Widespread activation of the DNA damage response in human pancreatic intraepithelial neoplasia

Abstract

Pancreatic intraepithelial neoplasia (PanIN) lesions are the most common non-invasive precursors of pancreatic adenocarcinoma. We postulated that accumulating DNA damage within the PanIN epithelium activates checkpoint mechanisms. Tissue microarrays were constructed from 81 surgically resected primary pancreatic adenocarcinomas and an independent set of 58 PanIN lesions (31 PanIN-1, 14 PanIN-2, and 13 PanIN-3). Immunohistochemical labeling was carried out using anti-gammaH2AX(Ser139), anti-phosphoATM(Ser1981), anti-phosphoChk2(Thr68), and anti-p53. A 'histologic score' combining area and intensity of labeling in the nuclear compartment was determined for each lesion. A progressive increase in gammaH2AX(Ser139) labeling, consistent with escalating DNA damage, was observed in the non-invasive precursor lesions (scores of 4.34, 6.21, and 7.50, respectively, for PanIN-1, -2, and -3), compared with the pancreatic ductal epithelium (score 2.36) (ANOVA, P<0.0001). In conjunction, activation of the ataxia telangiectasia mutated (ATM)-Chk2 checkpoint pathway was observed in all histological grades of PanIN lesions. Specifically, pATM(Ser1981) histologic scores for PanIN-1, PanIN-2, and PanIN-3 were 4.83, 5.14, and 7.17, respectively, versus 2.33 for the ductal epithelium (ANOVA, P<0.0001); the corresponding scores for pChk2(Thr68) were 5.43, 7.64, and 5.44 in PanINs-1, -2, and -3, respectively, versus 2.75 in the ductal epithelium (ANOVA, P<0.0001). In contrast, absent to minimal nuclear p53 was observed in the ductal epithelium, and in PanINs-1 and -2 (a histologic score of 0-1.86), with a significant upregulation (corresponding to mutational inactivation) seen only at the stage of PanIN-3 and invasive neoplasia (histologic scores of 4.00 and 4.22). Nuclear p53 accumulation in cancers was associated with attenuation of the ATM-Chk2 checkpoint and a restitution to 'baseline' levels. To conclude, activation of the ATM-Chk2 checkpoint pathway is commonly observed in PanINs, likely in response to the accumulating DNA damage from events such as oncogene mutations and telomere dysfunction. Loss of p53 function appears to be a critical determinant for bypassing this checkpoint and the subsequent progression to invasive adenocarcinoma.

Figure 1

Histograms illustrating the histologic scores for each of the four proteins analyzed in this study, including phospho-γH2AX^Ser139, phospho-ATM^Ser1981, phospho-Chk2^Thr68, and p53. The histologic scores are stratified by normal ductal epithelium, PanINs-1, -2 and -3, and invasive cancer. The mean histologic score and standard deviations are represented for each grade of lesion. See text for details and statistical analyses.

Figure 2

Representative photomicrographs illustrating expression of phospho-γH2AX^Ser139, phospho-ATM^Ser1981, phospho-Chk2^Thr68, and p53 in various histological grades of pancreatic ductal lesions.

Figure 3

A proposed model of DNA damage response (DDR) mediated by the ATM-Chk2 checkpoint in the pancreatic ductal epithelium. In response to double strand breaks, DDR can be observed in the earliest PanIN lesions, and the increase in ATM-Chk2 expression parallels the histological progression to high-grade PanIN-3. Inactivation of p53 function at the stage of PanIN-3 and beyond is associated with bypass of the DDR checkpoint, and progression to invasive cancer.