Pancreatic ductal adenocarcinoma mice lacking mucin 1 have a profound defect in tumor growth and metastasis

Abstract

MUC1 is overexpressed and aberrantly glycosylated in more than 60% of pancreatic ductal adenocarcinomas. The functional role of MUC1 in pancreatic cancer has yet to be fully elucidated due to a dearth of appropriate models. In this study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1-null (KCKO) or express human MUC1 (KCM). We show that KCKO mice have significantly slower tumor progression and rates of secondary metastasis, compared with both KC and KCM. Cell lines derived from KCKO tumors have significantly less tumorigenic capacity compared with cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared with mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor, platelet-derived growth factor, or matrix metalloproteinase 9. Further, significantly less KCKO cells entered the G(2)-M phase of the cell cycle compared with the KCM cells. Proteomics and Western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of mitogen-activated protein kinase (MAPK), as well as a significant decrease in nestin and tubulin-α2 chain expression in KCKO cells. Treatment with a MEK1/2 inhibitor, U0126, abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, suggesting that MUC1 is necessary for MAPK activity and oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro.

Figure 1. KCKO mice have lower tumor burden, metastasis, and levels of VEGF and PGE₂ with higher survival compared to KC and KCM mice

A. Pancreas weight and survival of KCKO, KC, and KCM mice. (pancreas weight: **p<0.01;***p<0.001 compared to KCKO; survival: *p<0.01 compared to KCM and KC). B. Circulating levels of PGE₂M and VEGF in C57Bl/6, KCKO, KC, and KCM mice (**p<0.01;***p<0.001 compared to KCKO). C. Representative IHC images of pancreas from KCKO, KC, and KCM mice as a function of age (200X). Representative H&E of lung metastasis (100 and 200X)

Figure 2. Significantly decreased tumor burden and increased survival in syngeneic C57/BL6 mice challenged with KCKO versus KCM cells

A. Tumor growth curve and tumor wet weight in 8–10wk and nine month old mice. Significantly higher tumor weight in mice injected with KCM versus KCKO cells (*p<0.05, ***p<0.001). B. Tumor growth curve and tumor wet weight in 9-month old mice. Significantly higher tumor weight in mice injected with KCM versus KCKO cells (**p<0.01, ***p<0.001). C. Survival curve of 8–10wk old mice challenged with KCKO and KCM cells. Significantly higher survival in mice injected with KCKO versus KCM tumors (***p<0.001). D. Tumor growth curve of KCM versus KCKO cells of mice in the survival study (***p<0.001).

Figure 3. Significantly altered rate of cell division and progression through the cell cycle in KCKO versus KCM cells

A. Histogram representing CFSE staining in KCM and KCKO cells by flow cytometry. At time zero, KCM cells had MFI of 2499, by 48hrs, CFSE had been diluted to an MFI of 96. Whereas, in KCKO cells, the initial MFI was 1500, and by 48hrs, CFSE had been diluted to MFI of 73. B. Graph representing average MFI of CFSE staining in KCM and KCKO cells as a function of time in 3 experiments. C. Representative histograms of cell cycle analysis of KCM and KCKO cells by flow cytometry at 12 and 24hrs. D. Percentage of KCM and KCKO cells in the G0/G1, S, and G2M phase of the cell cycle. Average of 3 experiments is shown.

Figure 4. Significantly lower proliferation and invasion index in KCKO versus KCM cells

A – C. Proliferation as measured by [³H]-thymidine uptake in response to A. EGF, B. PDGF, and C. MMP9. Significantly higher proliferation was observed in KCM versus KCKO cells (*p<0.05,***p<0.001). D. Percentage of KCM and KCKO cells that invaded in response to PDGF and MMP9 (***p<0.001).

Figure 5. Differential protein expression profile in MUC1-expressing vs Muc1-null cells

A. Western blotting analysis of KCM and KCKO cell lysates probed various proteins. B. Proteomics data from KCKO and KCM cell lysates (average of n=2 with each experiment conducted in triplicate). Data displayed as fold increase in KCM compared to KCKO cells. Only proteins that were changed >2-fold are shown.

Figure 6. Treatment with MEK1/2 inhibitor, U0126, abrogates proliferation of KCM but not KCKO cells

A. Western blotting analysis showing phosphorylation of MAPK was completely abrogated with the addition of U0126, the MEK 1/2 inhibitor B and C. Proliferation of KCM and KCKO cells 6, 12, and 24hrs post treatment with U0126 using the B. CFSE dilution assay and C. [³H]-thymidine uptake assay. Experiments were repeated three times in triplicate.