293FT cells. IBS000540, IBS001594, and IBS015625 increased phosphorylated TAZ and decreased unphosphorylated TAZ. Hence, these three compounds inhibited TAZ in the phosphorylation-dependent manner through the canonical Hippo pathway. We previously reported these three compounds as TAZ inhibitors (7Nagashima S. Maruyama J. Kawano S. Iwasa H. Nakagawa K. Ishigami-Yuasa M. Kagechika H. Nishina H. Hata Y. Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.Cancer Sci. 2016; 107: 791-802Crossref PubMed Scopus (3) Google Scholar). In contrast, IBS015181 did not increase the amount of phosphorylated TAZ. It implies that the effect of IBS015181 does not depend on the canonical Hippo pathway. In this article, we named IBS015181 (IUPAC name: 2,2-dichloro-N-(4-nitrophenyl)-3-phenylcyclopropane-1-carboxamide) as TAZ inhibitor 4 (TI-4) and characterized it. Chromosomal segregation 1 like (CSE1L) (also called cellular apoptosis susceptibility (CAS) and exportin-2; according to its official gene symbol (HGNC 2431; Entrez Gene 1434), we used CSE1L to describe both the protein (NP001307) and the gene in this article) was identified as the gene that rendered human breast cancer MCF-7 cells resistant to immunotoxins and turned out to be homologous to yeast CSE1 gene, which is involved in the regulation of chromatins (8Brinkmann U. Brinkmann E. Gallo M. Pastan I. Cloning and characterization of a cellular apoptosis susceptibility gene, the human homologue to the yeast chromosome segregation gene CSE1.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10427-10431Crossref PubMed Scopus (163) Google Scholar, 9Xiao Z. McGrew J.T. Schroeder A.J. Fitzgerald-Hayes M. CSE1 and CSE2, two new genes required for accurate mitotic chromosome segregation in Saccharomyces cerevisiae.Mol. Cell. Biol. 1993; 13: 4691-4702Crossref PubMed Scopus (111) Google Scholar, 10Irniger S. Piatti S. Michaelis C. Nasmyth K. Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast.Cell. 1995; 81: 269-278Abstract Full Text PDF PubMed Scopus (466) Google Scholar). CSE1L binds to importin α in the presence of RanGTP and is required for the recycling of importin α (11Kutay U. Bischoff F.R. Kostka S. Kraft R. Görlich D. Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor.Cell. 1997; 90: 1061-1071Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 12Herold A. Truant R. Wiegand H. Cullen B.R. Determination of the functional domain organization of the importin alpha nuclear import factor.J. Cell Biol. 1998; 143: 309-318Crossref PubMed Scopus (100) Google Scholar). CSE1L is implicated in cell proliferation and apoptosis in human cancers (13Wellmann A. Flemming P. Behrens P. Wuppermann K. Lang H. Oldhafer K. Pastan I. Brinkmann U. High expression of the proliferation and apoptosis associated CSE1L/CAS gene in hepatitis and liver neoplasms: Correlation with tumor progression.Int. J. Mol. Med. 2001; 7: 489-494PubMed Google Scholar). High expression of CSE1L is associated with a poor prognosis in cancers and is considered to be a prognostic marker (14Behrens P. Brinkmann U. Fogt F. Wernert N. Wellmann A. Implication of the proliferation and apoptosis associated CSE1L/CAS gene for breast cancer development.Anticancer Res. 2001; 21: 2413-2417PubMed Google Scholar, 15Alnabulsi A. Agouni A. Mitra S. Garcia-Murillas I. Carpenter B. Bird S. Murray G.I. Cellular apoptosis susceptibility (chromosome segregation 1-like, CSE1L) gene is a key regulator of apoptosis, migration and invasion in colorectal cancer.J. Pathol. 2012; 228: 471-481Crossref PubMed Scopus (28) Google Scholar, 16Chang C.C. Tai C.J. Su T.C. Shen K.H. Lin S.H. Yeh C.M. Yeh K.T. Lin Y.M. Jiang M.C. The prognostic significance of nuclear CSE1L in urinary bladder urothelial carcinomas.Ann. Diagn. Pathol. 2012; 16: 362-368Crossref PubMed Scopus (19) Google Scholar, 17Yuksel U.M. Dilek G. Dogan L. Gulcelik M.A. Berberoglu U. The relationship between CSE1L expression and axillary lymph node metastasis in breast cancer.Tumori. 2015; 101: 194-198Crossref PubMed Scopus (8) Google Scholar). However, the mechanism underlying of the oncogenic action of CSE1L is not fully understood. We identified CSE1L as the target of TI-4. We revealed that CSE1L is involved in the regulation of the nuclear import of TAZ. Moreover, we demonstrated that CSE1L increases the nuclear TAZ and enhances malignancy in cancer cells. The structure of TI-4 is not related to those of the previously reported TAZ inhibitors, IBS00540, IBS001594, and IBS015625 (Fig. 1A) (7Nagashima S. Maruyama J. Kawano S. Iwasa H. Nakagawa K. Ishigami-Yuasa M. Kagechika H. Nishina H. Hata Y. Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.Cancer Sci. 2016; 107: 791-802Crossref PubMed Scopus (3) Google Scholar). First, we confirmed the effect of TI-4 on the subcellular distribution of TAZ in U2OS cells expressing GFP-TAZ. TI-4 decreased the nuclear GFP-TAZ (Fig. 1B, an arrow). TI-4 also reduced endogenous TAZ in the nucleus in U2OS cells (Fig. 1C, an arrow). In the Phos-tag gel, TI-4 rather decreased the phosphorylated TAZ (Fig. 1D, arrows), which was detected with anti-phospho-TAZ (S89) antibody but did not increase the unphosphorylated TAZ (Fig. 1D, an arrowhead). We also applied TI-4 to the cells expressing GFP-TAZ S89A mutant (Fig. S1A). TI-4 shifted GFP-TAZ S89A to the cytoplasm, which further supports that the effect of TI-4 is independent of TAZ phosphorylation. In the previous study, we showed that TI-4 suppressed TAZ/TEAD-driven luciferase reporter activity in HEK293FT cells (7Nagashima S. Maruyama J. Kawano S. Iwasa H. Nakagawa K. Ishigami-Yuasa M. Kagechika H. Nishina H. Hata Y. Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.Cancer Sci. 2016; 107: 791-802Crossref PubMed Scopus (3) Google Scholar). In the reporter assay, we used the artificial reporter gene driven by eight repeats of the TEAD-responsive sequence. In this study, we examined the effect of TI-4 on endogenous TEAD target genes. quantitative RT-PCR demonstrated that TI-4 suppressed the expression of CTGF and CYR61 in H1299 cells (Fig. 2A). As TAZ and TEAD play a role in the cancer development, we next examined the effect of TI-4 on cancer cells. We applied TI-4 to A549, H1299, and U87MG cells. We previously confirmed that WWTR1 silencing compromises the cell viability in these cancer cells (7Nagashima S. Maruyama J. Kawano S. Iwasa H. Nakagawa K. Ishigami-Yuasa M. Kagechika H. Nishina H. Hata Y. Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.Cancer Sci. 2016; 107: 791-802Crossref PubMed Scopus (3) Google Scholar). Therefore, if TI-4 is a TAZ inhibitor, it should suppress the viability of these cancer cells. Indeed, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays showed that TI-4 suppressed cell proliferation in these cells (Fig. 2B). TI-4 also blocked colony formation in the soft agar (Fig. 2C). These findings support that TI-4 inhibits TAZ/TEAD target genes and exhibits the anticancer effect. As TI-4 did not affect the phosphorylation state of TAZ but recruited TAZ into the cytoplasm, we speculated that TI-4 regulates TAZ through an unknown mechanism, which is distinct from the canonical Hippo pathway. With this in mind, we attempted to identify a target molecule of TI-4. We immobilized TI-4 onto Sepharose beads through a photoaffinity linker, charged the cell lysates of U87MG cells, and identified proteins that specifically bound to the beads (Fig. 3A). Although several protein bands were detected on control beads (Fig. 3A, Ctrl), the protein with approximately 97 kDa was detected only on TI-4-affinity beads (Fig. 3A, TI-4, an arrow). The protein was subjected to MS analysis and identified peptides derived from CSE1L (Fig. S2). Next, we applied TI-4 to U2OS-GFP-TAZ cells after CSE1L silencing and examined whether CSE1L is required for TI-4 to recruit TAZ into the cytoplasm. CSE1L silencing abolished the effect of TI-4 (Fig. 3B). In the immunofluorescence, TI-4 decreased GFP-TAZ in the nucleus (Fig. 3C, siCtrl, arrowheads). CSE1L silencing itself reduced the nuclear GFP-TAZ (Fig. 3C, siCSE1L #1, an arrowhead), and TI-4 did not show any additional effect. We also examined in H1299 cells whether CSE1L silencing blocked the effect of TI-4 on the expression of CTGF and CYR61. CSE1L silencing itself suppressed the expression of CTGF and CYR61 in HEK293FT cells (Fig. 3D, the first and third bars), while TI-4 did not decrease the expression of CTGF and CYR61 in the CSE1L-negative background (Fig. 3D, the third and fourth bars). Furthermore, CSE1L silencing suppressed the cell viability of H1299 cells in MTT assay (Fig. 3E), whereas TI-4 did not show any additional effect in the CSE1L-negative background (Fig. 3E, siCSE1L #1). These results support that TI-4 requires CSE1L to inhibit TAZ/TEAD target genes and to show anticancer effect. We next examined whether and how CSE1L affects the subcellular distribution of TAZ. CSE1L silencing reduced the nuclear GFP-TAZ in U2OS-GFP-TAZ cells and the endogenous nuclear TAZ in U2OS cells (Fig. 4, A and B, arrows). Conversely, CSE1L overexpression increased the nuclear TAZ (Fig. 4C, an arrow). CSE1L silencing did not increase unphosphorylated TAZ (Fig. 4D, an arrow). Furthermore, we knocked down CSE1L in U2OS cells expressing GFP-TAZ S89A (Fig. S1B). CSE1L silencing shifted TAZ S89A mutant to the cytoplasm. These findings support that the effect of CSE1L on the subcellular localization of TAZ does not depend on the phosphorylation state of TAZ. We next performed fluorescence recovery after photobleaching (FRAP) assay. The nuclear GFP was photobleached in U2OS-GFP-TAZ cells. TI-4 delayed the recovery of the GFP signal in the nucleus (Fig. 5A, squares). Ivermectin, which was identified as the TAZ inhibitor in the drug screening and is thought to be an importin inhibitor, showed a similar effect (18Nishio M. Sugimachi K. Goto H. Wang J. Morikawa T. Miyachi Y. Takano Y. Hikasa H. Itoh T. Suzuki S.O. Kurihara H. Aishima S. Leask A. Sasaki T. Nakano T. et al.Dysregulated YAP1/TAZ and TGF-β signaling mediate hepatocarcinogenesis in Mob1a/1b-deficient mice.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E71-E80Crossref PubMed Scopus (97) Google Scholar) (Fig. 5A, triangles). As CSE1L silencing decreased nuclear GFP-TAZ, the basal intensity of the nuclear GFP signal before photobleaching was lower in CSE1L-depleted cells than in control cells. Even so, CSE1L silencing delayed the recovery (Fig. 5B). TI-4 did not show additional effect after CSE1L silencing (Fig. 5C). We investigated into the molecular mechanism, by which TI-4 delays the nuclear incorporation of TAZ. The alias of CSE1L is exportin-2. Importin α has a nuclear localization signal (NLS)-binding site, forms a complex with NLS-harboring proteins and importin β, and incorporates NLS-harboring proteins into the nucleus (19Oka M. Yoneda Y. Importin α: Functions as a nuclear transport factor and beyond.Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 2018; 94: 259-274Crossref PubMed Scopus (37) Google Scholar). In the nucleus, the complex is dissociated. Then CSE1L binds to the released importin α and exports it into the cytoplasm (11Kutay U. Bischoff F.R. Kostka S. Kraft R. Görlich D. Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor.Cell. 1997; 90: 1061-1071Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 12Herold A. Truant R. Wiegand H. Cullen B.R. Determination of the functional domain organization of the importin alpha nuclear import factor.J. Cell Biol. 1998; 143: 309-318Crossref PubMed Scopus (100) Google Scholar). Thus, CSE1L contributes to the recycling of importin α. Recently, Kofler et al. (20Kofler M. Speight P. Little D. Di Ciano-Oliveira C. Szászi K. Kapus A. Mediated nuclear import and export of TAZ and the underlying molecular requirements.Nat. Commun. 2018; 9: 4966Crossref PubMed Scopus (34) Google Scholar) have revealed that TAZ has a new class NLS motif in the C-terminal region and a nuclear efflux signal in the N-terminal region. We tested whether TAZ can interact with importin α. We coexpressed GFP-TAZ with various FLAG-tagged importin α proteins in HEK293FT cells and performed the coimmunoprecipitation experiment and detected the interaction between TAZ and importin α proteins (Fig. S4A). We also immunoprecipitated TAZ from HEK293FT cells and detected the interaction between endogenous TAZ and importin α5 (Fig. S4B) and focused on importin α5 in this study. First, we confirmed that CSE1L is involved in the export of importin α5 in U2OS cells. We knocked down CSE1L in U2OS cells expressing GFP-importin α5. CSE1L silencing increased nuclear GFP-importin α5 in immunofluorescence and the subcellular fractionation (Fig. S5, an arrow). We subsequently examined whether and how TI-4 affects the interaction between CSE1L and importin α5. The interaction between CSE1L and importin α5 was hardly detected at the basal state, but TI-4 made it detectable (Fig. 6A, an arrow). We next examined whether and how TI-4 influenced the subcellular localization of importin α5. Under the treatment of TI-4, the nuclear importin α5 was reduced (Fig. 6B). These findings suggest that importin α5 remains to bind to CSE1L and is not incorporated into the nucleus under the treatment with TI-4. CSE1L binds to the C-terminal region of importin α and interferes with the interaction between the NLS and armadillo repeats (12Herold A. Truant R. Wiegand H. Cullen B.R. Determination of the functional domain organization of the importin alpha nuclear import factor.J. Cell Biol. 1998; 143: 309-318Crossref PubMed Scopus (100) Google Scholar). Hence, importin α does not bind to an NLS-harboring protein, until it is released from CSE1L. Considering the possibility that CSE1L competes with TAZ for the binding to importin α, we examined the effect of TI-4 on the interaction between TAZ and importin α5. TI-4 reduced the interaction between importin α5 and TAZ (Fig. 6C, an arrow). In contrast, CSE1L silencing remarkably augmented the interaction (Fig. 6D, an arrow). TI-4 did not inhibit the interaction between TAZ and importin α5 in the CSE1L-negative background (Fig. 6D, an arrowhead). These findings suggest that TI-4 blocks the dissociation of importin α5 from CSE1L in the cytoplasm and eventually inhibits the complex formation including importin α5 and TAZ. CSE1L is involved in the recycling of importin α (11Kutay U. Bischoff F.R. Kostka S. Kraft R. Görlich D. Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor.Cell. 1997; 90: 1061-1071Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). CSE1L is required for the nuclear import of nuclear proteins including histone deacetylases (21Dong Q. Li X. Wang C.Z. Xu S. Yuan G. Shao W. Liu B. Zheng Y. Wang H. Lei X. Zhang Z. Zhu B. Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E4013-E4022Crossref PubMed Scopus (14) Google Scholar). Therefore, we considered that CSE1L may regulate the subcellular distribution of other nuclear proteins. CSE1L silencing decreased the nuclear endogenous YAP1 in U2OS cells (Fig. 4B). We used U2OS cells expressing GFP-YAP1 and conducted FRAP assay (Fig. S6A). In FRAP experiment, CSE1L silencing delayed the recovery of the nuclear GFP-YAP1. However, when we immunoblotted the samples of subcellular fractionation, CSE1L silencing did not affect the nuclear localization of endogenous β-catenin and FoxO3a (Fig. S6B). Thus, although the underlying mechanism is unknown, CSE1L is involved in the regulation of the limited population of nuclear proteins. In human cancers, CSE1L confers malignant properties to cancer cells and is regarded as a poor prognostic biomarker (22Jiang M.C. CAS (CSE1L) signaling pathway in tumor progression and its potential as a biomarker and target for targeted therapy.Tumour Biol. 2016; 37: 13077-13090Crossref PubMed Scopus (25) Google Scholar). We analyzed the public data of cancer patients with PrognoScan (http://gibk21.bse.kyutech.ac.jp/PrognoScan/index.html) (Fig. S7). Patients with high expression of CSE1L exhibited shorter survival in breast cancer (GSE4922, GSE11121), brain tumor (GSE4271), liposarcoma (GSE30929), lung cancer (GSE13213), and ovarian cancer (GSE9891). We raised a question whether and how TAZ contributes to CSE1L-mediated enhancement of malignancy. CSE1L promoted cell migration and invasiveness in A549 cells in transwell assays, whereas WWTR1 silencing attenuated the effect of CSE1L (Fig. 7A). Likewise, CSE1L promoted colony formation in the soft agar assay, but WWTR1 silencing blocked it (Fig. 7C). Conversely, we also observed that CSE1L silencing reduced TAZ-mediated malignant transformation in A549 cells (Fig. 7, B and D). We performed the same experiments using U87MG cells and obtained similar results (Fig. S8). Hence, it is reasoned that CSE1L increases TAZ in the nucleus and induces malignant transformation in cancer cells. On the other hand, it is likely that TAZ, even when expressed, fails to enhance malignancy in cancer cells lacking CSE1L. Finally, we wanted to know whether and how CSE1L expression correlates with TAZ expression in cancer patients. To this aim, we used human cancer microarray, evaluated CSE1L expression by using a semiquantitative scale of immunoreactive score and examined whether TAZ was detected in the nucleus in human cancers with high levels of CSE1L (Fig. S9). Of 45 lung adenocarcinoma and 16 lung squamous cell carcinoma cases, 33 and 12 cases were classified as CSE1L-high cases, respectively. All CSE1L-high cases express high levels of nuclear TAZ (Table 1). In contrast, nuclear TAZ expression was high in none of CSE1L-low adenocarcinoma and squamous cell carcinoma cases. In gastric and esophageal cancers, most cases did not express high levels of CSE1L (18% for gastric cancers and 15% for esophageal cancers). Nevertheless, CSE1L-high cancers tended to express TAZ at the high level in these cancers. Thus, high expression of CSE1L correlates with high expression of nuclear TAZ.Table 1Expression of CSE1L and nuclear TAZ in human cancersNuclear TAZ expressionHighLowSum(A) Lung cancer (ⅰ)AdenocarcinomaCSE1L expressionHigh33437Low088Sum331245 (ⅱ) Squamous cell carcinomaCSE1L expressionHigh12113Low033Sum12416(B) Gastric cancer CSE1L expressionHigh909Low202141 Sum294150(C) Esophageal cancer CSE1L expressionHigh437Low13940 Sum54247(A) (i) Chi square, 26.757, p = 0.0000002; (ii) chi square, 11.077, p = 0.0008741.(B) Chi square, 7.95, p = 0.005; (C) chi-square, 18.71, p = 0.00002. Open table in a new tab (A) (i) Chi square, 26.757, p = 0.0000002; (ii) chi square, 11.077, p = 0.0008741. (B) Chi square, 7.95, p = 0.005; (C) chi-square, 18.71, p = 0.00002. In this article, we reported the novel TAZ inhibitor, TI-4. TI-4 shifts TAZ to the cytoplasm from the nucleus independent of TAZ phosphorylation, which suggests that the target of TI-4 is not a component of the canonical Hippo pathway. We identified CSE1L as its target. CSE1L is a mammalian homologue of yeast Cse1, which associates with chromatin, and was identified as the gene that made MCF-7 cells resistant to immunotoxin (8Brinkmann U. Brinkmann E. Gallo M. Pastan I. Cloning and characterization of a cellular apoptosis susceptibility gene, the human homologue to the yeast chromosome segregation gene CSE1.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10427-10431Crossref PubMed Scopus (163) Google Scholar). CSE1L was also identified as an importin α−binding protein (11Kutay U. Bischoff F.R. Kostka S. Kraft R. Görlich D. Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor.Cell. 1997; 90: 1061-1071Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 12Herold A. Truant R. Wiegand H. Cullen B.R. Determination of the functional domain organization of the importin alpha nuclear import factor.J. Cell Biol. 1998; 143: 309-318Crossref PubMed Scopus (100) Google Scholar). To maintain the nuclear transport, importin α must return from the nucleus to the cytoplasm. CSE1L is involved in the nuclear export of importin α. A previous study revealed that the nuclear import of histone deacetylases is suppressed by CSE1L depletion (21Dong Q. Li X. Wang C.Z. Xu S. Yuan G. Shao W. Liu B. Zheng Y. Wang H. Lei X. Zhang Z. Zhu B. Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E4013-E4022Crossref PubMed Scopus (14) Google Scholar). Therefore, we suspected that CSE1L is also required for the nuclear import of TAZ. CSE1L silencing indeed decreased the nuclear TAZ, whereas CSE1L increased it. Despite the importance of the subcellular localization in the regulation of YAP1 and TAZ, our knowledge regarding the nuclear cytoplasmic transport of these proteins is yet limited. In Drosophila melanogaster, importin α1 binds to the N-terminal 55 amino acids of Yorkie, a homologue of YAP1/TAZ, and drives it into the nucleus (23Wang S. Lu Y. Yin M.X. Wang C. Wu W. Li J. Wu W. Ge L. Hu L. Zhao Y. Zhang L. Importin α1 mediates Yorkie nuclear import via an N-terminal non-canonical nuclear localization signal.J. Biol. Chem. 2016; 291: 7926-7937Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Although the N-terminal sequence of TAZ is different from that of Yorkie and TAZ has the NLS not in the N-terminal region but in the C-terminal region (20Kofler M. Speight P. Little D. Di Ciano-Oliveira C. Szászi K. Kapus A. Mediated nuclear import and export of TAZ and the underlying molecular requirements.Nat. Commun. 2018; 9: 4966Crossref PubMed Scopus (34) Google Scholar), we detected the coimmunoprecipitation of TAZ and importin α5. As CSE1L interferes with the binding of NLS to importin α, CSE1L is supposed to block the interaction between TAZ and importin α. Consistently, CSE1L silencing enhanced the interaction between TAZ and importin α5. TI-4 strengthened the interaction between CSE1L and importin α and weakened the interaction between TAZ and importin α depending on CSE1L. All these findings support the model that CSE1L facilitates TAZ nuclear incorporation through recycling importin α and that TI-4 prevents the formation of the complex including TAZ and importin α to block the nuclear entry of TAZ (Fig. 8). In human cancers, high expression of CSE1L is associated with a poor prognosis. CSE1L enhances drug resistance, invasion, and metastasis (13Wellmann A. Flemming P. Behrens P. Wuppermann K. Lang H. Oldhafer K. Pastan I. Brinkmann U. High expression of the proliferation and apoptosis associated CSE1L/CAS gene in hepatitis and liver neoplasms: Correlation with tumor progression.Int. J. Mol. Med. 2001; 7: 489-494PubMed Google Scholar, 14Behrens P. Brinkmann U. Fogt F. Wernert N. Wellmann A. Implication of the proliferation and apoptosis associated CSE1L/CAS gene for breast cancer development.Anticancer Res. 2001; 21: 2413-2417PubMed Google Scholar, 24Liao C.F. Luo S.F. Li L.T. Lin C.Y. Chen Y.C. Jiang M.C. CSE1L/CAS, the cellular apoptosis susceptibility protein, enhances invasion and metastasis but not proliferation of cancer cells.J. Exp. Clin. Cancer Res. 2008; 27: 15Crossref PubMed Scopus (40) Google Scholar). Serum CSE1L is regarded as a prognostic marker of cancers (25Tung M.C. Tsai C.S. Tung J.N. Tsao T.Y. Chen H.C. Yeh K.T. Liao C.F. Jiang M.C. Higher prevalence of secretory CSE1L/CAS in sera of patients with metastatic cancer.Cancer Epidemiol. Biomarkers Prev. 2009; 18: 1570-1577Crossref PubMed Scopus (29) Google Scholar, 26Stella Tsai C.S. Chen H.C. Tung J.N. Tsou S.S. Tsao T.Y. Liao C.F. Chen Y.C. Yeh C.Y. Yeh K.T. Jiang M.C. Serum cellular apoptosis susceptibility protein is a potential prognostic marker for metastatic colorectal cancer.Am. J. Pathol. 2010; 176: 1619-1628Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The mechanism, by which CSE1L confers malignant properties to cancer cells, is not yet fully understood. Various explanations are proposed. CSE1L is associated with a set of p53 target genes and suppresses them (27Tanaka T. Ohkubo S. Tatsuno I. Prives C. hCAS/CSE1L associates with chromatin and regulates expression of select p53 target genes.Cell. 2007; 130: 638-650Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). CSE1L mediates the silencing of methylated genes (21Dong Q. Li X. Wang C.Z. Xu S. Yuan G. Shao W. Liu B. Zheng Y. Wang H. Lei X. Zhang Z. Zhu B. Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E4013-E4022Crossref PubMed Scopus (14) Google Scholar). CSE1L suppresses tumor suppressor RASSF1 gene expression (28Lorenzato A. Martino C. Dani N. Oligschläger Y. Ferrero A.M. Biglia N. Calogero R. Olivero M. Di Renzo M.F. The cellular apoptosis susceptibility CAS/CSE1L gene protects ovarian cancer cells from death by suppressing RASSF1C.FASEB J. 2012; 26: 2446-2456Crossref PubMed Scopus (25) Google Scholar). CSE1L reduces the nuclear accumulation of RAD51 and is involved in the regulation of DNA repair (29Okimoto S. Sun J. Fukuto A. Horikoshi Y. Matsuda S. Matsuda T. Ikura M. Ikura T. Machida S. Kurumizaka H. Miyamoto Y. Oka M. Yoneda Y. Kiuchi Y. Tashiro S. hCAS/CSE1L regulates RAD51 distribution and focus formation for homologous recombinational repair.Genes Cells. 2015; 20: 681-694Crossref PubMed Scopus (8) Google Scholar). CSE1L is implicated in the activation of Ras/ERK, cAMP/PKA, and microphthalmia-associated transcription factor signals (30Lee W.R. Shen S.C. Wu P.R. Chou C.L. Shih Y.H. Yeh C.M. Yeh K.T. Jiang M.C. CSE1L links cAMP/PKA and Ras/ERK pathways and regulates the expressions and phosphorylations of ERK1/2, CREB, and MITF in melanoma cells.Mol. Carcinog. 2016; 55: 1542-1552Crossref PubMed Scopus (21) Google Scholar). We observed that TAZ is detected in the nucleus in CSE1L-expressing cancer cells and that WWTR1 silencing antagonizes the effect of CSE1L in cancer cells. Conversely, CSE1L silencing attenuates the effect of TAZ. These findings suggest that TAZ functions downstream of CSE1L, while CSE1L augments the effect of TAZ by shifting TAZ into the nucleus. One intriguing question is whether TI-4 is effective for epithelioid hemangioendothelioma (EHE) (31Lamar J.M. Motilal Nehru V. Weinberg G. Epithelioid hemangioendothelioma as a model of YAP/TAZ-driven cancer: Insights from a rare fusion sarcoma.Cancers (Basel). 2018; 10: 229Crossref Scopus (18) Google Scholar). EHE is a soft-tissue sarcoma caused by the fusion of TAZ and calmodulin-binding transcription activator 1 (CAMTA1). TAZ–CAMTA1 is released from the regulation by the Hippo pathway and constitutively activates TAZ-mediated transcription. If the nuclear import of TAZ–CAMTA1 depends on importin α, TI-4 may be useful to control EHE. In conclusion, we revealed through the analysis of a new TAZ inhibitor that CSE1L accumulates TAZ in the nucleus and induces malignant transformation in human cancers. pCIneoFLAG-His6 (FH), pCIneoGFP-TAZ, pCIneoGFP-YAP1, pLL3.7-EGFPC2-TAZ, pLL3.7-EGFPC2-TAZ S89A, pLL3.7-EGFP2-YAP1 were previously described (7Nagashima S. Maruyama J. Kawano S. Iwasa H. Nakagawa K. Ishigami-Yuasa M. Kagechika H. Nishina H. Hata Y. Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.Cancer Sci. 2016; 107: 791-802Crossref PubMed Scopus (3) Google Scholar, 32Kawano S. Maruyama J. Nagashima S. Inami K. Qiu W. Iwasa H. Nakagawa K. Ishigami-Yuasa M. Kagechika H. Nishina H. Hata Y. A cell-based screening for TAZ activators identifies ethacridine, a widely used antiseptic and abortifacient, as a compound that promotes dephosphorylation of TAZ and inhibits adipogenesis in C3H10T1/2 cells.J. Biochem. 2015; 158: 413-423Crossref PubMed Scopus (23) Google Scholar). 8xGTIIC-δ51LucII luciferase reporter, pcDNA-Myc-His6-CSE1L, pcDNA FLAG-Importin α1, α3, and α4 are generous gifts of Hiroshi Sasaki (Osaka University), Satoshi Tashiro (Hiroshima University), and Koh Nakayama (Tokyo Medical and Dental University) (29Okimoto S. Sun J. Fukuto A. Horikoshi Y. Matsuda S. Matsuda T. Ikura M. Ikura T. Machida S. Kurumizaka H. Miyamoto Y. Oka M. Yoneda Y. Kiuchi Y. Tashiro S. hCAS/CSE1L regulates RAD51 distribution and focus formation for homologous recombinational repair.Genes Cells. 2015; 20: 681-694Crossref PubMed Scopus (8) Google Scholar, 33Ota M. Sasaki H. Mammalian Tead proteins regulate cell proliferation and contact inhibition as transcriptional mediators of Hippo signaling.Development. 2008; 135: 4059-4069Crossref PubMed Scopus (279) Google Scholar). PCR was performed with primers (H3691, 5′-acgcgtatggaactcagcgatgcaaatctgcaa-3′ and H3692, 5′-agtcgacttaaagcagtgtcacactggctgcctg-3′) on pcDNA-Myc-His6-CSE1L. The PCR product was digested with MluI/Sall and ligated into the same sites of pCIneomCherry to generate pCIneomCherry-CSE1L. NheI/Sall fragment from pCIneomCherry-CSE1L was ligated into NheI/XhoI sites of pLL3.7-ires-puro to generate pLL3.7-mCherry-CSE1L-ires-puro. PCR was performed with primers (H3753, 5′-acgcgtgaattcatgcccaggaaaagagaa-3′ and H3754, 5′-actagtcgacttcaagaaaccttccatagg-3′) on human kidney and lung cDNAs (Clontech). The PCR product was ligated into MluI/SalI sites of pCIneoFH and pCIneoEGFPC2 to generate pCIneoFH- and pCIneoEGFPC2-importin α5. NheI/Sall fragment from pCIneoEGFPC2-importin α5 was ligated into NheI/XhoI sites of pLL3.7-ires-puro to generate pLL3.7-GFP-importin α5-ires-puro. Rabbit anti-phospho-TAZ antibody was previously described (34Yang Z. Nakagawa K. Sarkar A. Maruyama J. Iwasa H. Bao Y. Ishigami-Yuasa M. Ito S. Kagechika H. Hata S. Nishina H. Abe S. Kitagawa M. Hata Y. Screening with a novel cell-based assay for TAZ activators identifies a compound that enhances myogenesis in C2C12 cells and facilitates muscle repair in a muscle injury model.Mol. Cell. Biol. 2014; 34: 1607-1621Crossref PubMed Scopus (37) Google Scholar). Other antibodies and the reagents used in this study are as follows: mouse monoclonal anti-GFP (sc-9996), mouse monoclonal anti-poly (adp-ribose) polymerase (sc-8007), mouse monoclonal anti-CAS (H-2) (sc-271537), mouse monoclonal karyopherin α1 (187.1) (sc-101292) (Santa Cruz Biotechnology); mouse monoclonal anti-TAZ (560235) (BD Biosciences); rabbit polyclonal anti-α-tubulin (PM054) and rabbit polyclonal anti-β-actin (PM053) (Medical and Biological Laboratories Co Ltd); anti-DYKDDDDK-tag (014-22383) and anti-DYKDDDDK-beads (016-22784) (Wako Pure Chemical Industries); rabbit polyclonal anti-mCherry (GeneTex); rabbit monoclonal FoxO3a (75D8)(2497) and rabbit monoclonal YAP1(D8H1X)XP (14074) (Cell Signaling Technology); rabbit polyclonal importin α5 (18137-1-AP) and rabbit polyclonal TAZ (23306-1-AP) (Proteintech Group, Inc); mouse monoclonal β-catenin (13-8400) (Zymed Laboratory Inc); Protein G Sepharose 4 Fast Flow (17-0618-01) (GE Healthcare BioSciences); Rabbit IgG Isotype Control (31235) (Thermo Fisher Scientific); and TI-4 (2,2-dichloro-N-(4-nitrophenyl)-3-phenylcyclopropanecarboxsmide) (STK825627; Vitas-M Chemical Limited).

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