Identification of Cdk1–LATS–Pin1 As a Novel Signaling Axis in Anti

Published OnlineFirst March 9, 2018; DOI: 10.1158/1541-7786.MCR-17-0684

Signal Transduction Molecular Cancer Research Identiﬁcation of Cdk1–LATS–Pin1 as a Novel Signaling Axis in Anti-tubulin Drug Response of Cancer Cells Benjamin Yeung1, Prem Khanal1, Virja Mehta2, Laura Trinkle-Mulcahy2, and Xiaolong Yang1

Abstract

The Hippo pathway is a signaling cascade that plays impor- anti-tubulin drugs activated cyclin-dependent kinase 1 (CDK1), tant roles in organ size control, tumorigenesis, metastasis, stress which phosphorylates LATS2 at ﬁve S/T-P motifs that function- response, stem cell differentiation, and renewal during devel- ally interact with the WW domain of Pin1 and inhibit its opment and tissue homeostasis and mechanotransduction. antiapoptotic function. Thus, these data identify Cdk1 and Recently, it has been observed that loss of the Hippo pathway Pin1 as a novel upstream regulator and downstream mediator, core component LATS (large tumor suppressor) or overexpres- respectively, of LATS in antitubulin drug response. Further sion of its downstream targets YAP and its paralog TAZ causes studies on this novel Cdk1–LATS–Pin1 signaling axis will be resistance of cancer cells to anti-tubulin drugs. However, YAP important for understanding the molecular mechanisms of and TAZ mediates anti-tubulin drug-induced apoptosis inde- drug resistance and will provide useful information for target- pendent of its upstream regulator LATS and the Hippo path- ing of this pathway in the future. way. Thus, the underlying molecular mechanism of how LATS is involved in the anti-tubulin drug response remains Implications: This study provides new insight on the molecular unknown. Proteomic approaches, SILAC and BioID, were used mechanism of anti-tubulin drug resistance and suggests novel to identify the isomerase Pin1 as a novel LATS-interacting therapeutic targets for drug-resistant cancers. Mol Cancer Res; 1–11. protein after anti-tubulin drug treatment. Treatment with 2018 AACR.

Introduction signaling networks controlling drug response is of critical impor- tance in advancing current cancer therapies. Anti-tubulin drugs, which include taxanes and vinca alkaloids, The Hippo pathway is an emerging signaling pathway that have been a part of the cancer pharmacopoeia for decades and plays critical roles in organ size control, tumorigenesis, metas- are one of the most successful classes of chemotherapeutics to date tasis, stem cell differentiation and renewal, mechanotransduc- (1, 2). By inhibiting the polymerization dynamics of microtu- tion (4–7). Recent studies have implicated the Hippo pathway bules, anti-tubulin drugs are potent mitotic poisons that impede in playing an important role in chemotherapeutic resistance normal function of the mitotic spindle, leading to extended (8–10). Classic mammalian Hippo signaling consists of a core mitotic arrest and cell death (3). Cancer cells promote prolifer- kinase cascade in which Mst1/2 (mammalian Ste20-like 1/2) ation and invasion by means of an overactive cell cycle and serine/threonine (S/T) kinases phosphorylate and activate increased mitotic division, thus making them particularly suscep- LATS1/2 (large tumor suppressor 1/2) kinases, which subse- tible to microtubule disruption. Although anti-tubulin drugs have quently phosphorylates and inhibits transcriptional coactiva- proven to be clinically efﬁcacious, predicting patient response is tor/corepressors YAP (Yes-associated protein) and its paralog often difﬁcult and the development of drug-resistant tumor cells TAZ (transcriptional coactivator with PDZ-binding motif), pre- leading to relapse/patient mortality continues to be a major venting their translocation fromcytoplasmtonucleustobind challenge to successful treatment (1). Therefore, identifying novel TEAD family of transcription factors and induce expression of genes that promote cell proliferation, survival, differentiation, and migration (4, 11). Importantly this protein cassette inte- grates numerous upstream signals and translates them to 1Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada. 2Department of Cellular and Molecular Medicine, downstream effectors while simultaneously cross-talking with University of Ottawa, Ottawa, Canada. other signaling pathways to maintain homeostasis and prevents tumorigenesis (4, 11). Unsurprisingly, dysregulation of the Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Hippo pathway, especially the well-established tumor suppressor genes LATS1/2, can lead to various human cancers (5, 12). B. Yeung and P. Khanal contributed equally to this article. Recently, we and others have found that many of the core Corresponding Author: Xiaolong Yang, Queen's University, 88 Stuart Street, components or upstream regulators of the Hippo pathway (e.g., Kingston, Ontario K7L3N6, Canada. Phone: 613-533-6000, ext.75998; Fax: 613- RASSF1A, Expanded, MST, Ajuba, YAP/TAZ, and Vgll4) play 533-2970; E-mail: [email protected] pivotal roles in the resistance of cancer cells to chemothera- doi: 10.1158/1541-7786.MCR-17-0684 peutic drugs [e.g., taxol, doxorubicin, cisplatin, 5-FU, and 2018 American Association for Cancer Research. EGFR, and MEK inhibitors; refs. 10, 11, 13–17].

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Interestingly, studies by our laboratory and others showed that doxycycline for about 10 days, with media being changed every LATS plays a pivotal role in anti-tubulin drug resistance. Loss of 2–3 days and passed onto larger plates. A total of 1 106 cells LATS1/2 by siRNA knockdown renders HeLa cervical cancer cells were passed onto each 150-mm plate (five for control and resistant to taxol-induced cell death (18). In addition, LATS1 was ten each for drug-treated) and grown to approximately 40%– identified in a functional genomic screen looking for genes 50% confluence. Media were changed containing 50 mmol/L involved in taxol sensitivity of A549 lung cancer cells (19). biotin, 2 mg/mL doxycycline and either DMSO, 100 nmol/L However, although we and others found that YAP and TAZ are taxol, or 1 mg/mL nocodazole and cultured for 24 hours. also important in anti-tubulin drug response, this was indepen- Adherent (control) or floating (drug-treated) cells were har- dent of LATS1/2 or Hippo pathway regulation, suggesting that vested and protein was extracted using modified RIPA buffer YAP and TAZ are not the proteins mediating MST1/2 or LATS1/2 containing proteinase inhibitors/benzonase and sonicated. function in anti-tubulin drug response (8, 9, 20). Moreover, there Supernatants were checked for expression of LATS2 by Western is no change on LATS1 phosphorylation status on MST phos- blot analysis and protein concentration measured by Bio-Rad phorylation site T1089 after taxol treatment (ref. 8 and data not DC protein assay. Twenty micrograms of lysate was used for shown), suggesting that MST1/2 is not the kinase regulating LATS overnight pulldown at 4Cusing30mLofstreptavidin-sephar- in response to anti-tubulin drugs. Therefore, the upstream regu- ose beads, which were then washed 2 with modified RIPA lator(s) or downstream mediator(s) of LATS1/2 in anti-tubulin buffer, 2 with TAP lysis buffer (50 mmol/L Hepes-KOH, pH drug response remain unknown. 8.0, 100 mmol/L KCl, 10% glycerol, 2 mmol/L EDTA, and 0.1% In this study, we have identified Cdk1 and Peptidyl-prolyl NP-40), and 3 with 25 mmol/L ammonium bicarbonate (pH isomerase Pin1 as novel upstream regulator and downstream 8.0) and prepared for mass spectrometry analysis. mediator of LATS1/2, respectively, in response to anti-tubulin Proteomics analysis was performed at the Ottawa Hospital drugs. This novel Cdk1–LATS–Pin1 signaling axis may play Research Institute Proteomics Core Facility (Ottawa, Canada). important roles in anti-tubulin drug resistance and represent a Proteins were digested in-gel using trypsin (Promega) according novel target for drug-resistant cancer therapy in the future. to the method of Shevchenko (22). Peptide extracts were con- centrated by Vacufuge (Eppendorf). LC/MS-MS was performed Materials and Methods using a Dionex Ultimate 3000 RLSC nano HPLC (Thermo Scien- tific) and Orbitrap Fusion Lumos mass spectrometer (Thermo Plasmid construction and site-directed mutagenesis Scientific). Database searching (against the human Uniprot data- Plasmid construction and site-directed mutagenesis were per- base) and quantitation were performed using MaxQuant software formed as described in detail previously (21). Human PPIA, PPIB, v1.2.7.4.14. The following criteria were used: peptide tolerance ¼ and Pin1 cDNAs (obtained from Addgene) were subcloned into 10 ppm, trypsin as the enzyme (two missed cleavages allowed), pcDNA3.1-HA and pGEX4T1 vectors. Inducible TRIPZ-lentiviral and carboxyamidomethylation of cysteine as a fixed modifica- shRNA vector (Dharmacon) was modified by cloning of human tion. Variable modifications were oxidation of methionine and N- LATS2, Pin1, or BirA-R118G cDNAs (Addgene) into AgeI and MluI terminal acetylation. The heavy SILAC label was Lys6 (K6). site of TRIPZ vector. Minimum ratio count was 2, and quantitation was based on Cell culture and treatment of cells by drugs and transfection razor and unique peptides. Peptide and protein FDR was 0.01. Cos7 monkey embryonic kidney cells, HeLa cervical carcinoma, siRNA-mediated gene expression knockdown and H1299 lung carcinoma cell lines were purchased from the On-Target SMARTpool siRNA for Cyclin B1 along with an siRNA ATCC and cultured as instructed by ATCC. The cells were pur- with scrambled sequence (siControl) were purchased from Dhar- chased over 10 years ago. Passages 30–50 were normally used in macon. HeLa cells were transfected with 50 nmol/L of siRNAs our experiments. No authentication and Mycoplasma tests have using RNAiMax (Invitrogen) according to manufacturer's instruc- been performed. The cells were normally passed 1:10 and cultured tions. Two days posttransfection, cells were treated with 1 mg/mL less than 2 months. HeLa and H1299 cells were treated with anti- nocodazole or 100 nmol/L Taxol for 24 hours, followed by tubulin drugs and kinase inhibitors as described previously. protein extraction and Western blot analysis. Transient transfection of expression constructs was performed using PolyJet in vitro transfection reagent (SignaGen Laboratories) according to the manufacturer's protocol. GST fusion protein production, pull-down, and in vitro kinase assays Lentivirus production and establishment of stable cell lines GST and GST fusion proteins were produced and purified Lentivirus production and purification were performed as as previously described (21). For GST-pulldown assays, about described previously (8). H1299 cells stably expressing TRIPZ- 100–200 mg of each respective protein lysate was precleared LATS2-WT, -LATS2-5xA, or -BirA-LATS2-WT were established by overnight at 4 C with 20 mL glutathione sepharose 4B beads infection with lentivirus and subsequent selection with 1 mg/mL (GSB; GE Healthcare). Supernatants were transferred and mixed puromycin. For WPI-vector or -Pin1-WT overexpression, H1299 with 10 mg of appropriate GST fusion protein and incubated at TRIPZ-LATS2-WT or -5xA cells were infected again and expression 4 C for 2 hours. Twenty microliters of GSB was then added and was examined by Western blot analysis using anti-LATS2 or -Pin1 further incubated for another 1 hour. Beads were then washed antibodies. four times with lysis buffer (50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA and 1.0% Nonidet P-40), resus- SILAC labeling, BioID purification, and MS data analysis pended in 2 SDS sample buffer, boiled for 10 minutes, and SILAC Protein Quantitation kit was used and purchased from centrifuged. Resultant supernatants were subjected to SDS-PAGE Pierce. HeLa N-BirA-LATS2-TRIPZ cells were cultured in light or and Western blotting as described previously. For in vitro kinase 13 heavy (containing C6-L-Lysine-2HCl) media with 2 mg/mL assays, 20 U of Cdk1–cyclinB kinase (New England Biolabs,

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P6020S) and 1 mg of fusion protein were mixed and incubated at similarities of LATS1 and LATS2, we mainly focused the following 30C for 20 minutes. Phosphorylated proteins were alkylated by characterization study on LATS2, as its roles in anti-tubulin drug adding p-nitrobenzyl mesylate (final concentration 2.5 mmol/L) response are unclear. Similar to endogenous LATS2, we also found and incubated at room temperature for 2 hours. Samples were that FLAG-tagged LATS2 transfected into HeLa cells produced an prepared for Western blot analysis as described previously; phos- upshift upon treatment with various anti-tubulin drugs (Fig. 1B), phorylated proteins were detected by anti-thiophosphate ester 51- suggesting that the band-shift observed in HeLa cells is specificto 8 antibody (Abcam). LATS2. We further confirmed that the LATS2 mobility shift was due to phosphorylation as the band-shift can be reversed after Antibodies, Phos-tag and Western blot analysis, and treatment with lambda phosphatase (Fig. 1C). As we and others coimmunoprecipitation previously found that Cdk1 is a kinase that can complex with Anti-LATS2 antibody was from Bethyl Laboratories. LATS1, Cyclin B to phosphorylate other Hippo components under anti- phospho-Ser909 LATS1, phospho-Ser127-YAP, phospho-Tyr15- tubulin drug treatment (8–10, 20), we asked whether LATS2 was Cdk1 antibodies were from Cell Signaling Technology. Anti-Pin1, also phosphorylated under such control. Indeed, pretreatment HA F7 (for co-IP) and Cyclin B1 antibodies were from Santa Cruz with Cdk1 inhibitor RO3306 or transient knockdown of Cyclin B b Biotechnology. FLAG (M2) and -actin antibodies were from by siRNA was sufficient to reverse LATS2 phosphorylation under Sigma. Cdk1 and TAZ antibodies were from BD Transduction various anti-tubulin drug conditions (Fig. 1D and E). Finally, we Laboratories. Antibodies against phospho-Ser/Thr (MPM2) were tested whether LATS2 phosphorylation is specific for HeLa cells from Millipore and thiophosphate ester (51-8) from Abcam. and found that LATS2 is phosphorylated in a wide variety of Phos-tag was obtained from Wako Pure Chemical Industries human cancer cells after nocodazole or Taxol treatment (Supple- m m – and used at 40 mol/L along with 100 mol/L MnCl2 in 6% mentary Fig. S2). Taken together, these data strongly suggest that 8% SDS-acrylamide gels. Western blot analysis, coimmunopre- LATS2 can be phosphorylated by Cdk1 in apoptotic cells in a wide cipitation, and phosphatase treatment were done as described variety of cell types after treatment with anti-tubulin drugs. previously (8). Identification of LATS2 phosphorylation sites Analysis of apoptosis by caspase-3 activity measurement and To identify the phosphorylation site(s) on LATS2, we first Trypan blue exclusion assays performed in vitro kinase assays using purified Cdk1/Cyclin B as H1299 TRIPZ-LATS2-WT or -5xA cells were pretreated with or kinase and GST-fusion proteins expressing different regions of m without 2 g/mL doxycycline for 2 days prior to be seeded in LATS2 as substrates. All of the LATS2 deletion fusion proteins, triplicate for treatment with 100 nmol/L Taxol for 24 hours. excluding the 601–800 region, were significantly phosphory- Caspase-3 activity was assayed using the Caspase-Glo 3/7 kit lated by Cdk1/Cyclin B, suggesting that there are at least three (Promega, G8090) and measured by luminometer (Turner distinct Cdk1 phosphorylation sites in LATS2 (Fig. 2A). As BioSystems). Absolute RLU values were then normalized to Cdk1 is a proline (P)-directed S/T kinase, we scanned the protein concentration using Bio-Rad DC Protein Assay. Measure- sequence of each phosphorylated LATS2 fragment (1–200, ment of cell death/apoptosis by Trypan blue exclusion analysis 201–400, 401–600, and 801–1089) for SP or TP motifs and fl was as described previously (15). Brie y, H1299-stable lines were found fifteen potential Cdk1 sites (Fig. 2B). We constructed m pretreated with 2 g/mL doxycycline for 2 days before being plasmids expressing various deletions containing either conse- 3 seeded into 12-well plates in triplicate (2 10 cells/well). The cutive or point mutations of LATS2 (Fig. 2C–F), transfected next day, media was changed that contained DMSO or appropri- them into HeLa cells, and treated with Taxol for 24 hours. Using ate concentrations of Taxol and incubated for 24 hours. Alive and Phos-tag gels, we identified five potential Cdk1-phosphoryla- dead cells were collected and assayed as described before. The tion sites that are necessary for Taxol-induced mobility shift: – experiments were repeated 2 3 times and mean and SD of each S157, S342, T349, S598 and S1027 (Fig. 2B–F). Mutation of experiment were presented. these five sites (termed LATS2-5xA) completely abolished the mobility shift of LATS2 after nocodazole or Taxol treatment Results (Fig. 2G). Similar to LATS2, LATS1 is also phosphorylated by LATS2 is phosphorylated after anti-tubulin drug treatment Cdk1 (Supplementary Fig. S3). In summary, we identified five To further explore how LATS1/2 is involved in drug response, S/T-P motif phosphorylation sites that are responsible for we treated HeLa cells with DMSO (vehicle control) or various anti- LATS2 band-shift after anti-tubulin drug treatment. tubulin drugs for 24 hours and collected attached (DMSO) or floating (mitotic apoptotic) cells. Interestingly, treatment of HeLa Identification of Pin1 as a novel LATS2-interacting protein after cells with anti-tubulin drugs causes a mobility shift for LATS1 but anti-tubulin drug treatment using BioID-SILAC not LATS2 on traditional SDS-PAGE gels (Fig. 1A; Supplementary Previous studies by our laboratory and others found that under Fig. S1). Thus, to test for the presence of differential migration of Taxol treatment, the main Hippo pathway effector YAP could no LATS2, we ran collected lysates on low percentage, Phos-tag gels, longer be phosphorylated and regulated by LATS (8, 20), prompt- which can separate phosphorylated from unphosphorylated pro- ing us to seek novel proteins mediating LATS function under these teins more effectively. Surprisingly, treatment with anti-tubulin conditions. We employed an approach that combined proximity- drugs caused dramatic retardation of endogenous LATS2 as com- dependent labelling (BioID) and quantitative proteomics meth- pared with control (Fig. 1A). We also noted that there was no od called SILAC (stable isotope labeling by amino acids in cell change in endogenous levels of YAP or its phosphorylation at culture), which has been applied successfully in studying a variety S127 (a known LATS regulatory site; ref. 21), further supporting of proteins and processes (23–25). While BioID relies on the the notion that LATS1/2 is involved in drug response through fusion of a protein of interest to a mutant form of biotin ligase YAP-independent means (Fig. 1A). Because of the functional BirA, which can promiscuously biotinylate proximal proteins

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Figure 1. Phosphorylation of LATS2 after anti-tubulin drug treatment. A, Western blot analysis of HeLa cells after 24 hr treatment with nocodazole (NOC, 1 mg/mL), Taxol (TX, 100 nmol/L), docetaxol (DTX, 100 nmol/L), vinblastine (VBL, 100 nmol/L), or colchicine (CLC, 100 nmol/L). Total cell lysates were probed with the indicated antibodies on 6% Phos-tag or regular SDS-PAGE gels. B, HeLa cells were transfected with FLAG-tagged LATS2 and treated/blotted as indicated in A. C, LATS2-FLAG transfected lysates from B were treated with or without l-phosphatase ( or þPPase). D, HeLa cells were transfected with LATS2-FLAG along with scramble or Cyclin B siRNA. Forty-eight hours posttransfection, cells were treated with TX or nocodazole as before and blotted with LATS2, and Cyclin B antibodies. E, HeLa cells were transfected with LATS2-FLAG and pretreated with DMSO or 10 mmol/L Cdk1 inhibitor RO3306 for 2 hours and subsequently treated by anti-tubulin drugs and blotted as in A. Protein lysates were run on normal or phos-tag SDS-PAGE gels as indicated. All b-actin controls were run on normal SDS-PAGE gel.

irrespective of whether they interact indirectly or transiently, LATS2 interacting proteins in large scale using the same procedure SILAC uses "heavy" and "light" amino acids to label proteins of (Fig. 3A). two populations and quantify differential abundance of each After mass spectrometry analysis, we identified candidate pro- peptides of digested proteins by mass spectrometry. By coupling teins that have enhanced binding to LATS2 after TX or nocodazole Bio-ID with SILAC, we could then identify novel anti-tubulin treatment (Supplementary Table S1). Most interestingly, we drug-induced LATS2-interacting proteins with high confidence. found various prolyl-isomerases (e.g., PPIB, PPIG and FKBP4) The experimental workflow is briefly summarized in Fig. 3A. First, scattered in our identified list of proteins. Although Pin1 and PPIA we generated stably expressing doxycycline-inducible N-terminal were not detected in our screen most likely due to lack of efficient BirA-LATS2 in HeLa cells using lentivirus (Fig. 3B); C-terminal trypsin digestion, they are also members of the cis-trans isomerase BirA-LATS2 cells were also generated but was found to impair family and were included in our analysis. To validate our findings, LATS2 activity (data not shown). Next, we validated the system by we performed coimmunoprecipitation (Co-IP) assays with HeLa a small-scale purification of known LATS2-interacting proteins. cells transfected with FLAG-tagged LATS2 and HA-tagged iso- We treated HeLa-N-BirA-LATS2 cells with biotin alone or together merases in the presence of DMSO or Taxol. Interestingly, higher with Taxol or nocodazole and subsequently performed pulldown levels of LATS2-FLAG were immunoprecipitated with PPIA-HA, with streptavidin agarose. Since BirA-LATS2 is able to biotinylate PPIB-HA, or Pin1-HA (with anti-HA antibody) in the presence of itself, we could detect LATS2 on the beads only when biotin was Taxol (Fig. 3D). We further tested whether enhanced levels of present (Fig. 3C). Furthermore, we probed for other known LATS2 these isomerases induce Taxol resistance by overexpressing them interacting proteins (21, 26) and found that TAZ specifically binds in HeLa cells. Consistent with a previous publication (27), only to LATS2 when Hela-N-BirA-LATS2 cells were treated with Dox to overexpression of Pin1 causes resistance of cancer cells to Taxol- induce LATS2 in the absence or presence of TX or nocodazole (Fig. induced apoptosis (Supplementary Fig. S4). Therefore, we decid- 3C and data not shown), suggesting that this BioID system ed to further explore how interaction of LATS2 with Pin1 is worked for LATS2. We then proceeded to systematically profile involved in Taxol response.

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Figure 2. Mapping of LATS2 phosphorylation sites. A, In vitro kinase assay using 100 ng of Cdk1-CyclinB kinase complex and 1 mgofeachpurified GST-LATS2 fragment fusion proteins. B–F, Schematic of S/T-P sites in each LATS2 region (B). The potential Cdk1 phosphorylation sites are indicated by asterisks. Cdk1- phosphorylation sites are mapped by transfecting HeLa cells with different FLAG-LATS2 fragments/mutations with subsequent treatment of 100 nmol/LTaxol (TX) for 24 hours. Collected lysates were then run on Phos-tag gels for each region: LATS2-1-200, -201-400, -401-600, and -801-1089 (in C–F respectively). The positions of identified Cdk1 phosphorylation sites are indicated as framed numbers in B. G, Mutation of the identified five Cdk1 phosphorylation sites in HeLa cells transfected with LATS2 (5xA) abolishes mobility shift after 24-hour nocodazole (1 mg/mL) or Taxol (100 nmol/L) treatment. All protein lysates were run on phos-tag SDS-PAGE.

Interaction of LATS and Pin1 in vitro and in vivo were immunoprecipitated with HA antibody, LATS1 and LATS2 To test whether the interaction between LATS and Pin1 was not was found in the immune complex confirming its interaction (Fig. cell type specific, we performed binding assays using other cell 4C and D). We were unable to confirm the endogenous interac- lines. In addition, as LATS1 and LATS2 have similar function, we tion of LATS and Pin1. Nevertheless, as Pin1 binds to known also tested whether Pin1 also interact with LATS1. FLAG-tagged substrates via its WW domain, we asked whether this was also LATS1 or LATS2 expression vectors were transfected into Cos7 necessary for LATS1/2 interaction. To map the domains respon- cells and GST pull-down assays were used to assess direct inter- sible for Pin1 binding, we made expression vectors containing action in vitro. As expected, both LATS1 and LATS2 proteins were either the Pin1-WW and PPIase domain. When GST pull-down able to bind Pin1-GST (Fig. 4A and B). Next, we used co-IP assays assays were performed with cell lysates expressing FLAG-tagged to examine whether either LATS proteins interacted with Pin1 in LATS1 or LATS2 and Pin1 GST fusion proteins purified from vivo. FLAG-tagged LATS1 or -LATS2 and HA-tagged Pin1 were bacteria, the WW rather than PPIase domain of Pin1 was found to transfected alone or together into HEK293 cells. When cell lysates bind with LATS1/2 (Fig. 4E and F). This was further verified with

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Figure 3. Identiﬁcation of Pin1 as a LATS2- interacting protein after anti-tubulin drugs treatment by BioID-SILAC. A, Experimental workﬂow for BioID- SILAC in HeLa-BirA-LATS2 cells. B, Western blot analysis of LATS2 and b-actin in HeLa-BirA-LATS2 cells that were treated with (þ) or without () 2 mg/mL doxycycline for 2 days. C, HeLa-BirA-LATS2 cells from B were treated 50 mmol/L biotin and 100 nmol/L Taxol (TX) or 1 mg/mL nocodazole for 24 hours. After protein extraction, lysates were precleared and streptavidin-agarose beads were added for pulldown of biotinylated proteins, then eluted for detection by Western blot analysis with LATS2 or TAZ antibodies. D, HeLa cells were cotransfected with FLAG-LATS2 and HA-PPIA, -PPIB, or Pin1 and treated with 100 nmol/L Taxol (TX) for 24 hours 1-day posttransfection. Collected lysates were then immunoprecipitated with anti-HA antibody and agarose A beads. Pulldown proteins were eluted and subsequently detected by anti-FLAG antibody on Western blot analysis.

co-IP assays using lysates that were transfected with FLAG-tagged inhibitor) completely prevented the detection of FLAG-LATS2 LATS1 or -LATS2 and either HA-tagged Pin1-WT, WW, or PPIase after GST-pulldown with Pin1, despite treatment with anti-tubu- alone or together (Fig. 4G and H). In summary, these experiments lin drugs (Fig. 5E), suggesting that phosphorylation of LATS by confirm that Pin1 can bind to both LATS1 and LATS2 through its Cdk1 is essential for its interaction with Pin 1. Finally, we tested WW domain. the effect of the identified Cdk1-phosphorylation sites of LATS2 (Fig. 2B and G). Mutation of the five sites completely abolished Phosphorylation-dependent interaction of LATS2 and Pin1 LATS2 binding (FLAG-LATS2-WT vs. -5xA) to Pin1 by GST-pull- As the WW domain of Pin1 specifically recognizes phospho-S/ down assay (Fig. 5F). These results suggest that treatment with T-P motifs (28) and LATS2 is phosphorylated by Cdk1 after anti- anti-tubulin drugs promotes Cdk1 phosphorylation of LATS2 and tubulin drug treatment (Figs. 1 and 2), we tested whether the is necessary for priming it for recognition and binding to Pin1. binding between Pin1 and LATS is dependent on the phosphorylation status of LATS in the absence (endogenous CDK1 phos- Characterization of Cdk1–LATS–Pin1 signaling axis in anti- phorylation of LATS) of anti-tubulin drug. Treatment of calf tubulin drug-induced apoptosis intestinal phosphatase (CIP) on HEK293 lysates transfected with We next characterized the functional significance of Cdk1– FLAG-tagged LATS1 or LATS2 greatly reduced binding between LATS–Pin1 signaling axis. First, as LATS2 has roles in promoting LATS and GST-Pin1 according to GST pull-down assays (Fig. 5A apoptosis and drug sensitivity (5, 11, 18, 29), we examined the and B). Binding was not completely abolished due to residual effects of Cdk1 phosphorylation site mutations of LATS2 (5xA) LATS2 phosphorylation, as detected by anti-pS/T-P antibody (Fig. on its functions. We established H1299 lung cancer cell line 5A and B). Next, we tested whether lysates from HeLa cells treated (low endogenous LATS2) stably expressing doxycycline-inducible with various anti-tubulin drugs for 24 hours would increase LATS2-WT or -5xA (Fig. 6A) and tested the effects of 5xA interaction. In accordance, binding between FLAG-tagged LATS1 on LATS function. Using a caspase-3 activity assay and Taxol- or LATS2 with GST-Pin1 was markedly higher in the anti-tubulin treated conditions, LATS2-WT overexpression greatly increased drug treated samples as compared with DMSO control (Fig. 5C caspase-3 activity, as opposed to LATS2-5xA which saw approx- and D). Most significantly, pretreatment with RO3306 (Cdk1 imately half the level of activation (Fig. 6B). In addition, we

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Figure 4. Interaction of LATS1/2 and Pin1 in vitro and in vivo. A, B, GST-pulldown assays between Pin1 and LATS1 (A) or LATS2 (B). About 200 mg of protein lysates from COS7 cells expressing FLAG-tagged LATS1 or LATS2 was mixed with 10 mg of GST or Pin1-GST on beads. After washing and elution, binding proteins were subjected to Western blotting by anti-FLAG antibody. 1/10 input (20 mg) represents 1/10 of protein lysate used for pulldown. Ponceau S was used to stain for fusion proteins on the membrane. C and D, Coimmunoprecipitation assays between HA-Pin1 and FLAG-tagged LATS1 (C) or LATS2 (D). HEK293 cells were transfected alone or in combination with HA-Pin1, FLAG-LATS1, or –LATS2 and immunoprecipitated with anti-HA antibody, followed by Western blot analysis using anti-FLAG antibody to probe for binding. Expression levels of Pin1/LATS in cell lysates were also examined. E–H, Identiﬁcation of functional domains of Pin1 interacting with LATS1 or LATS2. GST-pulldown assays using GST or GST-Pin1-WT, -PPIase, or –WW domain fusion proteins to FLAG-LATS1 (E) or FLAG-LATS2 (F) COS7 lysates. Co-IP assays of HEK293 cells transfected with HA-Pin1-WT, -PPIase, or –WW in combination with FLAG-tagged LATS1 (G) or LATS2 (H). previously reported that siRNA knockdown of LATS1/2 can cause suggest that mutation of the ﬁve Cdk1-phosphorylation sites of resistance to Taxol treatment in HeLa cells (18). Interestingly, LATS2 greatly impairs its antitumorigenic functions and Taxol overexpression of LATS2-WT in H1299 sensitized the cells sensitivity. through a range of Taxol treatment, an effect that was lost in As Pin1 is known to regulate other tumor suppressor genes by LATS2-5xA overexpression cells (Fig. 6C). These results strongly binding to pS/P-T motifs (27, 28, 30), we examined the functional

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Figure 5. Phosphorylation-dependent interaction of LATS1/2 and Pin1. A and B, GST-pulldown assays using GST or GST-Pin1 mixed with HEK293 lysates expressing FLAG-LATS1 (A)or –LATS2 (B) with (þ) or without () treatment of calf intestinal phosphatase (CIP). Ponceau S was used to stain for equal loading of fusion proteins. Input lysates (10 mg) were probed with phospho-S/T antibody to check for CIP treatment. C and D, HeLa cells expressing FLAG-LATS1 (C)or–LATS2 (D) were treated with DMSO or anti-tubulin drugs as described in Fig. 1A and mixed with GST or GST-Pin1 for binding assay as in Fig. 4B. Expression levels of input were veriﬁed by Western blot analysis using FLAG-antibody and Ponceau S staining for fusion proteins. E, GST-pulldown assays of GST or Pin1-GST mixed with LATS2-FLAG– transfected HeLa cell lysates pretreated with Cdk1-inhibitor RO3306 for 2 hours and subsequently with anti-tubulin drugs (as in Fig. 1E). F, HeLa cells transfected with FLAG-LATS2-WT or -5xA were treated with DMSO or 100 nmol/L Taxol for 24 hours. After cell collection and protein extraction, lysates were mixed with Pin1-GST in pulldown assay.

consequences of overexpression of both LATS2 and Pin1. From 6E). Collectively, these ﬁndings indicate that Taxol induces apo- previously established H1299 TRIPZ-LATS2-WT cells, we stably ptosis through regulation of Cdk1–LATS1/2–Pin1 signaling axis expressed WPI vector control or Pin1-WT by infection with (Fig. 6F). lentivirus (Fig. 6D). As expected, overexpression of LATS2-WT rather than LATS2-5xSA alone sensitized cells to Taxol treatment, whereas overexpression of Pin1 caused resistance of cells to Taxol Discussion (Fig. 6E). Most signiﬁcantly, overexpression of LATS2-WT rather LATS is a well-characterized major tumor suppressor, whose than LATS2-5xSA inhibited Pin1-induced Taxol resistance (Fig. dysregulation can lead to various types of human cancers.

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Cdk1–LATS–Pin1 Signaling Mediates Anti-tubulin Drug Response

Figure 6. Functional interaction of LATS2 and Pin1 in anti-tubulin drug response. A, Western blot analysis of doxycycline (Dox)-inducible overexpression of LATS2-WT and -5xA in H1299 cells with (þ) or without () 2 days of 2 mg/mL doxycycline treatment. B, H1299 cell lines from A were treated with 100 nmol/L Taxol treatment for 24 hours. Collected lysates were measured for caspase-3 activity in triplicate using luminometer and normalized to untreated control. C, Trypan blue exclusion analysis for cell death of H1299 cell lines from A after 24-hour treatment with DMSO or 10–50 nmol/L Taxol. Alive and dead cells were collected in triplicate and counted as described previously. D, Western blot analysis of lysates from H1299 cells overexpressing WPI-vector or –Pin1-WT and Dox-inducible LATS2-WT or -5xA. E, Cells established from D were treated with 50 nmol/L Taxol for 24 hours and subject to Trypan blue dye exclusion assay as described in C. F, A model for Cdk1-LATS2-Pin1 signaling axis. , P < 0.05 (t test).

Although tremendous progress has been made in our understand- conditions that trigger the Hippo pathway, cAMP-dependent ing of how LATS controls organism homeostasis and prevents protein kinase (PKA) was found to phosphorylate LATS2 at cancer formation, its role in drug response, particularly to anti- S598 (along with three other different sites), an event that induces tubulin drugs, is not well defined. Our studies provide the first full activation of LATS2 activity (31). This could partially explain evidence that LATS can be specifically phosphorylated by Cdk1 why enhanced phosphorylation of S598 on LATS2 by Cdk1 independent of the upstream Hippo (MST1/2) signaling pathway. activates LATS2 function, whereas mutation to alanine reduced Significantly, we identified five sites (S157, S342, T349, S598, and LATS2 function, as revealed in our functional assays (Fig. 6B and S1027) that are responsible for enhanced LATS2 phosphorylation C), a mechanism similar to the Mst1/2 phosphorylation and by Cdk1 after anti-tubulin drug treatment (Fig. 2G). Interestingly, autophosphorylation sites on LATS2 (5). Aside from S598, the out of the five Cdk1 phosphorylation sites, we identified on other 4 sites are also phosphorylated by Cdk1 and the functional LATS2, only one has previously been reported (31). Under known significance of these sites is unclear. Therefore, it will be very

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Yeung et al.

interesting to further study how each individual phosphorylation mutation of these sites not only abolishes its interaction with Pin1 site affects both the LATS2 activity and function. Moreover, as but also significantly reduces its activation of anti-tubulin drug- reduced expression of LATS1/2 causes anti-tubulin drug resistance induced apoptosis (Fig. 6E). Most significantly, we found that (18) and mutation of Cdk1 phosphorylation sites (5xSA) signif- LATS2-WT rather than LATS2-5xA mutant lacking interaction with icantly reduces its activation of Taxol-induced cell death, it will be Pin1 is able to inhibit Pin1-induced anti-tubulin drug resistance also interesting to further examine the status of LATS2 phosphor- (Fig. 6E). The molecular mechanism by which LATS inhibits Pin1 ylation in anti-tubulin drug-resistant cells and tissues to see function is unknown. It has been previously shown that Pin1 is whether reduced LATS2 phosphorylation may be responsible for phosphorylated on several serine residues by a number of distinct anti-tubulin drug resistance. kinases (e.g., S65 by Plk1, S138 by MLK3, and S71 by DAPK, Previous studies suggest that phosphorylation of Hippo effec- respectively) that regulate Pin1 stability, substrate binding capa- tors YAP and its paralog TAZ by Cdk1 is important for both bility or isomerase activity (38 –40). Therefore, it is possible that normal mitotic progression during cell division and apoptosis LATS may inhibit Pin1 by phosphorylating Pin1. Intriguingly we induced by anti-tubulin drugs (8, 9, 20, 32). While moderate found that purified LATS2 is able to phosphorylate Pin1 in vitro in activation of Cdk1 during mitosis induces YAP/TAZ phosphory- the PPIase region (Supplementary Fig. S6) even though it does not lation/activation and subsequent mitosis progression, hyperacti- contain a consensus LATS phosphorylation motif. Future study to vation of Cdk1 by anti-tubulin drugs significantly inactivates their determine whether this phosphorylation occurs in vivo and its functions and causes apoptosis. Therefore, YAP/TAZ phosphory- potential functional consequences need to be examined. Our data lation levels control the balance between cell proliferation and also showed that LATS can interact with YAP/TAZ in the absence or cell death (8, 9, 20, 32). Therefore, it is also possible that LATS can presence of Taxol (data not shown). Therefore, it is possible that be phosphorylated on these 5 S/T sites during mitosis and play phosphorylation of LATS1/2 by Cdk1 causes conformational important role in cell proliferation. Consistent with this state- changes to LATS1/2 and switches their preferred substrates from ment, LATS1 has already been shown to be phosphorylated YAP/TAZ to Pin1. Moreover, it is also possible that Pin1 can during mitosis (33–35) and low levels of endogenous LATS2 inhibit LATS function as a negative feedback mechanism (Fig. 6F). phosphorylation/shift is also detected without drug treatment As Pin1 is a cis-trans isomerase and modulate proteins by changing (Fig. 1A). In addition, we also found that mutation of Cdk1 its conformation, it will be interesting to further explore whether phosphorylation sites on LATS2 (LATS2-5xA) significantly Pin1 regulates LATS in various biological functions. reduced LATS2-induced suppression of transformation in This study highlights a previously unrevealed mechanism H1299 lung cancer cells (Supplementary Fig. S5). Moreover, Pin1 through which LATS2 is phosphorylated and activated by Cdk1 was originally identified as a mitotic protein (27, 36). Therefore, in response to anti-tubulin drugs. Interestingly, we and others further elucidation of the roles of Cdk1–LATS–Pin1 signaling axis have previously shown that other core and regulatory Hippo in mitosis, cell proliferation, and transformation will be very pathway components (e.g., YAP, TAZ, Ajuba, Kibra, and Vgll4) significant in our understanding the molecular mechanism of are also phosphorylated by Cdk1 under various contexts. As Pin1 mitosis and its roles in cancer. recognizes pS/T-P phosphoproteins and is ubiquitously As our previous studies indicate that YAP/TAZ is not involved in expressed, it is likely that Pin1 could bind and modulate the anti-tubulin drug response downstream of LATS (8, 9), identifi- activity of these Hippo components, potentially the pathway as a cation of novel targets mediating LATS function is required to whole. Indeed, Pin1 has already been shown to be intricately elucidate the molecular mechanism underlying LATS-involved involved in other phosphorylation-dependent networks such as drug response. By performing a BioID-SILAC proteomic screen, mitosis, acting as an added layer of regulatory complexity (28, we have identified several proteins and validated several iso- 30). Further investigation into how Pin1 may affect other Hippo merases with enhanced interactions with LATS2 after anti-tubulin pathway components will provide insights into the underlying drug treatment. We have further characterized Pin1 as a critical mechanisms in cancer development and treatment. binding partner and downstream target of LATS. Pin1 is a pepti- Taken together, we have discovered a novel Cdk1–LATS–Pin1 dyl-prolyl cis/trans isomerase that interacts with numerous onco- signaling axis mediating anti-tubulin drug response in cancer genic or tumor suppressive phosphorylated proteins, causes con- cells. Further exploration of this signaling in the development of formational changes in target proteins, and eventually regulates cancer and drug resistance and targeting of this axis will have the activities of such proteins (27, 28, 30, 37). Pin1 binds to S/T-P significant implication for successful cancer therapy in the future. motif of its interacting protein through its WW domain. Over- expression and/or activated forms of Pin1 has been observed in a Disclosure of Potential Conflicts of Interest variety of cancers including breast, ovary, prostate, lung, gastric, No potential conflicts of interest were disclosed. cervical cancers, and melanoma (28, 37). Moreover, Pin1 activates numerous oncogenes and also inactivates tumor suppressors (27, Authors' Contributions 28, 30, 37). Recently, overexpression of Pin1 has been shown to Conception and design: B. Yeung, P. Khanal, X. Yang induce resistance of cancer cells to anti-tubulin drug Taxol (27). Development of methodology: B. Yeung, P. Khanal However, the upstream signaling pathway regulating Pin1 func- Acquisition of data (provided animals, acquired and managed patients, tion in anti-tubulin drug response has not been elucidated. In this provided facilities, etc.): B. Yeung, P. Khanal, V. Mehta study, we have provided the first in vitro and in vivo evidence that Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Cdk1-LATS is a novel signaling axis negatively regulating Pin1. We computational analysis): B. Yeung, P. Khanal, V. Mehta, L. Trinkle-Mulcahy have shown that both LATS1 and LATS2 can interact with WW Writing, review, and/or revision of the manuscript: B. Yeung, P. Khanal, X. Yang domain of Pin1 in a phosphorylation-dependent manner (Fig. 5). Administrative, technical, or material support (i.e., reporting or organizing In addition, we have shown that phosphorylation of 5 sites on data, constructing databases): B. Yeung, P. Khanal LATS2 by Cdk1 is essential for its interactions with Pin1 and Study supervision: P. Khanal, X. Yang

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Cdk1–LATS–Pin1 Signaling Mediates Anti-tubulin Drug Response

Acknowledgments The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in We would like to thank Canadian Institute of Health Research (#119325, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 148629; to X. Yang), the Canadian Breast Cancer Foundation/Canadian Cancer Society (to X. Yang) for their ﬁnancial support, and Yawei Hao for technical Received November 20, 2017; revised January 23, 2018; accepted February 7, support. 2018; published ﬁrst March 9, 2018.

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www.aacrjournals.org Mol Cancer Res; 2018 OF11

Identification of Cdk1−LATS−Pin1 as a Novel Signaling Axis in Anti-tubulin Drug Response of Cancer Cells

Benjamin Yeung, Prem Khanal, Virja Mehta, et al.

Mol Cancer Res Published OnlineFirst March 9, 2018.

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