Lower incidence of metastasis in PDA.Muc1KO mice compared to PDA.MUC1 mice: Evidence of EMT in PDA.MUC1 tumors
PDA.MUC1 and PDA.Muc1KO mice were sacrificed at ~36–40 weeks of age when all mice had primary pancreatic tumors. When organs were evaluated for macroscopic lesions, sixty percent (8/13 mice) of the PDA.MUC1 mice developed lung metastasis with ~40% (5/13) developing liver and ~20% (3/13) developing peritoneum metastasis (). This was in contrast to PDA.Muc1KO mice where only 1 out of 10 mice developed metastasis in all three organs. To investigate the mechanism by which MUC1 initiated higher metastasis, we evaluated if MUC1 plays a role in activating the EMT-associated proteins in the primary tumors. Tumor lysates from PDA.MUC1 and PDA.Muc1KO tumors (n=5) were analyzed by proteomics. Several proteins were differentially expressed; however, the most notable differences were in the levels of Vimentin and E-Cadherin. In the PDA.MUC1 tumors, there was a significant 5-fold increase in Vimentin with a concurrent decrease in E-Cadherin (). In contrast, the pattern was completely reversed in the PDA.Muc1KO tumors with significantly higher (4-fold) levels of E-Cadherin and lower levels of Vimentin (). The data was the first indication of possible induction of EMT in the MUC1-expressing tumors. Repression of E-Cadherin and induction of Vimentin in the PDA.MUC1 tumors directly correlated with increased metastasis in the PDA.MUC1 mice.
PDA mice lacking Muc1 have significantly lower incidence of secondary metastasis associated with decreased induction of EMT proteins in the tumors
MUC1 promotes induction of mesenchymal markers and functionally enhances the invasive capacity of pancreatic cancer cells in vitro
To further confirm the role of MUC1 in EMT, KCKO and KCM cell lines were generated from the PDA.Muc1KO and PDA.MUC1 mice respectively. MUC1 expression was confirmed by Western blotting () using MUC1 tandem repeat (TR, B27.29) and MUC1 CT (CT2) antibodies with high expression detected in KCM and no expression in KCKO cells (). B27.29 antibody detects the high molecular weight extracellular domain (>200 kD) and CT2 detects the low molecular weight CT domain (30kD). Both cell lines were subjected to an in vitro invasion assay, results of which showed significantly higher invasion index of the KCM compared to the KCKO cells (p<0.01, ), confirming MUC1 as the major contributor of increased motility and invasiveness. Furthermore, presence of MUC1 in KCM tumors clearly repressed E-Cadherin expression () and induced expression of transcription factors and proteins associated with the mesenchymal phenotype. These included Snail, Slug, N-cadherin, and Vimentin (), which potentially contributed to the contact inhibition and high invasion in vitro and in vivo (). Lastly, KCM cells expressed higher levels of vascular endothelial growth factor (VEGF) compared to KCKO cells (), another indicator of highly metastatic phenotype. Thus, we hypothesized that MUC1 induces EMT and initiates invasion in mouse pancreatic cancer cells.
Over expression of MUC1 in human pancreatic cancer cells promotes EMT and induces invasion: Role of tyrosines in the MUC1 CT
To further test our hypothesis and delineate the potential mechanism, we stably infected human pancreatic cancer cell lines, BxPC3 and Su86.86 with full length MUC1 or mutated MUC1 CT. Cells expressing full length MUC1 were designated BxPC3.MUC1 and Su86.86.MUC1 while cells expressing mutant MUC1 CT were designated BxPC3.Y0 and Su86.86.Y0. Cells infected with an empty vector containing the neomycin resistance gene were used as controls and designated BxPC3.Neo and Su86.86.Neo. The only difference between MUC1 and Y0 constructs was that in Y0 construct, all 7 tyrosines in MUC1 CT were replaced by phenylalanine. Expression of MUC1 was confirmed in both cell lines by Western blotting () using both the B27.29 and CT2 antibodies. The small shift in electrophoretic mobility noted in the Y0 cell lysate relative to MUC1 cells likely reflects a decrease in phosphorylation due to the mutations of the tyrosine residues in the Y0 cells (). An in vitro invasion assay showed significantly higher invasion index for MUC1 cells as compared to Y0 and Neo cells (p<0.01, ). These data suggest that the increased invasiveness of the MUC1-expressing cells may be attributed to the tyrosines in the MUC1 CT. To substantiate the role of the MUC1 CT tyrosines as an inducer of EMT, real-time PCR arrays were used to explore transcription of 72 genes implicated in EMT and metastasis. Genes whose transcription was altered by at least 2-fold compared to Neo-cells were considered significant. Several genes associated with tight junctions and epithelial phenotype was significantly reduced in BxPC3-MUC1 compared to the Neo cells. These included Tight Junction Proteins 1 and 3, Occludin, Laminin α5, β-and δ-catenin, Claudin, and E-cadherin (). In contrast, there was no such change in the Y0 cells (). Conversely, genes associated with the mesenchymal phenotype and invasion was significantly increased in the BxPC3 MUC1 and decreased in the Y0 cells compared to Neo cells. These included Vimentin, Twist, Slug, Snail, and Goosecoid, (). Both functional and molecular studies indicate that MUC1 initiates the process of EMT via signaling through the tyrosines in its CT and that no external factor is necessary to stimulate these changes.
Figure 2 Significantly higher invasion and transcription of genes associated with a mesenchymal phenotype coupled with significantly lower transcription of genes associated with an epithelial phenotype in MUC1 over-expressing human pancreatic cancer cells as compared (more ...)
Loss of epithelial and gain of mesenchymal markers in MUC1 but not in Y0 cells directly corresponds to their transcription of metastasis-associated genes
At the protein level, gain of mesenchymal markers such as Slug, Snail and Vimentin and loss of E-cadherin expression was apparent in the MUC1 cells (). These alterations in protein expression compared to Neo cells was not detected in the Y0 cells, implicating the importance of the tyrosines for effective oncogenic signaling and induction of EMT (). Downregulation of E-cadherin was further confirmed by confocal microscopy in BXPC3 MUC1 but not in the Y0 cells (). It is well established that Snail represses the expression of E-cadherin to induce EMT in several cancer cells (5
). Thus, data clearly suggests that MUC1 plays a critical role in inducing EMT in human pancreatic cancer cell lines via signaling through its CT tyrosines. Furthermore, genes associated with metastasis and angiogenesis such as VEGF, MMP-9, 3, & 2 and IL-6R were significantly increased in BxPC3 MUC1 but not in the Y0 cells compared to Neo controls (). Therefore, we propose that without functional tyrosines in MUC1 CT, EMT and metastasis may be blocked in human pancreatic cancer cells.
Repression of E-Cadherin and induction of Snail, Slug, and Vimentin in MUC1 cells coupled with significantly increased transcription of genes associated with metastasis. Complete reversal in MUC1-Y0 expressing cells
Circulating tumor cells (CTCs) detected in mice bearing the BxPC3-MUC1 but not Neo or Y0 tumors
To test tumor growth and metastasis in vivo, BxPC3-MUC1, Y0 and Neo cells were subcutaneously injected into nude mice. All cells formed tumors within 15 days post injection with tumor growth being significantly higher in BxPC3 MUC1 versus Y0 and Neo tumors ( (*p=0.02 – 0.05). BxPC3 MUC1 and Y0 tumors expressed high levels of MUC1 while Neo tumors expressed negligible amounts of MUC1 (). We were unable to detect gross metastatic lesions from these tumors; therefore we evaluated levels of CTCs in the blood of these mice. Within 2-weeks of culture, we were able to detect colonies of tumor cells in the blood collected from BxPC3-MUC1 but not from Neo or Y0 tumor-bearing mice (). The data signifies the role of MUC1 CT tyrosines during pancreatic cancer growth and extravasation of the cells into the blood stream for future metastasis.
Higher tumor burden with loss of E-Cadherin and gain of mesenchymal and metastatic proteins in BxPC3 MUC1 versus Y0 and Neo tumors
Detection of EMT in vivo in BxPC3-MUC1 tumors
Protein lysates from the tumors were analyzed for the epithelial and mesenchymal markers. Similar to the in vitro data in , the BxPC3 MUC1 tumor lysate showed downregulation of E-Cadherin () and upregulation of Slug, Snail, and Vimentin () clearly indicating the initiation of EMT in vivo. This process was completely abrogated when the tyrosines were mutated even though high levels of MUC1 protein was being expressed in the Y0 tumors ().
Significantly higher levels of pro-metastatic and pro-angiogenic growth factors in the BXPC3 MUC1 tumor microenvironment compared to Y0 or Neo tumors
To determine the effect of EMT on the tumor microenvironment itself, we tested the protein levels of some of the known mediators of metastasis and angiogenesis in the tumor lysates using the Ray Biotech protein array kit. Two-fold or more increase was considered significant and is represented (). Significant increases in levels of circulating VEGF (10-fold), insulin-like growth factor-1 (IGF-1, 2-fold), interleukin-6 (IL-6, 3-fold) and its receptor (IL-6R, 2.2-fold), stem cell factor (SCF, 2.5 fold), P-selectin (20-fold), and epidermal growth factor (EGF, 2.8-fold) were observed in the BxPC3 MUC1 tumors compared to the Neo and Y0 tumors. VEGF expression was also determined by IHC in the tumor sections with high levels detected in the BxPC3 MUC1 tumors versus Neo and Y0 tumors (). Thus, BxPC3 MUC1 xenografted tumors exhibited an aggressive phenotype consistent with the human tumors that express high MUC1. This included loss of E-cadherin, induction of Vimentin and upregulation of metastatic and angiogenic factors. It is striking that simply abrogating the signal transduction events by mutating the seven tyrosines can completely reverse the process of EMT and significantly alter the tumor microenvironment, subsequently leading to a less aggressive, less metastatic, and less angiogenic phenotype.
MUC1 interacts with β-catenin and translocates to the nucleus in the BxPC3-MUC1 but not in the Y0 cells
To delineate the underlying mechanism by which the Y0 tumors circumvent EMT, we tested the nuclear localization of MUC1 CT and β-catenin in the MUC1 and Y0 cells. It is known that tyrosine residues in MUC1 CT are critically important for nuclear localization of MUC1 CT and β-catenin (20
). Although β-catenin was present in the nuclear extracts in both cells, the levels were significantly higher in BxPC3 MUC1 cells compared to the Y0 cells (). Similar results were obtained with the KCM and KCKO cells where higher levels of β-catenin were detected in the nuclear extract of the KCM versus the KCKO cells (). More importantly, although both BxPC3 MUC1 and Y0 cells contained equal levels of MUC1 (), there was no detectable MUC1 CT in the nuclear extract of the Y0 cells while high levels was detected in the BxPC3 MUC1 cells (). The results were substantiated in Su86.86 MUC1 and Y0 cells (). The data clearly suggests that signaling through the tyrosines is critically important for efficient nuclear translocation of MUC1 CT and β-catenin. Purity of the nuclear extract was confirmed by the presence of lamin and the absence of IKK ().
MUC1 interacts with β-catenin and translocates to the nucleus in the MUC1-expressing cells. Complete reversal in the Y0 cells
Since tyrosine phosphorylation is required for MUC1 to bind β-catenin and translocate to the nucleus, we examined if β-catenin and MUC1 co-IP in these cells. β-catenin interacted with MUC1 only in the BxPC3-MUC1 cells but minimally in the Y0 cells (). The co-IP of β-catenin and MUC1 was confirmed in the KCM and KCKO cells and in the Su86.86 cells (). Thus, we confirm that binding of MUC1 to β-catenin appears to provide the signal required for movement of MUC1 to the nucleus and that the tyrosines in MUC1 CT have to be functional for the two proteins to interact.
Although it has been known for decades that MUC1 is aberrantly over-expressed in greater than 95% of metastatic PDA and is associated with poor prognosis, its precise role has remained obscure. We show for the first time in both mouse and human pancreatic tumors that 1) over-expression of MUC1 initiates the process of EMT and augments metastasis and 2) lack of tyrosines in the CT of MUC1 abrogates this process. The first evidence of EMT came from the proteomics data showing repression of E-Cadherin and induction of Vimentin expression in the PDA.MUC1 tumors when compared to the PDA.Muc1KO tumors. This correlated with significantly higher incidence of secondary metastasis in PDA.MUC1 mice as compared to the PDA.Muc1KO mice. The PDA mice are unique in that the pancreatic tumors arise spontaneously in an appropriate tissue background, within a suitable stromal and hormonal milieu, and in the context of a viable immune system (17
). The tumor progression and the histopathology of the tumors in the PDA mice mimic the human disease with the initial development of pancreatic intra-epithelial neoplastic (PanIN) lesions progressing to carcinoma-in-situ (CIS), and invasive adenocarcinoma. The process of EMT and increased invasiveness in MUC1-expressing pancreatic tumors was confirmed in cell lines generated from the PDA mice and validated in two human pancreatic cell lines. The underlying molecular mechanism in all cell lines pointed to the induction of transcription factors, Snail, and Slug which stimulated the expression of Vimentin and repressed E-Cadherin expression. E-Cadherin plays a key role in the establishment and maintenance of adherent junctions and loss of the same results in contact inhibition and cell motility. Interestingly, the molecular process of EMT was completely abrogated when all seven tyrosines in the CT of MUC1 were mutated to phenylalanine. Note that the cytoplasmic tail of MUC1 Y0 shows a shift in electrophoretic mobility relative to MUC1 WT; this likely reflects a decrease in phosphorylation due to mutation of the tyrosine residues and therefore change in the overall charge. This has been previously reported in other cell types (28
). We do not believe that the change in the electropheretic mobility is due to size since the delta between seven tyrosines and seven phenylalanines is ~67 daltons. Recent evidence shows that the phosphorylation of the tyrosines in the MUC1 CT are critical for the binding of β-catenin to MUC1 CT and that the MUC1 CT-β-catenin complex can be translocated to the nucleus to render its oncogenic signal (29
). We show that lack of tyrosines in the MUC1 CT regulates the interaction between MUC1 CT with β-catenin, and therefore its efficient translocation to the nucleus (). It is also known that β-catenin binds to the SXXXXXSSL motif in the CT of MUC1 (30
) and changes in tyrosine phosphorylation of MUC1 CT correlated with differences in cell adhesion (30
). Thus, we can speculate that the underlying mechanism for EMT might be the requirement of the tyrosines adjacent to the β-catenin binding domain (possibly at the TDRSPYEKV site) to be phosphorylated. C-src and PKCδ have been shown to phosphorylate the tyrosines at that site and increase the interaction between β-catenin and MUC1 (24
). We therefore propose that MUC1 may directly influence the transcriptional co-activator status of β-catenin, and up-regulate several genes that are associated with EMT such as Snail, Slug, Twist, Vimentin and Goosecoid (), possibly through interactions with TCF/LEF1 and/or other transcription factors. Snail in turn represses E-cadherin message and protein levels in MUC1-expressing cells (–), perhaps through direct binding to the three E-boxes in the E-cadherin promoter (6
). E-cadherin plays a key role in the establishment and maintenance of adherence junctions, repression of which leads to down regulation of Tight Junction Proteins 1 and 3, Occludin, Laminin α5, and β-and δ-catenins (). Dissolution of pancreatic epithelial cell junctions is hence brought about by a concerted activation of mesenchymal proteins and repression of epithelial proteins in vitro
and in vivo
leading to EMT and metastasis exemplified by the presence of CTCs in BxPC3.MUC1 tumors () and enhanced metastasis in the PDA.MUC1 mice ().
These molecular changes lead to an in vivo
microenvironment favoring increased angiogenic and pro-inflammatory cytokines in the MUC1 tumors (), leading to increased metastasis. The glycoprotein P-selectin, an adhesion molecule involved in the initiation of inflammatory process and induction of IL-6 and its receptors (33
) was increased by 20 fold in MUC1 versus Neo tumors (). VEGF, a potent inducer of angiogenesis and promoter of tumor progression (35
) was increased by 10-fold in the MUC1 tumors (). Lastly, SCF, EGF and IGF-1, factors known to induce growth and proliferation in pancreatic cancer cells (36
) were increased by 2–3 fold in MUC1 tumors (). Thus, it is indeed striking that the significant induction of these factors in the MUC1 tumors were completely abrogated when the tyrosines in MUC1 CT were deemed non-functional (). This once again signifies the critical requirement of the MUC1 CT tyrosines in pancreatic cancer oncogenesis and metastasis.
The regulation of MUC1 CT phosphorylation and parameters that affect its association with β-catenin are not fully understood, although interaction of MUC1 with ErbB1 (39
) has been shown to enhance the binding between c-src, β-catenin and MUC1 (20
) and increase ERK1/2 phosphorylation and NFκB activation (28
). Preliminary data from our laboratory shows that the active form of NFκB-p65 subunit translocation to the nucleus is negligible in the Y0 cells compared to the MUC1 cells (unpublished data). Our study exemplifies the functional consequences (EMT and metastasis) of signaling through MUC1 CT, and for the first time delineates the role of this protein in a unique mouse model of PDA that lacks Muc1. The data defines the role of MUC1 signaling (through its CT tyrosines) in the initiation of EMT perhaps by β-catenin-MUC1 interaction and translocation to the nucleus, leading to activation of the EMT-associated transcription factors Snail and Slug. This in turn enables the tumors to acquire a highly aggressive phenotype creating a pro-metastatic microenvironment in vivo
. Future studies will focus on identifying the particular tyrosine/s within MUC1 CT and determining if inhibiting its phosphorylation using small molecule kinase inhibitors may lead to a novel treatment modality for pancreatic cancer.