Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 https://doi.org/10.1186/s13046-021-01936-4

RESEARCH Open Access Activation of the clock TIMELESS by H3k27 acetylation promotes colorectal cancer tumorigenesis by binding to -9 Meng Cao1†, Yi Wang2†, Yijing Xiao3†, Dandan Zheng2, Chunchun Zhi4, Xin Xia2 and Xiaoqin Yuan2,5*

Abstract Background: Colorectal cancer (CRC) is a common tumor characterized by its high mortality. However, the underlying molecular mechanisms that drive CRC tumorigenesis are unclear. Clock have important roles in tumor development. In the present study, the expression and functions of clock gene TIMELESS (encoding the Timeless ) in CRC were investigated. Methods: Immunohistochemistry, cell proliferation, migration, invasion, EMT and xenograft tumor experiments were used to prove the function of Timeless in the tumorigenesis of CRC. Immunoprecipitation, mass spectrometry, Immunofluorescence and Chromatin immunoprecipitation (ChIP) were utilized to clarify the mechanism of Timeless in regulating CRC tumorigenesis. Results: We found that Timeless was upregulated in CRC tissues compared with corresponding normal tissues and its expression was closely associated with the TNM stages and overall survival of CRC patients. Functional studies demonstrated that Timeless promoted the proliferation, invasion, and EMT of CRC cells in vitro and in vivo. Mechanistic investigations showed that Timeless activated the β- signal pathway by binding to Myosin-9, which binds to β-catenin to induce its nuclear translocation. The upregulation of Timeless was attributed to CREB- binding protein (CBP)/p300-mediated H3K27 acetylation of the promoter region of Timeless. Conclusion: Timeless regulates the tumorigenesis of CRC by binding to and regulating myosin-9, suggesting Timeless might be a potential prognostic biomarker and therapeutic target for CRC. Keywords: Timeless, Myosin-9, Wnt/β-catenin pathway, Colorectal cancer

* Correspondence: [email protected] †Meng Cao, Yi Wang and Yijing Xiao contributed equally to this work. 2Department of Anatomy, Histology and Embryology, Nanjing Medical University, Xuehai Building, Rm D509, 101 Longmian Avenue, Jiangning District, Nanjing 211166, China 5Key Laboratory for Aging & Disease, Nanjing Medical University, Nanjing 211166, China Full list of author information is available at the end of the article

© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 2 of 17

Background Materials and methods Colorectal cancer (CRC) is a common tumor worldwide Clinical samples with a high mortality rate [1]. With an incidence ranking A total of 114 pairs of paraffin-embedded human CRC the third among all cancers, CRC has been proven as the specimens and corresponding adjacent normal mucosa most common cause of cancer death in the United samples were histopathologically diagnosed at the First Af- States [2]. In China, CRC is a commonly-diagnosed can- filiated Hospital of Nanjing Medical University and Nanjing cer with 521,490 cases (12.2% of total cancer cases) and Drum Tower Hospital. The detailed clinicopathologic char- 247,563 cancer-related deaths (8% of total cancer deaths) acteristics of the CRC patients are summarized in Table 1. reported in 2018 [3]. However, the underlying molecular This project was approved by the Research Ethics Commit- mechanisms that drive CRC tumorigenesis are poorly tee of Nanjing Medical University (Approval ID: (2016)640) understood. and Research Ethics Committee of Nanjing Drum Tower Accumulating evidence suggests that the dysregulation Hospital (Approval ID: 2020–378-02). of circadian clock including CLOCK1, CRY1, CRY2, and PER1–3 is associated with the in- Table 1 Association of Timeless expression with creased incidence and metastasis of cancers such as clinicopathological variables in patients with CRC (n = 114) colorectal cancer, breast cancer, and glioma [4–8]. Per- Variable Group Number Timeless expression P value turbations of circadian clock genes impact key pathways of cases High Low affecting cancer development and progression, including (%) cell-cycle control, apoptosis, metabolic regulation, Gender epithelial-to-mesenchymal transition, immunity, and male 66 (57.9) 33 33 1 DNA damage responses [8–11]. female 48 (42.1) 24 24 TIMELESS (TIM), an evolutionarily-conserved circa- Age (years) dian gene encoding the Timeless protein, interacts with Cryptochrome (CRY) and Period (PER) and acts ≥60 70 (61.4) 41 29 0.021* on the negative arm of the circadian cycle [12–15]. <60 44 (38.6) 16 28 Timeless, as a replication fork-associated factor, interacts Tumor Diameter (cm) with Timeless-interacting protein (Tipin) to form a > 5 43 (37.7) 19 24 0.334 stable complex that affects replication checkpoints and ≤5 71 (62.3) 38 33 normal DNA replication [16, 17]. Timeless also interacts Tumor Location with PARP-1 to promote DNA-damage repair [18, 19]. Recently, Timeless has been reported to be upregu- Colon 76 (66.7) 39 37 0.691 lated in various types of cancer and to be involved in Rectum 38 (33.3) 18 20 cancer development, progression, and associated with Clinical stage poor patient survival. For example, in breast cancer, I-II 55 (48.2) 18 37 0.000*** Timeless was significantly upregulated in tumor tissues III-IV 59 (51.8) 39 20 and patients with high Timeless levels had a poorer T stage prognosis than patients with low Timeless levels [20]. Timeless overexpression was associated with poor clin- T1–2 20 (17.5) 5 15 0.014* ical survival and lymph node metastasis in early-stage T3–4 94 (82.5) 52 42 cervical carcinoma [21]. Moreover, Timeless was an in- N stage dependent prognostic factor for overall survival and absent 57 (50) 17 40 0.000*** progression-free survival in nasopharyngeal carcinoma present 57 (50) 40 17 [22] and CRC tissues had higher levels of Timeless M stage mRNA compared with the corresponding healthy mu- cosa of CRC patients. In addition, high Timeless mRNA M0 100 (87.7) 47 53 0.087 levels were associated with TNM stages III-IV and M1 14 (12.3) 10 4 microsatellite instability [4]. However, the precise role of Vascular invasion Timeless in the progression of CRC is poorly absent 85 (74.6) 36 49 0.005** understood. present 29 (25.4) 21 8 In this study, we aims to investigate the expression of Perineural invasion Timeless in CRC tissues and demonstrate the function and mechanism of Timeless in the proliferation, migra- absent 71 (62.3) 34 37 0.562 tion, invasion and EMT of CRC cells. Our results may present 43 (37.7) 23 20 provide a diagnostic and prognostic biomarker of CRC. * P < 0.05, ** P < 0.001, *** P < 0.0001 Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 3 of 17

Immunohistochemistry (IHC) staining and evaluation TIM siRNA1: sense: 5′-GUAGCUUAGCCUUUCAAA IHC staining was performed on paraffin-embedded tis- TT-3′,antisense:5′-UUUGAAAGGACUAAGCUACTT- sues using anti-Timeless (Abcam, Cambridge, 3′; siRNA2: sense: 5′-AGAAGAGAAGGAAGAAGA UK) according to a method described previously [23]. ATT-3′,antisense:5′-UUCUUCUUCCUUCUCUU- IHC results were scored using the modified Histoscore CUTT-3′; and siRNA3: sense: 5′-GCCUACAUGU (H-score) [24]. Briefly, in five fields at × 400 magnifica- GCUAGAGAUTT-3′,antisense:5′-AUCUCUAGCA tion chosen randomly, the staining intensity of cell nu- CAUGUAGGCTT-3′.MYH9siRNA:sense:5′-GGAG clei was scored as 0, 1, 2 or 3, corresponding to negative, CGGAACACUGACCAGTT-3′,antisense:5′-CUGGUC weak, moderate and strong staining, respectively. The AGUGUUCCGCUCCTT-3′CBP siRNA: sense: 5′-GGAG total number of cells in each field and the number of CCAUCUAGUGCAUAATT-3′ antisense: 5′-UUAUGC cells stained at each intensity were counted. This semi- ACUAGAUGGCUCCTT-3′;p300siRNA:sense:5′- quantitative assessment was performed by two inde- AACCCCUCCUCUUCAGCACCATT-3′,antisense:5′- pendent investigators blinded to the clinical parameters UGGUGCUGAAGAGGAGGGGUUTT-3′. of the corresponding patients. The H-score was deter- mined according to the following formula: [(% of weak Generation of stable Timeless knockdown cells staining) × 1] + [(% of moderate staining) × 2] + [(% of A lentivirus harboring sh-TIM was constructed by strong staining) × 3], yielding a range from 0 to 300 [25]. Genechem (Shanghai, China), and HCT116 cells were The expression level of each protein was categorized as infected with an MOI of 20. After infection, 2 μg/ml low or high according to the median H-score. puromycin (Sigma) was used to select stably transduced cells. HCT116 cells infected with lentivirus expressing scrambled shRNA with puromycin resistance were used Cell culture as controls. The scrambled shRNA sequence and the The human CRC cell lines HCT116, RKO, HT29, CX-1, shRNA targeting Timeless were TTCTCCGAACGTGT SW620, Caco-2, and LoVo were purchased from the CACGT and GAAGGATCTGATCCGCTAT, Shanghai Institute of Cell Biology, Chinese Academy of respectively. Sciences. HCT116, RKO, CX-1, SW620, and LoVo cells were cultured in RPMI 1640 medium (HyClone, South CCK8 assay Logan, UT, USA), and other cell lines were maintained Cell growth was assessed using the Cell Counting Kit 8 in DMEM medium (HyClone), supplemented with 10% (MedchemExpress). In brief, 3000 cells transfected with FBS (Gibco, Grand Island, NY, USA), 100 U/mL penicil- siRNA or plasmid were seeded into 96-well plates per lin, and 0.1 mg/mL streptomycin (Gibco). All cell lines well in 100 μl culture medium. Cell growth was deter- were cultured at 37 °C in a humidified incubator of 5% mined at 0, 24, 48, 72, and 96 h following the manufac- CO . The cells were collected for further studies after 2 turer’s instructions. The absorbance at a wavelength of treatment with cycloheximide (CHX) (MedchemExpress, 450 nm was measured in a microplate reader (Bio-Tek, Monmouth Junction, NJ, USA), C646 (MedchemEx- Winooski, VT, USA). press), or MG132 (Sigma-Aldrich, St. Louis, MO, USA) for different periods. Colony formation assay The cells transfected with siRNA or plasmid were seeded Small interfering RNA and plasmid transfection into 6-well plates (1000 cells/well) and cultured in Timeless, Myosin-9, P300 and CBP were knocked down medium containing 10% FBS for 10 to 15 days, The by small interfering RNAs (siRNA) targeting Timeless medium was replaced every 3 days. Colonies were fixed (si-TIM), Myosin-9 (si-MYH9), P300 (si-P300) and CBP with 4% formaldehyde for 15 min and stained with 0.1% (si-CBP) (RiboBio, Guangzhou, China). Their scramble crystal violet solution (Keygene, Nanjing, China) for 15 controls (si-NC) (RiboBio) were transfected into CRC min. The crystal violet stained colonies were counted to cells with Lipofectamine RNAiMAX (Invitrogen, Wal- determine colony formation. tham, MA, USA). Timeless and Myosin-9 cDNA were cloned respectively into pcDNA3.1 expression vectors to Transwell assays construct pcDNA-TIM and pcDNA-MYH9, respectively. Cell migration and invasion assays were performed with A plasmid expressing β-catenin (pcDNA-Ctnnb1) was a 8-μm pore Transwell chambers (Corning, NY, USA) as gift from Dr. Yuping Lai (East China Normal University). previously described [23]. Overall, 5 × 104 or 8 × 104 cells The plasmids were transfected into CRC cells using Li- in 200 μl serum-free medium were seeded in the upper pofectamine 2000 (Invitrogen) according to the manu- chamber coated with or without Matrigel for migration facturer’s protocol. The siRNA sequences for Timeless, or invasion assays, respectively. The lower chambers Myosin-9, and CBP are as follows: were filled with 600 μL medium with 10% FBS for Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 4 of 17

migration or with 20% FBS for invasion. After 24–36 h, analyzed by Western blotting with anti-Flag (Sigma), the cells on the bottom surface of the membrane were anti-Myosin-9 (Abcam), anti-Timeless (Abcam), and fixed with 95% ethanol and stained with 0.1% crystal vio- anti-β-catenin (Abcam) . let. Five random fields (× 200) were counted using a To identify proteins that bind to Timeless, we immu- microscope to assess cell migration and invasion. noprecipitated cell lysates with Timeless antibody (Abcam) or IgG. Then, the proteins precipitated by Tumor formation and metastasis assays in nude mouse Timeless antibody and IgG were detected by mass spec- models trometry analyses (Shanghai Applied Protein Tech, Male nude BALB/c mice (4–5 weeks old) were Shanghai, China). purchased from the Model Animal Research Center of Nanjing University (Nanjing, China) and maintained on RNA extraction and real-time RT-PCR a 12-h light/12-h dark cycle under pathogen-free condi- Total RNA was extracted from cells with TRIzol reagent tions. For xenograft animal models, the mice were ran- (Invitrogen, Grand Island, NY, USA). RT-qPCR was car- domly divided into two groups, each consisting of five ried out with a PrimeScript™ 1st Strand cDNA Synthesis mice. After anesthetization, 5 × 106 Timeless-knockdown Kit (Takara Bio, Shiga, Japan) and SYBR green reagents stable HCT116 cells or control cells were injected sub- (Takara Bio) according to the user’s manual and de- cutaneously into the flank of mice. The size of tumors tected in an ABI 7300 sequence detector (Applied Bio- was measured twice a week for 4 weeks, and the tumor systems, Foster City, CA, USA). was used for volume was calculated according to the equation: vol- normalization. The PCR primers used in this study were: ume = length × width2 × 0.5. After 4 weeks, all mice were ACTIN-F: 5′-TCATGAAGTGTGACGTGGACAT-3′, sacrificed and the xenografts were dissected and ACTIN-R: 5′-CTCAGGAGGAGCAATGATCTTG-3′; weighed. For the in vivo lung metastasis assay, Timeless- TIM-F: 5′-CACCAGGACAAGCCTCTCTTT-3′, TIM- knockdown stable HCT116 cells and control cells (4 × R: 5′-TTAGGCAGATTGCCAAAACAGA-3′; CBP-F: 106) were injected into the tail vein of each nude mouse 5′-CGGCTCTAGTATCAACCCAGG-3′, CBP-R: 5′- (n = 5 for each group). These mice were sacrificed after TTTTGTGCTTGCGGATTCAGT-3′;p300-F: 5′-AATC 10 weeks and examined for lung metastases. Animal wel- CTTTCCATACCGAACC-3′, p300-R: 5′-GAGGGCAG fare and experimental procedures were approved by the TCAGAGCCATAC-3′; MYH9-F: 5′-CCTCAAGGAG Institutional Animal Care and Use Committee of CGTTACTACTCA-3′, MYH9-R: 5′-CTGTAGGCGG Nanjing Medical University (Approval ID 1601080). All TGTCTGTGAT-3′; AXIN2-F: 5′-GGTTTCCCCT protocols were performed in accordance with the Guide TGGACCTCG-3′, AXIN2-R: 5′-CCGTCGAAGTCTCA for the Care and Use of Laboratory Animals. CCTTTAATG-3′; CCND1-F: 5′-GCTGCGAAGT GGAAACCATC-3′, CCND1-R: 5′-CCTCCTTCTG Western blot analysis CACACATTTGAA-3′; LEF1-F: 5′-GAGAAAAGTG Protein lysates from cells or tumor tissues were CTCGTCACTGT-3′, LEF1-R: 5′-TGCCAAATATGAA- separated by sodium dodecyl sulfate polyacrylamide gel TAACGACCCA-3′. electrophoresis (SDS-PAGE), transferred onto polyvinyli- dene fluoride (PVDF) membranes, and blotted using the Nuclear and cytoplasmic protein extraction following antibodies: Timeless (Abcam, Cambridge, UK), Nuclear and cytoplasmic proteins were extracted using a Myosin-9 (Abcam), Snail (Abcam), E-cadherin (CST, Kit (Beyotime Biotechnology, Shanghai, China) following Danvers, MA, USA), N-cadherin (Abcam), β-catenin the manufacturer’s instructions. All samples were stored (Abcam), Histone H3 (CST), H3K27ac (Abcam), CBP at − 80 °C for further use. (Santa Cruz Biotechnology, CA, USA), P300 (Abcam), GAPDH (Santa Cruz Biotechnology), and Flag (Sigma- Immunofluorescence Aldrich). The cells were seeded on coverslips in 24-well plates and cultured for 24 h. Then, the cells were fixed with 4% Immunoprecipitation (IP) and mass spectrometry paraformaldehyde for 15 min, permeabilized with 0.2% HCT116 cells were transfected with Flag-tagged Triton X-100 in PBS, blocked with 5% bovine serum al- pcDNA-TIM, pcDNA-MYH9, or pcDNA-Ctnnb1, and bumin (BSA), followed by incubation with specific pri- pcDNA3.1 was used as a negative control. After trans- mary antibodies at 4 °C overnight or rhodamine fection, the cells were lysed with RIPA lysis buffer. Then, phalloidin (UE, Chicago, USA) incubated at room cell lysates were incubated with anti-Flag magnetic beads temperature in the dark for 20 min to detect F-actin. (Sigma-Aldrich) overnight at 4 °C. After incubation, the After washing, the cells were incubated with secondary beads were washed with lysis buffer. The precipitated antibodies at room temperature for 1 h followed by proteins were eluted with SDS sample buffer and counterstaining with DAPI. The secondary antibodies Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 5 of 17

were Alexa Fluor 488 goat anti-rabbit IgG (Proteintech, Timeless promotes cell proliferation and invasion in vitro Chicago, USA) and Fluor 647 goat anti-mouse IgG We next investigated the functions of Timeless in CRC (Fcmacs, Nanjing, China). Images were captured by con- cells. We measured Timeless levels in several CRC cell focal microscopy (Zeiss, Analytik Jena, Germany). lines (Fig. 2a) and selected HCT116 and RKO cell lines with a medium expression of Timeless for further stud- Chromatin immunoprecipitation (ChIP) ies. We transiently transfected specific siRNAs and ChIP assay was performed using a ChIP Kit (Abcam) fol- Timeless overexpressed plasmid (pcDNA-Tim) into lowing the manufacturer’s protocol. Briefly, the cells HCT116 and RKO cells. As shown in Fig. 2b, siRNAs were fixed with formaldehyde to generate DNA–protein specifically decreased the expression of Timeless cross-links. Then, the cell lysates were sonicated to shear whereas pcDNA-Tim transfection increased the expres- the DNA into fragments between 200 and 1000 bps and sion of Timeless. The results of CCK-8 assays revealed immunoprecipitated with H3K27ac antibody (Abcam) or that silencing Timeless significantly suppressed the pro- a negative control IgG antibody. Precipitated DNA was liferation of HCT116 and RKO cells. In contrast, the purified and analyzed by real-time PCR. overexpression of Timeless significantly increased cell viability (P < 0.001; Fig. 2c). Colony formation assays re- vealed that the knockdown of Timeless significantly Statistical analysis inhibited the proliferation of HCT116 and RKO cell Statistical analysis was performed using SPSS 20.0 (Ab- lines, whereas Timelessoverexpression cells showed an bott Laboratories, IL, USA). All data were presented as opposite effect (Fig. 2d). In transwell assays, siRNA- ’ t the mean ± SD. Student s -test was used to analyze data treated cells also showed a significant decrease in migra- between two groups. The Chi-Square test was used to tive and invasive potential, compared with control cells. examine the clinicopathological characteristics between However, Timeless-overexpressed CRC cells showed en- Timeless high- and Timeless low-expressing patients. hanced migration and invasion compared with control Overall survival curves were plotted using the Kaplan- cells (P < 0.01; Fig. 2e, f). Meier method and analyzed by the log-rank test. The ’ t Student s -test was used to examine the relationship be- Timeless promotes the EMT of CRC cells tween Timeless levels and clinicopathological character- During these experiments, we found that the cell shapes P istics. < 0.05 was considered statistically significant. changed upon Timeless expression. Therefore, we stained F-actin using immunofluorescence labeling with Results phalloidin. The cells with Timeless overexpression ex- Timeless is upregulated in CRC tissues and associated hibited spindle shapes and rearrangement of F-actin, with poor prognosis confirming to the typical morphological changes during To investigate the role of Timeless in CRC, we analyzed epithelial–mesenchymal transition (EMT) (Fig. 3a) The the expression of Timeless protein in 114 pairs of cells with Timeless knockdown showed an opposite paraffin-embedded, archived CRC specimens by immu- trend. Next, we examined whether Timeless played a nohistochemical staining. As shown in Fig. 1a, Timeless role in EMT. We investigated the expression of markers was expressed mainly in the nucleus and rarely in the associated with EMT in different Timeless-expressing cytoplasm. The level of Timeless expression in CRC tis- CRC cells. Western blotting showed that the knockdown sues was significantly increased, compared with that in of Timeless significantly downregulated the mesenchy- corresponding adjacent normal mucosa (Fig. 1b). Time- mal marker N-cadherin and the transcription factor less expression increased with the progression of CRC Snail, and upregulated the epithelial marker E-cadherin clinical stage (Fig. 1c, d). Moreover, patients with a (Fig. 3b). Conversely, the overexpression of Timeless sig- higher level of Timeless had a shorter overall survival nificantly downregulated E-cadherin and upregulated N- (Fig. 1e). Correlation analysis between Timeless expres- cadherin and Snail (Fig. 3b). Taken together, Timeless sion and clinical pathological features verified that promoted the EMT in CRC cells in vitro. Timeless upregulation was significantly correlated with age (P = 0.021), clinical stage (P < 0.0001), T stage (P = Timeless promotes the proliferation and metastasis of 0.014), N stage (P < 0.0001), and vascular invasion (P = CRC cells in vivo 0.005) (Table 1). However, no significant correlations To determine the effect of Timeless on tumorigenesis were observed between Timeless expression and gender, in vivo, HCT116 cells (sh-Tim) with stable Timeless tumor diameter, tumor location, and perineural invasion knockdown and control HCT116 cells (sh-Ctrl) were (Table 1). Collectively, Timeless was highly expressed in generated (Fig. 4a). Stable sh-Tim and sh-Ctrl cells were CRC and correlated with a poor outcome in patients injected subcutaneously into the flanks of 5-week-old with CRC. male nude mice. As shown in Fig. 4b-d, the growth, Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 6 of 17

a Normal Tumor b

Case 43 P < 0.0001 250

200 HC

fI 150

100

Case 81 Score o -

H 50

0 Normal Tumor (n=114) (n=114)

Case 113

c Clinical stage I/II Clinical stage III/IV d

250 P < 0.0001

200

150

100

H-Score of IHC 50

0 I/II(n=55) III/IV(n=59)

P=0.0167 e High expression of Timeless 1.0 (n=57) val i 0.8 Low expression of Timeless

rv (n=57) 0.6 lsu 0.4

0.2 Overal 0.0 0 20406080 Survival time (months) Fig. 1 Timeless is upregulated in CRC tissues and associated with poor prognosis. (a) Representative images from the immunohistochemical analyses of Timeless protein expression in normal colorectal tissues and colorectal cancer tissues. (b) Statistical analyses of the H-score of Timeless staining in CRC and corresponding normal tissues. (c) Representative images from immunohistochemical analyses of Timeless protein expression in CRC tissues at different clinical stages. (d) Statistical analyses of the H-score of Timeless staining in CRC tissues at different clinical stages. (e) Kaplan–Meier survival curves showing an association between Timeless and the overall survival in patients with CRC Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 7 of 17

a b si-NC si-Tim1 si-Tim2 si-Tim3 pcDNA3.1 pcDNA-Tim HCT116 RKOSW620 HT-29 LoVo Caco2 CX-1 HCT116 Timeless Timeless

0.55 0.38 0.78 0.15 0.34 0.33 0.20 0.33 0.09 0.09 0.06 0.35 0.96 GAPDH GAPDH

RKO Timeless 0.49 0.15 0.15 0.11 0.32 0.58 GAPDH

c 20 RKO 20 RKO si-NC 25 HCT116 d 20 HCT116 si-NC pcDNA3.1 l pcDNA3.1 d

l si-Tim1 si-Tim1 20 ** 15 15 ** pcDNA-Tim

fo pcDNA-Tim fold 15 ** ** si-Tim2

si-Tim2 h h 15 si-Tim3 10

si-Tim3 wt 10

10 growth fo

10 l l 5

5 e ll gro

5 C

5 Cell growth fold Ce Cell growt 0 0 0 0 h h h h H H H H 0 2 6h 0 4H 8 h H H H 24 48 7 9 2 4 72 96 0 6h 0 4H 8 2 6H 24h 48h 72h 9 2 4 7 9

HCT116 HCT116 s

d s 400 ** 400 e e i ** * si-NC si-Tim1 si-Tim2 si-Tim3 pcDNA 3.1 * on

l 300 300 o

fC 200 200 o ber of Coloni ber 100 100 m

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RKO RKO

s 500 * 500 ** e * ies 400 * n 400

300 Colo 300 f

RKO o 200 r 200

100 100 Numbe Number of Coloni 0 0 pcDNA3.1 pcDNA-Tim -NC im1 im2 Tim3 si i-T -T i- s si s

HCT116 e HCT116 ** ion

600 ** t 1000 a ation ** r **

si-NC si-Tim1 si-Tim2 si-Tim3 pcDNA 3.1 igr 800

400 m lmig l l 600

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Number of ce 0 C 1 2 3

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200 o 200 er

0 mb 0 Nu

Number of ce pcDNA3.1 pcDNA-Tim C -N im1 im2 m3 T T Ti si - - i- si si s

n HCT116 HCT116 o 800 ** ** 400 ** 600 si-Tim2 si-Tim3 pcDNA 3.1 sion ** f si-NC si-Tim1 va 300 in 400 l 200 HCT116 fcel 200 ro 100 0

Number of cell invasi pcDNA3.1 pcDNA-Tim 0 Numbe 1 2 3 -NC m m i Tim Ti s i-Ti i- i- s s s RKO RKO RKO ** 300 **

on ** i 100 ** s

va 80 200 n

ll i 60

40 100 of ce

er 20 b 0 m Number of cell invasion u 0 pcDNA3.1 pcDNA-Tim N C 1 2 3 N im im im i- T T s i-T i- i- s s s Fig. 2 Timeless promotes cell proliferation and invasion in vitro. (a) The expression levels of Timeless in seven CRC cell lines were analyzed by western blotting. (b) Timeless expression was detected by Western blotting in HCT116 and RKO cells transfected with the indicated siRNAs or pcDNA-Tim. (c) CCK-8 assay and (d) colony-forming growth assays were performed using HCT116 and RKO cells transfected with Timeless siRNAs or pcDNA-Tim. Transwell assays were performed to detect the migration (e) and invasion (f) of Timeless-knockdown and overexpressing cells after 36–48 h. Data represent the mean ± standard deviation from three independent experiments. *P < 0.05, **P < 0.01 Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 8 of 17

a pcDNA 3.1 si-NC si-Tim

HCT116 DAPI / Phalloidin

RKO

b HCT116 RKO si-NC si-Tim pcDNA3.1 pcDNA-Tim si-NC si-Tim pcDNA3.1 pcDNA-Tim N-cadherin

0.68 0.50 0.56 0.79 1.17 0.27 0.67 1.97 E-cadherin

1.03 1.39 0.62 0.41 0.58 2.06 1.25 0.90 Snail

1.01 0.59 0.73 1.02 1.02 0.77 0.76 1.18

GAPDH Fig. 3 Timeless promotes the EMT of CRC cells. (a) detected by Rhodamine phalloidin staining in Timeless overexpressing or knockdown HCT116 and RKO cells. Original magnification, × 630 (b) Expression of EMT related molecules detected by western blotting in Timeless overexpressing or knockdown HCT116 and RKO cells volume, and weight of tumor xenografts were signifi- Timeless activates Wnt/ß-catenin signaling cantly decreased in the sh-Tim group compared with Previous studies have reported that the Wnt/β-catenin the control group. The results of immunohistology pathway is involved in Timeless-induced chemoresis- showed that the mean expression level of Timeless in tance and EMT [22]. To investigate whether the Wnt/β- xenograft tumors from the sh-Tim group were lower catenin pathway was involved in CRC tumorigenesis than that from the control group (Fig. 4e). Likewise, ex- mediated by Timeless, we measured the expression of β- pression of the proliferation marker PCNA was signifi- catenin in CRC cells with Timeless knockdown and cantly decreased in tumors from the sh-Tim group Timeless overexpression. Upon Timeless knockdown, compared with tumors from the control group (Fig. 4e). the levels of cellular β-catenin and nuclear β-catenin Consistent with our previous findings, decreased N- were reduced in CRC cell lines, whereas Timeless cadherin and Snail and increased E-cadherin expression overexpression increased cellular β-catenin and nu- levels were shown in the Timeless-knockdown sh-Tim clear β-catenin levels (Fig. 5a, b). The results from group (Fig. 4f). the laser scanning confocal microscope also showed We then examined the effects of Timeless on CRC high expressed Timeless induced the nuclear location metastasis in vivo, control or Timeless-knockdown of β-catenin in CRC cells (Fig. 5c). To investigate HCT116 cells were injected into nude mice via the tail whether Timeless activated the Wnt/β-catenin path- vein. The results revealed that Timeless-knockdown sig- way, we examined the expressions of genes targeted nificantly decreased colorectal cancer pulmonary metas- by the Wnt/β-catenin pathway in CRC cells. As tasis (Fig. 4g). Further, H&E staining revealed that shown in Fig. 5d, the expressions of Cyclin D1, Timeless-knockdown HCT116 cells formed less nodules Axin2, and LEF1 were markedly reduced or increased in the lungs comparing to control HCT116 cells by Timeless knockdown or Timeless overexpression, (Fig. 4h). respectively. However, the mRNA level of β-catenin Collectively, the knockdown of Timeless repressed the (CTNNB1) was not affected by the expression of proliferation and metastasis of CRC in vivo. Timeless (Fig. 5d). Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 9 of 17

a b c d 1200 0.8 ) g sh-Ctrl sh-Tim ) ** 3

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du 20 Snail o * 0.75 0.79 0.43 0.30 15 sh-Ctrl tatic n s

a 10 t

GAPDH e m 5 N-cadherin 0 0.49 0.22 0.06 0.08 sh-Tim

Number of -Ctrl E-cadherin h h-Tim s s 0.20 0.20 0.51 0.98 GAPDH

Fig. 4 Timeless accelerates the growth and metastasis of CRC cells in vivo. (a) Expression of Timeless was detected in Timeless-knockdown stable HCT116 cells by western blotting. (b) Images of xenograft tumors from groups of BALB/c-nude mice 4 weeks after the subcutaneous injection of stable Timeless-knockdown HCT116 cells (sh-Tim) or control HCT116 cells (sh-Ctrl). (c) Tumor size was calculated using the equation volume = (length × width2)/2 every 2–3 days 1 week after injection. (d) Weights of xenograft tumors were measured. (e) Hematoxylin and eosin staining and immunohistochemistry for Timeless and PCNA in xenograft tumors. Original magnification, ×200. (f) Western blotting analysis of EMT-related Snail, E-cadherin, and N-cadherin in xenograft tumors. (g) Photos of tumor metastasis in lung tissues (left panel). The number of metastatic foci in lung tissues is quantified in the right. The white arrows show the metastases. (h) Hematoxylin and eosin staining of lung tissues. Original magnification, ×100. Error bars indicate the means ± SD. *P < 0.05, **P < 0.01

Given that Timeless participates in regulatory path- spectrometry. Among the identified proteins, myosin ways by binding to proteins, we investigated whether heavy chain (Myosin-9) was selected for verification Timeless interacted with β-catenin. Flag-tagged β- because of its good mass spectrometric data (Fig. 6a) catenin was used to immunoprecipitate Timeless; how- and its association with CRC [26]. We transfected Flag- ever, no bands specific to Timeless were observed with Timeless or Flag-Myosin-9 into HCT116 cells and an anti-Timeless antibody (Fig. 5e). Next, we performed extracted cell lysates for immunoprecipitation with anti- an IP assay to determine whether endogenous β-catenin Flag and immunoblotting with anti-Myosin-9 or anti- interacted with Timeless and found no bands specific for Timeless. IP analysis showed endogenous and exogenous β-catenin (Fig. 5f). There was no interaction between Myosin-9 could bind to Timeless (Fig. 6b); therefore, we Timeless and β-catenin, indicating other molecules detected the colocalization of Timeless and Myosin-9 in might mediate the regulation of Timeless on β-catenin. CRC cells by immunofluorescence (Fig. 6c). Timeless knockdown decreased the expression of Myosin-9, Timeless binds to Myosin-9 to maintain its stability whereas Timeless overexpression upregulated Myosin-9 To identify the unknown binding proteins of Timeless, levels (Fig. 6d). Nucleoplasm isolation showed Timeless proteins were extracted from lysates of HCT116 cells knockdown decreased both the nuclear and cytoplasmic and immunoprecipitated with a specific Timeless anti- levels of Myosin-9, whereas Timeless-overexpression body. The immunoprecipitate was analyzed by mass showed an opposite effect (Fig. 6e). To test whether the Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 10 of 17

Cytosol Nuclear a b Cytosol Nuclear HCT116 HCT116 ß-Catenin ß-Catenin

1.11 0.46 0.35 0.63 1.54 1.06 1.20 0.37 0.36 0.96 0.76 2.27 Timeless GAPDH 0.22 0.04 0.60 1.41 GAPDH Histone H3 RKO RKO ß-Catenin ß-Catenin 0.31 0.07 0.43 0.77 0.25 0.11 0.50 0.03 0.14 0.26 0.57 1.04 Timeless GAPDH 0.32 0.09 0.49 1.59 GAPDH Histone H3

c e f HCT116 RKO DAPI -catenin MERGE DAPI -catenin MERGE Input Input si-NC ß-Catenin Timeless FLAG si-Tim FLAG GAPDH

GAPDH pcDNA 3.1 IP: FLAG IP: FLAG ß-Catenin Timeless

FLAG FLAG d HCT116

l 1.5 si-NC 3000 * HCT116

eve n.s el l si-Tim 2500 ev A 1.0 l 2000 pcDNA-3.1 1500 1000 pcDNA-Tim ** ** RNA 0.5 ** ** m 4 * e 3 ** v ** i t 2 n.s 1

Relative mRN 0.0 ela

1 1 r 0 IM N2 F1 T I E X ND M 2 1 1 1 A C L I N B C T I CTNNB AX LEF CCND TNN C RKO n.s 2500 RKO 1.0 pcDNA-3.1 si-NC ** level 2000 pcDNA-Tim

si-TIM A 1500 ** ** 0.5 ** 3 * ** ** 2 ** n.s

ative mRNA level 1

relative mRN 0 Rel 0.0 1 1 1 2 1 1 M N2 B TI I EF TIM IN D ND L N AX C NN LEF C T AX CC C CTNNB1 Fig. 5 Timeless activates Wnt/β-catenin signaling. Western blotting was performed to detect total (a) and nuclear β-catenin (b) in Timeless knockdown and overexpressing CRC cells. (c) The laser scanning confocal microscope was performed to detect the subcellular location of β- catenin. Original magnification, × 400 (d) Expression levels of Axin2, CCND1, LEF1 and CTNNB1 were assayed by qRT-PCR in the indicated cells. An immunoprecipitation (IP) assay was performed to detect Flag-tagged β-catenin (e)or(f) Flag-tagged Timeless that interacted with Timeless or β- catenin in HCT116 cells. Error bars indicate the means ± SD. *P < 0.05, **P < 0.01 binding of Timeless to Myosin-9 affected Myosin-9 sta- rescued Myosin-9 protein expression (Fig. 6g) indicating bility, CHX treatment was performed, and the results Timeless might affect Myosin-9 protein stability. Taken showed that Myosin-9 stability was decreased by Time- together, Timeless bound to Myosin-9 and increased its less knockdown in CRC cells (Fig. 6f). MG132 treatment stability by a proteasome-dependent mechanism. Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 11 of 17

a b Input

Myosin-9 Timeless 0.77 1.23 0.96 1.06

FLAG FLAG

DAPI Myosin-9 Timeless MERG c GAPDH GAPDH E IP: FLAG HCT116 Myosin-9 Timeless

FLAG FLAG

RKO e Cytosol Nuclear Cytosol Nuclea d HCT116 HCT116 si-NC si-Tim pcDNA3.1 pcDNA-Tim Myosin-9 Timeless 0.43 0.22 1.36 0.42 0.27 0.52 0.76 1.65 0.48 0.20 0.60 1.49 Timeless Myosin-9 0.34 0.94 1.17 2.30 0.59 0.27 0.44 1.10 0.27 0.13 1.27 0.17 GAPDH GAPDH RKO Histone H3 Timeless RKO 0.42 0.12 0.37 0.60 Myosin-9 Myosin-9 1.04 0.49 0.80 1.21 0.52 0.27 0.92 0.20 0.36 0.57 0.38 1.34 GAPDH Timeless 0.40 0.21 0.88 0.11 0.48 2.17 1.10 2.55 GAPDH f Histone H3 sh-Ctrl sh-Tim CHX(min) 0 15 30 45 60 0153045 60 HCT116 g Myosin-9 MG132 -+ 0.62 0.38 0.44 0.47 0.31 0.21 0.15 0.05 0.01 0.06 HCT116 GAPDH Myosin-9 RKO 0.44 0.20 0.91 0.90 Myosin-9 GAPDH 0.59 0.54 0.34 0.38 0.32 0.52 0.29 0.09 0.04 0.11 GAPDH RKO Myosin-9 HCT116 1.5 1.5 0.41 0.15 0.79 0.87 n sh-Ctrl i RKO

e sh-Ctrl sh-Tim ein t GAPDH 1.0 1.0 sh-Tim

* 0.5 in-9 pro s 0.5 ** yosin-9 prot ** ** ** **

M ** Myo 0.0 0.0 020406080 020406080 CHX(min) CHX(min) Fig. 6 Timeless binds to Myosin-9 to maintain its stability. (a) Myosin-9 protein was identified by mass spectrometry. (b) Immunoprecipitation analyses of the interaction between Timeless and Myosin-9 in HCT116 cells. (c) Immunofluorescence costaining of Timeless (Red) and Myosin-9 (Green) to indicate colocalization in HCT116 (upper) and RKO (lower) cells. Original magnification, × 400. Western blotting was performed to detect total (d) and subcellular Myosin-9 (e) in Timeless knockdown and overexpressing CRC cells. GAPDH and Histone-3 were used as internal loading controls, respectively. (f) CRC cells with specific Timeless siRNA or control siRNA were treated with 100 μg/ml CHX and harvested at different time points as indicated. Myosin-9 was detected by western blotting and quantified by densitometry. (g) Timeless knockdown and control CRC cells were treated with 5 μM of MG132 for 12 h, and Myosin-9 protein was detected by western blotting. Error bars indicate the means ± SD. *P < 0.05, **P < 0.01 Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 12 of 17

Timeless activates β-catenin through the Myosin-9/β- H3K27ac was enriched in the promoter region of Time- catenin pathway less in HCT116 and CX-1 CRC cells (Fig. 8b). However, To investigate whether Myosin-9 mediated Timeless- the intensity of H3K27ac was markedly higher in induced CRC cell proliferation and invasion, we knocked HCT116 cells than in CX-1 cells. H3K27ac is known to down Myosin-9 expression in CRC cells with Timeless be catalyzed by the P300/ CBP (CREB binding protein, overexpression (Fig. 7a). Then, we performed CCK8 and CBP) complex. C646, a histone acetyltransferase (HAT) colony-forming assays, and found Myosin-9 knock-down inhibitor targeting P300, significantly decreased the rescued the growth of Timeless-overexpressed CRC cells mRNA and protein levels of Timeless in CRC cells (Fig. 7b, c). Transwell assay showed that a low expres- (Fig. 8c, d). We knocked down CBP or P300 with a spe- sion of Myosin-9 retarded the migration and invasion of cific siRNA (Fig. 8e, f) and performed ChIP with anti- Timeless-overexpressed CRC cells (Fig. 7d, e). Further- H3K27ac antibody. Both CBP knockdown and P300 more, Myosin-9 knockdown rescued changes in the knockdown significantly decreased the enrichment of expressions of EMT molecular markers, including E- H3K27ac in the Timeless promoter region (Fig. 8e, f). In cadherin, N-cadherin, and Snail (Fig. 7f). Taken together, addition, a low expression of CBP or P300 decreased Myosin-9 mediated the Timeless-induced proliferation, TIMELESS mRNA and protein levels in CRC cells invasion, and EMT of CRC cells. (Fig. 8g-j). These results suggested that the upregulation A previous study has reported that Myosin-9 controlls of Timeless via histone acetylation in the Timeless pro- β-catenin transcriptional activity by interacting with β- moter region was mediated by CBP/P300. Taken to- catenin to promote the proliferation, migration, invasion, gether, Timeless transcriptionally regulated by histone and sphere formation of pancreatic cancer cells in vitro acetylation activated the β-catenin pathway via binding and in vivo [27]. We investigated whether Myosin-9 me- to Myosin-9 to enhance its stability. This process then diated the Timeless-induced activation of β-catenin. We promoted the proliferation, migration, invasion, and found that Myosin-9 knockdown reduced the expression EMT of CRC cells (Fig. 9). of cellular (Fig. 7g) and nuclear β-catenin (Fig. 7h). Meanwhile, Myosin-9 knockdown rescued the impact of Discussion Timeless overexpression on β-catenin expression Circadian rhythm disorders may increase the risk of can- (Fig. 7i). In addition, IP assay demonstrated that β- cer, including colorectal cancer [4, 8]. Timeless, a core catenin was immunoprecipitated with Myosin-9, indicat- gene related to circadian clock, is upregulated in human ing they interacted in HCT116 cells (Fig. 7j). And this cancers including breast, lung, colon, prostate, bladder, interaction was decreased upon Timeless knockdown and pancreatic cancer [30–33]. Timeless is upregulated (Fig. 7k), indicating that Timeless is required to promote in human lung cancer cell lines and in clinical lung can- Myosin-9 /β-catenin interactions. Therefore, Myosin-9 cer tissues [31]. Furthermore, high Timeless expression may medicate the Timeless-induced β-catenin pathway. is correlated with the poor overall survival of patients Taken together, Timeless activated the β-catenin signal- with lung cancer [31]. Knockdown of Timeless can sup- ing pathway by binding to Myosin-9, which bound to β- press the self-renewal of cancer stem cells, and the inva- catenin and induced its nuclear translocation. sion and migration abilities of breast cancer cells [20]. In cervical cancer, high Timeless expression is correlated Timeless is transcriptionally activated by H3K27 with pelvic lymph node metastasis, lymphovascular acetylation space involvement, and unfavorable OS and DFS [21]. In Histone modifications is an important epigenetic mech- accord with these studies, the present study found Time- anism that deregulates cancer-related gene expression in less was highly expressed in CRC tissues compared with tumors [28] and histone H3 lysine 27 acetylation corresponding normal tissues. The Timeless expression (H3K27Ac) modification is upregulated in CRC tissues was closely associated with clinical stage, T classification, [29]. To investigate whether the increased expression of N category, and vascular invasion. Patients with a high Timeless in our CRC cell lines was associated with Timeless protein level had a worse overall survival than aberrant histone modifications, we performed a genome those with a low level. Furthermore, our functional study bioinformatics analysis (http://genome.ucsc.edu/). The showed Timeless promoted the proliferation, migration, promoter region of Timeless was highly enriched for and invasion of CRC cells as well as activated EMT H3K27ac (Fig. 8a). To confirm that the increased expres- in vitro and in vivo. sion of Timeless was associated with aberrant histone The mechanisms of Timeless upregulation in tumors modifications, we performed chromatin immunoprecipi- remain unclear. Previous studies have reported that the tation (ChIP) to compare histone modification around aberrant expression of Timeless in cancer is activated the Timeless promoter in HCT116 cells (High Timeless byERK signaling pathways [34], or by transcription fac- expression) and CX-1 cells (Low Timeless expression). tors such as E2F1 and E2F4 [35]. In addition, genetic Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 13 of 17

pcDNA3.1 pcDNA3.1 pcDNA-Tim pcDNA-Tim abpcDNA3.1 HCT116 c 25 +siRNA-NC +si-NC +si-MYH9 +si-NC +si-MYH9 pcDNA3.1 HCT116 20 +siRNA-MYH9 d ** **

ol pcDNA-Tim f +siRNA-NC * h 15 HCT116 Myosin-9 t

w pcDNA-Tim o

r +siRNA-MYH9 10 0.24 0.12 0.49 0.16 g l

Timeless Cel 5

0.09 1.23 1.20 0 0.08 RKO h h h h 0 4h 8 2 GAPDH 2 4 7 96

RKO RKO RKO 20 pcDNA3.1 HCT116 400 +siRNA-NC 300 ** * ** ** pcDNA3.1 * d ** onies Myosin-9 15 l 300 +siRNA-MYH9 * 200 ** * 0.52 0.35 pcDNA-Tim 200 0.35 0.14 10 +siRNA-NC 100 pcDNA-Tim 100 growth fol

Timeless l

+siRNA-MYH9 umber of Co

5 N 0 Number of Colonies 0 Cel .1 m m 1 m 3 3.1 i 0.63 0.64 1.44 1.37 3. 3.1 Ti Tim A -T C -Ti 9 A 9 - - 9 NA NC A A H H A A H Y DNA -NC N -NCN A- DN A-N 0 c cDN cD -MYH9 DN -M NA D NA D MY p pc cDN GAPDH p p iRN A p iRN pc A h h h h h iR A-MYpc R pc A- N s N 0 4 8 6 N +s + R 2 4 72 9 +s RN +si iR iR si si + +s +s +

pcDNA3.1 pcDNA3.1 pcDNA-Tim pcDNA-Tim HCT116 d RKO on f

+si-NC +si-MYH9 +si-NC +si-MYH9 ti 800 a

r 1000

g ** * ** HCT116 i ** 600 800 m ** ** ll e HCT116 ll migration 600

c 400 e Snail f

o 400 200 er 200 0.52 0.35 0.75 0.47 mb

0 mber of c u 0 E-cadherin N

m m Nu 1 RKO 3.1 i i . .1 im C A C 9 3 3 T Tim H9 A C A 9 - - 9 0.58 0.83 0.40 0.69 N A-T N N H A NC A H DNA3.1 DN Y NA-T - - Y Y c A-N M A MYH N A- N p pc - D DN cD M D M A c c A- p NA pcDN - c N RN p p iR A p iR pcD A- N-cadherin N siRN s N +si iR + +s iRN + R s s e + +siRN + +si 0.69 0.57 1.14 0.72 n

o RKO

HCT116 i GAPDH 600 80

** ** as v ** **

n 60 HCT116 i

invasion ** RKO

l 400 * cell 40

of Snail 200

er 20 b 0.30 0.17 0.66 0.54 m RKO 0 0 Nu E-cadherin Number of cel 1 1 1 1 . . im m . im im 3 3 Ti 3. T A C A 9 -T C - 9 A A3 9 -T - 9 N N H A N A NC A A H - Y - YH - -NC Y A N A A DN N cD -M DN -M 0.93 1.5 0.49 0.63 c -MYHD pcDN N p cD N c pcDN N p c A p iR p p iRNApcDN A-M N s NA siR NA s N +siR R + g + + R i iR i s s + +siR +s + N-cadherin HCT116 si-NC si-MYH9 h 0.50 0.44 0.59 0.49 Cytosol Nuclear Cytosol Nuclear GAPDH ß-Catenin HCT116 RKO 0.85 0.22 ß-Catenin i Myosin-9 0.50 0.35 0.84 0.22 0.46 0.28 0.86 0.30 HCT116 0.54 0.19 Myosin-9 GAPDH 0.23 0.19 0.63 0.20 0.47 0.31 1.14 0.14 ß-Catenin GAPDH RKO 0.25 0.13 0.62 0.53 GAPDH ß-Catenin Histone H3 RKO 0.26 0.13 ß-Catenin Myosin-9 j 0.50 0.28 0.66 0.48 0.14 0.04 Input k GAPDH GAPDH Myosin-9 Input FLAG IP si-NC si-Tim ß-Catenin IgG anti-MYH9 IgG anti-MYH9 GAPDH Timeless Myosin-9 IP: FLAG Myosin-9 ß-Catenin GAPDH FLAG

Fig. 7 Timeless activates β-catenin through the Myosin-9/β-catenin pathway. Timeless overexpressing HCT116 and RKO cells were transfected with Myosin-9 specific si-MYH9 (pcDNA-Tim + si-MYH9) or control si-NC (pcDNA-Tim + si-NC). (a) Expression levels of Timeless and Myosin-9 in HCT116 and RKO cells were measured by western blotting. CCK8 (b) and colony-forming growth assays (c) were used to determine the proliferation rate of the indicated CRC cells. Transwell assays were performed to test the migration (d) and invasion (e) of the indicated CRC cells. (f) EMT-associated proteins in the indicated CRC cells were measured by western blotting. Expression level of total β-catenin (g) and subcellular β-catenin (h) in Myosin-9 knockdown cells were detected with Western blot. (i) Expression level of β-catenin was detected in CRC cells with Timeless overexpression and Myosin-9 knockdown. (j) Immunoprecipitation (IP) assay was performed to detect interactions between Myosin-9 and β-catenin. (k) Immunoprecipitation (IP) with anti-Myosin-9 was performed to detect the level of β-catenin in the cells with Timeless knockdown. Error bars indicate the means ± SD. *P < 0.05, **P < 0.01 Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 14 of 17

a

bc d HCT116 RKO HCT116 of 25 ** 1.5 1.5 RKO Timeless

nput) 20 fi vel of Tim * ** 15 e 1.0 1.04 0.45 1.13 0.39 ichment 1.0 %o

enr 10 H3K27ac ac ( 0.5 0.5 ive 5 1.69 0.26

K27 1.29 0.70 at ive mRNA level of Tim el

0 t H3 0.0 0.0 lative mRNA l R

HCT116 CX-1 e 6 46 Histone H3

R SO SO 64 Rela 6 M C C D DM 1.21 1.12 2.59 2.69 GAPDH

efHCT116 g f

o 40 t

t) 40 HCT116 n ** HCT116 RKO u

ent of 1.5

np 30 el 1.5 me el i 30 ** si-NC

m si-NC v ev e ch l of si-CBP l si-CBP ch 20 ** ** ** % 20 1.0 1.0 ** nri NA e ac ( mRNA 10 10 mR ve e 0.5 e 0.5 ative enri iv l tiv 3K27ac (% of input) e

0 0 at H H3K27 l Relati si-NC si-CBP R si-NC si-P300 Rela 0.0 Re 0.0 CBP TIMELESS CBP TIMELESS CBP P300

h i HCT116 RKO HCT116 l 1.5 1.5 RKO

ve si-NC vel si-NC e ** ** si-P300 ** ** si-P300 1.0 1.0 CBP 0.44 0.20 1.59 1.07 0.5 0.5 Timeless lative mRNA l

Relative mRNA le 0.0 Re 0.0 1.31 0.67 P300 TIMELESS P300 TIMELESS 0.50 0.23 H3K27ac j HCT116 RKO 1.16 0.63 0.73 0.40 GAPDH

P300 0.20 0.09 0.55 0.25 Timeless 0.41 0.03 0.45 0.13 H3K27ac

0.42 0.17 0.80 0.32 GAPDH

Fig. 8 Timeless is transcriptionally activated by H3K27 acetylation. (a) Genome bioinformatics analysis showed that the Timeless promoter was highly enriched for H3K27ac.(cited from http://genome.ucsc.edu/)(b) Chromatin immunoprecipitation (ChIP) was performed to measure the H3K27ac enrichment in HCT116 cells with high a Timeless level and CX-1 cells with a low Timeless level. Timeless expression was measured by RT-qPCR (c) and protein levels of Timeless, H3K27ac, and total Histone-3 were measured by western blotting (d) in HCT116 and RKO cells treated with C646 or DMSO for 48 h. The enrichment of H3K27ac in the Timeless promoter was assessed via ChIP assay in HCT116 cells silenced by CBP (e) and P300 (f). Timeless, CBP (g) and P300 (h) mRNA expressions were measured by RT-qPCR and protein levels of CBP (i), P300 (j), Timeless, and H3K27ac in HCT116 and RKO cells were measured by western blotting and epigenetic regulatory mechanisms such as hypome- the promoter region of Timeless was highly enriched for thylation of the Timeless promoter, may mediate the ex- H3K27ac, and ChIP assay confirmed H3K27ac enrich- pression of Timeless in breast cancer [30]. Histone ment in CRC cells with high Timeless expression, com- acetylation modification has been proven as an import- pared with CRC cells with low Timeless expression. The ant epigenetic mechanism that regulates cancer-related results of histone acetyltransferase (HAT) inhibitor C646 genes [28]. In our study, bioinformatic analysis showed treatment and CBP or P300 knock-down confirmed that Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 15 of 17

Fig. 9 Scheme showing the proposed mechanisms involved in the effect of Timeless in CRC. H3K27 acetylation-induced Timeless bound to and stabilized Myosin-9, thereby activating the β-catenin signal pathway, which promoted colorectal cancer tumorigenesis histone acetylation regulated Timeless expression. These interactions between Timeless and β-catenin, indicating results suggest that the high expression of Timeless in that other molecules probably mediate the activation of β- CRC may arises from the enhancement of histone catenin pathway by Timeless. To reveal the mechanism of acetylation. how Timeless is involved in CRC, we performed IP with Timeless is mainly involved in the proliferation, apop- anti-Timeless antibodies and mass spectrometry followed tosis, and metastasis of tumor cells via multiple signaling by IP verification. We found that Timeless bound to pathways. Timeless boosts the progression of breast can- Myosin-9, which improved its stability. cer through activating MYC [20]. Furthermore, Timeless Myosin-9 encodes nonmuscle myosin IIA (NMIIA), a confers nasopharyngeal carcinoma with cisplatin resist- myosin II superfamily of motor proteins involved in im- ance by activating the Wnt/β-catenin signaling pathway portant functions, including cytokinesis, cell motility, and promoting epithelial mesenchymal transition [22]. and the maintenance of cell shape. The role of Myosin-9 Moreover, a recent study has shown that Timeless up- in cancer is controversial and depends on tumor type. regulation together with Claspin can protect cancer cells Several studies have reported that Myosin-9 is an onco- from oncogene-induced replication stress depending on gene that promotes cell proliferation, invasion, and a checkpoint-independent mechanism [36]. We also metastasis in various cancers [26, 27, 40, 41]. For ex- found that Timeless overexpression increased the levels ample, Myosin-9 expression is significantly correlated of total and nuclear β-catenin and its downstream pro- with the depth of wall invasion, lymph node metastasis, teins, including Cyclin D1, LEF1, and Axin2. However, distant metastasis, and TNM stage in gastric cancer [42]. the knockdown of Timeless reversed these changes. Myosin-9 knockdown with a specific siRNA inhibits Wnt/β-catenin pathway, also termed as canonical Wnt CRC cell growth and invasion [26]. In contrast, a study pathway, is involved in diverse cellular processes, using a direct in vivo RNA interference (RNAi) strategy including embryonic development, cell proliferation, dif- has found Myosin-9 knockdown triggers the formation ferentiation, migration, and survival [37, 38]. Hyperacti- of invasive squamous cell carcinoma in tumor- vation of Wnt/β-catenin signaling is implicated in susceptible backgrounds by regulating p53 stability, numerous cancers, including CRC [38]. The level and suggesting Myosin-9 is a tumor suppressor [43]. In the cellular location of β-catenin are critical in the canonical present study, Myosin-9 knockdown inhibited and res- Wnt pathway [39]. Our results together with other stud- cued Timeless-induced CRC proliferation, migration, in- ies, demonstrated that Timeless increased β-catenin ex- vasion, EMT, and β-catenin signaling, indicating it is an pression and nuclear translocation to activate the Wnt/ oncogenic and guards the cancer-promoting effects of β-catenin signaling pathway. Timeless. Myosin-9 exerts its oncogenic effect through Several studies have shown that Timeless mediates its promoting β-catenin nuclear translocation [27]. In the effects by binding to other proteins such as Timeless- present study, we showed Myosin-9 bound to β-catenin interacting protein (Tipin) to form a stable complex [17]. and induced its nuclear translocation, and this inter- However, in the present study, we did not detect any action was promoted by Timeless. In our study, Timeless Cao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:162 Page 16 of 17

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