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Background Esophageal adenocarcinoma (EAC) is characterized by poor prognosis and low survival rate. Chronic gastroesophageal reflux disease (GERD) is the main risk factor for the development of Barrett’s esophagus (BE), a preneoplastic metaplastic condition, and its progression to EAC. Yes-associated protein 1 (YAP1) activation mediates stem-like properties under cellular stress. The role of acidic bile salts (ABS) in promoting YAP1 activation under reflux conditions remains unexplored. Methods A combination of EAC cell lines, transgenic mice, and patient-derived xenografts were utilized in this study. mRNA expression and protein levels of APE1 and YAP1 were evaluated by qRT-PCR, western blot, and immunohistochemistry. YAP1 activation was confirmed by immunofluorescence staining and luciferase transcriptional activity reporter assay. The functional role and mechanism of regulation of YAP1 by APE1 was determined by sphere formation assay, siRNA mediated knockdown, redox-specific inhibition, and co-immunoprecipitation assays. Results We showed that YAP1 signaling is activated in BE and EAC cells following exposure to ABS, the mimicry of reflux conditions in patients with GERD. This induction was consistent with APE1 upregulation in response to ABS. YAP1 activation was confirmed by its nuclear accumulation with corresponding up-regulation of YAP1 target genes. APE1 silencing inhibited YAP1 protein induction and reduced its nuclear expression and transcriptional activity, following ABS treatment. Further investigation revealed that APE1-redox-specific inhibition (E3330) or APE1 redox-deficient mutant (C65A) abrogated ABS-mediated YAP1 activation, indicating an APE1 redox-dependent mechanism. APE1 silencing or E3330 treatment reduced YAP1 protein levels and diminished the number and size of EAC spheroids. Mechanistically, we demonstrated that APE1 regulated YAP1 stability through interaction with β-TrCP ubiquitinase, whereas APE1-redox-specific inhibition induced YAP1 poly-ubiquitination promoting its degradation. Conclusion Our findings established a novel function of APE1 in EAC progression elucidating druggable molecular vulnerabilities via targeting APE1 or YAP1 for the treatment of EAC.

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.

In this short communication we report a quick, cost-free method of purification of DNA fragments from agarose gel. Unlike those procedures that involve commercial kits, this method uses glass wool or absorbent cotton to filter agarose gel during a quick spinning-down of DNA, thus significantly simplifying the routine practice of many molecular biologists and decreasing the cost.

In this short communication we report a quick, cost-free method of purification of DNA fragments from agarose gel. Unlike those procedures that involve commercial kits, this method uses glass wool or absorbent cotton to filter agarose gel during a quick spinning-down of DNA, thus significantly simplifying the routine practice of many molecular biologists and decreasing the cost.