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, 8 (10), e3147

Calreticulin Promotes EGF-induced EMT in Pancreatic Cancer Cells via Integrin/EGFR-ERK/MAPK Signaling Pathway

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Calreticulin Promotes EGF-induced EMT in Pancreatic Cancer Cells via Integrin/EGFR-ERK/MAPK Signaling Pathway

Weiwei Sheng et al. Cell Death Dis.

Abstract

Our previous study showed that Calreticulin (CRT) promoted the development of pancreatic cancer (PC) through ERK/MAPK pathway. We next investigate whether CRT promotes EGF-induced epithelial-mesenchymal transition (EMT) in PC via Integrin/EGFR-ERK/MAPK signaling, which has not been reported yet to our knowledge. EGF simultaneously induced EMT and activated Integrin/EGFR-ERK/MAPK signaling pathway in 3 PC cells. However, CRT silencing significantly inhibited EGF function, including inhibiting EGF-induced EMT-like cell morphology, EGF-enhanced cell invasion and migration, and EGF induced the decrease of E-cadherin, ZO-1, and β-catenin and the increase of the key proteins in Integrin/EGFR-ERK/MAPK signaling (pEGFR-tyr1173, Fibronectin, Integrinβ1, c-Myc and pERK). Conversely, CRT overexpression rescued the change of EMT-related proteins induced by EGF in CRT silencing PC cells. Additionally, CRT was co-stained with pEGFR1173 (with EGF), Fibronectin and Integrinβ1 by IF under confocal microscopy and was co-immunoprecipitated with Fibronectin, Integrinβ1 and c-Myc in both PC cells, all of which indicating a close interaction of CRT with Integrin/EGFR-ERK/MAPK signaling pathway in PC. In vivo, CRT silencing inhibited subcutaneous tumor growth and liver metastasis of pancreatic tumor. A positive relationship of CRT with Fibronectin, Integrinβ1, c-Myc and pERK and a negative association of CRT with E-cad was also observed in vivo and clinical samples. Meanwhile, overexpression of the above proteins was closely associated with multiple aggressive clinicopathological characteristics and the poor prognosis of PC patients. CRT promotes EGF-induced EMT in PC cells via Integrin/EGFR-ERK/MAPK signaling pathway, which would be a promising therapy target for PC.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
CRT location by IF and its silencing construction in PC cells. (a) IF staining of CRT (FITC, green) and nuclear (Hoechst, blue) in 4 PC cell lines. (bd) CRT protein level in sg1-CRT, sg2-CRT and scramble-infected Capan-2 (b), AsPC-1 (c) and BxPC-3 (d) cell lines detected by WB. White bars: CRT protein expression in scramble groups. Black bars: CRT protein expression in the sg1-CRT and sg2-CRT groups. **P<0.01 compared with the control
Figure 2
Figure 2
Cell morphology in sg1-CRT, sg2-CRT and scramble-infected Capan-2 and AsPC-1 cells with or without EGF (50 ng/ml) treatment. (a and b) Under EGF treatment, the fibroblastoid-like phenotype in Capan-2 (a) and AsPC-1 (b) cells with scramble groups was much more apparent compared with that in the sg1-CRT and sg2-CRT groups
Figure 3
Figure 3
The change of EMT and Integrin/EGFR-ERK/MAPK signaling-related proteins in sg1-CRT, sg2-CRT and scramble-infected (a) Capan-2 and (b) AsPC-1 cells with or without EGF (50 ng/ml) treatment. White bars: target protein expression in scramble groups with or without EGF treatment. Black bars: target protein expression in the sg1-CRT and sg2-CRT groups with or without EGF treatment. Bars indicate±S.E.*P<0.05; **P<0.01 compared with control
Figure 4
Figure 4
Cell invasion and migration in sg1-CRT, sg2-CRT and scramble-infected PC cells with or without EGF treatment. (a and b) Cell invasion (a) and migration (b) in sg1-CRT, sg2-CRT and scramble-infected Capan-2 cells with or without EGF (50 ng/ml) treatment. (c and d) Cell invasion (c) and migration (d) in sg1-CRT, sg2-CRT and scramble-infected AsPC-1 cells with or without EGF (50 ng/ml) treatment. Black bars: cell invasion or migration in the scramble, sg1-CRT and sg2-CRT groups with EGF treatment. White bars: cell invasion or migration in the scramble, sg1-CRT and sg2-CRT groups without EGF treatment. Bars indicate±S.E.*P<0.05; **P<0.01 compared with control
Figure 5
Figure 5
Transfection efficiency of PC cells transfected with CRT-GFP and corresponding rescue experiment. (a) AsPC-1 cells showed low transfection efficiency with CRT-GFP. (b) Capan-2 cells showed high transfection efficiency with CRT-GFP. (c) The change of EMT-related proteins induced by EGF in the sg1-CRT+GFP and sg2-CRT+GFP groups was rescued by CRT ovrerexpression in the sg1-CRT+CRT-GFP and sg2-CRT+CRT-GFP groups. Bars indicate±S.E.*P<0.05; **P<0.01 compared with control
Figure 6
Figure 6
The effect of CRT silencing to pEGFR expression and the co-staining of CRT and pEGFR1173 in PC cells with or without EGF (50 ng/ml) treatment. (a and b) pEGFR1173, pEGFR1068 and pEGFR845 protein levels in the scramble, sg1-CRT and sg2-CRT groups with or without EGF treatment in Capan-2 (a) and AsPC-1 (b) cells. (c and d) Under confocal microscope, the expression and co-staining of CRT and pEGFR1173 in Capan-2 (c) and AsPC-1 (d) cells by IF with or without EGF (50 ng/ml) treatment. White bars: target protein expression in the scramble group with or without EGF treatment. Black bars: target protein expression in the sg1-CRT and sg2-CRT groups with or without EGF treatment. Bars indicate±S.E.*P<0.05; **P<0.01 compared with control
Figure 7
Figure 7
The co-staining of CRT with Fibronectin, Integrinβ1 and E-cad in PC cells under conforcal microscope. (ac) CRT was co-stained with Fibronectin (a) and Integrinβ1 (b) in predominant cytoplasm of Capan-2 cells, but not with E-cad (c). (df) CRT was co-stained with Fibronectin (d) and Integrinβ1 (e) in predominant cytoplasm of AsPC-1 cells, but not with E-cad (f)
Figure 8
Figure 8
Interaction between CRT, Fibronectin, Integrinβ1 and c-Myc by immunoprecipitation. (a) Capan-2 lysates were immunoprecipitated and WB. (b) AsPC-1 lysates were immunoprecipitated and WB. Input and IgG bands were used as positive and negative control, respectively
Figure 9
Figure 9
Differences of tumor growth, liver metastasis and the expression of CRT, Fibronectin, Integrinβ1, c-Myc, pERK, E-cad and Vimentin in the sg-CRT and scramble groups in vivo. (a) Tumor volumes in nude mice transplanted with sg-CRT and scramble-infected Capan-2 cells. (b) The primary tumors were diagnosed by HE staining. (c) The average number of liver metastases in nude mice implanted with sg-CRT and scramble-infected AsPC-1 cells. (d) Liver metastases were diagnosed by HE staining. (e) CRT, Fibronectin, Integrinβ1, c-Myc, pERK, E-cad and Vimentin expression in subcutaneous pancreatic tumors in the sg-CRT and scramble groups by IHC. Bars indicate±S.E. *P<0.05 compared with control
Figure 10
Figure 10
Overexpression of CRT, Fibronectin, Integrinβ1, c-Myc, pERK, E-cad and Vimentin in PC samples. (a) High expression of CRT, positive expression of Fibronectin, Integrinβ1, c-Myc, pERK and Vimentin and normal E-cad expression in PC tissues. (b) The expression of CRT, Fibronectin, Integrinβ1, c-Myc, pERK, E-cad and Vimentin in one same sample tissue, respectively. (c) The expression of CRT, Fibronectin, Integrinβ1, c-Myc, pERK, E-cad and Vimentin in another one same sample tissue, respectively
Figure 11
Figure 11
The relationship of CRT, Fibronectin and Integrinβ1 with the survival of 68 postoperative PC patients in Kaplan–Meier analysis. (a) High and low expression of CRT was plotted against overall survival time. (b) Positive (+) and negative (−) expression of Fibronectin was plotted against overall survival time. (c) Positive (+) and negative (−) expression of Integrinβ1 was plotted against overall survival time. (d) Co-expression of CRT and Fibronectin was plotted against overall survival time. (e) Co-expression of CRT and Integrinβ1 was plotted against overall survival time

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