CSE1L promotes nuclear accumulation of transcriptional coactivator TAZ and enhances invasiveness of human 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.
<!>TI-4 suppresses cell proliferation and soft agar colony formation of A549, H1299, and U87MG cells<!><!>CSE1L silencing attenuates the effect of TI-4<!><!>CSE1L regulates the nuclear localization of YAP1<!><!>Discussion<!><!>Discussion<!>DNA constructions and virus production<!>Antibodies and reagents<!>Cell cultures and gene introduction<!>Chemical compound screening<!>Preparation of control and TI-4-affinity beads<!>Isolation of interacting molecules on TI-4-affinity beads<!>MS<!>Quantitative RT-PCR<!>RNA interferences<!>Subcellular fractionation<!>Immunoprecipitation<!>Phosphate-affinity SDS-PAGE<!>FRAP assay<!>MTT and soft agar assays<!>Cell invasive assay<!>Immunohistochemistry of human cancers<!>Statistical analysis<!>Data availability<!>Supporting information<!>Conflict of interest<!>Supporting information
<p>Edited by Alex Toker</p><p>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 (1). 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 (2). Phosphorylation by LATS kinases also causes protein degradation (3, 4). 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 (5, 6). 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.</p><p>We screened for TAZ inhibitors by means of a cell-based assay (7). 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 (7). 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.</p><p>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 (8, 9, 10). CSE1L binds to importin α in the presence of RanGTP and is required for the recycling of importin α (11, 12).</p><p>CSE1L is implicated in cell proliferation and apoptosis in human cancers (13). High expression of CSE1L is associated with a poor prognosis in cancers and is considered to be a prognostic marker (14, 15, 16, 17). However, the mechanism underlying of the oncogenic action of CSE1L is not fully understood.</p><p>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.</p><p>The effect of TI-4 on the subcellular localization and the phosphorylation of TAZ.A, the structural formula of TI-4. B, U2OS cells expressing GFP-TAZ were plated at 5 × 105 cells/10-cm dish and 24 h later, treated with DMSO or 10 μM TI-4. Forty-eight hours later, the subcellular fractionation was performed. The comparable amounts of the whole-cell lysate (W), the cytoplasmic fraction (C), and the nuclear fraction (N) were immunoblotted with the indicated antibodies. PARP and α-tubulin were used as the nuclear and cytoplasmic markers, respectively. TI-4 decreased the nuclear GFP-TAZ (an arrow). C, the subcellular fractionation was performed by using U2OS cells. Endogenous TAZ was detected and analyzed as in panel B. D, U2OS cells expressing GFP-TAZ were plated at 1 × 105 cells/well in a 6-well plate and 24 h later, treated with DMSO or 10 μM TI-4. Twenty-four hours later, the whole-cell lysates were run on Phos-tag gels and immunoblotted with anti-phospho-Ser89 (P-TAZ(S89)) and GFP antibodies. As control, the cells were exposed to 5 μM ethacridine for 24 h or 1 mM H2O2 for 30 min. Ethacridine decreased phosphorylated TAZ and increased unphosphorylated TAZ (the first lane). H2O2 treatment increased phosphorylated TAZ and decreased the unphosphorylated TAZ (the fourth lane). The experiments in panels B, C, and D were repeated more than three times. DMSO, dimethyl sulfoxide; GFP-TAZ, GFP-tagged TAZ; PARP, poly (adp-ribose) polymerase; TAZ, transcriptional coactivator with PDZ-binding motif; TI-4, TAZ inhibitor 4.</p><p>The effect of TI-4 on cancer cells.A, H1299 cells were plated at 1 × 105 cells/well in a 6-well plate. Twenty-four hours later, the cells were treated with DMSO or 10 μM TI-4 for 48 h. Total RNA was extracted, and quantitative RT-PCR was performed. The experiments were performed three times. B, A549, H1299, and U87MG cells were plated at 3000 cells/well in a 96-well plate. Twenty-four hours later, the cells were treated with DMSO or 10 μM TI-4 and cultured for 48 h and 96 h. The colorimetric assay was performed. The experiments were performed three times. C, A549, H1299, and U87MG cells were treated with DMSO or 10 μM TI-4. The cells were plated in the soft agar. On day 14, the colony area was evaluated with ImageJ. The mean value for DMSO-treated cells was set at 1.0. The experiments were performed three times. In panels A–C, data are shown as the mean ± SD. ∗∗p < 0.01; and ∗∗∗p < 0.001. The experiments were repeated three times. DMSO, dimethyl sulfoxide; TI-4, TAZ inhibitor 4.</p><!><p>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 (7). 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.</p><!><p>CSE1L is a putative target of TI-4.A, the cell lysates of U87MG cells were incubated on either control or TI-4-conjugated beads as described in Experimental procedures. The arrow indicates the protein that specifically binds to TI-4-conjugated beads. B and C, U2OS-GFP-TAZ cells were plated at 1.5 × 105 cells/well in a 6-well plate and transfected with control siRNA or CSE1L siRNA. Forty-eight hours later, the cells were replated at 3 × 103 cells/well in a 96-well plate and cultured with or without 10 μM TI-4 for 48 h. The cells were fixed, and the nuclei were visualized with Hoechst 33342. GFP signals inside and outside the nucleus were measured, and the ratio of the nuclear GFP over the cytoplasmic GFP (N/C ratio) was calculated. In panel B, the distributions of N/C ratio in 300 cells are depicted as cumulative curves. In the CSE1L-negative background, TI-4 failed to shift the distribution. In panel C, TI-4 and CSE1L silencing reduced the nuclear GFP-TAZ (arrowheads). D, H1299 cells were plated at 1.5 × 105 cells/well in a 6-well plate and transfected with control siRNA or CSE1L siRNA. Twenty-four hours after transfection, TI-4 was added to a final concentration of 10 μM, and the cells were further cultured for 48 h. Total RNA was extracted, and quantitative RT-PCR was performed to measure CTGF and CYR61 mRNAs. Data are shown as the mean ± SD. ∗∗∗p < 0.001. E, H1299 cells were plated at 1.5 × 105 cells/well in a 6-well plate and transfected with control siRNA or CSE1L siRNA. Twenty-four hours later, the cells were plated at 3000 cells/well in a 96-well plate. Twenty-four hours later, the cells were treated with DMSO or 10 μM TI-4 and cultured for 48 h and 96 h. The MTT assay was performed. Data are shown as the mean ± SD. ∗∗∗p < 0.001. F, the efficiency of CSE1L silencing was confirmed by immunoblotting and quantitative RT-PCR. The experiments were repeated twice for those mentioned in panel A and three times for those mentioned in panels B–E. CSE1L, chromosomal segregation 1 like; DMSO, dimethyl sulfoxide; GFP-TAZ, GFP-tagged TAZ; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; n.s., not significant; TI-4, TAZ inhibitor 4.</p><!><p>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.</p><!><p>The effect of CSE1L on the subcellular distribution of TAZ.A and B, U2OS-GFP-TAZ or U2OS cells were plated at 4.0 × 105 cells/6-cm dish and transfected with control siRNA or CSE1L-siRNA. Seventy-two hours later, the subcellular fractionation was performed. PARP and α-tubulin were used as nuclear (N) and cytoplasmic (C) markers, respectively. The nuclear GFP-TAZ or endogenous TAZ was reduced by CSE1L silencing (arrows). Endogenous YAP1 was also decreased by CSE1L silencing (arrows). C, U2OS-GFP-TAZ cells were plated at 4 × 105 cells/6-cm dish. Twenty-four hours later, the cells were transfected with control pCIneomCherry or pCIneomCherry-CSE1L. Forty-eight hours later, the subcellular fractionation was performed. The nuclear GFP-TAZ was increased by CSE1L expression (an arrow). The numbers under the immunoblottings indicate the ratio of cytoplasmic and nuclear TAZ in the indicated image. Other two experiments showed the similar results as shown in Fig. S3A. D, U2OS-GFP-TAZ cells were plated at 1.5 × 105 cells/well in a 6-well plate and transfected with control siRNA or CSE1L siRNA. The whole-cell lysates were run on Phos-tag gels and immunoblotted with the indicated antibodies. As the control, the cells were treated with 10 μM ethacridine for 24 h and with 1 mM H2O2 for 30 min. Upper bands were detected with anti-phospho-TAZ (S89) and anti-GFP antibodies (arrowheads), while the lower band was detected only with anti-GFP antibody. Ethacridine increased unphosphorylated TAZ, whereas H2O2 reduced it (an arrow). The experiments were performed three times. CSE1L, chromosomal segregation 1 like; GFP-TAZ, GFP-tagged TAZ; PARP, poly (adp-ribose) polymerase; TAZ, transcriptional coactivator with PDZ-binding motif.</p><p>CSE1L silencing delays the recovery of nuclear GFP-TAZ after photobleaching.A, the effect of TI-4 and ivermectin. B, the effect of CSE1L silencing. C, the effect of TI-4 in the CSE1L-negative background. Representative images of FRAP experiments are demonstrated on the left. The bleached areas are demarcated with white lines. Fluorescent recovery rates were calculated as described in Experimental procedures. About 5~10 cells for each condition were measured for each experiment. Three independent experiments were performed. The scale bars represent 50 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. CSE1L, chromosomal segregation 1 like; GFP-TAZ, GFP-tagged TAZ; FRAP, fluorescence recovery after photobleaching; NS, not significant.</p><p>TI-4 strengthens the interaction between CSE1L and importin α5 and weakens the interaction between TAZ and importin α5.A and C, HEK293FT cells were plated at 8 × 105 cells/well in a 6-well plate. Sixteen hours later, cells were transfected with pCIneoFH-importin α5, pCIneoGFP-CSE1L, and pCIneoGFP-TAZ as indicated. Twenty-four hours later, the cells were treated with DMSO or 10 μM TI-4 and cultured for 48 h. The immunoprecipitation was performed with anti-DYKDDDDK-beads. A, CSE1L was coimmunoprecipitated with importin α5 under TI-4 treatment (an arrow). C, TI-4 treatment reduced the amount of coimmunoprecipitated TAZ with importin α5 (an arrow). B, U2OS-GFP-importin α5 cells were plated at 1 × 105 cells/dish in a 35-mm dish, and 24 h later, treated with DMSO or 10 μM TI-4. Forty-eight hours later, the cells were fixed, and the nuclei were visualized with Hoechst 33342. TI-4 reduced the nuclear GFP-importin α5. D, HEK293FT cells were plated at 1 × 106/dish in a 6-cm dish and transfected with control siRNA or CSE1L siRNA. Twenty-four hours later, the cells were replated at 8 × 105 cells/well in a 6-well plate. Twenty-four hours later, the cells were transfected with pCIneoFH-importin α5 and pCIneoGFP-TAZ, cultured for 24 h, and then treated with DMSO or 10 μM TI-4. Forty-eight hours later, the immunoprecipitation was performed with anti-DYKDDDDK-tag beads. CSE1L silencing strengthened the interaction between importin α5 and TAZ (an arrow). Under this condition, TI-4 failed to inhibit the interaction between importin α5 and TAZ (an arrowhead). The experiments were performed three times. The numbers under the immunoblottings indicate the signal intensity of GFP-CSE1L and GFP-TAZ in the indicated image. Other two experiments showed the similar results as shown in Fig. S3, B and C. CSE1L, chromosomal segregation 1 like; DMSO, dimethyl sulfoxide; GFP-TAZ, GFP-tagged TAZ; TAZ, transcriptional coactivator with PDZ-binding motif; TI-4, TAZ inhibitor 4.</p><!><p>CSE1L is involved in the recycling of importin α (11). CSE1L is required for the nuclear import of nuclear proteins including histone deacetylases (21). 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.</p><!><p>CSE1L and TAZ co-operatively promote cell invasiveness, motility, and colony formation in A549 cells. A549, A549-mCherry-CSE1L, and A549-GFP-TAZ cells were plated at 1.5 × 105 cells/well in a 6-well plate and transfected with control siRNA, WWTR1 siRNA, or CSE1L siRNA as indicated. In panels A and B, 24 h later, cells were transferred to the serum-free medium. Twenty-four hours later, cells were replated at 4 × 104 cells/insert in 8.0-μm Transparent PET Membrane Insert, and invasion and migration assays were performed as described in Experimental procedures. Forty hours later, cells were stained by crystal violet. The migrating or invading cells were observed under the microscope in three independent fields for each condition and quantified using ImageJ. In panels C and D, 24 h later, cells were replated 3000 cells/well in 12-well plate for the soft agar assay as described in Experimental procedures. Two weeks later, three independent images for each sample were obtained with a microscope BZ-X700 (Keyence) and analyzed by BZ-H3C Hybrid Cell Count Module. Data are shown as the mean ± SD. ∗∗p < 0.01; and ∗∗∗p < 0.001. The experiments were performed three times. E, the cells were immunoblotted to confirm the expression of mCherry-CSE1L and GFP-TAZ and to show the efficiency of WWTR1 silencing and CSE1L silencing. CSE1L, chromosomal segregation 1 like; GFP-TAZ, GFP-tagged TAZ; TAZ, transcriptional coactivator with PDZ-binding motif.</p><p>Expression of CSE1L and nuclear TAZ in human cancers</p><p>(A) (i) Chi square, 26.757, p = 0.0000002; (ii) chi square, 11.077, p = 0.0008741.</p><p>(B) Chi square, 7.95, p = 0.005; (C) chi-square, 18.71, p = 0.00002.</p><!><p>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.</p><!><p>The putative mechanism by which TI-4 inhibits the nuclear incorporation of TAZ. TAZ (blue symbols) forms a complex with importin α (orange symbols) and importin β (gray symbols) and is incorporated into the nucleus. The complex is dissociated in the nucleus. CSE1L (yellow symbols) binds to the released importin α and recruits it to the cytoplasm. Importin α is dissociated from CSE1L in the cytoplasm and is used for the next cycle to import TAZ into the nucleus. TI-4 blocks the dissociation of importin α from CSE1L. Therefore, importin α is accumulated in the cytoplasm, and TAZ fails to bind to importin α, so that TAZ cannot enter the nucleus. CSE1L, chromosomal segregation 1 like; TAZ, transcriptional coactivator with PDZ-binding motif; TI-4, TAZ inhibitor 4.</p><!><p>In human cancers, high expression of CSE1L is associated with a poor prognosis. CSE1L enhances drug resistance, invasion, and metastasis (13, 14, 24). Serum CSE1L is regarded as a prognostic marker of cancers (25, 26). 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 (27). CSE1L mediates the silencing of methylated genes (21). CSE1L suppresses tumor suppressor RASSF1 gene expression (28). CSE1L reduces the nuclear accumulation of RAD51 and is involved in the regulation of DNA repair (29). CSE1L is implicated in the activation of Ras/ERK, cAMP/PKA, and microphthalmia-associated transcription factor signals (30). 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.</p><p>One intriguing question is whether TI-4 is effective for epithelioid hemangioendothelioma (EHE) (31). 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.</p><p>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.</p><!><p>pCIneoFLAG-His6 (FH), pCIneoGFP-TAZ, pCIneoGFP-YAP1, pLL3.7-EGFPC2-TAZ, pLL3.7-EGFPC2-TAZ S89A, pLL3.7-EGFP2-YAP1 were previously described (7, 32). 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) (29, 33). 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.</p><!><p>Rabbit anti-phospho-TAZ antibody was previously described (34). 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).</p><!><p>HEK293FT, U2OS, H1299, A549, and U87MG cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 10 mM Hepes-NaOH, pH 7.4, under 5% CO2 at 37 °C. Cell authentication was performed by using short-tandem repeat profile (GenePrint 10 System) (Promega). Mycoplasma infection was periodically checked by using e-Myco Mycoplasma PCR Detection Kit (ver.2.0) (#25235, iNtRON Biotechnology). When infection was detected, Mycoplasma elimination was performed by Plasmocin treatment (#ant-mpt-1, InvivoGen). DNA transfection was performed with Lipofectamine 2000 (Life Technologies). Stable transformant cells were prepared by using lentivirus vectors, pLL3.7-EGFPC2-TAZ, pLL3.7-EGFPC2-TAZ S89A, pLL3.7-EGFP2-YAP1, pLL3.7-GFP-importin α5, and pLL3.7-mCherry-CSE1L.</p><!><p>TI-4 was identified in the chemical compound screening that was previously reported (7). Briefly, U2OS-GFP-TAZ cells were plated at 3000 cells/well in 96-well plates. Cells were treated with 10 μM compounds for 16 h. The nucleus was visualized with Hoechst 33342. GFP signals inside and outside the nucleus were measured by ArrayScan VTI in 300 cells for each compound (Thermo Fisher Scientific). Robust Z-scores (|x-median(x)|/normalized interquartile range) were calculated. 86 compounds with a robust Z-score less than 3.0 were selected. 32 compounds were omitted because of the cytotoxicity. We evaluated by using TAZ/TEAD reporter assays in HEK293FT cells 33 compounds that reduced the nuclear TAZ to less than 90% in the second screening. We focused on five compounds (IBS000540, IBS008420, IBS001594, IBS015181 (renamed as TI-4 in this study), and IBS015625) and previously characterized three compounds (IBS000540, IBS001594, and IBS015625). IBS008420 later dropped out because of autoimmunofluorescence.</p><!><p>TI-4-affinity column beads were prepared by the use of photoaffinity linker–coated Sepharose beads. Detailed protocol is available (35).</p><!><p>U87MG cells were plated at 3 × 106 cells in a 10-cm dish. Twenty-four hours later, the cells were harvested from four dishes with PBS, transferred into one 1.5-ml microtube and centrifuged at 800g for 5 min at 4 °C. The cell pellets was resuspended in 1 ml of buffer A (20 mM Tris-HCl pH7.4, 5 mM MgCl2, 5 mM CaCl2, 1 mM EDTA, 1 mg/l (p-amidinophenyl)methanesulfonyl fluoride hydrochloride (APMSF), 1 mg/l leupeptin and 1 mg/l pepstatin A), incubated for 30 min on ice, then homogenized with 40 strokes in a glass homogenizer, and centrifuged at 100,000g for 20 min at 4 °C. The supernatant was incubated with 5 μl of the control beads for 1 h and centrifuged at 2300g for 1 min at 4 °C. The half aliquots were transferred into two 1.5-ml microtubes. Five microliter of either control or TI-4-conjugated beads was added to each tube, incubated for 24 h at 4 °C, and centrifuged at 2300g for 1 min. The beads were washed four times with buffer A and finally resuspended in 30 μl of the buffer. Fifteen microliter of the SDS sample buffer (180 mM Tris-HCl pH 6.8, 6% (w/v) SDS, 30% (v/v) glycerol, 0.06% (w/v) bromophenol blue, and 15% (v/v) β-mercaptoethanol) was added and boiled. Thirty microliter of each sample was charged and analyzed on SDS-PAGE.</p><!><p>The parameters of MS were summarized in Table S1. The immunoprecipitated samples separated by SDS-PAGE were stained with Coomassie brilliant blue (Quick Blue Staining Solution, #DS500, BioDynamics Laboratory Inc). Gel pieces were washed with 5% acetic acid and 50% methanol and then dehydrated with 66% acetonitrile and 17 mM NH4HCO3. The samples were incubated with 10 mM DTT at 60 °C for 1 h and blocked with 50 mM iodoacetamide at room temperature (RT) for 45 min, followed by digestion with 1 pmol of sequencing-grade trypsin (Promega). The peptides were injected into a nano-UHPLC system (Bruker Daltonics). Mass analysis was performed on a maXis-4G-CPR mass spectrometer equipped with a nano-ESI source. Magic C18AQ UHPLC Nano-Trap column (5-μm particle size, 200 Å pore diameter) (Biologica Co) and L-column ODS (0.1 × 150 mm., 3-μm particle size, Chemicals Evaluation and Research Institute) were used. The peptides were eluted from the column using a 10% (v/v) to 35% (v/v) acetonitrile gradient over 50 min. The eluted peptides were electrosprayed into the spectrometer. MS/MS spectra were acquired in a data-dependent mode.</p><!><p>Quantitative RT-PCR analysis was performed by using SYBR Green (Roche) and ABI7500 Real-Time PCR system (Applied Biosystems). The used primers are human GAPDH, 5′-ccactcctccacctttgac-3′ and 5′-accctgttgctgtagcca-3′; human CTGF, 5′-ccaatgacaacgcctcctg-3′ and 5′-tggtgcagccagaaagctc-3′; human CYR61, 5′-agcctcgcatcctatacaacc-3′, and 5′-ttctttcacaaggcggcactc-3′; human WWTR1, 5′-tatcccagccaaatctcgtg-3′, and 5′-ttctgctggctcagggtact-3′; and human CSE1L, 5′-cggttcaaacacaatagcaagtg-3′ and 5′-ctgattcaggaggcatgtgct-3′.</p><!><p>RNA interferences were performed with Lipofectamine RNAiMAX (Thermo Fisher Scientific). The dsRNAs (Ambion) were as follows: s24787 for human WWTR1, s3590 for human CSE1L, and Silencer Select negative control No.2 #4390846 (Thermo Fisher Scientific); and sc-29908 CAS siRNA for human CSE1L (Santa Cruz Biotechnology). The validity of the knockdown was confirmed by quantitative RT-PCR or immunoblotting.</p><!><p>U2OS or U2OS-GFP-TAZ cells were plated at 5 × 105/dish in a 10-cm dish. Twenty-four hours later, cells were harvested by scraping in PBS and transferred into 1.5-ml microtubes. After centrifugation at 800g for 5 min, the pellets were resuspended five times by 200-μl yellow pipette in 200-μl extraction buffer (10 mM Hepes-NaOH pH7.4, 1.5 mM MgCl2, 10 mM KCl, 0.34 M sucrose, 10% (v/v) glycerol, 0.05% (v/v) NP-40, and 10 mg/l APMSF) and incubated for 5 min on ice. Seventy microliter of the mixture was saved as the whole cell lysate. The remaining mixture was centrifuged at 800g for 5 min, and the supernatant was saved as the cytoplasmic fraction. The pellet was washed once, resuspended in 130-μl extraction buffered, and used as the nuclear fraction.</p><!><p>HEK293FT cells were plated at 8 × 105 cells/well in a 6-well plate. Sixteen hours later, cells were transfected with various combinations of expression vectors. The total amount of DNA was adjusted to 2 μg for all the points. Twenty-four hours later, cells were treated with dimethyl sulfoxide or 10 μM TI-4, further cultured for 48 h, and then lysed in 500-μl lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 10 mM MgCl2, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 10 mg/l APMSF, and 10 mg/l leupeptin). The supernatant was collected after centrifugation at 20,000g for 10 min. Forty microliter of the supernatant was stocked as the input, and 400 μl of the supernatant was used for the immunoprecipitation with 5-μl anti-DYKDDDDK-tag beads. To detect the coimmunoprecipitation of endogenous TAZ and importin α5, CSE1L was silenced in advance. HEK293FT cells were plated at 3 × 106 cells/dish in a 10-cm dish and were transfected with CSE1L siRNA. Seventy-two hours later, the cells were lysed in the lysis buffer. The supernatant was collected after centrifugation at 20,000g for 10 min. Four hundred microliter of the supernatant was incubated with 1 μg of rabbit control IgG or TAZ polyclonal antibody for 2 h and then with 5-μl Protein G Sepharose 4 Fast Flow protein G beads. Proteins were detected in the inputs and the immunoprecipitates with appropriate antibodies.</p><!><p>Phosphate-affinity SDS-PAGE was performed with Phos-tag acrylamide and polyvinylidene difluoride membranes (Millipore) as described previously (31). We used ethacridine and H2O2 as the control. Ethacridine dephosphorylates and activates TAZ (31). H2O2 activates mammalian Ste20-like kinases and phosphorylates TAZ.</p><!><p>The FRAP assay was performed by using a confocal laser-scanning microscope (LSM 510 Meta, Carl-Zeiss). U2OS-cells expressing GFP-TAZ or GFP-YAP1 were plated at 1 × 105 cells/dish in a μ-Dish 35-mm (ib81156, NIPPON Genetics Co Ltd). Cells were stained with DRAQ5 (BioStatus Ltd) to visualize nuclei 30 min before observation. Selected cells were photobleached inside the nucleus by laser pulses at 488 nm (100% power, once capture) at 37 °C. Thereafter, fluorescence recovery was monitored for 130 s at 1.3-s intervals. The fluorescent recovery was evaluated according to the published method (36). Briefly, GFP signals were measured in the nucleus (N) and the whole cell (W) before (pre) and after the photobleaching (t). The cytoplasmic GFP (Cyto) signals were measured only before the photobleaching. The formula (F) to estimate the recovery was defined as follows.ThenumeratorofF(t)=(N(t)−N(0))×Cyto(pre)×W(0)ThedenominatorofF(t)=W(t)×W(pre)×W(t)</p><p>As photobleaching annihilated the nuclear GFP-TAZ and reduced the total amount of GFP-TAZ, W(t) was corrected by multiplying with W(pre)/Cyto(pre). W(0)/W(t) was a correction factor to compensate GFP-TAZ that was newly produced during observation.</p><!><p>MTT and soft agar assays were performed as described previously (7). Cells were plated 3000 cells/well in a 96-well plate for the MTT assay. The colorimetric assay was performed with thiazolyl blue tetrazolium bromide. Insoluble formazan was measured by iMark Microplate Absorbance Reader (Bio-Rad) at 595 nm. For the soft agar assay, 500 μl DMEM containing 10% (v/v) FBS, 0.5% (w/v) agarose, and 10 μM TI-4 was plated in one well of a 12-well plate. Then, 500-μl DMEM containing 3000 cells, 10% (v/v) FBS, 0.3% (w/v) agarose, and dimethyl sulfoxide or 10 μM TI-4 was overlaid and kept for 1 h at RT. After that, 250-μl DMEM containing 10% (v/v) FBS was further overlaid and cultured for 2 weeks. To evaluate the effect of the silencing of CSE1L or WWTR1, A549 and U87MG cells were transfected with either control siRNA or WWTR1 siRNA. Twenty-four hours later, the cells were used for the soft agar assay. The images were obtained with a fluorescence microscope BZ-X700 (Keyence) and analyzed by BZ-H3C Hybrid Cell Count Module. The areas of cells were measured from three independent fields.</p><!><p>Cell migration and invasive assays were performed with 6.5-mm Transwell with 8.0-μm Transparent PET Membrane Insert (353097) (Corning Inc). The inserts were incubated with the fibronectin solution of 66 μg/ml at 4 °C for 24 h. The inserts used for invasion assay was further coated with 30-μg Matrigel in 100 μl of 10 mM Tris HCl, pH 8.0, containing 0.7% (w/v) NaCl. After centrifugation at 1000g for 10 min, the plate was incubated at 37 °C for 30 min, and 4 × 104 A549 or U87MG cells were suspended in 100 μl DMEM, added to each plate, and incubated at 37 °C. Ten minutes later, 600-μl DMEM containing 10% FBS was added to the lower chamber. Forty hours later, the medium and Matrigel were removed by a cotton swab from the top of permeable support. The cells on the lower surface were fixed in 70% (v/v) ethanol and stained with 0.2% (w/v) crystal violet. The permeable supports were dried. The migrating or invading cells were observed under the microscope and quantified using ImageJ (https://imagej.net/).</p><!><p>This study was conducted according to the Declaration of Helsinki guidelines, and the study design of using human pathology archives has been approved by the Institutional Review Board of Hamamatsu University School of Medicine (20-011 for H. S.). Paraffin-embedded tissue samples from 235 cases of adenocarcinoma of the lung and 161 cases of squamous cell carcinoma of the lung who had undergone surgery at Hamamatsu University Hospital were used for the tissue microarray block. To explain in further detail, the tissue microarray block was prepared by transferring a cylinder of 3-mm-diameter tumor portion from each block containing resected tumor by using a microarrayer (KIN-1, Azumaya). The histopathological diagnosis of lung cancer was confirmed in Diagnostic Division of Hamamatsu University Hospital. This study was conducted with the approval of the Institutional Review Board of Hamamatsu University School of Medicine. The images were evaluated by two independent observers and were classified into high and low groups.</p><!><p>Two samples were compared with the Student's t test. ANOVA with Tukey's test was used for the multiple comparison.</p><!><p>The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD024194 and 10.6019/PXD024194.</p><!><p>This article contains supporting information.</p><!><p>The authors declare that they have no conflicts of interest with the contents of this article.</p><!><p>Supplemental Figures S1–S9 and Table S1</p>