WD repeat-containing protein 1 maintains β-Catenin activity to promote pancreatic cancer aggressiveness

Abstract

Background: The molecular signature underlying pancreatic ductal adenocarcinoma (PDAC) progression may include key proteins affecting the malignant phenotypes. Here, we aimed to identify the proteins implicated in PDAC with different tumour-node-metastasis (TNM) stages.

Methods: Eight-plex isobaric tags coupled with two-dimensional liquid chromatography-tandem mass spectrometry were used to analyse the proteome of PDAC tissues with different TNM stages. A loss-of-function study was performed to evaluate the oncogenic roles of WD repeat-containing protein 1 (WDR1) in PDAC. The molecular mechanism by which WDR1 promotes PDAC progression was studied by real-time qPCR, Western blotting, proximity ligation assay and co-immunoprecipitation.

Results: A total of 5036 proteins were identified, and 4708 proteins were quantified with high confidence. Compared with normal pancreatic tissues, 37 proteins were changed significantly in PDAC tissues of different stages. Moreover, 64 proteins were upregulated or downregulated in a stepwise manner as the TNM stages of PDAC increased, and 10 proteins were related to tumorigenesis. The functionally uncharacterised protein, WDR1, was highly expressed in PDAC and predicted a poor prognosis. WDR1 knockdown suppressed PDAC tumour growth and metastasis in vitro and in vivo. Moreover, WDR1 knockdown repressed the activity of the Wnt/β-Catenin pathway; ectopic expression of a stabilised form of β-Catenin restored the suppressive effects of WDR1 knockdown. Mechanistically, WDR1 interacted with USP7 to prevent ubiquitination-mediated degradation of β-Catenin.

Conclusion: Our study identifies several previous functional unknown proteins implicated in the progression of PDAC, and provides new insight into the oncogenic roles of WDR1 in PDAC development.

Fig. 1. The differentially expressed proteins identified by iTRAQ–2DLC–MS/MS.

a Flowchart of the experimental set-up. Fresh PDAC and the corresponding normal tissue samples were harvested from newly diagnosed PDAC patients. Samples were pooled and subsequently identified by mass spectrometry, and data were then normalised for subsequent data analysis. b Gene ontology analysis of differentially expressed proteins with fold change ≥1.2 and P value ≤ 0.05. c Gene ontology analysis of differentially expressed proteins with fold change < 1.2 and P value ≤ 0.05. d Heatmap and Venn diagram showing the 37 upregulated or downregulated proteins across all stages of PDAC tissues. e IPA analysis showed that the 10 differentially expressed proteins were involved in tumorigenesis. Selected upregulated proteins are marked in red and downregulated proteins are marked in green.

Fig. 2. The expression pattern of WDR1 in pancreatic cancer.

a Combined data from TCGA dataset and GTEx portal (http://gemini.cancer-pku.cn/) showed that the mRNA expression level of WDR1 in pancreatic cancer (n = 179) was significantly higher than that in normal pancreas tissues (n = 175). b Western blotting shows the protein expression levels of WDR1 in normal pancreas and pancreatic cancer with different TNM stages. c Quantitative analysis of the expression levels (relative band density) of WDR1 in normal pancreas (N), and pancreatic cancer with different TNM stages (IIA, IIB, III and IV). d Representative images of WDR1 staining in normal pancreas and PDAC tissues with increasing TNM stages. e The frequency distribution of WDR1 expression in low-stage PDAC tissues (n = 40), high-stage PDAC tissues (n = 41) and normal pancreas tissues (n = 44). f Kaplan–Meier analysis of the overall survival of PDAC patients based on the protein expression of WDR1. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. WDR1 knockdown suppresses tumour growth and metastasis of pancreatic cancer.

a Western blotting analysis of WDR1 protein expression in PDAC cells. b The shRNA-mediated WDR1-knockdown efficiency in PANC1 and AsPC1 cells was analysed by Western blotting. c The effect of WDR1 knockdown on in vitro PDAC cell proliferation was determined by plate colony-formation assay. d The effect of WDR1 knockdown on in vivo PDAC tumour growth was studied by PANC1-derived subcutaneous xenograft model. e The tumour volume and weight of sh-Ctrl and sh-WDR1 PANC1-derived subcutaneous xenograft tumour tissues. f Representative immunohistochemical images of Ki67 staining from sh-Ctrl and sh-WDR1 PANC1-derived tumour tissues. g The effect of WDR1 knockdown on in vitro PDAC cell invasion was determined by transwell assay. h The effect of WDR1 knockdown on in vivo PDAC tumour metastasis; the sh-Ctrl and sh-WDR1 PANC1 cells were injected into the tail vein of BALB/c nude mice. After 30 days, lung metastasis was measured by an in vivo imaging system. i Representative immunohistochemical images of MMP9 staining from sh-Ctrl and sh-WDR1 lung metastasis tumour tissues. *P < 0.05, **P < 0.01.

Fig. 4. WDR1 correlates with Wnt/β-Catenin signalling activity in pancreatic cancer.

a Gene set enrichment analysis of RNA-seq data of sh-Ctrl and sh-WDR1 PANC1 cells. b Relative TOP/FOP activities of sh-Ctrl and sh-WDR1 PANC1 and AsPC1 cells. c Real-time qPCR analysis of the effect of WDR1 knockdown on the expression of downstream targets of Wnt/β-Catenin signalling. d Correlation analysis of WDR1 with c-Myc, MMP9 and CCND1 in the TCGA PAAD cohort. e The effect of WDR1 knockdown on in vitro PDAC cell proliferation and invasion upon transfection of β-Catenin S33Y mutant plasmids. *P < 0.05, **P < 0.01.

Fig. 5. WDR1 interacts with USP7 and β-Catenin in pancreatic cancer.

a Co-immunoprecipitation (Co-IP) analysis of the interaction between WDR1 and USP7 in PANC1 and AsPC1 cells. b In situ proximity ligation assay (PLA) analysis of the interaction between WDR1 and USP7 in PANC1 and AsPC1 cells. c Correlation analysis of WDR1 with USP7 in the TCGA PAAD cohort. d Co-IP analysis of the interaction between WDR1 and β-Catenin in PANC1 and AsPC1 cells. e Western blotting analysis of the effects of WDR1 or USP7 on β-Catenin expression in PANC1 and AsPC1 cells. f Relative TOP/FOP activities of sh-Ctrl and sh-USP7 PANC1 and AsPC1 cells. g The effect of USP7 knockdown on in vitro PDAC cell proliferation was determined by plate colony- formation assay. h The effect of USP7 knockdown on in vitro PDAC cell invasion was determined by transwell assay. *P < 0.05, **P < 0.01.

Fig. 6. WDR1 and USP7 regulate β-Catenin ubiquitination in pancreatic cancer.

a Cell lysates from sh-Ctrl and sh-USP7 PANC1 and AsPC1 cells were immunoprecipitated with anti-β-Catenin antibody, and the immunocomplexes were immunoblotted with antibodies against UB and β-Catenin. b Co-IP analysis of the interaction between USP7 and β-Catenin in sh-Ctrl and sh-WDR1–1 PANC1 and AsPC1 cells. c The sh-Ctrl and sh-USP7 PANC1 and AsPC1 cells were treated with 100 μg/ml CHX for the indicated times; then, the cell extracts were harvested, and subjected to immunoblotting with the indicated antibodies. d In the presence or absence of HBX19818, PANC1 and AsPC1 cells were treated with 100 μg/ml CHX for the indicated times; then, the cell extracts were harvested and subjected to immunoblotting with the indicated antibodies. e A schematic diagram shows the mechanism by which WDR1 interacts with USP7 to promote β-Catenin deubiquitination in pancreatic cancer. WDR1 might act as a scaffold to bridges Usp7 and β-Catenin and enhance USP7-mediated β-Catenin deubiquitination.