Optimized dosing schedule based on circadian dynamics of mouse breast cancer stem cells improves the anti-tumor effects of aldehyde dehydrogenase

Although malignant phenotypes of triple-negative breast cancer (TNBC) are subject to circadian alterations, the role of cancer stem cells (CSC) in defining this circadian change remains unclear. CSC are often characterized by high aldehyde dehydrogenase (ALDH) activity, which is associated with the malignancy of cancer cells and used for identification and isolation of CSC. Here we show that the popultation of ALDH-positive cells in a mouse 4T1 breast tumor model exhibits pronounced circadian alterations. Alterations in the number of ALDH-positive cells was generated by time-dependent increases and decreases in the expression of Aldh3a1 . Importantly, circadian clock genes were rhythmically expressed in ALDH-negative cells, but not in ALDH-positive cells. Circadian expression of Aldh3a1 in ALDH-positive cells was dependent on the time-dependent release of Wingless-type mmtv integration site family 10a (WNT10a) from ALDH-negative cells. Furthermore, anti-tumor and anti-metastatic effects of ALDH inhibitor N,N-diethylaminobenzaldehyde were enhanced by administration at the time of day when ALDH activity was increased in 4T1 tumor cells. Our findings reveal a new role for the circadian clock within the tumor microenvironment in regulating the circadian dynamics of CSC. These results should enable the development of novel therapeutic strategies for treatment of TNBC with ALDH inhibitors. Significance: This seminal report reveals circadian dynamics of cancer stem cells are regulated by the tumor microenvironment, and provides a proof of principle of its implication for chronotherapy in TNBC. of DEAB at ZT2 did not result in significant anti-tumor as well as anti-metastatic activity. These data reveal a significant relationship previous findings that circadian host disruption accelerates the progression of many types of cancer, including those without any clock or barely detectable Bmal1 expression and shorten survival (46,47). Disruption of circadian rhythms in host animal also accelerates tumor growth and angiogenesis/stromagenesis through the mediation of Wnt signaling pathway (48). In contrast, the reinforcement of the host circadian clock or that of cancer tissues slows proliferation of tumor cells in relation to the timing of meals or kinase inhibitor administration (49,50). Consequently, the maintenance of circadian clock function in microenvironment may be important to suppress overpopulation of CSCs. Our present findings suggest that the effectiveness of anti-cancer drugs vary with the circadian dynamics of CSCs, which are regulated by the tumor microenvironmental factors. However, many drugs are still administered without regard to the time of day. Identification of rhythmic markers for detection of the circadian dynamics of CSCs in tumors should enable their use in chronotherapy, in which chemoradiation and/or high-dosage treatments are administered at a time of day when CSCs are most vulnerable. Furthermore, circadian machinery in tumor microenvironment may be a therapeutic target of TNBC, because the machinery was participated in the regulation of tumor malignancies.


INTRODUCTION
According to the World Health Organization, breast cancer is the most common cancer in patients worldwide (1). However, current treatment strategies cannot eliminate the majority of breast cancers. In particular, triple-negative breast cancer (TNBC) is highly aggressive (2).
TNBC is usually resistant to chemotherapeutic drugs and has also been incriminated in recurrence after chemotherapy, radiation treatment, and resection surgery (3).
One approach for increasing the efficacy of pharmacotherapy is to administer drugs at the time of day when they are most effective and/or best tolerated. A chronopharmacological strategy can enhance the effects of drugs and/or attenuate their toxicity (4,5). Circadian variations in biological functions, such as gene expression and protein synthesis, are thought to be important factors affecting the efficacy of drugs. Experimental chronopharmacology studies have successfully guided the development of chronotherapy schedules with 5-fluorouracil, leucovorin, and oxaliplatin in human colorectal cancer. Chronomodulated chemotherapy regimens have also produced the highest tumor response rates and the longest survival reported in multicenter randomized trials (6)(7)(8)(9). The circadian rhythm in the tolerability and the efficacy of docetaxel and doxorubicin in mice bearing syngeneic mammary cancer tumors derived from MA13/C cells is investigated as a prerequisite for the development of chronotherapy schedules with these drugs in human breast cancer (10) .
However, there is no previous study evaluating chronotherapy for treatment of TNBC.
Tumor masses are composed of heterogeneous cells, and this heterogeneity is relevant to resistance to chemotherapy and a high risk of recurrence. Cancer stem-like cells (CSCs) represent a distinct proportion in cancer cells, but they play a key role in driving tumor growth, progression, and metastasis owing to their self-renewal and differentiation capacities. CSCs in TNBC tumor mass are also considered to be responsible for metastasis, recurrence, and resistance against chemotherapy and radiation treatment (11,12). Because CSCs are characterized by specific cell surface markers (13,14), this subpopulation of cells can be isolated from mixed tumorigenic and nontumorigenic cells using different immune selection methods (15). However, a limitation of surface marker recognition approaches is that the Research.
on July 23, 2018. © 2018 American Association for Cancer cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Author Manuscript Published OnlineFirst on May 7, 2018; DOI: 10.1158/0008-5472.  4 results are ascribed to the specific studied population (16). In addition, because the characteristics of CSCs are complex, general studies of CSCs should be more thorough and provide more data to confirm the population as CSCs (17,18). Therefore, a more useful method involving detection of the activity of a specific protein in CSCs has been developed to identify and/or isolate CSCs in both basic research and the clinical setting (19). High aldehyde dehydrogenase (ALDH) activity is often detected in cells with stem-like properties, suggesting that this enzyme can be used as a marker to isolate CSCs (20); the ALDEFLUOR assay measures ALDH enzyme activity via cleavage of a fluorescent substrate, BODIPY-aminoacetaldehyde, and is a commonly used method to identify and isolate CSCs (21,22) .
In this study, we used the ALDEFLUOR assay to investigate the ALDH activity in mice implanted with murine TNBC 4T1 cells. The number of ALDH-positive cells (high ALDH activity cells) in a mouse breast tumour model exhibited pronounced circadian alterations, which was caused by the time-dependent release of Wingless-type mmtv integration site family 10a (WNT10a) from ALDH-negative cells. Therefore, we investigated whether anti-tumor and anti-metastatic effects of ALDH inhibitor were improved by changing the dosing schedule. Research.

Cells and treatments
4T1 mouse breast cancer cells were purchased from American Type Culture Collection.
Cells were cultured under a 5% CO 2 environment at 37°C in roswell park memorial institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in two-dimensional (2D) CELLSTAR cell culture flasks (Greiner Bio-One, Monroe, NC) or synthetic three-dimensional (3D) scaffold biomaterials (VECELL 3D-inserts; VECELL Inc., Kitakyusyu, Japan). We confirmed that there was no microbial in this cell lines using TaKaRa PCR Mycoplasma Detection Set. We confirmed that cell lines were authenticated by each cell bank using short tandem repeat-polymerase chain reaction (PCR) analysis, and these cell lines were used in less than 3 months from frozen stocks. We confirmed that there was no microbial growth in both cell lines using fluorochrome staining. To downregulate the Aldh3a1 gene, 4T1 cells were infected with lentiviral vectors expressing small hairpin (sh) ribonucleic acid (RNA) against the mouse Aldh3a1 gene (pGFP-C-shAldh3a1 Lenti Vector; Origene Technologies, Inc., Rockville, MD). After infection of cells with lentiviral vectors, cells were maintained in medium containing 2 µg/mL puromycin.
Green fluorescent protein (GFP)-expressing cells were selected by sorting using fluorescence activated cell sorting (FACS) (BD Biosciences, Franklin Lakes, NJ). Downregulation of the Aldh3a1 gene was confirmed by reverse transcription (RT)-PCR. Aldh3a1::Luc-expressing 4T1 cells were prepared using luciferase reporter vectors under the control of the mouse Aldh3a1 promoter. The mouse Aldh3a1 promoter region spanning from 660 to 675 bp (the distance in base pairs from the putative transcription start site, +1) was amplified by PCR, and the product was ligated into the pGL4.18 luciferase reporter vector (Promega, Madison, WI).

ALDEFLUOR assay
ALDH-positive (ALDH-high activity) and ALDH-negative (ALDH-low activity) cells were gated based on the ALDEFLUOR assay (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions. Briefly, dissociated single cells from cell lines or tumor specimens were suspended in ALDEFLUOR assay buffer containing an ALDH substrate, BODIPY-aminoacetaldehyde at 1.5 µM; this was followed by incubation for 40 min at 37°C. A specific inhibitor of ALDH, DEAB, was used at a 10-fold molar excess as a negative control. FACS (BD Biosciences) was performed on more than 1 × 10 6 cells under low pressure in the absence of ultraviolet light. The data were analyzed using BD FACSDiva software V6.1.3 (BD Biosciences).

Animals and treatments
Five-week-old female BALB/c mice (Kyudo Co., Ltd., Saga, Japan) were housed under a standardized light-dark cycle at 24 ± 1°C and 60% ± 10% humidity with food and water ad libitum. Thirty microliters medium containing 5 × 10 4

Quantitative RT-PCR analysis
Total RNA was extracted using RNAiso (Takara Bio Co., Ltd., Shiga, Japan) or a QIAGEN RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) and amplified by PCR. Real-time PCR analysis was performed on diluted cDNA samples using the THUNDERBIRD SYBR qPCR Mix (Toyobo) with the 7500 Real-time PCR system (Applied Biosystems, Foster City, CA). Data were normalized using 18s and β-actin mRNAs as controls because spinal expression of these MRAs is constant throughout the day. Primer sequences are listed in Supplementary Table   1.

Chromatin immunoprecipitation analysis
Cross-linked chromatin extracted from cells of the spinal cord was sonicated on ice, and nuclear fractions were obtained by centrifugation at 10,000 × g for 5 min. Supernatants were incubated with antibodies against β-catenin (#8480; Cell Signaling Technology, Beverly, MA), Transcription factor 7-like 2 (TCF7L2) (#2569; Cell Signaling Technology), or rabbit IgG (sc66931; Santa Cruz Biotechnology). DNA was purified using a DNA purification kit (Promega) and amplified by PCR for the surrounding β-catenin/TCF binding site in the upstream region of the Aldh3a1 gene. The primer sequences for the amplification of the surrounding β-catenin/TCF binding site were as follows: 5ʹ-CCTGGGTGATACAGAGAGGA-3ʹ and 5ʹ-CACAACCTACTGGTTGGAGA-3ʹ. The quantitative reliability of PCR was evaluated through kinetic analysis of the amplified products to ensure that signals were only derived from the exponential phase of amplification. This analysis was also performed in the absence of an antibody or in the presence of rabbit IgG as negative controls; no PCR products were detected with ethidium bromide staining in any samples.

In vivo bioluminescence monitoring
An in vivo imaging system (IVIS Spectrum; Caliper Life Sciences Inc., Hopkinton, MA) was used for in vivo bioluminescence monitoring (24). Mice implanted with isoflurane anesthesia was approximately 20 min for each experiment. After data were obtained, mice recovered from isoflurane anesthesia within 1 to 2 min.

In vitro bioluminescence monitoring
The bioluminescence of cultured Aldh3a1::Luc-expressing 4T1 cells was recorded using a real-time monitoring system (Lumicycle; Actimetrics, Wilmette, IL). The ALDH-positive populations of Aldh3a1::Luc-expressing 4T1 cells were cultured on VECELL 3D inserts. The 3D inserts were placed in 35-mm dishes, in which ALDH-negative 4T1 cells were seeded and stimulated with 100 nM dexamethasone for synchronization of their circadian clocks. The amplitude of bioluminescence derived from Aldh3a1::Luc was calculated using Lumicycle analysis software (Actimetrics). Bioluminescence images at the cellular level were acquired using the LV200 LuminoView microscope system (Olympus, Tokyo, Japan).

Statistical and data analyses
The values presented are expressed as means ± standard errors of the means. The significance of the 24-h variations in each parameter was tested by one-way analysis of variance (ANOVA). The statistical significance of differences between groups was analyzed by one-way or two-way ANOVA, followed by Tukey-Kramer post-hoc tests and Dunnett's test.
Equal variances were not formally tested. A 5% level of probability was considered significant.

Circadian variations in the number of ALDH-positive cells in a mouse 4T1 breast tumor model.
To investigate the malignancy of ALDH-positive cells, which indicated high activity of ALDH, isolated from mouse 4T1 breast cancer cell lines, we implanted ALDH-positive cells into the hind footpads of female BALB/c mice (Supplementary Fig. 1A). The ALDH-positive cells exhibited profound tumor formation as well as enhanced pulmonary metastasis ( Supplementary Fig. 1B). Furthermore, the expression levels known CSC biomarkers,
Next, we elucidated whether ALDH-positive cells, which indicated high activity of ALDH, masses. The mRNA levels for Aldh3a1, but not for Aldh6a1, in 4T1 cell-implanted mice showed a significant circadian oscillation (F 5,30 = 2.836, P = 0.033; one-way ANOVA; Fig. 1B left panel, Supplementary Fig. 2). A similar rhythmic variation was also detected in the protein levels of ALDH3a1 (F 5,12 = 7.355, P = 0.002; one-way ANOVA, Fig. 1B right panel), suggesting that the rhythmic expression of the Aldh3a1 gene was related to circadian alterations in ALDH activity in 4T1 tumor masses.
To investigate this possibility, we prepared Aldh3a1-downregulated 4T1 cells via introduction of lentivirus vectors expressing a specific shRNA (Supplementary Fig. 3A). The growth and pulmonary metastasis of tumors formed by Aldh3a1-downregulated 4T1 cells was significantly slower than those observed in mice implanted with control shRNA-expressing 4T1 cells (Supplementary Fig. 3B and C). Furthermore, the proportion of ALDH-positive cells in Aldh3a1-downregulated 4T1 tumors did not show significant circadian alterations (Fig.   1C). These data suggest that the Aldh3a1 gene is responsible for generating circadian alterations in ALDH activity in 4T1 tumor masses. This circadian alteration in ALDH activity appeared to be reflected in the time-dependent changes in the number of ALDH-positive cells in 4T1 tumor masses.

Circadian regulation of Aldh3a1 expression in 4T1 tumors by Wnt/β-catenin signaling
Next, we investigated the mechanism through which Aldh3a1 was expressed in a circadian manner in 4T1 tumor cells. In mammalian cells, circadian rhythms in gene expression are generated by a molecular oscillator driven by a transcriptional-translational feedback loop consisting of clock genes (29). Therefore, we assessed the temporal  (Fig. 1C). Three criteria were applied to select circadian cycle-dependent genes that regulate Aldh3a1 expression: (1) the expression of genes in ALDH-negative cells being greater than that in ALDH-positive cells, (2) the expression of genes in ALDH-negative cells at ZT0 being greater than that in ALDH-negative cells at ZT12, (3) the expression of genes in ALDH-negative cells at ZT12 being greater than that in ALDH-negative cells at ZT0. From this analysis, we identified 618 candidate circadian time-dependent genes in ALDH-negative cells (Supplementary Table 2). Among the genes involved in pathways related to cancer progression, we focused on those that encode WNT proteins, because WNT proteins are secreted molecules that act on cell-surface receptors, and also WNT signal transduction has been implicated in sustaining the stemness of CSCs (30). To determine whether WNT signaling is involved in the circadian regulation of Aldh3a1, the mRNA levels of Aldh3a1 were assessed in 4T1 tumor cells implanted in mice after the intratumoral injection of a canonical WNT signal inhibitor, Wnt-C59, at ZT2 and ZT14. Four hours after the injection of Wnt-C59 at each time point, the mRNA levels of Aldh3a1 did not exhibit a significant time-dependent variation (Fig. 2B), suggesting that WNT signaling is involved in the circadian regulation of Aldh3a1 expression in 4T1 tumor cells.
Extracellular WNT stimulates several intracellular signal transduction cascades, resulting in the activation or repression of a variety of genes (31). The major effector of these transduction cascades is a bipartite transcription factor formed by β-catenin and a member of the TCF protein family, such as TCF7L2 (32). A consensus DNA sequence of the β-catenin/TCF binding site CTTTGA is located between 532 and 538 bp from the transcription start site of the mouse Aldh3a1 gene (Fig. 2C left panel). The DNA sequence of the β-catenin/TCF binding site has also been found at a similar location in all mammals examined, including mice, rats, monkeys, and humans (Supplementary Fig. 4A). Thus, the luciferase reporter of the mouse Aldh3a1 promoter containing the motif CTTTGA (Aldh3a1::Luc) responded to β-catenin and TCF7L2 (Supplementary Fig. 4B). The results of chromatin immunoprecipitation also revealed that both β-catenin and TCF7L2 bound to the promoter region of the Aldh3a1 gene in 4T1 tumors, and the amount of bound protein was increased at ZT14 compared with that at ZT2 (Fig. 2C right panel).
To determine whether the upstream region containing the β-catenin/TCF binding site was responsible for the circadian expression of Aldh3a1, we prepared 4T1 cells that stably  Supplementary Fig. 5A). After confirming that the luciferase activity of the Aldh3a1::Luc-expressing cells was mainly driven by ALDH-positive populations ( Supplementary Fig. 5B), we implanted these cells into the hind footpads of mice. In vivo imaging analysis results revealed that the bioluminescence from tumors formed by Aldh3a1::Luc-expressing 4T1 cells also showed significant circadian oscillation, with peak levels during the early dark phase (F 5,34 = 15.427, P < 0.001, one-way ANOVA ; Fig. 2D). The rhythmic pattern of the bioluminescence from Aldh3a1::Luc-expressing 4T1 tumors resembled the overall rhythm of the expression of ALHD3a1 (Fig. 1C).

Circadian oscillation of Aldh3a1 expression in ALDH-positive cells by temporal enhancement of WNT10a release from microenvironmental cells
Several genes encoding WNT ligands were highly expressed in ALDH-negative 4T1 cells (Supplementary Fig. 6). Among these, the expression of Wnt10a mRNA exhibited profound circadian oscillation only in ALDH-negative cells (F 5,12 = 17.117, P < 0.001, one-way ANOVA; Fig. 3A). In vitro promoter analysis revealed that the transcription of Wnt10a was controlled by the main components of the circadian clock (Supplementary Fig. 7). The CLOCK/BMAL1-mediated transactivation of Wnt10a was repressed by PER2 and CRY1, suggesting that PER and CRY proteins periodically repress the CLOCK/BMAL1-mediated transactivation of the Wnt10a gene. Positive and negative regulation by the products of circadian clock genes appeared to generate a circadian rhythm in the mRNA and protein expression of Wnt10a. Although the expression of the WNT10a protein in ALDH-positive 4T1 cells was not detected by western blot analysis, its protein levels in ALDH-negative populations of 4T1 tumors exhibited a significant time-dependent variation (F 1.14 = 9.699, P = 0.036, two-way ANOVA; Fig. 3B). In contrast, the ALDH3a1 protein was difficult to detect in ALDH-negative populations of 4T1 tumors; however, the protein levels showed a significant To investigate this possibility, we prepared Wnt10a-downregulated Aldh3a1::Luc-expressing 4T1 cells (Supplementary Fig. 8A and B, Supplementary Fig. 9) and implanted them into the hind footpads of mice. The bioluminescence from tumors formed by control Aldh3a1::Luc-expressing 4T1 cells (expressing the control shRNA) showed a significant time-dependent variation (P < 0.01, Fig. 3C). However, the variation in the bioluminescence from Aldh3a1::Luc-expressing 4T1 tumors was dampened by the downregulation of Wnt10a. The intensity of the bioluminescence from Wnt10a-downregulated 4T1 tumors remained low at both the light and dark phases. Consistent with these observations, the downregulation of Wnt10a in 4T1 cells decreased the number of ALDH-positive cells and dampened their circadian oscillation (Fig. 3D), supporting the notion that WNT10a is a major regulator of the circadian expression of the Aldh3a1 gene in 4T1 tumor cells.
In addition to analysis of the 4T1 tumor mass, we also detected significant circadian accumulation of the WNT10a protein in the culture medium of ALDH-negative 4T1 cells after synchronizing their circadian clocks by dexamethasone treatment (F 13,28 = 6.325, P < 0.001, one-way ANOVA; Fig. 4A). This finding suggests that tumor microenvironmental cells enhance the release of WNT10a in a circadian fashion. The rapid degradation of WNT protein has been reported previously (33). The half-life of the WNT10a protein in the medium was approximately 4 h (Fig. 4B). Consequently, circadian accumulation of WNT10a in the culture medium of ALDH-negative 4T1 cells may be associated with both its time-dependent synthesis and rapid degradation.
To determine whether the time-dependent release of WNT10a from tumor scaffold (34). The 3D scaffold chambers were inserted into wells in which ALDH-negative 4T1 cells were seeded on the bottom (Fig. 4C left panel). Aldh3a1::Luc-expressing ALDH-positive 4T1 cells showed a significant time-dependent oscillation of bioluminescence when co-cultured with dexamethasone-treated ALDH-negative 4T1 cells (Fig. 4C right panel,   Supplementary Movie 1); however, this oscillation did not occur when they were co-cultured with dexamethasone-untreated ALDH-negative 4T1 cells (Supplementary Movie 2). These results suggest that the time-dependent enhancement of Wnt10a released from tumor microenvironmental cells regulates the circadian expression of the Aldh3a1 gene in ALDH-positive 4T1 cells.

Dosing time-dependent change in the anti-tumor and anti-metastatic effects of ALDH inhibitor DEAB on 4T1 tumor-bearing mice
Since the number of ALDH-positive cells showed significant circadian variation in 4T1 tumor-bearing mice (Fig. 1), we investigated whether anti-tumor and anti-metastatic effects of ab ALDH inhibitor were changed by optimizing dosing schedule. DEAB is commonly used as a selective inhibitor of ALDH in CSCs (35). The intraperitoneal administration of DEAB (50 mg/kg) at ZT14, when the number of ALDH-positive cells was abundant, significantly suppressed the growth and pulmonary metastasis of 4T1 tumor cells in mice ( Fig. 5A and B).
In contrast, administration of the same dosage of DEAB at ZT2 did not result in significant anti-tumor as well as anti-metastatic activity. These data reveal a significant relationship  5C). This circadian interaction between ALDH-positive and ALDH-negative cells suggested a potential therapeutic targets as chronotherapy for treatment of TNBC.
ALDHs exhibit a wide taxonomic distribution from bacteria to humans. They catalyze the conversion of aldehydes to corresponding acids via an NAD(P) + -dependent irreversible reaction (36). The family of ALDH contributes to sustain the stemness of cancer cells; therefore their activity is used as a marker for CSCs (20). Implantation of ALDH-negative cells to mice showed slow tumor growth and poor metastasis. Furthermore, inhibition of ALDH activity resulted not only in the disruption of circadian variations in the number of ALDH-positive cells, but also in the prevention of tumor growth and metastasis. ALDH has the ability to ameliorate oxidative stress in tumor cells (37). Therefore, development of method selectively inhibiting ALDH activity in CSCs would be useful for treatment of malignant cancers including TNBC.
The circadian clock machinery in immature cells, e.g., ES cells and stem-like cells, is functional after differentiation (38,39). Low level expression of clock genes in those immature cells is thought to be important for sustaining their stemness (11). The expressions of clock gene in ALDH-positive cells were lower than those in ALDH-negative cells, suggesting that dysfunction of circadian machinery is also required for sustaining the stemness of CSCs in 4T1 tumors. Overexpression of Aldh3a1 in 4T1 cells slightly increased the mRNA levels of Bmal1 and Rev-Erbα, but in contrary decreased the expression of Clock (Supplementary Fig.   10). Although we were unable to clarify whether ALDH was indispensable for suppression of circadian clock machinery in CSCs, low level expression of these clock genes in ALDH-positive cells was unlikely due to the elevation of ALDH enzymatic activity. Recent study has demonstrated that pharmacological activation of REV-ERBs is specifically lethal in cancer cells (40). However, such strategy may be ineffective to CSCs in 4T1 tumors because of low expression of clock genes including Rev-erbα. It has been well known that microenvironment surrounding CSCs is composed by tumor-associated fibroblasts, macrophages, myeloid-derived suppressor cells, and/or regulatory T-cells (42). In addition, stromal fibroblasts are also involved in microenvironmental constitutive cells, which is capable of releasing WNT proteins (43,44). Because gain of β-catenin activity allows stem cell overpopulation and cancer development (45), disruption of the circadian machinery in microenvironment cells may lead to arrhythmic expression of WNT10a therefore enhancing malignancies of TNBC. This notion is also supported by previous findings that circadian host disruption accelerates the progression of many types of cancer, including those without any clock or barely detectable Bmal1 expression and shorten survival (46,47). Disruption of circadian rhythms in host animal also accelerates tumor growth and angiogenesis/stromagenesis through the mediation of Wnt signaling pathway (48). In contrast, the reinforcement of the host circadian clock or that of cancer tissues slows proliferation of tumor cells in relation to the timing of meals or kinase inhibitor administration (49,50). Consequently, the maintenance of circadian clock function in microenvironment may be important to suppress overpopulation of CSCs.   Values are means ± SEMs (n = 4). **P < 0.01, significant difference between the two time points (F 1,6 = 751.527, P < 0.001 for Control shRNA; F 1,6 = 340.149, P < 0.001 for Wnt10a shRNA; two-way ANOVA with Tukey-Kramer post-hoc tests). ## P < 0.01, significant difference between the two groups (F 1,6 = 306.436, P < 0.001 for ZT14; two-way ANOVA with Tukey-Kramer post-hoc tests).