A working model is illustrating how oncogenic K-Ras and loss of Smad4 cooperate to cause an invasive phenotype.

<p>Oncogenic K-Ras signaling induces EGFR expression and K-Ras induced EGFR expression is normally suppressed by Smad4-dependent TGF-β signaling. Loss of Smad4 therefore leads to optimal up-regulation of K-ras induced EGFR expression. The increased expression and signaling by EGFR activates PI3K which induces nuclear translocation of NF-κB that in turn drives the expression of MMP9 and uPA.</p

Nuclear translocation of NF-κB sub-unit.

<p><b>A</b>. Western blot analysis for the NF-κB (p65) expression level in different genetically modified HPNE cells. <b>B</b>. Western blot analysis showing constitutive nuclear expression of p65 in cells expressing oncogenic K-Ras and loss of Smad4. <b>C</b>. Effect of EGF on nuclear translocation of NF-κB (p65). HPNE/K-Ras/ShSmad4 cells are treated with EGF (50 ng/ml) and harvested at indicated time points. Western blot analyses were performed with both nuclear and cytoplasmic extracts. A parallel Coomassie blue stained gel was presented as a loading control. <b>D</b>. Nuclear translocation of p65 is partially blocked by treatment of inhibitors described above in Figure 2. <b>E</b>. NF-B mediated regulation of uPA and MMP9. ChIP assay was performed to show the binding of NF-κB to the promoters of uPA and MMP9. The details for ChIP assay and PCR primers are described in material and methods section. </p

Expression level and activity of MMP9 and uPA was determined after treating HPNE/K-Ras/ShSmad4 cells with different inhibitors.

<p><b>A</b>. Western blots analysis of MMP9 and uPA after treating cells with AG1478 (10 μM), Bay11-7082 (10 μM) or Ly294002 (10 μM). <b>B</b>. These inhibitors also blocked the secreted levels of MMP9 and uPA as determined by enzymatic activity in gelatin and casein zymography. <b>C</b>. Inhibition of the expression level of ph-EGFR by ph-EGFR, NF-κB and PI3K blockers, AG1478, Bay11-7082 and Ly294002 respectively at above concentrations. <b>D</b>. Western blot analysis was performed to monitor the cyclin D2 expression for the HPNE/K-Ras/ShSmad4 cells treated with Bay11-7082 to confirm the specific effect of Bay11-7082 [42] on NF-κB signaling. <b>E</b>. Western block showing that Ly294002 (10 μM) blocks AKT phosphorylation.</p

Characterization of genetically modified HPNE cell lines.

<p><b>A</b>. Expression of oncogenic K-Ras and knockdown of Smad4 cooperate to induce EGFR, MMP9 and uPA expressions in HPNE and other K-Ras and Smad4 modified HPNE cells. <b>B</b>. The densitometry values were presented as the measurement of EGFR, MMP9 and uPA expression levels. The values were determined from multiple Western blots (n=3) analyses compared to the density of β-actin that was used as a loading control. The relative expression level of a particular protein was calculated by comparison to the density of the same protein in HPNE cells. Statistical significance value **p< 0.01 was calculated using student’s T-tests. <b>C</b>. Immunostaining followed by images from fluorescent microscopy showed the expression of EGFR, MMP9 and uPA in HPNE and genetically modified HPNE cells. Nuclei were visualized by staining with DAPI (blue). <b>D</b>. Samples (secreted proteins in serum free medium) were collected after 16 hours incubation of the cells in serum free medium. Gelatin and casein zymography analyses were performed to determine MMP9 and uPA activity. </p

Knocking down of p16 enabled the HPNE/K-ras cells to overcome senescence.

<p>(A) Representative fields of senescence-associated β-galactosidase (SA-β-gal) activity in HPNE cell lines. (B) Quantification of SA-β-gal staining in HPNE cells, data are showed as percentage of positive cells **<i>P</i><0.01 versus the corresponding control groups. (C) The expression of cyclin-dependent inhibitors – p15, p21 and p27 was analyzed by Western blot in HPNE cell lines. β-Actin was used as loading control.</p

Molecular analysis of HPNE cell lines offers insight into malignant transformation in human pancreatic cancer.

<p>(A) Activation of the Akt signaling pathway by K-ras in HPNE cell lines as detected by Western blot analysis. (B) Activation of the MAPK signaling pathway by K-ras in HPNE cell lines. The expression of total and phosphorylated-Erk and p38 was detected by Western blot analysis. β-Actin was used as loading control. (C) Expression of p16 was decreased after siRNA depletion of HBP1, as detected by real-time PCR. (D) Relative quantification of SA-β-gal staining cells after siRNA depletion of HBP1 in HPNE/K-ras cells (E) Expression of EGF and TGFα was increased in HPNE/Kras cells as detected by real-time PCR. (F) Expression of TGFα was increased in HPNE-iKras cells as detected by real-time PCR. (G) The proposed model for transformation of HPNE cell line. hTERT enabled cell to acquire replicating immortality; oncogenic K-ras rendered HPNE cells able to sustain proliferative signaling, and thus increased cell proliferation and cell growth; and inactivation of p16 by shRNA silencing enabled HPNE cells to evade growth suppressors, disrupt the senescence checkpoint, and ultimately induce transformation of HPNE cells.</p

SMAD4 Regulates Cell Motility through Transcription of N-Cadherin in Human Pancreatic Ductal Epithelium

<div><p>Expression of the cellular adhesion protein N-cadherin is a critical event during epithelial-mesenchymal transition (EMT). The SMAD4 protein has been identified as a mediator of transforming growth factor-β (TGF-β) superfamily signaling, which regulates EMT, but the mechanisms linking TGF-β signaling to N-cadherin expression remain unclear. When the TGF-β pathway is activated, SMAD proteins, including the common mediator SMAD4, are subsequently translocated into the nucleus, where they influence gene transcription via SMAD binding elements (SBEs). Here we describe a mechanism for control of <i>CDH2</i>, the gene encoding N-cadherin, through the canonical TGFβ–SMAD4 pathway. We first identified four previously undescribed SBEs within the <i>CDH2</i> promoter. Using telomerase immortalized human pancreatic ductal epithelium, we found that TGF-β stimulation prompted specific SMAD4 binding to all four SBEs. Luciferase reporter and SMAD4-knockdown experiments demonstrated that specific SMAD4 binding to the SBE located at −3790 bp to −3795 bp within the promoter region of <i>CDH2</i> was necessary for TGF-β-stimulated transcription. Expression of N-cadherin on the surface of epithelial cells facilitates motility and invasion, and we demonstrated that knockdown of SMAD4 causes decreased N-cadherin expression, which results in diminished migration and invasion of human pancreatic ductal epithelial cells. Similar reduction of cell motility was produced after <i>CDH2</i> knockdown. Together, these findings suggest that SMAD4 is critical for the TGF-β-driven upregulation of N-cadherin and the resultant invasive phenotype of human pancreatic ductal epithelial cells during EMT.</p></div

Activation of K-ras and silencing of p16 in HPNE cells increased cell proliferation and growth.

<p>(A) The cell growth curve of HPNE cell lines as detected by using a cell counter. (B) Cell cycle analysis of HPNE cell lines. The percentage of cells in each phase of the cell cycle is shown. (C) Anchorage-independent cell growth of HPNE cell lines in soft agar assay. A representative field of soft agar for each cell line is shown. (D) Cells were incubated with gemcitabine for 72 h, and cell viability was measured by MTT assay. (E) The expression of cell cycle proteins cyclins and c-Myc was increased by mutant K-ras in HPNE cell lines, as determined by Western blot analysis. β-Actin was used as loading control. (F) Relative mRNA levels of E2F target genes in HPNE/K-ras and HPNE/K-ras/p16sh cells. All data are presented as mean ± SD (n = 3 independent experiments). **<i>P</i><0.01 versus the corresponding control groups.</p

Activation of K-ras in HPNE cells increased cell invasion.

<p>(A) The quantification of stained HPNE cell lines per field in a transwell-matrigel penetration assay. Representative micrographs for each cell line are shown. Data are presented as mean ± SD (n = 3 independent experiments). **<i>P</i><0.01 versus the HPNE control cells. (B) The expression of EMT markers cytokeratin-19, E-cadherin, vimentin, and N-cadherin was detected in HPNE cell lines by Western blot analysis. (C) The expression of invasion-related proteins MMP2 and uPA in HPNE cell lines was detected by Western blot analysis. β-Actin was used as loading control.</p

HPNE cells with activation of K-ras and inactivation of p16 induced tumorigenesis <i>in vivo</i>.

<p>(A) <i>In vivo</i> bioluminescence imaging of tumor growth by HPNE, HPNE/K-ras and HPNE/K-ras/p16shRNA cells at 8 weeks' after injection in NOD/SCID mice is shown. (B) The rates of tumor formation and metastasis in NOD/SCID mice. (C) Representative micrographs showing the histology of the orthotopic tumors formed by HPNE/K-ras/p16shRNA cells as revealed by H&E staining: (i) undifferentiated ductal carcinoma with sarcomatoid features, (ii) necrosis, (iii) liver metastasis, and (iv) spleen metastasis. (D) Immunohistochemical analysis of the expression of HER-2 and EGFR in tumors formed <i>in vivo</i> by the HPNE/K-ras/p16shRNA cells compared with those in human pancreatic cancer and human normal pancreatic duct. Scale bar: 100 µm.</p