MUC4 potentiates invasion and metastasis of pancreatic cancer cells through stabilization of fibroblast growth factor receptor 1

Abstract

MUC4 is a type-1 transmembrane mucin differentially expressed in multiple cancers and has previously been shown to potentiate progression and metastasis of pancreatic cancer. In this study, we investigated the molecular mechanisms associated with the MUC4-induced invasion and metastasis in pancreatic cancer. Stable silencing of MUC4 in multiple pancreatic cancer cells resulted in the downregulation of N-cadherin and its interacting partner fibroblast growth factor receptor 1 (FGFR1) through downregulation of partly by pFAK, pMKK7, pJNK and pc-Jun pathway and partly through PI-3K/Akt pathway. The downregulation of FGFR1 in turn led to downregulation of pAkt, pERK1/2, pNF-κB, pIkBα, uPA, MMP-9, vimentin, N-cadherin, Twist, Slug and Zeb1 and upregulation of E-cadherin, Occludin, Cytokeratin-18 and Caspase-9 in MUC4 knockdown BXPC3 and Capan1 cells compared with scramble vector transfected cells. Further, downregulation of FGFR1 was associated with a significant change in morphology and reorganization of the actin-cytoskeleton, leading to a significant decrease in motility (P < 0.00001) and invasion (P < 0.0001) in vitro and decreased tumorigenicity and incidence of metastasis in vivo upon orthotopic implantation in the athymic mice. Taken together, the results of the present study suggest that MUC4 promotes invasion and metastasis by FGFR1 stabilization through the N-cadherin upregulation.

Fig. 1.

Strategy for shRNA-mediated silencing of oncogenic MUC4 in Capan1 and BxPC3 cells. (A) Analysis of MUC4 expression in BxPC3/Capan1-shMUC4, BxPC3/Capan1-Scr by immunoblotting shows significant inhibition in protein expression (70–80%), respectively. β-actin was used as a loading control. (B) Real-time PCR analysis using primers that specifically amplify the MUC4 gene. Capan1 and BxPC3 pooled population cells show a 50–60% decrease in the expression of MUC4 at the mRNA level compared with the control population. No amplification was observed in HPDE, which is negative for the MUC4 expression. (C and D). Confocal analysis showed a decreased expression of MUC4 on the cell membrane in Capan1 and BxPC3-shMUC4 cells compared with scramble cells. (E and F) A morphological comparison between the Capan1/BxPC3-shMUC4 and Capan1/BxPC3-Scr cells. The MUC4 knockdown Capan1 and BxPC3 cells grow in aggregates compared with the scramble cells. (G and H) The phalloidin-rhodamine (phalloidin-RITC) staining of actin–cytoskeleton. MUC4-expressing Capan1 and BxPC3-Scr cells revealed the presence of more microspikes, lamellopodia and filopodia-like cellular projections compared with the MUC4 knockdown Capan1/BxPC3 cells.

Fig. 2.

Role of MUC4 in the EMT process. (A) The immunoblot analysis showed significant upregulation of epithelial markers such as E-cadherin, Occludin and CK-18, and downregulation of mesenchymal markers, such as N-cadherin, Twist, uPA and vimentin, in Capan1/BxPC3-shMUC4 cells compared with Capan1/BxPC3-Scr vector cells. (B) Confocal microscopy showed increased staining for E-cadherin and CK-18 and a faint expression of N-cadherin and vimentin was observed in Capan1/BxPC3-shMUC4 compared with Capan1/BxPC3-Scr vector cells. FITC-conjugated goat anti-mouse IgG for secondary antibody and DAPI was used for nuclear staining. β-actin was used as a loading control in immunoblotting. (C) Real-time PCR analysis using primers that specifically amplify the Slug and Zeb1 genes showed reduced expression Slug and Zeb1 in MUC4 knockdown Capan1 and BxPC3 cells.

Fig. 3.

MUC4-mediated upregulation of N-cadherin through FAK signaling. (A and B) Western blot analysis showed a significant upregulation of pHer2, pScr, pFAK, pMKK7, pJNK, pc-Jun and upregulate N-cadherin in Capan1/BxPC3-Scr vector cells as compared with Capan1/BxPC3-shMUC4. The total form of FAK, MKK7, JNK1/2, c-Jun molecules remains unchanged. β-actin was used as a loading control.

Fig. 4.

Effect of MUC4 knockdown on key signaling molecules in PC cells (A and B) involved in apoptosis (caspase 9), metastasis and invasion (pScr, phospho and total FAK, phospho and total Akt and phospho and total ERK1/2, NFkB, FGFR1, MMP-9) in Capan1/BxPC3-shMUC4 and Capan1/BxPC3-Scr vector cells. The results showed upregulation of Caspase-9 and downregulation of pSrc, pFAK (Tyr576/577, Tyr925), pERK1/2, PAkt, NFkB, FGFR1 in Capan1/BXPC3-shMUC4 compared with scramble vector transfected cells. The total form of FAK, Akt and ERK molecules remains unchanged. β-actin was used as a loading control. (C) Reciprocal co-immunoprecipitation analysis to show the interactions between N-cadherin and FGFR1. Lysates from the MUC4-expressing Capan1/BxPC3 cell lines were utilized for immunoprecipitation with N-cadherin and FGFR1 antibodies. The immunoprecipitates were electrophoretically resolved on 10% polyacrylamide gel and immunoblotted with anti-N-cadherin or anti-FGFR1 antibodies. The isotype antibodies were used as controls. (D) Real-time PCR analysis using primers that specifically amplify the FGFR1 and N-cadherin genes showed reduced expression of FGFR1 and N-cadherin in MUC4 knockdown Capan1and BxPC3 cells lines were utilized for immunoprecipitation with N-cadherin and FGFR1 antibodies.

Fig. 5.

N-cadherin mediated stabilization of FGFR1. (A) Western blot analysis showed that significant upregulation of E-cadherin, downregulation of FGFR1, uPA, pERK1/2, in N-cadherin knockdown Capan1/BxPC3 cells as compared with vector controls (Capan1/BxPC3-scr/ShN-cadherin). The total form of ERK1/2 remains unchanged. β-actin was used as a loading control. (B) Confocal microscopy shows increased staining for E-cadherin in N-cadherin knockdown BxPC3-cells and diminished staining in BxPC3-Scr vector cells (scale bar 20 µm) (FITC-conjugated goat anti-mouse IgG for secondary antibody and DAPI was used for nuclear staining). (C). Capan1 and BxPC3 cells (1×106) were seeded on noncoated or matrigel-coated membranes for motility incubated for 24h. The lower chamber was filled with DMEM medium containing 10% fetal bovine serum used as a chemo attractant. The cells that migrate through trans-well membrane were fixed, stained and photographed for 10 random fields under bright-field microscopy (Magnification X10). The decreased motility and invasion were observed in N-cadherin knockdown Capan1/BxPC3 cells compared to scramble controls (***P < 0.00001 and ***P < 0.00001). (D) Effect of oncogenic MUC4 knockdown on size of orthotopically grown primary tumors. The mean weight of tumors formed by Capan1-shMUC4 cells was significantly less than that formed by the scrambled cells (P < 0.05). (E and F) are the hemotoxalin and eosin staining of primary tumors and (G–I) metastatic lesions from liver, intestine and spleen. (Δ indicates tumor, * indicates normal tissue). (J) Real-time PCR analysis using primers that specifically amplify the N-cadherin gene showed reduced expression N-cadherin in N-cadherin knockdown Capan1and BxPC3 cells.

Fig. 5.

N-cadherin mediated stabilization of FGFR1. (A) Western blot analysis showed that significant upregulation of E-cadherin, downregulation of FGFR1, uPA, pERK1/2, in N-cadherin knockdown Capan1/BxPC3 cells as compared with vector controls (Capan1/BxPC3-scr/ShN-cadherin). The total form of ERK1/2 remains unchanged. β-actin was used as a loading control. (B) Confocal microscopy shows increased staining for E-cadherin in N-cadherin knockdown BxPC3-cells and diminished staining in BxPC3-Scr vector cells (scale bar 20 µm) (FITC-conjugated goat anti-mouse IgG for secondary antibody and DAPI was used for nuclear staining). (C). Capan1 and BxPC3 cells (1×106) were seeded on noncoated or matrigel-coated membranes for motility incubated for 24h. The lower chamber was filled with DMEM medium containing 10% fetal bovine serum used as a chemo attractant. The cells that migrate through trans-well membrane were fixed, stained and photographed for 10 random fields under bright-field microscopy (Magnification X10). The decreased motility and invasion were observed in N-cadherin knockdown Capan1/BxPC3 cells compared to scramble controls (***P < 0.00001 and ***P < 0.00001). (D) Effect of oncogenic MUC4 knockdown on size of orthotopically grown primary tumors. The mean weight of tumors formed by Capan1-shMUC4 cells was significantly less than that formed by the scrambled cells (P < 0.05). (E and F) are the hemotoxalin and eosin staining of primary tumors and (G–I) metastatic lesions from liver, intestine and spleen. (Δ indicates tumor, * indicates normal tissue). (J) Real-time PCR analysis using primers that specifically amplify the N-cadherin gene showed reduced expression N-cadherin in N-cadherin knockdown Capan1and BxPC3 cells.

Fig. 6.

Proposed model for MUC4-mediated signaling events that promote progression and metastasis of pancreatic cancer. MUC4-mediated activation of FAK induces N-cadherin expression via pMKK7, pJNK and pc-Jun activation. N-cadherin in turn interacts with FGFR1 at the plasma membrane and prevents its internalization and degradation promoting continuous activation of downstream signaling molecules Akt, ERK1/2, AP1 and NF-kB. Continuous activation of Akt, ERK1/2, AP1 and NF-kB will promote cell proliferation, survival, migration and metastasis. Moreover, MUC4-mediated upregulation of NF-kB can repress E-cadherin expression. The upregulated AP1 and NF-kB also induces expression of uPA, which promotes cancer cell invasion and metastasis.