Genome-Wide Screening Reveals an EMT Molecular Network Mediated by Sonic Hedgehog-Gli1 Signaling in Pancreatic Cancer Cells

Aims

The role of sonic hedgehog (SHH) in epithelial mesenchymal transition (EMT) of pancreatic cancer (PC) is known, however, its mechanism is unclear. Because SHH promotes tumor development predominantly through Gli1, we sought to understand its mechanism by identifying Gli1 targets in pancreatic cancer cells.

Methods

First, we investigated invasion, migration, and EMT in PC cells transfected with lentiviral Gli1 interference vectors or SHH over-expression vectors in vitro and in vivo. Next, we determined the target gene profiles of Gli1 in PC cells using cDNA microarray assays. Finally, the primary regulatory networks downstream of SHH-Gli1 signaling in PC cells were studied through functional analyses of these targets.

Results

Our results indicate there is decreased E-cadherin expression upon increased expression of SHH/Gli1. Migration of PC cells increased significantly in a dose-dependent manner within 24 hours of Gli1 expression (P<0.05). The ratio of liver metastasis and intrasplenic miniature metastasis increased markedly upon activation of SHH-Gli1 signals in nude mice. Using cDNA microarray, we identified 278 upregulated and 59 downregulated genes upon Gli1 expression in AsPC-1 cells. The data indicate that SHH-Gli1 signals promote EMT by mediating a complex signaling network including TGFβ, Ras, Wnt, growth factors, PI3K/AKT, integrins, transmembrane 4 superfamily (TM4SF), and S100A4.

Conclusion

Our results suggest that targeting the molecular connections established between SHH-Gli1 signaling and EMT could provide effective therapies for PC.     

Regeneration of the exocrine pancreas is delayed in telomere-dysfunctional mice

Introduction

Telomere shortening is a cell-intrinsic mechanism that limits cell proliferation by induction of DNA damage responses resulting either in apoptosis or cellular senescence. Shortening of telomeres has been shown to occur during human aging and in chronic diseases that accelerate cell turnover, such as chronic hepatitis. Telomere shortening can limit organ homeostasis and regeneration in response to injury. Whether the same holds true for pancreas regeneration in response to injury is not known.

Methods

In the present study, pancreatic regeneration after acute cerulein-induced pancreatitis was studied in late generation telomerase knockout mice with short telomeres compared to telomerase wild-type mice with long telomeres.

Results

Late generation telomerase knockout mice exhibited impaired exocrine pancreatic regeneration after acute pancreatitis as seen by persistence of metaplastic acinar cells and markedly reduced proliferation. The expression levels of p53 and p21 were not significantly increased in regenerating pancreas of late generation telomerase knockout mice compared to wild-type mice.

Conclusion

Our results indicate that pancreatic regeneration is limited in the context of telomere dysfunction without evidence for p53 checkpoint activation.     

Tenascin-C enhances pancreatic cancer cell growth and motility and affects cell adhesion through activation of the integrin pathway

Background

Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells.

Methods

Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition.

Results

Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt.

Conclusion

TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and might therefore play a role in PDAC spreading and metastasis in vivo.     

Anti-tumor activity of a novel compound-CDF is mediated by regulating miR-21, miR-200, and PTEN in pancreatic cancer

Background

The existence of cancer stem cells (CSCs) or cancer stem-like cells in a tumor mass is believed to be responsible for tumor recurrence because of their intrinsic and extrinsic drug-resistance characteristics. Therefore, targeted killing of CSCs would be a newer strategy for the prevention of tumor recurrence and/or treatment by overcoming drug-resistance. We have developed a novel synthetic compound-CDF, which showed greater bioavailability in animal tissues such as pancreas, and also induced cell growth inhibition and apoptosis, which was mediated by inactivation of NF-κB, COX-2, and VEGF in pancreatic cancer (PC) cells.

Methodology/Principal Findings

In the current study we showed, for the first time, that CDF could significantly inhibit the sphere-forming ability (pancreatospheres) of PC cells consistent with increased disintegration of pancreatospheres, which was associated with attenuation of CSC markers (CD44 and EpCAM), especially in gemcitabine-resistant (MIAPaCa-2) PC cells containing high proportion of CSCs consistent with increased miR-21 and decreased miR-200. In a xenograft mouse model of human PC, CDF treatment significantly inhibited tumor growth, which was associated with decreased NF-κB DNA binding activity, COX-2, and miR-21 expression, and increased PTEN and miR-200 expression in tumor remnants.

Conclusions/Significance

These results strongly suggest that the anti-tumor activity of CDF is associated with inhibition of CSC function via down-regulation of CSC-associated signaling pathways. Therefore, CDF could be useful for the prevention of tumor recurrence and/or treatment of PC with better treatment outcome in the future.     

Secretome-based identification of ULBP2 as a novel serum marker for pancreatic cancer detection

Background

To discover novel markers for improving the efficacy of pancreatic cancer (PC) diagnosis, the secretome of two PC cell lines (BxPC-3 and MIA PaCa-2) was profiled. UL16 binding protein 2 (ULBP2), one of the proteins identified in the PC cell secretome, was selected for evaluation as a biomarker for PC detection because its mRNA level was also found to be significantly elevated in PC tissues.

Methods

ULBP2 expression in PC tissues from 67 patients was studied by immunohistochemistry. ULBP2 serum levels in 154 PC patients and 142 healthy controls were measured by bead-based immunoassay, and the efficacy of serum ULBP2 for PC detection was compared with the widely used serological PC marker carbohydrate antigen 19-9 (CA 19-9).

Results

Immunohistochemical analyses revealed an elevated expression of ULPB2 in PC tissues compared with adjacent non-cancerous tissues. Meanwhile, the serum levels of ULBP2 among all PC patients (n = 154) and in early-stage cancer patients were significantly higher than those in healthy controls (p<0.0001). The combination of ULBP2 and CA 19-9 outperformed each marker alone in distinguishing PC patients from healthy individuals. Importantly, an analysis of the area under receiver operating characteristic curves showed that ULBP2 was superior to CA 19-9 in discriminating patients with early-stage PC from healthy controls.

Conclusions

Collectively, our results indicate that ULBP2 may represent a novel and useful serum biomarker for pancreatic cancer primary screening.     

Sonic Hedgehog-Gli1 Signaling Pathway Regulates the Epithelial Mesenchymal Transition (EMT) by Mediating a New Target Gene,S100A4, in Pancreatic Cancer Cells

Aims

The hedgehog signaling pathway plays an important role in EMT of pancreatic cancer cells, but the precise mechanisms remain elusive. Because S100A4 as a key EMT moleculer marker was found to be upregulated upon Gli1 in pancreatic cancer cells, we focused on the relationship between Shh-Gli1 signals and S100 genes family.

Methods

On the base of cDNA microarray data, we investigated regulating mechanism of Gli1 to some members of S100A genes family in pancreatic cancer cell lines firstly. Then, the regulation of Gli1 to S100A4 gene was studied by molecular biology assays and the pro-metastasis effection of Gli1-dependent S100A4 was investigated in vitro. Finally, the expressions of Shh, Gli1, S100A4 and E-cadherin in pancreatic cancer tissues were studied by using immunohistochemistry assays.

Results

Five members of the S100 genes family, S100A2, S100A4, S100A6, S100A11, and S100A14 were found to be downregulated significantly upon Gli1 knockdown. Gli1 enhancer prediction combining with in vitro data demonstrated that Gli1 primarily regulates S100A family members via cis-acting elements. Indeed, the data indicate S100A4 and vimentin genes were upregulated significantly by Shh/Gli1-expression increasing and E-cadherin was significantly reduced at the same time. Migration of PC cells was increased significantly in a dose-dependent manner of Gli1 expression (P<0.05) and siS100A4 significantly reversed the response of PC cells induced by L-Shh transduction (P<0.01).

Conclusion

Our data establish a novel connection between Shh-Gli1 signaling and S100A4 regulation, which imply that S100A4 might be one of the key factors in EMT mediated by Shh-Gli1 signaling in pancreatic cancer.     

Recruitment and activation of pancreatic stellate cells from the bone marrow in pancreatic cancer: a model of tumor-host interaction

Background and Aims

Chronic pancreatitis and pancreatic cancer are characterised by extensive stellate cell mediated fibrosis, and current therapeutic development includes targeting pancreatic cancer stroma and tumor-host interactions. Recent evidence has suggested that circulating bone marrow derived stem cells (BMDC) contribute to solid organs. We aimed to define the role of circulating haematopoietic cells in the normal and diseased pancreas.

Methods

Whole bone marrow was harvested from male β-actin-EGFP donor mice and transplanted into irradiated female recipient C57/BL6 mice. Chronic pancreatitis was induced with repeat injections of caerulein, while carcinogenesis was induced with an intrapancreatic injection of dimethylbenzanthracene (DMBA). Phenotype of engrafted donor-derived cells within the pancreas was assessed by immunohistochemistry, immunofluorescence and in situ hybridisation.

Results

GFP positive cells were visible in the exocrine pancreatic epithelia from 3 months post transplantation. These exhibited acinar morphology and were positive for amylase and peanut agglutinin. Mice administered caerulein developed chronic pancreatitis while DMBA mice exhibited precursor lesions and pancreatic cancer. No acinar cells were identified to be donor-derived upon cessation of cerulein treatment, however rare occurrences of bone marrow-derived acinar cells were observed during pancreatic regeneration. Increased recruitment of BMDC was observed within the desmoplastic stroma, contributing to the activated pancreatic stellate cell (PaSC) population in both diseases. Expression of stellate cell markers CELSR3, PBX1 and GFAP was observed in BMD cancer-associated PaSCs, however cancer-associated, but not pancreatitis-associated BMD PaSCs, expressed the cancer PaSC specific marker CELSR3.

Conclusions

This study demonstrates that BMDC can incorporate into the pancreas and adopt the differentiated state of the exocrine compartment. BMDC that contribute to the activated PaSC population in chronic pancreatitis and pancreatic cancer have different phenotypes, and may play important roles in these diseases. Further, bone marrow transplantation may provide a useful model for the study of tumor-host interactions in cancer and pancreatitis.     

Evaluation of a gene-directed enzyme-product therapy (GDEPT) in human pancreatic tumor cells and their use as in vivo models for pancreatic cancer

Background

Gene-directed enzyme prodrug therapy (GDEPT) is a two-step treatment protocol for solid tumors that involves the transfer of a gene encoding a prodrug-activating enzyme followed by administration of the inactive prodrug that is subsequently activated by the enzyme to its tumor toxic form. However, the establishment of such novel treatment regimes to combat pancreatic cancer requires defined and robust animal model systems.

Methods

Here, we comprehensively compared six human pancreatic cancer cell lines (PaCa-44, PANC-1, MIA PaCa-2, Hs-766T, Capan-2, and BxPc-3) in subcutaneous and orthotopical mouse models as well as in their susceptibility to different GDEPTs.

Results

Tumor uptake was 83% to 100% in the subcutaneous model and 60% to 100% in the orthotopical mouse model, except for Hs-766T cells, which did not grow orthotopically. Pathohistological analyses of the orthotopical models revealed an infiltrative growth of almost all tumors into the pancreas; however, the different cell lines gave rise to tumors with different morphological characteristics. All of the resultant tumors were positive for MUC-1 staining indicating their origin from glandular or ductal epithelium, but revealed scattered pan-cytokeratin staining. Transfer of the cytochrome P450 and cytosine deaminase suicide gene, respectively, into the pancreatic cancer cell lines using retroviral vector technology revealed high level infectibility of these cell lines and allowed the analysis of the sensitivity of these cells to the chemotherapeutic drugs ifosfamide and 5-fluorocytosine, respectively.

Conclusion

These data qualify the cell lines as part of valuable in vitro and in vivo models for the use in defined preclinical studies for pancreas tumor therapy.     

Zebrafish ProVEGF-C Expression,Proteolytic Processing and Inhibitory Effect of Unprocessed ProVEGF-C during Fin Regeneration

Background

In zebrafish, vascular endothelial growth factor-C precursor (proVEGF-C) processing occurs within the dibasic motif HSIIRR₂₁₄ suggesting the involvement of one or more basic amino acid-specific proprotein convertases (PCs) in this process. In the present study, we examined zebrafish proVEGF-C expression and processing and the effect of unprocessed proVEGF-C on caudal fin regeneration.

Methodology/Principal Findings

Cell transfection assays revealed that the cleavage of proVEGF-C, mainly mediated by the proprotein convertases Furin and PC5 and to a less degree by PACE4 and PC7, is abolished by PCs inhibitors or by mutation of its cleavage site (HSIIRR₂₁₄ into HSIISS₂₁₄). In vitro, unprocessed proVEGF-C failed to activate its signaling proteins Akt and ERK and to induce cell proliferation. In vivo, following caudal fin amputation, the induction of VEGF-C, Furin and PC5 expression occurs as early as 2 days post-amputation (dpa) with a maximum levels at 4–7 dpa. Using immunofluorescence staining we localized high expression of VEGF-C and the convertases Furin and PC5 surrounding the apical growth zone of the regenerating fin. While expression of wild-type proVEGF-C in this area had no effect, unprocessed proVEGF-C inhibited fin regeneration.

Conclusions/Significances

Taken together, these data indicate that zebrafish fin regeneration is associated with up-regulation of VEGF-C and the convertases Furin and PC5 and highlight the inhibitory effect of unprocessed proVEGF-C on fin regeneration.     

Insights into Pancreatic Cancer Etiology from Pathway Analysis of Genome-Wide Association Study Data

Background

Pancreatic cancer is the fourth leading cause of cancer death in the U.S. and the etiology of this highly lethal disease has not been well defined. To identify genetic susceptibility factors for pancreatic cancer, we conducted pathway analysis of genome-wide association study (GWAS) data in 3,141 pancreatic cancer patients and 3,367 controls with European ancestry.

Methods

Using the gene set ridge regression in association studies (GRASS) method, we analyzed 197 pathways identified from the Kyoto Encyclopedia of Genes and Genomes database. We used the logistic kernel machine (LKM) test to identify major contributing genes to each pathway. We conducted functional enrichment analysis of the most significant genes (P<0.01) using the Database for Annotation, Visualization, and Integrated Discovery (DAVID).

Results

Two pathways were significantly associated with risk of pancreatic cancer after adjusting for multiple comparisons (P<0.00025) and in replication testing: neuroactive ligand-receptor interaction, (Ps<0.00002), and the olfactory transduction pathway (P = 0.0001). LKM test identified four genes that were significantly associated with risk of pancreatic cancer after Bonferroni correction (P<1×10⁻⁵): ABO, HNF1A, OR13C4, and SHH. Functional enrichment analysis using DAVID consistently found the G protein-coupled receptor signaling pathway (including both neuroactive ligand-receptor interaction and olfactory transduction pathways) to be the most significant pathway for pancreatic cancer risk in this study population.

Conclusion

These novel findings provide new perspectives on genetic susceptibility to and molecular mechanisms of pancreatic cancer.     

Involvement of host stroma cells and tissue fibrosis in pancreatic tumor development in transgenic mice

Introduction

Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development.

Methods

Pancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFβ/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells.

Results

Inhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development.

Conclusions

The utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice.     

Irrelevance of Microsatellite Instability in the Epidemiology of Sporadic Pancreatic Ductal Adenocarcinoma

Background and Aims

Pancreatic cancer risk is increased in Lynch syndrome (LS) patients with mismatch repair gene defects predisposing to colonic and extracolonic cancers with microsatellite instability (MSI). However, the frequency of MSI pancreatic cancers has never been ascertained in consecutive, unselected clinical series, and their contribution to the sporadic and inherited burden of pancreatic cancer remains to be established. Aims of the study were to determine the prevalence of MSI in surgically resected pancreatic cancers in a multicentric, retrospective study, and to assess the occurrence of pancreatic cancer in LS.

Methods

MS-status was screened by a panel of 5 mononucleotide repeats (Bat26, Bat25, NR-21, NR-24 and NR-27) in 338 consecutive pancreatic ductal adenocarcinoma (PDAC), resected at two Italian and one German referral centres. The personal history of pancreatic cancer was assessed in an independent set of 58 probands with LS and in 138 first degree relatives who had cancers.

Results

Only one PDAC (0.3%) showed MSI. This was a medullary type cancer, with hMLH1-deficiency, and no identified germ-line mutation but methylation of hMLH1. Pancreatic cancer occurred in 5 (2.5%) LS patients. Histological sampling was available for 2 cases, revealing PDAC in one case and an ampullary cancer in the other one.

Conclusions

MSI prevalence is negligible in sporadic, resected PDAC. Differently, the prevalence of pancreatic cancer is 2.5% in LS patients, and cancers other than PDAC may be encountered in this setting. Surveillance for pancreatic cancer should be advised in LS mutation carriers at referral centers.     

RLIP76 regulates PI3K/Akt signaling and chemo-radiotherapy resistance in pancreatic cancer

Purpose

Pancreatic cancer is an aggressive malignancy with characteristic metastatic course of disease and resistance to conventional chemo-radiotherapy. RLIP76 is a multi-functional cell membrane protein that functions as a major mercapturic acid pathway transporter as well as key regulator of receptor-ligand complexes. In this regard, we investigated the significance of targeting RLIP76 on PI3K/Akt pathway and mechanisms regulating response to chemo-radiotherapy.

Research Design and Methods

Cell survival was assessed by MTT and colony forming assays. Cellular levels of proteins and phosphorylation was determined by Western blot analyses. The impact on apoptosis was determined by TUNEL assay. The anti-cancer effects of RLIP76 targeted interventions in vivo were determined using mice xenograft model of the pancreatic cancer. The regulation of doxorubicin transport and radiation sensitivity were determined by transport studies and colony forming assays, respectively.

Results

Our current studies reveal an encompassing model for the role of RLIP76 in regulating the levels of fundamental proteins like PI3K, Akt, E-cadherin, CDK4, Bcl2 and PCNA which are of specific importance in the signal transduction from critical upstream signaling cascades that determine the proliferation, apoptosis and differentiation of pancreatic cancer cells. RLIP76 depletion also caused marked and sustained regression of established human BxPC-3 pancreatic cancer tumors in nude mouse xenograft model. RLIP76 turned out to be a major regulator of drug transport along with contributing to the radiation resistance in pancreatic cancer.

Conclusions/Significance

RLIP76 represents a mechanistically significant target for developing effective interventions in aggressive and refractory pancreatic cancers.     

TNF-Like Weak Inducer of Apoptosis (TWEAK) Promotes Beta Cell Neogenesis from Pancreatic Ductal Epithelium in Adult Mice

Aim/Hypothesis

The adult mammalian pancreas has limited ability to regenerate in order to restore adequate insulin production from multipotent progenitors, the identity and function of which remain poorly understood. Here we test whether the TNF family member TWEAK (TNF-like weak inducer of apoptosis) promotes β-cell neogenesis from proliferating pancreatic ductal epithelium in adult mice.

Methods

C57Bl/6J mice were treated with Fc-TWEAK and pancreas harvested at different time points for analysis by histology and immunohistochemistry. For lineage tracing, 4 week old double transgenic mice CAII-CreER^TM: R26R-eYFP were implanted with tamoxifen pellet, injected with Fc-TWEAK or control Ig twice weekly and analyzed at day 18 for TWEAK-induced duct cell progeny by costaining for insulin and YFP. The effect of TWEAK on pancreatic regeneration was determined by pancytokeratin immunostaining of paraffin embedded sections from wildtype and TWEAK receptor (Fn14) deficient mice after Px.

Results

TWEAK stimulates proliferation of ductal epithelial cells through its receptor Fn14, while it has no mitogenic effect on pancreatic α- or β-cells or acinar cells. Importantly, TWEAK induces transient expression of endogenous Ngn3, a master regulator of endocrine cell development, and induces focal ductal structures with characteristics of regeneration foci. In addition, we identify by lineage tracing TWEAK-induced pancreatic β-cells derived from pancreatic duct epithelial cells. Conversely, we show that Fn14 deficiency delays formation of regenerating foci after Px and limits their expansion.

Conclusions/Interpretation

We conclude that TWEAK is a novel factor mediating pancreatic β-cell neogenesis from ductal epithelium in normal adult mice.     

Analysis of PALB2 Gene in BRCA1/BRCA2 Negative Spanish Hereditary Breast/Ovarian Cancer Families with Pancreatic Cancer Cases

Background

The PALB2 gene, also known as FANCN, forms a bond and co-localizes with BRCA2 in DNA repair. Germline mutations in PALB2 have been identified in approximately 1% of familial breast cancer and 3–4% of familial pancreatic cancer. The goal of this study was to determine the prevalence of PALB2 mutations in a population of BRCA1/BRCA2 negative breast cancer patients selected from either a personal or family history of pancreatic cancer.

Methods

132 non-BRCA1/BRCA2 breast/ovarian cancer families with at least one pancreatic cancer case were included in the study. PALB2 mutational analysis was performed by direct sequencing of all coding exons and intron/exon boundaries, as well as multiplex ligation-dependent probe amplification.

Results

Two PALB2 truncating mutations, the c.1653T>A (p.Tyr551Stop) previously reported, and c.3362del (p.Gly1121ValfsX3) which is a novel frameshift mutation, were identified. Moreover, several PALB2 variants were detected; some of them were predicted as pathological by bioinformatic analysis. Considering truncating mutations, the prevalence rate of our population of BRCA1/2-negative breast cancer patients with pancreatic cancer is 1.5%.

Conclusions

The prevalence rate of PALB2 mutations in non-BRCA1/BRCA2 breast/ovarian cancer families, selected from either a personal or family pancreatic cancer history, is similar to that previously described for unselected breast/ovarian cancer families. Future research directed towards identifying other gene(s) involved in the development of breast/pancreatic cancer families is required.     

Bmi1 Enhances Tumorigenicity and Cancer Stem Cell Function in Pancreatic Adenocarcinoma

Background

Bmi1 is an integral component of the Polycomb Repressive Complex 1 (PRC1) and is involved in the pathogenesis of multiple cancers. It also plays a key role in the functioning of endogenous stem cells and cancer stem cells. Previous work implicated a role for cancer stem cells in the pathogenesis of pancreatic cancer. We hypothesized that Bmi1 plays an integral role in enhancing pancreatic tumorigenicity and the function of cancer stem cells in pancreatic ductal adenocarcinoma.

Methods

We measured endogenous Bmi1 levels in primary human pancreatic ductal adenocarcinomas, pancreatic intraepithelial neoplasias (PanINs) and normal pancreas by immunohistochemistry and Western blotting. The function of Bmi1 in pancreatic cancer was assessed by alteration of Bmi1 expression in several cell model systems by measuring cell proliferation, cell apoptosis, in vitro invasion, chemotherapy resistance, and in vivo growth and metastasis in an orthotopic model of pancreatic cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human pancreatic cancer xenografts after Bmi1 silencing.

Results

Bmi1 was overexpressed in human PanINs, pancreatic cancers, and in several pancreatic cancer cell lines. Overexpression of Bmi1 in MiaPaCa2 cells resulted in increased proliferation, in vitro invasion, larger in vivo tumors, more metastases, and gemcitabine resistance while opposite results were seen when Bmi1 was silenced in Panc-1 cells. Bmi1 was overexpressed in the cancer stem cell compartment of primary human pancreatic cancer xenografts. Pancreatic tumorspheres also demonstrated high levels of Bmi1. Silencing of Bmi1 inhibited secondary and tertiary tumorsphere formation, decreased primary pancreatic xenograft growth, and lowered the proportion of cancer stem cells in the xenograft tissue.

Conclusions

Our results implicate Bmi1 in the invasiveness and growth of pancreatic cancer and demonstrate its key role in the regulation of pancreatic cancer stem cells.