A novel tumor suppressor SPINK5 targets Wnt/β‐catenin signaling pathway in esophageal cancer

Abstract Esophageal cancer is one of the most common tumor in the world, and the morbidity rate is as high as 100/100 000 in some parts of China. Therefore, it is important and urgent to explore the pathogenesis of esophageal cancer and find new therapeutic targets for esophageal cancer. In this study, we found that a novel tumor suppressor SPINK5 is significantly reduced in the development of esophageal cancer, and is closely related to the pathological differentiation and lymph node metastasis of esophageal cancer via bioinformatics analysis and esophageal cancer tissue array. Further studies have found that SPINK5 is closely related to Wnt/β‐catenin signaling pathway by bioinformatics analysis and western blot. In esophageal cancer cells, SPINK5 overexpression can inhibit Wnt/β‐catenin signaling pathway. Combined with LiCl or MG‐132 treatment, SPINK5 can inhibit GSK3β phosphorylation and promote β‐catenin protein degradation, thus inhibit Wnt/β‐catenin signaling pathway. In vivo study, SPINK5 overexpression can significantly inhibit the growth of esophageal cancer cells. Our study shows that SPINK5 can inhibit the proliferation, migration, and invasion of esophageal cancer cells by inhibiting Wnt/β‐catenin signaling pathway, and thus plays an important role in the development of esophageal cancer, and may serve as a treatment target of esophageal cancer.


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WANG et Al. morbidity rate is as high as 100/100 000, which has become a world-recognized area with high incidence of esophageal cancer. In these areas, esophageal cancer has become the second largest cancer killer. 4 Therefore, it is an urgent research topic to continue to explore the pathogenesis of esophageal cancer and to find potential therapeutic targets for esophageal cancer.
The serine protease inhibitor kazal type (SPINK) family is the largest branch of the serine protease inhibitor family. 5 Its family members include SPINK1, SPINK2, SPINK4, SPINK5, SPINK5L2, SPINK5L3, SPINK6, SPINK7, and SPINK9. 6 Later, SPINK 8, SPINK 10, SPINK11, and SPINK12 were found in the rat's epididymis. 7 The SPINK family consists of the Kazal domain. A typical Kazal domain contains 3 pairs of disulfide bonds, 2 a-helices, and 3 anti-parallel β-sheets. 8 The SPINK5 gene is located in the 5q32 region of the chromosome and is composed of 15 functional regions. The SPINK5 gene encodes a lymphoid epithelial-associated inhibitor LEKTI, a serine protease inhibitor. 9 This study has found that dysfunction of SPINK5 is mainly related to Netherton Syndrome (NS). NS is caused by loss of expression or dysfunction of LEKTI due to mutation of SPINK5 gene. 10 There were few studies exploring the relationship between SPINK5 and human cancer. There was mRNA microarray analysis showed that SPINK5 was downregulated in esophageal squamous cell carcinoma. 11 However, the mechanism of action of SPINK5 in the development of esophageal cancer is still unclear.
In this study, we first explored the mechanism of action of SPINK5 in the development of esophageal cancer. We found that SPINK5 acted as a tumor suppressor in esophageal cancer to inhibit proliferation, migration, and migration of esophageal cancer cells via inhibiting Wnt/β-catenin signaling pathway, which provided an inspiration for exploring the mechanism of action of SPINK5 in tumorigenesis and development, and provides a theoretical basis for the search for new therapeutic targets for esophageal cancer.

| Tissue sample and cell culture
A total of 2 esophageal tissue microarrays were used in this study. One tissue microarray containing 12 esophageal cancer tissues and their matched esophageal cancer tissues were purchased from Alenabio Company (Xi'an, China). The other tissue microarray contains 205 cases of esophageal cancer tissue which was from the tissue samples library of our laboratory. KYSE510, ECA109, and HEK293T cells were purchased from the China Center for Type Culture Collection (CCTCC; Chinese Academy of Sciences, Shanghai, China). KYSE510 cells were cultured in RPMI1640 medium (HyClone, USA) supplemented with 10% fetal bovine serum (Gibco). ECA109 and HEK293T cells were cultured in DMEM medium (HyClone, USA) supplemented with 10% fetal bovine serum (Gibco).

| Plasmids, siRNAs, antibodies, and construction of stable cell line
The SPINK5 overexpression plasmid was cloned into the pflag-CMV vector by nested PCR using the CDS sequence of the SPINK5 gene (NM_001127698.1). The primers of plasmid construction could be seen in Table S1. The TOP/FOP flash reporter plasmids containing wild-type (TOP flash) or mutated (FOP flash) TCF/LEF DNA binding sites were conserved in our laboratory.
The siRNAs of SPINK5 were purchased from Genepharma Company (Shanghai, China) and contained 3 pairs of sequences, which were siRNA1 at nucleotides 1065 bp, siRNA2 at nucleotides 2046 bp, and siRNA3 at nucleotides 2910 bp. The sequences could be seen in Table S1. Transfection of plasmid and siRNA was carried out using Lipofectamine 2000 reagent (Invitrogen Co., Ltd.). The procedure of transfection was according to the instructions of Lipofectamine 2000 reagent. In the case of transfection of siRNA, siRNA1, siRNA2, and siRNA3 were mixed in equal proportions and then transfected into cells as the siRNA group.

| Cell proliferation assay
The ability of cell proliferation was measured by CCK-8 assay and plate colony formation assay. The procedure of the CCK8 assay was as follows. The cells in the logarithmic growth phase were seeded at a density of 1 × 10 3 per well in 96-well plates, and the proliferation of cells in each treatment group was detected at 0h, 24h, 48h using the CCK8 detection kit. For the plate colony formation assay, the cells in the logarithmic growth phase were inoculated into the 6-well plate at a density of 200 per well. After further culture for 1 week, the cells were stained with crystal violet solution, and the growth of the cells in each treatment group was observed. Cell counting was performed by image J software.

| Cell migration and invasion assay
Cell migration and invasion ability was measured by Transwell assay. For cell migration assay, cells in logarithmic growth phase were seeded at the upper transwell chamber insert (Corning, USA) at a density of 2 × 10 4 cells per well. The chamber was placed in a 24-well plate in which the upper chamber contained serum-free cell culture medium and the lower chamber contained 10% FBS complete medium. The culture was continued for 24 hours. The medium was discarded, and stained with a crystal violet solution to observe the number of migrated cells. For cell invasion assay, Matrigel gel was uniformly applied to the upper chamber in advance, and then the cells were inoculated. The other procedures were the same as those in the cell migration experiment.

| TOP/FOP flash assay
The TOP/FOP flash assay is a reporter gene assay used to detect Wnt/β-catenin signaling pathway activity. 12 The TOP flash reporter plasmid contains a wild-type (CCTTTGATC) TCF/LEF binding site, and the FOP flash reporter plasmid contains a muted (CCTTTGGCC) TCF/LEF binding site. The cells in the logarithmic growth phase were seeded in 24well plates, and the target gene overexpression plasmid or siRNA was co-transfected into the cells with TOP flash or FOP flash using Lipofectamine 2000 reagent. After 48 hours, the Dual Luciferase Reporter Assay Kit (Promega) was used to detect the reporter gene activity of each group of cells.

| Western blot
The cell proteins were extracted from each group of cells by RIPA lysate and sonicator, and the concentration of each group of protein samples was detected by BCA protein concentration detection kit to prepare protein samples. An equal amount of protein sample (40 μg) was added to a 10% SDS-PAGE gel and electrophoresed at 60 and 110 V, respectively. After the electrophoresis, the protein in the SDS-PAGE gel was transferred to the PVDF membrane at a moderate current. The PVDF membrane was then blocked with 5% skim milk for 1 hour, and the corresponding protein antibody was separately incubated overnight according to the molecular weight of the protein of interest. Then, the PVDF membrane was separately incubated with the corresponding HRP-labeled secondary antibody, and finally observed and photographed by the chemiluminescence imaging system (Tanon, Shanghai, China).

| Immunohistochemistry
Immunohistochemistry was used to detect the expression level of the protein in esophageal tissue. The tissue chip was dewaxed, and after dehydration, the endogenous peroxidase in the tissue was inactivated using 1 mmol/L sodium citrate buffer (pH 6.0). After blocking with goat serum for 30 minutes, the antibodies of the protein of interest were separately incubated overnight at 4°C. The next day, the biotin-labeled secondary antibody was incubated with the tissue chip for 1 hour at room temperature, followed by incubation with streptavidin-labeled horseradish peroxidase for 1 hour and finally with 3, 3-diaminobenzidine (DAB), and the depth of the color represents the expression level of the target protein. The Pannoramic MIDI automatic digital slide scanner (3DHISTECH Ltd., Budapest, HUNGARY) was used for image capture. Quantification of target protein staining was performed using IHC profiler in ImageJ. 13

| Nude mouse orthotopic transplantation tumor models
The tumorigenic ability of esophageal cancer cells ECA109 after SPINK5 overexpression was determined by orthotopic transplanted tumor model in nude mice. A nude mouse model of orthotopic transplanted tumor was established by subcutaneous injection of ECA109 stable cell line (1 × 10 8 cell/ mL) in 4-6 weeks old Balb/c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.). After the nude mice were sacrificed, the tumor weight and tumor volume were observed and recorded. All the animal protocols were approved by Zhang Zhongjing College of Traditional Chinese Medicine, Nanyang institute of Technology, China.

| Statistical analysis
The data were presented as the mean ± standard deviation (SD). Each experiment was repeated at least 3 times. The variance analysis between groups was performed using a one-way ANOVA. Chi squared-tests were used to detect the significance of the relationship between expression of SPINK5 and clinicopathologic features of esophageal cancer tissue microarray. P < 0.05 was considered statistically significant.

| SPINK5 expression is decreased in esophageal cancer tissues and is associated with clinicopathological features of esophageal cancer
There are no studies to determine the difference in the expression levels of SPINK5 in normal esophageal and esophageal cancer tissues. Therefore, we detected the expression level of SPINK5 protein in 12 cases of esophageal cancer tissues and their matched normal esophageal tissues by immunohistochemistry. The results showed that the expression of SPINK5 protein was significantly reduced in 11 cases (11/12) of esophageal cancer, compared with normal esophageal tissues ( Figure 1A). To further investigate the relationship between the expression level of SPINK5 protein and the clinicopathological features of esophageal cancer, we examined the expression level of SPINK5 protein in 205 cases of esophageal cancer by immunohistochemistry, the results showed that the expression level of SPINK5 protein was closely related to lymph node metastasis and pathological differentiation of esophageal cancer ( Figure  1B,C, Table 1). When the expression level of SPINK5 protein was lower, the pathological differentiation of esophageal cancer patients was lower, and lymph node metastasis was more likely F I G U R E 1 SPINK5 is significantly downregulated in human esophageal cancer tissues. (A) Compared to normal esophageal tissues, SPINK5 protein expression was upregulated in esophageal cancer, which was detected in 12 cases of human esophageal cancer tissue microarray by immunohistochemistry. (B) The protein levels of SPINK5 in esophageal cancer tissues which occurs lymph node metastasis were lower than that in esophageal cancer tissues which have not lymph node metastasis. (C) The protein levels of SPINK5 in poor differentiation of esophageal cancer tissues were lower than that in moderate and well differentiation of esophageal cancer tissues. (D) The expression of SPINK5 was analyzed by bioinformatics in GEPIA database. (E) The relationship of SPINK5 expression with disease free survival percent of esophageal cancer patient was analyzed by bioinformatics in GEPIA database to occur. We analyzed the GEPIA database and found that the expression level of SPINK5 mRNA in esophageal cancer tissues was significantly lower than that in normal esophageal tissues ( Figure 1D). In addition, although the expression level of SPINK5 mRNA was not significantly different from the disease-free survival rate of patients with esophageal cancer, however, based on the analysis results, we can find that the higher the expression level of SPINK5 mRNA, the better the prognosis of patients with esophageal cancer ( Figure 1E). These results showed that SPINK5 was downregulated in esophageal cancer, and maybe related to the development of esophageal cancer.

| SPINK5 significantly inhibits the proliferation, migration, and invasion of esophageal cancer cells
We have found that the expression level of SPINK5 is significantly reduced during the development of esophageal cancer, so what role does SPINK5 play in the development of esophageal cancer? We first constructed the overexpression plasmid and siRNA of SPINK5 and then transfected into esophageal cancer cells KYSE510 and ECA109 ( Figure  2A). Then, CCK-8 assay and plate cloning assay were used to detect the effect of SPINK5 on the proliferation of esophageal cancer cells. The results showed that overexpression of SPINK5 significantly inhibited the proliferation of esophageal cancer cells KYSE510 and ECA109 ( Figure 2B,D), while knockdown of SPINK5 significantly promoted proliferation of esophageal cancer cells KYSE510 and ECA109 ( Figure 2C,E). In addition, metastasis is the leading cause of death in patients with esophageal cancer. We examined the effect of SPINK5 on the migration and invasion of esophageal cancer cells by Transwell assay. Transwell migration assay showed that overexpression of SPINK5 significantly inhibited the migration of esophageal cancer cells KYSE510 and ECA109 ( Figure 3A), while knockdown of SPINK5 significantly promoted the migration of esophageal cancer cells KYSE510 and ECA109 ( Figure 3B). Transwell invasion experiments were consistent with Transwell migration experiments ( Figure 3C-D). These results indicate that SPINK5 acts as a tumor suppressor gene and participates in the development of esophageal cancer by inhibiting cell proliferation, migration, and invasion.

| SPINK5 inhibits Wnt/β-catenin signaling pathway in esophageal cancer
In the development of esophageal cancer, a variety of signaling pathways are involved, among which Wnt/β-catenin signaling pathway is one of the most classical oncogenic signaling pathways. 14 Its overactivation can promote the expression of target gene c-myc and cyclin D1 which in turn to promote the proliferation, migration, and invasion of tumor cells. In esophageal cancer cells KYSE510 and ECA109, we transfected SPINK5 overexpression plasmid and siRNA, respectively, and then detected the expression levels of key factors of Wnt/β-catenin signaling pathway by western blot. The results showed that overexpression of SPINK5 inhibited the protein expression levels of β-catenin, p-GSK3β(S9), c-myc, and cyclin D1 and upregulated p-β-catenin (S33/37) in esophageal cancer cells KYSE510 and ECA109 ( Figure 4A). Knockdown of SPINK5 significantly inhibited the protein expression level of p-β-catenin (S33/37) and upregulated the protein expression levels of β-catenin, p-GSK3β (S9), c-myc and cyclin D1 ( Figure 4B). The TOP/FOP flash assay was SPINK5 knockdown enhances the proliferation of KYSE510 and ECA109 cells which was detected by the colon formation assay. Values represent the mean ± SD from 3 independent measurements. *P < 0.05, **P < 0.01, ***P < 0.001 used to detect Wnt/β-catenin signaling pathway activity. In esophageal cancer cells KYSE510 and ECA109, overexpression of SPINK5 significantly inhibited TOP flash reporter activity ( Figure 4C), while knockdown of SPINK5 significantly increased TOP flash reporter activity ( Figure  4D), suggesting that overexpression of SPINK5 inhibits Wnt/β-catenin signaling pathway activity, while SPINK5 knockdown is able to activate Wnt/β-catenin signaling pathway. To further validate the association of SPINK5 with Wnt/β-catenin signaling pathways in esophageal cancer, we used bioinformatics to analyze the correlation between the expression levels of SPINK5 and Wnt/β-catenin  Figure 4E). These results indicate that SPINK5 is able to inhibit the Wnt/β-catenin signaling pathway in esophageal cancer.

| SPINK5 can affect the degradation of β-catenin through GSK3β pathway in esophageal cancer cell
The cytoplasmic CK1, AXIN, APC, GSK3β, and other proteins form a degradation complex, which promotes the phosphorylation of the ser33 and ser37 of β-catenin by binding to β-catenin protein, which leads to the ubiquitination degradation of β-catenin. 15 In previous studies, we found that overexpression of SPINK5 in esophageal cancer cells inhibited the expression of p-GSK3β (S9), whereas knockdown of SPINK5 upregulated the expression of p-GSK3β (S9), suggesting that SPINK5 can affect the activity of GSK3β. LiCl is often used as an inhibitor of GSK3β which promotes GSK3β phosphorylation and inactivation. 16 We found that knockdown of SPINK5 in esophageal cancer cells has a similar effect to LiCl and upregulates the expression level of p-GSK3β (S9) ( Figure 5A). Overexpression of SPINK5 significantly reversed the upregulation of β-catenin and p-GSK3β (S9) protein expression by LiCl treatment ( Figure 5B). TOP/ FOP flash assay showed that overexpression of SPINK5 significantly reversed the activation of Wnt/β-catenin signaling pathway by LiCl treatment ( Figure 5C). In esophageal cancer cells, we overexpressed β-catenin (GFP-β-catenin) in combination with protease inhibitor MG132, the results showed that MG132 significantly upregulated the expression of GFP-β-catenin and reversed the overexpression of SPINK5 to inhibit GFP-β-catenin ( Figure 5D). These results suggest that SPINK5 can promote β-catenin protein degradation by inhibiting GSK3β phosphorylation in esophageal cancer cells.

| SPINK5 inhibits the proliferation and migration of esophageal cancer cells through Wnt/β-catenin signaling pathway and inhibits the growth of esophageal cancer cells in nude mice
In esophageal cancer cells, we have determined that SPINK5 regulates the Wnt/β-catenin signaling pathway. Furthermore, we verified whether SPINK5 affects the proliferation and migration of esophageal cancer cells via the Wnt/β-catenin signaling pathway. We used LiCl to activate the Wnt/β-catenin signaling pathway, and then observed the effect of overexpression of SPINK5 on the proliferation and migration of esophageal cancer cells by plate colony formation assay and Transwell assay. The results showed that the proliferation and migration ability of esophageal cancer cells increased significantly after treatment with 30 mmol/L LiCl, while overexpression of SPINK5 significantly inhibited the proliferation and migration of esophageal cancer cells, and reversed the promotion of LiCl treatment on the proliferation and migration of esophageal cancer cells ( Figure 6A-B). These results indicate that SPINK5 can inhibit the proliferation and migration of esophageal cancer cells through the Wnt/β-catenin Values represent the mean ± SD from 3 independent measurements. **P < 0.01, ***P < 0.001 signaling pathway. In addition, we further verified the effect of SPINK5 on the growth of esophageal cancer cells in nude mice. We constructed a SPINK5 overexpressing ECA109 esophageal cancer cell line, which was then implanted subcutaneously in nude mice. The results showed that overexpression of SPINK5 significantly inhibited the growth of esophageal cancer cells in nude mice ( Figure 6C-D). These results indicate that SPINK5 significantly inhibits the proliferation and migration of esophageal cancer cells and has potential as a therapeutic target for esophageal cancer.

| DISCUSSION
The role of SPINK family genes in tumorigenesis and development is still unclear. Studies have shown that SPINK1 is highly expressed in prostate cancer, 17 colorectal cancer, 18 liver cancer, 19 and other tumor tissues, 20 and promotes tumors occurrence and development by activating PI3K/AKT, MAPK/ERK, and other signaling pathways. 21 SPINK7 is elevated in normal adult esophageal tissue, but decreased or lost in esophageal and paracancerous tissues. SPINK7 is downregulated in esophageal cancer, and inhibits invasion, and migration of tumor cells through uPA/plasmin, uPAR/ FPRL1, and other signaling pathways. 22 However, the relationship between SPINK5 and tumors is not clear. In this study, we found that the protein expression level of SPINK5 was significantly reduced in esophageal cancer tissues relative to normal esophageal tissues, which was consistent with the results of previous mRNA microarray analysis. 11 In addition, in vitro studies showed that SPINK5 overexpression significantly inhibited the proliferation, migration, and invasion of esophageal cancer cells. These results suggest that SPINK5 may be involved in the development of esophageal cancer as a tumor suppressor gene. The occurrence and development of malignant tumors, such as esophageal cancer, are closely related to downregulation or inactivation of tumor suppressor genes, upregulation or activation of oncogene expression, activation of oncogenic signaling pathways, and inactivation of tumor suppressor signaling pathways. 23 The Wnt/β-catenin signaling pathway is one of the most classical oncogenic signaling pathways. Excessive activation of the Wnt/β-catenin signaling pathway is often accompanied by the development of malignant tumors such as esophageal cancer. 24 Although the Wnt/β-catenin signaling pathway is essential for embryonic The volume and weight of xenografts in SPINK5 group and NC group were counted and analyzed. Values represent the mean ± SD from 3 independent measurements. **P < 0.01, ***P < 0.001 development, cell division and other physiological processes, however, when the Wnt/β-catenin signaling pathway is overactivated, the expression of target genes such as c-myc and cyclin D1, which are related to cell proliferation and migration, is upregulated, which promotes the cell proliferation and migration, leading to malignant transformation of cells. 25 Therefore, exploring the upstream and downstream regulatory mechanisms of Wnt/β-catenin signaling pathway is a hot spot in current cancer research. In this study, we found through bioinformatics analysis that the expression level of SPINK5 is closely related to Wnt/β-catenin signaling pathway in esophageal cancer. The results of the In vitro study further demonstrated that SPINK5 significantly inhibits the activity of the Wnt/β-catenin signaling pathway in esophageal cancer cells.
The Wnt/β-catenin signaling pathway is a complex and precise signaling pathway in which the localization and expression level of β-catenin protein is a key point. When the Wnt/β-catenin signaling pathway is inactivated, CK1, AXIN, APC, GSK3β, and other proteins in the cytoplasm form a degradation complex, which promotes the ser33 and ser37 phosphorylation of β-catenin by binding to β-catenin protein, leading to the degradation of β-catenin by ubiquitination. 26 When the Wnt/β-catenin signaling pathway is activated, the Wnt family protein binds to the receptor Frizzle protein, as well as the co-receptors LRP5 and LRP6 on the cell membrane, and transmit an activation signal to the cytoplasm through the DVL protein, leading to the dissociation of degradation complexes in the cytoplasm, the degradation of β-catenin protein is inhibited, and the β-catenin protein in the cytoplasm is aggregated, which then enters the nucleus and binds to the TCF/LEF transcriptional regulatory complex to activate the transcriptional expression of the target gene. 27 Therefore, the expression level and localization of β-catenin protein in cells determines the activity of the Wnt/β-catenin signaling pathway. In this study, we found that SPINK5 overexpression significantly inhibited the expression of β-catenin, while knockdown of SPINK5 promoted the expression of β-catenin. GSK3β is one of the key factors in the degradation complex, which can promote the phosphorylation of serine at positions 33 and 37 of βcatenin protein. The kinase activity of GSK3β is closely related to the phosphorylation of ser9. 28 GSK3β is inactivated by phosphorylating at Ser9, so the expression level of p-GSK3β (S9) in cells is one of the indicators to measure the activity of degradation complex. 29 In this study, we found that SPINK5 inhibits the expression level of p-GSK3β (S9), but has no significant effect on the expression level of GSK3β. Furthermore, using the GSK3β inhibitor LiCl in combination with SPINK5 overexpression or knockdown, we further determined that SPINK5 can activate GSK3β and inhibit the activity of Wnt/β-catenin signaling pathway by affecting the GSK3β/β-catenin degradation complex.
In the process of tumor development, activation of Wnt/βcatenin signaling pathway can upregulate cell proliferation and migration-related target genes, such as c-myc and cyclin D1, which promote cell proliferation and migration. 30 Therefore, we further demonstrated that SPINK5 overexpression could reverse the effect of LiCl treatment, which confirmed that SPINK5 could indeed inhibit the proliferation and migration of esophageal cancer cells by inhibiting the activity of Wnt/β-catenin signaling pathway. In addition, the inhibitory effect of SPINK5 overexpression on the growth of esophageal cancer cells was further verified in nude mice, suggesting that SPINK5 may be a new therapeutic target for esophageal cancer.
In conclusion, in this study, we first explored the role of SPINK5 as a tumor suppressor gene in the development of esophageal carcinoma, and further found that SPINK5 can inhibit the Wnt/β-catenin signaling pathway to play a role in the proliferation, migration, and invasion of esophageal cancer cells. In addition, in this study, we also found that SPINK5 is mainly expressed on the cell membrane in normal esophageal tissues, while SPINK5 is mainly in the cytoplasm and nucleus in esophageal cancer tissues. In general, the position of protein determines its function. Therefore, the correlation between the transposition of SPINK5 and its function is one of the directions of future research.