Research ArticleExperimental Studies
Open Access

Role of the Circadian Clock Gene DEC1 in Chemosensitivity and Apoptosis in Colorectal Cancer

SOICHIRO MINAMI, NORIKATSU MIYOSHI, SHIKI FUJINO, MASAYOSHI YASUI, MASAYUKI OHUE, MITSUNOBU TAKEDA, YUKI SEKIDO, TSUYOSHI HATA, ATSUSHI HAMABE, TAKAYUKI OGINO, MAMORU UEMURA, HIROFUMI YAMAMOTO, YUICHIRO DOKI and HIDETOSHI EGUCHI
Anticancer Research June 2025, 45 (6) 2309-2328; DOI: https://doi.org/10.21873/anticanres.17605

Abstract

Background/Aim: Disruptions in the circadian rhythm are linked to various diseases. The clock gene DEC1 is related to the progression and recurrence of various types of cancer; however, its role in colorectal cancer has not been determined. Therefore, we aimed to evaluate the significance of DEC1 expression level in colorectal cancer and its relationship with prognosis.

Materials and Methods: Using quantitative reverse transcription-polymerase chain reaction and immunohistochemistry, we compared DEC1 mRNA and protein expression in clinical samples. We compared colorectal cancer cell lines and organoids using cell proliferation, wound healing, chemosensitivity, and apoptosis assays. We also performed RNA sequencing to investigate whether changes in DEC1 expression influence the expression of other genes, thereby affecting drug sensitivity and apoptosis.

Results: Based on data obtained from The Cancer Genome Atlas database and clinical samples, high DEC1 expression was associated with a poor prognosis. However, in vitro and in vivo experiments revealed that DEC1 knockdown in colorectal cancer cell lines does not significantly affect cell proliferation or migration. Modulating DEC1 expression levels altered the sensitivity of cells to 5-fluorouracil, indicating that DEC1 plays a role in treatment response. The suppression of DEC1 expression led to an increase in cell apoptosis. RNA sequencing, analyses of data from The Cancer Genome Atlas database, and Metascape analysis revealed seven genes related to DEC1 associated with apoptosis.

Conclusion: DEC1 expression is related to the circadian rhythm in colorectal cancer cells, and several other genes contribute to this relationship. Overall, DEC1 may function beyond circadian rhythm regulation, potentially affecting the development and progression of colorectal cancer.

Keywords:

Introduction

Colorectal cancer (CRC) is the second most fatal and third most commonly diagnosed malignancy worldwide (1), with high morbidity and mortality rates. It is projected that the number of new CRC cases will rise dramatically by 60% by 2030 (2), posing a significant and ongoing public health concern. The etiology of CRC is multifaceted, involving genetic and environmental factors (3). Considerable progress has been made in understanding the complex genetic landscape associated with CRC, with emphasis on the role of cancer-related genes in CRC initiation, progression, and treatment response.

Circadian rhythms, present in all living organisms, have been implicated in the development of various diseases, including cancer, diabetes, and cardiovascular disorders (4, 5). Disruptions in these rhythms increase the risk of breast and prostate cancers (6-8). The functions of clock genes, associated with circadian rhythms, are not fully understood. Moreover, while clock genes have been implicated in cancer cells, with effects on proliferation and metastasis, a consensus on their roles has not been reached (9, 10).

The expression of circadian rhythm-associated clock genes has been reported in cancer cell lines (11, 12). The mechanisms through which clock genes associate with circadian rhythms may have implications for cancer treatment (9, 13, 14); however, their clinical application is limited (15, 16). Although the expression of clock genes can be analyzed in cell lines and animal specimens, comparisons between normal and cancerous tissues within a living organism are extremely challenging.

Differentiated embryo chondrocyte 1 (DEC1), a clock gene, has been implicated in cancer progression and recurrence, with the association varying among cancer types including oral squamous cell carcinoma, breast cancer, and gastric cancer (17-21). However, the precise role of DEC1 in colorectal cancer remains unclear. Therefore, in our previous study, we established an in vivo cancer model using 2D organoids (2DOs) (22). 2D organoids are primary cultured cells that retain the characteristics of the original clinical specimens while maintaining cellular diversity. They can be analyzed and evaluated as adherent cells, preserving the heterogeneity of the tumor microenvironment. In the present study, we aimed to evaluate clock gene expression under circadian rhythms by cultivating 2DOs from clinical samples and compare gene expression profiles between 2DOs derived from clinical samples (primary cultured CRC cell lines) and normal colorectal epithelial cells using RNA sequencing (RNA-Seq), with a focus on DEC1. Our results are expected to provide insights into the significance of DEC1 expression in clinical samples and relationship between DEC1 expression and prognosis, as well as contribute to the development of novel cancer therapies targeting clock genes.

Materials and Methods

Clinical samples. The patients with CRC (n=135) underwent rectal resection or amputation surgery at the Osaka International Cancer Institute between November 1, 2011 and December 31, 2013. The medical records of patients were accessed between April 1, 2023 and June 30, 2023. The exclusion criteria were as follows: 1) recurrent surgery; 2) multiple primary cancers; 3) preoperative chemoradiotherapy; 4) transanal endoscopic microsurgery; and 5) insufficient pathological findings or preoperative laboratory data.

The following information was collected from the medical records of the patients: age, sex, location of the primary tumor, size of the primary tumor, carcinoembryonic antigen (CEA) level, degree of differentiation, depth of tumor invasion, lymph node metastasis, distant metastasis, pathological stage, venous invasion, and lymphatic vessel invasion. Clinicopathological factors were assessed according to the tumor, node, and metastasis classifications of the International Union Against Cancer and were classified according to the 9th edition of the Union for International Cancer Control Tumor–Node–Metastasis (TNM) Classification. All patients were followed up postoperatively according to the Japanese treatment guidelines. Tumor markers were evaluated every three months. Computed tomography (CT) was performed every 3-6 months and colonoscopy was performed every 1-2 years.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was extracted from cultured cells using TRIzol® RNA Isolation Reagent (Thermo Fisher Scientific, Waltham, MA, USA), as previously described (23). RNA quality was checked (RNA concentration >0.5 μg/μl and OD260/280=1.8-2.0). cDNA was synthesized from 10 ng of total RNA using a High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Polymerase chain reaction was performed using the Light Cycler™ 2.0 System (Roche Applied Science, Tokyo, Japan) and Thunderbird® SYBR® qPCR mix (Toyobo Life Science, Osaka, Japan). Relative expression levels were calculated using a CT-based calibrated standard curve method and normalized to GAPDH mRNA. The experiment was performed in triplicate with three independent biological replicates. Shown are means with standard deviations (n=3). Statistical analysis was performed using the Mann–Whitney U-test (p<0.05), and JMP version 17 (SAS Institute Inc., Cary, NC, USA).

Commercially available primers. GAPDH (PrimePCR™ SYBR® Green Assay, Desalt 200R Wet-Validated, Predesign. 10025637, qHsacID0038674) (Bio-Rad, Hercules, CA, USA); DEC1 (BHLHE40) (PrimePCR™ SYBR® Green Assay, Desalt 200R Wet-Validated, Predesign. 10025637, qHsacI D0010785) (Bio-Rad); DEC2 (BHLHE41) (PrimePCR™ SYBR® Green Assay, Desalt 200R Wet-Validated, Predesign. 10025636, qHsacID0006964) (Bio-Rad).

Immunohistochemistry. Immunohistochemistry (IHC) was performed on tissues obtained from surgically resected primary lesions. For IHC staining, tissue sections were incubated overnight with an anti-DEC1 rabbit antibody (HPA028922, 1:400 dilution; Sigma-Aldrich, St. Louis, MO, USA) at 4°C. A normal esophagus served as a positive DEC1 control in accordance with the instructions provided in the antibody package insert. Intensity scores were assigned based on nuclear staining as follows: 2+ for intensely stained nuclei (positive control), 1+ for weaker staining, and 0 for unstained nuclei. Groups were subsequently categorized as DEC1-low (score 0) or DEC1-high (score 1+ or 2+) (Figure S1).

CRC cell culture. Human CRC cell lines (RKO and SW480) were a gift from Dr. Bert Vogelstein (Johns Hopkins University). The cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM; Wako, 048-29763) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 1% GlutaMAX-I (Thermo Fisher Scientific), and 1% penicillin/streptomycin/amphotericin B (Wako Pure Chemical Co., Osaka, Japan). The cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

Establishment and culture of human organoids. CRC tissues were sectioned into small fragments and dissociated using 1 mg/ml collagenase (C6885; Sigma-Aldrich) in DMEM. They were agitated using a bioshaker (BR-13FP; Taitec Co., Saitama, Japan) at 6 × g for 15 min at 37°C. After dissociation, the tissue was sieved with a custom-made filter (Sansho Co., Tokyo, Japan), followed by centrifugation at 400 × g for 5 min at room temperature (20°C-25°C). The cell pellets were resuspended in a culture medium (modified stem cell culture medium) (22). Suspended human organoids (iCC603 and iCC129) were seeded on Matrigel-coated plates (Corning Inc., Corning, NY, USA). The medium was changed every 2-3 days. When the cells reached >70% confluence, they were passaged using Accutase (Nacalai Tesque, Kyoto, Japan) for approximately 5 min. The cells were collected, resuspended in a culture medium, and seeded on Matrigel-coated plates (24).

shRNA transfection of cultured cells. DEC1-specific small hairpin RNAs in the pLKO puromycin-resistant vector [MISSION shRNA TRCN0000013249 (described as sh1) and TRCN0000232187 (described as sh2)] were purchased from Sigma-Aldrich (SIGMA Mission shRNA). As a negative control, a non-targeting shRNA control (MISSION Non-Target shRNA Control, Cat# SHC001), which contains a non-mammalian sequence and does not target any known mammalian gene, in the same vector was also obtained from Sigma-Aldrich. Trans-IT-293 Transfection Reagent (TAKARA Bio, Shiga, Japan) was used to transfect 293T cells to create a lentivirus constitutively expressing the shDEC1 sequence. After 48 h, the lentivirus supernatant was collected, and the purified supernatant was used to infect CRC cells at 50% confluence. The proper multiplicity of infection of lentivirus was added to the cells and incubated for 96 h. The medium was changed to fresh medium containing 4 μg/ml puromycin. Knockdown of DEC1 was confirmed using RT-PCR and western blotting before further analysis.

Over-expression of DEC1 in cultured cells. The DEC1 plasmid gifted from Silvia Monticelli (Addgene plasmid # 149714; http://n2t.net/addgene:149714; RRID: Addgene_149714)) was transfected using the Neon Transfection System (Thermo Fisher Scientific) at a 5 μg per well according to the manufacture’s protocol. The vector backbone, including that used for the control, was pLVX-FlagHA-IRES-ZsGreen1. Two μg/ml doxycycline (Clontech, Palo Alto, CA, USA) was used to induce over-expression.

Western blotting. Cells were lysed with RIPA lysis buffer. Equal amounts of protein were loaded into 10% TGX polyacrylamide gels (Bio-Rad Laboratories). The proteins were electrophoresed and transferred to polyvinylidene difluoride membranes. Immunoblots were detected using iBind Flex Western Device (Thermo Fisher Scientific).

The primary antibodies used were as follows:

anti-BHLHE40 (1:400; rabbit. HPA028922; Atlas Antibodies, Bromma, Sweden), and anti-histone H3 (1:1,000; rabbit. no. 4499; Cell Signaling Technology, Danvers, MA, USA). The iBind Western System was run overnight with an HRP-linked secondary antibody (1:2,000; cat. no. 7074; Cell Signaling Technology). Density levels were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Protein expression was normalized to histone H3 levels.

Cell proliferation and wound healing assays. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), following the manufacturer’s instructions. Cells (5×103 cells/well) were cultured in 96-well plates. After 24 h, 10% CCK-8 was added to each well and incubated at 37°C for 2 h. Absorbance of the samples at 450 nm was measured using a microplate reader (Bio-Rad Laboratories). Cell proliferation was determined by calculating relative absorbance values, measured by plate reader, at 24, 48, and 72 h, using the 24-h value as a baseline. Statistical significance between the control and DEC1 knockdown groups was determined at each time point (24, 48, and 72 h) using the Mann–Whitney U-test. The test was performed once daily for four days, and the experiment was performed in triplicate with three independent biological replicates.

For the wound healing assay, 1×105 cells/well were cultured in six-well plates. After 24 h, the complete medium was replaced with a serum-free medium. When the cells reached 90% confluence, wounds were created with a 200-μl pipette tip across each well. The cells were gently washed twice with phosphate-buffered saline to remove loose cells, and a serum-free medium was added. To ensure uniformity in the wound area, multiple positioning marks were made at the center of the denuded surface. Scratch zones were photographed using an inverted microscope at 0, 24, and 48 h. The Axio Vision Rel. 4·8 software was used for measurements to determine the migratory ability of cancer cells.

The experiment was performed in triplicate with three independent biological replicates, and each wound area was measured three times.

Induction of circadian rhythms. We modified a previously published experimental method to induce circadian rhythms in DEC1 expression in colon cancer cell lines and 2DOs (26). In the first experiment, colon cancer cell lines and 2DOs were seeded at 5×105 cells/10 cm dish in DMEM medium supplemented with 5% serum and cultured in a 37°C incubator. After seven days, the medium was substantially depleted of growth factors and nutrients and the cells had reached approximately 80% confluency. The medium was replaced with pre-warmed DMEM containing 50% serum and incubated at 37°C for 2 h, at which point (time 0), the medium was replaced with pre-warmed serum-free DMEM. It has been known for many years that treatment of serum-starved cells with high concentrations of serum induces the transient expression of several immediate early genes. Then, DEC1 expression was measured every 6 h.

Chemosensitivity assay. Cells (2×103 cells/well) and organoids (5×103 cells/well) were seeded and cultured in 96-well plates. When the cells reached 60%-70% confluence, they were treated with 5-fluorouracil (5-FU) (0.3-150 μg/ml for cell lines and 0.003-300 μg/ml for 2DOs). The 2DOs used in these experiments were human tumor cells isolated from surgical specimens and passaged 5-8 times. After seeding in 96-well plates, the cells were incubated for 24, 48, and 72 h. Absorbance of the samples was measured at 450 nm following treatment with 10% CCK-8 at 37°C for 2 h. Cell viability was calculated as the ratio of optical density values of the drug-treated samples to those of controls. IC50 was calculated as the drug concentration that reduced cell viability by 50%, determined through nonlinear regression analysis of the curve.

Flow cytometric analysis. The expression of surface proteins was determined using flow cytometric analysis. Cells were dissociated with trypsin without ethylenediaminetetraacetate and subsequently incubated with components of the Annexin V-DY-634 propidium iodide (PI) Apoptosis Staining/Detection Kit (Abcam, Cambridge, UK). The proportion of apoptotic cells was determined using a flow cytometer (FACScan, BD Biosciences, Franklin Lakes, NJ, USA). Relative fluorescence intensities were measured using an SH800 cell sorter (Sony Corporation, Tokyo, Japan). Data were analyzed using the FlowJo software, version 10.2 (FlowJo, Ashland, OR, USA).

Apoptosis assay. Apoptotic cells were detected using Annexin V, APC conjugate (cat. no. A35110; Thermo Fisher Scientific Inc.), and PI (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were exposed to 5-FU for 72 h, followed by staining with Annexin V/PI. Data were collected 1 h after transfection. The samples were evaluated using an SH800 cell sorter (Sony Corporation). Data analysis was performed using the FlowJo software (FlowJo).

Xenograft model. Stable transfected cell lines were established based on our previous methodology, which utilized lentiviral vectors carrying specific shRNAs for knockdown and cDNA constructs for over-expression (22). To evaluate tumor growth and treatment response in an immunodeficient environment, preventing rejection of the human cells, 1×105 cells, stably transfected, were suspended in Matrigel (BD Biosciences) and subcutaneously transplanted into the dorsal flanks of 6-week-old, male, nonobese, diabetes/severe combined immunodeficiency mice (NOD/ShiJic-scidJcl, CLEA, Tokyo, Japan). Transplanted animals were monitored three times per week (Figure S2).

The mice were euthanized on day 13 after transplantation, through cervical dislocation under general anesthesia using a mixture of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg). The anesthetic was administered via intraperitoneal injection. The Osaka International Cancer Institute Review Board and Animal Research Committee approved this study (R6030619-1).

Tumor growth was monitored by measuring the tumors with digital calipers. The tumor volume (V) was calculated using the following calculation:

Embedded Image

where L is the longest tumor diameter and W is the diameter perpendicular to L. This calculation assumes that the tumor approximates an ellipsoidal shape. Measurements were performed at regular intervals throughout the study to assess tumor progression.

RNA sequencing. RNA sequencing was carried out as previously described (27), utilizing RNA extracted and submitted to the Genome Analysis Laboratory at the Bioinformatics Center, Research Institute for Microbial Diseases, Osaka University. Single-read sequencing was performed on a next-generation sequencer. Mapping of reads was conducted with Hisat 2, and subsequently, expression levels were quantified using Cufflinks according to the software’s manual. For bioinformatics analysis, gene expression was analyzed employing iDEP.96 and GSEA software, based on the FPKM values obtained.

Ethics. The study was conducted in accordance with the tenets of the Declaration of Helsinki and approved by the Institutional Review Board of Osaka International Cancer Institute (protocol code 1711015224-23 and date of approval, April 27, 2023). The animal study protocol was approved by the Osaka International Cancer Institute Review Board and Animal Research Committee (protocol code 24021917 and date of approval, March 3, 2022). Written informed consent has been obtained from the patients to publish this paper.

Statistical analysis. Continuous variables are expressed as mean±standard deviation. Differences in clinicopathological factors between the DEC1 groups were analyzed using the chi-square test or Fisher’s exact test. Continuous variables with parametric distributions were analyzed using Student’s t-test or analysis of variance. Overall survival (OS) and disease-free survival (DFS) curves were plotted using the Kaplan–Meier method and compared using the generalized log-rank test. Univariate and multivariate analyses were performed using a Cox proportional hazards regression model to identify the independent risk factors for OS and DFS. Statistical significance was defined as two-sided p<0.05. All statistical analyses were performed using JMP version 17 (SAS Institute Inc., Cary, NC, USA). The specific statistical tests employed for each comparison are detailed within the figure legends. This approach ensures clarity and allows for precise identification of the analytical methods used for each dataset presented.

Results

DEC1 is significantly over-expressed in CRC tissues. DEC1 and DEC2 expression data (cancer vs. normal) obtained from The Cancer Genome Atlas (TCGA) database indicated that DEC1 expression is significantly higher in CRC tissues than in the adjacent normal tissues (p<0.001 and 0.023, Figure 1A). Moreover, in CRC clinical samples (n=135) of colorectal cancer and adjacent normal tissues, qRT-PCR analysis revealed that DEC1 expression was higher in the colorectal cancer tissues than in the adjacent normal tissues (Figure 1B).

Figure 1.
Figure 1.
Figure 1.

DEC1 expression is associated with prognosis in colorectal cancer. A) High DEC1 and DEC2 expression is significantly associated with a poor prognosis, as revealed by The Cancer Genome Atlas (TCGA) database analysis. B) DEC1 mRNA is significantly over-expressed in colorectal cancer. DEC1 mRNA expression levels are significantly higher in cancerous tissues than in the adjacent normal tissues in clinical samples. Statistical analysis was performed using the log-rank test. C) High DEC1 mRNA expression is associated with poorer disease-free survival (DFS) and overall survival (OS). DFS: pStage I-III + pStage IV (R0), n=117; OS: pStage I-IV, n=135. Statistical analysis was performed using the log-rank test. D) High DEC1 protein expression is associated with poor disease-free survival (DFS) and overall survival (OS). DFS: pStage I-III + pStage IV (R0), n=108; OS: pStage I-IV, n=126. Statistical analysis was performed using the log-rank test.

High DEC1 expression is associated with a poorer disease-free survival and overall survival in patients with CRC. High DEC1 expression significantly correlated with distant metastasis (Table I, p=0.004). The correlation between DEC1 expression and DFS in patients with CRC did not differ significantly between the low- and high-DEC1 expression groups (n=117; Figure 1C). Conversely, in terms of OS, the high-DEC1 expression group had a significantly poorer prognosis than the low-DEC1 expression group (n=135; Figure 1C). The multivariate analysis indicated that lymph node metastasis is an independent prognostic factor for DFS and OS in patients with CRC (Table SI, p=0.003; Table SII, p=0.021). Consistent with the findings of TCGA database analysis, IHC revealed stronger nuclear staining for DEC1 in tumor tissues than in normal tissues. We classified patients with CRC into two groups based on DEC1 nuclear staining intensity: high expression (scores 1+ or 2+) and low expression (score 0).

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Table I.

Clinicopathological characteristics of patients with colorectal cancer with high or low DEC1 expression.

High DEC1 protein expression is associated with a poor OS. The study included 126 cases from a total of 135 clinical samples, after excluding nine cases where the pathological tissue blocks were unsuitable for staining. High DEC1 expression significantly correlated with preoperative CEA levels and distant metastasis (Table II, p=0.006 and 0.007, respectively). Notably, DFS did not differ significantly between the high- and low-DEC1 expression groups (n=108; Figure 1D). Conversely, the high-DEC1 expression group had a significantly poorer OS than the low-DEC1 expression group (n=126; Figure 1D). The multivariate analysis indicated that lymph node metastasis is an independent prognostic factor for DFS in patients with CRC (Table SIII, p=0.003). However, DEC1 expression was identified as an independent prognostic factor for OS in patients with CRC (Table III, p=0.039). These results indicate that DEC1 is highly expressed in tumors and is associated with the prognosis in CRC. The correlation between DEC1 mRNA and protein expression was examined in a limited number of tumor samples. The frequency of high DEC1 expression was in accordance with the results for DEC1 mRNA expression. The RT-PCR confirmed that 49 of the 60 tumors with high protein expression levels, also had higher DEC1 mRNA expression levels, whereas 54 of the 67 tumors with low protein expression had lower mRNA levels, indicating that high expression of DEC1 mRNA was associated with DEC1 protein expression (Table SIV, p<0.05).

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Table II.

Clinicopathological characteristics of patients with colorectal cancer and high or low DEC1 protein expression.

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Table III.

Kaplan–Meier analysis of overall survival (OS) according to DEC1 protein expression.

DEC1 does not promote CRC cell migration and tumor formation in vitro or in vivo. In vitro and in vivo experiments were used to evaluate the molecular mechanisms underlying the involvement of DEC1 in CRC development. DEC1 expression was elevated in RKO and SW480 colon cancer cell lines. The knockdown experiments conducted using specific shRNAs (control, shDEC1 #1, and shDEC1 #2) and DEC1 plasmids in RKO and SW480 cells revealed the down-regulation of DEC1 expression at both mRNA and protein levels (Figure S3). The CCK-8 assay revealed that DEC1 knockdown did not suppress CRC cell proliferation in vitro or in the xenograft model (Figure 2A and D). Given DEC1’s known association with the circadian rhythm and its potential regulation by hormonal fluctuations, we used only male mice to minimize confounding factors. Future investigations including female mice will be necessary to explore possible sex-related influences on DEC1 expression. The wound healing assay showed that DEC1 knockdown did not significantly inhibit wound healing (Figure 2B and C).

Figure 2.
Figure 2.
Figure 2.

DEC1 expression does not promote cell proliferation and tumor formation in colorectal cancer cells in vitro and in vivo. A) In vitro cell proliferation assay using RKO and SW480 cells. B) Wound healing assay in RKO cells. C) Wound healing assay in SW480 cells. D) Cell proliferation assay in the xenograft model: RKO and SW480. Sample size for each experiment was six (n=6). The relative rates were determined by setting the values at day 0 as 1. Statistical analysis was performed using the Mann–Whitney U-test; however, no statistically significant differences were observed between treatment conditions, and thus, p-values are not included.

DEC1 mRNA expression and circadian rhythm. Comparisons of DEC1 mRNA expression levels in RKO cells and iCC129 transfected with shRNA (control) or two types of DEC1 shRNA (shDEC1 #1 and #2) revealed that the circadian rhythm was disrupted in shDEC1-transfected cells, accompanied by decreased DEC1 mRNA expression (Figure 3A). DEC2 mRNA expression remained unchanged after DEC1 knockdown (Figure 3B and Figure S4). Given the involvement of various DEC subtypes in circadian rhythms (28), we considered the possibility that DEC1 may be associated with poor prognosis in CRC through mechanisms independent of its role in circadian rhythm. Furthermore, since DEC2 expression remained unchanged upon DEC1 modulation, we hypothesized that DEC1 alone might contribute to CRC progression and poor prognosis.

Figure 3.
Figure 3.
Figure 3.
Figure 3.

DEC1 affects circadian rhythm and influences chemotherapy resistance in colorectal cancer cells both in vitro and in vivo. A) Circadian rhythms in RKO cells and iCC129 organoids. B) Changes in DEC2 expression associated with the regulation of DEC1 expression. C) Sensitivity of cell lines (RKO and SW480) and organoids (iCC603 and iCC129) to 5-FU. D) Sensitivity of cell lines (RKO and SW480) to 5-FU. E) Proliferation assay following intraperitoneal administration of 5-FU in the xenograft model. The number of samples was 6 (n=6). Statistical analysis was performed using the Mann–Whitney U-test.

DEC1 expression influences chemotherapy resistance in CRC cells in vitro and in vivo. The chemosensitivity analysis with 5-FU demonstrated that shDEC1-transfected cells exhibited higher sensitivity to 5-FU than cells transfected with negative control shRNA in the cell lines (RKO and SW480) and organoids (iCC603 and iCC129) (Figure 3C). The CCK-8 assay revealed that the viability of sh-DEC1-transfected cells was significantly lower than that of the control cells. Conversely, RKO and SW480 cells, in which DEC1 expression was restored following DEC1 over-expression after shRNA-mediated knockdown, exhibited reduced sensitivity to 5-FU compared to cells with DEC1 knockdown alone (Figure 3D). These results indicate that forced over-expression of DEC1 after shRNA-mediated knockdown led to decreased sensitivity to 5-FU, highlighting the potential role of DEC1 in modulating chemotherapy response. We generated a subcutaneous xenograft model using the SW480 cell line. After confirming tumor formation, 5-FU was administered intraperitoneally into the model mice, resulting in reduced tumor proliferation in shDEC1 cells compared with that in the control group cells (Figure 3E and Table SV).

DEC1 expression and clinicopathological characteristics of stage III and IV CRC. We examined the relationship between DEC1 expression and survival in patients with stage III and IV CRC. Based on OS, the high-DEC1 expression group had a significantly poorer prognosis than the low-DEC1 expression group (n=86) (Figure S5).

Association between chemosensitivity and apoptosis. The fluorescence-activated cell sorting apoptosis assay revealed that RKO and SW480 cells transfected with DEC1 shRNA exhibited a significantly higher proportion of apoptotic cells after treatment with 5-FU than cells transfected with the negative control shRNA (Figure 4A and B). The results of the apoptosis assay for control and shDEC1 in RKO and SW480 are shown in Figure S6.

Figure 4.
Figure 4.
Figure 4.

Association between chemosensitivity and apoptosis examined using RNA sequencing analysis. A) Apoptosis assay in RKO cells treated with 5-FU. Following 5-FU treatment, cells with shDEC1 knockdown exhibit a higher population of apoptotic cells, as indicated by increased cell distribution in Q2 and Q4 quadrants. B) Apoptosis assay in SW480 cells treated with 5-FU. C) RNA sequencing analysis.

Identification of genes associated with chemosensitivity and apoptosis. DEC1 knockdown led to increased sensitivity to chemotherapy and a higher number of apoptotic cells than in the control group, whereas DEC1 rescue exhibited the opposite effect; therefore, we used RNA-Seq to identify genes associated with both chemosensitivity and apoptosis. We extracted data of genes exhibiting more than a 10-fold change in mRNA expression in shDEC1 cell lines compared with those in the control. We performed a Metascape analysis to identify genes that are significantly associated with a poor OS (n=67) and a prognostic analysis using data from TCGA. We found that DEC1 knockdown significantly altered the expression of seven genes related to apoptosis (Figure 4C).

Discussion

Clock genes, including PER, CRY, CLOCK, and BMAL, have established roles in regulating circadian rhythm, immune response, carcinogenesis, low oxygen response, and tissue differentiation (25, 29). They are also involved in processes such as apoptosis and epithelial–mesenchymal transition in cancer cells (30). The DEC family includes DEC1 and DEC2, which are complementary genes, with undetermined molecular functions (28, 31).

DEC1 is associated with circadian rhythms, cell differentiation, cancer, apoptosis induction, cell growth regulation, circadian rhythm maintenance, cancer progression, and hypoxia response (32-34). The correlation between DEC1 expression and tumor progression or recurrence varies among cancer types, and despite numerous reports on the roles of DEC1 in other cancers, its role is not well defined in CRC (17-21). In this study, we focused on DEC1, which exhibited significantly higher expression in the human organoids established in our previous study than in normal tissues.

The mRNA expression level of DEC1 varies across cancer types, including gastric and breast cancers (20, 21). In the present study, we observed higher DEC1 expression in tumor tissues from patients with CRC than in the adjacent normal tissues. We stratified the patient cohort into subgroups based on high and low DEC1 mRNA and protein expression levels. Notably, the subgroup with elevated DEC1 expression exhibited a worse prognosis. This trend persisted even when the analysis was confined to patients with stage III and IV CRC, who underwent chemotherapy; the group with high DEC1 protein expression still displayed a poorer prognosis. Although DEC1 expression correlated with lymph node metastasis, survival analysis did not show a significant difference, indicating potential involvement of additional factors (Figure S7 and Table SVI). These results suggest that DEC1 expression may be an indicator of prognosis and the necessity for adjuvant chemotherapy.

DEC1 knockdown did not result in significant changes in cellular characteristics compared to control cells. Altered DEC1 expression has been reported in various cancers (35). In gastric cancer cells, DEC1 promotes survivin expression and enhances anti-apoptotic activity under hypoxic conditions (22). In colon cancer, DEC1 promotes the phosphorylation of STAT3, a key factor in the JAK-STAT signaling pathway activated by IL-6, increasing cancer cell survival and proliferation (35). However, DEC1, as a clock gene, operates in a feedback loop with other genes. Therefore, we hypothesized that alterations in DEC1 expression alone do not directly affect cellular function. In future studies, we plan to explore the relationship between DEC1 expression and established pathways as well as its interactions with oncogenes and tumor suppressor genes. DEC1, a gene associated with circadian rhythms, exhibits oscillatory expression patterns as previously reported. While shRNA-mediated knockdown of DEC1 effectively suppresses overall expression levels, but the amplitude of the rhythm, though diminished, persists. However, the precise relationship between these changes in rhythmic expression and decreased malignancy or altered treatment sensitivity remains to be elucidated. Further research is warranted to fully understand these interactions. The circadian regulation of DEC1 may be influenced by metabolic conditions, particularly glucose availability. Previous studies suggest that DEC1 expression declines when glucose levels are depleted, indicating a potential metabolic regulation of its circadian rhythm. Additionally, AMPK, a key energy sensor, has been reported to interact with DEC1, potentially linking cellular energy status to its circadian dynamics. These findings suggest that DEC1 may integrate metabolic cues into the regulation of circadian rhythms, which could have implications for its role in cancer progression (36).

DEC1 knockdown with shRNA resulted in enhanced sensitivity of cancer cells to 5-FU chemotherapy, and an increased apoptosis rate. Notably, paclitaxel up-regulates DEC1 expression in breast cancer cells and promotes apoptosis (37). Additionally, DEC1 enhances cisplatin-induced apoptosis in highly differentiated esophageal cancer cells (33). These findings suggest that the effects of DEC1 may vary depending on the anticancer drug, cancer type, and differentiation status.

In this study, we specifically employed 5-FU, which is a chemotherapeutic agent commonly used for CRC; it is known for its capacity to induce cell death during the S phase. This is the first study to report the relationship between DEC1 expression, chemotherapy sensitivity, and apoptosis in CRC. The results suggest that the down-regulation of DEC1 expression in CRC cells could augment drug sensitivity and induce apoptosis, indicating that DEC1 is a potential treatment target. As associations between DEC1 expression and angiogenesis have been established (38), future investigations should explore relationships with other drugs, including oxaliplatin and angiogenesis inhibitors.

As DEC1 knockdown enhanced drug sensitivity and the number of apoptotic cells, we performed RNA-Seq to identify genes associated with DEC1-mediated alterations in chemotherapy sensitivity, revealing the up-regulation of seven apoptosis-related genes. Following DEC1 knockdown, BID, CASP7, CASP8, FZD6, OMA1, and PTHRH2 were up-regulated, while BIRC5 was down-regulated, supporting the role of DEC1 in apoptosis regulation. Among these genes, PTRH2 is known to regulate phosphoinositide 3-kinase (PI3K)/AKT and extracellular-signal-regulated kinase (ERK) signaling. PTRH2 has been implicated in mitochondrial energy metabolism and apoptosis (39). However, the specific association of DEC1 with cancer, as well as its role in colorectal cancer (CRC), remains unclear and warrants further investigation. While there are occasional reports on the relationship between DEC1 and apoptosis in some cancers, no studies have yet explored its role in CRC (22, 32, 34). DEC1 may represent a promising therapeutic target in CRC.

Study limitations. First, only 5-FU was used to assess drug sensitivity; thus, future studies should investigate sensitivity to oxaliplatin and irinotecan, which are currently employed in the treatment of CRC. Second, DEC1 is associated with circadian rhythms; while we confirmed this observation in vitro, we were unable to conduct animal experiments to explore circadian rhythms, drug sensitivity, and other treatment-related parameters. Finally, our RNA-Seq analysis of CRC cells expressing DEC1 revealed that DEC1 regulates seven apoptosis-associated genes. While direct mechanistic validation was not performed, our findings provide a foundation for future investigations into the precise molecular mechanisms underlying DEC1-mediated apoptosis in CRC. Considering the interactions of DEC1 with other clock genes and its current status as an improbable therapeutic target, we intend to explore the relationship between DEC1 and these genes, as well as their therapeutic value, in future studies.

As previously discussed, DEC1 has the potential to influence both tumor promotion and suppression, depending on the specific conditions (20, 21). Research indicates that DEC1 expression is regulated by various pathways, which may either promote or suppress tumorigenesis. The outcome depends on the particular pathway involved and DEC1’s interaction with it, potentially leading to either tumor progression or regression (40, 41). For example, DEC1 acts as a downstream effector of the TGF-β/Smad signaling pathway, playing roles in both tumor promotion and suppression. Transforming growth factor β (TGF-β) is a dual-function growth factor that can either inhibit or stimulate cell proliferation in a context-dependent manner (41). TGF-β regulates numerous processes during normal development, and disturbances in TGF-β signaling are linked to the initiation of various cancers (41). Thus, DEC1 may function differently in different types of cancer, and further research is warranted. Given that DEC1 has been reported to be involved in the TGF-β/Smad signaling pathway in breast cancer, and this pathway is known to play a significant role in colorectal cancer progression and prognosis, we hypothesize that DEC1 may similarly exert a profound influence on treatment outcomes and patient survival in this malignancy (42).

Our findings suggest that DEC1 does not directly affect the characteristics of CRC cells; however, its down-regulation is associated with chemosensitivity and apoptosis. This down-regulation was associated with changes in the expression of apoptosis-related genes, as observed in our results. These findings have crucial implications for the treatment and prognosis of patients with CRC. Given DEC1’s association with chemosensitivity and survival, it may serve as a predictive biomarker for chemotherapy response in patients with CRC. Future clinical studies should validate its utility in stratifying patients for personalized treatment approaches, potentially guiding therapeutic decisions based on DEC1 expression levels.

Acknowledgements

The Authors thank the staff of the Department of Innovative Oncology Research and Regenerative Medicine, Osaka International Cancer Institute, for their engagement and support during the trial.

Footnotes

  • Authors’ Contributions

    SM, NM, and SF conceived and designed the study. NM and SF supervised the study. SM conducted the experiments. SM and NM prepared and wrote the article and participated in data analysis. SM, NM, SF, MT, YS, TH, AH, TO, MU, HY, YD, and HE participated in the study design and trial. SM and NM were responsible for trial management, including the execution and oversight of preliminary feasibility experiments. They also contributed to data interpretation, as well as manuscript writing and proofreading. SM, NM, SF, MT, YS, TH, AH, TO, MU, HY, YD, and HE participated in patient recruitment.

  • Supplementary Material

    Supplementary materials are available at: https://github.com/NorikatsuMiyoshi/supples-Role-of-the-Circadian-Clock-Gene-DEC1-in-Chemosensitivity-and-Apoptosis-in-CRC

  • Conflicts of Interest

    The Authors declare that they have no competing interests in relation to this study.

  • Funding

    This research was funded by JSPS KAKENHI, grant number 23K08211, and JSS Clinical Investigation Project Award.

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received April 8, 2025.
  • Revision received April 19, 2025.
  • Accepted April 22, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Role of the Circadian Clock Gene DEC1 in Chemosensitivity and Apoptosis in Colorectal Cancer
SOICHIRO MINAMI, NORIKATSU MIYOSHI, SHIKI FUJINO, MASAYOSHI YASUI, MASAYUKI OHUE, MITSUNOBU TAKEDA, YUKI SEKIDO, TSUYOSHI HATA, ATSUSHI HAMABE, TAKAYUKI OGINO, MAMORU UEMURA, HIROFUMI YAMAMOTO, YUICHIRO DOKI, HIDETOSHI EGUCHI
Anticancer Research Jun 2025, 45 (6) 2309-2328; DOI: 10.21873/anticanres.17605
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