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

Abstract

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.

Figure 1. The expression of integrin β6 in non-tumorous bile duct tissues and cholangiocarcinoma tissues.

(A–C) Negative β6 expression in non-tumorous bile duct tissues. (D–F) Moderate expression of integrin β6 in cholangiocarcinoma tissues without lymph node metastasis. (G–I) Strong staining of β6 in cholangiocarcinoma tissues with lymph node metastasis.

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 β6 expression in cholangiocarcinoma tissues and adjacent non-tumorous tissues. β6 expression was significantly upregulated in cholangiocarcinoma tissues compared to adjacent non-tumorous tissues. (A) β6 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.

Figure 3. β6 silencing markedly decreased the proliferation, migration and invasion of cholangiocarcinoma cells.

(A) Real-time PCR showed that integrin β6 expression was high in both RBE and QBC939 cells; β6 expression was significantly suppressed after β6-specific siRNA transfection. (B) Western blotting showed that β6 expression was significantly suppressed after β6 siRNA transfection in RBE and QBC939 cells at the protein level. (C) CCK8 assays revealed that silencing of β6 significantly decreased cell viability in RBE and QBC939 cells. (D) After transfection of β6 siRNA, MTT assays were conducted to evaluate the proliferation of RBE and QBC939 cells. (E) RBE and QBC939 cells transfected with β6 siRNA were used for wound-healing assays. (F,G) RBE (F) and QBC939 (G) cells transfected with β6 siRNA were used for Transwell migration assays and invasion assays. Five fields of cells in the lower chamber were counted (×200 magnification). Data represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01.

Figure 4. β6 overexpression markedly promoted the proliferation, migration and invasion of cholangiocarcinoma cells.

(A) Real-time PCR showed that integrin β6 expression was significantly upregulated after transfection of the β6 overexpression plasmid. (B) Western blotting showed that β6 expression was significantly increased after transfection with the β6 overexpression plasmid in RBE and QBC939 cells at the protein level. (C) CCK8 assays revealed that overexpression of β6 significantly promoted cell viability in RBE and QBC939 cells. (D) After β6 overexpression, MTT assays were conducted to evaluate the proliferation of RBE and QBC939 cells. (E) RBE and QBC939 cells transfected with the β6 overexpression plasmid were used for wound-healing assays. (F,G) RBE (F) and QBC939 (G) cells transfected with the β6 overexpression plasmid were used for Transwell migration assays and invasion assays. Five fields of cells were counted in the lower chambers (×200 magnification). Data represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01.

Figure 5. Integrin β6 increased MMP9 expression at both the mRNA and protein levels.

(A,B) MMP9 mRNA was significantly decreased in RBE cells (A) and QBC939 cells (B) after transfection with β6 siRNA, while the expression of uPA, MMP2 and MMP3 showed no significant changes. (C,D) MMP9 mRNA was significantly increased in RBE cells (C) and QBC939 cells (D) after β6 overexpression, while the expression of uPA, MMP2 and MMP3 showed no significant changes. (E) The protein level of MMP9 was detected by ELISA in RBE and QBC939 cells transfected with β6 siRNA. (F) The protein level of MMP9 was detected by ELISA in RBE and QBC939 cells after β6 overexpression. Data represent the mean ± SD from three independent experiments. *P < 0.05; **P < 0.01.

Figure 6. Integrin β6 promoted the invasiveness of cholangiocarcinoma cells by activating Rac1.

(A) RBE cells treated with different concentrations of NSC23766, a Rac1 inhibitor, were used for migration and invasion assays. (B) After treatment with NSC23766, RBE and QBC939 cells were transfected with the β6-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 β6-overexpressing plasmid were used for migration and invasion assays. (D) After transfection with β6 siRNA or β6-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.

Figure 7. Integrin β6 induced F-actin polymerization and MMP9 expression by activating Rac1.

(A) After transfection of β6 siRNA, F-actin in RBE and QBC939 cells was stained by TRITC-conjugated phalloidin and observed with laser confocal microscopy. The % decrease in F-actin = [(F-actin in untreated cells–F-actin in NC or β6 siRNA treated cells)/F-actin in untreated cells] ×100. Scale bar, 10 μm. (B) After treatment with NSC23766, RBE and QBC939 cells were transfected with β6 siRNA or β6-overexpressing plasmid, and F-actin was stained by TRITC-conjugated phalloidin and observed with laser confocal microscopy. (C) MMP9 mRNA expression was significantly decreased in RBE cells and QBC939 cells after transfection with Rac1 siRNA, while the expression of uPA, MMP2 and MMP3 showed no significant changes. (D) The protein level of MMP9 was detected by ELISA in RBE and QBC939 cells after Rac1 silencing. (E) The protein level of MMP9 was detected by ELISA in RBE (C) and QBC939 cells (D) pretreated with NSC23766 and β6 siRNA or β6-overexpressing plasmid. Data represent the mean ± SD from three independent experiments. *P < 0.05; **P < 0.01.

Figure 8. Integrin β6 effectively promoted tumor growth and MMP9 expression of subcutaneous xenograft tumors.

The growth curves of tumors from the LV-NC, LV-siβ6 and LV-β6 groups. Tumors were isolated and measured. The mean volume of tumors in the LV-siβ6 group (n = 6) was significantly smaller than that of the control, while the LV-β6 group (n = 6) had a larger mean tumor volume. (A) The mean tumor weight was significantly lower in the LV-siβ6 group (n = 6) and higher in the LV-β6 group (n = 6) compared to the control. (B) The expression of integrin β6 and MMP9 was detected by IHC in paraffin-embedded tumor tissue sections. (C) IHC sum scores were used to determine the expression of integrin β6 and MMP9 in the xenograft tumor tissues. Data represent the mean ± SD. *P < 0.05; **P < 0.01.