Abstract 880: Analysis of NF-κB as a prediction marker of 5-FU based adjuvant chemotherapy for adenocarcinoma of the stomach.
Authors: Endo Fumitaka , Nishizuka Satoshi , Ishida Kazushige , Kume Kohei , Ide Taishi , Katagiri Hirokatsu , Ishida Kaoru , Iwaya Takeshi , Fujiwara Hisataka , Koeda Keisuke , Wakabayashi Go 
Published: August 2014

Introduction: The relapse rate of gastrointestinal cancer patients who underwent R0 resection followed by postoperative 5-FU based adjuvant chemotherapy is approximately 40%. Therefore, it is important to identify prediction markers of the chemotherapeutic efficacy. Recently, we identified NF-κB as a candidate relapse prediction biomarker from a cell-based screening followed by an immunohistochemical retrospective study (Ishida, et al PLoSONE, 2012). To evaluate the biological significance of NF-κB in the context of 5-FU-based chemotherapy, we analyzed the biological response of NF-κB stimulated by 5-FU.

Materials and Methods: Human gastric cancer cell lines (MKN45, MKN74, KE39, GSS, KatoIII) were used. Induction and localization of NF-κB in response to 5-FU by western blot and immunohistochemistry were observed. Transcriptional products induced by 5-FU were investigated using a DNA microarray. Knockdown of p65 and p53 genes was performed to examine their transcriptional machinery. Both DNA binding domain mutation and codon72 polymorphism in p53 gene were also investigated. Pro/Pro variant from codon72 affects the binding efficacy for NF-κB subunit p65.

Results: By applying 5-FU, both total p65 and phosphorylated-p65 levels were increased in the nucleus. By DNA microarray analysis, 10 transcript products induced by 5-FU were identified. Interestingly, 5 of 10 transcripts have been known as that of p53 downstream. Theoretical prediction of promoter binding sites of p53 and NF-κB revealed that p53 binding sequences were located closer to the transcription start site than those of NF-κB. However, the density of the consensus sequences of NF-κB was higher than that of p53. Interestingly, there were many binding sequences of NF-κB in TP53 promoter, but only a few of those of p53 in p65 promoter. Knockdown of p65 gene also decreased p53 protein as well as its downstream gene expression, such as p21. In contrast, NF-κB was not affected by TP53 knockdown. MKN45 and GSS, whose codon72 variant is Pro/Pro are relatively resistant to 5-FU, but Arg/Arg variant of KE39 and MKN74 is &gt;10times sensitive to 5-FU. KatoIII with a large deletion of TP53 is resistant to 5-FU. These facts may suggest that NF-κB and p53 binding is relevant to 5-FU sensitivity.

Conclusion: Present results suggest that: (i) NF-κB plays a more important role than p53 in response to 5-FU; and (ii) NF-κB may play a compensatory function in p53 mutant cells. From a viewpoint of predicting 5-FU based chemotherapeutic efficacy, NF-κB is considered to be a 5-FU-chemosensitivity prediction marker.

Citation Format: Fumitaka Endo, Satoshi Nishizuka, Kazushige Ishida, Kohei Kume, Taishi Ide, Hirokatsu Katagiri, Kaoru Ishida, Takeshi Iwaya, Hisataka Fujiwara, Keisuke Koeda, Go Wakabayashi. Analysis of NF-κB as a prediction marker of 5-FU based adjuvant chemotherapy for adenocarcinoma of the stomach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 880. doi:10.1158/1538-7445.AM2013-880