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.     

Pancreatic cancer cells enhance the ability of collagen internalization during epithelial-mesenchymal transition

Background

Extracellular matrix (ECM) remodeling is predominantly mediated by fibroblasts using intracellular and extracellular pathways. Although it is well known that extracellular degradation of the ECM by proteases derived from cancer cells facilitates cellular invasion, the intracellular degradation of ECM components by cancer cells has not been clarified. The aim of this study was to characterize collagen internalization, which is the initial step of the intracellular degradation pathway in pancreatic cancer cells, in light of epithelial–mesenchymal transition (EMT).

Methodology/Principal Findings

We analyzed the function of collagen internalization in two pancreatic cancer cell lines, SUIT-2 and KP-2, and pancreatic stellate cells (PSCs) using Oregon Green 488-gelatin. PSCs had a strong ability for collagen uptake, and the pancreatic cancer cells also internalized collagen although less efficiently. The collagen internalization abilities of SUIT-2 and KP-2 cells were promoted by EMT induced by human recombinant transforming growth factor β1 (P<0.05). Expression of Endo180, a collagen uptake receptor, was high in mesenchymal pancreatic cancer cell lines, as determined by EMT marker expression (P<0.01). Quantitative RT-PCR and western blot analyses showed that Endo180 expression was also increased by EMT induction in SUIT-2 and KP-2 cells. Endo180 knockdown by RNA interference attenuated the collagen uptake (P<0.01) and invasive abilities (P<0.05) of SUIT-2 and KP-2 cells.

Conclusions/Significance

Pancreatic cancer cells are capable of collagen internalization, which is enhanced by EMT. This ECM clearance system may be a novel mechanism for cellular invasion and a potential therapeutic target in pancreatic cancer.     

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.     

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions

Background

Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear.

Methodology/Principal Findings

Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype.

Conclusions/Significance

These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.     

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.     

Loss and recovery of Mgat3 and GnT-III Mediated E-cadherin N-glycosylation is a mechanism involved in epithelial-mesenchymal-epithelial transitions

Background

N-acetylglucosaminyltransferase-III (GnT-III) is a glycosyltransferase encoded by Mgat3 that catalyzes the addition of β1,4-bisecting-N-acetylglucosamine on N-glycans. GnT-III has been pointed as a metastases suppressor having varying effects on cell adhesion and migration. We have previously described the existence of a functional feedback loop between E-cadherin expression and GnT-III-mediated glycosylation. The effects of GnT-III-mediated glycosylation on E-cadherin expression and cellular phenotype lead us to evaluate Mgat3 and GnT-III-glycosylation role during Epithelial-Mesenchymal-Transition (EMT) and the reverted process, Mesenchymal-Epithelial-Transition (MET).

Methodology/Principal Findings

We analyzed the expression profile and genetic mechanism controlling Mgat3 expression as well as GnT-III-mediated glycosylation, in general and specifically on E-cadherin, during EMT/MET. We found that during EMT, Mgat3 expression was dramatically decreased and later recovered when cells returned to an epithelial-like phenotype. We further identified that Mgat3 promoter methylation/demethylation is involved in this expression regulation. The impact of Mgat3 expression variation, along EMT/MET, leads to a variation in the expression levels of the enzymatic product of GnT-III (bisecting GlcNAc structures), and more importantly, to the specific modification of E-cadherin glycosylation with bisecting GlcNAc structures.

Conclusions/Significance

Altogether, this work identifies for the first time Mgat3 glycogene expression and GnT-III-mediated glycosylation, specifically on E-cadherin, as a novel and major component of the EMT/MET mechanism signature, supporting its role during EMT/MET.     

CD166/ALCAM Expression Is Characteristic of Tumorigenicity and Invasive and Migratory Activities of Pancreatic Cancer Cells

Background

CD166, also known as activated leukocyte cell adhesion molecule (ALCAM), is expressed by various cells in several tissues including cancer. However, the role of CD166 in malignant tumors is controversial, especially in pancreatic cancer. This study aimed to clarify the role and significance of CD166 expression in pancreatic cancer.

Methods

We performed immunohistochemistry and flow cytometry to analyze the expression of CD166 in surgical pancreatic tissues and pancreatic cancer cell lines. The differences between isolated CD166+ and CD166- pancreatic cancer cells were analyzed by invasion and migration assays, and in mouse xenograft models. We also performed quantitative RT-PCR and microarray analyses to evaluate the expression levels of CD166 and related genes in cultured cells.

Results

Immunohistochemistry revealed high expression of CD166 in pancreatic cancer tissues (12.2%; 12/98) compared with that in normal pancreas controls (0%; 0/17) (p = 0.0435). Flow cytometry indicated that CD166 was expressed in 33.8–70.2% of cells in surgical pancreatic tissues and 0–99.5% of pancreatic cancer cell lines. Invasion and migration assays demonstrated that CD166- pancreatic cancer cells showed stronger invasive and migratory activities than those of CD166+ cancer cells (p<0.05). On the other hand, CD166+ Panc-1 cells showed a significantly stronger colony formation activity than that of CD166- Panc-1 cells (p<0.05). In vivo analysis revealed that CD166+ cells elicited significantly greater tumor growth than that of CD166- cells (p<0.05) in both subcutaneous and orthotopic mouse tumor models. mRNA expression of the epithelial-mesenchymal transition activator Zeb1 was over-expressed in CD166- cells (p<0.001). Microarray analysis showed that TSPAN8 and BST2 were over-expressed in CD166+ cells, while BMP7 and Col6A1 were over-expressed in CD166- cells.

Conclusions

CD166+ pancreatic cancer cells are strongly tumorigenic, while CD166- pancreatic cancer cells exhibit comparatively stronger invasive and migratory activities. These findings suggest that CD166 expression is related to different functions in pancreatic cancer cells.     

CSF N-Glycan Profiles to Investigate Biomarkers in Brain Developmental Disorders: Application to Leukodystrophies Related to eIF2B Mutations

Background

Primary or secondary abnormalities of glycosylation have been reported in various brain diseases. Decreased asialotransferrin to sialotransferrin ratio in cerebrospinal fluid (CSF) is a diagnostic marker of leukodystrophies related to mutations of genes encoding translation initiation factor, EIF2B. We investigated the CSF glycome of eIF2B-mutated patients and age-matched normal individuals in order to further characterize the glycosylation defect for possible use as a biomarker.

Methodology/Principal Findings

We conducted a differential N-glycan analysis using MALDI-TOF/MS of permethylated N-glycans in CSF and plasma of controls and eIF2B-mutated patients. We found in control CSF that tri-antennary/bisecting and high mannose structures were highly represented in samples obtained between 1 to 5 years of age, whereas fucosylated, sialylated structures were predominant at later age. In CSF, but not in plasma, of eIF2B-mutated patient samples, we found increased relative intensity of bi-antennary structures and decreased tri-antennary/bisecting structures in N-glycan profiles. Four of these structures appeared to be biomarker candidates of glycomic profiles of eIF2B-related disorders.

Conclusion

Our results suggest a dynamic development of normal CSF N-glycan profiles from high mannose type structures to complex sialylated structures that could be correlated with postnatal brain maturation. CSF N-glycome analysis shows relevant quantitative changes associated with eIF2B related disorders. This approach could be applied to other neurological disorders involving developmental gliogenesis/synaptogenesis abnormalities.     

Proteomics profiling identify CAPS as a potential predictive marker of tamoxifen resistance in estrogen receptor positive breast cancer

Background

Despite the success of tamoxifen since its introduction, about one-third of patients with estrogen (ER) and/or progesterone receptor (PgR) - positive breast cancer (BC) do not benefit from therapy. Here, we aim to identify molecular mechanisms and protein biomarkers involved in tamoxifen resistance.

Results

Using iTRAQ and Immobilized pH gradient-isoelectric focusing (IPG-IEF) mass spectrometry based proteomics we compared tumors from 12 patients with early relapses (<2 years) and 12 responsive to therapy (relapse-free > 7 years). A panel of 13 proteins (TCEAL4, AZGP1, S100A10, ALDH6A1, AHNAK, FBP1, S100A4, HSP90AB1, PDXK, GFPT1, RAB21, MX1, CAPS) from the 3101 identified proteins, potentially separate relapse from non-relapse BC patients. The proteins in the panel are involved in processes such as calcium (Ca²⁺) signaling, metabolism, epithelial mesenchymal transition (EMT), metastasis and invasion. Validation of the highest expressed proteins in the relapse group identify high tumor levels of CAPS as predictive of tamoxifen response in a patient cohort receiving tamoxifen as only adjuvant therapy.

Conclusions

This data implicate CAPS in tamoxifen resistance and as a potential predictive marker.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9080-y) contains supplementary material, which is available to authorized users.     

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.     

Hypermethylation of tumor suppressor genes involved in critical regulatory pathways for developing a blood-based test in breast cancer

Background

Aberrant DNA methylation patterns might be used as a biomarker for diagnosis and management of cancer patients.

Methods and Findings

To achieve a gene panel for developing a breast cancer blood-based test we quantitatively assessed the DNA methylation proportion of 248 CpG sites per sample (total of 31,248 sites in all analyzed samples) on 10 candidate genes (APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P16, P21 and TIMP3). The number of 126 samples consisting of two different cohorts was used (first cohort: plasma samples from breast cancer patients and normal controls; second cohort: triple matched samples including cancerous tissue, matched normal tissue and serum samples). In the first cohort, circulating cell free methylated DNA of the 8 tumor suppressor genes (TSGs) was significantly higher in patients with breast cancer compared to normal controls (P<0.01). In the second cohort containing triple matched samples, seven genes showed concordant hypermethylated profile in tumor tissue and serum samples compared to normal tissue (P<0.05). Using eight genes as a panel to develop a blood-based test for breast cancer, a sensitivity and specificity of more than 90% could be achieved in distinguishing between tumor and normal samples.

Conclusions

Our study suggests that the selected TSG panel combined with the high-throughput technology might be a useful tool to develop epigenetic based predictive and prognostic biomarker for breast cancer relying on pathologic methylation changes in tumor tissue, as well as in circulation.     

Mutational Profiling of Kinases in Human Tumours of Pancreatic Origin Identifies Candidate Cancer Genes in Ductal and Ampulla of Vater Carcinomas

Background

Protein kinases are key regulators of cellular processes (such as proliferation, apoptosis and invasion) that are often deregulated in human cancers. Accordingly, kinase genes have been the first to be systematically analyzed in human tumors leading to the discovery that many oncogenes correspond to mutated kinases. In most cases the genetic alterations translate in constitutively active kinase proteins, which are amenable of therapeutic targeting. Tumours of the pancreas are aggressive neoplasms for which no effective therapeutic strategy is currently available.

Methodology/Principal Findings

We conducted a DNA-sequence analysis of a selected set of 35 kinase genes in a panel of 52 pancreatic exocrine neoplasms, including 36 pancreatic ductal adenocarcinoma, and 16 ampulla of Vater cancer. Among other changes we found somatic mutations in ATM, EGFR, EPHA3, EPHB2, and KIT, none of which was previously described in cancers.

Conclusions/Significance

Although the alterations identified require further experimental evaluation, the localization within defined protein domains indicates functional relevance for most of them. Some of the mutated genes, including the tyrosine kinases EPHA3 and EPHB2, are clearly amenable to pharmacological intervention and could represent novel therapeutic targets for these incurable cancers.     

Selected Cytokines in Patients with Pancreatic Cancer: A Preliminary Report

Background/Aims

Recent experimental studies have suggested that various cytokines may be important players in the development and progression of pancreatic cancer. However, these findings have not yet been verified in a clinical setting.

Methods

In this study, we examined the levels of a broad panel of cytokines, including interleukin (IL)-1, IL-6, IL-8, IL-10, IL-12, IL-17, and IL-23, as well as tumor necrosis factor alpha (TNFα) and granulocyte-colony stimulating factor (G-CSF) in patients with pancreatic adenocarcinoma (n = 43), other pancreatic malignancies (neuroendocrine [n = 10] and solid pseudopapillary tumors [n = 3]), and healthy individuals (n = 41).

Results

We found that there were higher levels of IL-6, IL-8, IL-10 and TNFα in patients with pancreatic cancer compared to healthy controls (for all, at least p<0.03). Cancer patients had lower IL-23 concentrations than healthy individuals and patients diagnosed with other types of malignancies (for both, p = 0.002). Levels of IL-6, IL-8, IL-10, and IL-23 were significantly associated with the direct number of circulating bone marrow (BM)-derived mesenchymal or very small embryonic/epiblast-like stem cells (SCs) in patients with pancreatic cancer. Moreover, our study identified a potential ability of IL-6, IL-8, IL-10, IL-23, and TNFα levels to enable discrimination of pancreatic cancer from other pancreatic tumors and diseases, including acute and chronic pancreatitis and post-pancreatitis cysts (with sensitivity and specificity ranging between 70%–82%).

Conclusions

Our study i) supports the significance of selected cytokines in the clinical presentation of pancreatic cancer, ii) highlights numerous associations between selected interleukins and intensified BMSCs trafficking in patients with pancreatic cancer, and iii) preliminarily characterizes the diagnostic potential of several cytokines as potential novel clinical markers of pancreatic cancer in humans.     

Comparison of CpG Island Methylator Phenotype (CIMP) Frequency in Colon Cancer Using Different Probe- and Gene-Specific Scoring Alternatives on Recommended Multi-Gene Panels

Background

In colorectal cancer a distinct subgroup of tumours demonstrate the CpG island methylator phenotype (CIMP). However, a consensus of how to score CIMP is not reached, and variation in definition may influence the reported CIMP prevalence in tumours. Thus, we sought to compare currently suggested definitions and cut-offs for methylation markers and how they influence CIMP classification in colon cancer.

Methods

Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), with subsequent fragment analysis, was used to investigate methylation of tumour samples. In total, 31 CpG sites, located in 8 different genes (RUNX3, MLH1, NEUROG1, CDKN2A, IGF2, CRABP1, SOCS1 and CACNA1G) were investigated in 64 distinct colon cancers and 2 colon cancer cell lines. The Ogino gene panel includes all 8 genes, in addition to the Weisenberger panel of which only 5 of the 8 genes included were investigated. In total, 18 alternative combinations of scoring of CIMP positivity on probe-, gene-, and panel-level were analysed and compared.

Results

For 47 samples (71%), the CIMP status was constant and independent of criteria used for scoring; 34 samples were constantly scored as CIMP negative, and 13 (20%) consistently scored as CIMP positive. Only four of 31 probes (13%) investigated showed no difference in the numbers of positive samples using the different cut-offs. Within the panels a trend was observed that increasing the gene-level stringency resulted in a larger difference in CIMP positive samples than increasing the probe-level stringency. A significant difference between positive samples using ‘the most stringent’ as compared to ‘the least stringent’ criteria (20% vs 46%, respectively; p<0.005) was demonstrated.

Conclusions

A statistical significant variation in the frequency of CIMP depending on the cut-offs and genes included in a panel was found, with twice as many positives samples by least compared to most stringent definition used.     

Metformin Inhibits the IL-6-Induced Epithelial-Mesenchymal Transition and Lung Adenocarcinoma Growth and Metastasis

Objective

Epithelial-mesenchymal transition (EMT) plays an important role in cancer tumorigenesis. However, the underlying mechanisms of EMT in lung adenocarcinoma, and how this process might be inhibited, remain to be explored. This study investigated the role of IL-6 in lung adenocarcinoma cell EMT and explored the potential effects of metformin on this process.

Methods

Invasion assay and MTT assay was performed to determine cell invasion and cell proliferation. Western blotting, immunofluorescence, real-time PCR, ELISA, and immunohistochemistry were performed to detect the expression of IL-6, E-cadherin, Vimentin, and p-STAT3.

Results

We discovered that IL-6, via STAT3 phosphorylation, could promote lung adenocarcinoma cell invasion via EMT in vitro. This was supported by the inverse correlation between E-cadherin and IL-6 expression, positive correlation between IL-6 and vimentin mRNA expression and between STAT3 phosphorylation and IL-6 expression in tumor tissues. Importantly, metformin inhibited tumor growth and distant metastases in tumor-bearing nude mice and reversed IL-6-induced EMT both in vitro and in vivo. Furthermore, we found that blockade of STAT3 phosphorylation might be the underlying mechanism of metformin inhibition of IL-6-induced EMT.

Conclusions

Collectively, our present results show that enhanced IL-6 expression, via STAT3 phosphorylation, is a mechanism of EMT in lung adenocarcinoma. We found that metformin could inhibit IL-6-induced EMT possibly by blocking STAT3 phosphorylation.     

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.