Urocortin-1 Promotes Colorectal Cancer Cells Migration, Proliferation and Inhibits Apoptosis via Inhibition of p53 Signaling Pathway

doi:10.21203/rs.3.rs-3216797/v1

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Research Article

Urocortin-1 Promotes Colorectal Cancer Cells Migration, Proliferation and Inhibits Apoptosis via Inhibition of p53 Signaling Pathway

https://doi.org/10.21203/rs.3.rs-3216797/v1

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Journal Publication

published 28 Mar, 2024

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Purpose

To investigate the effect of urocortin-1 (UCN-1) on the growth, migration and apoptosis of colorectal cancer (CRC) in vivo and vitro and mechanism of UCN-1 modulating CRC cells in vitro.

Methods

The correlation between UCN-1 and CRC was evaluated by Cancer Genome Atlas (TCGA) database and the tissues microarray. The expression of UCN-1 in CRC cells was explored by quantitative real-time polymerase chain reaction (RT-qPCR) or western blot. In vitro, the influence of UCN-1 on proliferation, apoptosis and migration HCT-116 and RKO cells were explored by celigo cell counting assay, flow cytometry and wound healing assay or transwell, respectively. In vivo the effect of UCN-1 on CRC tumor growth and progression was evaluated in the nude mice. The downstream pathway behind UCN-1 regulating CRC was found by phospho-kinase profiler array in RKO cells. Expression of UCN-1 in cells was knocked down or upregulated using lentivirus.

Results

Both of the results of TCGA database and the tissues microarray shown that UCN-1 strongly expressed in tissues of CRC patients. Furthermore, the tissues microarray results showed that expression of UCN-1 was higher in male CRC patients than that in female patients, and high expression of UCN-1 was associated with higher risk of lymphatic metastasis and later pathological stage. Additionally, knockdown of UCN-1 in CRC cells caused a reduction in cell proliferation, migration, and colony formation as well as an increase in apoptosis. In xenograft experiments, tumors generated from RKO cells with UCN-1 knockdown exhibited declined tumor volume and weight. Reduction of the expression of Ki67 in xenograft tumors reflected that knockdown of UCN-1 curbed the growth of CRC tumors. Furthermore, the human phospho-kinase array showed that p53 signal pathway participated in UCN-1-mediated CRC development. The suppression in migration and proliferation caused by UCN-1 knockdown was reversed by inhibitors of p53 signal pathway, while the increase of cell apoptosis was withdrawn. On the other hand, overexpression of UCN-1 promoted the proliferation and migration and inhibited apoptosis of CRC cells. Overexpression of p53 reversed the effect of UCN-1 overexpression on CRC development.

Conclusion

UCN-1 promotes the migration, proliferation and inhibits apoptosis via inhibition of p53 signaling pathways.

Urocortin-1

p53

ERK1/2

HCT-116

RKO

Colorectal cancer

The increasing incidence of colorectal cancer (CRC) making it becomes the third most common malignant tumor in the world, which seriously endangers human health and increases the social burden (Hasbullah and Musa 2021). Early detection and treatment of CRC are important in effectively improving the CRC patients’ survival rate and the life quality (Lewandowska et al. 2022). However, CRC is usually first diagnosed at an advanced stage (Or et al. 2016), because CRC at an early stage is difficult to detect. Therefore, systemic chemotherapy after surgical resection is generally used for patients with advanced CRC. Clinical recurrence resulting from incomplete surgical resection and metastasis of CRC are the main causes of death related to CRC, there is an urgent need to find effective method to inhibit CRC proliferation and migration to inhibit the tumor recurrence after surgery to improve the survival rate of CRC patients (Peters, Bien, and Zubair 2015).

Urocortin (UCN) belongs to the corticotropin-releasing-hormone (CRH) family, which is involved in lot of physiological actions (Haznadar et al. 2018). It is widely expressed in cardiovascular, pancreas, central nervous and digestive tract of rats and human (Wang et al. 2021; Zhou, Thompson, et al. 2022; Gao et al. 2021; Vuppaladhadiam et al. 2020). UCN binds to G protein-coupled receptor, which known as corticotropin-releasing factor receptor (CRF-R), to mediate stress response (Haznadar et al. 2018), inflammation (Yang, Xu, and Li 2008) and tumorigenesis (Cheng et al. 2014). Increasing evidence have been showed the critical role of UCN participated in diverse malignant tumors of human. For example, researchers have found that UCN can affect the migration of hepatoma carcinoma (Wang et al. 2008; Zhu et al. 2014) and endometrial cancer (Florio et al. 2006). UCN contains three isoforms, UCN-1, UCN-2 and UCN-3. UCN-1, the first UCN discovered, is reported participated in endometrial cancer (Owens et al. 2017), stress-induced gastrointestinal dysfunction (Ayyadurai et al. 2017), the gastrointestinal motor function and visceral pain (Martinez et al. 2004; Yin, Dong, and Yin 2008). There is a large amount of literature supporting that UCN-1 is important in intestinal tract (Tache et al. 2004; Koido et al. 2014), especially in stimulated colonic motor activity (Maillot et al. 2000; Martinez et al. 2002; Maillot et al. 2003; You et al. 2015; Saruta et al. 2004). However, the role of UCN-1 in CRC development have not yet been investigated.

In this study, UCN-1 expression level was detected in both Cancer Genome Atlas (TCGA) database and the tissues microarray. UCN-1 was strongly expressed in CRC patients. Furthermore, the tissues microarray showed that high expression of UCN-1 was associated with gender, higher risk of lymphatic metastasis and later pathological stage. Then UCN-1 expression level was detected in CRC cells. We found UCN-1 was upregulated in CRC cells. Then lentivirus mediated siRNA was used to knock down UCN-1. Subsequently, the effect of UCN-1 knockdown on CRC development in vitro and in vivo were employed and analyzed. The results showed that down-regulation of UCN-1 was critical to the decrease the proliferation and migration of CRC cells as well as an increase in apoptosis. Furthermore, we used the human phospho-kinase array to explore the downstream pathway of UCN-1 regulating CRC. Accordingly, we found that p53 signal pathway participated in UCN-1-mediated CRC development. Inhibitors of p53 signaling pathway reserved the effect of UCN-1 knockdown on CRC development. Meanwhile, overexpression of p53 reserved the effect of UCN-1 up-regulation on CRC development. Therefore, UCN-1 may participate in the progression of CRC via inhibiting the p53signaling pathway, which maybe a direction for the future treatment of CRC.

Cell Culture

Fetal human colon cell (FHC), human colon cancer cell HCT-116, RKO and HT29 purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640 with 10% fetal bovine serum (FBS) (both from Gibco, Grand Island, NY, USA), and 1% penicillin (100 U/ml)/streptomycin (100 µg/ml) at 37˚C in an atmosphere with 5% CO₂.

Human tissue microarray and Immunohistochemistry

A total of 180 cases of normal tissues (87 cases) and different malignant CRC tumor tissues (93 cases) were purchased from Shanghai Outdo Biotech (Shanghai, China). These cases of tissue chip immunohistochemistry experiments were also commissioned to BioSCI Res (Shanghai, China). All the participants have signed an informed consent and provided their relevant clinical data. The work carried out was in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The experiments were approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (NO.: 2022-KY-0083-03).

The CRC tissue microarrays and xenograft tumor tissue slides were soaked by xylene and rehydrated by series of alcohol. And then, the tissue microarrays and slides were repaired by citrate buffer, followed by blocking treatment with H₂O₂ and serum. The specimens were incubated with the primary antibody against UCN-1 (1:100, Proteintech, 19731-1-AP), Ki-67 (1:100, Abcam, ab16667) and secondary antibody (1:200, Abcam, ab97080). The tissue microarrays and slides were stained by DAB and hematoxylin and photographed. All specimens were examined by two pathologists independently and randomly. The positive cell score was classified as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). Immunohistochemistry (IHC) results were reached by multiplying the positive cell score and the staining intensity. The staining index < 6 was defined as low UCN-1 expression, while the staining index ≥ 6 as high UCN-1 expression.

The cancer genome atlas (TCGA) database analysis

Informative data about UCN-1 in CRC was obtained from TCGA (https://cancergenome.nih.gov/) and processed as well as analyzed by R statistical software TCGAbiolinks.

Cell transfection

To knock down UCN-1 in RKO cells, three short hairpin RNAs (shRNA) were designed to target the UCN-1 gene: sh-RNA_1: 5’-GACAACCCTTCTCTGTCCATT-3’, sh-RNA_2: 5’-CGAGCAGAACCGCATCATATT-3’, sh-RNA_3: 5’-CTCAATCTTGGACCGTACAGA-3’. They were inserted into the BR-V-121 lentiviral vector (Sangon Biotech, Shanghai, China) as well as no-silencing RNA (5’-TTCTCCGAACGTGTCACGT-3’) used as negative control. The double-stranded shRNA was inserted into the plasmid containing GFP gene and then linearized with EcoR I and Age I. Plasmids used to overexpress UCN-1 and p53 and lentivirus packaging were also constructed by Sangon Biotech (China). Human embryonic kidney (HEK) 293T cells (American Type Culture Collection, Manassas, VA, USA) were co-transfected with different plasmids by Lipofectamine 2000 Thermo Fisher Scientific, USA) following the instruction. After transfection, the cells were cultured for 48 h and then the lentivirus was harvested and concentrated. For lentivirus infection, HCT-116 and RKO cell were co-cultured with lentivirus-containing medium at MOI of 10. The infection efficiency and knockdown or overexpression efficiency were evaluated by fluorescence microscopy, RT-PCR and western blot post-infection.

Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) Assays

Total RNA was extracted from the cell lines by using TRIzol (Sigma) according to manufacturer’s instructions. The cDNA was synthesized from the total RNA using Hiscript QRT supermix for qPCR (Vazyme). The relative gene expression level was measured by AceQ qPCR SYBR Green master mix (Vazyme) and quantified by the comparative 2 − ΔΔCt method. GAPDH mRNA was the endogenous control. Primer sequences were shown as follows: GAPDH forward primer 5’-TGACTTCAACAGCGACACCCA-3’, GAPDH reverse primer 5’-CACCCTGTTGCTGTAG CCAAA-3’; UCN-1 forward primer 5’-CGGGACAACCCTTCTCTGTC-3’; UCN reverse primer 5’-TGCCCACCGAGTCGAATATG-3’.

Western Blot

Protein lysates from the cells were derived by using RIPA lysis buffer with protease inhibitors on ice. BCA Protein Assay Kit (HyClone-Pierce) was used to determine the total protein concentration. And then equal quantities of denatured protein were subjected to SDS-PAGE and transferred to PVDF membranes. Following blocking with 5% skim milk for 1 hour, the PVDF membranes were incubated with specific primary antibodies: anti-UCN-1 (1:500, Proteintech, 19731-1-AP), anti-GAPDH (1:30000, Proteintech, 60004-1-lg), anti-p53 (1:2000, Proteintech, 10442-1-AP), anti-p-p53 (1:1000, Proteintech, 67900-1-Ig). Subsequently, the membranes were washed and blotted with the following secondary antibodies: Goat Anti-Rabbit (1:3000, Beyotime, A0208), Goat Anti-Mouse (1:3000, Beyotime, A0216). GAPDH was seen as internal control.

Celigo Cell Counting Assay

Lentivirus-infected HCT-116 and RKO cells were seeded at a 96-well plate with 3000 cells per well and cultured at 37◦C with 5% CO2. The number of cells was continuously evaluated by Celigo (Nexcelom, Bioscience, Lawrence, MA, USA) for green fluorescence for 5 days at the same time. Cell proliferation rate was analyzed according to the number of cells with green fluorescence.

Cell counting kit8 (CCK‑8)

Lentivirus-infected HCT-116 and RKO cells were seeded into 96-well plates at 5000 cells per well. After 24 h, the cells were treated with 20 uM Pifithrin-α (Selleck) or not for another 24 h. 10 µl of CCK-8 (Sigma) solution was added into each well for 2 h at 37℃ before assay. Optical density (OD) was detected at 450 nm to measure the cell viability.

Flow Cytometry

Lentivirus-infected colon cancer cells were cultured in 6-well plates (2 mL/well). Then, cells of different treatment were collected and cell precipitation was washed with the 4◦C pre-cooled D-Hanks (pH = 7.2 ~ 7.4). Annexin V and PI Apoptosis Detection Kit (southern biotech) was applied to assess cell apoptosis rates according to the kit protocol. Briefly, the collected cells were incubated with Annexin V-APC and PI for indicated time at room temperature and then detected by flow cytometry.

Colony Formation Assay

Lentivirus-infected HCT-116 and RKO cells were cultured in 6-well plates (2 mL/well) at a density of 500 cells per well at initial. The cells were cultured for up to 8 days and every 3 days the medium was changed. Then visible colonies were fixed with 4% paraformaldehyde for 30–60 min and stained with 500ul Giemsa for 10–20 min before recorded by fluorescence microscope (Olympus, Tokyo, Japan).

Wound Healing Assay

HCT-116 and RKO cell infected with lentivirus were seeded into 96-well plates at 50000 cells per well with 100 µl of complete medium to each well. When the fusion of the cell reached about 90%, a scratch was made in the middle of the well. The cells were washed with PBS and incubated with serum free medium. Cells were cultured in an incubator at 37°C with 5% CO₂.Wound healing was observed under a microscope at 0 h and 24 h. Wound closure was calculated by Image J 7.0.

Cell Migration Assay

HCT-116 and RKO cells from different groups in logarithmic growth phase were seeded on Transwell upper chamber (Corning Incorporated, Corning, NY, USA) inserted in 24-well plates at the density of 50000 per well. Transwell chambers were coated with 8.0 µm polycarbonate membrane. A total of 100 µL cell suspension with FBS-free medium was supplemented into apical chamber. And the basolateral chamber was added with 600 µl 1640 medium containing 30% FBS. After incubation for 48 h, the cells adhering to the lower surface were treated with 4% paraformaldehyde fixation and stained with Giemsa, followed by photographed under a microscope.

Xenograft Animal Model

Nude mice (8 weeks, male) were obtained from Gempharmatech (Nanjing, China). The authors were accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work were appropriately investigated and resolved. Experiments were performed under a project license (NO.: 2022-KY-0083-03) granted by the Animal Ethics Committee of The First Affiliated Hospital of Zhengzhou University. Animals were maintained in a sterile environment, while their cages, food and bedding were sterilized by autoclaving. The mice were free to consume food and fresh water. RKO cells infected with shCtrl or shUCN-1 were implanted subcutaneously on the right forelimb armpit of the nude mice. Each group included 10 mice. Tumor weight and tumor growth of post-injected mice were measured at indicated time. Tumor volume was calculated using the following formula: π/6×L×S×S, where L represented long axis, S mean short axis. 21 days after injection, all of the mice were sacrificed and tumor samples were collected for the further analysis.

Human Phospho-Kinase Array (proteome profiler)

The Human Phosphorylation Array was used to explore the effects of down-regulation of UCN-1 on the phosphorylation-related proteins in RKO cells without UCN-1 knockdown and RKO cells with UCN-1 knockdown. Human Phospho-Kinase Array Kit (R&D Systems, Minnesota, USA) was performed following the manufacturer’s protocol. Cell lysate were incubated with the array membranes overnight. Then the arrays were washed and incubated with the detection antibody cocktails. Finally, the membranes were incubated with streptavidin conjugated to horseradish peroxidase, followed by chemiluminescence detection.

Statistical Analysis

All the data were analyzed by GraphPad Prism 8 or SPSS 13.0. Expression patterns in colorectal cancer tissues and para-carcinoma tissues were analyzed by Sign test. Relationship between UCN-1 expression and the characteristics of tumor in patients with colorectal cancer was analyzed by Mann-Whitney U and Spearman rank correlation analysis. The data were analyzed by t test, one-way analysis of variance (ANOVA) with Tukey’s test, and two-way ANOVA with Tukey’s post hoc multiple comparisons test. Data exhibited as the mean ± SEM from at least three independent replications. *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant.

Expression of UCN-1 was upregulated in CRC Tissues

To assess the clinical relevance of UCN-1 in CRC, we first interrogated the TCGA database (CRC tissues = 635, para-cancerous normal tissues = 51). The results of bioinformatics analysis showed that the mRNA expression of UCN-1 was significantly increased in CRC tissues (Fig. 1a) compared with the normal tissues. Then a tissue microarray, including 93 CRC tissues and 87 para-cancerous normal tissue tissues, was performed. Compared with para-cancerous normal tissues, most of the tumor tissues were immunoreactive for UCN-1 (Fig. 1b). Overall, 51 of 93 (54.8%) cases presented high UCN-1 expression in CRC tissues, whereas 3 of 87 (3.4%) cases exhibited high UCN-1 levels in para-carcinoma tissue (Table. 1). According to the sign test analysis, the expression of UCN-1 in CRC tissues was significantly higher than that in para-cancerous tissues (Table. 1), which indicating that UCN-1 can be used for subsequent statistical analysis of clinicopathological data such as tumor diameter, tumor infiltration tumor, lymph node metastasis and clinical stage. According to Mann-Whitney U analysis of the tissue microarray result, there was a significant difference in gender, tumor lymph node metastasis and clinical stage between low expression and high expression of UCN-1 (Table. 2). Based on this result, we performed Spearman rank correlation analysis for lymph node metastasis, gender and clinical stage. We found that CRC patients with abundant expression of UCN-1 harbored more higher risk of lymphatic metastasis and later pathological stage than those patients with low levels of UCN-1 (Table. 3). Interestingly, high expression of UCN-1 was more likely to occur in male patients (Tables. 2 and 3). These results implied that UCN-1 might act as a cancer promoting factor in CRC development.

Knockdown of UCN-1 Decreased Proliferation and Migration but Increased Apoptosis of CRC Cells in vitro

The analysis of clinicopathological data indicated that UCN-1 was related with CRC development. Next, we examined the expression of UCN-1 in CRC cell lines. We found that UCN-1 was all high expressed in different CRC cell lines, including HCT-116, HT29 and RKO cell lines (Fig. 2a) compared with that in normal human colon cell line (FHC cells). Given that UCN-1 was abundantly expressed in RKO and HCT-116 cell lines, we selected these two cell lines for subsequent experiments. As the expression of UCN-1 was positively correlated with tumor lymph node metastasis, UCN-1 may act as a promote factor in CRC cell migration. To obtain further evidence for the above hypothesis, we down-regulated UCN-1 expression by using a lentiviral shRNA approach. Initially, RKO cells were transduced with 3 different shRNAs (shRNA_1, shRNA_2 and shRNA_3) targeting UCN-1 (shUCN) or with non-silencing control shRNA (shCtrl). Stable knockdown of UCN-1 mRNA was achieved in shRNA_2 group, whereas silencing was not successful in shRNA_1 and shRNA_3 group (Fig. S1). As the best knockdown effect of shRNA_2, we chose shRNA_2 to silence UCN-1 to study the function of UCN-1 in RKO and HCT-116 cell lines. To evaluate the infective efficiency of shRNAs, the green fluorescent gene was linked to shRNAs (Fig. 2b). Silencing UCN-1 did not affect RKO cell morphology. Furthermore, both mRNA and protein of UCN-1 in HCT-116 cell and RKO cell were detected. Our results showed that UCN-1 were significantly knocked down in HCT-116 cell and RKO cell (Figs. 2c and 2d).

The results of celigo cell counting assay demonstrated that proliferative abilities of HCT-116 and RKO cell lines were weakened after UCN-1 knockdown (Figs. 3a, 3b and Fig. S2). The results of colony forming assay further confirmed that suppression of UCN-1 expression attenuated proliferation of CRC cells (Figs. 3c). In addition, knockdown of UCN-1 obviously attenuated migration of HCT-116 cells and RKO cells based on the results of transwell assay and wound healing assay (Figs. 3d and 3e). Furthermore, we found that knocking down the UCN-1 not only markedly suppressed the abilities of migration and proliferation but also promoted cell apoptosis. Compared with controls, the apoptotic rates of HCT-116 cells with UCN-1 knockdown were increased from 4.40% ± 0.04–10.40% ± 0.20% (Fig. 3f), whereas the apoptotic rates of RKO cells with UCN-1 knockdown were increased from 3.64% ± 0.02–7.92% ± 0.31% (Fig. 3f). These results indicated that UCN-1 may promote proliferation and migration but suppress apoptosis of CRC cells.

Knockdown of UCN-1 Inhibited CRC Growth In Vivo

To analyze the effect of the UCN-1 knockdown on CRC development in vivo, we established the nude mouse xenograft model with RKO cells. Mice injected with RKO cells UCN-1 un-knockdown and UCN-1 knockdown served as control group (shCtrl) and experimental group (shUCN), respectively. In vivo imaging was performed 5–7 days after injection, and mice were sacrificed on 28th. The tumor volume and weight were observed. Tumors with variable size were present in both shCtrl and shUCN groups (Fig. 4a). We found the number of the tumors formed in shUCN group was fewer than that in shCtrl group (Figure. 4a). Tumors in shCtrl group were about 2.15-fold heavier and 2-fold larger than that in shUCN group (Figs. 4b and 4c). Histologically, the protein expression of UCN-1 of the tumors formed in shUCN group significantly decreased compared with that in shCtrl group, suggesting our knockdown strategy was successful (Figure. 4d). Unexpectedly, the morphological of the tumors formed in shUCN groups did not differ from controls (Fig. S3). To observe the growth of these tumors, Ki-67, a proliferation marker, was strained in both shUCN group and shCtrl group (Figure. 4d and 4e). Based on result of Ki67, proliferate rate was significantly higher in shCtrl tumors than that in shUCN tumors (Fig. 4d and 4e), indicating that knockdown of UCN-1 decreased the ability of proliferation of CRC cells in vivo.

p53 signaling pathway participated in UCN-1-mediated CRC development

To further explore the downstream pathway behind UCN-1 regulating CRC, phosphokinase protein chip technology was used in RKO cell lines with UCN-1 knockdown. Our result demonstrated that knockdown of UCN-1 increased the expression of p53(S46), p70S6K(T389), p70S6K(T4211/T424), PYK2(Y402), RSK1/2(S221/S227), STAT1(Y701), STAT2(Y705) and STAT3(S727), as well as decreased p-kinases including ERK1/2(T202/Y204, T185/Y187), c-jun(S63), Hsp27(S78/S82), Src(Y419) and Yes(Y426) (Figs. 5a-5c).

It has been reported that CRC represents the cancer entity with the highest prevalence of p53 mutations (Liebl and Hofmann 2021; Zhou, Shu, et al. 2022; Li et al. 2015), indicating that it was associated with p53 signaling pathway. The phosphorylation level of p53 at Ser46 (p-p53) increased greatly after knocking down UCN-1 (Fig. 5), which was further supported by the result of western blot (Fig. 6a and 6b). Based on these results, we believed UCN-1 was negatively correlated with p-p53 and may regulate CRC progression through the inactivation of the p53 signaling pathway. In order to further explore the above hypothesis, pifithrin-α (20 uM), a small molecule inhibitor of p53 pathway was used. Compared with controls, the protein expression of p53 and p-p53 in shUCN group were increased but decreased again after pifithrin-α treatment (Figs. 6a and 6b). Not only protein expression but also the proliferate rate of HCT-116 cells and RKO cells of was affected. The proliferation of HCT-116 cells and RKO cells of was decreased by UCN-1 knockdown. However, this effect was reversed by pifithrin-α treatment (Figures. 6c and 6d). The apoptosis of HCT-116 cells and RKO cells in shUCN group was increased from 3.37% ± 0.12% and 6.19% ± 0.16–10.52% ± 0.25% and 11.62% ± 0.47%, respectively. With the pifithrin-α treatment, the apoptosis of HCT-116 cells and RKO cells decreased to 6.57% ± 0.05% and 8.01% ± 0.56%, respectively (Figures. 6e and 6f). In summary, the effects on proliferation level and apoptosis caused by UCN-1 knockdown were reversed by application of inhibitor of p53 signaling pathway, indicating that UCN-1 could promote CRC cells proliferation and suppress apoptosis via inhibiting p53 signaling pathway in vitro.

To further prove the above conclusion, we over-expressed UCN-1 expression by using a lentiviral cDNA approach. Compared with controls, the protein expression of p53 and p-p53 were decreased in UCN-1 over-expression group (UCN (oe)) but re-induced by p53 over-expressed plasmid (UCN (oe) + p53(oe)) (Figures. 7Aa and 7b). Not only protein expression but also the proliferation and migration of HCT-116 cells and RKO cells was up-regulated by over-expression of UCN-1 (Figures. 7c-7e and Fig. S4). The phenomenon was reversed in UCN (oe) + p53(oe) group (Figures. 7c-7e). The apoptotic rates of HCT-116 cells and RKO cells in UCN-1(oe) group were decreased from 7.03% ± 0.87% and 8.69% ± 1.11–3.96% ± 0.33% and 5.13% ± 0.52%, respectively (Figure. 7f). It was re-increased to 6.05% ± 1.01% and 7.38% ± 0.62% in UCN-1(oe) + p53(oe) group (Figure. 7f). The above results indicated that UCN-1 over-expression promoted CRC cells proliferation and migration and inhibited apoptosis. However, this function was reserved by over-expression of p53, indicating that the functions of UCN-1 promoting proliferation and migration and decreasing apoptosis of CRC cells were inhibited by over-expression of p53 signaling pathway in vitro.

In this study, we demonstrated the UCN-1 participated in CRC development via inhibition of p53 signaling pathway. Both the TCGA database analysis and the tissue microarray showed that overexpression of UCN-1 was found in CRC patients. Furthermore, the result of tissue microarray conformed that UCN-1 the importance of UCN-1 in CRC stage, gender and lymphatic metastasis. Based on these findings, we further investigated the biological functions of UCN-1in CRC progression. We found that UCN-1 acted as a crucial regulator of in CRC development and verified it may significantly enhance proliferate and migrate abilities of CRC cells, as well as suppress cell apoptosis. In the xenograft tumor model experiment, UCN-1 facilitated tumor outgrowth in CRC. Next, we used the human phosphorylation array to explore the detailed mechanism and found p53 acting as downstream signaling way. The suppression in proliferation and migration and increase of cell apoptosis caused by UCN-1 knockdown was reversed with the treatment of pifithrin-α. On the contrary of UCN-1 knockdown, overexpression of UCN-1 promoted the proliferation and migration and decrease of cell apoptosis. This function could be reserved by overexpressing of p53. Therefore, UCN-1 could promote CRC cells proliferation and migration and restrain apoptosis via inhibiting p53 signaling pathway.

Abundant of studies have revealed that UCN-1 was involved in the development of several cancer types (Balogh et al. 2022; Owens et al. 2017; Kamada et al. 2012). However, the contribution of UCN-1 on CRC development has not been elucidated so far. The relationship between CRC and UCN-1 is still unclear. Our study found that UCN-1 was over-expressed in CRC and positively correlated with the malignancy of the disease based on IHC analysis and TCGA database. Similarly, the biological actions of UCN-1 exerted by interactions with two distinct CRH receptor subtypes, CRH receptor 1 (CRHR1) and CRH receptor 2 (CRHR2). Both CRH receptors and UCN-1 are distributed in the colon and played an important role in stimulated colonic motor activity (Maillot et al. 2000; Martinez et al. 2002; Maillot et al. 2003; You et al. 2015; Saruta et al. 2004). All of these indicated that UCN-1 expressed in the colon and was associated with CRC. On the other hand, further tissue microarray analysis showed that UCN-1 expression was associated with gender. The proportion of high expression of UCN-1 in male patients was higher than that in female patients. Interestingly, the incidence of CRC is also higher in men than in women (Lewandowska et al. 2022). This result indicated that UCN-1 may be an important factor that involved in the correlation of gender and CRC. This is an interesting and meaningful discovery, because UCN-1 is not a sex hormone. Why high UCN-1 expression was more likely to occur in male patients? One possible explanation for this phenomenon is that UCN-1 and sex hormones may be regulated with each other. The sex and estrogen decreased the distribution of UCN-1 in the nucleus of the solitary tract (Ciriello 2015). Deletion of CRHR1, the UCN-1 receptors, selectively impairs maternal, but not intermale aggression (Gammie, Seasholtz, and Stevenson 2008). These studies indicated that estrogen can reduce the distribution of UCN-1 in some tissues, and UCN-1 affects estrogen secretion. The high CRC risk and mortality are more likely to appear in males(Lu et al. 2020). The relationship between UCN-1 and estrogen may serve as the explain for the sex differences in CRC.

Although the mechanism of these observations is unknown, they may be an interesting research direction to study the mechanism of UCN-1 regulating CRC. Lymph node metastasis is the common pattern of tumor metastasis. Our tissue microarray analysis showed that UCN-1 expression was associated with lymphatic metastasis. This result indicated that UCN-1 may be related with the ability of CRC migration. It has been reported that UCN-1 affects migration of hepatic cancer cell (Zeng et al. 2016) and endometrial cancer cells in vitro (Brenner, Kloor, and Pox 2014). However, the effect of UCN-1 on tumor migration may be related to the type of tumor. For example, UCN-1 increased migration of hepatic cancer cell (Zhu et al. 2014) but suppressed the migrate ability of endometrial cancer cells (Owens et al. 2017).

To explore the role of UCN-1 on CRC migration, CRC cells were used. Our results showed that knockdown of UCN-1 suppressed the migrate ability, and over-expression of UCN-1 enhanced the migrate ability. To ensure the rigor of the experiment, we used two CRC cell lines, RKO cells and HCT-116 cells. Knockdown of UCN-1 reduced the migration rate of these two cell lines by 71.4% and 84.2%, respectively. In addition, the effect of UCN-1 on the proliferation and apoptosis of CRC tumor cells were investigated. Knockdown of UCN-1 decreased the proliferate rate and increased the apoptotic of CRC cells. This result was also verified in animal experiments. Knockdown of UCN-1 reduced the number and size of tumors formed in nude mice. All the results indicated that UCN-1 may act as a target for CRC therapy.

Our results showed that UCN-1 involved in CRC, but how UCN-1 modulated CRC was still unclear. To solve this problem, phosphokinase protein microarray was applied. Our study showed UCN-1 knockdown resulted in increasing the expression of p53 and phosphorylated p53. p53 is an important tumor suppressor that participating in preventing cancer development. It is worth noting that p53 signaling pathway was associated with CRC (Liebl and Hofmann 2021; Liu et al. 2022; Liu et al. 2021). There was an interesting phenomenon in our experiment. The p53 phosphorylation at Ser 46 was significantly increased after UCN-1 knockdown, but the phosphorylation at Ser15 and Ser392 was not significantly changed. As we all known, the enhanced stability and transcriptional activity of p53 depend on phosphorylation modification. p53 has multiple phosphorylation sites, including Ser15 and Ser46 in the N-terminal domain and Ser392 in the C-terminal domain. Phosphorylated p53 at different sites plays different physiological roles. Phosphorylation of p53 at Ser 46 is mainly involved in apoptosis (Liebl and Hofmann 2019). The upregulated expression of phosphorylation of p53 at Ser 46 after UCN-1 knockdown indicated us that UCN-1 might suppress apoptosis via promoting the phosphorylation of p53 at S46. As expected, after treatment with pifithrin-α, the increase of phosphorylation of p53 at Ser 46 and cell apoptosis caused by UCN-1 knockdown were withdrawn. Overexpression of p53 also significantly attenuated the suppression of p-p53 at Ser 46 and apoptosis caused by UCN-1 overexpression. Therefore, UCN-1 involved in apoptosis of CRC cells via promoting the expression of phosphorylation of p53 at Ser46. On other hand, UCN-1 knockdown resulted in suppression in proliferation and migration of CRC cells. The treatment with pifithrin-α reserved the suppression of migration and proliferation caused by UCN-1 knockdown. Similarly, the promote effects of overexpression of UCN-1 on migration and proliferation were reserved by overexpression of p53. However, phosphorylation of p53 at Ser46 has not been reported to mediate proliferation and migration of cells so far. These effects on cell proliferation and migration are likely to act through phosphorylation at other sites. As p53 has more than 30 phosphorylation sites, UCN-1 may regulate proliferation and migration via other sites. Unfortunately, we only examined three phosphorylation sites in this study. It needs to perform more experiments to identify the phosphorylation sites that involved in proliferation and migration. Besides activation of p53 caused by the phosphorylation suppresses CRC initiation and progression (Wang et al. 2019), inactivation of p53 by altering p53 regulators or mutations in p53 also occur frequently in CRC(Huang et al. 2018; Zhou, Hao, and Lu 2019). Ablation of mutant p53 in CRC inhibits Stat3-mediated tumor growth and invasion (Schulz-Heddergott et al. 2018). Loss of p53 function plays a crucial role in treatment resistance in a variety of neoplastic diseases, including CRC. Although our study did not pay much attention to the effect of p53 mutants on CRC, mutant p53 also played an important role in CRC.

In addition, we speculated UCN-1 may inhibit CRC by regulating the immune response via p53 signaling pathway. Several studies have clearly shown CRH signaling pathway participates in chronic intestinal disorders, including inflammatory bowel disease (IBD) (Buckinx et al. 2011; Chatoo et al. 2018). The immune imbalance (Chang et al. 2011), such as IBD (Keller et al. 2019; Mattar et al. 2011), is considered the predisposition factors for developing CRC. It has been reported UCN-1 modulates gastrointestinal (GI) tract via regulating intestinal immune environment (Balkwill and Mantovani 2001). On the other hand, p53 has been reported to regulate the inflammatory tumor microenvironment (Uehara and Tanaka 2018) and immune factors (Cooks et al. 2018; Li, Li, and Wang 2020). Based on these studies, UCN-1 may inhibit CRC by regulating the immune response via p53 signaling pathway. This part is not addressed in this study, and it will be a direction for our follow-up studies. In summary, our study demonstrated the function of UCN-1 on CRC development and explored whether UCN-1 modulated CRC via p53 signaling pathway. We found high expression of UCN-1 in CRC was likely to appear in CRC patients with a worse pathological prognosis. Knockdown of UCN-1 decreased but over-expression of UCN-1 increased migration and proliferation in vitro. Besides, knockdown of UCN-1 induced apoptosis in vitro. In vivo, knockdown tumors were smaller and less proliferative than controls. Furthermore, the effect of UCN-1 knockdown on CRC development was reversed by inhibition of p53 signaling pathway. On the other hand, UCN-1 overexpression increased the proliferation and migration and decreased the apoptosis. It was reversed by p53 overexpression. Based on our study, UCN-1 participates in the regulation of CRC progression via inhibition of p53 signaling pathway. This finding has never been reported before and may provide a new target for CRC treatment.

Acknowledgments

We thank the First Affiliated Hospital of Zhengzhou University for the funding.

Statements and Declarations

Funding

This study was supported by 2019 Henan Medical Science and technology research plan (joint construction) project (LHGJ20190066).

Conflict of Interest

The authors declare no competing interest.

Author Contributions

X.L. G. designed all experiments, performed the experiments, analyzed data and drafted the manuscript. Y.L. assisted in molecular and behavioral assays. The manuscript was revised by X.L. G., X.Y. C and B.H. S. All experiments were supervised by X.L. G., Y.L., X.Y. C and B.H. S All authors approved the final edited version.

Data Availability

The datasets generated during the current study are not publicly available. Because this is a basic study and data sharing is not necessary. But they are available from the corresponding author on reasonable request.

Ethics approval

Experiments were performed under a project license (NO.: 2022-KY-0083-03) granted by the Animal Ethics Committee of The First Affiliated Hospital of Zhengzhou University.

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UCN-1 expression	Tumor tissue		Para-carcinoma tissue		p value
UCN-1 expression	Cases	Percentage	Cases	Percentage	<0.0001***
Low	42	45.2%	84	96.6%
High	51	54.8%	3	3.4%

TABLE 1 | Expression patterns in colorectal cancer tissues and para-carcinoma tissues revealed in immunohistochemistry analysis

Features	No. of patients	UCN-1 expression		p value
Features	No. of patients	low	high	p value
All patients	93	42	51
Age (years)				0.925
≥68	46	21	25
＜68	47	21	26
Gender				0.043*
Male	44	15	29
Female	49	27	22
Tumor size				0.828
≥5	37	17	20
＜5	55	24	31
Stage				0.001**
1	4	2	2
2	46	28	18
3	31	6	25
Grade				0.706
1	1	0	1
2	77	35	42
3	14	6	8
4	1	1	0
T Infiltrate				0.356
T1	1	1	0
T2	4	1	3
T3	70	30	40
T4	16	9	7
lymphatic metastasis（N）				<0.0001***
N0	58	34	24
N1	26	6	20
N2	8	1	7

TABLE 2 | Relationship between UCN-1 expression and tumor characteristics in patients with colorectal cancer

		UCN-1
lymprhatic metastasis（N）	Spearman coefficient	0.373**
	Signification (double-tailed)	0.000***
	N	92
Gender	Spearman coefficient	-0.211*
	Signification (double-tailed)	0.043*
	N	93
Stage	Spearman coefficient	0.372**
	Signification (double-tailed)	0.001**
	N	81

TABLE 3 | Relationship between UCN-1 expression and tumor characteristics in patients with colorectal cancer

No competing interests reported.

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Urocortin-1 Promotes Colorectal Cancer Cells Migration, Proliferation and Inhibits Apoptosis via Inhibition of p53 Signaling Pathway

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Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Materials and method

Cell Culture

Human tissue microarray and Immunohistochemistry

The cancer genome atlas (TCGA) database analysis

Cell transfection

Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) Assays

Western Blot

Celigo Cell Counting Assay

Cell counting kit8 (CCK‑8)

Flow Cytometry

Colony Formation Assay

Wound Healing Assay

Cell Migration Assay

Xenograft Animal Model

Human Phospho-Kinase Array (proteome profiler)

Statistical Analysis

Result

Expression of UCN-1 was upregulated in CRC Tissues

Knockdown of UCN-1 Decreased Proliferation and Migration but Increased Apoptosis of CRC Cells in vitro

Knockdown of UCN-1 Inhibited CRC Growth In Vivo

p53 signaling pathway participated in UCN-1-mediated CRC development

Discussion

Declarations

References

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Supplementary Files

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