Abstract
Abstract Background: The incidence of esophageal adenocarcinoma (EAC) has rising rapidly in the US and western countries during past three decades. However, it remains very lethal disease with an overall five-year survival rate less than 20%, due to later stages on diagnosis and lack of efficient therapeutic approaches. The development of alternative therapeutic strategies based on updated biological and molecular discoveries are urgently needed to improve clinical outcome. NFE2-related factor 2 (NRF2) is a major regulator of cellular anti-oxidant properties that maintains cell viability and cellular homeostasis. We have found that NRF2 is constitutively overexpressed in EAC. Here we show that overexpression of NRF2 in EAC promotes tumor growth and targeting NRF2 may have therapeutic efficacy in EAC. Methods and Results: Using western blotting analysis, we observed overexpression of NRF2 protein level in EAC cell lines comparing to cell lines originating from normal esophagus and Barrett's esophagus. We confirmed overexpression of NRF2 in primary EAC tissue samples using immunohistochemistry (IHC) staining on a tissue microarray. Knocking down of NRF2 using NRF2 specific siRNAs (validated by downregulation of NRF2 target genes like HO-1, GR, NQO1) significantly inhibited tumor cell growth in FLO1 and OE33 tumor cells by colony formation assay. To target constitutively overexpressed NRF2, we used a potent NRF2 specific inhibitor, Brusatol. We first confirmed that Brusatol could significantly inhibit NRF2 ARE reporter activity in less than 100 nM range and subsequently downregulated NRF2 target genes like HO-1 and GR. Our data shows that EAC cancer cells are sensitive to Brusatol with IC 50 within 100 nM (using Colony formation assay and ATP-Glo assay), whereas Barrett's cells (CPA) and a normal esophageal fibroblast cells (hEF) are relatively resistant to Brusatol (IC 50 = 0.31 µM and 0.32 µM, respectively). Cisplatin (CDDP) is one of the first line chemotherapy drug for EAC. But drug-resistance usually emerges that leads to failure of treatments. Of note, Brusatol could kill cancer cells efficiently (with IC 50 less than 50 nM) in CDDP resistant cells, such as OE19 of which the IC 50 to CDDP is more than 10 µM. Moreover, Brusatol in combination with CDDP could significantly sensitize cancer cells to CDDP treatments. It is known that cancer cells usually have higher than normal ROS level and NRF2 is the master regulator of cellular stress favoring cell survival. Our results displayed that inhibition of NRF2 by Brusatol in combination with CDDP significantly increased cellular ROS level than using the drugs alone, and this sharp increase of ROS levels were associated with significant cell death as determined by flow cytometry of annexin V. In summary, our data indicate that targeting constitutively overexpressed NRF2 in EAC may have potential therapeutic efficacy alone or in combination with other therapeutic reagents. Citation Format: Dunfa Peng, Heng Lu, Tianling Hu, Kannappan Sriramajayam, Wael El-Rifai. Targeting constitutively overexpressed NRF2 in esophageal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1938.
