Block of proliferation 1 promotes cell migration and invasion in human colorectal cancer cells via the JNK pathway

Abstract Metastasis is one of the most common causes of death in patients with colorectal cancer (CRC). Block of proliferation 1 (BOP1) regulates tumorigenesis, epithelial‐to‐mesenchymal transition, migration, metastasis, and drug resistance in several tumor types. However, the role of BOP1 in the regulation of colorectal cancer cell migration and invasion is still largely unclear. In this study, the results of immunohistochemistry showed that BOP1 was upregulated in our cohort of CRC patients. BOP1 knockdown inhibited the migration and invasion of CRC cells, confirmed by the downregulation of the mRNA levels of MMP‐2 and MMP‐9. The overexpression of BOP1 in CRC cells exerted the opposite effect. SP600125, an inhibitor of JNK signaling, partially abolished the BOP1 overexpression‐mediated increase in the migratory and invasive ability of CRC cells. Our results indicated that BOP1 is an important regulator of CRC cell invasion and migration, predominantly through the JNK signaling pathway.


| Clinical specimens
Forty-six pairs of CRC tissue samples from patients who underwent surgery and were pathologically diagnosed at Nantong University Maternal and Child Health Hospital were recruited. None of the CRC patients in this study received preoperative chemotherapy or radiotherapy. The Union for International Cancer Control classification was used to determine the clinical stage. The study protocol was approved by the Ethics Committee of Nantong University Maternal and Child Health Hospital. Written informed consent procedures were obtained from each patient.

| Cell transfection
Two BOP1 siRNAs and scramble siRNA were purchased from GeneChem Co., Ltd. The vector pcDNA3.1 containing the full-length BOP1 cDNA sequence (BOP1) and control vector (vector) were obtained from GeneCopoeia Inc. siRNA or plasmid transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the recommended instructions.

| Western blot analysis
Western blot for BOP1, P-JNK, and JNK protein expression was performed as previously described. 15,16 Briefly, 30 μg of total protein was separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). Anti-human BOP1 (Invitrogen), anti-human P-JNK (Abcam), and anti-human JNK antibodies (Abcam) were incubated with the membranes overnight at 4°C. Then, the membranes were incubated with HRP-conjugated secondary antibodies (CST) and visualized with a chemiluminescent detection system (Beyotime). Protein levels were quantified by density analysis using ImageJ software (National Institutes of Health).

| RNA extraction and quantitative real-time polymerase chain reaction
Total RNA was extracted from cultured cells using TRIzol rea-

| Migration and invasion assays
The cell migration and invasion assays were performed as previously described. 17,18 For the migration assay, HT29 or SW480 cells in serum-free medium were seeded into a 24-well Insert System with an 8-μm pore size polyethylene terephthalate membrane (BD Biosciences). Complete medium was placed in the lower chambers.
For the cell invasion assay, HT29 or SW480 cells in serum-free medium were seeded into the chambers precoated with a matrix gel (BD Biosciences). Cells that had migrated or invaded through the membrane were fixed using 10% methanol, stained with 0.1% crystal violet, and quantified using a light microscope (Olympus). Migrated and invasive cells were counted from 5 randomly selected fields of each sample.

| Statistical analysis
All results in this study are presented as the mean ± SEM. Statistical analysis was performed using GraphPad Prism 6.0 software by Student's t test. For clinical data analysis, the correlation between 2 groups was analyzed by the Spearman correlation test. A P-value <.05, .01, or .001 was considered significant.

| BOP1 protein expression in CRC tissues and correlation between BOP1 and clinicopathological features of patients with CRC
To examine BOP1 protein expression in CRC tissues, we performed IHC analysis on both tumor tissue samples and matched adjacent nontumor tissue samples. Compared with that in paired adjacent normal tissue samples, BOP1 expression was significantly upregulated in tumor tissues ( Figure 1A and 1B). Furthermore, we analyzed the association between the protein level of BOP1 and the clinicopathological features of CRC patients. BOP1 expression in tissues of CRC patients was associated with TNM stage, lymph node metastasis, and distant metastasis (

| BOP1 knockdown inhibits the migration and invasion of HT29 cells
The protein levels of BOP1 in multiple CRC cell lines with different metastatic potential were analyzed by Western blot. The level of BOP1 was higher in the highly metastatic CRC cell lines (HT29 and SW620) than in the low-metastatic CRC cell lines (HCT116 and CaCO2) (Figure 2A). The BOP1 expression level was highest in HT29 cells (Figure 2A). Two specific BOP1 siRNAs significantly downregulated both the mRNA and protein levels of BOP1 in HT29 cells ( Figure 2B and 2C). BOP1 siRNAs could decrease the migratory and invasive activity of HT29 cell ( Figure 2D). MMP-2 and MMP-9 have been reported to be involved in metastasis processes of CRC. 19,20 Herein, we used these two proteins as metastasis markers to evaluate the effect of BOP1 on the migration and invasion of CRC cells.
We observed that BOP1 knockdown downregulated the mRNA and protein levels of MMP-2 and MMP-9 ( Figure 2E and 2F).

| BOP1 knockdown inhibits the JNK signaling pathway
A previous study reported that BOP1 could activate the JNK signaling pathway and further affect migration in CRC. 10 Hence, we hypothesized that BOP1 knockdown may reduce the migratory and invasive activity of CRC cells via the JNK signaling pathway.
As shown in Figure 4A, Western blot analysis indicated that the p-JNK level in HT29 cells transfected with the two specific BOP1 siRNAs was significantly decreased. In contrast, the p-JNK level in HCT116 cells transfected with the BOP1 vector was significantly increased ( Figure 4B). Moreover, SP600125, a JNK signaling pathway inhibitor, decreased the p-JNK level induced by BOP1 overexpression ( Figure 4B).

| JNK signaling inhibitor reduces BOP1 overexpression-induced migration and invasion of HCT116 cells
To further confirm that JNK signaling was involved in BOP1 overex-

| D ISCUSS I ON
Multiple genetic changes in tumor suppressor genes and oncogenes are involved in the progression of CRC. [21][22][23] A previous study showed that abnormal BOP1 expression was observed in hepatocellular carcinoma clinical specimens. 11 In the present study, we performed IHC analysis of both tumor tissue samples and matched adjacent nontumor tissue samples to analyze the expression level of BOP1 in patients with CRC. BOP1 expression was significantly upregulated in CRC tumor tissues ( Figure 1). Furthermore, BOP1 expression in the tissues of CRC patients was associated with TNM stage, lymph node metastasis, and distant metastasis (  is involved in the Wnt/β-catenin pathway-induced epithelial-to-mesenchymal transition, cell migration, and experimental metastasis of CRC cells. 10  RhoA activation in hepatocellular carcinoma. 11 Moreover, BOP1 was found to be involved in the Wnt/β-catenin/JNK signaling pathway-mediated experimental metastasis and migration of CRC cells. 10 Therefore, we assumed that BOP1 could promote CRC cell invasion and migration through the JNK signaling pathway. BOP1 knockdown significantly decreased the p-JNK level in HT29 cells, while BOP1 overexpression significantly increased the p-JNK level in HCT116 cells. Moreover, SP600125 decreased the p-JNK level induced by BOP1 overexpression. It is important that SP600125 reversed the BOP1 overexpression-mediated increase in the migratory and invasive ability of HCT116 cells. Overall, BOP1 promoted the invasion and migration of CRC cells through the JNK signaling pathway.
In conclusion, our results indicated that BOP1 is a key regulator of CRC cell invasion and migration, predominantly through the JNK signaling pathway. BOP1 may be a key component of the JNK prometastatic signaling network in CRC cells.

ACK N OWLED G M ENTS
None.

AUTH O R CO NTR I B UTI O N S
Xiaoxi Chen conceived and designed the experiments and provided experimental guidance in the laboratory; Yu Zhao analyzed the data and wrote the article.