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Cancer Research 67, 5201, June 1, 2007. doi: 10.1158/0008-5472.CAN-06-4647
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Platelet-Derived Growth Factor Receptor ß–Mediated Phosphorylation of MUC1 Enhances Invasiveness in Pancreatic Adenocarcinoma Cells

Pankaj K. Singh1,2, Yunfei Wen1,2, Benjamin J. Swanson1, Kandavel Shanmugam3, Andrius Kazlauskas4, Ronald L. Cerny5, Sandra J. Gendler3 and Michael A. Hollingsworth1,2

1 Eppley Institute for Research in Cancer and Allied Diseases and 2 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska; 3 Department of Biochemistry/Molecular Biology Program, Mayo Clinic College of Medicine, Scottsdale, Arizona; 4 Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; and 5 University of Nebraska-Lincoln, Lincoln, Nebraska

Requests for reprints: Michael A. Hollingsworth, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. Phone: 402-559-8343; Fax: 402-559-3339; E-mail: mahollin{at}unmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MUC1 is a heterodimeric transmembrane glycoprotein that is overexpressed and aberrantly glycosylated in ductal adenocarcinomas. Differential phosphorylation of the MUC1 cytoplasmic tail (MUC1CT) has been associated with signaling events that influence the proliferation and metastasis of cancer cells. We identified a novel tyrosine phosphorylation site (HGRYVPP) in the MUC1CT by mass spectrometric analysis of MUC1 from human pancreatic adenocarcinoma cell lines. Analyses in vitro and in vivo showed that platelet-derived growth factor receptor ß (PDGFRß) catalyzed phosphorylation of this site and of tyrosine in the RDTYHPM site. Stimulation of S2-013.MUC1F cells with PDGF-BB increased nuclear colocalization of MUC1CT and ß-catenin. PDGF-BB stimulation had no significant effect on cell proliferation rate; however, it enhanced invasion in vitro through Matrigel and in vivo tumor growth and metastases. Invasive properties of the cells were significantly altered on expression of phosphorylation-abrogating or phosphorylation-mimicking mutations at these sites. We propose that interactions of MUC1 and PDGFRß induce signal transduction events that influence the metastatic properties of pancreatic adenocarcinoma. [Cancer Res 2007;67(11):5201–10]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pancreatic cancer is associated with high mortality and poor prognosis, in part because it is known to metastasize early during disease progression. Several studies have shown that the MUC1 glycoprotein contributes to growth and metastasis of pancreatic adenocarcinomas. MUC1 protein has been detected in >90% of pancreatic tumors examined by immunohistochemistry (1), in the pancreatic juice of pancreatic adenocarcinoma patients by proteomic analysis, and in most pancreatic cancer cell lines (2, 3). Sialylated MUC1 is overexpressed by invading and metastatic pancreatic cancer cells but not by normal pancreas or in cases of chronic pancreatitis or pancreatic ductal hyperplasia (4). Previous studies from our laboratory have established a role for MUC1 in invasion and metastasis in pancreatic cancer (5, 6).

MUC1 is a type I transmembrane protein that consists of a large extracellular subunit comprised by a mucin-type tandem repeat and a smaller subunit that includes a small extracellular domain and a transmembrane domain plus a 72–amino acid cytoplasmic tail (MUC1CT). These two subunits are generated from the intracellular autocatalytic proteolytic cleavage of a single precursor polypeptide chain (7–9). The larger extracellular subunit consists of a heavily O-glycosylated tandem repeat unit of 20 amino acids that is repeated from 18 to over 100 times in different alleles (10–12).

The MUC1CT is phosphorylated and involved in different signaling pathways (13). Initial studies suggested that changes in phosphorylation of MUC1CT were correlated with differences in cell adhesion (14–17). An SXXXXXSSL motif in the MUC1CT interacts with ß-catenin and may compete with E-cadherin for binding with ß-catenin (18, 19). This interaction is abrogated by mutation of a tyrosine to phenylalanine at the DRSPYEKV sequence, a site that is phosphorylated by c-Src, epidermal growth factor receptor (EGFR), or Lyn kinases. A fragment of MUC1CT is translocated to the nucleus in association with ß-catenin and may influence its activity as a transcriptional coactivator (19). Phosphorylated YEKV is believed to serve as a binding site for Src SH2 domains (20). Phosphorylation of tyrosine in the YTNP sequence in the MUC1CT is postulated to enhance binding to the adaptor protein Grb2 and its associated small G protein activator SOS, providing a potential activation mechanism for Ras and its effectors (21, 22). Thus, there is substantial evidence that phosphorylation of the MUC1CT modulates signaling related to proliferation and metastasis.

Previous investigations into the phosphorylation of MUC1 have been based on expression of constructs harboring mutations at specific tyrosine residues and analysis of changes in degrees of phosphorylation of all tyrosine residues on the MUC1CT by Western blot analysis with anti-phosphotyrosine antibodies. Here, we directly evaluated the phosphorylation status of MUC1 in pancreatic cancer cell lines by mass spectrometry (MS)-based sequence analysis of the MUC1CT, which revealed a novel tyrosine phosphorylation site at the HGRYVPP sequence in MUC1CT. We showed that platelet-derived growth factor receptor ß (PDGFRß) is a kinase for the site and that stimulation of the pancreatic cancer cell line S2-013.MUC1F with PDGF-BB enhanced phosphorylation of MUC1CT, increased nuclear localization of MUC1CT and its association partner, ß-catenin, and enhanced the invasiveness of S2-013.MUC1F cells without significantly affecting their proliferation rate. Nuclear localization of MUC1CT and invasiveness of the cells were significantly diminished by tyrosine to phenylalanine mutations at these sites, whereas mutations of the tyrosine residues to glutamic acid enhanced nuclear localization and invasiveness. These results provide compelling evidence that PDGFRß-mediated phosphorylation of the MUC1CT regulates invasiveness of pancreatic adenocarcinoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents. Panc1 was obtained from the American Type Culture Collection. S2-013 is a cloned subline of a human pancreatic tumor cell line (SUIT-2) derived from a liver metastasis (23). Armenian Hamster monoclonal antibody (mAb) CT2 against the MUC1CT was kindly provided by Dr. Sandra J. Gendler (Mayo Clinic, Scottsdale, AZ). The mAb anti-ß-actin was purchased from Sigma. Horseradish peroxidase (HRP)-conjugated AffiniPure Goat Anti-Armenian Hamster IgG(H+L) was purchased from Jackson ImmunoResearch Laboratories, Inc. Rabbit polyclonal IgG against the kinase insert domain of PDGFRß has been described elsewhere (24). HRP-conjugated rabbit anti-goat IgG was purchased from Pierce. Recombinant human PDGF-BB was purchased from PeproTech. Recombinant active PDGFRß and c-Src were purchased from Upstate Cell Signaling Solutions. MUC1F-wt, MUC1.FHPM (having tyrosine at YHPM mutated to phenylalanine), MUC1.FVPP (having tyrosine at YVPP mutated to phenylalanine), MUC1.EHPM (having tyrosine at YHPM site mutated to glutamate), and MUC1.EVPP (having tyrosine at YVPP site mutated to glutamate) were created by using the QuikChange II XL Site-Directed Mutagenesis kit (Stratagene) and cloned in pLNCX.1.

Cell culture. FLAG epitope-tagged MUC1 (MUC1F) transfectants of the S2-013 cell line (S2-013.MUC1F), Panc1 cell line (Panc1.MUC1F), and cytoplasmic tail deleted MUC1 transfectants of the S2-013 cell line (S2-013.CT3) were cultured as described previously (25). HPAF-2 cells were maintained in Eagle's MEM (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS), nonessential amino acids, sodium pyruvate, and penicillin/streptomycin under similar conditions. To express MUC1F-wt and the mutant MUC1 constructs, retroviral transductions were done essentially as described previously (26).

Western blotting and immunoprecipitation. Cell lysate proteins were resolved on 10% or 14% Novex Tris-glycine denaturing polyacrylamide gels (Invitrogen) in a 1x SDS-PAGE buffer (1 g/L SDS, 3 g/L Tris base, 14.4 g/L glycine). Western blotting and immunoprecipitations were done as described previously (19).

In vitro kinase assay. Peptides (1 µg) in 10 µL reaction buffer [PDGFRß reaction buffer: 40 mmol/L MOPS (pH 7.5), 1 mmol/L EGTA; Src reaction buffer: 100 mmol/L Tris-HCl (pH 7.2), 125 mmol/L MgCl2, 25 mmol/L MnCl2, 2 mmol/L EGTA, 0.25 mmol/L sodium orthovanadate, 2 mmol/L DTT] were incubated at 37°C for 30 min along with 10 µCi [{gamma}-32P]ATP and 500 ng of active human recombinant PDGFRß or 20 units of active human recombinant c-Src. The reaction was stopped by boiling the samples in reducing SDS sample buffer. Samples were resolved on 16% Tricine gel. The gel was then dried and by phosphorimager analysis.

Tandem MS analysis of MUC1CT. Immunoprecipitated MUC1CT from cell lysates or in vitro phosphorylated MUC1CT peptides were resolved on 14% Novex Tris-glycine denaturing polyacrylamide gels (Invitrogen) and silver stained, and then bands corresponding MUC1CT were excised and digested by a published method (27). Eluted peptides were analyzed using a Q-TOF Ultima tandem mass spectrometer (Micromass/Waters) with electrospray ionization. Analyses were done using data-dependent acquisition with the following variables: 1-s survey scan (380–1,900 Da) followed by up to three 2.4-s MS/MS acquisitions (60–1,900 Da) at a mass resolution of 8,000 Da. The instrument was calibrated using fragment ion masses of doubly protonated Glu-fibrinopeptide.

The MS/MS data were processed using MassLynx software (Micromass) to produce peak lists that were input to the search engine MASCOT (Matrix Science) and searched against the National Center for Biotechnology Information nonredundant database. Search variables were as follows: mass accuracy 0.1 Da, enzyme specificity trypsin, fixed modification carboxyamidomethylcysteine, and variable modification oxidized methionine. Protein identifications were based on random probability scores with a minimum value of 25.

Immunofluorescence microscopy. Cells were cultured on glass coverslips (Fisherbrand Microscope cover glass: 12-545-100 18CIR-1) at 4.5 x 105 per well for 12 h. Cells were rinsed once with serum-free medium, starved of serum for 24 h, and then treated with PDGF-BB (50 ng/mL) for 2 h or were left untreated in the serum-free medium. Immunofluorescence staining was done as described previously (19).

Matrigel invasion assay. In vitro invasive potential of cells under PDGF-BB stimulation was assayed using the Biocoat Matrigel Invasion Chamber (Becton Dickinson Labware) as described previously (6). Cells (5 x 104) were seeded onto the upper chamber of double-structured matrix gel chamber. The lower chamber contained DMEM with or without 50 ng/mL PDGF-BB or 1% FBS.

Tumor growth studies. Congenitally athymic female National Cancer Institute nude mice (NCr-nu/nu) were purchased from the National Cancer Institute. Animals were maintained in pathogen-free conditions and fed sterile water and food ad libitum. Mice were treated in accordance with the Institutional Animal Care and Use Committee guidelines. Cells (1 x 105) were used for orthotopic injections of cells into the pancreas of nude mice as described previously (5). Mice (12–14) were used for each cell type. The tumor size was measured postsurgery after the end of 4 weeks. Internal organs, including lungs, liver, and all accessible mesenteric and mediastinal lymph nodes, were dissected from sacrificed mice, fixed, and sectioned. The identity of metastases within the dissected organs was confirmed by staining the tissue sections with H&E before quantifying the numbers of metastases.

Statistical analysis. Results are expressed as mean ± SE of three to five independent experiments, each treatment done in triplicate. Nonparametric Kruskal-Wallis tests were used to compare differences between cell lines. If the overall Kruskal-Wallis test indicated a difference in cell lines, a Wilcoxon rank sum test was done for each pairwise comparison of interest. Analyses were done using the Statistical Package for the Social Sciences or Statistical Analysis System for Windows version 8.2 (SAS Institute). Student's t test was used when appropriate. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of a novel in vivo MUC1 phosphorylation site. Two human pancreatic adenocarcinoma cell lines, Panc1 and S2-013, stably expressing recombinant FLAG epitope-tagged full-length MUC1 (Panc1.MUC1F and S2-013.MUC1F, respectively) were used for characterization of the phosphorylation status of MUC1. Panc1 is a poorly differentiated human pancreatic adenocarcinoma cell line that expresses low levels of endogenous MUC1 and relatively low levels of other glycoproteins (28). S2-013 is a moderately differentiated human pancreatic tumor cell line that is known to express abundant O-glycosylated mucin-like proteins, including low levels of endogenous MUC1 (23). cDNA constructs encoding FLAG epitope-tagged forms of MUC1 have been described previously (12).

Cell lysates of S2-013.MUC1F and Panc1.MUC1F were immunoprecipitated with CT2, a mAb raised against a peptide containing the COOH-terminal 17 amino acids of MUC1CT, separated by SDS-PAGE, and silver stained with a MS-compatible silver stain procedure. A duplicate gel was Western blotted and probed with CT2 antibody. Silver-stained bands of MUC1CT were identified at ~30 kDa by comparison with bands obtained from the parallel Western blotting experiment. These bands were sliced from the gel, subjected to in-gel trypsin digestion, and analyzed by tandem MS for identification of phosphorylation sites. In repeated experiments, we detected tryptic fragments for the entire MUC1CT and observed a single and novel phosphorylation site at the first tyrosine residue in the YVPPSSTDRSPYEK peptide fragment in both Panc1.MUC1F and S2-013.MUC1F cells (Fig. 1A and B ). We did not detect phosphorylation of any other tyrosine residues in these analyses (although phosphorylation of several serine residues was detected and will be the subject of a separate report).


Figure 1
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  		Figure 1. MS/MS spectra of in vivo phosphorylated pYVPPSSTDRSPYEK. MUC1CT immunoprecipitated from Panc1.MUC1F and S2-013.MUC1F cells was resolved on SDS-PAGE and silver stained, and appropriate bands were excised and subjected to trypsin digestion and tandem MS analysis to identify phosphorylation sites. MS/MS spectra of pYVPPSSTDRSPYEK from Panc1.MUC1F cells (A) and S2-013.MUC1F cells (B).

 
Identification of the corresponding kinase with motif scanning programs. Because the kinase that phosphorylates tyrosine at the HGRYVPP site was not known, the MUC1CT sequence was evaluated with Scansite,6 a homology-based algorithm that searches for motifs within proteins that are likely to be phosphorylated by specific protein kinases (29). Optimal phosphorylation sites for protein kinases are predicted by determining a matrix of selectivity values for amino acids at each position relative to the phosphorylation site as determined from oriented peptide library techniques. The Scansite program identified PDGFRß as a potential kinase for the tyrosine residue at the HGRYVPP site (score: 0.3498). As a control for purposes of comparison, Scansite identified a c-Src SH2 domain affinity for tyrosine at the DRSPYEKV motif (score: 0.2045), which is a known kinase for that site. Searches at either moderate or high stringency did not identify other kinases for these sites. PDGFRß and c-Src were both identified under conditions of low stringency.

In vitro kinase assays. To test the prediction that PDGFRß phosphorylates the MUC1CT at HGRYVPP, a series of in vitro kinase assays were undertaken with recombinant human PDGFRß and synthetic peptides spanning the MUC1CT. Reactions from these assays were resolved by SDS-PAGE, which were dried and imaged on a phosphorimager screen.

MUC1CTp-66 (Fig. 2A, top ), a 66-residue peptide spanning most of the MUC1CT region, was phosphorylated by recombinant human PDGFRß as determined by phosphorimages of the products of in vitro kinase assays (Fig. 2A, bottom left). A protein tyrosine kinase substrate, Raytide EL, used as a positive control, was also phosphorylated by recombinant PDGFRß. As a second control, we did in vitro kinase assays with recombinant c-Src, which also catalyzed phosphorylation of the MUC1CT (Fig. 2A, bottom right).


Figure 2
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  		Figure 2. In vitro phosphorylation of MUC1CT by PDGFRß. A, MUC1CTp-66 is a 66-residue synthetic peptide corresponding to the cytoplasmic tail of human MUC1. MUC1CTp-1, MUC1CTp-2, and MUC1CTp-3 are three overlapping peptides, together spanning the whole cytoplasmic tail region of human MUC1. Each peptide was designed to span sequences that included one or two tyrosines and to include one or two tyrosines in each overlap, thereby including all seven tyrosines from the MUC1CT in three peptides. Autoradiograms from 32P-labeled in vitro kinase reactions of MUC1CT peptides with PDGFRß (bottom left) and c-Src (bottom right). Phosphorylated substrate peptides were confirmed by MS analysis. A protein tyrosine kinase substrate, Raytide EL, was used as a control for tyrosine kinase activity. B to D, MUC1CTp-66 peptides phosphorylated by in vitro kinase assays with PDGFRß (B and C) or c-Src (D) were sequenced by tandem MS and showed phosphorylation at DTpYHPMSEYPTYHTHGR (B) and pYVPPSSTDRSPYEK by PDGFRß (C). MUC1CTp-66 was phosphorylated by c-Src at YVPPSSTDRSPpYEK (D).

 
To further evaluate the region of MUC1CT phosphorylated by recombinant PDGFRß, in vitro kinase assays were done with three overlapping peptides spanning the whole MUC1CT: MUC1CTp-1 (a 30-residue peptide), MUC1CTp-2 (a 32-residue peptide), and MUC1CTp-3 (a 29-residue peptide). The sequences encoded by these peptides are shown in Fig. 2A. The HGRYVPP sequence was included in only the MUC1CTp-2 fragment. MUC1CTp-1 and MUC1CTp-2 were phosphorylated by recombinant PDGFRß; however, MUC1CTp-3 was not phosphorylated by PDGFRß (Fig. 2A).

MS analysis of in vitro phosphorylated MUC1CTp-66. MS sequence analysis of in vitro PDGFRß-phosphorylated MUC1CTp-66 revealed two phosphorylation sites: tyrosines at RDTYHPM (Fig. 2B) and HGRYVPP (Fig. 2C) in the MUC1CT. In addition, the tyrosine at DRSPYEKV was phosphorylated by c-Src (Fig. 2D). We also conducted in vitro assays with recombinant human EGFR and c-Met (hepatocyte growth factor receptor), which did not phosphorylate MUC1 at the YVPP site but did phosphorylate other sites that will be the subject of a separate report (data not shown).

PDGFRß-mediated in vivo phosphorylation of MUC1CT. Labeling of S2-013.MUC1F cells with [32P]orthophosphate following stimulation with 50 ng/mL PDGF-BB induced significant increases in phosphorylation of MUC1CT compared with unstimulated cells (Fig. 3A ). This enhancement in phosphorylation was abrogated by the PDGFR-specific phosphorylation inhibitor AG1295 (10 µmol/L; Fig. 3A). The stimulation of phosphorylation was dependent on the duration of incubation with PDGF-BB, as there were increasing intensities of phosphorylated MUC1CT bands on the autoradiograms when cells were treated for 15 min, 30 min, and 1 h, respectively (Fig. 3B and C).


Figure 3
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  		Figure 3. In vivo phosphorylation of MUC1 by PDGFRß. A, serum-starved S2-013.MUC1F cells were incubated for 15 min with [32P]orthophosphate (250 µCi/mL) in the presence or absence of human recombinant PDGF-BB (50 ng/mL) or the PDGF inhibitor AG1295 (10 µmol/L). Immunoprecipitates (IP) with mAb CT2 were resolved on 14% SDS-PAGE and immunoblotted (IB) with CT2 (bottom), and then the membrane was subjected to autoradiography (top). B, autoradiography of CT2 immunoprecipitates from S2-013.MUC1F cells incubated with PDGF-BB (50 ng/mL) and [32P]orthophosphate (250 µCi/mL) for increasing periods (15 min, 30 min, and 1 h), with unstimulated cells as controls (top). Bottom, a Western blotting experiment was done in parallel to determine the amounts of immunoprecipitated MUC1CT. Representative of experiments repeated thrice. C, average pixel intensity for each band was normalized for pixel intensity in MUC1CT bands and plotted against fold increase compared with unstimulated cells. D, nuclear (N) and cytoplasmic (C) localization of MUC1CT phosphorylated at tyrosines at the YVPP and YHPM sites determined by Western blotting with phosphorylation site-specific antibodies. The polyvinylidene difluoride membrane was reprobed with antibodies against MUC1CT (CT2 mAb), histone2B (H2B), a marker for nuclear proteins, and ß-actin as controls for protein loading.

 
PDGF stimulation causes nuclear localization of MUC1CT phosphorylated at tyrosines in the YVPP and YHPM motifs. We produced rabbit polyclonal antibodies specific for phosphorylated tyrosines at the YVPP and YHPM sites, which were validated for specificity by ELISA and Western blotting analysis with 66-residue MUC1CT peptides phosphorylated at the these sites (unphosphorylated 66-residue MUC1CT peptide was used as a control; data not shown). These antibodies were used to evaluate the effect of PDGF stimulation on phosphorylation of these sites in the MUC1CT. Western blotting of nuclear and cytoplasmic extracts from PDGF-BB–stimulated S2-013.MUC1F cells revealed a significant increase in phosphorylation of tyrosines in the YVPP and YHPM motifs that was concordant with increasing duration of PDGF stimulation and was mostly observed in the nuclear fraction (Fig. 3D). MS analysis of independent cell lysates confirmed phosphorylation but is not a quantitative method (data not shown). A single band was observed with the anti-pYVPP antibody; however, two bands were observed with the anti-pYHPM antibody, whose appearance was dependent on the duration of PDGF stimulation. The two bands could represent different MUC1CT fragments phosphorylated at one or both tyrosines in the YHPM and YVPP motifs or tyrosine phosphorylated only in the YHPM motif but showing a distinct mobility perhaps because of other post-translational modifications that are not defined at this time.

PDGF-BB stimulation enhances nuclear localization of ß-catenin and MUC1CT. We evaluated the effects of PDGFRß-mediated phosphorylation of MUC1CT on colocalization with ß-catenin. Immunofluorescence analysis with the CT2 mAb (raised against the MUC1CT) and a mAb against ß-catenin was done on S2-013.MUC1F cells treated with 50 ng/mL PDGF-BB for 2 h (30). The localization patterns of MUC1CT and ß-catenin were compared with serum-starved S2-013.MUC1F cells.

Serum-starved S2-013.MUC1F cells showed localization of MUC1CT and ß-catenin at the periphery of cells (Fig. 4A, top ). Some but not all MUC1 and ß-catenin were colocalized at the periphery; presumably, ß-catenin at cell junctions is colocalized with E-cadherin (31), and MUC1 is found in areas of cell other than at these junctions. PDGF-BB stimulation induced extensive nuclear localization of ß-catenin and nuclear and diffuse cytoplasmic staining of MUC1CT (Fig. 4A, bottom). These results show that PDGFRß-mediated phosphorylation of MUC1CT enhanced nuclear localization of MUC1CT.


Figure 4
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  		Figure 4. A, effect of PDGF-BB stimulation on the subcellular localization of ß-catenin and MUC1CT in S2-013.MUC1F cells. S2-013.MUC1F cells were serum starved for 24 h, stimulated for 2 h or left unstimulated, fixed, and permeabilized before incubation with mAbs CT2, anti-ß-catenin, and the nuclear dye propidium iodide. Fluorescence signals from each scan were acquired sequentially. mAb CT2 (anti-MUC1CT) was visualized as blue and detected by using a Cy5-conjugated secondary antibody, mAb anti-ß-catenin was identified with FITC-conjugated secondary antibody and visualized as green, and nuclei were visualized in red. White arrows, triple-merge of blue, green, and red (MUC1CT/ß-catenin/Nucleus) showed colocalization of MUC1CT and ß-catenin at cell surfaces under serum-starved conditions. B, effect of tyrosine mutations on nuclear localization of MUC1. S2-013.MUC1.FHPM, S2-013.MUC1.EHPM, S2-013.MUC1.FVPP, and S2-013.MUC1.EVPP cells were stained for MUC1CT (mAb CT2) and nucleus [propidium iodide (PI)]. Bar, 25 µm. Magnification, x63. Results represent three to four individual cell scanning observations.

 
To confirm that phosphotyrosines in the YHPM and YVPP motifs contributed to the nuclear localization of MUC1CT, phosphorylation-abrogating (Y to F) and phosphorylation-mimicking (Y to E) mutations were generated for the YHPM and YVPP motifs of MUC1CT cloned into the pLNCX.1 retroviral expression vector. Retrovirally transduced S2-013 cells were tested for stable expression of the recombinant MUC1 proteins by Western blotting with mAb CT2. These S2-013 transfectants, named S2-013.MUC1.FHPM, S2-013.MUC1.EHPM, S2-013.MUC1.FVPP, and S2-013.MUC1.EVPP, were used to evaluate the nuclear localization of MUC1CT under serum-starved and PDGF-BB–stimulated conditions. As expected, the S2-013.MUC1.FHPM and S2-013.MUC1.FVPP cells expressing the inactivating mutations showed significant localization of MUC1CT to the cell periphery, which was not dramatically affected by stimulation with PDGF-BB (Fig. 4B). S2-013.MUC1.EHPM and S2-013.MUC1.EVPP cells expressing the activating mutations showed significant constitutive nuclear localization of MUC1 even under serum-starved conditions (Fig. 4B).

PDGFRß-mediated phosphorylation of MUC1 enhances invasiveness of pancreatic adenocarcinoma cells. Overexpression of MUC1 and enhanced nuclear localization of MUC1CT and ß-catenin are associated with increased metastasis and poor prognosis for different adenocarcinoma. Hence, Matrigel invasion assays were done to evaluate the effect of PDGFRß-mediated phosphorylation of MUC1 on invasiveness of pancreatic adenocarcinoma cells (Fig. 5 ). S2-013.MUC1F cells had significantly higher invasion rates compared with S2-013.CT3 (P < 0.0001) or S2-013.Neo cells (P < 0.0001; Fig. 5). The invasive potential of cytoplasmic tail-truncated MUC1 containing S2-013 cells was not significantly different from control-transfected cells (P > 0.3). Stimulation of S2-013.MUC1F with PDGF-BB (50 ng/mL) significantly enhanced the invasiveness of these cells compared with unstimulated cells (P < 0.02). The PDGF-BB–mediated increase in invasive potential of S2-013.MUC1F cells was ablated by treatment of cells with a PDGFRß kinase inhibitor (10 µmol/L). There was no significant effect of PDGF-BB stimulation on the invasive potential of S2-013.CT3 and S2-013.Neo cells in response to the PDGF-BB stimulation (P = 0.3 and 0.7, respectively).


Figure 5
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  		Figure 5. Effect of PDGF-BB on Matrigel invasion of S2-013 cells transfected with vector control (S2-013.Neo), cytoplasmic tail-truncated (S2-013.CT3), or full-length constructs of MUC1 (S2-013.MUC1F). Number of S2-013 cells invading through Matrigel and an 8-µm-pored membrane after 24 h of culture with or without PDGF-BB (50 ng/mL). Cells (5 x 104) were seeded in the upper compartment. The lower chamber contained DMEM with or without PDGF-BB (50 ng/mL). Columns, mean of invading cell number from three independent experiments done in triplicate; bars, SE.

 
Role of tyrosines in the YHPM and YVPP motifs of MUC1CT in the invasive potential of S2-013.MUC1F cells. After observing that PDGF-BB stimulation increased invasiveness of MUC1-expressing cells, we sought to establish the role of phosphotyrosines in the YHPM and YVPP motifs of MUC1CT on the invasiveness of MUC1-expressing cells. Cell lines expressing inactivating and activating mutations at these motifs (S2-013.MUC1.FHPM, S2-013.MUC1.EHPM, S2-013.MUC1.FVPP, and S2-013.MUC1.EVPP) and control cells (S2-013.MUC1F and S2-013.Neo) were evaluated in Matrigel invasion assays. S2-013 cells expressing either of the phosphorylation-abrogating tyrosine mutations (S2-013.MUC1.FHPM or S2-013.MUC1.FVPP) showed reduced in vitro invasiveness compared with S2-013.MUC1F cells, suggesting that these residues contribute to the invasiveness of S2-013.MUC1F cells (both P < 0.001; Fig. 6A ). In contrast, increased in vitro invasiveness was observed by S2-013 cells expressing either of the phosphorylation-mimicking tyrosine mutations (S2-013.MUC1.EHPM or S2-013.MUC1.EVPP) compared with S2-013.MUC1F cells (P < 0.05 and 0.001; Fig. 6A).


Figure 6
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  		Figure 6. Effect of tyrosine mutations of MUC1CT on invasion and metastasis. A, effect of tyrosine mutations of MUC1CT on in vitro Matrigel invasion. Cells were seeded onto the upper chamber in serum-free DMEM, whereas the lower chamber contained DMEM with 1% FBS. B, effect of tyrosine mutations on in vivo tumor growth rate. Cells (1 x 105) were orthotopically implanted into athymic female nude mice, and the dimensions of the tumor were detected by excising and measuring the tumor after sacrificing mice at the end of 4th week. Columns, mean tumor diameter plotted in terms of area unit obtained by multiplication of the two largest cross sections; bars, SE. Effect of tyrosine mutations on lymph node (C) and lung metastases (D). The incidences of metastases were detected by microscopic observation of the H&E-stained tissue sections obtained from the orthotopically implanted athymic female nude mice. The mice were sacrificed after the 4th week of orthotopic implantation of tyrosine mutant or MUC1F or mock vector–expressing S2-013 cells. Columns, mean number of metastases as determined by counting of metastases by microscopic examination of formalin-fixed and H&E-stained tissue sections; bars, SE. *, P < 0.05, compared with the S2-013.MUC1F cells; ***, P < 0.001, compared with the S2-013.MUC1F cells. Asterisk(s) over horizontal lines, statistical significance of the events for the cells represented by the vertical bars underneath the line.

 
We evaluated the effect of these MUC1 tyrosine mutations on the tumorigenic and metastatic potential of S2-013.MUC1 cells by orthotopic injection into nude mice. Mice were sacrificed after 4 weeks. Mean tumor size was analyzed postsurgery after the end of 4 weeks (Fig. 6B). S2-013.MUC1.EHPM and S2-013.MUC1.EVPP cells showed larger tumors than S2-013.MUC1F, whereas S2-013.MUC1.FHPM and S2-013.MUC1.FVPP showed slightly smaller tumors; however, the differences did not achieve statistical significance (Fig. 6B). Internal organs and lymph nodes from sacrificed mice were dissected, fixed, sectioned, and stained with H&E to identify and quantify metastases. The incidence of lymph node metastases was significantly lower in mice implanted with S2-013.MUC1.FHPM and S2-013.MUC1.FVPP cells compared with S2-013.MUC1F (P < 0.05 and 0.001, respectively; Fig. 6C). Mice implanted with S2-013.MUC1.EHPM showed significantly higher rates of lymph node metastases compared with S2-013.MUC1F (P < 0.05). About lung metastases, only mice implanted with S2-013.MUC1.FHPM showed statistically significant differences (reduced; P < 0.05; Fig. 6D). There were no significant differences in incidences of liver and diaphragm metastases (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Proteomics methods were used to identify sites of tyrosine phosphorylation on the MUC1CT produced by pancreatic tumor cells. Phosphorylation of tyrosine at the HGRYVPP sequence was observed in two different human pancreatic carcinoma cells, S2-013.MUC1F and Panc1.MUC1F, suggesting that this modification is associated with pancreatic cancer. This is the first report of phosphorylation of this site, the first report to directly show the sites of tyrosine phosphorylation on the MUC1CT by the use of MS, and the first report to confirm that these occur by the use of site-specific antibodies (previous studies used mutated constructs and showed a decrease in total phosphorylation).

We detected and sequenced peptides spanning the entire cytoplasmic tail, which shows that the proteomics methods used here were sufficiently sensitive to detect multiple peptide species from the MUC1CT. Our failure to detect phosphorylation at tyrosine sites other than HGRYVPP raises the possibility that pancreatic tumor cells cultured under the growth conditions analyzed here predominantly phosphorylate the tyrosine at HGRYVPP and that phosphorylation of other sites is highly regulated. It remains possible that there were low levels of other modifications present in the extracts that were undetectable by the techniques used here. Previous results reporting phosphorylation at DRSPYEKV were obtained with breast cancer cell lines or cell lines derived from other organ sites, which raises another possibility: that there are organ-specific or cell-specific differences in the phosphorylation of the MUC1CT that reflect differences in the expression or activity of receptor tyrosine kinases or phosphatases in these cells. In this regard, it is notable that we detected phosphorylation of tyrosine in the DRSPYEKV site in vitro by c-Src (Fig. 2). In any case, there should be further definitive determination of specific sites on the MUC1CT that are phosphorylated by different normal and tumor cell types.

Analysis by the Scansite algorithm revealed PDGFRß as a putative kinase that catalyzed phosphorylation at the HGRYVPP site. In vitro kinase assays with MUC1CTp-66, a 66-residue synthetic peptide of the MUC1CT, clearly showed that PDGFRß phosphorylated MUC1CT (Fig. 2A) and subsequent analysis showed that tyrosines at sequences RDTYHPM and HGRYVPP in the MUC1CT were phosphorylated in vitro by PDGFRß (Fig. 2B–D). It should be noted that phosphorylation of tyrosine at the RDTYHPM sequence by PDGFR was predicted previously by Wreschner et al. (32). Wang et al. (33) previously presented evidence of phosphorylation of tyrosine at the RDTYHPM motif in a CD8/MUC1 chimeric protein synthesized from a recombinant chimeric construct expressed in Chinese hamster ovary cells, on stimulation with CD8 antibody, using phenylalanine substitution mutants and anti-phosphotyrosine antibodies to detect phosphorylation. It is also notable that both of these sites are within a region of the MUC1CT postulated to mediate association with p53 (34).

We detected significant expression of PDGFRß in multiple human pancreatic adenocarcinoma cell lines representing different differentiation states of pancreatic adenocarcinoma and detected phosphorylation of the tyrosine in the YVPP motif in FG, Colo357, and Capan-2 (data not shown). PDGFB, PDGFR{alpha}, and PDGFRß are highly expressed in pancreatic cancer and involved in signaling cascades that regulate the proliferation of pancreatic acinar cells and to be a key player in ductal cell carcinogenesis of pancreatic cancer as well as other cancers (35–38).

We observed an enhancement in MUC1CT phosphorylation on stimulation of S2-013.MUC1F cells with PDGF-BB, a ligand for PDGFRß, which supports the hypothesis that PDGFRß stimulation influences MUC1-mediated signaling. This enhancement of phosphorylation was abolished by a PDGFR-specific kinase inhibitor, tyrphostin (AG1295), which has been used to specifically abrogate the activation of PDGFR (39–42). Tyrphostin has shown promising results for the treatment of proliferative vitreoretinopathy, aortic allograft vasculopathy, and restenosis. Western blotting with antibodies specific for the phosphotyrosines at HGRYVPP and RDTYHPM confirmed PDGF-BB stimulation-dependent in vivo phosphorylation of the MUC1CT at tyrosines in both these sites.

Our studies showed that PDGF-BB stimulation induced PDGFRß-mediated phosphorylation of the MUC1CT, which in turn enhanced the nuclear localization of MUC1CT and ß-catenin and decreased localization of these proteins at cell surfaces. Activation of other receptor tyrosine kinases, such as EGFR, disrupts cadherin-ß-catenin interactions at the cell surface and increases nuclear signaling by ß-catenin (43). PDGFRß activation can lead to activation of a plethora of tyrosine kinases, including c-Src, which directly phosphorylate ß-catenin and disrupt its cell surface localization (44). Both MUC1CT and ß-catenin can be localized to the nucleus, and nuclear localization of ß-catenin has been correlated with increased proliferation and pathologic grades of tumors. The increased nuclear localization of MUC1CT and ß-catenin led us to investigate whether PDGFRß-mediated phosphorylation of the MUC1CT influenced the proliferative and metastatic activity of pancreatic adenocarcinoma cells. We observed no significant differences in proliferation in vitro among controls or cells overexpressing recombinant MUC1 at any time points under PDGF-BB stimulation compared with unstimulated cells as determined by flow cytometric cell cycle analysis (data not shown). These findings were confirmed by results of thymidine incorporation assays after 24 and 48 h of PDGF-BB stimulation (data not shown).

In contrast, results of in vitro invasion assays clearly indicated that PDGF-BB stimulation significantly enhanced the invasive potential of MUC1-overexpressing S2-013 cells. The increased invasive potential was ablated by the PDGFRß kinase inhibitor AG1295, indicating that these effects were directly related to the kinase activity of PDGFRß. Independently, Hwang et al. (36) have shown a reduction in growth and metastasis of a human pancreatic carcinoma in an orthotopic nude mouse model by inhibiting PDGFR phosphorylation with STI571 (Gleevec). That there was no significant PDGF-BB–mediated increase in the invasive potential in control S2013.Neo or S2013.CT3 cells indicates that PDGFRß-mediated phosphorylation of MUC1CT is critical for the observed enhancement in invasive potential. Furthermore, expression of phosphorylation-abrogating mutations in the MUC1CT diminished the invasive potential in both in vitro and in vivo assays. In contrast, both invasion and metastasis were significantly enhanced by phosphorylation-mimicking mutations, supporting our hypothesis that phosphorylation at these sites regulates the tumorigenic and metastatic potential of pancreatic cancer cells.

How is PDGFRß-mediated phosphorylation of the MUC1CT regulated in normal pancreatic cells versus pancreatic adenocarcinoma cells? One obvious factor is the requirement for tissue- or cell-specific coexpression of MUC1, PDGFRß, and PDGFB; however, simple coexpression of MUC1 and receptor tyrosine kinases, such as PDGFRß or EGFR, in a given cell does not guarantee that these proteins will interact and signal in a meaningful way. Differential accessibility of the substrate (MUC1CT) to the kinase (PDGFRß) can be regulated spatially in polarized epithelial cells and is likely to contribute to regulation of these phosphorylation events, which we postulate are functional in different types of normal cells and tumor cells (13). We do not know what brings MUC1 and receptor tyrosine kinases together. Normal epithelial cells express receptor tyrosine kinases (such as EGFR and PDGFRß) at the basal surfaces, whereas MUC1 is expressed at the apical surface (45). This spatial separation would be predicted to preclude interaction of these molecules. One characteristic of oncogenic transformation is the loss of apical-basal polarity, which in theory would enable membrane-associated mucins, such as MUC1, and growth factor receptors to reside in physical proximity as has been shown in the mammary gland (30) and thereby enable their association. Such a mechanism may explain the phosphorylation of MUC1 by PDGFRß in pancreatic adenocarcinomas, which have lost some aspects of apical-basal cell polarity and overexpress MUC1. Does such an event represent aberrant signaling? Perhaps, but it is also possible to envisage a model for the interaction of MUC1 and growth factor receptors, such as PDGFR or EGFR, in normal epithelia that had been injured or damaged and had lost some aspects of cellular polarity or differentiated architecture. Damage or alterations in epithelia that resulted in disrupted cell polarity and enabled the association and phosphorylation of MUC1 by PDGFRß may send signals to the cell, informing it of this loss of differentiated structure or function. Such loss of cellular structure in normal epithelia would require activation of cellular repair mechanisms that may involve cell proliferation and motility, both of which are functions previously associated with MUC1 in cancer. Thus, signaling mediated by MUC1, PDGFR, and other receptor tyrosine kinases in pancreatic cancer may be another example of tumors appropriating a normal cellular function for purposes of survival, proliferation, and invasion.

In summary, the findings presented here show that MUC1 is phosphorylated by PDGFRß, which leads to increased nuclear localization of MUC1CT and ß-catenin, and enhances the invasive potential of pancreatic adenocarcinoma cells. These results also support the hypothesis that phosphorylation events mediate MUC1-mediated tumor-associated signaling. Blockage of both MUC1 and PDGFRß signaling pathways might be of clinical relevance for the treatment of pancreatic cancer and should be investigated further. The fact that we did not detect phosphorylation at other tyrosine sites in the MUC1CT raises the possibility that there are differences among tissues or tumors in site-specific phosphorylation of the MUC1CT or that phosphorylation of other sites is regulated by factors not reflected in pancreatic tumor cell lines. In any case, it is important that additional studies be carried out to decipher the role of phosphorylation of the MUC1CT in signal transduction.


   Acknowledgments
 
Grant support: Graduate Studies Assistantships (P.K. Singh, Y. Wen, and B.J. Swanson) and NIH grants 5R01 CA57362 and T32 CA09476. The MS facility is supported in part by NIH grant P20 RR15635 from the COBRE Program of the National Center for Research Resources, National Cancer Institute Cancer Center Support grant P30 CA36727, and the Nebraska Research Initiative.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Thomas Caffrey, Dr. Judith K. Christman, and Janice Taylor (Confocal Laser Scanning Microscopy Core Facility) for their assistance.


   Footnotes
 
6 http://scansite.mit.edu Back

Received 12/19/06. Revised 3/22/07. Accepted 3/28/07.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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