Overexpression of S100A4 in pancreatic ductal adenocarcinomas is associated with poor differentiation and DNA hypomethylation

Abstract

Using the National Center for Biotechnology Information Serial Analysis of Gene Expression database, we found that S100A4, a calcium-binding protein previously implicated in metastasis, was expressed in five of seven pancreatic carcinoma libraries but not in the two normal pancreatic duct libraries. We confirmed the overexpression of S100A4 using reverse transcriptase-polymerase chain reaction, which demonstrated that 18 of 19 (95%) pancreatic carcinoma cell lines expressed S100A4. Using immunohistochemistry, we found that 57 of 61 invasive pancreatic carcinomas (93%), 3 of 18 high-grade pancreatic intraepithelial neoplasia lesions (17%), and 0 of the 69 low-grade pancreatic intraepithelial neoplasia lesions expressed S100A4 protein, whereas normal pancreatic tissue and tissue affected by chronic pancreatitis did not label. Expression of S100A4 was associated with poor differentiation of the pancreatic adenocarcinomas (P = 0.001). We found that three CpG sites in the first intron of the S100A4 gene were approximately 90% methylated in microdissected normal pancreatic duct cells using bisulfite-modified sequencing and in two cell lines and three primary pancreatic carcinomas with a reduced or absent expression of S100A4. In contrast, these CpGs were 100% hypomethylated in 11 of 12 pancreatic cancer cell lines by methylation-specific polymerase chain reaction. The association between the expression of S100A4 and hypomethylation of the first intron of S100A4 was statistically significant (P = 0.002). These data suggest that the majority of pancreatic carcinomas undergo selection for hypomethylation and overexpression of S100A4. Because most pancreatic carcinomas express S100A4, it may be a useful target for early detection strategies.

Figure 1.

RT-PCR analysis of 19 pancreatic carcinoma cell lines and 2 colorectal carcinoma cell lines (SW480 and RKO). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a RNA control. All cancer cell lines but Hs766T and RKO strongly express S100A4. Treatment of Hs766T with 5-aza-2-deoxycytidine restores the expression of S100A4.

Figure 2.

Immunostaining of pancreatic tissue with anti-S100A4 polyclonal antibody. A: Strong labeling of pancreatic adenocarcinoma cells with an absence of labeling of the normal pancreatic cells. B: Pancreatic adenocarcinoma not labeled for S100A4. C: Pancreatic adenocarcinoma with strong labeling of the poorly differentiated area (bottom) whereas the well-differentiated area (top) does not label. D: Reactive ductal change in chronic pancreatitis showing an absence of labeling of duct cells. Original magnifications: ×160 (A, B, D); ×100 (C).

Figure 3.

Bisulfite-modified sequencing of the first intron of the S100A4 gene showing the methylation status of two CpG sites (+315, +331). NP 133 is a normal pancreas sample with 90% methylation; Hs766T pancreatic cancer cell line is completely methylated; and PC 19 is a primary pancreatic adenocarcinoma, labeled for S100A4 on immunohistochemistry, showing a 90% conversion of the cytosine residues to thymine.