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Cell Cycle. Nov 1, 2011; 10(21): 3692–3700.
Published online Nov 1, 2011. doi:  10.4161/cc.10.21.17895
PMCID: PMC3266007
Caveolin-1 promotes pancreatic cancer cell differentiation and restores membranous E-cadherin via suppression of the epithelial-mesenchymal transition
Ahmed F Salem,1,2,3 Gloria Bonuccelli,1 Generoso Bevilacqua,2 Hwyda Arafat,4 Richard G Pestell,1 Federica Sotgia,1 and Michael P Lisanticorresponding author1
1Departments of Stem Cell Biology and Regenerative Medicine and Cancer Biology; Kimmel Cancer Center; Philadelphia, PA USA
4Department of Surgery; Jefferson Pancreatic, Biliary and Related Cancer Center; Thomas Jefferson University; Philadelphia, PA USA
2Division of Surgical, Molecular and Ultrastructural Pathology; University of Pisa; Pisa, Italy
3Department of Molecular Drug Evaluation; Division of Biochemistry; National Organization for Drug Control and Research; Giza, Egypt
corresponding authorCorresponding author.
Correspondence to: Michael P. Lisanti; Email: Michael.Lisanti/at/kimmelcancercenter.org
Received August 24, 2011; Accepted August 29, 2011.
Pancreatic cancer is one of the deadliest cancers due to early rapid metastasis and chemoresistance. Recently, epithelial to mesenchymal transition (EMT) was shown to play a key role in the pathogenesis of pancreatic cancer. To understand the role of caveolin-1 (Cav-1) in EMT, we overexpressed Cav-1 in a pancreatic cancer cell line, Panc 10.05, that does not normally express Cav-1. Here, we show that Cav-1 expression in pancreatic cancer cells induces an epithelial phenotype and promotes cell-cell contact, with increased expression of plasma membrane bound E-cadherin and β-catenin. Mechanistically, Cav-1 induces Snail downregulation and decreased activation of AKT, MAPK and TGFβ-Smad signaling pathways. In vitro, Cav-1 expression reduces cell migration and invasion, and attenuates doxorubicin-chemoresistance of pancreatic cancer cells. Importantly, in vivo studies revealed that Cav-1 expression greatly suppresses tumor formation in a xenograft model. Most interestingly, Panc/Cav-1 tumors displayed organized nests of differentiated cells that were totally absent in control tumors. Confirming our in vitro results, these nests of differentiated cells showed reexpression of E-cadherin and β-catenin at the cell membrane. Thus, we provide evidence that Cav-1 functions as a crucial modulator of EMT and cell differentiation in pancreatic cancer.
Key words: caveolae, caveolin-1, epithelial-mesenchymal transition, E-cadherin, pancreatic cancer, cell differentiation, chemoresistance
Caveolin-1 (Cav-1) is the main constituent molecule of caveolae, which are omega-shaped invaginations found at the cell membrane.1 Caveolae are thought to function as signaling platforms regulating the activation of several signaling pathways. As an example, many receptor and non-receptor tyrosine kinases are localized in caveolae, such as hepatocyte growth factor (HGF) receptor, epidermal growth factor (EGF) receptor, transforming growth factorβ (TGFβ) receptor and c-Src.2,3 Cav-1 binds and tonically inhibits the activation of such signaling proteins. As a consequence, many studies recognize Cav-1 as a modulator of cell transformation, proliferation and metastasis.4,5 Depending on the tissue context, Cav-1 can act either as a tumor suppressor, for example in pulmonary,6,7 mammary,8,9 colon10 and ovarian11 cancers, or as a tumor promoter, for example in prostate,12 bladder13 and renal14 cancers.
Epithelial-mesenchymal transition (EMT) is defined as the ability of epithelial cells to convert from a polarized morphology to a loose mesenchymal phenotype.15 This transition occurs through numerous cellular and molecular alterations and allows the transient cell to degrade the basement membrane leading to increased cell mobility. As a consequence, EMT is thought to play an important role during cancer cell invasion. Furthermore, Arumugam et al. demonstrated that cells undergoing EMT are more resistant to cytotoxic drugs.16 Secreted growth factors, notably TGFβ, HGF, EGF and PDGF, are responsible for the activation of downstream pathways, such as PI3K/AKT, ERK/MAPK and Smads pathways leading to the activation of EMT effectors, namely Snail, Slug, zinc finger E-box-binding homeobox 1 (ZEB1) and ZEB2.1720 During the EMT process, membranous E-cadherin is endocytosed and downregulated, which causes cells to lose their cell junctions and to release β-catenin into the cytoplasm. β-catenin translocates to the nucleus, promoting invasiveness and chemoresistance.
In this study, we present evidence that Cav-1 is a crucial modulator of EMT and cell differentiation in pancreatic cancer cells. First, we assessed Cav-1 mRNA and protein levels in eight pancreatic cell lines. Then, using a retroviral approach we expressed Cav-1 in the Panc 10.05 cancer cell line that showed the least Cav-1 expression. As a consequence of Cav-1 expression, Panc 10.05 cells acquired an epithelial morphology and displayed more cell-cell contacts than control cells. Also, Cav-1 expression was sufficient to increase the levels of E-cadherin and β-catenin and to promote their localization at the cell membrane. Mechanistically, the expression of the E-cadherin inhibitor Snail was greatly diminished by Cav-1. In addition, Cav-1 expressing cells displayed decreased activation of ERK, Smad2 and AKT pathways as compared with control cells. In vitro, Cav-1 expression potently decreased pancreatic cancer cell migration and invasion, and sensitized cancer cells toward doxorubicin-induced cell death. In an in vivo xenograft model, Cav-1 expression greatly blocked tumor formation of pancreatic cancer cells. More interestingly, Cav-1 expressing tumors possessed nests of differentiated cells, which were totally absent in control tumors. These nests of differentiated cells exhibited strong E-cadherin and β-catenin expression at the plasma membrane with a specific absence of Snail.
Assessment of Cav-1 expression.
Cav-1 expression was evaluated in a panel of pancreatic cell lines by real time PCR (Fig. 1A) and protein gel blot analysis (Fig. 1B). Although Panc 10.05 cells display detectable Cav-1 mRNA levels, they failed to express Cav-1 protein when compared with other cell lines (Fig. 1A and B). So, to study Cav-1 function on pancreatic cancer cells, Cav-1 overexpression was established in Panc 10.05 cells using a retroviral approach (Panc10/Cav-1) (Fig. 1C). Control cells carrying the empty vector (Panc10/pBabe) were established in parallel.
Figure 1
Figure 1
Cav-1 overexpression in human Panc10.5 pancreatic cancer cells. (A and B) Cav-1 expression is undetectable in Panc10.5 cells. (A) Real-time PCR was performed to assess Cav-1 mRNA levels on the indicated eight pancreatic cell lines mRNA. Normalization (more ...)
Cav-1 induces epithelial differentiation, with increased expression of membrane bound E-cadherin and β-catenin.
We immediately noticed that Panc10/Cav-1 exhibited a different morphology than Panc10/pBabe control cells. Panc10/Cav-1 exhibited cell-cell contacts, and an epithelial-like polygonal shape. In contrast, Panc10/pBabe cells grew individually as single cell and exhibited a fibroblastic shape (Fig. 2A). To directly assess if Cav-1 expression induced an epithelial morphology, we evaluated the expression of epithelial markers, such as E-cadherin and β-catenin. Protein gel blot analysis demonstrated that Panc10/Cav-1 cells display elevated expression levels of E-cadherin and β-catenin (Fig. 2B). Furthermore, immunofluorescence analysis demonstrated that E-cadherin is localized at the plasma membrane in Panc10/Cav-1 cells (Fig. 2C). As a consequence of E-cadherin expression, β-catenin expression was restored and stabilized at the plasma membrane of Panc10/Cav-1 cells (Fig. 2C). This data indicate that Cav-1 expression profoundly alters cellular morphology, induces an epithelial phenotype and thus may suppress EMT.
Figure 2
Figure 2
Cav-1 alters pancreatic cancer cell morphology and restores the expression of E-cadherin and β-catenin at the plasma membrane. (A) Phase contrast microscopy. Control Panc10/pBabe cells display spindle-shape morphology, and preferentially grow (more ...)
Cav-1 suppresses EMT pathways.
To investigate the mechanisms by which Cav-1 restores E-cadherin expression and suppresses EMT, we performed immunoblotting analysis with several critical molecules that trigger EMT in pancreatic cancer cells. Previous studies have highlighted the role of ERK and Smads in EMT induction.21 Interestingly, Cav-1 expression suppresses the activation of phospho-ERK and phospho-Smad2 (Fig. 3A). Studies have also shown that the AKT pathway has a profound influence on EMT induction.18 Mainly, AKT regulates the expression of the transcriptional factor, Snail, which downregulates E-cadherin and initiates EMT. Interestingly, the levels of phospho-AKT and total AKT were also decreased in Panc10/Cav-1 cells (Fig. 3B). In addition, Snail levels were downregulated in cells expressing Cav-1 compared with control cells (Fig. 3B).
Figure 3
Figure 3
Cav-1 suppresses the activation of key pathways triggering EMT. Protein gel blot analysis was performed on Panc10/pBabe and Panc10/Cav-1 cell lysates with antibodies against key regulators of EMT. (A) The levels of phospho-ER K and phospho-Smad2 are decreased (more ...)
To directly evaluate AKT activity, we performed an AKT activity assay on Panc10/Cav-1 and Panc10/pBabe cells. The AKT activity assay demonstrates reduced phospho-GSK-3 levels in Panc10/Cav-1 cells compared with Panc10/pBabe cells (Fig. 3C), suggesting reduced AKT activity in Cav-1 expressing cells.
Cav-1 attenuates migration and invasion, and reduces drug resistance of pancreatic cancer cells.
AKT and ERK pathways play an important role in cancer cells invasion and migration. As Cav-1 suppresses the activation of AKT and ERK pathways, we next asked if Cav-1 hinders cell migration and invasion potentials. To this end, Panc10/Cav-1 and Panc10/pBabe cells were seeded over 8 µm-pore Transwells, which were not coated or coated with Matrigel for migration and invasion, respectively. As expected, Cav-1 expression dramatically attenuated migration and invasion capacity by approximately 2.5 and 16-fold, respectively, when compared with Panc10/pBabe (Fig. 4A).
Figure 4
Figure 4
Cav-1 attenuates pancreatic cancer cells migration, invasion and drug resistance. (A) Cav-1 suppresses migration and invasion potentials of Panc10.5 cells by 2.5- and 16-fold respectively. Column, average of migrating or invading number of cells per low (more ...)
Doxorubicin is a promising cytotoxic drug, especially when combined with AKT inhibitors.22 As we observed above that Cav-1 inhibits AKT activity, we next attempted to evaluate if Cav-1 expression altered chemosensitivity to doxorubicin. To this end, increasing concentrations of doxorubicin were added to Panc10/Cav-1 cells or vector alone control cells and cell survival was detected using an MTT assay. Interestingly, Panc10/Cav-1 cells displayed significantly higher sensitivity to doxorubicin than Panc10/pBabe cells (Fig. 4B), suggesting that Cav-1 expression significantly reduces drug resistance of pancreatic cancer cells.
Cav-1 expression blocks tumorigenesis.
To evaluate if Cav-1 impairs the tumorigenic potentials of Panc 10.05 cell line, Panc10/Cav-1 cells and Panc10/pBabe cells were injected into the flanks of nude mice. After 7 weeks, tumors were extracted, measured and weighed. Surprisingly, Cav-1 expression decreased tumor weight and volume by about 6- and 4-fold, respectively, compared with control tumors (Fig. 5).
Figure 5
Figure 5
Cav-1 blocks tumor formation. Panc10/pBabe and Panc/Cav-1 cells were injected into the flank of nude mice (n = 9). After 7 weeks, tumors were collected, measured and weigh. Note that Cav-1 expression significantly decreases tumors weight and volume by (more ...)
Cav-1 expression restores cells differentiation.
In order to assess if Cav-1 expression restores differentiation of pancreatic cancer cells in vivo, we subjected Panc10/Cav-1 and Panc10/pBabe tumors to detailed histological evaluation. Astonishingly, H&E staining demonstrated that Panc10/Cav-1 tumors displayed multiple nests of organized cells displaying semi differentiation toward squamous architecture (Fig. 6A). These nests of organized cells were surrounded by eosinophilic fibroblasts, giving a structure of epithelial cells surrounded by a basement membrane. These nests were found in 7 out of 9 (80%) Cav-1 expressing tumors, and were totally absent in Panc/pBabe tumors. These findings clearly indicate that Cav-1 plays a critical role in restoring cell differentiation and also that these differentiated cells are able to recruit fibroblasts from the environment to act as a basement membrane.
Figure 6
Figure 6
Cav-1 promotes cell differentiation and restores the expression of E-cadherin and β-catenin at the plasma membrane. (A) H&E staining of Panc10/pBabe and Panc10/Cav-1 tumors shows that Cav-1 restores cancer cell differentiation. Note that (more ...)
Cav-1 restores E-cadherin and β-catenin expression in differentiated tumor nests.
To directly examine the differentiation status of these nests of organized cells, Panc10/Cav-1 and Panc10/pBabe tumors were analyzed by immunohistochemistry with E-cadherin and β-catenin antibodies. Importantly, differentiated cells in Cav-1 expressing tumors showed high membranous staining of E-cadherin and β-catenin (Fig. 6B). Moreover, Snail expression was absent in the nest cells compared with the surrounding undifferentiated cells. All of the in vivo results are consistent with in vitro results, and clearly indicate the critical role of Cav-1 in suppression of EMT in pancreatic cancer.
Pancreatic cancer is considered to be the fourth leading cause of cancer related death due to its drug resistance and rapid metastasis.23 The aim of this study was to evaluate the role of Cav-1 in the regulation of epithelial to mesenchymal transition (EMT) in pancreatic cancer. Importantly, EMT has been shown to play an important role in aggressive pancreatic cancer progression.23 EMT is a biological and molecular process in which epithelial cells lose cell polarity and gain a fibroblastic spindle-shape morphology allowing them to infiltrate tissues and invade organs.24
Interestingly, here we show that enforcing Cav-1 expression induced profound alterations in the morphology of Panc 10.05 pancreatic cancer cells. Panc 10.05 cells expressing Cav-1 displayed cell-cell adherens, which were absent in the spindle-shape control cells. To better characterize the phenotype, we then examined the levels of E-cadherin, which is responsible for cell adherence and tight junctions. Protein gel blot and immunofluorescence analysis indicated that E-cadherin expression was undetectable in Panc10/pBabe cells but was readily restored in Panc10/Cav-1 cells. Of great interest, E-cadherin was localized at the cell membrane in Panc10/Cav-1 cells. Restoration of E-cadherin expression is critical, as loss of E-cadherin correlates with undifferentiated and anaplastic pancreatic tumors leading to worse prognosis as compared with differentiated pancreatic tumors that express E-cadherin.25
It is well known that β-catenin-dependent transcription plays an important role in the initiation of EMT and invasion of cancer cells.26 Interestingly, we show here that Cav-1 expression was sufficient to restore the expression of β-catenin at the plasma membrane. Consistent with these findings, we have previously reported that Cav-1 stabilizes β-catenin at the cell membrane, preventing the formation of the complex of β-catenin with the transcription factor Lef-1, β-catenin nuclear translocation and activation of gene transcription.27
β-catenin-dependent transcription is also promoted by Snail, that has a reciprocal correlation with E-cadherin expression.28,29 Snail transcriptionally represses E-cadherin by recruiting histone deacetylases (HDAC) to the E-boxes of the E-cadherin promoter.30 Interestingly, Yin et al. showed that Snail overexpression correlated with lymph node invasion and distant metastasis in human pancreatic cancer.31 Also, Snail overexpression in Panc-1 pancreatic cancer cells enhanced metastatic potential after orthotopic injection in the pancreas of nude mice and promoted chemoresistance to 5-fluorouracil or gemcitabine. Here, we present compelling evidence showing that Cav-1 is sufficient to downregulate Snail expression, and to sensitize pancreatic cancer cells to chemotherapy.
To mechanistically understand how Cav-1 affects Snail expression, we investigated the three main molecules responsible for Snail activation, namely ERK, Smad2 and AKT, which are involved in the mitogenactivated protein kinase (MAPK), TGFβ and phosphatidylinositol-3-kinase (PI3K)/AKT pathways, respectively.18,3234 All three pathways are key for Snail activation and EMT initiation and maintenance. Li et al. demonstrated that E-cadherin re-expression in breast cancer cells was not sufficient to revert the EMT process unless ERK was suppressed.35 Moreover, previous studies have shown that MAPK modulates the role of TGFβ to act as a tumor promoter in human pancreatic cancer cells.36 TGFβ pathway phosphorylates and activates Smad2, inducing Smad2 nuclear translocation and binding to transcriptional factors, such ZEB1 or ZEB2, that control EMT.37 AKT pathway is not only an EMT inducer, but also it is a pro-survival and metastasis promoter molecule.18 Remarkably, our results show that Cav-1 greatly decreases the activation of ERK and Smad2 and AKT, restoring epithelial cell morphology.
We also found that Cav-1 expression altered certain key features associated with EMT, such as migration, invasion and chemoresistance. Consistent with our current results, a previous study has demonstrated that Cav-1 suppresses migration and invasion in pancreatic cancer cells by inhibition of MAPK signaling pathway.38
Chemoresistance is a serious concern in the clinical management of pancreatic cancer patients. Many previous studies indicated that the pro-survival AKT pathway is responsible for doxorubicin resistance in breast, lung, gastric and uterine cancer.22,3942 Moreover, recent studies implied that drug resistance in pancreatic cancer inversely correlates with E-cadherin expression and re-expression of E-cadherin sensitizes pancreatic cancer cells to cytotoxic drugs.16 Importantly, we show here that Cav-1 downregulated AKT expression and activation, and sensitized pancreatic cancer cells to doxorubicin-induced cell death. Also, Arumugam et al. indicated that EMT induces chemoresistant toward gemcitabine and 5-flurouracil in pancreatic cancer cells.16 Consistent with these data, we show here that Cav-1 expression inhibits EMT process and leads to cancer cell chemosensitization.
Previous studies demonstrated that Cav-1 inhibits tumor growth.5,38 Our current results show that Cav-1 expression greatly blocks tumor formation in an in vivo xenograft model. More interestingly, Cav-1 expressing tumors displayed nests of differentiated cells, which were completely absent in control tumors. These nests of differentiated cells expressed high levels of E-cadherin and β-catenin at the plasma membrane. These remarkable findings suggest a critical role of Cav-1 in cell differentiation and epithelial cell plasticity.
Although our results describe Cav-1 as a tumor suppressor in pancreatic cancer, clinical data portrays Cav-1 as a tumor promoter and high Cav-1 expression correlates with high tumor grade.43 However, Cav-1 is not an independent prognostic factor in pancreatic cancer and predicts survival only when combined with other biomarkers, such as FASN.43 This discrepancy may be explained by the fact that in higher tumor grades Cav-1 loses its tumor suppression role or gains an oncogenic function, potentially by genetic mutations.44 A biphasic differential expression of Cav-1 was previously suggested in other types of human cancers, including oral cancers, where Cav-1 is highly expressed in early stage disease, but lost in metastatic and advanced lesions.45 Another possible explanation is that E-cadherin expression is necessary for Cav-1 to act as a tumor suppressor, as E-cadherin inhibits the β-catenin-TCF oncogenic pathway.46 Loss of E-cadherin in high grade tumors may promote Cav-1 interaction with other partners, such as FASN, and induce an oncogenic switch in Cav-1 function. However, the relation of Cav-1 and E-cadherin appears to be important to determine the behavior of Cav-1, which in turn deeply affects the behavior of pancreatic cancer cells.
In conclusion, our data demonstrate that Cav-1 plays a critical role in promoting pancreatic cancer cells differentiation, and implicate that Cav-1 may be a promising therapy for pancreatic cancer. We showed that Cav-1 restored the epithelial status of pancreatic cancer cells, cell differentiation and maintained E-cadherin at plasma membrane. Delivery of Cav-1 by gene therapy or by peptide administration may hold the promise to effectively treat or retard pancreatic cancer progression. For example, systemic administration of a cell-permeable Cav-1 peptide has been employed to ameliorate signs of lung fibrosis in a pre-clinical model of scleroderma, in whose pathogenesis a loss of Cav-1 plays a crucial role. Thus, restoration of Cav-1 function by treatment with a Cav-1 peptide may be a novel therapeutic approach for pancreatic cancer.
Cell culture.
Panc 10.05, Mia Paca, BxPC3, Aspc-1, HPAF II and HS766T cell lines were purchased from American Type Culture Collection (ATCC). PK9 cells were a kind gift of Scott Kern (John Hopkins University). Human pancreatic duct epithelial (HPDE) cell were a kind gift of Dr. Ming-Sound Tsao (University Health Network, Canada). All cell lines were maintained at 37°C in 5% CO2 and grown in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS), except Panc 10.05 cells which were also supplemented with 10 IU/ml of human recombinant insulin (Sigma-Aldrich). ATCC routinely performs DNA profiling to authenticate their cell lines. For all the in vitro and in vivo experiments, only early passages of these cells (passages 5–6) were used.
Real-time PCR analysis.
mRNA was extracted from all cell lines using Trizol (Sigma-Aldrich). cDNA was synthesized from the purified mRNA using SuperScript III First-Strand (Invitrogen) according to the manufacture's instruction. Cav-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes were purchased as “assays on demand” (Applied Biosystems) and GAPDH was used as housekeeping gene. cDNA was prepared, and subjected to real-time PCR using the TaqMan technology (7500 Sequence Detector; Applied Biosystems). Experiments were performed in duplicates.
Stable retroviral transfection.
Full-length Cav-1 gene was subcloned into the pBabe retroviral vector using standard PCR techniques.47 Then, Phoenix amphotropic packaging cells were transfected with pBabe vectors using a modified calcium phosphate method.48 48 h after transfection, viral supernatants were collected, filtered and added to Panc 10.05 cells. Two cycles of infection were performed every 12 h. For selection, puromycin was added at a final concentration of 2.5 µg/ml. Finally, Cav-1 expression was confirmed by protein gel blot analysis.
Protein gel blotting.
Cells were lysed using RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Sodium Deoxycholate, 1.0% Triton X-100, 0.1% SDS), plus protease inhibitors (Roche Diagnostics) and phosphatase inhibitors (Sigma-Aldrich). Cell lysates were centrifuged to remove cell debris. Protein quantification was achieved by BCA protein assay (Pierce). Proteins (50 µg) were separated by 10–12% SDS-PAGE electrophoresis and transferred overnight at 4°C to nitrocellulose membrane (Whatman). Antibodies used for immunoblotting were as follow: rabbit polyclonal anti-Cav-1 (N-20), and mouse monoclonal anti-GAPDH from Santa Cruz Biotechnology; mouse monoclonal anti-E-cadherin, mouse monoclonal anti-β-catenin and mouse monoclonal anti-Akt from BD Bioscience; rabbit polyclonal anti-Snail, rabbit polyclonal anti-Smad2, rabbit polyclonal antiphospho-Smad2 (465/7), rabbit polyclonal anti-Erk1/2, rabbit polyclonal anti-phospho-Erk1/2 (202/4), and rabbit polyclonal anti-phospho-Akt (Ser473) from Cell Signaling Technologies. The same antibodies were also used for immunofluorescence and immunohistochemistry.
Immunofluorescence.
Panc10.05 cells were plated on coverslips in 12-well plates and cultured for two days. Then, cells were rinsed with PBS with 0.1 mM CaCl2 and 1 mM MgCl2 (PBS/CM), and fixed with 2% paraformaldehyde in PBS/CM for 30 min. After fixation, cells were washed three times with PBS/CM and permeabilized with IF buffer (PBS/CM with 0.1% Triton-X100 and 0.2% bovine serum albumin) for 10 min. Then, cells were quenched with 50 mM NH4Cl in PBS/CM for 10 min, rinsed and incubated with anti-Cav-1 (1:500), or anti-E-cadherin (1:200), or anti-β-catenin (1:200) antibodies for 1 h at room temperature. Then, cells were washed with IF buffer and incubated with fluorescent secondary antibodies (Molecular Probes) for 30 min. After washing, cells were incubated with Hoechst 33342 nuclear staining (Molecular Probes), rinsed and mounted with ProLong Gold anti-fade (Molecular Probes). Images were acquired with a Zeiss LSM510 Meta confocal microscope system and analyzed with Zeiss LSM Browser (3,5,0,359; Zeiss).
Akt activity.
Akt kinase activity was measured using a nonradioactive AKT Kinase assay kit (Cell Signaling), according to the manufacture's instruction. Briefly, cell lysates were incubated with immobilized Akt antibody beads overnight at 4°C. Next day, samples were gently centrifuged and pellets were washed twice. Pellets were resuspended in kinase buffer and incubated with the glycogen synthase kinase-3 (GSK-3) fusion protein in the presence of 10 mM ATP for 30 min at 30°C. The reaction was stopped with 25 µl of 3x SDS sample buffer. Then, samples were analyzed by protein gel blotting with phospho-GSK-3 (Ser21/9) antibody (Cell Signaling).
Migration and invasion assays.
Cell migration and invasion were measured in vitro using a modified Boyden chamber assay.49,50 Briefly, 2.5 × 104 Panc10/Cav-1 and Panc10/pBabe cells were resuspended in 0.5 ml of serum-free RPMI-1640, and added to the 8 µm-pore upper chamber (BD Biosciences). The upper chambers were either coated with Matrigel (for invasion assay) or not coated (for migration assay). The lower chambers containing RPMI-1640 medium supplemented with 10% FBS served as a chemoattractant. Cells were incubated at 37°C for 10 h or 20 h for migration or invasion, respectively. The non-migrating/non-invasive cells on top of the upper chamber were carefully removed using a cotton swap, and the remaining cells were stained with 0.5% crystal violet dissolved in methanol for 30–60 min. Chambers were rinsed with water, dried and then examined under a bright-field microscope. Invasion and migration potentials were evaluated by counting 5 fields for each experimental condition. The assays were performed in triplicates at different time points.
Drug sensitivity assay.
Cells were plated in 96-well dishes. After 24 h, doxorubicin hydrochloride (Sigma-Aldrich) was added to the cells at the indicated concentrations and incubated for 48 h. Cells survival was detected using CellTiter 96 non-Radioactive Cell proliferation Assay kit (MTT assay, Promega). Cells survival was detected at a 570 nm wavelength according to the manufacture's instruction. Samples were run in triplicates.
Animal studies.
Animal studies were conducted according to the guidelines of the National Institutes of Health and the Institutional Animal Care and Use Committee. Panc10/Cav-1 and Panc10/pBabe cells (1 × 106 cells) were resuspended in 100 µl sterile PBS and injected into the left flank of male nude mice (8 weeks of age obtained from NIH, N=9 mice for each group). After 7 weeks, tumors were harvested, weighted and measured. Volumes were calculated by assuming that a tumor is an ellipsoid using the formula V = 4/3 π a b2, where V is the tumor volume, a is the length of the long axis, and b is the length of the short axis. Finally, tumors were formalin-fixed and embedded in paraffin blocks for further histological and immunohistochemical analysis.
Immunohistochemistry.
Tumor sections were processed as we described before.44 Briefly, 5 µm sections were prepared from paraffin-embedded tumors. Then, sections were deparaffinized with xylene and rehydrated with graded concentration of ethanol. Antigen retrival was performed with 10mM sodium citrate buffer pH 6.0 for 10 min using an electric pressure cooker. Then, sections were incubated with 3% H2O2 for 10 min, washed and blocked with 10% goat serum in PBS for 1 h at room temperature (RT). After blocking, slides were incubated with anti-E-cadherin (1:400) or anti-b-catenin (1:200) or anti-Snail (1:250) antibodies in blocking solution overnight at 4°C. Then, slides were washed and incubated with biotinylated secondary antibodies for 30 min at RT. After washing with PBS, slides were incubated with streptoavidin-horseradish peroxidase reagent for 30 min at RT. Slides were then washed and incubated with the 3,3-diaminobenzidine reagent until developing a brown color. Finally, slides were washed, counterstained with hematoxylin, dehydrated, and mounted.
Statistical analysis.
Statistical significance was examined by Student t-test. Values of p < 0.05 were considered significant.
Acknowledgments
M.P.L. and his laboratory were supported by grants from the NIH/NCI (R01-CA-080250; R01-CA-098779; R01-CA-120876; R01-AR-055660), and the Susan G. Komen Breast Cancer Foundation. F.S. was supported by grants from the W.W. Smith Charitable Trust, the Breast Cancer Alliance (BCA), and a Research Scholar Grant from the American Cancer Society (ACS). Funds were also contributed by the Margaret Q. Landenberger Research Foundation (to M.P.L.). R.G.P. was supported by grants from the NIH/NCI (R01-CA-70896, R01-CA-75503, R01-CA-86072, and R01-CA-107382) and the Dr. Ralph and Marian C. Falk Medical Research Trust. The Kimmel Cancer Center was supported by the NIH/NCI Cancer Center Core grant P30-CA-56036 (to R.G.P.).
This project is funded, in part, under a grant with the Pennsylvania Department of Health (to M.P.L. and F.S.). The Department specifically disclaims responsibility for any analyses, interpretations or conclusions.
Abbreviations
Cav-1caveolin-1
EGFepidermal growth factor
EMTepithelial to mesenchymal transition
HGFhepatocyte growth factor
TGFβtransforming growth factorβ,

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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