PLEK2 promotes gallbladder cancer invasion and metastasis through EGFR/CCL2 pathway

Abstract

Background: Gallbladder cancer (GBC) is an extremely malignant tumor with a high mortality rate. Little is known about its invasion and metastasis mechanism so far.

Methods: To identify the driver genes in GBC metastasis, we performed a mRNA microarray of metastatic GBC and paired non-tumor samples, and found PLEK2 was markedly upregulated in GBC tissues. Next, the expression of PLEK2 in GBC were examined in a larger cohort of patients by qRT-PCR, western blot and IHC staining. The clinicopathologic correlation of PLEK2 was determined by statistical analyses. The biological involvement of PLEK2 in GBC metastasis and the underlying mechanisms were investigated.

Results: In this study, we found that PLEK2 had higher expression in GBC tumor tissues compared to non-cancerous adjacent tissues and cholecystolithiasis tissues. The clinicopathologic analyses showed PLEK2 expression was positively correlated with tumor TNM stage, distant metastasis and PLEK2 was an independent predictor of overall survival (OS) in GBC patients. The cellular function assays showed PLEK2 promoted GBC cells migration, invasion and liver metastasis in mouse model via the regulation of epithelial-mesenchymal transition (EMT) process. Our mass spectrum and co-immunoprecipitation (co-IP) assays demonstrated that PLEK2 could interact with the kinase domain of EGFR and suppress EGFR ubiquitination mediated by c-CBL, leading to constitutive activation of EGFR signaling. Furthermore, RNA-sequencing and qRT-PCR results demonstrated chemokine (C-C motif) ligand 2 (CCL2), a target gene downstream of PLEK2/EGFR signaling, mediated the motility-promoting function of PLEK2.

Conclusions: On the basis of these collective data, we propose that PLEK2 promotes the invasion and metastasis of GBC by EGFR/CCL2 pathway and PLEK2 can serve as a potential therapeutic target for GBC treatment.

Keywords: CCL2; EGFR; Gallbladder Cancer; Metastasis; PLEK2.

Fig. 1

PLEK2 was up-regulated in gallbladder cancer and correlated with overall survival. a Heatmap results of a mRNA microarray consisted of six pairs of metastatic GBC and non-tumor samples. b, c The relative expression levels of PLEK2 in GBC tumor tissues (Tumor or T) and matched normal tissues (Non-Tumor or N) were detected by qRT-PCR (b) and western blot (c). d Representative IHC staining images of PLEK2 in cholecystolithiasis and GBC tissues, and quantification of PLEK2 expression according to IHC scores. e Representative IHC staining images of different scores calculated by intensity and percentage of stained cells and overall survival for the low and high PLEK2 expression groups. f Multivariate analysis of the factors associated with overall survival of GBC patients. All experiments were repeated at least three times, and data were analyzed using student’s t-test. *P < 0.05. Error bars indicate SEM

Fig. 2

PLEK2 promoted the migration, invasion and metastasis of GBC cells. a, c Migration and invasion ability of NOZ and GBC-SD cells with stable PLEK2 knockdown (a) and PLEK2 overexpression (c) were measured by transwell migration and matrigel invasion assays. b, d Represent images of mouse livers of NOZ cells with stable PLEK2 knockdown (b) and PLEK2 overexpression (d) are shown and the numbers of metastatic nodes per liver were measured. e Morphological change of NOZ cells upon PLEK2 knockdown or overexpression. f Expression of epithelial marker E-cadherin, mesenchymal markers Fibronectin, Vimentin and N-cadherin were detected by western blot. All experiments were repeated at least three times, and data were analyzed using student’s t-test. * P < 0.05. Error bars indicate SEM

Fig. 3

PLEK2 interacted with EGFR. a Total cell lysates extract from FLAG-PLEK2 stably expressed cells were subjected to affinity purification and mass spectrometry analysis of PLEK2-associated proteins were performed. b Interaction between exogenous PLEK2 and EGFR by Co-IP analyses in 293 T cells. c Interaction between endogenous PLEK2 and EGFR by Co-IP analyses in NOZ cells. d, e Mapping of the binding site of PLEK2/EGFR by Co-IP analyses. f Immunofluorescence assays in GBC-SD cells. The localization of PLEK2 was detected by confocal laser scanning microscopy as indicated. g Co-IP of overexpressed Flag-PLEK2 and EGFR in 293 T cells treated with 50 ng/mL EGF at indicated times

Fig. 4

PLEK2 suppressed EGFR degradation. a Protein levels of EGFR in PLEK2 knockdown and overexpression cells were detected by western blot. b, c Alterations of EGFR degradation in PLEK2 knockdown cells in response to 100 nM CHX treatment for the indicated time were detected by western blot. d EGFR expression in PLEK2 knockdown cells in response to 10 μM MG132 treatment for 6 h were detected by western blot. e The ubiquitination level of EGFR with increasing ectopic PLEK2 expression were detected by co-IP. f Co-IP of overexpressed PLEK2, c-CBL and EGFR in 293 T cells. g Co-IP of Flag-c-CBL and EGFR, together with increasing amount of Myc-PLEK2 in 293 T cells. All experiments were repeated at least three times, and data were analyzed using student’s t-test. * P < 0.05. Error bars indicate SEM

Fig. 5

Oncogenic effects of PLEK2 depended on EGFR pathways. a, b Protein levels of EGFR downstream signaling in indicated GBC cell lines with either PLEK2 knockdown or overexpression were detected by western blot. c Migration and invasion ability of PLEK2 knockdown NOZ cells with increasing ectopic EGFR expression were measured. d Protein levels of EGFR downstream signaling in PLEK2 knockdown cells with increasing ectopic EGFR expression were detected by western blot. e Migration and invasion ability of PLEK2 overexpression NOZ cells with EGFR inhibitor erlotinib treatment were measured. f Protein levels of EGFR downstream signaling in PLEK2 overexpression cells with 1 mmol/L erlotinib treatment for 12 h were detected. All experiments were repeated at least three times, and data were analyzed using student’s t-test. * P < 0.05. Error bars indicate SEM

Fig. 6

CCL2 was a target gene downstream of PLEK2/EGFR signaling. a Differential gene pathway analyses of PLEK2 knockdown cells by mRNA sequencing. b, c The relative mRNA levels of cytoskeleton organization genes were detected by qRT-PCR. d Protein levels of CCL2 in indicated cell lines with either PLEK2 knockdown or overexpression were detected by western blot. e Protein levels of CCL2 in PLEK2 overexpression cells with EGFR knockdown were detected by western blot. f The protein levels of secreted CCL2 in cellular supernatant were detected by ELISA analysis. g Migration and invasion ability of PLEK2 knockdown NOZ cells with 0.1μg/mL CCL2 treatment were measured by transwell. h Protein levels of EMT markers in PLEK2 knockdown cells with 0.1μg/mL CCL2 treatment for 24 h were detected by western blot. i The relative mRNA levels of EMT related transcription factors in PLEK2 knockdown cells with 0.1μg/mL CCL2 treatment for 24 h were detected by qRT-PCR. All experiments were repeated at least three times, and data were analyzed using student’s t-test. * P < 0.05. Error bars indicate SEM

Fig. 7

The prognostic value of combination of PLEK2 and EGFR. a Representative immunostaining images of PLEK2, EGFR and CCL2 in GBC tissues. b Pearson’s correlation analysis between PLEK2 and EGFR/CCL2 in GBC tumour samples. c The overall survival for the low and high EGFR expression groups. d The overall survival of the combination cohort was stratified by PLEK2 and EGFR expression levels