The study participants were divided into a CRC group and healthy group (control group). With respect to the CRC group, all patients were hospitalized in the gastrointestinal surgery department of the Second Affiliated Hospital of Zunyi Medical University from January 2020 to January 2021. A total of 143 patients, ranging from 21 to 84 years old, with an average age of 62.01 years old, were diagnosed with CRC via colonoscopy and pathology testing. The 143 patients comprised 89 males, ranging from 21 to 84 years old, with an average age of 60.28 years and 54 females, ranging from 32 to 76 years, with an average age of 64.74 years. For the control group, a total of 143 healthy subjects were selected after physical examination at our hospital during the same period. The participants’ ages ranged from 22 to 78 years, with an average age of 61.87 years. The control group comprised 89 males, aged between 22 and 77 years, with an average age of 61.2 years, and 54 females, ranging from 30 to 78 years old, with an average age of 63.57 years. There were no significant differences in age or sex between the two groups (P > 0.05). The serum from these subjects was stored at −80°C. All experimental procedures were carried out in accordance with the Chinese legislation regarding medical ethics and were approved by the Medical Ethics Committee of Zunyi Medical University [(2019) H-008].

To test the concentration of serum NP, a 0.5 mL serum sample was mixed with 4 mL of n-hexane-ether extract solution (volume ratio of 7:3), after which the mixture was vortexed for 30 s and allowed to stand for 15 min. The supernatant was then dried in a 50°C water bath. The samples were dissolved in 0.5 mL acetonitrile and detected by high performance liquid chromatography (HPLC).

COLO205, SW480, and HCT116 cells were obtained from iCell (Shanghai, China). COLO205 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), HCT116 cells were cultured in dulbecco’s modified eagle medium supplemented with 10% FBS, and SW480 cells were cultured in Leibovitz’s L-15 medium supplemented with 10% FBS. All cells were cultured in an incubator at 37°C with 5% CO₂. All media and FBS were purchased from Thermo Fisher Scientific (Shanghai, China).

Nonylphenol (N109556, Aladdin, Shanghai, China) was dissolved in Dimethyl sulfoxide (DMSO) to 100 mM and diluted to different concentrations (10^–7, 10^–6, and 10^–5 M) to treat the cells.

The ERK1/2 inhibitor (PD98059, MCE, Shanghai, China, the chemical structure showed in Supplementary Figure 1) was dissolved in DMSO to 10 mM and diluted to 20 μM to treat the cells.

Lentiviruses carrying CCDC80 and a negative control (1 × 10⁹ pfu) were generated by GeneChem (Shanghai, China). Before the experiments, the infected COLO205 and SW480 cells were treated with G418 (800 ng/mL) for 2 weeks.

Infected COLO205 and SW480 cells were seeded in 24-well plates at a density of 1 × 10⁵ cells/well for 24 h. After treatment with NP (10^–6 M) or DMSO (n = 3 per group) for 24 h, the cells were treated with 5-ethynyl-2′-deoxyuridine (EdU) and counted using the BeyoClickTMEdU-488 cell proliferation kit (Beyotime, Hangzhou, China) according to the manufacturer’s instructions. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole. They were then visualized under a fluorescence microscope, photographed, and the number of positive cells (green fluorescence) were analyzed using the ImageJ software.

Cell apoptosis was analyzed using flow cytometry. Cells were seeded into 6-well plates at a density of 5 × 10⁵ cells/well for 24 h and treated with NP (10^–6 M) for 24 h. Cells were then collected and stained using the Annexin V-FITC/PI cell apoptosis kit (Jiancheng, Nanjing, China).

Infected COLO205 and SW480 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well for 24 h. After treatment with NP (10^–6 M) or DMSO (n = 6 per group) for 24 h, 10 μL of cell counting kit-8 assay (CCK8) reagent (TransGen, Beijing, China) was added to the cells, after which they were incubated for 4 h in an incubator at 37°C with 5% CO₂. Absorbance was measured at 450 nm using a microplate analyzer.

COLO205 cells were treated with NP (10^–6 M) for 24 h, and then digested with trypsin. The samples were sent to Personalbio (Shanghai, China) for second-generation sequencing. Gene expression was normalized using fragments per kilobase of transcript per million fragments (FPKM). The differentially expressed genes between the control and NP treatment groups were selected by DESeq, with a cutoff of |log2FoldChange| > 1, P-value < 0.05. The raw data were uploaded in Gene Expression Omnibus (GEO) as the series number is GSE18222.

COLO205, SW480, and HCT-116 cells were exposed to NP (10^–7, 10^–6, and 10^–5 M) and collected 24 h after treatment. Total RNA was extracted using TRIzol reagent (Invitrogen, United States). After reverse transcription, the expression of CCDC80, matrix metallopeptidase 19 (MMP19), and Sulfiredoxin-1 (SRXN1) was tested using the QuantStudio^TM 6 Flex Real-Time PCR System (Applied Biosystems) using the YBR Green PCR kit (KAPA Biosystems, United States). The relative expression of these genes was analyzed using 2^–ΔΔCt method, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.

The cancer genome atlas (TCGA) analysis of differentially expressed genes after NP treatment was performed using the UALCAN system (Chandrashekar et al., 2017).

Tumor tissue (n = 9) and para-cancerous tissue (n = 9) were fixed with 4% paraformaldehyde and embedded in paraffin. Immunohistochemistry (IHC) and hematoxylin-eosin (HE) staining were performed after sectioning. The primary antibody used was anti-CCDC80 (1:50, Bioss, Beijing, China). Cells with yellow or brown staining in the nucleus and/or cytoplasm were deemed positive for immunoreactivity. The immunoreactivity was scored as average optical density (IOD) using Image-Pro Plus 6.0 software (IPP6.0).

The protein expression of treated cells was analyzed using western blotting, as previously described (Kim et al., 2015). The primary antibodies used were anti-GAPDH (Abcam, United Kingdom), anti-ERK1/2, anti-p-ERK1/2, anti-proliferating cell nuclear antigen (PCNA), anti-c-MYC, anti-cyclin D1, anti-Bad, anti-Bcl-2, and anti-Cleaved Caspase-3 (Santa Cruz Biotechnology, United States). The IOD was analyzed using IPP6.0.

The mice (BALB/c athymic nude mice, male, 5–6-week-old) information remains the same as previously described (Kim et al., 2015). All experimental procedures were carried out in accordance with the Chinese legislation regarding experimental animals and were approved by the Animal Experiment Ethics Committee of Zunyi Medical University [(2019) A-004]. SW480 cells were digested and resuspended at a concentration of 1.0 × 10⁷ cells/mL. To construct xenograft models, 20 μL of cells were injected into the left posterior flank of each mouse (n = 6 per group). Five days after injection, the mice were divided into four groups: (1) control group, (2) CCDC80 group, (3) NP group, and (4) NP + CCDC80 group. Fifty microliters of lentivirus (5 × 10⁷ pfu) carrying CCDC80 or negative control was injected intratumorally every week for 3 weeks (Xie et al., 2019; Park et al., 2020). NP treatment [17 mg/(kg…d)] and other treatments were consistent with our previous study (Kim et al., 2015).

Three parallel experiments were performed for each treatment. Data were analyzed using GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, United States). Data are presented as mean ± SD. Differences between multiple groups were analyzed using one-way analysis of variance (ANOVA) with Dunnett’s post-test. Differences between two groups were analyzed using Student’s t-test. Statistical significance was set at P < 0.05.

Serum NP levels in healthy patients (n = 143) and patients with CRC (n = 143) were tested. We found a higher concentration of serum NP in patients with CRC than in healthy individuals in both men and women (Figure 1). This result suggests that the concentration of serum NP is associated with CRC occurrence.

We used different concentrations of NP (0, 10^–7, 10^–6, and 10^–5 M) in treating COLO205 and SW480 cells. The Edu assay suggested that NP-induced CRC cell proliferation in a dose-dependent manner (Figure 2A and Supplementary Figure 2A). Furthermore, flow cytometry showed that NP inhibited apoptosis in a dose-dependent manner in CRC cells (Figure 2B). To explore the mechanism of NP on CRC cell progression, we performed transcriptome sequencing to analyze the transcript changes after NP exposure in CRC cells. We found 16 upregulated genes and 12 downregulated genes in COLO205 cells after NP treatment (Figure 3A). Gene ontology (GO) enrichment analysis suggested that NP treatment significantly influenced the processes associated with the extracellular matrix and cell cycle (Figure 3B). Quantitative PCR (qPCR) was performed to validate the sequencing results. The results showed that CCDC80 was downregulated and MMP19 and SRXN1 were upregulated in HCT116, SW480, and COLO205 cells after treatment with different concentrations of NP (Figure 3C).

After TCGA analysis for the differentially expressed genes, we found that the expression of CCDC80, a gene downregulated by NP treatment, was significantly lower in tumor tissues than in normal tissues (Figures 4A,B). The expression of CCDC80 in CRC tissues and para-cancerous tissues confirmed the results of the TCGA analysis (Figures 4C,D).

To explore the roles of CCDC80 in NP-induced CRC cell growth, we constructed CCDC80 overexpressed stable infected CRC cell lines and then treated them with NP. EdU and CCK-8 analyses showed that overexpression of CCDC80 significantly suppressed CRC cell proliferation, even after NP treatment (Figures 5A,B and Supplementary Figure 2B). The results of the colony forming assay showed that CRC cell growth was significantly enhanced after NP treatment and suppressed by CCDC80 overexpression (Figure 5C). CCDC80 overexpression also significantly reduced NP-induced cell growth. Flow cytometry showed that overexpression of CCDC80 significantly induced cell apoptosis, even after NP treatment (Figure 5D). Western blot assay showed that, while NP treatment could induce the expression of proliferation-related proteins PCNA, c-MYC, and CyclinD1, CCDC80 overexpression could reduce it (Figure 5E). Furthermore, the expression of proapoptotic proteins (Bad and cleaved Caspase-3) was significantly decreased, and the expression of anti-apoptotic protein (Bcl-2) was significantly increased by NP treatment. Conversely, the overexpression of CCDC80 increased the expression of pro-apoptotic proteins and decreased the expression of anti-apoptotic proteins (Figure 5E and Supplementary Figures 3A,B). The overexpression of CCDC80 attenuated the effect of NP treatment on the expression of proliferation-related and apoptosis-related proteins.

Our previous study revealed that NP activated ERK1/2 (Xie et al., 2019). Therefore, we explored the relationship between the NP treatment, expression of CCDC80, and activation of ERK1/2. Western blot assay showed a decreased expression of phosphorylated ERK1/2, but its expression did not significantly change with the overexpression of CCDC80 after NP treatment (Figure 6A). We further investigated the effect of extracellular regulated protein kinases (ERK) inhibitor (PD98059) combined with NP in infected COLO205 and SW480 cells (Figure 6B). Treatment with the ERK inhibitor alone significantly inhibited the proliferation of CRC cells and induced apoptosis. The effect of the ERK inhibitor was attenuated by NP treatment but was not significantly enhanced by the overexpression of CCDC80 (Figures 6C–E, 7A). In addition, we performed western blot assay to analyze the expression of proliferation-related and apoptosis-related proteins and the activation of ERK1/2 after treatment with an ERK inhibitor combined with NP in infected CRC cells. Treatment with NP alone increased the expression of proliferation-related proteins (PCNA, c-MYC, and CyclinD1) and Bcl-2, while overexpression of CCDC80, especially when combined with an ERK inhibitor treatment, decreased the expression of these proteins. Furthermore, NP treatment induced the expression of anti-apoptotic proteins (Bad and cleaved Caspase-3), whereas this was reduced by the overexpression of CCDC80 combined with an ERK inhibitor (Figure 7B and Supplementary Figures 3C,D).

A mouse xenograft model of human CRC cells was established to study the effect of CCDC80 on NP-induced tumor growth in vivo. The results showed that NP treatment significantly increased tumor growth, and that the overexpression of CCDC80 significantly suppressed tumor growth, even when co-treated with NP (Figures 8A–C and Supplementary Figure 4). IHC results showed lower expression of the proliferative marker Ki67 in the CCDC80 overexpression group (Figure 8D). Western blot assay showed similar effects of CCDC80 overexpression and NP treatment on the expression of proliferation-related proteins (PCNA, c-MYC, and CyclinD1), apoptosis-related proteins (Bcl-2, Bad, and cleaved Caspase-3), and activation of ERK1/2 in in vitro experiments (Figure 8E). These results revealed that CCDC80 inhibited NP-induced tumor growth.