CHI3L1 promotes proliferation and improves sensitivity to cetuximab in colon cancer cells by down‐regulating p53

Abstract Background Chitinase 3‐like protein 1 (CHI3L1) is most likely a malignant tumor metastasis‐associated gene. However, the functions of CHI3L1 in colon cancer cell proliferation and its cetuximab sensitivity are still unclear. We aimed to investigate the mechanism of CHI3L1 in promoting colon cancer cell proliferation and its sensitivity to cetuximab. Methods The expression of CHI3L1 in colon cancer and adjacent tissues were detected by immunohistochemistry. CHI3L1 was overexpressed in colon cancer cell lines by lentiviral technology. Cell proliferation and sensitivity to cetuximab were measured by MTT assay, cell cycle was analyzed by flow cytometry, and expression of cell cycle‐related proteins was analyzed by immunoblotting. Results The results showed that the level of CHI3L1 in colon cancer tissue was significantly higher than that in adjacent tissue, which was also correlated with overall survival. The cell proliferation rate was significantly increased after overexpression of CHI3L1, and the sensitivity to cetuximab was significantly increased. The expression of p53 was down‐regulated while the EGFR was up‐regulated significantly in CHI3L1 overexpressed cells. When rescued the expression of p53 in HCT116‐CHI3L1 cells, the cell proliferation and sensitivity to cetuximab could be restored. Conclusion High levels of CHI3L1 are associated with poor prognosis and accelerate the proliferation of colon cancer cells and increase the sensitivity to cetuximab. Its mechanism of increasing the cell proliferation and sensitivity to cetuximab may be explained by down‐regulating p53 expression and then, up‐regulating the expression of EGFR.


| INTRODUC TI ON
In spite of the decreased incidence of colon cancer (CC) in recent 10 years, it remains the fourth most frequently diagnosed cancer and the second leading cause of cancer death in the United States. 1 But in China, colon cancer is on the rise, which is the fourth most commonly diagnosed cancers among men and the third one among women. 2 Approximately 20%-25% of patients diagnosed with colon cancer present with metastatic disease and result in a poor prognosis. [3][4][5] Cetuximab is a chimeric monoclonal antibody directed against EGFR that inhibits its downstream signaling pathways. It has been studied in combination with chemotherapy as an initial therapy option for treatment of metastatic colon cancer and provides a clear clinical benefit in patients with RAS wild-type gene mutation. 6,7 Unfortunately, cetuximab is only effective in approximately 10%-20% of patients with colon cancer, 8,9 implying the sense of identification of biomarkers that predict which patients are likely to be sensitive to cetuximab beyond RAS mutation and BRAF mutation status.
Chitinase 3-like protein 1 (CHI3L1) (also known as YKL-40) was discovered in human osteosarcoma cell line MG63 by Johansen et al 10 in 1992. CHI3L1 is a highly conserved glycoprotein with a coding gene of about 8 kb located on human chromosome 1q32.1. 11,12 CHI3L1 is highly expressed in embryonic tissues with rapid proliferation and differentiation characteristics and adult tissue cells with high cell activity. 13,14 Several studies have demonstrated that plasma YKL-40 is a potential prognostic marker in different solid tumors, such as breast cancer, ovarian cancer, prostate cancer, and colon cancer. [15][16][17][18][19][20][21] Besides, CHI3L1 has been shown to promote cancer angiogenesis in breast, colon, and glioblastoma cancer cells in vivo. [22][23][24] These results suggest that CHI3L1 is most likely a malignant tumor metastasis-associated gene. However, the functions of CHI3L1 in colon cell proliferation and its sensitivity to cetuximab are still unclear.
In the present study, we aimed to investigate the mechanism of CHI3L1 in promoting proliferation and sensitivity to cetuximab in colon cancer cells.

| Clinical data
The relative expression levels of CHI3L1, as well as clinicopathologi-

| Cell culture
Monolayer cells were cultured in DMEM medium containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C with 5% CO 2 , and the cells were sub-cultured every 2-3 days at a ratio of 1:3-1:5. Cell growth at the logarithmic phase was used in experiments.

| Lentivirus packaging and infection
293T cells were cultured in DMEM containing 10% fetal calf serum (FBS), 100 U/mL ampicillin, and 100 U/mL streptomycin based on a 37°C 5% CO 2 incubator. The culture solution was changed to DMEM medium 2 hours before the transfection. About 2-3 μg of recombinant lentiviral plasmid pCDH-CMV-CHI3L1-GFP-puro (pCDH-CMV-GFPpuro plasmid used as control), lentiviral packaging plasmid △8.2, and VSV-G were mixed at a weight ratio of 10:10:1. About 6 μL of transfection reagent and 100 μL of Opti-MEM medium (Serum-free and antibiotic-free) was added into the mixture, which was then incubated at room temperature for 20 minutes. The mixture was added to the cultured 293T cells, and after transfection for 6 hours, the medium was replaced with complete medium. After 48 hours of transfection, the fluorescence was observed, and the supernatant virus was collected and pass through a 0.45 μm filter to SW620 and HCT116 cells. The cells were screened with puromycin to obtain CHI3L1 stably expressed cell line. Quantitative PCR (qPCR) and Western blotting were used to detect the expression of CHI3L1 in overexpressed and control cells.

| Real-time PCR
To detect CHI3L1 mRNA expression in different cell lines. Cell total RNA was extracted with Trizol (method according to the instruction) and was

| Western blot
Removed the cells from CO 2 incubator, discarded the medium and put the cells on the ice, rinsed cells by cold PBS for two times, drained the remaining PBS, and lysed cell on the ice in the RIPA lysate. The cell lysate was passed through a 1 mL syringe to make DNA fracture, and 3 μL of cell lysate was taken for BCA quantification after incubated at 95°C for 5-10 minutes. About 40 μg of protein with the sample buffer was loaded in each lane for electrophoresis in 10% SDS-page gel.
Protein was then electric transferred to PVDF membrane by 200 mA constant-current for 1.5 hours. PVDF membrane was blocked using 5% skim milk for 1 hour and incubated with primary antibody at 4°C overnight. The membrane was washed with TBST for three times, added with the secondary antibody and incubated at room temperature for 1 hour Then, the membrane was washed with TBST for three times. The membrane was exposed to enhanced chemiluminescence substrates, and blots were photographed. The relative expression of proteins was quantified by ImageJ software.

| MTT assay
Using MTT assay to detect cell sensitivity to cetuximab. About 24 hours before drug treatment, HCT116 and SW620 cells were

| Cell cycles analysis
When the cells were grown to a confluence of 80%, the cell cul-

| Statistical analysis
The intergroup comparison of clinicopathologic variables was performed with the chi-square test. Overall survival (OS) was calculated from the date of diagnosis to the date of death for any cause. Survival was analyzed using the Kaplan-Meier method. The association between each of the potential prognostic factors and differences between the curves were analyzed by the log-rank test. Multivariate analysis was performed using the Cox regression model. The statistical test was two-sided, and P < .05 was considered statistically significant. Statistical analyses were conducted by SPSS 20.0 (IBM, SPSS) and GraphPad Prism.

| CHI3L1 levels are up-regulated in colon cancer and correlated with unfavorable prognosis of colon cancer patients
By using the UCSC Xena platform, we demonstrated that the expression level of CHI3L1 was significantly increased in colon cancer tissues (n = 285, P < .0001). In the 25 pair-matched samples, CHI3L1 was markedly up-regulated in cancer tissues compared with adjacent normal tissues (n = 25, P < .0001; Figure 1A). The CHI3L1 immunohistochemistry staining in 15 colon cancer samples and corresponding adjacent normal tissues showed that CHI3L1 mainly distributed in the cytoplasm and its positive rate was much higher in cancer tissues than adjacent normal tissues (P = .005; Figure 1B).
Besides, CHI3L1 mRNA expression was also higher in three colon cell lines than the normal intestinal epithelium cell line HIEC except for the colon cell line GEO by using qRT-PCR analysis ( Figure 1C).
These results indicated that the upregulation of CHI3L1 was a frequent molecular event in colon cancer.

| Construction of CHI3L1 stably transfected cell line
The CHI3L1 stably transfected cell line was constructed by lentiviral technology. The pCDH-CHI3L1 overexpression plasmid was successfully constructed by sequencing. CHI3L1 was overexpressed in SW620 and HCT116 by lentiviral technology, and the cell infection rate was 98% from the fluorescence results (Figure 2A). qPCR results showed that the level of CHI3L1 mRNA was significantly increased in SW620 and HCT116 cells after transfection of the expression plasmid ( Figure 2B). The results of immunoblotting showed that the expression of CHI3L1 was significantly increased compared with the control (Figure 2C), which indicates that CHI3L1 Stable transfected cell lines were successfully constructed.

| Increased sensitivity to cetuximab by overexpression of CHI3L1
The cell proliferation curve was examined, and the results showed that the relative proliferation rate of cells increased after overexpression of CHI3L1 ( Figure 3A). The sensitivity of the cells to cetuximab was examined. The results showed that the inhibition of cetuximab by HCT116 overexpressed CHI3L1 was significantly enhanced ( Figure 3B). About 31.25, 62.5, 125, 250, and 500 ng/mL of exogenous CHI3L1 protein were added to the two colon cancer cells, and the sensitivity of which to cetuximab was detected. The results showed that the sensitivity of cells to cetuximab increased with the increase of CHI3L1 in the concentration range of 31.25-250 ng/mL, indicating that high level of CHI3L1 will increase the sensitivity of colon cancer cells to cetuximab ( Figure 3C). The effect of cetuximab on the cell cycle of colon cancer cells before and after CHI3L1 overexpression was detected by flow cytometry. Compared with the control group, the proportion of G0/G1 phase cells was increased in the CHI3L1 overexpressed group both in the presence and absence of cetuximab treatment ( Figure 3D and 3). TA B L E 2 Cox regression analysis of Chitinase 3-like protein 1 (CHI3L1) expression as survival predictor

| Mechanism exploration
The changes in cell cycle-related proteins were detected by immunoblotting. The results showed that with the increase of cetuximab, the expression of EGFR up-regulated while the expression of p53 protein was significantly down-regulated ( Figure 4A and 4). It is speculated that CHI3L1 may increase the sensitivity of cells to cetuximab by down-regulating p53 and up-regulating EGFR. To confirm this hypothesis, p53 overexpression plasmid was transfected into HCT116-CHI3L1 cells to increase the expression level of p53. Its proliferation and sensitivity to cetuximab were detected, and the expression levels of p53 and EGFR were also detected by Western blot. The results showed that p53 was down-regulated and EGFR was up-regulated after overexpression of CHI3L1, while the expression of EGFR was decreased after the overexpression of p53 ( Figure 4C). At the same time, the relative proliferation rate ( Figure 4D) and the sensitivity of cells to cetuximab were significantly decreased ( Figure 4E) after the overexpression of p53 compared with the control group. These indicated that CHI3L1 could increase the cell sensitivity to cetuximab and promote the proliferation of cell by down-regulating p53 and up-regulating EGFR. The p53 gene is one of the most frequently studied tumor suppressor genes, which is associated with malignant transformation as well as tumorigenesis and development. There is a high incidence of mutations or deletions of this gene in most tumor types. 32 Also, studies indicated that downregulation of p53 could accelerate the proliferation in cancer cells with various mechanisms. [33][34][35] In our study, overexpression of CHI3L1 in colon cancer cells stimulated cell proliferation, which could be partly explained by down-regulation of p53. EGFR was a key target for molecular therapy, involved in the regulation of cell metabolism, growth, migration, and differentiation, which was overexpressed in various tumors such as prostate cancer, breast cancer, and colon cancer. 36 Meanwhile, a previous study showed that loss of p53 could cause the increased activity of EGFR promoter, and further increase the expression of EGFR. 37 In consistence with the result, our study showed that EGFR was up-regulated accompanied by down-regulation of p53 in CHI3L1 overexpressed cells. Further, when the p53 overexpression plasmid was transferred to rescue the level of p53, cell proliferation and sensitivity to cetuximab were significantly restored.

| D ISCUSS I ON
This study revealed that high levels of CHI3L1 could increase the sensitivity of cetuximab by down-regulating p53 and up-regulating EGFR, which might provide valuable drug guidance for cetuximab in patients with colon cancer.
In conclusion, high levels of CHI3L1 are associated with poor prognosis in colon cancer patients. Overexpression of CHI3L1 can promote the proliferation of colon cancer cells and increase the sensitivity to cetuximab by down-regulating the p53 expression F I G U R E 3 Effect of Chitinase 3-like protein 1 (CHI3L1) overexpression on cell proliferation and drug sensitivity. A, CHI3L1 overexpression promotes cell proliferation. B, The inhibition rates of SW620 and HCT116 cells after treatment with different concentrations of cetuximab. C, The sensitivity of cells to cetuximab (1mg/ml) increased after the addition of different concentrations of exogenous CHI3L1. D, Effect of cetuximab on cell cycle after overexpression of CHI3L1. E, Proportion of each phase of the cell cycle. *, P < .05, in HCT116 group; △ , P < .05, in SW620 group and up-regulating the expression of EGFR. In the future, we will further verify the role of CHI3L1 in guiding the personalized medicine of cetuximab.

This study was supported by grants from the Medical Technology
Project of Ningbo (number 2018A01).