promotes colon cancer tumorigenesis in a mitotic -dependent manner and through CDK8-mediated YAP activation

Jiuli Zhoua,b,c, Yongji Zenga,b,c, Lian Cuid, Xingcheng Chena,b,c, Seth Stauffera,b,c, Zhan Wanga,b, Fang Yue, Subodh M. Lelec, Geoffrey A. Talmonc, Adrian R. Blacka,b, Yuanhong Chena,b, and Jixin Donga,b,1

aEppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198; bFred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198; cDepartment of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198; dDepartment of Dermatology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China; and eDepartment of Biostatistics, College of Public Health, University of Nebraska Medical Center, Omaha, NE 68198

Edited by Kun-Liang Guan, University of California at San Diego, La Jolla, CA, and accepted by Editorial Board Member Rakesh K. Jain June 11, 2018 (received for review January 11, 2018) Zyxin is a member of the focal adhesion complex and plays a which CDK8 regulates β-catenin remain obscure. CDK8 mRNA critical role in actin filament polymerization and cell motility. is up-regulated in malignant melanoma by loss of a transcrip- Several recent studies showed that Zyxin is a positive regulator of tional repressor called the histone variant macroH2A, which Yki/YAP (Yes-associated ) signaling. However, little is functions as a tumor suppressor in melanoma (18). In addition, known about the mechanisms by which Zyxin itself is regulated CDK8 protein levels are also controlled by S-phase as- and how Zyxin affects Hippo–YAP activity. We first showed that sociated protein 2 (Skp2)-mediated degradation of macroH2A1 Zyxin is phosphorylated by CDK1 during . Depletion of protein, and these three work together to regulate G2/M Zyxin resulted in significantly impaired colon cancer cell prolifera- transition and tumorigenesis in breast cancer (19). CDK8 exerts its tion, migration, anchorage-independent growth, and tumor for- oncogenic function mainly through phosphorylation of substrates. mation in xenograft animal models. Mitotic phosphorylation is Several substrates for CDK8 have been identified, including the required for Zyxin activity in promoting growth. Zyxin regulates Notch intracellular domain, SMAD complexes, E2F1, STAT1, and YAP activity through the colon cancer oncogene CDK8. CDK8 the C-terminal domain of RNA polymerase II (16). These studies knockout phenocopied Zyxin knockdown in colon cancer cells, highlight an important oncogenic function of CDK8 kinase activity. while ectopic expression of CDK8 substantially restored the tumor- A connection between CDK8 and YAP, the critical transcriptional igenic defects of Zyxin-depletion cells. Mechanistically, we showed coactivator of Hippo signaling, has not been established. Here, we that CDK8 directly phosphorylated YAP and promoted its activa- report that Zyxin promotes colon cancer cell growth, and its tion. Fully activated YAP is required to support the growth in oncogenic activity is partially controlled by mitotic phosphory- CDK8-knockout colon cancer cells in vitro and in vivo. Together, lation. We further showed that Zyxin regulates YAP activity these observations suggest that Zyxin promotes colon cancer tu- through CDK8 in colon cancer cells. In addition, we identified morigenesis in a mitotic-phosphorylation-dependent manner and YAP as a direct substrate of CDK8 and CDK8-mediated phos- through CDK8-mediated YAP activation. phorylation that promotes YAP activity in vitro and in vivo.

Zyxin | CDK8 | Hippo–YAP pathway | mitosis | colon cancer Significance – yxin is a LIM domain protein concentrated at sites of cell Zyxin is a member of the cell–cell adhesion complex and con- Zcell adhesion, where it is proposed to dock proteins involved trols cell cytoskeleton and motility. Analysis from clinical in cytoskeletal dynamics and signaling (1). Zyxin has also been samples revealed that Zyxin is highly expressed in colon cancer implicated in sensing the mechanotransduction signal (2, 3). Of compared with normal tissue, suggesting an oncogenic role for interest, Zyxin contains a nuclear export signal and shuttles be- Zyxin in cancer. Depletion of Zyxin resulted in significantly tween the nucleus and sites of cell adhesion (4), although the impaired colon cancer cell proliferation, migration, cellular underlying mechanisms and function of its localization dynamics transformation, and tumor formation in xenograft animal are unclear. Several recent studies showed that Zyxin is a posi- models. We also showed that Zyxin is phosphorylated by tive regulator of tissue growth through Hippo–Yorkie/Yki sig- CDK1 during mitosis. Mitotic phosphorylation is required for naling in Drosophila (5–8). Zyxin regulates Yki-mediated tissue Zyxin activity in promoting colon cancer growth. Zyxin regu- growth by promoting F-actin polymerization independent of lates YAP activity through the colon cancer oncogene CDK8. Hippo/Warts (5). This mechanism is conserved in We further showed that CDK8 directly phosphorylates YAP and mammalian cancer cells (9, 10). Furthermore, in human breast promotes its activation. These observations identify the Zyxin– cancer cells, Zyxin promotes growth and tumorigenesis by mod- CDK8–YAP axis as a potential therapeutic target in cancer. ulating Hippo–yes-associated protein (YAP) signaling activity (11), which plays an important role in the development and pro- Author contributions: J.Z. and J.D. designed research; J.Z., Y.Z., L.C., X.C., S.S., Z.W., and – Y.C. performed research; G.A.T. and A.R.B. contributed new reagents/analytic tools; J.Z., gression of many human malignancies (12 15). Despite the Y.Z., L.C., F.Y., S.M.L., G.A.T., A.R.B., Y.C., and J.D. analyzed data; and J.Z. and J.D. wrote growth-promoting role of Zyxin, however, little is known about the the paper. mechanisms by which Zyxin itself is regulated and how Zyxin af- The authors declare no conflict of interest. fects Hippo–YAP (and/or other signaling) activity in cancer cells. This article is a PNAS Direct Submission. K.-L.G. is a guest editor invited by the The protein kinase cyclin-dependent kinase 8 (CDK8) is a Editorial Board. component of the mediator complex that functions as a bridge Published under the PNAS license. between basal transcription machinery and -specific tran- 1To whom correspondence should be addressed. Email: [email protected]. scriptional factors (16). CDK8 is amplified and overexpressed in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. colon cancer and exerts its oncogenic activity partially through 1073/pnas.1800621115/-/DCSupplemental. regulating β-catenin activity (17). The precise mechanisms by Published online July 2, 2018.

E6760–E6769 | PNAS | vol. 115 | no. 29 www.pnas.org/cgi/doi/10.1073/pnas.1800621115 Downloaded by guest on September 26, 2021 Results extent) directly phosphorylated GST–Zyxin proteins in vitro Zyxin Is Phosphorylated by CDK1 in Vitro During Mitosis. Others and (Fig. 1D). Zyxin is not a suitable substrate for MAPK-p38α, we have shown that several Hippo–YAP components are regu- MEK1, ERK1, and JNK1/2 kinases in vitro (Fig. 1D), although lated and implicated in mitosis (20–26). These studies suggest these kinases recognize the same phosphorylation sequence that the Hippo–YAP pathway controls tumorigenesis through consensus as CDK1. dysregulation during mitosis. Given the connection between Zyxin Is Phosphorylated by CDK1 in Cells During Mitosis. CDK1 Zyxin and Hippo–YAP signaling, we tested the possibility that phosphorylates an S/TP consensus sequence (27). Database Zyxin might contribute to tumorigenesis through regulating cell- analysis identified three SPs (S281, S308, and S344) as possible cycle progression, especially mitosis. main phosphorylation sites in Zyxin during mitosis (28). We As shown in Fig. 1A, treatment with taxol or nocodazole— — generated phospho-specific antibodies against these sites. both agents that bind to microtubules and arrest cells in mitosis Nocodazole or taxol treatment significantly increased phos- significantly increased mobility upshift of Zyxin on a Phos-tag gel phorylation of S281, S308, and S344 of endogenous Zyxin (Fig. A (Fig. 1 ). Lambda phosphatase treatment completely converted 1E). Phosphopeptide, but not regular nonphosphopeptide, in- all slow-migrating bands to fast-migrating bands, confirming cubation completely blocked the phospho-signal, suggesting that that Zyxin is phosphorylated in mitosis induced by spindle this antibody detects the phosphorylated form of Zyxin (SI Ap- B poisons (Fig. 1 ). pendix, Fig. S1 A–C). Exogenous Zyxin was also phosphorylated Next, we used various kinase inhibitors to identify the candi- during mitotic arrest, and mutating serines to nonphosphorylatable date kinase for Zyxin phosphorylation. Of interest, treatments alanines abolished the phosphorylation (SI Appendix, Fig. S1D). with RO3306 (CDK1 inhibitor) or Purvalanol A (CDK1/2/5 in- The siRNA-mediated depletion of Zyxin significantly blocked hibitor) significantly inhibited mobility shift and phosphorylation the phospho-signal, confirming the specificity of these phospho- (Fig. 1C, lanes 5 and 6). These data suggest that CDK1, a well- antibodies (Fig. 1F). Phosphorylation of Zyxin occurred in dose- known master mitotic kinase, is likely the relevant kinase for dependent and time course-dependent manners (SI Appendix, Zyxin phosphorylation. Fig. 1D shows that purified CDK1/cyclin Fig. S1 E and F). Using kinase inhibitors, we demonstrated that B kinase complex (CDK2 and CDK5/p25 kinases to a lesser phosphorylation of Zyxin is CDK1 kinase-dependent (Fig. 1G, CELL BIOLOGY ABC Noco - + + λppase - - +

Noco DMSO Taxol Zyxin -100 Zyxin -100 (Phos-tag) DMSO DMSO VX680 MK5108 RO3306 Purvalanol A BI2536 U0126 MK2206 SB203580 Nocodazole - + + + + + + + + + (Phos-tag) -70 ∗∗ -70 Zyxin (Phos-tag) -70 Zyxin -70 Zyxin -70 Zyxin -70 Cyclin B Cyclin B -55 -55 Cyclin B -55 β-Actin β-Actin -40 -40 β-Actin -40

DEGST-Zyxin

DMSO Noco Taxol DMSO Noco Taxol DMSO Noco Taxol -170 kinase - Fig. 1. CDK1 phosphorylation of Zyxin during mi- α CDK1 CDK2 CDK5 p38 MEK1 ERK1 JNK1 JNK2 -130 -100 totic arrest. (A) Taxol (100 nM for 16 h) or nocoda- -130 zole (100 ng/mL for 16 h; Noco) treatments in HeLa 32P- -70 -100 Zyxin -55 cells caused mobility upshift of Zyxin on Phos-tag p-S281 Zyxin p-S344 Zyxin SDS-polyacrylamide gels. (B) HeLa cells were treated

p-S308 Zyxin -40 -130 -30 with nocodazole (Noco) as indicated and cell lysates GST -100 were further treated with (+) or without (−) λ Zyxin Zyxin Zyxin ------70 phosphatase (ppase). (C) HeLa cells were treated MBP + + + + + + + + + with taxol together with or without various kinase Cyclin B Cyclin B Cyclin B -55 32P- inhibitors as indicated. Inhibitors were added (with β-Actin β β MBP -15 -Actin -Actin -40 MG132 to prevent cyclin B from degradation and cells from exiting from mitosis) 1.5 h before harvesting the

FGsiControl + + - - Hcells. (D) In vitro kinase assays with purified kinases siZyxin - - #1 #2 siControl + + - using bacterially purified GST–Zyxin (Abnova) and Nocodazole - + + + siCyclin B1 - - + MBP (myelin basic protein) (Sigma) proteins as sub- Taxol - + + p-S281 Zyxin -70 DMSO DMSO VX680 MK5108 RO3306 Purvalanol A BI2536 U0126 MK2206 SB203580 strates. MBP proteins were included as positive con- Noco - + + + + + + + + + p-S281 -70 trols, confirming the kinases are active. (E)Zyxinis p-S281 phosphorylated at multiple sites during mitotic arrest. p-S308 Zyxin -70 -70 p-S308 -70 HeLa cells were treated as in A and total cell lysates were probed with the indicated antibodies. (F)Vali- p-S308 p-S344 Zyxin -70 -70 dation of phospho-specific antibodies. HeLa cells were p-S344 -70 transfected with 40 nM scramble (control) or 40 nM p-S344 siRNA against Zyxin for 48 h and were further treated Zyxin -70 -70 Zyxin -70 with nocodazole as indicated. (G)HeLacellswere Zyxin treated as in C. Total cell lysates were subjected to Cyclin B -55 -70 Cyclin B -55 Western blotting with the indicated antibodies. (H) Cyclin B -55 HeLa cells were transfected with 40 nM scramble (control) β-Actin β-Actin or siRNA against Cyclin B1 for 48 h and were further -40 β-Actin -40 -40 treated with taxol as indicated.

Zhou et al. PNAS | vol. 115 | no. 29 | E6761 Downloaded by guest on September 26, 2021 lanes 5 and 6). Furthermore, knockdown of Cyclin B1 or Zyxin Expression Is Induced During Mitosis. During our experiments, CDK1 largely blocked Zyxin phosphorylation (Fig. 1H and SI we noticed that, in addition to mobility shift/phosphorylation, Appendix, Fig. S1G). Enhanced expression of active CDK1/ Zyxin protein levels were also increased during taxol or Cyclin B1 was sufficient to stimulate Zyxin phosphorylation (SI nocodazole-induced mitotic arrest (Fig. 2D) and in normal mi- Appendix,Fig.S1H). tosis (SI Appendix, Fig. S4D). Quantitative RT-PCR (qRT-PCR) Immunofluorescence staining revealed that a strong signal was confirmed that Zyxin mRNA levels were induced by taxol or detected in nocodazole-arrested prometaphase cells for anti- nocodazole (Fig. 2E) and in normal mitosis (Fig. 2F). YAP bodies against Zyxin S281 and S344 (SI Appendix,Fig.S2A and B, targets survivin and Cyr61, but not YAP itself, were induced in Upper, white arrows). Very low or no signal was detected in interphase mitosis (Fig. 2F and SI Appendix, Fig. S4 C–E). A previous study cells (SI Appendix,Fig.S2A and B, yellow arrows). Again, phos- showed that Zyxin is a direct target of TAZ (a paralog of YAP)/ phopeptide, but not nonphosphopeptide, incubation completely TEAD (30), and we determined whether Zyxin induction in blocked the signal, suggesting that these antibodies specifically detect mitosis is YAP/TAZ-dependent. Indeed, addition of verteporfin phosphorylated Zyxin (SI Appendix,Fig.S2A and B). The specificity [(VP) a YAP/TEAD inhibitor (31)] or knockdown of YAP of the antibodies was further confirmed by siRNA knockdown of largely abolished Zyxin mRNA increase induced by taxol or Zyxin (SI Appendix, Fig. S2 C and D). Addition of RO3306 largely nocodazole treatment (Fig. 2G and SI Appendix, Fig. S4F). Of abolished the signals detected by p-Zxyin S281 and S344 antibodies interest, inhibition of CDK1 by Purvalanol A also blocked Zyxin in mitotic cells, further indicating that the phosphorylation is expression, suggesting that Zyxin expression in mitosis is induced CDK1-dependent (SI Appendix,Fig.S2A and B, Lower). in a CDK1-dependent manner (Fig. 2G). To determine whether mitotic phosphorylation of Zyxin occurs during unperturbed/normal mitosis, we performed immunofluo- Zyxin Is Required for Colon Cancer Cell Growth in Vitro and in Vivo. rescence staining on cells collected from a double thymidine block We next explored the biological significance of Zyxin in cancer and release (29). Very weak signals were detected in interphase or cell growth. We analyzed the Oncomine and TCGA online da- telophase/cytokinesis cells (Fig. 2 A and B, yellow arrows, and SI tabases and found Zyxin to be highly expressed in colon cancer Appendix,Fig.S3A and B). The phospho-signal increased in (Fig. 3A). We performed immunohistochemistry (IHC) staining prometaphase and peaked in cells (Fig. 2 A and B,white on tissue microarrays (TMAs) (n = 66 cancer/tumor, n = 35 normal) arrows, and SI Appendix,Fig.S3A and B). After being released and confirmed that Zyxin protein levels were significantly increased from double thymidine block, cells entered into mitosis at 10–12 h in colon cancer samples compared with normal tissue (Fig. 3 B and (21, 24) (revealed by increased p-S10 H3 levels), and p-Zyxin C). The specificity of the Zyxin IHC was validated by using Zyxin- S281 and S344 signals also increased in these cells (Fig. 2C). Mi- knockdown tumor samples (Fig. 3D). These findings highlight a totic phosphorylation of Zyxin occurred in freely cycling cells in a critical role of Zyxin in tumorigenesis in colon cancer. CDK1-dependent manner (SI Appendix,Fig.S4A and B). These To directly determine the role of Zyxin in colon cancer, we observations indicate that Zyxin is phosphorylated by CDK1 during depleted Zyxin (with two independent shRNAs) in various colon mitosis. cancer cells (Fig. 3 E–G). Knockdown of Zyxin greatly reduced

ABp-Zyxin S281 β-tubulin p-Zyxin S344 β-tubulin D

Control Taxol Nocodazole

Zyxin -70

Cyclin B -55 β -Actin -40 20 um 20 um 7.0 ∗∗ E Fig. 2. Zyxin is phosphorylated and induced during 6.0 unperturbed mitosis. (A) HeLa cells were synchro- 5.0 ∗∗ nized by a double thymidine (DT) block and release 4.0 method. Cells were stained with antibodies against p-Zyxin S281 or β-tubulin, or with DAPI. A 40× ob- 3.0 jective lens was used to view various phases of the 2.0 cells in a field. (B) Experiments were done similarly as 1.0 in A with p-Zyxin S344 antibodies. White and yellow Relative Zyx mRNA (fold) Relative Zyx mRNA 0.0 arrows (in A and B) mark the metaphase and in- DAPI Merged DAPI Merged Control Noc Taxol terphase cells, respectively. (C) HeLa cells were syn- C DT block and release FG chronized by a DT block and release method. Total 5.0 Time (h) 0 7.5 9.5 10.5 12 24 cell lysates were harvested at the indicated time Zyxin 3.0 Survivin p-S281 points and subjected to Western blotting analysis. -70 ∗∗ ∗∗∗ 4.0 Zyxin (D) Zyxin protein levels increased during mitotic ar- 2.0 2.5 3.0 rest. (E) Zyxin mRNA levels were induced during mi- p-S344 -70 2.0 totic arrest. (F) Zyxin mRNA levels were increased Zyxin 1.5 1.5 2.0 ∗∗ during normal mitosis. HeLa cells were treated as in C Zyxin -70 1.0 ∗∗ and harvested at 10 h post release. Survivin serves as 1.0 1.0 a positive control. (G) HeLa cells were treated with

p-S10 H3 0.5 (fold) Relative Zyx mRNA taxol and inhibitors were added 2 h before harvest- -17 (fold) Relative mRNA 0.5 0.0 ing the cells. qRT-PCR was performed to measure 0.0 0.0 Taxol - + + + β-Actin -40 G1/S G2/M G1/S G2/M Inhibitors - - PA VP Zyxin mRNA levels. Data were expressed as the mean ± SEM of three independent experiments (E– 1 2 3 4 5 6 G). **P < 0.01; ***P < 0.001 (t test).

E6762 | www.pnas.org/cgi/doi/10.1073/pnas.1800621115 Zhou et al. Downloaded by guest on September 26, 2021 ABCD +++ p=4.8e-06 ++ 3% 13% 3 N=483 N=41 - 48% + 36% 2 Normal (N=35) Normal (+) Tumor (+++) Control-tumor - 4% + 17% 1 +++ 45%

++ 34% Rel. Zyxin expression (log2) 0 Tumor Normal Tumor (N=66) Tumor (+) Tumor (++) shZyxin-tumor

EFGKHCT116 1000

shControl shZyxin-1 shZyxin-2 shControl shZyxin-1 shZyxin-2 shControl shZyxin-1 shZyxin-2 Fig. 3. Zyxin levels are increased in colon cancer Zyxin Zyxin -70 -70 Zyxin -70 patients and knockdown of Zyxin inhibits colon 500 ∗∗∗ ∗∗∗ cancer cell growth. (A) Zyxin mRNA levels are in- β β Colony number -actin -40 β-actin -40 -actin -40 creased in colon cancer samples. RNA-sequencing RKO SW480 HCT116 0 data from The Cancer Genome Atlas (TCGA) were shRNA Ctrl Zyx-1 Zyx-2 ∗∗∗ downloaded from NCBI’s Omnibus HIJ0.90 ∗∗∗ ∗∗ ∗∗ 0.95 ∗∗∗∗∗∗ (GEO) with the accession number of GSE62944 (the 0.85 L SW480 0.9 Student t test was used for statistical evaluation). (B 0.80 0.85 and C) IHC staining analysis of Zyxin on colon cancer 1000 0.8 TMAs (P < 0.001, Wilcoxon rank sum test). Represen- 0.75 0.75 tative staining images were shown (C). Staining and 0.70 0.7 500 scoringweredoneaswedescribed(63).−: negative; +: 0.65 ∗∗∗ ++ +++ CELL BIOLOGY 0.65 low expression; : moderate expression; : strong 0.6 Colony number ∗∗∗ Relative wound width Relative wound width Relative wound width expression. (D) Validation of the Zyxin antibody in IHC 0.60 0.55 0 shRNA Ctrl Zyx-1 Zyx-2 shRNA Ctrl Zyx-1 Zyx-2 shRNA Ctrl Zyx-1 Zyx-2 shRNA Ctrl Zyx-1 Zyx-2 specificity using control and Zyxin-knockdown tumors. – MNO∗∗∗ (E G) Establishment of colon cancer cell lines with 30 15 ∗∗∗ stable knockdown of Zyxin. (H–J)Cellmigration ) ∗∗∗ ) 5

RKO-shControl 5 5 25 SW480-shControl HCT116-shControl (wound healing) assays of cell lines established in E–G. RKO-shZyxin-1 ∗∗∗ SW480-shZyxin-1 ∗∗∗ 20 HCT116-shZyxin-1 20 RKO-shZyxin-2 10 SW480-shZyxin-2 15 HCT116-shZyxin-2 (K and L) Anchorage-independent growth (colony as- 15 ∗∗∗ ∗∗∗ says on soft agar) of HCT116 and SW480 cell lines with 10 10 ∗∗∗ 5 ∗∗∗ knockdown of Zyxin (F and G). (M–O) Cell proliferation ∗∗∗ 5 5 ∗∗ assays in cell lines established in E–G. Data were Cell number ( x10 ) Cell number ( number X10 Cell Cell number ( number X10 Cell 0 0 0 expressed as the mean ± SEM of three independent Days Days 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 Days 0 2 4 6 experiments (H–O). **P < 0.01; ***P < 0.001 (t test).

migration (as shown by the wound healing assay) (Fig. 3 H–J and inhibited by cofilin, for which activity is negatively controlled by SI Appendix, Fig. S5 A–C), anchorage-independent growth (Fig. phosphorylation at Serine3 (35–37). We found that phospho-S3 cofilin 3 K and L and SI Appendix, Fig. S5 D and E), and proliferation levels were reduced and F-actin formation/cytoskeleton was strongly (Fig. 3 M–O) in RKO, SW480, and HCT116 cells. Importantly, inhibited upon Zyxin knockdown in colon cancer cells (SI Appendix, reexpression of shRNA-resistant wild-type Zyxin completely Fig. S6 D and E), suggesting that Zyxin might control YAP activity rescued all these phenotypes in RKO cells (Fig. 4 A–C). How- and subsequent cell migration and proliferation through ever, expression of nonphosphorylatable mutant Zyxin-3A F-actin remodeling. (S281A/S308A/S344A) only partially restored these characteris- To explore the functional interaction between Zyxin and YAP, tics (Fig. 4 B and C), suggesting that mitotic phosphorylation is we reexpressed YAP–S127A in Zyxin knockdown cells (SI required for Zyxin oncogenic activity in colon cancer cells. Appendix,Fig.S6F and G). Enhanced expression of YAP–S127A Similar results were observed in SW480 cells (Fig. 4 D–F). As completely rescued the effects (migration and proliferation) of expected, Zyxin knockdown also greatly reduced tumor growth Zyxin knockdown in both SW480 and HCT116 cells (SI Appendix, in animals, and cells bearing Zyxin-3A formed much smaller Fig. S6 H–K), suggesting that Zyxin promotes colon cancer cell tumors compared with cells expressing wild-type Zyxin (Fig. 4 G growth through YAP. and H). Knockdown efficiency was confirmed, and reexpression levels between wild-type and mutant Zyxin (Zyxin-3A) were similar Zyxin Regulates CDK8 to Promote Migration and Proliferation in in tumors (SI Appendix,Fig.S5F). These results suggest a critical Colon Cancer Cells. To elucidate the underlying mechanisms in role of Zyxin and its mitotic phosphorylation in colon cancer. which Zyxin regulates YAP activity and controls colon cancer cell growth, we performed an array analysis involving 84 cell- Zyxin Regulates YAP Activity in Colon Cancer Cells. Zyxin has been cycle regulators in control and Zyxin-knockdown cells. Five shown to be a positive regulator of YAP/Yki activity (5–8, 11). were down-regulated and one gene up-regulated (CDKN2B) in We confirmed that Zyxin knockdown increased p-YAP S127 levels Zyxin-depleted cells compared with control cells (Fig. 5A). Most and promoted YAP cytoplasmic localization (SI Appendix,Fig.S6A (except for NBN) were confirmed by qRT-PCR in an independent and B). Consistent with reduced YAP activity, expression of the target Zyxin-knockdown cell line (SI Appendix,Fig.S7A). Among these gene CTGF was significantly reduced upon Zyxin knockdown in both genes, CDK8 (cyclin-dependent kinase 8) attracted us, since it RKO and SW480 cells (SI Appendix,Fig.S6C). F-actin cytoskeletal functions as an oncogene in colon cancer (17) and in melanoma remodeling was shown to control YAP/Yki activity in mammalian (18). We confirmed that CDK8 protein levels were also reduced in cells and in Drosophila (5–9, 32–34). F-actin polymerization was Zyxin-knockdown RKO and SW480 cells (Fig. 5B) and restored by

Zhou et al. PNAS | vol. 115 | no. 29 | E6763 Downloaded by guest on September 26, 2021 ABC

) 25 shControl 0.85 5 shZyxin 20 shZyxin+Res-Zyxin-WT shControl shZyxin-1 shZyxin-1 shZyxin-1 shZyxin+Res-Zyxin-3A ∗∗∗ Res-Zyxin - - WT 3A ∗∗∗ 0.75 15 RKO Zyxin -70 10 ∗∗ 0.65 5 β ( number X10 Cell -actin -40 Relative wound width 0.55 0 shZyxin - + + + Days 0 2 4 6 RKO Res-Zyxin - - WT 3A

D EFshControl

) shZyxin

∗∗ 5 0.85 30 shZyxin+Res-Zyxin-WT Fig. 4. Mitotic phosphorylation of Zyxin is required shZyxin+Res-Zyxin-3A shControl shZyxin-1 shZyxin-1 shZyxin-1 for colon cancer tumorigenesis. (A) Establishment of Res-Zyxin - - WT 3A ∗∗ 0.75 20 SW480 Zyxin-knockdown cell lines expressing shRNA-resistant Zyxin (Res) Zyxin or Zyxin-3A in RKO colon cancer cells. 3A: -70 0.65 10 ∗∗ S281A/S308A/S344A. (B and C) Cell migration (wound

Cell number ( number X10 Cell healing) and proliferation assays in cell lines estab-

β Relative wound width -actin -40 0.55 lished in A.(D) Establishment of Zyxin-knockdown cell shZyxin - + + + 0 SW480 Res-Zyxin - - WT 3A Days 0246lines expressing shRNA-resistant (Res) Zyxin or Zyxin-3A in SW480 colon cancer cells. 3A, S281A/S308A/S344A. (E and GHshControl (N=5) F) Cell migration (wound healing) and proliferation as- 3 shZyxin (N=5) says in cell lines established in D. Data were expressed as 8 the mean ± SEM of three independent experiments (B, C, shZyxin+Res-Zyxin-WT (N=5) shControl E,andF). **P < 0.01; ***P < 0.001 (Res-Zyxin-WT vs. Res- 6 shZyxin+Res-Zyxin-3A (N=5) shZyxin Zyxin-3A) (t test). (G and H)Zyxin-3Afailedtorescuetu- mor growth in Zyxin-knockdown RKO cells. RKO cell lines 4 from A were s.c. inoculated into athymic nude mice (both ∗∗∗ shZyxin+Res-Zyxin-WT left and right sides) and the representative tumors in each 2 ∗∗∗ ∗∗ group were excised and photographed at the endpoint

Tumor volume (x100mm ) Tumor shZyxin+Res-Zyxin-3A 0 (H). Yellow asterisks mark the comparisons between Res- Days 10 14 17 21 25 29 Zyxin-WT and Res-Zyxin-3A. **P < 0.01; ***P < 0.001 post injection (t test).

reexpression of exogenous Zyxin (SI Appendix,Fig.S7B). The in RKO and SW480 cells in which Zyxin was depleted and Zyxin-3A mutant had similar activity to wild-type Zyxin in rescuing CDK8 reexpressed (Fig. 6 E–H), suggesting that YAP is essential CDK8 levels, suggesting that mitotic phosphorylation of Zyxin is for CDK8-driven tumorigenesis in colon cancer. Thus, we identified dispensable in controlling CDK8 expression (SI Appendix,Fig. CDK8 as a positive regulator of YAP activity. S7B). Importantly, ectopic expression of CDK8, but not the kinase- dead form (CDK8-KD), substantially restored the migratory CDK8 Phosphorylates and Promotes YAP Activity. CDK8 is a mem- (assayed by wound healing) and proliferating ability of Zyxin- ber of the mediator complex, which couples transcriptional depleted RKO and SW480 cells (Fig. 5 C–F). Moreover, regulators to control basal transcription (16). Since CDK8 is a siRNA-mediated transient knockdown (two independent siRNAs) kinase and regulates YAP transcriptional activity, we tested or CRISPR/double nickase-mediated knockout (KO; two inde- whether CDK8 directly phosphorylates YAP. In vitro kinase pendent gRNAs) (38) of CDK8 significantly impaired cell mi- assays showed that purified CDK8/Cyclin C complex readily gration, proliferation, and anchorage-independent growth in phosphorylated GST- or His-tagged YAP proteins (Fig. 6 I and J). SW480 cells (Fig. 5 G–L). CDK8 KO also reduced proliferation CDK8 could also undergo autophosphorylation under these con- in DLD-1 cells (Fig. 5 M and N). Of note, both mRNA and ditions (SI Appendix,Fig.S8). CDKs phosphorylate at S/T-P mini- protein levels of CDK8 were up-regulated in colon tumors mal consensus sequences. YAP contains seven conserved S/TPs SI Appendix C D compared with normal tissue ( , Fig. S7 and ). We (T119, S128, S138, S217, S289, S367, and T412; the T143, T154, and found that CDK8 and Zyxin protein levels were positively cor- S340 sites do not exist in mouse and were excluded from further related in metastatic colon cancer samples (SI Appendix, Fig. S7 E F analysis). Of interest, mutating four amino acid positions (T119, and ). Together, these data suggest that Zyxin promotes S128, S289, and S367) to alanines (YAP-4A) completely blocked colon cancer cell growth via CDK8. phosphorylation (Fig. 6J), suggesting that CDK8 phosphorylates In line with the above functional studies, reexpression of these sites in YAP in vitro. CDK8 largely restored the YAP phosphorylation and target gene Given that both Zyxin (Figs. 1 and 2) and YAP are phos- (CTGF) expression in Zyxin knockdown cells (Fig. 6 A and B), phorylated during mitosis (21, 39), we next explored whether CDK8 suggesting that Zyxin regulates YAP activity through CDK8. phosphorylates YAP in mitosis. Interestingly, S128 phosphorylation CDK8 Requires YAP to Promote Cell Growth in Colon Cancer. Based was significantly increased in nocodazole-arrested mitotic cells (Fig. K on our observations that Zyxin regulates YAP through CDK8 6 ). Consistent with our previous study (21), phosphorylation of (Figs. 5 and 6 A and B), we reasoned that CDK8 also controls T119 and S289 was also increased in mitotic cells (Fig. 6K). Im- YAP activity. Indeed, we found that S127 phosphorylation of portantly, CDK8 deletion largely blocked the phosphorylation of all YAP was increased in CDK8-KO cells (Fig. 6C). Consistent with three sites induced by mitotic arrest, suggesting that CDK8 is a decreased activity of YAP in CDK8-KO cells, the expression of relevant YAP kinase in vivo, specifically in mitosis (Fig. 6K). Using YAP target gene CTGF was inhibited upon CDK8 deletion (Fig. reporter assays, we next examined whether phosphorylation of YAP 6D). Furthermore, CRISPR-mediated KO (two independent affects its activity. Mutating these four sites to alanines greatly gRNAs) of YAP strongly inhibited migration and proliferation suppressed YAP–S127A activity (Fig. 6L). These observations suggest

E6764 | www.pnas.org/cgi/doi/10.1073/pnas.1800621115 Zhou et al. Downloaded by guest on September 26, 2021 ABC Gene F.C. F-CDK8 - - WT KD - - WT KD CCND2 -3.82 shZyxin - + + + - + + + shControl shZyxin-1 shZyxin-2 shControl shZyxin-1 shZyxin-2 Zyxin CDK8 -2.94 Zyxin -70 -70 CDKN2B 2.08 MAD2L1 -2.03 Flag NBN -2.27 CDK8 -55 -55 SKP2 -2.15 Magnitude of log2 (F. C.) β β-actin -actin -40 -40 RKO SW480 -1.934 0 1.934 SW480 RKO

0.90 RKO ∗∗∗ ∗∗∗ DE30 F40 )

) shControl

shControl 5 0.80 5 25 shZyxin shZyxin shZyxin+CDK8 30 shZyxin+CDK8 20 shZyxin+CDK8-KD shZyxin+CDK8-KD 0.70 15 20 SW480 RKO ∗∗∗ ∗∗∗ Fig. 5. Zyxin regulates migration and proliferation _ 0.60 10 ∗∗∗ _ ∗∗∗ 10 _ through CDK8 in colon cancer cells. (A) Expression

5 Cell number ( X10 Cell number ( X10 _ Relative wound width 0.50 profiling of regulators identified multiple 0 0 shRNA Ctrl Zyx Zyx Zyx Days Days 0 2 4 6 genes including CDK8 that are regulated by Zyxin. CDK8 - - WT KD 0 2 4 6 The RT2 cell cycle arrays (Qiagen) were used and ex- periments were performed following manufacturer’s GH∗∗∗ I M 0.90 siControl instructions. F.C., fold change. (B) CDK8 protein levels 5 15 siCDK8-1 0.85 ∗∗∗ siCDK8-2 ∗∗ were decreased in Zyxin-knockdown cells. (C) Estab- siControl siCDK8-1 siCDK8-2 0.80 10 ∗∗∗ Control CDK8-KO-1 CDK8-KO-2 lishment of Zyxin-knockdown cells expressing Flag- CDK8 -55 0.75 CDK8 -55 tagged CDK8 or CDK8-KD (kinase dead, D173A). 0.70 5 (D–F) Migration (wound healing) and proliferation β β assays in cell lines from C. Ectopic expression of -actin -40 0.65 ∗∗∗ -actin -40 Relative wound width 0.60 Cell number ( x10 ) 0 CDK8, but not CDK8-KD, rescued growth defects of SW480 DLD-1 siRNA Ctrl CDK8-1CDK8-2 Days 0 2 4 6 Zyxin-knockdown cells. (G–I) Transient knockdown

(two independent siRNAs) of CDK8 inhibited SW480 CELL BIOLOGY JK0.90 ∗∗ L N ∗∗ ∗∗∗ Control SW480 Control 30 cell migration (wound healing) and proliferation. (J)

0.85 30 5 CDK8-KO-1 5 CDK8-KO-1 Establishment of CDK8 KO cell lines. Two indepen- 25 CDK8-KO-2

0.80 CDK8-KO-2 ∗∗

Control CDK8-KO-1 CDK8-KO-2 20 dent gRNAs were used. (K and L) Cell migration 20 ∗∗∗ 0.75 SW480 DLD-1 (wound healing) assays and proliferation curves of CDK8 -55 ∗∗∗ 15 0.70 10 10 ∗∗∗ cell lines from J.(M and N) CDK8 KO inhibited pro- β -actin -40 0.65 5 liferation in DLD-1 cells. Data were expressed as the Cell number ( x10 ) Cell number ( x10 )

Relative wound width ± SW480 0 0 mean SEM of three independent experiments (D, F, CDK8 Ctrl KO-1 KO-2 Days 0246Days 0246H, I, K, L, and N). **P < 0.01; ***P < 0.001 (t test).

that CDK8 directly phosphorylates and promotes YAP transcriptional Like YAP–S127A or YAP-4D, enhanced expression of activity, identifying YAP as a direct substrate for CDK8. β-catenin–S33Y (active form) had little effect on CDK8-KO cell growth (Fig. 7B and SI Appendix, Fig. S10 A, B, and D), sug- YAP Phosphorylation Is Required for CDK8-Driven Migration and gesting that active β-catenin alone is not sufficient to rescue Proliferation. To further explore the functional relationship be- CDK8-KO–mediated cell growth. Coexpression of β-catenin- tween CDK8 and YAP, we performed complementary experi- S33Y with YAP–S127A/4D did not further increase growth ments. Ectopic expression of the constitutively active form of compared with YAP–S127A/4D alone in CDK8-KO cells (Fig. 7 YAP (YAP–S127A) had little impact on proliferation and mi- B–I and SI Appendix, Fig. S10 D and E). Our data suggest that gration in SW480 and DLD-1 CDK8-KO cells (SI Appendix, Fig. CDK8 promotes YAP activation through a twofold mechanism: S10 A and B). Expression of phospho-mimetic mutant YAP-4D canonical S127 phosphorylation and direct phosphorylation. failed to rescue proliferation and migration in CDK8-KO cells Both YAP and β-catenin are required for CDK8-driven tu- (Fig. 7 A and B). However, expression of YAP-S127A/4D morigenesis in colon cancer. Altogether, our findings indicate that (highest activity) substantially (but not completely) restored Zyxin promotes colon tumorigenesis in a mitotic phosphorylation- CDK8-KO–mediated migration, proliferation, and anchorage- dependent manner and through CDK8-mediated YAP activation. independent growth, suggesting that CDK8-mediated phos- Discussion phorylation is essential for full activation of YAP (Fig. 7 B–E and Our study revealed that the cell-adhesion protein Zyxin pro- SI Appendix, Fig. S10 C–E), which is consistent with our earlier motes colon cancer cell growth by controlling CDK8 expression. observations (Fig. 6). CDK8 KO significantly inhibited tumor It is currently not known how Zyxin regulates CDK8 expression. growth in vivo in both SW480 and DLD-1 cells (Fig. 7 F–I). – CDK8 mRNA expression is negatively regulated by the histone Ectopic expression of YAP S127A/4D also largely restored tu- variant macroH2A (18). A recent study demonstrated that the F–I mor growth of CDK8-KO cells in animals (Fig. 7 ). E3 , Skp2, promotes macroH2A ubiquitination and deg- A previous report showed that CDK8 functions as a colon radation (19). Interestingly, in our array analysis, Skp2 is one of β oncogene through regulating -catenin (17). Consistent with this the five genes down-regulated by Zyxin depletion (Fig. 5 and SI β study, the inhibitory phosphorylation of -catenin was increased, Appendix, Fig. S7A). Therefore, it is possible that Zyxin controls and target gene (LEF1) expression was inhibited in CDK8-KO CDK8 expression through the Skp2–macroH2A axis. Since both cells (SI Appendix,Fig.S9A and B). Interestingly, KO of β-catenin Zyxin and CDK8 levels are increased in colon cancer patients also greatly impaired migration and proliferation in RKO cells with (Fig. 3 and SI Appendix, Fig. S7 C and D) and possess oncogenic Zyxin depletion and CDK8 reexpression (SI Appendix,Fig.S9C–E). roles in colon cancer development (17), it will be interesting to These findings suggest that CDK8 controls YAP and β-catenin ac- explore the biological significance of Skp2 and macroH2A in tivity, both of which are critical regulators in colon cancer. colon cancer and their functional interactions with Zyxin/CDK8.

Zhou et al. PNAS | vol. 115 | no. 29 | E6765 Downloaded by guest on September 26, 2021 ABF-CDK8 - - + C shZyxin - + + SW480 ∗∗ 1.2 1.5 RKO ∗∗ CDK8-KO - #1 #2 - #1 #2 Zyxin -70 ∗∗∗ 1.0 ∗∗∗ CDK8 -55 p-S127 YAP -70 0.8 1.0 0.6 p-S127 YAP -70 YAP -70 0.4 0.5 YAP -70 Flag -55 0.2

Relative CTGF mRNA β-actin β 0.0 0.0 -40 -actin -40 shZyxin - + + - + + CDK8 - - + - - + DLD-1 SW480 RKO Fig. 6. CDK8 phosphorylates YAP and promotes its 20 DEF1.2 shZyxin+CDK8_ shZyxin+CDK8 ∗∗∗ SW480 1.2 activation. (A and B) Zyxin regulates YAP activity DLD-1 YAP-KO - #1 #2 shZyxin+CDK8+YAP-KO-1

1.0 5 through CDK8. Ectopic expression of CDK8 inhibited 1.0 15 shZyxin+CDK8+YAP-KO-2 p-YAP S127 (A) and rescued CTGF (YAP target gene) 0.8 0.8 YAP -70 RKO expression (B) in Zyxin-knockdown cells. (C and D) 0.6 ∗∗∗ 0.6 10 Flag ∗∗∗ CDK8 regulates YAP activity. CDK8 KO increased YAP 0.4 0.4 ∗∗∗ -55 ∗∗∗ ∗∗∗ 5 phosphorylation (S127) (C) and inhibited CTGF ex- 0.2 0.2 Cell number ( x10 ) β-actin pression (D). (E–H) YAP is required for CDK8-driven Relative CTGF mRNA -40 0.0 0.0 0 proliferation in colon cancer cells. (E and G) Estab- CDK8-KO - #1 #2 - #1 #2 RKO Days 0 2 4 6 lishment of YAP KO cell lines. CRISPR/double nickase- ∗∗∗ G shZyxin+CDK8_ HIJ16 shZyxin+CDK8 GST-YAP + + + mediated (two independent gRNAs were used) KO of CDK8 - + + His-YAP WT WT 4A YAP-KO - #1 #2 shZyxin+CDK8+YAP-KO-1 CDK8 - + + YAP in RKO and SW480 cells where Zyxin was de- 5 12 shZyxin+CDK8+YAP-KO-2 32 P-YAP- -100 32 -100 pleted and CDK8 reexpressed. (F and H) Cell pro- YAP -70 32 P-CDK8- P-CDK8- SW480 32 P-YAP- -70 8 liferation assays for cell lines from E and G.(I) In vitro ∗∗∗ Autoradiography kinase assays using GST-YAP as substrates. Two CDK8 -55 Autoradiography 4 GST-YAP- -100 -100 batches of CDK8 kinases (SignalChem) were used. (J) GST-CDK8- His-YAP- Cell number ( x10 ) -70 In vitro kinase assays with His-YAP-WT and -YAP-4A β-actin -40 -70 0 (T119A/S128A/S289A/S367A) proteins as substrates. SW480 Days 0 2 4 6 -100 GST-Cyc C- -55 GST-CDK8- (K) CDK8 phosphorylates YAP in mitosis. SW480 -70 K CDK8-KO - - + + CDK8-KO - - + + control and CDK8-KO cells were treated with DMSO Nocodazole - + - + Nocodazole - + - + GST-Cyc C- -55 or nocodazole for 16 h. Endogenous YAP proteins p-S128 YAP-SE p-T119 YAP were immunoprecipitated and probed with -70 -70 phospho-antibodies against YAP T119, S128, 1.0 0.31 L 150 S289 and total YAP antibodies. Relative phosphory- p-S128 YAP-LE ∗∗∗ -70 p-S289 YAP lation (fold changes in signal intensity) was quanti-

-70 IP: YAP 1.0 0.26 IP: YAP fied by Image J and shown below the blots from 1.0 0.32 100 three independent experiments. LE, long exposure; YAP -70 YAP -70 SE, short exposure. (L) CDK8-mediated phosphoryla- 50 tion stimulates YAP transcriptional activity. Lucifer- CDK8 -55 CDK8 -55 ase reporter assays were done as we described (60,

Rel. luciferase activity 64). Data were expressed as the mean ± SEM of three Lysate

β Lysate -Actin -40 β 0 < -Actin -40 biological repeats (B, D, F, H, and L). **P 0.01; Vector S127A S127A/4A ***P < 0.001 (t test).

CDK8 positively regulates β-catenin transcriptional activity in activation and is required for CDK8-KO cell growth (Fig. 7). colon cancer cells (17), although the precise mechanism remains These observations not only added additional layers of YAP obscure. It may involve the phosphorylation of E2F1 and relieve regulation, but also raised complex questions. For example, E2F1-mediated repression on β-catenin/TCF signaling activity S128 has been shown to be phosphorylated by Nemo-like kinase (17, 40, 41). The kinase activity of CDK8 is necessary for (42, 43), and T119/S289/S367 are phosphorylated by CDK1 (21). β-catenin–driven transcription (17). Furthermore, a dominant- How these kinases and phosphorylation are coordinated in cells negative TCF construct, previously shown to inhibit β-catenin– requires further investigation. Another question is: How does induced cellular transformation, also only partially abrogated CDK8 regulate YAP S127 phosphorylation and localization? CDK8 activity (17). This notion is supported by the observations YAP S127 is largely mediated by Lats1/2 kinases in the Hippo that several profiling experiments failed to identify the broad pathway; however, we did not observe significant changes of deregulation of β-catenin signaling in CDK8-disturbed cells (16). Lats1/2 kinase activity (revealed by phosphorylation levels of These results strongly indicate that CDK8 controls additional their activation loop) in CDK8-KO cells and -intact cells, sug- factor(s) also outside the β-catenin signaling pathway to promote gesting a Hippo-independent regulation of YAP activity. colon cancer cell growth (i.e., other factors in addition to Our study showed that the active β-catenin mutant (β-catenin– β-catenin exist downstream of CDK8). Although our data (SI S33Y) had little effect on rescuing the phenotypes of CDK8 KO Appendix, Fig. S9 D and E) and previous studies (17) showed that in colon cancer cells (SI Appendix, Fig. S10). This implies that β-catenin–KO suppressed the CDK8-driven cell migration and additional target(s) exists or, alternatively, that β-catenin–S33Y proliferation, restoration of β-catenin in CDK8-KO cells was not (like YAP–S127A) is not fully active in CDK8-deleted cells. The sufficient to support colon cancer cell growth (SI Appendix, Fig. similarity between YAP and β-catenin prompted us to reason S10), further suggesting that β-catenin signaling is not the main that β-catenin is a direct substrate for CDK8. Indeed, β-catenin (or only) effector of CDK8. Our study highlights the importance sequence analysis revealed three S/TP phospho-consensus (S191/ of YAP in mediating CDK8-driven tumorigenesis in colon can- S246/S605) as potential sites for CDK8 phosphorylation, and cer (Figs. 6 and 7). CDK8 regulates YAP activity through a these sites have been shown to be important for β-catenin cel- twofold mechanism: phosphorylation on S127/localization and lular localization and activity (44). direct phosphorylation at T119/S128/S289/S367 (Figs. 6 and 7). The global burden of colon cancer is rising steadily. Although CDK8-mediated direct phosphorylation promotes YAP further much progress has been made in therapeutic approaches, for

E6766 | www.pnas.org/cgi/doi/10.1073/pnas.1800621115 Zhou et al. Downloaded by guest on September 26, 2021 AB∗ C ∗∗∗ SW480

β-Cat-CA Vector S127A YAP-4D 127A/4D S127A/4D+β-Cat-CA 0.85 40

) Control Control

CDK8-KO 5 CDK8-KO-2 ∗∗∗ 0.80 30 CDK8-KO-2+S127A/4D YAP -70 CDK8-KO-2+S127A/4D + β -Cat-CA

β -Cat-CA ∗∗∗ 0.75 20 CDK8 -55 ∗∗ SW480 0.70 Flag 10

-100 Cell number ( X10 (β-Cat-CA) Vector Control YAP-S127A/4D YAP-4D YAP-S127A Relative wound width 0.65 0 YAP-S127A/4D+ β-actin β -Catenin-CA Days 0 2 46 -40 0.60 SW480 CDK8-KO - + + + + + +

DF8 Control G ∗ CDK8-KO-2 15 ∗∗ CDK8-KO-2+YAP-S127A/4D Control ∗∗∗ 6 CDK8-KO-2+YAP-S127A/4D + β -Cat-CA ∗∗ 10 CDK8-KO-2 4 CDK8-KO-2+YAP-S127A/4D 5 2 ∗ β CDK8-KO-2+YAP-S127A/4D+ -Cat-CA CELL BIOLOGY Vector YAP-S127A/4D YAP-S127A/4D + β -Cat-CA Control

Tumor volume(x100 mm3) volume(x100 Tumor ∗ Colony number (x100) 0 0 CDK8-KO - + + + 10 20 30 40 50 SW480 Days post injection (SW480) EH I n.s. ∗∗∗ 300 10 Control CDK8-KO-1 ∗∗∗ Control 8 CDK8-KO-2 ∗∗∗ CDK8-KO-2+YAP-S127A/4D n.s. 200 CDK8-KO-2+YAP-S127A/4D 6 CDK8-KO-2 +β-Cat-CA ∗∗∗ ∗∗ ∗∗ 4 ∗∗ 100 ∗ ∗∗ CDK8-KO-2+YAP-S127A/4D Control Vector YAP-S127A/4D 2

Colony number ∗∗ YAP-S127A/4D + β -Cat-CA 0 (x100 mm3) volume Tumor 0 CDK8-KO-2+YAP-S127A/4D+β-Cat-CA CDK8-KO - + + + 6 9 12 15 18 21 24 DLD-1 Days post injection (DLD-1)

Fig. 7. YAP phosphorylation is required for CDK8-driven tumorigenesis in colon cancer cells. (A) Establishment of cell lines expressing various YAP mutants and/or β-catenin in CDK8 KO SW480 cells. CA, constitutive active form (S33Y mutant). (B–D) Functional assays of cell lines established in A. Cell migration (wound healing) assays in various cell lines as indicated (B). Cell proliferation curves of cell lines with various gene expression status (C). Anchorage-independent growth (colony assays in soft agar) in cell lines as indicated (D). Data (B–D) are from three to four independent experiments and expressed as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (t test). (E) Anchorage-independent growth in various DLD-1 cells lines as indicated. Data are from three independent experiments and expressed as the mean ± SEM. ***P < 0.001. n.s., not significant. (F and G) Tumor growth curves and images of all tumors (at the endpoint) from SW480 cell lines. 2.5 × 106 cells (1:1 mixed with Matrigel) were s.c. inoculated into athymic nude mice on both flanks. n = 5 mice and 10 injections per group. Only three tumors were formed at the last two time points in the CDK8-KO group and the other three groups formed seven to eight tumors (G). Red asterisks mark the comparisons between control and CDK8-KO-2; Blue asterisks mark the comparisons between YAP-S127A/4D and CDK8-KO-2. *P < 0.05; **P < 0.01 (t test). (H and I)Tumor growth curves and representative images of tumors from DLD-1 cell lines. 1.0 × 106 cells (1:1 mixed with Matrigel) were s.c. inoculated into athymic nude mice on both flanks (5 mice and 10 injections/group, 10 tumors were averaged for each group at each time point). Red asterisks mark the comparisons between control and CDK8-KO-2; Blue asterisks mark the comparisons between YAP-S127A/4D and CDK8-KO-2. *P < 0.05; **P < 0.01; ***P < 0.001 (t test). n.s., not significant.

patients with locally advanced primary or recurrent colon cancer, selective inhibitors have been developed (16, 46, 47). Furthermore, often unresectable for cure, survival remains poor (45). Further- animal studies confirmed the potential efficacy of CDK8 inhibitors more, available treatment options for colon cancer are very limited (48), although interpretation of CDK8 inhibitor data requires cau- due to lack of validated targets. Our study has demonstrated the tion since CDK8 may also have a tumor-suppressive function during critical role and regulatory mechanisms of the Zyxin–CDK8–YAP/ early stages of colon tumorigenesis(49).VPwasrecentlyidentified β-catenin signaling in colon cancer. These studies suggest that this as an effective compound for preventing YAP activity (31). In- axis is a potential therapeutic target and provide several options for terestingly, VP (without light activation) significantly inhibited YAP targeted therapy using small-molecule inhibitors in colon cancer. activity in vitro and suppressed YAP-driven tumorigenesis in the For example, CDK8 function requires its kinase activity, and several mouse liver (31). Subsequent studies further confirmed that VP is

Zhou et al. PNAS | vol. 115 | no. 29 | E6767 Downloaded by guest on September 26, 2021 Table 1. β-catenin guide sequences immunizing (phosphorylated) peptides (1 μg/mL for 1 h at room temperature). The antibody (containing the phospho-peptide) was then used for staining in Oligo name Sequence parallel with staining using antibodies with no peptide or non-phospho-peptide. β-catenin-1A-Fwd CACCGGACGGTCGGACTCCCGCGG β-catenin-1A-Rev aaacCCGCGGGAGTCCGACCGTCC Cell Proliferation, Cell Migration (Wound Healing), and Colony Formation Assays. Colony formation assays in soft agar were performed as described β-catenin-1B-Fwd CACCGCGACCGTCCTCGACCTGCGG (60). Cells (5,000 per well) were seeded in six-well plates, and colonies were β-catenin-1B-Rev aaacCCGCAGGTCGAGGACGGTCGC counted by ImageJ online. For cell proliferation assays, cells (SW480 and β CACCGCGAGGACGGTCGGACTCCCG -catenin-2A-Fwd HCT116: 100,000 per well; RKO and DLD-1: 50,000 per well) were seeded in a β aaacCGGGAGTCCGACCGTCCTCGC -catenin-2A-Rev six-well plate in triplicate. Cells were counted by the automated cell counter β-catenin-2B-Fwd CACCGACCTGCGGTGGCGGCTCGG (Invitrogen) at indicated time points and proliferation curves were made β-catenin-2B-Rev aaacCCGAGCCGCCACCGCAGGTC based on the cell number in each well from at least three independent ex- periments. For wound healing (migration) assays, the cell monolayer was Fwd, forward; Rev, reverse. scratched by a P200 pipet tip when cells were confluent. Cells were washed three times with PBS and incubated with 2 mL of serum-free medium for an additional 24 h. The wound width was measured by QCapture Software (QImaging). also effective in inhibiting tumor growth in prostate cancer (50), uveal melanoma (51, 52), and colon cancer in vivo (53), suggesting that qRT-PCR. Total RNA isolation, RNA reverse transcription, and qRT-PCR were inhibiting YAP is a viable strategy in cancer treatment. Cyclin D1 is done as we have described (61). frequently deregulated in colon cancer and is a known target of β-catenin signaling (17, 54). Interestingly, Cyclin D1 was also induced IHC Staining and Scoring. The TMA slides consisted of 66 colon adenocarci- by YAP activation in the intestine (55) and was identified as a direct nomas and 35 normal adjacent tissues collected from colon cancer patients at target of YAP/TEAD in mesothelioma cells (56). Therefore, Cyclin the University of Nebraska Medical Center treated during 2008–2009. Slide D1isacommontargetofYAPandβ-catenin. Cyclin D1 is up- deparaffinization, antigen retrieving, and blocking were performed as we regulated (probably through YAP/β-catenin activation) in the have described (60). Tumors from RKO xenografts (control and Zyxin knock- Min/+ Min/+ Apc mice, and deletion of Cyclin D1 blocks/reduces Apc - down) were also used for Zyxin antibody validation. The sections were then driven adenoma formation (57). Acute deletion of Cyclin D1 in adult stained with anti-Zyxin antibody (at 1:100 dilutions; Cell Signaling Technology) ’ by using a Histostain-Plus IHC kit following the manufacturer’s instructions mice does not affect the animal s health (58). These observations (Invitrogen). The TMAs were also stained with anti-CDK8 antibody (IHC-00704, at suggest that targeting Cyclin D1 (e.g., with Palbociclib/PD-0332991, 1:300 dilutions; Bethyl Laboratory). Cell nuclei were stained with hematoxylin. which is a Food and Drug Administration-approved selective inhibitor Ventana iScan HT (Roche) was used for slide scanning with a 20× objective lens. of Cyclin D1–CDK4/6) would be a viable strategy in colon cancer, The staining results were independently evaluated by three researchers, in- especially in CDK8 and YAP/β-catenin–activated subtypes. cluding one pathologist (S.M.L.). The staining intensity (a scale of 0–3 was used: 0, negative; 1, weak; 2, moderate; and 3, strong) was scored (62). Materials and Methods CRISPR/Double Nickase-Mediated KO. To construct the all-in-one CRISPR/Cas9n Animal Studies. For in vivo xenograft studies, RKO cells (shControl, shZyxin, (double nickase) plasmid targeting β-catenin, the sense and antisense oli- shZyxin with Res-Zyxin-WT, or Res-Zyxin-3A) (1.0 × 106 cells each injection, gonucleotides from Table 1 were synthesized, annealed, and Golden Gate mixed with Matrigel at 1:1 ratio) were s.c. injected into the left or right flank assembled into the pX330A_D10A-1 × 2-EGFP (38) and pX330S-2 vectors as of 6-wk-old male athymic nude mice (Nude-Foxn1nu; Envigo). Five animals described (59). After the two vectors were generated, a final Golden Gate (10 injections) were used per group. Similar approaches were used for DLD-1 assembly was performed to generate the all-in-one vector as described (59). (1.0 × 106 cells per injection) and SW480 (2.5 × 106 cells per injection) cells in The resulting pX330A_D10A-1 × 2-EGFP–β-catenin-1 or -2 construct was Fig. 7. Tumor sizes were measured at the indicated time points. Tumor transfected into cells, and GFP-positive clones were selected by flow volume (V) was calculated by the formula: V = 0.5 × length × width2 (60). cytometry-based cell sorting. CDK8 and YAP CRISPR/double nickase KO Mice were euthanized at the end of the experiments, and the tumors were plasmids were purchased from Santa Cruz Biotechnology, and transfection excised for subsequent analysis. The animals were housed in pathogen-free and clone selection were done as instructed by the manufacturer. facilities. All animal experiments were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. Phos-Tag and Western Blot Analysis. Phos-tag was obtained from Wako Pure Chemical Industries, Ltd. (catalog no. 304-93521) and used at a concentration Statistical Analysis. Statistical significance was evaluated by using a two- μ μ of 20 M (with 100 M MnCl2) in 8% SDS-acrylamide gels. Before trans- tailed, unpaired Student’s t test. The Wilcoxon rank sum test was used to ferring, the gels were equilibrated in transfer buffer containing 10 mM compare the IHC staining data between groups. A P value of <0.05 was EDTA two times, each for 10 min. The gels were then soaked in transfer considered as indicating statistical significance. buffer (without EDTA) for another 10 min. Western blotting and lambda phosphatase treatment assays were done as described (20). ACKNOWLEDGMENTS. We thank Dr. Shian Wu (Nankai University, China) for the Zyxin shRNA constructs, Dr. Eek-hoon Jho (University of Seoul, Korea) Immunofluorescence Staining and Confocal Microscopy. Cell fixation, per- for the p-S128 YAP antibody, and Xiaolong Yang (Queen’s University, Can- meabilization, fluorescence staining, and microscopy were done as described ada) for the TetOn-shCDK1 cell line. We thank Dr. Wayne Xu (University of (29). The stained cells were mounted with Fluoromount (Vector Laborato- Manitoba, Canada) for analyzing the mRNA expression levels of Zyxin and ries) and visualized on an LSM710 Zeiss fluorescence microscope (Carl Zeiss). CDK8 from TCGA. We also thank Dr. Joyce Solheim for critical reading and comments on the manuscript. All fluorescence images were acquired by Zeiss The Slidebook 4.2 software (Intelligent Imaging Innovations) was used for LSM 710 or LSM 800 confocal microscopes at the Advanced Microscopy Core analyzing and processing all immunofluorescence images. F-actin was at the University of Nebraska Medical Center. The core is supported in part stained by using rhodamine-labeled phalloidin (Cytoskeleton). YAP cellular by National Institutes of Health (NIH) Grant P30 GM106397. Research in the localization was visualized by YAP immunofluorescence staining with an Alexa J.D. laboratory is supported by Fred and Pamela Buffett Cancer Center Sup- Fluor 647-conjugated YAP antibody (Cell Signaling Technology). For peptide port Grant P30 CA036727; NIH Grants P30 GM106397 and R01 GM109066; blocking, the phospho-Zyxin antibodies were first neutralized by an excess of and Department of Defense Health Program Grant W81XWH-14-1-0150.

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