Molecular Analysis of Precursor Lesions in Familial Pancreatic Cancer

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S100A4 Contributes to the Suppression of BNIP3 Expression, Chemoresistance, and Inhibition of Apoptosis in Pancreatic Cancer

International audiencePancreatic ductal adenocarcinoma (PDAC) is a devastating disease that is characterized by a particularly marked resistance to chemotherapy. We previously showed an association between decreased expression of BNIP3 and chemoresistance in PDAC cell lines. To further explore the molecular basis of chemoresistance in PDAC, we analyzed microarray data obtained from normal pancreas and PDAC tumor samples to identify genes exhibiting a negative correlation with the expression profile of BNIP3. This analysis identified several S100 family proteins, of which two, S100A2 and S100A4, showed in vitro the ability to repress exogenous BNIP3 promoter activity. We subsequently showed that RNA interference-mediated S100A4 knockdown resulted in an elevated expression of BNIP3 in PDAC cell lines that possess an unmethylated BNIP3 promoter, suggesting that, in addition to hypermethylation, S100A4 overexpression may represent an alternative mechanism for inhibiting BNIP3 function in PDAC. S100A4 knockdown also resulted in an increased sensitivity of PDAC cell lines to gemcitabine treatment, which was coupled with an increase in apoptosis and cell cycle arrest. To investigate the underlying mechanisms mediating these effects, we studied the effect of silencing the expression of S100A4 on the induction of apoptosis, cell cycle arrest, and the activation of apoptotic mediators. Knockdown of S100A4 clearly induced apoptosis with increased fragmentation of DNA and phosphatidyl serine externalization; activation of caspase-3, caspase-9, and poly(ADP-ribose) polymerase; and release of cytochrome c into the cytosol. These findings provide evidence that supports a novel role for S100A4 as a prosurvival factor in pancreatic cancer

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Confirmation of gene expression profiling.

<p>QRT-PCR analysis validated the differential expression for AGR2, S100P, FOS and EGR1 in primary PanIN lesions: PanA1, B1-3 and C1,2 represent non-X families, while PanX1 and 3 belong to Family X samples; PDAC1-7 represent seven different PDAC specimens.</p

Localisation and expression of BCL6 and HMGB1.

<p><b>(A)</b> Representative images of BCL6 positive cells (brown staining) in the stroma in the vicinity of PanIN lesions are shown in the top two panels (both magnified ×100); two bottom images show inflammatory infiltrate with BCL6 immunoreactive cells in two PDAC cases (magnification ×100 and ×50, respectively). <b>(B)</b> HMGB1 nuclear expression (brown staining) was seen in all pancreatic compartments, including stromal immune infiltrate: top panels show PanIN-1 (left) and -2 (right) (magnification ×50, insert and second panel x100); bottom panels show PanIN-3 (left) and PDAC (right) (magnification ×100 and ×50, respectively).</p

Expression of TFF1 in familial PanIN lesions.

<p>Panel <b>(A)</b> shows PanIN-1 with no TFF1 immunoreactivity, <b>(B)</b> and <b>(C)</b> PanIN-2 and <b>(D)</b> PanIN-3 lesion in the centre with strong TFF1 expression (all magnified x100).</p

Clinical information.

<p>M = male; F = female; HgA1c = Hemoglobin A1c (normal range 4.0–6.0); IDDM = insulin dependent diabetes mellitus; AODM = adult onset diabetes mellitus; NA = not available; Nl = normal; Abn =  abnormal; CP = chronic pancreatitis; EUS = endoscopic ultrasound; ERCP = endoscopic retrograde cholangiopancreatography; PDAC = pancreatic adenocarcinoma; FU = follow up.</p

Histology of PanIN lesions.

<p>The top panel shows the histology of three members of non-X-families (A1, B1 and C2). Images at the top show PanIN-1 and -2 lesions (magnification ×100); and images at the bottom show PanIN-3 lesions from family B and C; magnification ×200). The lower panel shows the histology of three different members of Family X: X1, X5 and X6 at the top show their gross appearance (magnification ×20); images at the bottom show PanIN-1 from X1 sample (magnification ×100); and PanIN-3 lesions from X5 and X6 (magnification ×200). * indicates adjacent histologically normal appearing tissue.</p