Significant association between 53 BP1 expression and grade of intraepithelial neoplasia of esophagus: Alteration during esophageal carcinogenesis

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Abstract

Background

Abnormal DNA damage response (DDR) leads to genomic instability and carcinogenesis. P53-binding protein 1 (53 BP1), a DDR molecule, is known to accumulate at the sites of DNA double-strand breaks. The aim of this study was to analyze the expression pattern of 53 BP1-nuclear foci (NF) in esophageal neoplasms in order to visualize the state of DDR in esophageal carcinogenesis and to clarify its significance in the molecular pathology of the disease.

Methods

A total of 61 lesions from 22 surgically resected samples of esophageal cancer, including histologically normal squamous epithelium, low-grade intraepithelial neoplasia (LG-IN), high-grade intraepithelial neoplasia (HG-IN), carcinoma in situ (CIS), and invasive squamous cell carcinoma (SCC), were included in the study. 53 BP1 and Ki-67 expression were analyzed by double-labeled immunofluorescence.

Results

The number of discrete 53 BP1-NF increased as the tumor progressed from normal epithelium through LG-IN, HG-IN, CIS, and SCC. 53 BP1-NF larger than 1 μm in diameter (large foci), indicating intensive DDR, also showed a stepwise increase during the progression of carcinogenesis. Of note, large foci of 53 BP1 were found in significantly higher numbers in HG-IN than in LG-IN. Furthermore, localization of 53 BP1-NF in Ki-67-positive cells, indicating the abnormal timing of DDR, also increased with malignancy progression.

Conclusions

53 BP1-NF accumulation increases during cancer progression from LG-IN to HG-IN to CIS to SCC. Detection of 53 BP1-NF by immunofluorescence, especially large foci, is a feasible method of estimating DNA instability and the malignant potential of esophageal intraepithelial neoplasia.

Introduction

Esophageal cancer is the sixth most common cause of cancer-related deaths worldwide [1]. There are two major subtypes of esophageal cancer: squamous cell carcinoma (SCC) and adenocarcinoma. While adenocarcinomas are prevalent in western countries, SCC is more common in Asian countries, such as Japan. The development of esophageal SCC is considered a multistep process that progresses from normal squamous epithelium to intraepithelial neoplasia to carcinoma [2], a process that has been genetically confirmed by whole-genome sequencing (WGS) [3]. Intraepithelial neoplasia in the esophagus is further classified into low-grade intraepithelial neoplasia (LG-IN) and high-grade intraepithelial neoplasia (HG-IN); LG-IN refers to when the neoplasia occupies less than half of the epithelium, whereas HG-IN refers to lesions involving more than half of the epithelium (WHO classification, 2010) [2]. When the full thickness of the epithelium is involved, it is classified as carcinoma in situ (CIS). HG-IN, CIS, and early SCC limited to the lamina propria are suitable for endoscopic treatment [4,5]. As the prognosis of advanced esophageal SCC is poor, it is of clinical interest to detect lesions suitable for treatment during the early phase. Identifying the extent of DNA damage response (DDR) occurring during SCC development may provide beneficial implications for early diagnosis and identifying appropriate therapeutic strategies.

Genomic instability (GIN) is thought to play an essential role in cellular transformation during SCC pathogenesis [3]. Tumor protein-53-binding protein 1 (53 BP1) is a large (350 kD) multi-domain protein that belongs to a family of evolutionarily conserved DDR proteins with BRCA1 C-terminus (BRCT) domains [[6], [7], [8], [9]]. 53 BP1 is a nuclear protein that localizes rapidly to sites of DNA double-strand breaks (DSBs) and activates p53, along with other kinases [8,[10], [11], [12], [13], [14]]. Activated kinases play a critical role in DDR, including, the induction of cell cycle arrest, DNA repair, and apoptosis [15,16]. 53 BP1 has also been well described as a marker for DDR. It has been well documented (via immunofluorescence) that 53 BP1 exhibits diffuse nuclear expression in untreated primary cells and is localized at DSB sites, forming discrete nuclear foci (NF) after radiation exposure in vitro [8,10,17,18]. In addition to activation by exogenous stimuli, 53 BP1 also forms NF during endogenous DDR during malignant transformation. Since GIN is triggered by intensive endogenous DDR [19,20], 53 BP1 expression can serve as a potential marker for estimating the level of GIN. We previously demonstrated a correlation between the appearance of 53 BP1 and the malignant potential of human tumors, including thyroid [21], skin [22], and uterine cervical cancers [23]. It appears that the presence of 53 BP1-NF larger than 1.0 μm in diameter (large foci: LF) represents prolonged DDR and is closely associated with a higher level of cervical carcinogenesis [23]. In the current study, we attempted to clarify the utility of 53 BP1 expression as a molecular marker of DNA instability in esophageal squamous neoplasms.

Section snippets

Patients and histological evaluation

This study was conducted retrospectively in accordance with the Declaration of Helsinki and was approved by the Institutional Ethical Committees for Medical Research at Nagasaki University (approval date: July 24, 2015; #15,062,617).

A total of 61 lesions from 22 surgically resected, formalin-fixed, paraffin-embedded (FFPE) esophageal cancer tissues were selected for the study; these included (1) normal squamous epithelium without nuclear or cellular atypia outside the margin of the tumor (12

Expression of p53 and total 53 BP1 in esophageal lesions

To confirm the accuracy of pathological diagnosis, p53 expression was first assessed in normal epithelium, LG-IN, HG-IN, and CIS samples (Fig. 1, Table 1). Normal epithelium did not exhibit p53 expression, whereas the basal layers of LG-IN were positive for p53 (11.1%). HG-IN showed p53 staining from the basal layers to the surface of the epithelium (55.6%), and a similar p53 expression pattern was observed in CIS (58.3%), consistent with the enhanced expression of mutated p53 during

Discussion

The present study used 53 BP1 expression levels to demonstrate distinct DNA damage patterns during esophageal carcinogenesis (Fig. 6). We found that the number of discrete 53 BP1-NF—a cytological marker of GIN [19]—in esophageal epithelium progressively increased as the tumor progressed from normal epithelium through LG-IN, HG-IN, CIS, and finally SCC. 53 BP1 is classified as an adaptor/mediator within the DDR mechanism, which is required for processing of the DDR signal and as a platform for

Conclusions

Our study demonstrated a stepwise increase in 53 BP1-NF during carcinogenesis. We also revealed the difference in GIN between LG-IN and HG-IN based on 53 BP1-LF expression. As 53 BP1-NF reflects DNA DSBs, we hypothesize that it represents the level of GIN in squamous cell neoplasms in the esophagus.

Authors’ contributions

NU, SM, and TI performed the experiments; NU and YA drafted the manuscript; KM and HK provided critical input regarding the manuscript; HK performed the statistical analyses; MN conceptualized the study and conducted the experiments. All authors read and approved the final version of the manuscript prior to submission.

Funding sources

This work was supported in part by the Atomic Bomb Disease Institute of Nagasaki University, a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture (Grant no. 23790406) and the Program of the Network-Type Joint Usage/Research Center for Radiation Disaster Medical Science.

Declaration of Competing Interest

The authors have no competing interests.

Acknowledgments

We wish to thank Editage (www.editage.jp) for English language editing.

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    These authors contributed to equally to this work.

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