Circadian Clock Gene Bmal1 Inhibits ...mechanisms underlying Bmal1 functional involvements in dif-ferent tumor types remain largely unknown. Substantial evidences indicated that the

Therapeutics, Targets, and Chemical Biology

Circadian Clock Gene Bmal1 InhibitsTumorigenesis and IncreasesPaclitaxel Sensitivityin Tongue Squamous Cell CarcinomaQingming Tang, Bo Cheng, Mengru Xie, Yatao Chen, Jiajia Zhao, Xin Zhou,and Lili Chen

Abstract

Circadian clock genes regulate cancer development and che-motherapy susceptibility. Accordingly, chronotherapy based oncircadian phenotypes might be applied to improve therapeuticefficacy. In this study, we investigated whether the circadian clockgene Bmal1 inhibited tumor development and increased pacli-taxel sensitivity in tongue squamous cell carcinoma (TSCC).Bmal1 expression was downregulated and its rhythmic patternof expression was affected in TSCC samples and cell lines. EctopicBmal1 inhibited cell proliferation,migration and invasion in vitro,and tumor growth in mouse xenograft models of TSCC. Afterexposure to paclitaxel, Bmal1-overexpressing cells displayed a

relative increase in apoptosis and were more susceptible topaclitaxel treatment in vivo. Mechanistic investigations suggesteda regulatory connection between Bmal1, TERT, and the oncogenictranscriptional repressor EZH2 (enhancer of zeste homolog 2),the recruitment of which to the TERT promoter increased pacli-taxel-induced apoptosis and cell growth inhibition. Clinically,paclitaxel efficacy correlated positively with Bmal1 expressionlevels in TSCC. Overall, our results identified Bmal1 as a noveltumor suppressor gene that elevates the sensitivity of cancer cellsto paclitaxel, with potential implications as a chronotherapytiming biomarker in TSCC. Cancer Res; 77(2); 532–44. �2016 AACR.

IntroductionMost physiological, biochemical, and behavioral processes in

mammals follow circadian rhythm, which is generated by anendogenous clock system with an approximately 24-hour cycle(1, 2). Molecularly, mammalian circadian clock comprises aseries of interlocking transcription–translation feedback loopsin every cell (3). In mammals, Bmal1 is required for normalcircadian rhythm maintenance. Notably, in Bmal1-deficientmouse model, Bmal1 was shown to be the only clock geneaffecting all rhythmic behaviors (4). In addition to the func-tional roles in biologic rhythm, increasing evidences indicatethat Bmal1 participates in the hallmarks of cancer, includingproliferative signaling maintenance, cell death resistance, repli-cative immortality, invasion and metastasis activation, andenergy metabolism reconfiguration (5–9). Chronic circadianmisalignment, such as fragmented sleep, significantly acceleratesthe incidence of various tumors (10, 11). Also, it has beenreported that Bmal1 has antitumor roles in ovarian cancer

(12), colorectal cancer (13), hematologic malignancies (7), andhead and neck squamous cell carcinoma (14). Nevertheless, themechanisms underlying Bmal1 functional involvements in dif-ferent tumor types remain largely unknown.

Substantial evidences indicated that the circadian clock canmodulate the efficacy of anticancer therapy. Thus far, more than15 different anticancer drugs have been reported to exhibitstrong time-of-administration effects on their efficacy (15–17),leading to the innovative idea of chronotherapy. Recent studiesdemonstrated that circadian system regulates critical molecularevents rhythmically, including drug metabolism and detoxifica-tion, molecular targets, cell cycle, DNA repair, and apoptosis(16, 18–21). Being orchestrated by the circadian system, thehierarchical organization of these processes determines the ther-apeutic effects (15). Although chronotherapeutic approach pro-vided encouraging results, it has not been routinely deployed inclinical practice due to the lack ofmechanistic basis of appropriatetherapeutic timing.

Head and neck squamous cell carcinoma (HNSCC) is rankedas the sixth most prevalent type of cancer worldwide (22).Particularly, tongue squamous cell carcinoma (TSCC) is a com-mon formofHNSCC. In spite of progresses in various therapeuticmethods during past 30 years, the overall 5-year survival rate ofTSCC patients remains less than 50% for all stages (23, 24).Paclitaxel, an antineoplastic agent, has been used as the standardfirst-line chemotherapy drug for TSCC cancer (25). But the out-come of patients receiving paclitaxel treatments remains compli-cated by unpredictable efficacy and toxicities. By improving theefficacy and the tolerability of agents through administration oftreatments according to biological rhythms, chronotherapeuticsmay help improving the therapeutic effects of paclitaxel. Unfortu-nately, the mechanisms underlying circadian clock gene Bmal1

Department of Stomatology, Union Hospital, Tongji Medical College, HuazhongUniversity of Science and Technology, Wuhan, Hubei, P.R. China

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Q. Tang and B. Cheng contributed equally to this article.

Corresponding Author: Lili Chen, Department of Stomatology, Union Hospital,Tongji Medical College, Huazhong University of Science and Technology, 1277JiefangAvenue,Wuhan430022, P.R. China. Phone: 8627-8572-6949; Fax: 8627-8572-6949; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-16-1322

�2016 American Association for Cancer Research.

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regulation of TSCC tumorigenesis and paclitaxel chronotherapyremain vague, which hindered appropriate timing of paclitaxelapplication.

In this study, we demonstrated that Bmal1 inhibits TSCC cellsproliferation, migration, and invasion. In xenograft models, weobserved that Bmal1 suppresses tumor growth.We next identifiedTERT as a downstream gene of Bmal1. In TSCC cells, transcriptionof TERT can be regulated by Bmal1. Furthermore, we found thatTERT mediates Bmal1-driven sensitivity to paclitaxel in TSCCcancer. Paclitaxel efficacy was highly consistent with the expres-sion level of Bmal1, suggesting that Bmal1 may represent a noveldiagnostic biomarker of host-specific paclitaxel therapeutic tim-ing in TSCC.

Materials and MethodsPatient tissue samples

A total of 53 cases of TSCC tissue specimens andpaired adjacentnoncancerous tissue specimens were obtained frompathological-ly diagnosed TSCC patients, which had not received chemother-apy and radiotherapy, at the Union Hospital of Tongji MedicalCollege, Huazhong University of Science and Technology(Wuhan, China) from June 2013 to September 2015. Fifty casesof normal tongue tissue specimens were obtained from noncan-cerous patientswhohadundergoneoperative resectionduring thesame period. All patients signed an appropriate consent form forbiobanking. As all operations were first-start elective cases, tissuespecimens were obtained at a consistent time point, between10:00 am and 3:00 pm. Clinical pathologic characteristics includ-ing age, sex, bodymass index, fasting blood glucose, TNM staging,tumor differentiation, and lymph node metastasis are listed inSupplementary Table S1. This study was approved by the Insti-tutional Research Ethics Committee of Tongji Medical College(Wuhan, China).

Cell cultureHuman TSCC cell lines (SCC9, SCC25, and CAL27) obtained

from ATCC 3 years ago were maintained at 37�C in 5% CO2 andair humidified incubator. All cell lines were tested and authen-ticated semiannually by short tandem repeat DNA fingerprint-ing in China Center for Type Culture Collection (Wuhan,China), with the recent test done in September 2016. In theconclusions of recent test, all the locations of SCC9, SCC25, andCAL27 were exactly matched with the locations found in ATCCcell banks.

Isolation, culture, and identification of keratinocytesHuman tongue keratinocytes (KC) were isolated and cul-

tured as previously described (26). Briefly, human tongueepithelial tissues were obtained from tongue noncancerousdisease patients (16–30 years old) during operative resectionunder informed consent. The samples were washed severaltimes in sterile PBS (HyClone) containing 1% penicillin–strep-tomycin (HyClone). The samples were separated from under-lying muscle or connective tissue and minced using sterilemicrosurgical instruments, then treated with 4 mg/mL dispaseII (Gibco) overnight at 4�C to separate epidermis. The epider-mal layer was lifted and digested with Trypsin-EDTA (Invitro-gen) for 20 minutes to isolate KCs. The isolated cells werecultured at 37�C in 5.0% CO2, with medium (Promocell)changed every 2 to 3 days, after which the KCs were authen-

ticated by immunofluorescence assay with rabbit antibodies ofanti-cytokeratin 19 (Abcam) and anti-vimentin (Abcam).

Circadian rhythm inductionThe medium was removed from confluent cultures of TSCC

cells or KCs in 6-well plates and replaced with DMEM:Ham's F-121:1 medium containing 1% penicillin–streptomycin supple-mented with 1 mmol/L dexamethasone (Sigma). The cells wereexposed to inductive agents for 1 hour, followed by replacingwithfresh medium (set this timing as circadian time 0, CT0). The cellsdid not receive any further medium changes from this pointonward until the time of harvest. Individual plate was harvestedfor total RNA at CT0, CT4, CT8, CT12, CT16, CT20, CT24, CT28,CT32, CT36, CT40, CT44, and CT48.

Plasmids and transfectionGuide RNAs targeting for human Bmal1 gene were designed

using Optimized Crispr Design (http://crispr.mit.edu/). Synthe-sizedDNAoligoswere inserted into CRISP/Cas9 vectorU6-gRNA.SCC9 cells were transiently transfected with a pool of plasmids,which encode Cas9 nuclease and guide RNAs targeting Bmal1 orthe vector for wild-type control. To overexpress Bmal1, TERT, andEZH2 in TSCC cells, an expression construct was generated bysubcloning PCR-amplified full-length human Bmal1, TERT, orEZH2 cDNA into a pcDNA3.1-Myc-His(þ) vector (Genechem);an empty vector was used as a negative control. To stably knockdown TERT or EZH2 in TSCC cells, small-hairpin RNA wasdesigned and inserted into pLKO.1 vector (Genechem); a scram-ble shRNAwasused asnegative control. All primer sequences usedin this study are shown in Supplementary Table S2. Multiplemonoclonal single-cell clones were picked and cultured individ-ually in separate wells. The final efficiencies for knockdown oroverexpression were assessed by Western blot analysis.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assays were con-

ducted using Chromatin Immunoprecipitation Kit (Millipore)following the manufacturer's protocol. Briefly, TSCC cells werecross-linked and lysed. Cell lysates were sonicated into fragmentsof an average size of 200 to 500 bp and then precleared withProteinA/GAgarose beads for 1hour. After centrifugation, 10%ofsupernatants were stored as "Input," and the remaining super-natants were divided into three parts, one for Bmal1 antibodydetection, one for EZH2 antibody detection (or for anti-RNAPolymerase II, positive control), and the rest for IgG reactivity(negative control). The next day, immune complexes were pre-cipitated and rinsed. All samples including inputs were incubatedat 65�C for 4 to 6 hours in the presence of proteinase K and RNaseA for reverse crosslinks of protein–DNA complexes. DNA waspurified in a final volume of 50 mL and subjected to PCR.

After reviewing previous literatures and searching GenBankdatabase, we screened the 50-flanking region of human TERT geneand designed four PCR primer sets to cover the putative Bmal1-binding sites in its promoter (Supplementary Table S2). Theamount of immunoprecipitated DNA was calculated in referenceto a standard curve and normalized to input DNA.

Luciferase reporter assaySCC9 cells that stably transfected with pcDNA3.1-Bmal1,

pcDNA3.1-EZH2, EZH2-shRNA, or empty vehicle were platedat 1 � 105 cells per well in 24-well plates. Firefly luciferase

Bmal1 Inhibits Tumorigenesis and Increases PTX Sensitivity

www.aacrjournals.org Cancer Res; 77(2) January 15, 2017 533




reporter vectors (100 ng) and Renilla luciferase reporter vectorspRL-SV40 (10 ng; Promega) were cotransfected into cells usingLipofectamine 2000 (Invitrogen) as instructed. Cell extractswere prepared 48 hours after transfection. Firefly and Renillaluciferase activity were consecutively measured using Dual-Luciferase Reporter Assay System (Promega). For TERT pro-moter activity, the luciferase signal was normalized by firefly/Renilla ratio.

RNA sequenceTotal RNA was extracted from SCC9/Bmal1 and SCC9/vehicle

cells. Then, the sequencing library of each RNA sample wasprepared using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scien-tific). The final Template-Positive Ion PI Ion Sphere particles wereenriched and loaded onto the Ion PITM chip. Later, raw reads�50bp that passed the filtering threshold were used for mapping.RPKM values were used for gene quantification and the upperquartile was used for correction.

Xenografted tumor modelA cohort of 180 male BALB/c nude mice (4 weeks old) was

purchased from the Beijing HFK Bioscience Co. Ltd. All mice weremaintained under 12/12-hour light/dark cycles with the lights onfrom 8 am (zeitgeber time 0, ZT0) to 8 pm (ZT12) and fed withantibiotic-free food and water ad libitum. All animal-related pro-cedures were performed according to the ethical guidelines andwere approved by the Institutional Animal Care and Use Com-mittee of Tongji Medical College (IACUC number: 483). Two invivo experiments were designed. In experiment 1, the mice wererandomly divided into SCC9/wild-type, SCC9/Bmal1 (Bmal1overexpression), and SCC9/Bmal1-KO (Bmal1 knockdown)groups (n ¼ 30 per group), and tumor cells were inoculated intonudemice by subcutaneous injection of 300-mL saline containing3 � 106 cells in the right upper back. Five weeks later, the micewere sacrificed at indicated time points (ZT2, ZT6, ZT10, ZT14,ZT18, andZT22) to compare tumorweight and volume. Similarly,in experiment 2, the mice were randomly divided into SCC9/Bmal1, SCC9/Bmal1-KO, and SCC9/wild-type groups (n¼ 30 pergroup). Two weeks after cell inoculation, paclitaxel (SelleckChemicals) was administered at a dose of 20 mg/kg at aboveindicated time points twice a week for 4 weeks. On day 42, themice were euthanized and the neoplasms were excised andweighed. All tumors were measured weekly, the length and widthof the tumors were obtained with calipers, and the volume wascalculated.

Other methodsWestern blot analysis, IHC, and immunofluorescence, quanti-

tative real-timeRT-PCRanalysis, scratchwound-healing assay, cellproliferation and cytotoxicity assays,Matrigel invasion assay,flowcytometry, and coimmunoprecipitation assay were performedusing standard protocol. See Supplementary Methods for moredetails.

Statistical analysisUnless otherwise stated, all data are shown asmean� SEM. The

SPSS version 18.0 statistical software was used for statisticalanalysis. Statistically significant differences between groups weredetermined by a two-tailed, paired Student t test, or ANOVA. AP value of < 0.05 was considered with statistical significance inall cases.

ResultsBmal1 expression level and rhythmic pattern are affected inTSCC patients and TSCC cell lines

Compared with the normal tongue epithelial tissues at eachtime point, as shown in Fig. 1A, the average mRNA levels ofBmal1were significantly reduced in both TSCC tissues and pairedadjacent noncancerous tissues. Similarly, the levels of Bmal1protein expressions were also downregulated compared withTSCC tissues and paired adjacent noncancerous tissues (Fig. 1Cand D). Consistently, Bmal1 mRNA and protein levels in threeTSCC cell lines were lower than that in the normal tongue KCsafter synchronization (Fig. 1B, E, and F). Compared with KCs,we also noticed that human tongue cancerous cells displayeddifferent Bmal1 expression rhythmic pattern characterized byshorter cycle and weaker amplitude (Fig. 1B).

Bmal1 overexpression inhibits TSCC cell proliferation,migration, and invasion in vitro

To determine the effects of Bmal1 expression on TSCC cellproliferation, migration, and invasion, we either overexpressedBmal1 in SCC9, SCC25, andCAL27 cells or knocked down Bmal1in SCC9 cells (Fig. 2A and C). We found that overexpression ofBmal1 caused significantly decreased cell proliferation, slowerwound-healing rate, and fewerMatrigel cells invasion in all SCC9,SCC25, and CAL27 cells (Fig. 2B, E, and G). We next performedadditional expression analyses to confirm the effects of Bmal1overexpression. Accordingly, EMT-associated transcription factorsZeb1/2, Twist, and Snail1 were all significantly downregulated inBmal1-overexpressing TSCC cells (Fig. 2I). In contrast, Bmal1knockdown resulted in significant increases of SCC9 cells prolif-eration, migration, and invasion (Fig. 2D, F, H, and J).

Bmal1 overexpression suppresses tumor growth in xenograftmodels

To determine whether Bmal1 has the tumor-suppressive effectin vivo, wild-type, Bmal1 knockdown, and Bmal1-overexpressingSCC9 cellswere subcutaneously implanted into the right back sideof nude mice. Toward this end, we observed that the xenograftsderived from Bmal1 knockdown SCC9 cells grew significantlyfaster than those from wild-type SCC9 cells. However, the xeno-grafts derived fromBmal1-overexpressing cellsweremuch smallerthan those fromwild-type SCC9 cells (Fig. 2K–M). Together, thesephenotypic results suggested that Bmal1 might act as a tumorsuppressor gene in TSCC.

Bmal1 overexpression increases paclitaxel sensitivity in vitroTo determine whether Bmal1 could enhance paclitaxel anti-

cancer effects on TSCC cells, we examined apoptosis and relatedsignal pathways in Bmal1-overexpressing SCC9, SCC25, andCAL27 cells and Bmal1-knockdown SCC9 cells. We observed thatBmal1-overexpressing cells were more sensitive to paclitaxel thancontrol cells (Fig. 3A; Supplementary Fig. S1A and S1B). Instead,the Bmal1 knockdown SCC9 cells were more resistant to pacli-taxel (Fig. 3B). Flow cytometry results showed that overexpressionof Bmal1 led to increase of apoptosis in all three cell lines treatedwith paclitaxel (Fig. 3C; Supplementary Fig. S1C and S1D),whereas knockdown of Bmal1 caused a decrease in apoptosis inSCC9 cells treatedwith paclitaxel (Supplementary Fig. S2A). Next,we examined S-phase and G2–M phase cell number of Bmal1-overexpressing cells treated with paclitaxel. We observed signif-icant cell number increase in S-phase and moderate cell number

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increase in G2–M phase (Fig. 3D; Supplementary Fig. S1E andS1F). In contrast, knockdown of Bmal1 resulted in cell numberdecrease in S-phase compared with wild-type cells (Supplemen-tary Fig. S2B). Western blot assay further demonstrated that theexpression levels of proapoptotic protein BAX, BAD, cleavedcaspase-3, and AIFwere increased in Bmal1-overexpressing SCC9,SCC25, and CAL27 cells, whereas the expression levels of anti-apoptosis protein XIAP and BCL2 were decreased (Fig. 3E andSupplementary Fig. S1G and S1H). However, knockdown ofBmal1 in SCC9 cells manifested an opposite trend for theseapoptosis-related proteins (Supplementary Fig. S2C).

TERT expression level is significantly downregulated inBmal1-overexpressing TSCC cells

In an effort to identify affected biological processes in Bmal1-overexpressing cells, we performed genome-wide RNA-seq toacquire transcriptional profile (Fig. 4A). We found that a totalof 3,747 genes expression were significantly affected in SCC9/Bmal1 cells (Fig. 4B and Supplementary Table S3). GO analysisrevealed that the differentially expressed genes were enrichedin biological processes including cell division, cell differentia-tion, cell migration, cell apoptosis, cell cycle, and proliferation(Fig. 4C). To identify potential Bmal1 target genes, we next

Figure 1.

Bmal1 expression level and rhythmicpattern are affected in TSCC. A and B,The mRNA level of Bmal1 in TSCCsamples (A) and TSCC cell lines (B) ateach time point. ANT, adjacentnoncancerous tissues; Normal, normaltongue epithelial tissues; KCs, humantongue keratinocytes. C,Representative Western blot analysisand densitometry graph show Bmal1protein expressions in TSCC samples.GAPDH was used as the loadingcontrol. (C, cancer; A, ANT; N, normal).D, Immunofluorescence assay forBmal1 (green) in TSCC samples. Nucleiwere stained by DAPI (blue). Scalebars, 100 mm. E,Western blot analysisof Bmal1 protein expression in threeTSCC cell lines and KCs. F,Immunofluorescence assay for Bmal1in three TSCC cell lines and KC cells.�� , P < 0.01.






focused on the selected gene set (Fig. 4D) and performedqRT-PCR to verify the expression downregulation of selectedgenes. In the group of genes we tested, TERT expression wassignificantly downregulated (Fig. 4E). Furthermore, differen-

tially expressed genes were mapped to the reference canonicalpathways in the KEGG database to probe Bmal1-related intra-cellular signaling in an unbiased fashion. The KEGG pathwayanalysis revealed that cellular signaling pathways involved in

Figure 2.

Bmal1 inhibits TSCC cell proliferation, migration, and invasion in vitro and in vivo. A, Western blot analysis of Bmal1 protein expressions in TSCC cellstransfected with empty vector (vehicle) and Bmal1. B, Bmal1 overexpression inhibited cell growth of TSCC cells. C, Western blot analysis of Bmal1 proteinexpressions in SCC9/Bmal1-KO cells. D, Bmal1 knockout promoted SCC9 cell growth. E and F, Representative microphotographs of wound closureassays (magnification, �100). Black lines mark migration fronts. G and H, Representative microphotographs of invasion assays (magnification, �200).I and J, The mRNA levels of EMT-associated transcription factors Zeb1/2, Twist, and Snail1 in Bmal1-overexpressing TSCC cells (I) and SCC9/Bmal1-KO cells (J).K–M, Bmal1 suppressed tumor growth in xenograft models. Representative image of tumors formed (K), mean tumor weights (L), and tumor volumegrowth curves (M) are shown. The experiments were performed independently for three times. � , P < 0.05; �� , P < 0.01.

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Figure 3.

Bmal1 overexpression increases paclitaxel sensitivity in vitro. A and B, Dose-dependent growth inhibition in response to paclitaxel (PTX) in SCC9/Bmal1 (A) andSCC9/Bmal1-KO cells (B). C, Apoptosis was evaluated by flow cytometry of SCC9/Bmal1 and SCC9/vehicle cells stained with Annexin V and PI. D, Cell-cyclephases were determined by flow cytometry after treatment with paclitaxel (0, 10, or 20 nmol/L, 48 hours). E, Western blot analysis of Bmal1, BAX, BAD, caspase-3–cleaved, AIF, XIAP, BCL2, and GAPDH (for loading controls) in SCC9/Bmal1 and SCC9/vehicle cells treated with 10 or 20 nmol/L paclitaxel. All experimentswere performed at least three times. � , P < 0.05; ��, P < 0.01; ��, P < 0.001.






Figure 4.

TERT expression level is significantly downregulated in Bmal1-overexpressing TSCC cells. A, Hierarchical clustering of quantitative genes expressionprofiling for SCC9/Bmal1 cells and SCC9/vehicle cells. B, Volcano plot of differentially expressed genes (DEG) between SCC9/Bmal1 cells and SCC9/vehiclecells. C, Gene ontology (GO) assignment of the top 15 biological processes (BP) associated with differentially expressed genes (ranked by P value). D,Heatmap shows some differentially expressed genes that were related to paclitaxel sensitivity. E, Confirmation of the differentially expressed genesby qRT-PCR. F, Interactome network shows that Bmal1 was largely involved in regulating TERT. �� , P < 0.001.

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the feedback regulation of TERT, such as Wnt pathway, NF-kBpathway, TGFb pathway, and PI3K-Akt pathway, variated inSCC9/Bmal1 cells (Fig. 4F). Taken together, these results sug-gested a regulatory connection between Bmal1 and TERT inTSCC.

TERT negatively correlates with Bmal1 in paclitaxel sensitivitymodulation

To gain insight into the mechanisms by which Bmal1improves paclitaxel sensitivity, we analyzed a panel of 53primary TSCC tissues and found a significantly negative corre-lation between Bma11 and TERT expression levels (Fig. 5A).Indeed, the level of TERT protein was greatly downregulated inBmal1-overexpressing TSCC cells (Fig. 5B). To determine wheth-er TERT also participates in paclitaxel sensitivity, we assayed theTERT overexpression effects on apoptosis and S-phase arrest inpaclitaxel-treated SCC9 cells. We observed that paclitaxel-induced apoptosis enhancement and S-phase arrest were signif-icantly reversed (Fig. 5C and D). Moreover, compared withBmal1-overexpressing cells, expression level changes of proa-poptotic protein BAX, BAD, cleaved caspase-3, AIF, and anti-apoptosis protein BCL2 were all reversed in paclitaxel-treatedBmal1 and TERT co-overexpressing cells (Fig. 5E). In addition,we found that shRNA-mediated human TERT knockdown couldupregulate cleaved caspase-3 and downregulate BCL2 in SCC9cells simultaneously (Supplementary Fig. S3A). Knockdown ofTERT rendered SCC9 cells more sensitive to paclitaxel treatment(Supplementary Fig. S3B and S3C). Taken together, these obser-vations indicated that TERT negatively correlates with Bmal1 inpaclitaxel sensitivity modulation.

Bmal1-mediated TERT expression inhibition requires EZH2As a widely acknowledged well-known transcription factor

(27), Bmal1 fulfills its function by activating the transcription ofdownstream target genes. Surprisingly, instead, we found thatoverexpression of Bmal1 led to TERT downregulation. To addressthis discrepancy, ChIP analysis was deployed to examine Bmal1-binding site within TERT promoter region. Our results indicatedthat Bmal1 selectively bound to the promoter (–1,857/–1,590,268 bp) of TERT (Fig. 6A), but not to the two putative Bmal1E-box (CACGTG) sites located at positions �184 and þ25 (datanot shown). A recent study has shown that EZH2 was requiredfor Bmal1 to suppress the transcription of Period 1 and Period 2,and was important for the maintenance of circadian rhythms(28). In this study, we further confirmed that EZH2 was recruit-ed to the same region of TERT promoter (–1,857/–1,590, 268bp; Fig. 6B). We then examined whether there is protein–proteininteraction between Bmal1 and EZH2. As shown in Fig. 6D, EZH2could interact with Bmal1 in SCC9, SCC25, and CAL27 celllines. Double immunofluorescence staining showed that Bmal1and EZH2 colocalized well in nucleus (Fig. 6C).

In Figure 6E, we showed that overexpression of Bmal1inhibited activity of TERT promoter, but not those of the TERTpromoter mutants with Bmal1 binding site being either deletedor mutated. To determine whether EZH2 is required for Bmal1-mediated inhibition of TERT expression, we assayed the activityof TERT promoter in the background of either overexpressionor knockdown of EZH2. Overexpression of EZH2 failed toaffect Bmal1-mediated inhibition of the activity of wild-typeTERT promoter. However, knockdown of EZH2 can fullyreverse the Bmal1-mediated TERT promoter inhibition and

enhanced TERT promoter activity (Fig. 6E). Together, thesefindings suggested that Bmal1-mediated TERT expression inhi-bition requires EZH2.

Paclitaxel chronotherapy efficacy is correlated with fluctuationof Bmal1 mRNA level

On the basis of previous reports, chronotherapy could poten-tially augment therapeutic efficacy of paclitaxel (29). Neverthe-less, we do not know whether or not there is correlation betweencircadian fluctuation of clock gene Bmal1 and paclitaxel efficacy.We first characterized the circadian features of Bmal1 expressionin human TSCC cell lines by examining their expression patternsin xenografts generated fromBmal1-overexpressing SCC9 (SCC9/Bmal1), Bmal1 knockdown SCC9 (SCC9/Bmal1-KO), and SCC9wild-type cells (SCC9/wild type). In SCC9/Bmal1 group, Bmal1mRNA level reached its peak around ZT10 and the trough aroundZT22 (Fig. 7A). In SCC9/wild-type group, the peak and the troughof Bmal1 mRNA level were around ZT6 and ZT18, respectively(Fig. 7B). In SCC9/Bmal1-KOgroup, however, we noticed that theBmal1 circadian phenotype was dubious (Fig. 7C). In addition,we also observed that the circadian pattern of TERT expressionlevel was contrary to Bmal1 in all three groups (Fig. 7A–C).

To determine the circadian correlation between Bmal1 andpaclitaxel efficacy at animal level, above TSCC cells were injectedsubcutaneously into BALB/c nude mice. We observed that thevolume and the weight of SCC9/Bmal1-derived tumors weresmallest in three groups. The xenografts growth was slowest inSCC9/Bmal1 group treated with paclitaxel at Bmal1 expressionpeak point ZT10 (Fig. 7D, G, and J). In SCC9/wild-type group, thexenografts growth was lowest with paclitaxel treatment at Bmal1expressionpeakpoint ZT6 (Fig. 7E,H, andK). In SCC9/Bmal1-KOgroup, there was no significant fluctuation in the efficacy kineticsamong all the experiment time points (Fig. 7F, I, and L). Weconcluded that paclitaxel efficacy can be boosted with drugadministering at the time point of peak Bmal1 expression.

DiscussionTo provide new insights into the mechanism by which the

circadian system influences tumorigenic process and treatment,we performed the study and showed that Bmal1 expression leveland rhythmic patternwere affected in both TSCC samples and celllines. Similar to the results in ovarian cancer (12), colorectalcancer (13), and pancreatic tumors (30), Bmal1 overexpressionsignificantly inhibited proliferation, migration, and invasion ofTSCC cells, suggesting that Bmal1might act as an anti-TSCC gene.Bmal1 could recruit EZH2 to the promoter of TERT to inhibitTERT transcription in TSCC cells. Furthermore, our experimentsdemonstrated that TERT negatively correlates with Bmal1 inpaclitaxel sensitivity modulation in TSCC. Paclitaxel chronother-apy efficacy is highly correlated with Bmal1 expression pattern.

Bmal1 level is significantly decreased in TSCC and adjacentnoncancerous tissues compared with normal tongue epithelialtissues. This is consistent with the results of previous studies,expression of clock genes in HNSCC (14, 31), and pancreatictumors (30) were significantly decreased. However, it was alsoreported that the expression of Bmal1 is upregulated in malig-nant pleural mesothelioma (5). This deficiency could beexplained by different roles of circadian genes in differenttumor types. These results highlight the complicated mechan-isms of cancer development.






Figure 5.

TERT negatively correlates with Bmal1 in paclitaxel sensitivity modulation. A, The correlation between Bmal1 and TERT expression levels in TSCC tissues. B,Western blot analysis of Bmal1, TERT, and GAPDH (for loading controls). C, Apoptosis was evaluated by flow cytometry of cells, which were treated with 10 nmol/L(top) or 20 nmol/L (bottom) paclitaxel (PTX) for 48 hours. D, Cell-cycle phases were determined by flow cytometry of cells after treatment with 10 nmol/L (top)or 20 nmol/L (bottom) paclitaxel (PTX) for 48 hours. E,Western blot analysis of Bmal1, TERT, and apoptosis-related genes in SCC9/Bmal1, SCC9/Bmal1þTERT, andSCC9/vehicle cells after treatment with paclitaxel at 10 or 20 nmol/L. All experiments were performed at least three times. � , P < 0.05; �� , P < 0.01.

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Figure 6.

Bmal1-mediated TERT expression inhibition requires EZH2. A and B, ChIP assays were performed with antibody for Bmal1, EZH2 (B), and antibodies for IgGor RNA Polymerase II (RP II) as negative or positive controls, respectively. Fwd, forward primer; Rev, reverse primer; TSS, transcription start site; M, marker.C, Confocal images of SCC9, SCC25, and CAL27 cells stained for EZH2 (red), Bmal1 (green), and DAPI (blue; magnification, �400). Colocalization isindicated by the merged images showing white immunofluorescence. D, Coimmunoprecipitation was performed with anti-Bmal1 or anti-EZH2 antibodyor IgG (served as negative control) and detected by Western blot analysis with anti-Bmal1 or anti-EZH2 antibody. IP, coimmunoprecipitation. E,Luciferase reporter assay was performed to measure the activities of the wild-type TERT promoter (�2004/þ39) and its deletion mutant (�1583/þ39)and the mutant with mutated Bmal1-binding site in SCC9 cells stably transfected with Bmal1, EZH2, or EZH2-shRNA, empty vector (vehicle), and thoseas indicated. Filled black circle, Bmal1-binding site. Filled crossed black circle, mutation of Bmal1-binding site. All experiments were performed at leastthree times. � , P < 0.05; �� , P < 0.01.






Figure 7.

Paclitaxel chronotherapy efficacy is correlated with fluctuation of Bmal1 mRNA level in vivo. A–C, Bmal1 (solid lines) and TERT (dotted lines) expressionin xenografts generated from SCC9/Bmal1 (A), SCC9/wild-type (B), and SCC9/Bmal1-KO (C) cells at indicated times, determined by RT-PCR analysis.D–L, SCC9/Bmal1, SCC9/wild-type, and SCC9/Bmal1-KO cells were subcutaneously injected into mice. Two weeks after cell inoculation, mice were treatedwith paclitaxel (20 mg/kg, twice a week) or normal saline for 4 weeks at indicated time points. NS, normal saline. D–F, Representative image oftumors formed after paclitaxel chronotherapy. G–I, Columns indicate tumor weight. J–L, Tumor volume growth curves in mice that were treatedwith paclitaxel at indicated times of Bmal1 expression (peak or trough); mice treated with normal saline were used as control group. � , P < 0.05; �� , P < 0.01.M, A model illustrating how Bmal1 inhibits tumorigenesis and increases paclitaxel sensitivity in TSCC.

Tang et al.





Several studies have assessed the Bmal1 effect on anticancerdrugs. Irinotecan cytotoxicity, cyclophosphamide, and oxalipla-tin sensitivity were reported to be directly linked to clock geneBmal1 expression (13, 21, 32). Specifically, Bmal1 was found toimpact on anticancer drugmetabolismand related B-cell response(32). Also, Bmal1 was reported to regulate G2–M phase arrest byactivating the ATM signaling pathway (13). The circadian clockgenes display circadian rhythms in their expression and generateoscillations in several membrane transporters and enzymesinvolved in anticancer drugs metabolism and detoxification,molecular targets, cell cycle, DNA repair, and apoptosis (16,18–21). Our previous study also demonstrated that Bmal1 playsa role in paclitaxel effects in vitro and in vivo. However, theunderlying mechanism and molecular targets of Bmal1 remainsto be identified. In this study, we found that Bmal1 is associatedwith the inhibition of TERT. TERT has extraordinarily high activityin >85% human cancers, attracting great interest due to itscorrelation with extended telomere length and out-of-controlcancer cell divisions (33, 34). Downregulated TERT is expectedto be beneficial to medical chemotherapy (35, 36).

We found that Bmal1 could directly recruit EZH2 to bind to theTERT promoter. EZH2, one of the most studied histone-modify-ing enzymes, is the catalytic subunit of polycomb repressivecomplex 2 (PRC2). Being one of the two major classes of poly-comb complexes, PRC2 is responsible for maintaining its targetgenes in a transcriptionally repressed state through tri-methyla-tion of lysine 27onhistoneH3 (H3K27me3; refs. 37, 38). A recentstudy showed that EZH2 coimmunoprecipitates with Bmal1throughout the circadian cycle in liver nuclear extracts (28).Human TERT promoter contains two classic CLOCK-Bmal1 bind-ing E-boxes with CACGTG sequences (39). Our data revealed thatBmal1 does not bind to the E-boxes near the transcription startsite, but to recruit EZH2 to CAAGTG sequences 1,752-bpupstream. This binding site is not a canonical E-box. After EZH2knockdown, Bmal1 showed dose-dependent transcriptional acti-vation of TERT (Fig. 6E). This could be explained by direct E-boxesbinding of Bmal1 upon depletion of EZH2 (40).

The current trend in chemotherapy is personalized medicineand chronotherapy. Chronotherapy aims at improving the toler-ability and the efficacy of medication through the administrationof treatments according to biological rhythms. The physiologicrhythm–based treatment delivery can improve therapeutic out-comes in tumor-bearing mice (41) and patients with cancer (15).Identifying biomarkers and therapeutic targets is a major focus ofchronotherapy research. Here we systematically explored thecorrelations between Bmal1 expression level and paclitaxel sen-sitivity in TSCC. We found that Bmal1 expression level in xeno-graft is associated with the size of xenograft. Importantly, theexpression level of Bmal1 is contingent upon the appropriatecircadian timing for paclitaxel administration.

Paclitaxel, a commonly used chemotherapeutic drug, has beenused as the first-line therapy for various cancers (42, 43). Patientswho initially respond to paclitaxel therapy often develop resis-tance to paclitaxel later (44–46). Therefore, improvement ofpaclitaxel sensitivity is urgently needed in the field. Some reportsindicated that nanoparticle (nab) paclitaxel can improve the drugbiodistribution and therapeutic efficacy (47). Inhibiting expres-sions of certain proteins or upregulating the transcription ofcertain miRNAs, including TERT (36), interleukin-1 receptor-associated kinase 1 (IRAK1; ref. 48), spleen tyrosine kinase (STK;ref. 49), and miR200 (50), can reverse paclitaxel resistance andpotentiate paclitaxel-induced cytotoxicity. Unfortunately, theoutcomes of these strategies are uncertain and cost-effective.Chronotherapy has been considered as an innovative way tomaximize the benefits and to minimize adverse effects of anti-cancer therapy simultaneously. It is appealing to increase thepaclitaxel sensitivity through adjusting the biological rhythm–

based administration timing of treatments.In summary, our results demonstrated that circadian clock gene

Bmal1 plays an important role in tumor development and associ-ates with TSCC paclitaxel sensitivity increase. Thus, our studyprovided strong evidences that Bmal1 is a novel therapeutic targetfor human TSCC and paclitaxel therapeutic timing biomarker forpatients receiving the paclitaxel-based chemotherapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: B. Cheng, L. ChenDevelopment of methodology: Q. Tang, B. Cheng, J. ZhaoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Q. Tang, B. Cheng, M. Xie, Y. Chen, X. ZhouAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Q. Tang, L. ChenWriting, review, and/or revision of themanuscript:Q. Tang, B. Cheng, L. ChenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Q. Tang, J. Zhao, L. ChenStudy supervision: L. Chen

Grant SupportThis work was supported by Major Project from the National Natural

Science Foundation of China (no. 31110103905 to L.L. Chen) and(no. 81370405 to B. Cheng), and National Outstanding Youth Science Fundof China (no. 31422022 to L.L. Chen).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 12, 2016; revised October 27, 2016; accepted November 1,2016; published OnlineFirst November 7, 2016.

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Tang et al.




2017;77:532-544. Published OnlineFirst November 7, 2016.Cancer Res Qingming Tang, Bo Cheng, Mengru Xie, et al. Paclitaxel Sensitivity in Tongue Squamous Cell Carcinoma

Inhibits Tumorigenesis and IncreasesBmal1Circadian Clock Gene

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