Abstract 5025: PAX5 methylation as a novel biomarker of esophageal cancer for both potential tumor malignancy and cisplatin sensitivity
Authors: Kurimoto Keisuke , Hayashi Masamichi , Koike Masahiko , Kanda Mitsuro , Nishikawa Yoko , Iwata Naoki , Niwa Yukiko , Takami Hideki , Kobayashi Daisuke , Tanaka Chie , Yamada Suguru , Nakayama Goro , Sugimoto Hiroyuki , Fujiwara Michitaka , Fujii Tsutomu , Rafael Guerrero-Preston , Kodera Yasuhiro 
Published: July 2016

Abstract
Therapeutic strategy of esophageal cancer largely depends on histopathological assessment just like tumor differentiation and TNM stage. In order to make appropriate choice for individual case, we have to pay attention to background molecular characteristics about tumor malignancy or chemotherapy sensitivity.
In order to find extremely tumor-specific marker of esophageal cancer, we picked several methylation markers from what we previously used in the research of head and neck cancer, which has the same background of smoking and alcohol and the same tissue-type. As a result, we identified PAX5 gene methylation as a very tumor-specific marker also in surgically resected 90 esophageal cancer cases. Then, we continued to examine PAX5 expression, function and other functions.
Quantitative MSP (QMSP) assay revealed significantly higher methylation score (100x[PAX5 QMSP]/[ACTB QMSP]) in tumor tissues (16.2±2.4) than in normal tissues (0.4±0.2) (P&lt;0.001, Mann-Whitney U test). Also, 77/90 (85.6%) of these cases showed tumor-specific QMSP elevation. Similarly, all 9 esophageal cancer cell lines (TE1, TE2, TE3, NUEC1, NUEC2, NUEC3, T.T, T.Tn and WSSC) showed high QMSP scores over 10. The inverse correlation of PAX5 methylation and expression was confirmed by Quantitative RT-PCR of clinical samples and 5-aza-dC treated cell lines. PAX5 downregulated cases indicated significantly poor overall survival (P = 0.037, log-rank test). For functional analysis, we examined WST1 assay and BrdU assay using ectopically PAX5 knock down NUEC1 cell line, which has secondly high PAX5 expression. The result showed significantly high cell proliferation and cell cycle acceleration. Since PAX5 is reported to bind p53 promoter and regulate its expression, we hypothesized epigenetically downregulated PAX5 might affect p53 expression and therefore cisplatin sensitivity of esophageal cancer. Actually, PAX5 downregulated NUEC1 cell line acquired significantly cisplatin-resistant character after siRNA transfection. Moreover, among postoperatively cisplatin-treated cases (25 cases), PAX5 hyper-methylated clinical cases (n = 6) demonstrated significantly worse survival than PAX5 hypo-methylated cases (n = 19) (P = 0.002, log-rank test). We also surveyed another downstream mechanism of PAX5 dysregulation. Human Cancer Pathway Finder PCR Assay identified three key molecules including SLC2A1 (Fold change: 4.6), CCL2 (12.5) and IGFBP5 (-5.0).
Epigenetic inactivation of PAX5 gene may affects malignant potential of esophageal cancers and cisplatin-based chemotherapy through p53 dysregulation or other carcinogenic pathways. It could be utilized for planning individual multidisciplinary therapy.
Citation Format: Keisuke Kurimoto, Masamichi Hayashi, Masahiko Koike, Mitsuro Kanda, Yoko Nishikawa, Naoki Iwata, Yukiko Niwa, Hideki Takami, Daisuke Kobayashi, Chie Tanaka, Suguru Yamada, Goro Nakayama, Hiroyuki Sugimoto, Michitaka Fujiwara, Tsutomu Fujii, Guerrero-Preston Rafael, Yasuhiro Kodera. PAX5 methylation as a novel biomarker of esophageal cancer for both potential tumor malignancy and cisplatin sensitivity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5025.