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MUC1 is over expressed and aberrantly glycosolated in >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 the present 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 to both KC and KCM. Cell lines derived from KCKO tumors have significantly lower tumorigenic capacity compared to cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared to mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or matrix metalloproteinase-9 (MMP9). Further, significantly fewer KCKO cells entered the G2M phase of the cell cycle compared to the KCM cells. Proteomics and western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of MAPK, as well as a significant decrease in Nestin and Tubulin α-2 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 in order to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro.
Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related deaths in the United States . It is one of the most deadly cancers due to its aggressive nature and relatively few treatment options. With a 5-year survival rate of only 5%, it has the poorest prognosis among all cancers. To date, the only potential curative is surgical resection, of which only 20% of patients are eligible. Alternative therapies, such as radiotherapy and chemotherapy remain largely ineffective. The development and evaluation of novel targeted therapeutic agents for improving the outcome of patients are of paramount importance.
Importantly, MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surfaces of normal glandular epithelia but is over expressed and aberrantly glycosylated in >60% of human PDA and in 100% of metastatic lesions [2–3]. In human cancers, MUC1 is commonly detected in high grade but not in low-grade pancreatic intraepithelial neoplasia (PanIN) [4–5]. Thus MUC1 may play an important role in the development and progression of PDA [6–8]. Over expression of MUC1 in pancreatic cancer has been known for quite some time; however its function has not been clearly elucidated mainly due to the lack of an appropriate pancreatic cancer model.
Activating mutations in the KRAS proto-oncogene are found in over 90% of invasive pancreatic ductal adenocarcinoma and are thought to represent an initiating event. Recently a transgenic mouse model has been created that expresses physiological levels of oncogenic KRAS with a glycine to aspartate substitution at codon 12, in the progenitor cells of mouse pancreas . These mice, designated as KC or Cre-LSL-KRASG12D, develop the full spectrum of pancreatic ductal adenocarcinoma. We have further crossed the KC mice to the human MUC1 transgenic (MUC1.Tg) mice which express MUC1 in a pattern and level consistent with that in humans (designated KCM) . KC mice were also crossed with the Muc1KO mice (designated KCKO), creating a Muc1-null PDA model. These mouse models provide us with a unique opportunity to fully evaluate the role of MUC1 in pancreatic cancer development.
This study is the first to utilize a model of pancreatic cancer that is Muc1-null in order to fully elucidate the oncogenic role of MUC1. In this study, we show that lack of Muc1 significantly decreased proliferation, invasion, and mitotic rates both in vivo and in vitro. Importantly, treatment with MEK1/2 inhibitor, U0126, completely abrogated the enhanced proliferation of the KCM cells. These data, therefore, may have implications in the future design of MUC1-targeted therapies for pancreatic cancer.
KC mice were generated in our laboratory on the C57BL/6 background by mating the P48-Cre with the LSL-KRASG12D mice [10–11]. They were further mated to the MUC1.Tg mice to generate KCM mice [3, 12] or to the Muc1KO mice  to generate KCKO mice. Tumors were excised at predetermined time points and weighed. Gross metastasis was evaluated in the lung, liver and peritoneum. Tumors were dissociated using collagenase IV (Worthington Biochemical) and cell lines generated in our laboratory. Cell lines are designated KCKO for those cells lacking Muc1 and KCM for those cells expressing MUC1. Since we have been unable to generate the KC cell lines, we have compared KCM with KCKO. The cells were maintained in complete DMEM (Invitrogen) supplemented with 10% FBS (HyClone), 1% glutamax (Invitrogen), and 1% penicillin/streptomycin.
PGE2 levels in the tumor lysate were determined using a specific ELISA kit for PGE2 metabolite (PGE-M) (Cayman Pharmaceuticals). VEGF levels were also determined by specific ELISA (RayBiotech).
KCM and KCKO cells were serum-starved for 24hrs and treated for 30 mins with rm-PDGF-CC (Peprotech), rm-EGF (Peprotech), and rm-MMP9 (R&D systems) at a concentration of 50ng/mL, or MEK1/2 inhibitor at 10µM concentration. Cell proliferation was determined by using [3H]-thymidine incorporation, in which 1µCi of [3H]-thymidine was added per well for 24hrs prior to harvesting. Incorporated thymidine was evaluated using the Topcount micro-scintillation counter. All determinations were performed in triplicate.
Cells were stained using the CellTrace CFSE Kit (Molecular Probes). CFSE was added to the cells at a final concentration of 2µM, incubated for 15min at 37°C, and 0.5×106 removed for initial positive staining. The remaining cells were plated in triplicate. Cells were harvested at predetermined time-points and CFSE dilution was determined by flow cytometry (Beckman Coulter). Analysis was conducted using FlowJo (Treestar, Ashland, OR).
Cells were harvested, fixed by resuspending in 10ml of 70% ethanol for 30min, and washed in ice-cold PBS. The pellets were resuspended in 0.5ml PBS, and 1mL of DNA extraction buffer was added. Cells were incubated, washed and resuspended in DNA staining solution containing 20µg/ml propidium iodide (Sigma-Aldrich) and 100µg/ml RNase (Invitrogen). DNA content was determined by flow cytometry and analyzed using FlowJo.
Cells (serum starved for 24hrs) were treated with rm-PDGF, rm-EGF, or rm-MMP-9 for 30min, trypsinized, washed, and resuspended in SFM. Cells were plated over transwell inserts (BD Biosciences) pre-coated with growth-factor reduced Matrigel™ (BD Biosciences), and permitted to invade towards serum contained in the bottom chamber for 48hrs. Percent invasion was determined as described in .
Western blotting was carried out as previously published . MUC1 CT antibody CT2, was made in Mayo Clinic Immunology Core . MUC1 TR antibody was provided by Dr. Joyce Taylor-Papadimitriou. ERK1/2antibodies: phospho-p44/42 and p44/42 were purchased from Cell Signaling Technologies. All other antibodies (p53, CDKN1A, c-myc, TGFβ-R1, MEK1, Cyclin B1, Wee 1, Cdc2-p34, Cdc-25c, and β-actin) were purchased from Santa Cruz Biotechnologies and used according to manufacturer's recommendations. Proteomics analysis was determined as previously described .
Ten week and nine month old mice were injected with 1×106 KCM or KCKO cells (in 50ul of PBS combined with 50uL of growth factor-reduced Matrigel™) into the flank of the mice (n=8). Mice were palpated starting at 6 days post tumor injection. Tumor weight was calculated according to the formula: grams=(length in centimeters × (width)2)/2 . Upon sacrifice, the tumors were weighed, prepared for lysates, and fixed for immunohistochemistry.
Tissues were fixed in 10% neutral-buffered formalin. Paraffin-embedded blocks were prepared by the Histology Core at The Mayo Clinic and 4-micron thick sections were cut for staining. Slides were H&E stained, and images taken at 100X and 200X-magnification.
Data were analyzed using GraphPad software. Results are expressed as mean±SEM and are representative of greater than or equal to three separate experiments. Comparison of groups was performed using one-way or two-way ANOVA followed by the Bonferroni post-test for multiple comparisons (*p<0.05, **p<0.01, ***p<0.001).
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 (Figure 1A). 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 . Most importantly, pancreas weight did not increase from 6 to 40wks of age in mice lacking Muc1 suggestive of a stable disease (Figure 1A). Further, KCKO mice showed a significant survival benefit compared to the KC and KCM mice (Figure 1A).
We have previously shown that MUC1-expressing PDA have higher levels of VEGF and PGE-M , leading to higher angiogenesis and metastasis . 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 (Figure 1B). PGE2 is an end-product of the cyclooxygenase-2 (COX-2) pathway and is known to induce tumor cell proliferation and increase motility [16–17].
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 (Figure 1C). 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 (Figure 1C). This data confirms our previously published analysis of the PanIN lesions in KC and KCM pancreas as a function of age . Most notably, pancreas from KCKO mice did not show high-grade PanIN lesions even at 24 and 40-weeks of age (Figure 1C). 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 Figure 1C.
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 (Figure 2A). 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 (Figure 2A).
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 (Figure 2B, 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 (Figure 2B). 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 Figure 2A and B), but that KCKO growth remained consistent.
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 (Figure 2C). 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 (Figure 2C). 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 (Figure 2D). These data recapitulate the data from the spontaneous model of PDA in Figure 1.
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 (Figure 3A). 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 (Figure 3B).
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 (Figure 3C). In contrast, KCKO cells that lack Muc1 had a significantly different distribution at both 12hr and 24hr time points (p<0.001, Figure 3D). 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 (Figure 3C). 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.
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 (Figure 4 A–C). With regards to invasion, the basal level invasion index of the KCKO cells was found to be significantly lower than KCM cells (Figure 4D, p<0.001). More importantly, KCKO cells did not respond to any of the exogenous factors to increase its invasion index (Figure 4D). 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.
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 (Figure 5A). Furthermore, there was a significant down-regulation of levels of phosphorylated MAPK p44/42 in the KCKO cells compared the KCM cells (Figure 5A). 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.
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 Figure 5B and C. 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 (Figure 5B). 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.
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 (Figure 6A). 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 (Figure 6A). 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 (Figure 6A). 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 (Figure 6B). Within 6hrs, KCM cells have already undergone rapid cell division as compared to KCKO cells (Figure 6B, 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 (Figure 6B). 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, Figure 6B). 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.
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 (Figure 6C, 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.
MUC1 is aberrantly over expressed in pancreatic cancer . Over expression of MUC1 is detectable during the early stages of pancreatic cancer development and is further increased in invasive carcinoma in humans and mice [3–4]. However, it is not known if pancreatic cancer cells are dependent on MUC1 for their growth and survival. Using appropriate mouse models, we show unequivocally for the first time that pancreatic cancer cells depend on MUC1 to grow and survive, by directly suppressing p53 and its major transcriptional target p21Cip (Figure 5) while activating MAPK, cdc-25-c, tubulin-α-2, and Nestin (Figure 5). This in turn stimulates proliferation and mitosis (Figure 3 and and4).4). Further, for survival, MUC1 expression causes the up-regulation of several multidrug resistance proteins and pro-survival factors that protect them from undergoing apoptosis (Figure 5). This is in stark contrast to what occurs in Muc1-null cells, in which the oncogenic signaling is shut down even though the KRas oncogene remains active. Muc1 deficiency leads to the failure to form tumors in vivo (Figure 2) and decreased proliferation and invasion (Figures 1–4).
The first evidence that MUC1 is required for pancreatic tumor growth came from the observation that the pancreas tumor weight remained unchanged between 6 and 40wks of age in Muc1-null mice, whereas, the tumor weight increased significantly in wild type mice (Figure 1A). This lack of tumor growth in the Muc1-null mice was further substantiated when cell lines generated from tumors in these mice resulted in stable disease when injected in vivo (Figure 2A–D) and had low proliferative index in vitro even when supplemented with exogenous growth factors known to enhance tumor cell proliferation (Figure 3 A–C). Further, MUC1 was shown to regulate cell cycle, as Muc1 deficiency lead to fewer cells entering the (G2M) phase of the cell cycle (Figure 4B). In addition, MUC1 drove Cdc-25c expression, a tyrosine phosphatase that triggers entry into mitosis. Since activated ERK1/2 is known to interact with Cdc-25c during interphase and phosphorylate Cdc-25c during mitosis , increased levels of ERK activation may be partially responsible for the up-regulation of cdc-25c and thereby enhanced mitosis and entry into the G2M phase of the cell cycle. To that effect, treatment with U0126, reduced proliferation of MUC1-expression cells to that of Muc1-null cells (Figure 6A–B).
MUC1 regulation of cell cycle checkpoints, proliferation, and invasion could also be attributed to strikingly higher levels of Tubulin α-2 chain and Nestin proteins (Figure 5B–C). Nestin, a marker for proliferating and invading cells, is differentially expressed during the cell cycle and promotes cell proliferation [19–20]. Nestin is also known to interact with Cyclin-dependent kinase 5 (CDK5), a kinase which phosphorylates MEK1. Thus, we hypothesize that MUC1 regulation of Nestin may activate CDK5 and thereby enhance phosphorylation of MEK1. Interestingly, inhibition of the ERK pathway has been shown to suppress the expression of nestin , once again, making the MAPK/ERK pathway an especially attractive target. Tubulin α-2 is a major constituent of microtubules and is required for mitotic spindle organization, mitosis, growth, and cell migration, thus emphasizing another important role of MUC1 in cell division. Currently, small molecule inhibitors of tubulin are clinically used as anti-mitotic drugs . Importantly, these anti-mitotic drugs have shown efficacy against multidrug-resistant tumors.
We postulate that the strong oncogenic signaling motifs reside in the cytoplasmic tail of MUC1 (MUC1 CT). MUC1 CT is a trans-membrane receptor that is known to function as an oncoprotein [22–24]. The tyrosines in MUC1 CT are essential for the oncogenic signal to occur in pancreatic cancer cells  and contains a YTNP site that, when phosphorylated, interacts with the proteins of the MAPK pathway [10, 11]. Our data and the above findings enable us to postulate that MUC1 contributes to pancreatic cancer cell growth and survival by promoting activation of the MAPK pathway, as pharmaceutically inhibiting this pathways inhibited proliferation in MUC1-expressing cells.
Finally, we have previously shown that treatment with a MUC1-based vaccine in combination with celecoxib was extremely effective in halting tumor progression in the KCM mice . Further, we have recently shown that the tyrosines in MUC1 CT was essential for epithelial to mesenchymal transition and invasion . Thus, targeting MUC1 CT may be an attractive approach, especially since the activation of MAPK pathway in pancreatic cancer cells may occur in part via MUC1 CT phosphorylation and interaction with β-catenin .
We acknowledge the funding provided by NIH CA R01CA118944. We acknowledge all the technicians in the animal facility for their assistance in maintaining our colonies.