Gastroenterology

Gastroenterology

Volume 137, Issue 1, July 2009, Pages 361-371.e5
Gastroenterology

Basic—Liver, Pancreas, and Biliary Tract
E-Cadherin Regulates Metastasis of Pancreatic Cancer In Vivo and Is Suppressed by a SNAIL/HDAC1/HDAC2 Repressor Complex

https://doi.org/10.1053/j.gastro.2009.04.004Get rights and content

Background & Aims

Early metastasis is a hallmark of pancreatic ductal adenocarcinoma and responsible for >90% of pancreatic cancer death. Because little is known about the biology and genetics of the metastatic process, we desired to elucidate molecular pathways mediating pancreatic cancer metastasis in vivo by an unbiased forward genetic approach.

Methods

Highly metastatic pancreatic cancer cell populations were selected by serial in vivo passaging of parental cells with low metastatic potential and characterized by global gene expression profiling, chromatin immunoprecipitation, and in vivo metastatic assay.

Results

In vivo selection of highly metastatic pancreatic cancer cells induced epithelial-mesenchymal transition (EMT), loss of E-cadherin expression, and up-regulation of mesenchymal genes such as Snail. Genetic inactivation of E-cadherin in parental cells induced EMT and increased metastasis in vivo. Silencing of E-cadherin in highly metastatic cells is mediated by a transcriptional repressor complex containing Snail and histone deacetylase 1 (HDAC1) and HDAC2. In line, mesenchymal pancreatic cancer specimens and primary cell lines from genetically engineered KrasG12D mice showed HDAC-dependent down-regulation of E-cadherin and high metastatic potential. Finally, transforming growth factor β-driven E-cadherin silencing and EMT of human pancreatic cancer cells depends on HDAC activity.

Conclusions

We provide the first in vivo evidence that HDACs and Snail play an essential role in silencing E-cadherin during the metastatic process of pancreatic cancer cells. These data link the epigenetic HDAC machinery to EMT and metastasis and provide preclinical evidence that HDACs are promising targets for antimetastatic therapy.

Section snippets

Generation of Primary Murine PDAC Cell Lines

Tumors from Ptf1a/p48ex1Cre/+;LSL-KRASG12D/+ or Ptf1a/p48ex1Cre/+;LSL-KRASG12D/+;TP53lox/lox mice were removed and digested in 10 mL Dulbecco's modified Eagle medium (DMEM) containing 150 U/mL collagenase Type 2 (Worthington, Lakewood, NJ) as described.20 Digested tumors were dispersed into single cell suspension, resuspended in DMEM medium containing 10% fetal calf serum (FCS), and cultivated as described.21, 22

Chromatin Immunoprecipitation Assay

Chromatin immunoprecipitation (ChIP) assays were performed as recently described23

In Vivo Selection for Lung Metastasis of Pancreatic Cancer Cells

To detect pathways involved in metastasis of PDAC, we used an established in vivo selection model19 as shown in Supplementary Figure S1A. Murine TD-2EGFP-fLuc-TVA pancreatic cancer cells that exhibited high tumorigenicity at the site of injection while having low metastatic potential to the lung21 were injected into the tail vein of nude mice, and the metastatic process was longitudinally monitored by in vivo bioluminescence imaging (Supplementary Figure 1B). At necropsy, metastases were

Discussion

In this study, we demonstrate the importance of E-cadherin silencing for EMT and the metastatic process of PDAC in vitro and in vivo using different murine and human pancreatic cancer models. Conceivably, E-cadherin could be silenced during tumor progression and metastasis by somatic mutations, promoter methylation, or transcriptional repression. However, a recent study detected no CDH1 gene mutations in human PDAC.18 Although hypermethylation of the CDH1 promoter was observed in the pancreatic

Acknowledgments

J.v.B. and S.E. contributed equally to this work.

The authors thank Dr Bates, Dr Li, and Dr Hughes for providing plasmids; Dr Jacks and Dr Tuveson for LSL-KRASG12D mice, Dr Berns for TP53lox/lox mice, and Dr Nakhai for Ptf1a/p48ex1Cre/+ mice; Dr Greten for TD-2 cells; U. Götz, M. Werb, and M. Göbel for excellent technical assistance; and the Microarray Core Unit headed by Dr R. Lang for Affymetrix GeneChip analysis.

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by Deutsche Krebshilfe (project 107143, to D.S.) and Deutsche Forschungsgemeinschaft (SCHN 959/1-1) and SFB456 (to G.S.).

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