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 performed using anti- γH2AXSer139, anti-phosphoATMSer1981, anti-phosphoChk2Thr68, 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 γH2AXSer139 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 to pancreatic ductal epithelium (score 2.36) (ANOVA, P<0.0001). In conjunction, activation of the ATM-Chk2 checkpoint pathway was observed in all histological grades of PanIN lesions. Specifically, pATMSer1981 histologic scores for PanIN-1, PanIN-2, and PanIN-3 were 4.83, 5.14, and 7.17, respectively, versus 2.33 for ductal epithelium (ANOVA, P<0.0001); the corresponding scores for pChk2Thr68 were 5.43, 7.64, and 5.44 in PanINs-1, -2, and -3, respectively, versus 2.75 in ductal epithelium (ANOVA, P<0.0001). In contrast, absent to minimal nuclear p53 was observed in ductal epithelium, and in PanINs-1 and 2 (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 score of 4.00 and 4.22). Nuclear p53 accumulation in cancers was associated with attenuation of the ATM-Chk2 checkpoint and a restitution of “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.

Functional p38 MAPK Identified by Biomarker Profiling of Pancreatic Cancer Restrains Growth through JNK Inhibition and Correlates with Improved Survival

“…For example, short term activation of JNK may promote cell survival (21,39), whereas long term activation may lead to increased apoptosis such as through the E3 ubiquitin ligase ITCH that promotes c-Jun degradation (23,40,41). Of interest, USP9x has recently been identified as a tumor suppressor in pancreatic cancer that also exerts it effects though regulation of ITCH expression (24,42), lending support to the notion that ITCH-dependent JNK signaling that leads to apoptosis, a consequence of chronic JNK activation by ongoing DNA damage (25,43), may be one of the cellular functions targeted.…”

Genome-Wide CpG Island Profiling of Intraductal Papillary Mucinous Neoplasms of the Pancreas

“…The remaining steps were performed using an automated immunostainer (DakoCytomation).Immunolabeled sections were lightly counterstained in with hematoxylin, dehydrated in ethanol, and cleared in xylene. Immunohistochemical labeling of sox17 protein was scored using a previously described histologic score quantifying both the area and intensity of labeling (33). Labeling Intensity was designated as 0–2 for absent, weak, and strong, and the area of labeling was designated as 0–4 for <5%, 5–25%, 26–50%, 51-75, and >76%, respectively (33).…”

Imaging DNA Damage Allows Detection of Preneoplasia in the BALB-neuT Model of Breast Cancer

“…Extensive published evidence has shown that the DDR is activated during development of many tumor types (17). Several reports have shown immunohistochemical evidence that DDR proteins are highly expressed in precancerous lesions but less so in invasive cancers or corresponding normal tissues (4)(5)(6)(7)(8). It is therefore logical to explore these molecules as biomarkers of early cancer development and reasonable to hypothesize that visualization of components of the DDR might provide an excellent means of early cancer detection (9).…”

“…Early reports of DDR activation during bladder, colon, breast, skin, and lung cancer development showed that it was accompanied by the activation of signaling proteins downstream of phosphorylated ATM (pATM), including pChk2 and the phosphorylated histone H2A variant H2AX, gH2AX (4,5). DDR activation has since been observed to occur during the development of many other types of cancer including pancreatic (6), ovarian (7), and hepatocellular carcinomas (8). Given the near-ubiquitous nature of DDR activation during oncogenesis, it has been suggested that markers of DNA replication stress and constitutive DDR activation might distinguish neoplastic growth from normal tissues.…”