In the absence of Muc1, pancreatic tumor burden and secondary metastasis are decreased
Mice were sacrificed at 6, 16, 26, and 40 weeks of age. The pancreas weight was used as the indicator of tumor weight. At 6wks of age, there was no statistical difference between KCKO and either KC or KCM. However, by 16wks of age and thereafter, KC and KCM mice had significantly higher tumor burden than KCKO mice (). It must be noted that the KCM mice had significantly higher tumor burden than KC mice confirming our previous results that over expression of MUC1 augments pancreatic tumor progression [3
]. Most importantly, pancreas weight did not increase from 6 to 40wks of age in mice lacking Muc1 suggestive of a stable disease (). Further, KCKO mice showed a significant survival benefit compared to the KC and KCM mice ().
KCKO mice have lower tumor burden, metastasis, and levels of VEGF and PGE2 with higher survival compared to KC and KCM mice
We have previously shown that MUC1-expressing PDA have higher levels of VEGF and PGE-M [3
], leading to higher angiogenesis and metastasis [15
]. Therefore, we evaluated the circulating levels by specific ELISA. Both VEGF and PGE-M levels were significantly lower in the KCKO mice compared to KC and KCM mice and most notably the levels did not increase with age in the KCKO mice as noted in KC and KCM mice (). PGE2
is an end-product of the cyclooxygenase-2 (COX-2) pathway and is known to induce tumor cell proliferation and increase motility [16
H&E stained pancreas sections were examined from 6, 24, and 40-week old KC, KCM and KCKO mice. Clearly, abnormal duct with low grade PanIN lesions were visualized in the KCM pancreas as early as 6-weeks of age (). At this time-point, the pancreas from KC and KCKO mice looked relatively normal. By 24 and 40-weeks of age, both KC and KCM pancreas showed PanIN lesions of varying grades with KCM pancreas showing signs of higher grade PanIN lesions and adenocarcinoma (). This data confirms our previously published analysis of the PanIN lesions in KC and KCM pancreas as a function of age [3
]. Most notably, pancreas from KCKO mice did not show high-grade PanIN lesions even at 24 and 40-weeks of age (). The data from these spontaneous models clearly point toward the critical role of MUC1 in the progression of pancreatic cancer. Further, pancreas sections from 26-week old KCM mice showed increased expression of VEGF, MMP9, EGFR, and PDGF as compared to age-matched KC and KCKO pancreas (Supplemental Figure 1
). Data demonstrates the highly aggressive nature of MUC1-expressing tumors which is substantiated by increased Proliferating Cell Nuclear Antigen (PCNA) staining in KCM and KC pancreas as compared to the KCKO pancreas (data not shown).
At 36–40wks of age, mice were euthanized and lungs, liver and peritoneum were evaluated for macroscopic gross lesions. Interestingly, 61% of KCM mice developed lung metastasis, 33% developed liver metastasis and 23% developed peritoneal metastasis (n=13). This is in stark contrast to the KCKO mice which had merely 10% of mice develop metastasis in any of the three organs (n=10). Thirty percent of the KC mice (n=13) had developed lung metastasis, 20% had developed liver metastasis, and 10% had developed peritoneal metastasis. As an example, a representative H&E image of a lung showing clear metastatic lesion is provided in .
Muc1-null tumor cells have significantly lower tumorigenic capacity compared to their MUC1-expressing counterpart
To further decipher the underlying mechanism of enhanced proliferation and progression in MUC1-expresssing tumors, we generated several cell lines from the KCM and KCKO tumors and first studied their tumor forming ability in vivo in both young and old mice. In the 8–10 week old mice (n = 4), both cell lines formed palpable tumors by 6d post-injection. By 12d post tumor challenge, KCM tumors grew faster (p<0.001), and continued the same trend until sacrifice at 21d post injection (). By 21d, the tumor burden in mice injected with KCM (n=5), as determined by caliper measurement had grown to an average of 1017mg whereas; those mice injected with KCKO had a tumor burden of merely 461mg (n=6). During necropsy, tumors were excised and weighed. KCM tumors weighed on an average of 700mg whereas KCKO remained at 500mg ().
Significantly decreased tumor burden and increased survival in syngeneic C57/BL6 mice challenged with KCKO versus KCM cells
Because the median age of pancreatic cancer patients is >65 years of age, we assessed whether this observation would hold true in aged mice. In nine month old mice (, n=5 mice per group), tumor burden was again significantly higher with KCM versus KCKO cells starting at 12d post-injection (p<0.05) and continued until 21d reaching a tumor weight of 1300mg for KCM versus 300mg for KCKO (). It must be noted that in the aged mice, the KCM cells grew more aggressively than in the younger mice and reached a much higher tumor burden at 21d (compare ), but that KCKO growth remained consistent.
Survival is significantly higher in mice injected with pancreatic cancer cells lacking MUC1
In order to assess survival, mice were injected with KCM and KCKO cells and tumors were allowed to grow until reaching 10% of the body weight or until ulcerations developed, whichever came first (). Survival was significantly increased in mice injected with KCKO compared to KCM cells (p<0.001). By 25d post tumor challenge, none of the mice injected with KCM cells survived (n=7), while 100% of mice injected with KCKO cells (n=6) survived at that age (). Mice injected with KCKO survived until ~40d post tumor injection. Tumor weight, derived from caliper measurements, shows a steady growth rate of tumors injected with KCM, while the KCKO tumor growth remains stunted and does not exceed ~500mgs (). These data recapitulate the data from the spontaneous model of PDA in .
Cell division and cell cycle progression is significantly altered in pancreatic cancer cells lacking Muc1/MUC1
To further analyze the effects of MUC1 on the in vitro kinetics of cellular division, KCKO and KCM were subjected to the CFSE dilution assay, which fluorescently labels cells and is depleted as they divide. Initial staining of KCM cells with CFSE resulted in a MFI of 2499. After 48hrs, CFSE had already been diluted to a MFI of 96. Initial staining of KCKO cells resulted in a MFI of 1500. After 48hrs, CFSE had been diluted to a MFI of 73 (). Although the KCM cells initially stained with greater intensity than did the KCKO cells, the CFSE was diluted much faster as can be seen by the slope of the line displaying MFI dilution over time ().
Significantly altered rate of cell division and progression through the cell cycle in KCKO versus KCM cells
Since we observed that MUC1 affects cell division, we next investigated how the cell cycle was affected by MUC1 expression. Cells were stained with Propidium iodide (PI) and the DNA content was determined by flow cytometry. KCM cells progress through the cell cycle at a steady rate. At 12hrs post plating, 26.9% of KCM cells were in the G0/G1, 34.3% in S, and 32.5% in G2/M phase of the cell cycle (). In contrast, KCKO cells that lack Muc1 had a significantly different distribution at both 12hr and 24hr time points (p<0.001, ). At 12hrs post plating, KCKO cells had 31.1% of cells in G0/G1, 52.9% in S, and 13.3% of cells in G2/M phase. This distribution remained relatively similar, in both cell types by 24hrs post plating (). KCKO cells clearly enter and accumulate in the S-phase where the DNA doubling occurs more rapidly than KCM cells but thereafter, KCKO cells do not progress to the G2 and mitotic phase as efficiently as KCM cells.
KCKO cells fail to proliferate or invade in response to EGF, PDGF, and MMP9
To assess if KCKO cells would respond to growth factors known to induce cell division, proliferation and invasion of cancer cells, KCKO and KCM cells were subjected to an in vitro proliferation assay, as determined by [3H]-thymidine uptake. First, KCM cells displayed a significantly higher rate of proliferation compared to KCKO cells. Stimulation with EGF, PDGF, or MMP9, did not induce proliferation in KCKO cells (). With regards to invasion, the basal level invasion index of the KCKO cells was found to be significantly lower than KCM cells (, p<0.001). More importantly, KCKO cells did not respond to any of the exogenous factors to increase its invasion index (). It should be noted that neither did the KCM cells, however that may be because of the high basal invasion index. Taken together the data suggests a failure of the KCKO cells to respond to exogenous EGF, PDGF or metalloproteinase.
Significantly lower proliferation and invasion index in KCKO versus KCM cells
Complete loss of cdc-25c expression and decreased phosphorylation of MAPK in KCKO cells may account for lower mitosis, proliferation, and invasion
Once it was confirmed that cell cycle progression and proliferation was altered in cells lacking MUC1, we began to investigate what specific proteins and markers were altered to cause such drastic differences. KCKO and KCM cells were subjected to both western blot analysis and proteomics. We probed for those proteins typically involved in cell cycle regulation pathways. Most notable was the complete loss of the tumor suppressor proteins, p53 and downstream p21, in the KCM cells but not in the KCKO cells. Associated with this was the complete loss of the M phase inducer phosphatase, cdc-25c, in the KCKO cells (). Furthermore, there was a significant down-regulation of levels of phosphorylated MAPK p44/42 in the KCKO cells compared the KCM cells (). Cdc-25c is a tyrosine phosphatase that directs dephosphorylation of cyclin B-bound CDC2 and triggers entry into mitosis. Thus, it becomes plausible to speculate that KCKO cells do not enter the mitotic phase efficiently because of the absence of Cdc-25c expression. Cdc-25c is also known to suppress p53-induced growth arrest which possibly explains why cells lacking Cdc25-c and lacking Muc1 do not lose p53 and p21 expression, do not phosphorylate MAPK and therefore do not divide, proliferate and invade effectively.
Differential protein expression profile in MUC1-expressing vs Muc1-null cells
MUC1 increases expression of Tubulin α-2 chain and Nestin protein
These alterations in cell cycle regulation were coupled with differential transcription of genes associated with proliferation and metastasis. Proteomics analyses of a total of 2874 cancer progression-associated proteins showed down regulation of 757 proteins in KCKO versus KCM cells. Genes with a two-fold decrease and below were considered to be significant and are shown in . It is extremely relevant that the most pronounced down regulation was seen in Tubulin α-2 chain and Nestin in KCKO cells, and therefore these proteins were highly up regulated in KCM cells (). Tubulin α-2 is a major constituent of microtubules and is required for mitotic spindle organization, mitosis, growth and cell migration. Similarly, Nestin is a marker of proliferating and migrating cells and highly expressed in mitotically active cells.
Treatment with MEK1/2 inhibitor, U0126, completely abrogates the enhanced proliferation in KCM cells
Since MEK1 phosphorylation is a critical signaling event for proliferation of KCM cells, we treated KCKO and KCM cells with U0126. As a positive control, cells were treated with 20% FBS. As was expected, the basal level of activated MEK1 was higher in KCM cells than in KCKO cells (). Similar to the cell lines, lysates from primary tumors of 26wk old KCKO mice showed reduced phosphorylation of MAPK versus tumors from the KCM mice (). This confirmed that activated MEK is a function of MUC1 expression and is critical in the progression of pancreatic cancer. When cell lines were treated with U0126, activation of MEK1/2 was completely abolished (). To assess if MEK1/2 is responsible for MUC1-enhanced cellular division and proliferation, KCM and KCKO cells treated with U0126 were subjected to CFSE dilution and [3H]-Thymidine uptake assays. CFSE dilution assay results are displayed as change in mean fluorescence intensity (MFI) at 6, 12, and 24hrs (). Within 6hrs, KCM cells have already undergone rapid cell division as compared to KCKO cells (, p<0.001) and treatment with the inhibitor did not significantly reduce cell division in either cell lines. However, at 12hrs and 24hrs post treatment, cell division was significantly lower in KCM cells with treatment (p<0.001 as compared to basal cell division) but there was no significant change in the KCKO cells with U0126 treatment (). Both sets of cells supplemented with 20% FBS have significantly increased cell division albeit KCM always showed significantly rapid cell division compared to KCKO cells (p<0.001, ). It is noteworthy that addition of the MEK1/2 inhibitor reduced cell division of the KCM cells to the level of KCKO cells at all time points.
Treatment with MEK1/2 inhibitor, U0126, abrogates proliferation of KCM but not KCKO cells
Similar results were obtained using the [3H]-Thymidine uptake assay. At 6 and 12hrs post treatment with U0126, proliferation of KCM cells was significantly decreased from its basal level proliferation (, p <0.001) and reached the level of KCKO cells. At 24hrs after treatment, there was no statistical significance between KCKO treated with U0126 and KCM treated with U0126. KCKO on the other hand did not respond to the inhibitor such that the basal proliferation and inhibitor treated proliferation remained similar suggesting that these cells do not require MEK1 activation. As expected, KCM cells were significantly more proliferative than KCKO cells (p<0.001) at all time points.