Mo1298 GITR STIMULATION ENHANCES THE ANTI-TUMOR IMMUNE RESPONSE IN A MOUSE MODEL OF ESCC

Background: Esophageal adenocarcinoma (EAC) is the most common type of esophageal cancer (>80%) in the United States.Unfortunately, approximately 80% of patients are diagnosed at advanced stages (III or IV) with a five-year survival rate less than 5%.According to American Cancer Society, smoking is a major and important risk factor of EAC.Understanding mechanisms in which smoking promotes EAC and the development of rationale therapeutic options that address biological and molecular basis of EAC tumorigenesis are urgently needed to improve clinical outcome.WEE1 is a nuclear kinase with a known function of phosphorylating CDC2/cyclin B kinase and negatively regulating cell cycle in normal biological conditions.Methods and Results: Using Immunohistochemistry (IHC) staining of WEE1 in normal esophageal (NE) or esophageal adenocarcinoma (EAC) tissue samples, our data indicated that WEE1 was significantly overexpressed in EAC compared with NE samples.Western blot data demonstrated that WEE1 protein was remarkably highly expressed in EAC cell lines and 3D organotypic culture of EAC OE19 cells.Western bolt data showed that nicotine treatment induced WEE1 expression, and CDC2 phosphorylation (p-CDC2, Y15), a well-established function of WEE1, in both FLO-1 and OE33 cells.To further validate our findings, we treated EAC cells with Nicotine-derived nitrosamine ketone (NNK), which is one of the most important and most potent carcinogens in tobacco and tobacco smoke.Western blot results indicated that NNK treatment induced WEE1 and p-CDC2 expression in both FLO-1 and OE33 cells.To best simulate aqueous smoking effect on EAC cells, we treated EAC cells with 2% cigarette smoke extract (CSE) as reported before.Consistent with nicotine and NNK treatment, 2% CSE induced WEE1 expression in both FLO-1 and OE33 cells detected by Western blot.Using immunofluorescence (IF) staining, the results confirmed our findings that both CSE and NNK significantly increases WEE1 expression in FLO-1 compared with control cells.Our data also indicated that miR-497-5p reconstitution decreased WEE1 protein expression in FLO-1 cells, while smoking significantly decreased miR-497-5p expression in EAC cells.To further study the therapeutic relevance of WEE1, we tested the response of EAC to cisplatin with or without WEE1 inhibition.Interestingly, WEE1 inhibitor significantly sensitized FLO-1 cells to cisplatin treatment.The knockdown of WEE1 in FLO-1 cells significantly decreased clonogenic cell survival challenged with a low level of cisplatin (CDDP) treatment compared with scramble siRNA cells.Conclusion: Our data demonstrated, for the first time, that smoking induced WEE1 overexpression in EAC could play an important role in EAC drug resistance.WEE1 inhibition is a promising therapeutic method to overcome the drug resistant and treatment refractory cancer cells.