PCDH1 promotes progression of pancreatic ductal adenocarcinoma via activation of NF-κB signalling by interacting with KPNB1

Abstract

Uncontrolled growth, distant metastasis and chemoresistance are critical characteristics of pancreatic ductal adenocarcinoma (PDAC), and they result in high mortality; however, the mechanisms triggering these effects have not been fully investigated. In this study, we analysed a dataset in the Cancer Genome Atlas (TCGA) and identified PCDH1, a rarely studied transmembrane protein, as a novel prognostic marker in PDAC patients. We demonstrated that PCDH1 expression was upregulated in PDAC tissues, and its expression levels were associated with the depth of tumour invasion and lymph node metastasis. Patients with high PCDH1 levels showed poor overall survival (OS). We also investigated the biological significance of PCDH1 in PDAC cell growth, metastasis, and side population (SP) phenotype acquisition and explored the internal molecular mechanisms of PCDH1 action. Our results demonstrated that PCDH1 enhanced p65 nuclear localization by interacting with KPNB1, a well-characterized nuclear transporter, thereby activating the NF-κB signalling pathway and increasing its functional effects during PDAC progression. Hence, our results indicate that PCDH1 can be used as a negative prognostic marker and may be a potential therapeutic target for PDAC patients.

Fig. 1. PCDH1 expression is upregulated in PDAC and predicts poor survival.

A High expression of PCDH1 in the TCGA dataset was correlated with worse prognosis in PDAC. B Heatmap of 32 genes of the protocadherin family in PDAC and noncancerous tissues. C In the HPDE6-C7 normal pancreatic ductal epithelial cell line and four PDAC cell lines, the expression of PCDH1 was detected by Western blotting. D Immunohistochemical (IHC) analysis of PCDH1 expression in 197 PDAC tissues and adjacent normal pancreatic tissues. The data were assessed by paired-samples t test. E Kaplan–Meier survival analysis of the correlation between PCDH1 expression and OS (log-rank test). F Multivariate Cox regression analysis of the prognostic factors of PDAC patients. ***P < 0.001.

Fig. 2. PCDH1 promotes the growth, metastasis and side population phenotype acquisition of PDAC cells in vitro.

A Knockdown efficiency and enforced expression of PCDH1 in PDAC cell lines was evaluated by Western blotting. B An MTT assay was performed to detect the effect of PCDH1 on the proliferation of PDAC cell lines. C Representative images showing the effects of PCDH1 expression knockdown (left) or overexpression (right) on colony formation. The number of colonies containing more than 50 cells was counted. D The effects of PCDH1 knockdown (left) or overexpression (right) on migration and invasion were evaluated by Transwell assays. E Representative images showing the effects of PCDH1 knockdown on SP phenotype acquisition. The results are presented as the mean ± SD of three independent experiments. The data were assessed by Student’s 2-tailed t test. *P < 0.05, **P < 0.01.

Fig. 3. PCDH1 promotes the growth and metastasis of PDAC cells in vivo.

PCDH1-downregulated PDAC cells were injected into nude mice on the basis of the different model established ((A) refers to the xenograft model; (B) refers to the hepatic metastasis model; (C) refers to the lung metastasis model; and (D) refers to the lymph node metastasis model). Each group contained 10 nude mice. A The growth of the subcutaneous xenograft tumours derived from the PDAC cells implanted in the nude mice (left, representative images showing the xenografts removed on day 28; right, volume of the tumours at different times). B Representative microscopy images of livers and haemoxylin and eosin (HE)-stained metastatic nodules (left). Number of metastases per liver (right). C Representative microscopy images of lungs and HE-stained metastatic nodules (left). Number of metastases per lung (right). D Lymph node metastases in the nude mice after intraplantar injection (left, representative images; right, the number and ratio of mice with lymph node metastasis). The results are presented as the mean ± SD of three independent experiments. The data were assessed by Student’s 2-tailed t test. *P < 0.05, **P < 0.01.

Fig. 4. PCDH1 activates the NF-κB pathway in PDAC cells.

A Representative heatmaps showing the differentially expressed genes (fold change > 2) after PCDH1 silencing by RNA interference in Panc-1 cells and the control group. The expression levels are shown as log2-transformed values. B GSEA of mRNA profiles revealed inhibited expression of NF-κB target genes after PCDH1 silencing by RNA interference in Panc-1 cells compared with the control group. C Quantitative RT–PCR showing that NF-κB regulated IL-6, IL-8 and TNFα mRNA expression in Panc-1 cells with in PCDH1 silenced by RNA interference and the control group. D Dual-luciferase assays were performed to detect the effects of PCDH1 on the NF-κB signalling pathways in PDAC cell lines. Western blot assay (E) and immunofluorescence assays (F) were performed to detect the effect of PCDH1 on the nuclear localization of p65. For the PCDH1 silencing groups shown in Figs. 4D, 4E and 4F, TNF-α (20 ng/ml) (Sigma–Aldrich, GF314) pretreatment was performed for 10 min. The results are presented as the mean ± SD of three independent experiments. The data were assessed by Student’s 2-tailed t test. *P < 0.05, **P < 0.01.

Fig. 5. Correlation between the expression of PCDH1 and NF-κB downstream effectors in 197 PDAC tissues.

A Representative IHC staining for PCDH1 and IL-6 is shown in two PCAC tissues. Correlations were detected between IL-6 (B), IL-8 (C), TNF-α (D) and PCDH1 expression by Pearson’s test in 197 PDAC tissues.

Fig. 6. PCDH1 activates the NF-κB pathway during PDAC progression by interacting with KPNB1.

A Coimmunoprecipitation (co-IP) and Western blot assays showing exogenous PCDH1 and KPNB1 expression in Panc-1 cells. B Co-IP and Western blot assays showing endogenous PCDH1 and KPNB1 expression in Panc-1 cells. Western blot assay (C) and immunofluorescence assays (D) were performed to detect the effect of PCDH1 on the nuclear localization of p65 after KPNB1 silencing. MTT (D) and Transwell assays (E) were performed to detect the effects of PCDH1 expression on PDAC cell proliferation and migration after KPNB1 silencing. The results presented are the mean ± SD of three independent experiments. The data were assessed by Student’s 2-tailed t test. *P < 0.05, **P < 0.01.