The Role of K67 As a Radio-sensitizer in Esophageal Cancer

Abstract

The roles of p-p62-Keap1-Nrf2 axis in the origin and development of cancers have been widely studied in multiple carcinomas. But its role in the radioresistance of esophageal cancer has not been revealed yet. This might be one of the targets for radiotherapy of esophageal cancer. We collected the tissue samples of 164 patients with locally advanced esophageal cancer who received concurrent chemoradiotherapy (CCRT) as first-line therapy and we analyzed the association of expression of p-p62 and nuclear Nrf2 with patients’ prognosis. Besides, we studied the function of K67, a compound blocking the interaction of p-p62 and Keap1, as a radio-sensitizer in TE-1 cells and its underlying mechanism. Higher expression of p-p62 and nuclear Nrf2 were associated with lower response rate (p=0.005 and <0.001 respectively). The expression of p-p62 and nuclear Nrf2 were independent prognostic factor for OS (p=0.018 and 0.02 respectively). Besides, the expression of p-p62 showed positive correlation with that of nuclear Nrf2, especially in patients with no response to CCRT (rho=0.754, p<0.001). We then studied the function of K67. Western blot showed that the expression of p-p62 and nuclear Nrf2 in TE-1 cells were dramatically increased after radiation. And CCK8 assay showed that K67 had no cytotoxicity to TE-1 cells, which made K67 a potential radio-sensitizer. The results of clone formation assay and flow cytometry suggested that K67 inhibited cell proliferation and meanwhile promoted apoptosis induced by radiation. Also, K67 increased the DNA double strand breaks and production of reactive oxygen species induced by radiation in TE-1 cells. And K67 promoted the G2/M cell cycle arrest, which was one of the effects of radiation. Further study showed that K67 dramatically decreased the level of Nrf2 entering the nucleus. Consequently, the expression of antioxidant enzymes transcribed by Nrf2, such as NQO1 and HO1, was also decreased. The result of CoIP showed that K67 blocked the interaction of p-p62 and Keap1 and meanwhile promoted the combination of Keap1 and Nrf2. The p-p62-Keap1-Nrf2 axis affects the radiosensitivity of esophageal cancer. And the compound K67 can increase the radiosensitivity of esophageal cancer via blocking the interaction of p-p62 and Keap1.