1. Volume 137, Issue 1, Pages 361-371.e5 (July 2009)
E-Cadherin Regulates Metastasis of Pancreatic Cancer In Vivo and Is Suppressed by a SNAIL/HDAC1/HDAC2 Repressor Complex 
Johannes von Burstin, Stefan Eser, Mariel C. Paul, Barbara Seidler, Martina Brandl, Marlena Messer, Alexander von Werder, Annegret Schmidt, Jörg Mages, Philipp Pagel, Angelika Schnieke, Roland M. Schmid, Günter Schneider, Dieter Saur 
Gastroenterology 
Volume 137, Issue 1, Pages e5 (July 2009)
DOI: /j.gastro
Copyright © 2009 AGA Institute Terms and Conditions

2. Figure 1
Epithelial-mesenchymal transition (EMT) and loss of E-cadherin expression in the highly metastatic TD-2EGFP-fLuc-TVA sublines P-1 and P-2. (A) Top: Phase contrast images of parental TD-2EGFP-fLuc-TVA cells (left panel: epithelial phenotype) and in vivo selected highly lung metastatic P-2 cells (right panel: mesenchymal phenotype). Bottom: Immunocytochemistry for E-cadherin expression. (B) Western blot analysis of E-cadherin expression in TD-2EGFP-fLuc-TVA, P-1, and P-2 cells. Membranes were stripped and probed for β-actin to ensure equal protein loading. (C and D) Quantitative E-cadherin (C) and Snail (D) mRNA expression. Total RNA was prepared from TD-2EGFP-fLuc-TVA, P-1, and P-2 cells. E-cadherin and Snail mRNA levels were quantified using real-time RT-PCR.
Gastroenterology  , e5DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

3. Figure 2
Epigenetic silencing of E-cadherin expression by the histone deacetylase (HDAC) machinery in highly metastatic P-1 and P-2 cells. (A) P-1 and P-2 cells were treated with Trichostatin A (TsA; 0.3 μmol/L) or DMSO as control for 24 hours. E-cadherin mRNA levels were quantified using real-time RT-PCR analysis. Results shown are the mean ± SD of 3 independent experiments, each done in triplicate (Student t test: **P < .001, TsA vs control). (B) Western blot analysis of E-cadherin expression in parental TD-2EGFP-fLuc-TVA, P-1, and P-2 sublines after treatment with 0.1 μmol/L and 0.3 μmol/L TsA for 24 hours or the demethylating agent 5′-Aza-2′-deoxycytidine (5-Aza; 1 μmol/L) for 48 hours and 96 hours. Nontreated and DMSO-treated cells were used as controls. Membranes were stripped and probed for β-actin to ensure equal protein loading. (C) Chromatin immunoprecipitation analysis of the proximal E-cadherin (CDH1) and albumin (control) promoter. Chromatin of TD-2EGFP-fLuc-TVA, P-1, or P-2 cells treated for 24 hours with DMSO as control or 0.3 μmol/L TsA was immunoprecipitated with RNA-polymerase II, Snail, HDAC1, HDAC2, HDAC3, acetylated histone H4, and mSin3A antibodies. Precipitation with an IgG antibody served as negative control. Precipitated DNA or 10% of chromatin input was amplified with gene-specific primers for the proximal CDH1 or the albumin promoter. (D) Coimmunoprecipitation of Snail, HDAC1, HDAC2, and mSin3A in TD-2EGFP-fLuc-TVA, P-1, and P-2 cells. Antibodies against Snail, HDAC1, HDAC2, HDAC3, and mSin3A were used for immunoprecipitation followed by Western blotting. One percent of lysate used for precipitation served as input control. (E) Avidin-biotin-complex-DNA binding assay using double-stranded 5′ biotinylated oligonucleotides corresponding to E-box1 and E-box2 of the proximal CDH1 promoter. Whole-cell extracts of TD-2EGFP-fLuc-TVA, P-1, and P-2 cells were incubated with the respective oligonucleotides and probed with a Snail antibody. Ten percent of whole-cell extracts were used as input control.
Gastroenterology  , e5DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

4. Figure 3
Inactivation of E-cadherin in parental TD-2EGFP-fLuc-TVA cells induces EMT and lung metastasis in vivo. TD-2EGFP-fLuc-TVA cells were infected with RCASBP(A) (replication competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor, Bryan-RSV polymerase gene and subgroupA envelope)-dominant-negative-E-cadherin (dn-E-cad) or RCASBP(A)-lacZ (control) retroviruses, and tail vein metastatic assays in nude mice were performed. (A) Western blot analysis of endogenous E-cadherin and dn-E-cad protein levels in TD-2EGFP-fLuc-TVA cells infected with RCASBP(A)-lacZ (control, left panel) or RCASBP(A)-dn-E-cad viruses (middle panel). Maintenance of dn-E-cad expression and loss of endogenous E-cadherin was verified in RCASBP(A)-dn-E-cad-infected TD-2EGFP-fLuc-TVA (P1) cells (right panel) isolated from lung metastases after tail vein injection. (B) Phase contrast images of RCASBP(A)-lacZ control (left panel: epithelial phenotype) and RCASBP(A)-dn-E-cad (right panel: mesenchymal phenotype)-infected TD-2EGFP-fLuc-TVA cells. (C) Lung metastases of intravenously injected TD-2EGFP-fLuc-TVA cells transduced with RCASBP(A)-dn-E-cad were removed, digested into single cell suspensions, and cultured in selection medium containing geneticin to generate P-1 cells. Phase contrast image of P-1 cells showing a mesenchymal phenotype.
Gastroenterology  , e5DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

5. Figure 4
EMT and down-regulation of E-cadherin in a genetically engineered KrasG12D-dependent mouse model of metastatic PDAC. (A–C) H&E-stained sections of primary PDAC showing well-differentiated ductal cancer cells in the center of the tumor (A) and poorly differentiated mesenchymal cancer cells at the invasive front of the primary tumor (B) and in the corresponding liver metastases (C) of a Ptf1a/p48ex1Cre/+;LSL-KRASG12D/+ mouse (#16992). (D–L) Corresponding paraffin sections stained for E-cadherin (D–F), CK-19 (G–I), or Snail (J–L) by immunohistochemistry (D, G, J: well-differentiated primary PDAC; E, H, K: poorly differentiated tumor cells at the invasion front; F, I, L: liver metastasis).
Gastroenterology  , e5DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

6. Figure 5
Epigenetic silencing of E-cadherin in highly metastatic mesenchymal Ptf1a/p48ex1Cre/+;LSL-KRASG12D/+ pancreatic cancer cells. (A) Phase contrast image of low passaged (P < 4) cell line isolated from the metastatic primary pancreatic tumor of Ptf1a/p48ex1Cre/+;LSL-KRASG12D/+ mouse #16992 (see Figure 4) in culture showing a mesenchymal phenotype. (B) Relative mRNA expression levels of the indicated genes associated with an epithelial or mesenchymal phenotype in cells compared with primary Ptf1a/p48ex1Cre/+;LSL-KRASG12D/+ PDAC cells with an epithelial phenotype. mRNA expression levels were quantified using real-time RT-PCR and normalized using cyclophilin. (C–K) Mesenchymal primary PDAC cells were orthotopically implanted into the pancreas of nude mice, and metastasis was assessed by macroscopic and microscopic pathologic examination. (C–E) H&E-stained paraffin sections of orthotopic PDAC (C), liver (D), and lung (E) metastases. (F–H) Immunohistochemistry for E-cadherin expression in the primary tumor (F), liver (G), and lung (H) metastases. (I–K) Immunohistochemistry of Snail expression in orthotopic PDAC (I), liver (J), and lung (K) metastases shows nuclear expression of Snail. (L) Quantitative real-time RT-PCR of E-cadherin mRNA expression in cells treated with DMSO (control) or Trichostatin A (TsA) for 24 hours. (Student t test: **P < .001 vs control [DMSO]). (M) Western blot analysis of E-cadherin protein levels in cells treated with TsA for 24 hours. Nontreated and DMSO-treated cells were used as controls. Membranes were probed for β-actin to ensure equal protein loading. (N) Quantitative RT-PCR of Snail, Slug, and Twist1 expression in cells treated with DMSO (control), 0.1 μmol/L or 0.3 μmol/L TsA for 24 hours.
Gastroenterology  , e5DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

7. Figure 6
HDAC activity contributes to E-cadherin silencing in murine and human PDAC. (A) Phase contrast images of 4 early passage mesenchymal murine PDAC cell lines isolated from primary pancreatic tumors (3202, 3250, W22) and lymph node metastases (3202LN) of Ptf1a/p48ex1Cre/+;LSL-KRASG12D/+ or Ptf1a/p48ex1Cre/+;LSL-KRASG12D/+;TP53lox/lox mice. (B) Relative mRNA expression levels of the indicated genes in mesenchymal 3202, 3202LN, 3250, and W22 PDAC cells compared with 4 primary Ptf1a/p48ex1Cre/+;LSL-KRASG12D/+ and Ptf1a/p48ex1Cre/+;LSL-KRASG12D/+;TP53lox/lox PDAC cell lines with an epithelial phenotype. (C) Western blot analysis of Snail and Twist1 protein expression in the indicated mesenchymal PDAC cell lines. (D) Western Blot analysis of E-cadherin protein levels of untreated or DMSO- or TsA-treated mesenchymal PDAC cells after 24 hours. (E) Western blot analysis of E-cadherin expression in the indicated mesenchymal cell lines 24 hours after treatment with TsA (0.3 μmol/L) or the HDAC class I selective inhibitor valproic acid (VPA; 1.5 mmol/L). (F) Western blot analysis of E-cadherin protein levels in TGF-β-induced EMT of human Panc1 pancreatic cancer cells. Panc1 cells were stimulated with TGF-β (10 ng/mL) for 48 hours to induce EMT. Cell lysates of nontreated, TGF-β-stimulated, or TGF-β- and VPA (1.5 mmol/L)-treated cells were analyzed for E-cadherin expression.
Gastroenterology  , e5DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

8. Supplementary Figure 1
In vivo selection of TD-2EGFP-fLuc-TVA pancreatic cancer cells for metastatic potential to the lung. (A) In vivo selection model for lung metastasis: the murine pancreatic cancer cell line TD-2EGFP-fLuc-TVA with stable expression of enhanced green fluorescent protein (EGFP), firefly luciferase (fLuc), and tumor virus A (TVA) was injected into the tail vein of nude mice to select for highly lung metastatic cells. Lung metastases were harvested, digested into single cell suspensions, and cultured in medium containing geneticin to generate sublines for reinjection to select for cells with enhanced metastatic potential. (B–D) Monitoring of lung metastasis formation by in vivo bioluminescence imaging (BLI) and ex vivo fluorescence imaging (FLI) in nude mice injected with TD-2EGFP-fLuc-TVA cells. (B) Stable expression of firefly luciferase by TD-2EGFP-fLuc-TVA cells enables monitoring of the in vivo selection process by BLI. (C and D) Macroscopic view of the lung of nude mouse injected with TD-2EGFP-fLuc-TVA cells. EGFP expression of TD-2EGFP-fLuc-TVA metastases was visualized by fluorescence stereomicroscopy. White light (C) and corresponding merged white light/fluorescent (D) image of lung metastases. (E) Histologic verification of lung metastases at necropsy using H&E-stained, paraffin-embedded lung sections from TD-2EGFP-fLuc-TVA injected nude mouse.
Gastroenterology  , e5DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

9. Supplementary Figure 2
Knockdown of Snail or HDAC2 induces E-cadherin mRNA expression in highly metastatic P-2 cells. (A) Snail mRNA levels of control or Snail siRNA-transfected P-2 cells were quantified 48 hours after transfection using real-time RT-PCR analysis and normalized to cyclophilin expression levels. (B) E-cadherin mRNA levels of control or Snail siRNA-transfected P-2 cells were quantified 48 hours after transfection using real-time RT-PCR analysis and normalized to cyclophilin expression levels. (C) HDAC2 mRNA levels of control or HDAC2 siRNA-transfected P-2 cells were quantified 48 hours after transfection using real-time RT-PCR analysis and normalized to cyclophilin expression levels. (D) E-cadherin mRNA levels of control or HDAC2 siRNA-transfected P-2 cells were quantified 48 hours after transfection using real-time RT-PCR analysis and normalized to cyclophilin expression levels.
Gastroenterology  , e5DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions