Integrin β6 serves as an immunohistochemical marker for lymph node metastasis and promotes cell invasiveness in cholangiocarcinoma

Cholangiocarcinoma is a devastating malignancy that is notoriously difficult to diagnose and is associated with a high mortality. Despite extensive efforts to improve the diagnosis and treatment of this neoplasm, limited progress has been made. Integrin β6 is a subtype of integrin that is expressed exclusively on the surfaces of epithelial cells and is associated with a variety of tumors. In the present study, we investigated the expression and roles of integrin β6 in cholangiocarcinoma. β6 upregulation in cholangiocarcinoma was correlated with lymph node metastasis and distant metastasis. Moreover, integrin β6 was identified as a biomarker for the diagnosis of cholangiocarcinoma and an indicator of lymph node metastasis. Integrin β6 significantly promoted the proliferation, migration and invasion of cholangiocarcinoma cells. Furthermore, integrin β6 increased Rac1-GTPase, resulting in the upregulation of metalloproteinase-9 (MMP9) and F-actin polymerization. Taken together, our results indicate that integrin β6 promotes tumor invasiveness in a Rac1-dependent manner and is a potential biomarker for tumor metastasis. Integrin β6 may help to improve the diagnostic accuracy, and targeting β6 may be a novel strategy for the treatment of cholangiocarcinoma.

demonstrated that there is a direct link between extracellular signal-regulated kinase (ERK2) and β 6 integrin, which is essential for β 6-mediated ERK2 activation and the corresponding downstream effects 20,21 . Furthermore, we synthesized β 6-targeted immunoliposomes and demonstrated that they had favorable antitumor efficacy and insignificant systemic toxicity in colon cancer 22 . Unsurprisingly, the exclusive expression of integrin β 6 in epithelium-derived tumors and its important effects make it a novel therapeutic target for the treatment of many tumors. As cholangiocarcinoma originates from the ductular epithelium of the biliary tree, the expression and roles of integrin β 6 in cholangiocarcinoma should be further explored.
Rac1 is a member of the Rho family of small GTPases. It is highly expressed in many cancer cell lines and has been implicated in a wide variety of cellular processes, including cytoskeletal reorganization and gene transcription, and its overexpression is correlated with poor prognosis in cholangiocarcinoma 23,24 . Furthermore, Rac1 also regulates various downstream effector molecules related to tumor invasiveness, such as MMP9 and uPA, and it is considered a central regulator of tumor malignancy 25,26 .
In the present study, for the first time, we demonstrated that integrin β 6 is overexpressed in cholangiocarcinoma and is associated with lymph node metastasis. Our data also indicate that integrin β 6 is crucial in the malignant progress of cholangiocarcinoma. Moreover, integrin β 6 promotes the development of cholangiocarcinoma by activating Rac1. Our data indicated that integrin β 6 may contribute to improving the diagnostic accuracy and treatment of cholangiocarcinoma and should be further explored.

Results
Integrin β6 expression was associated with lymph node metastasis and distant metastasis in cholangiocarcinoma. To investigate the expression of integrin β 6 in cholangiocarcinoma, we performed an immunohistochemistry (IHC) analysis using a total of 95 paraffin-embedded cholangiocarcinoma samples and corresponding adjacent normal tissues. As shown in Fig. 1, integrin β 6 staining was negative in non-tumorous tissues ( Fig. 1A-C), while moderate expression was observed in tumor tissues (Fig. 1D-F). Furthermore, strong β 6 expression was observed in specimens with lymph node metastasis ( Fig. 1G-I). Integrin β 6 was predominantly expressed in the membrane of the tumor cells. To further explore the clinical significance of β 6 expression in cholangiocarcinoma, we analyzed the correlation between β 6 expression and various clinicopathological factors. As shown in Table 1, no significant association was observed between β 6 expression and age (P = 0.5226), gender (P = 0.6637), CA19-9 level (P = 0.2648), CEA level (P = 0.6670), depth of invasion (P = 0.4396) and tumor differentiation (P = 0.3684). However, β 6 expression was significantly associated with lymph node metastasis (P = 0.0003) and distant metastasis (P = 0.0126), indicating that β 6 may be involved in the progression of cholangiocarcinoma. Integrin β6 was a promising biomarker for diagnosis of cholangiocarcinoma and prediction of lymph node metastasis. As shown in Fig. 2A, β 6 staining was negative in almost all non-tumorous tissues, while moderate to strong staining was observed in cholangiocarcinoma tissues, suggesting that β 6 can be used for the diagnosis of cholangiocarcinoma. The IHC results showed that β 6 expression was significantly upregulated in tumor tissues with lymph node metastasis compared to that of samples without lymph node metastasis (Fig. 2B).
To determine the diagnostic value of β 6 expression in cholangiocarcinoma, we constructed receiver operator characteristic (ROC) curves. To assess the capacity of β 6 expression (IHC sum scores) to diagnose cholangiocarcinoma or to predict lymph node metastasis, we calculated the area under the curve (AUC). The ROC curves suggested that the AUC value for the diagnosis of cholangiocarcinoma was up to 0.876 (Fig. 2C, CI (95%): 0.822 to 0.929, P < 0.001), with an estimated sensitivity and specificity of 78.95% and 93.68%, respectively, for β 6 expression as a predictor of cholangiocarcinoma (Supplementary Table S1). Moreover, the AUC value for predicting lymph node metastasis in cholangiocarcinoma was 0.701 (Fig. 2D, CI (95%): 0.599 to 0.791, P < 0.001), with an estimated sensitivity and specificity of 77.55% and 63.04%, respectively (Supplementary Table S2).
Integrin β6 markedly promoted the proliferation, migration and invasion of cholangiocarcinoma cells. As integrin β 6 was highly expressed in cholangiocarcinoma, the functional roles of integrin β 6 in human cholangiocarcinoma cells were investigated. β 6-specific siRNA was used in RBE and QBC939 cells. First, the efficiency of β 6 silencing in these cells was determined. As shown in Fig. 3A,B, β 6 expression was significantly decreased at both the mRNA level and protein level after transfection with β 6-specific siRNA. CCK8 assays were used to evaluate the effects of integrin β 6 on the viability of cholangiocarcinoma cells. The results showed that β 6 silencing in RBE and QBC939 cells markedly suppressed cell viability 48 hours after transfection in a time-dependent manner (Fig. 3C). Then, MTT assays were conducted, and the results indicated that β 6 downregulation in RBE and QBC939 cells was associated with decreased cell proliferation (Fig. 3D). Wound-healing assays showed that the migration of RBE and QBC939 cells was markedly suppressed after downregulation of β 6 ( Fig. 3E). Then, we performed Transwell migration and invasion assays in cholangiocarcinoma cells after β 6 silencing. β 6 silencing had no obvious effect on cell viability within 12 hours, which eliminated the potential confounding influence of β 6 silencing-induced cell proliferation on cell migration and invasion. The results showed that the migration and invasion capacities of both RBE and QBC939 cells were significantly suppressed by β 6 silencing ( Fig. 3F,G). To further confirm the effects of β 6 on cell proliferation, migration and invasion in cholangiocarcinoma cells, we repeated the aforementioned experiments after upregulation of β 6 using a β 6 overexpression plasmid. As shown in Fig. 4A,B, β 6 expression was significantly increased after transfection. The results showed that β 6 overexpression markedly promoted the proliferation, migration and invasion of cholangiocarcinoma cells ( Fig. 4C-G).
These data suggested that integrin β 6 could promote the proliferation, migration and invasion of cholangiocarcinoma cells.
Integrin β6 induced the expression of MMP9 in cholangiocarcinoma cells. To identify the mechanism underlying the effects of β 6 on invasion in cholangiocarcinoma cells, we measured the mRNA expression of uPA, MMP2, MMP3, and MMP9 using quantitative real-time PCR after silencing or overexpressing β 6. We found that β 6-specific siRNA significantly downregulated MMP9 mRNA expression in cholangiocarcinoma cells, and β 6 overexpression markedly increased MMP9 mRNA expression. No effect was observed on uPA, MMP2 and MMP3 ( Fig. 5A-D).
We further explored the effect of β 6 on secreted MMP9 in the supernatant of cholangiocarcinoma cells by ELISA. Consistent with the results from the mRNA analysis, the data showed that the secreted MMP9 in the supernatant of cholangiocarcinoma cells was significantly decreased after suppression of β 6 expression (Fig. 5E), while β 6 overexpression increased MMP9 secretion in the supernatant (Fig. 5F). Taken together, these results revealed that integrin β 6 could induce MMP9 expression at both the mRNA and protein levels.
Integrin β6 activated Rac1 in cholangiocarcinoma cells. As previous studies showed that Rac1 activity is involved in the malignant behaviors of cancer cells 24 , we hypothesized that integrin β 6 may promote invasiveness by activating Rac1. To confirm our hypothesis, we performed four experiments. First, using the specific Rac1 activity inhibitor NSC23766, we found that Rac1 was essential for the migration and invasion of cholangiocarcinoma cells, as NSC23766 suppressed migration and invasion of RBE cells in a dose-dependent manner (Fig. 6A). Then, we demonstrated that overexpression of integrin β 6 could effectively promote the migration and Figure 2. Upregulation of β6 in more aggressive cholangiocarcinoma tissues and ROC curves to assess the diagnostic value of β6 expression in cholangiocarcinoma. IHC sum scores were used to assess integrin β 6 expression in cholangiocarcinoma tissues and adjacent non-tumorous tissues. β 6 expression was significantly upregulated in cholangiocarcinoma tissues compared to adjacent non-tumorous tissues. (A) β 6 expression was markedly higher in cholangiocarcinoma tissues with lymph node metastasis than those without lymph node metastasis. (B) The ROC curves showed strong separation between cholangiocarcinoma tissues and adjacent non-tumorous tissues, with an AUC of 0.989 (P < 0.001). (C) The ROC curves showed strong separation between the patients with and without lymph node metastasis, with an AUC of 0.741 (P < 0.001). * * * P < 0.001. invasion capacity of cholangiocarcinoma cells, whereas NSC23766 significantly inhibited this effect (Fig. 6B). Moreover, the β 6-mediated promotion of migration and invasion could be inhibited by specific Rac1 siRNA (Fig. 6C). In addition, we found that β 6 silencing significantly decreased Rac1-GTPase activity (Fig. 6D), while β 6 overexpression increased Rac1 activity in cholangiocarcinoma cells (Fig. 6E). All these results demonstrated that β 6 promoted migration and invasion by activating Rac1.

The effects of integrin β6 were mediated through Rac1 downstream effectors, F-actin polymerization and MMP9 expression in cholangiocarcinoma cells. Previous studies found that Rac1 activity
is important for microfilament dynamics, which directly regulate cell motility 27 . In the present study, we explored the effect of integrin β 6 on the microfilament dynamics in cholangiocarcinoma cells. Figure 7A shows that in the siNC group, F-actin was organized into the cytoskeleton, while in the β 6-silenced group, F-actin was depolymerized in most cells. The results revealed that F-actin was significantly depolymerized after treatment with β 6 siRNA. Moreover, β 6 overexpression increased F-actin polymerization, and after treatment with NSC23766, this effect was abolished (Fig. 7B), indicating that β 6 promoted F-actin polymerization by activating Rac1.
Our previous work showed that β 6 induced the expression of MMP9 and promoted the invasiveness of cholangiocarcinoma cells in a Rac1-dependent manner. We thus hypothesized that integrin β 6 induced the expression of MMP9 via the Rac1 pathway. To test our hypothesis, we explored the effect of Rac1 on β 6-induced MMP9 expression. Rac1 silencing significantly decreased the expression of MMP9 at both the mRNA and protein level (Fig. 7C,D). Moreover, β 6 silencing decreased MMP9 expression, while β 6 overexpression increased the expression of MMP9. However, after pretreatment with a Rac1 activity inhibitor, the effect of both β 6 silencing and overexpression was abolished (Fig. 7E). These results confirmed our hypothesis and indicated that integrin  bearing RBE cells. RBE cells were infected with the lentiviral vectors LV-negative control, LV-siβ 6 or LV-β 6. Consistent with our in vitro findings, our results showed that silencing of β 6 markedly suppressed the growth of RBE xenograft tumors compared with that of the NC group, while β 6 overexpression promoted the growth of tumors (Fig. 8A). The overall mean tumor volume and tumor weight of the LV-siβ 6 group were significantly smaller than those of the LV-NC group, whereas tumors in the LV-β 6 group had a relatively larger volume and weight (Fig. 8B,C). As shown in Fig. 8D,E, compared with the LV-NC group, β 6 silencing markedly decreased the MMP9 expression in the xenograft tumors, while β 6 overexpression significantly increased the MMP9 expression. These results indicated that integrin β 6 promoted the growth and MMP9 expression of cholangiocarcinoma xenograft tumors.

Discussion
Cholangiocarcinoma is a predominantly fatal cancer, which is usually difficult to diagnose and to treat 7 . Therefore, elucidating the mechanisms underlying the malignant behavior of cholangiocarcinoma may facilitate the development of clinical strategies. In the present study, β 6 expression was markedly upregulated in cholangiocarcinoma and was associated with lymph node metastasis. Integrin β 6 promoted the migratory and invasive capacities of cholangiocarcinoma cells by activating Rac1, indicating that integrin β 6 may be a potential target for the treatment of cholangiocarcinoma.
The epithelial-restricted integrin β 6 is generally upregulated in tissues that have undergone malignant transformation 28,29 . Increased expressions of β 6 were previously reported in carcinomas of the colon, breast, stomach and pancreas, and increased β 6 expression is associated with advanced tumor stage and poor prognosis 13,17,18,30 . Patsenker et al. reported that integrin β 6 is a highly specific immunohistochemical marker for the differential diagnosis of hepatocellular carcinoma (HCC) and cholangiocarcinoma, as β 6 is strongly expressed in cholangiocarcinoma, and in HCC, β 6 staining is negative 31 . Here, we reported that the increased β 6 expression was related to lymph node metastasis and distant metastasis, suggesting that in addition to an immunohistochemical marker, β 6 staining in the surgical specimens could be used to evaluate the risks of lymph node metastasis. Additionally, integrin β 6 may also be involved in the development of cholangiocarcinoma.
Integrin β 6 promotes malignant behaviors, such as cell proliferation, migration, invasion and metastasis, in many types of malignancies 15,17,21,30 . However, the role of integrin β 6 in cholangiocarcinoma cells has not been elucidated. Our results demonstrated that β 6 could significantly promote cell proliferation and invasion.
Previous studies showed that β 6 increases the expression and secretion of MMP2, MMP3 and MMP9, resulting in the degradation of the extracellular matrix and increased tumor cell invasiveness in colon cancer, pancreatic cancer and ovarian cancer 32,33 . MMP9 is highly expressed in tissues, and it was shown to be a valuable prognostic indicator in cholangiocarcinoma 34 . As β 6 also promoted the invasiveness of cholangiocarcinoma cells, we explored the effect of β 6 on MMP expression in cholangiocarcinoma. Additionally, the effect of β 6 on uPA was assessed due to its role in cell invasiveness, and uPA was highly expressed in cholangiocarcinoma cells. Our results showed that β 6 induced the expression of MMP9 but not that of uPA, MMP2 or MMP3 in cholangiocarcinoma cells. The detailed mechanism by which β 6 promotes tumor development is complicated. Integrin β 6 may functions through the protein kinase C (PKC) pathway in colon cancer, while β 6 may also bind ERK and subsequently activate the ERK-Ets1 pathway in colon cancer and pancreatic cancer 35 . The SDF-1/CXCR4 axis and IL-8 are also involved in the β 6-regulated tumor cell migration 15,36 . Rac1 plays a vital role in the development of After treatment with NSC23766, RBE and QBC939 cells were transfected with the β 6-overexpressing plasmid, and the migration and invasion capacities were measured by Transwell assays. (C) RBE and QBC939 cells co-transfected with Rac1-specific siRNA and β 6-overexpressing plasmid were used for migration and invasion assays. (D) After transfection with β 6 siRNA or β 6-overexpressing plasmid, cell lysates of RBE and QBC939 were used for PAK-GST pull down assays. Activated Rac1 as well as total Rac1 pulled down in the lysates were detected by western blot analysis. Data are shown as the mean ± SD, and the results shown are representative of 3 independent experiments. Five fields of cells in the lower compartment were counted in Transwell assays (200× magnification). * P < 0.05; * * P < 0.01; * * * P < 0.001. cholangiocarcinoma 24,37 . Rac1 is a member of the Rho family of small GTPases and has been implicated in a wide variety of cellular processes, especially cell motility and cytoskeleton polymerization. Rac1 could also induce the expression and secretion of MMPs 23,25,38,39 . Therefore, targeting Rac1 may be a promising therapeutic approach for the treatment of cholangiocarcinoma. MMP9 was reported to contribute to the cholangiocarcinogenesis in a Rac1-dependent manner 40 . Consistent with these results, our data demonstrated that β 6 promoted the invasiveness of cholangiocarcinoma cells by activating Rac1, as both NSC23766 and Rac1 siRNA could abolish the effect of β 6. In addition, β 6 could activate Rac1, but it had no effect on total Rac1 expression. Mechanistically, we demonstrated that β 6 facilitated F-actin polymerization and induced MMP9 expression by activating Rac1. These Previous studies reported that integrin β 6 could localize to the cytoplasm and activate ERK via a specific binding site. Then, activated ERK translocates to the nucleus and activates Ets-1, leading to the proliferation, migration, and invasion of tumor cells 20,32,35 . Here, we demonstrated that β 6 promoted tumor cell malignant behaviors by activating Rac1. Rac1 is important in regulating vesicular transport. Therefore, the effect of Rac1 on the internalization of β 6 needs to be further explored.
Cholangiocarcinoma is a severe disease with poor prognosis. Molecular targeting strategies to improve treatment and reduce the side effects of conventional chemotherapy and radiotherapy are needed. In the present study, we demonstrated that integrin β 6 upregulation was an unfavorable indicator in cholangiocarcinoma. By targeting and activating Rac1, β 6 promotes the development and progression of cholangiocarcinoma. The perturbation of integrin β 6 may present a potential strategy for the treatment of patients suffering from cholangiocarcinoma.

Materials and Methods
Tissue samples. A total of 95 human cholangiocarcinoma specimens and the corresponding adjacent normal specimens were collected from Qilu Hospital of Shandong University. Among the 95 cholangiocarcinoma specimens, 61 cases had lymph node metastasis, while 34 cases had no lymph node metastasis. Clinicopathological classification and staging were determined according to the American Joint Committee on Cancer (AJCC) criteria 41 . All samples were fixed in 40 g/L formaldehyde and were embedded in paraffin for histological diagnosis and immunohistochemical studies. The protocols in the study were carried out in accordance with the approved guidelines. All the experimental protocols were approved by the Ethics Committee of Shandong University, China. Written informed consent was obtained from all subjects.
Western blotting. After 48 hours of transfection, cells were subjected to protein extraction using cell lysis buffer containing 1% protease inhibitors. Protein concentrations of the homogenized lysates were measured using a BCA protein assay kit (Sangon, Shanghai, China). Aliquots containing 30 μ g of protein were separated by 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal antibody against integrin β 6 (1:1000; Millipore, CA, USA), mouse monoclonal antibody against Rac1 (1:300; Abcam, Hong Kong), or anti-β -actin (1:1000; ZSGB-Bio, Beijing, China), followed by secondary antibodies (1:2000; goat-anti rabbit or mouse IgG, ZSGB-Bio) conjugated to peroxidase for 1 hour at room temperature. After washing, immunoreactivity was visualized using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Immunohistochemistry (IHC). IHC was performed using paraffin-embedded tissue sections. The sections were dewaxed and hydrated, followed by antigen retrieval (in 0.01 mol/L citrate buffer solution, pH 6.0, heated to boiling for 2-3 min in a stainless steel pressure cooker). Endogenous peroxidase was blocked using a 3% hydrogen peroxide solution. The section was incubated with the blocking goat serum for 15 min and then immunostained with rabbit antibody against β 6 (dilution 1:500) at 4 °C overnight. Secondary staining was performed with HRP-conjugated anti-rabbit IgG using a MaxVision Kit and a 3, 5-diaminobenzidine (DAB) peroxidase substrate kit (Maixin Co, Fuzhou, China). The sections were then counterstained with hematoxylin.
Evaluation of immunohistochemical staining. Immunohistochemical staining was independently evaluated by two experienced pathologists in a blinded manner. Staining was semi-quantitatively scored based on both the staining intensity (0, negative; 1, weak; 2, moderate; 3, strong) and the percentage of positively stained cells (0, 0%; 1, 1-25%; 2, 26-50%; 3, 51-75%; 4, 76-100%). Both scores for each specimen were then combined to obtain the final score of β 6 expression. The cut-off point for the sum of the scores was defined as follows: 0-2, low expression; 3-7, high expression. The appropriateness of the cut-off point was validated by ROC analysis.

Cell viability assays.
A total number of 2500 cells was seeded in 96-well plates in triplicate wells and cultured for the indicated times. Cell viability was evaluated using the Cell Counting Kit-8 (CCK8) (Beyotime, Haimen, China) assay according to the manufacturer's instructions. The absorbance was determined at 450 nm. Each time point was replicated in three wells, and the experiment was independently performed at least three times.
Cell proliferation assays. Cell proliferation assays were performed using MTT (3-(4,5-dimet hylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). A total number of 2500 cells was seeded in 96-well plates. Then, 24 hours after β 6 silencing or overexpression, MTT at 5 mg/ml was added and incubated for 4 hours. Finally, the medium was aspirated, DMSO was added, and the absorbance was determined at 490 nm. Each time point was repeated in three wells, and the experiment was independently performed at least three times.
Wound-healing assays. The monolayer wound-healing assay was used to assess cell migration. A total of 5 × 10 5 cells were seeded in 6-well plates, incubated overnight, and then pretreated with β 6 siRNA or the β 6 overexpression plasmid. After achieving 90% confluency, the cell monolayer was scratched with a sterile pipette tip, the floating cells were removed with PBS, and the cells were cultured for 24 hours. Photographic images were taken at 0 and 24 hours along the scrape line using a microscope. The results are expressed as the actual wound closure distance.

Transwell assays for cell migration and invasion. Tumor cell migration assays were analyzed in 24-well
Boyden chambers with 8-μ m pore size polycarbonate membranes (Costar, Acton, USA). For invasion assays, the membranes were precoated with 50 μ g Matrigel (BD Biosciences, San Diego, USA) to simulate matrix barriers. Then, 1 × 10 5 cells were resuspended in 200 μ l serum-free medium and seeded into the upper chamber, and the lower compartments were filled with 600 μ l medium with 10% FBS. After incubation (incubation time for migration is 10-12 hours and for invasion is 24 hours), the upper surface of the Transwell membrane was wiped gently with a cotton swab to remove the non-migrating cells. The membranes were fixed with methanol and stained with 0.1% crystal violet for twenty minutes. The stained cells were counted under a light microscope at × 200 magnification in at least five fields. NSC23766 (Calbiochem, San Diego, USA), a specific Rac1 inhibitor, was used to inhibit Rac1 activity in several experiments.

MMP activity assays.
A total of 2 × 10 5 cells were cultured in a six-well culture plate and then treated with siNC, siβ 6, mock plasmid or β 6 overexpression plasmid for 24 hours. The levels of secreted MMP-9 in the culture supernatant were determined using an enzyme-linked immunosorbent assay (ELISA) following the manufacturer's ELISA kit guidelines (R&D). Samples were assayed in triplicate and calibrated against a standard curve.