HMGB1 Release by Urothelial Carcinoma Cells is Required for the In Vivo Antitumor Response to Bacillus Calmette-Guerin

Purpose: Prior series showed that a portion of urothelial carcinoma cells exposed to bacillus Calmette-Guerin undergoes nonapoptotic cell death and release of the chemokine HMGB1. We evaluated the role of tumor cell derived HMGB1 in mediating the in vivo antitumor effect of bacillus Calmette-Guerin. Materials and Methods: The murine urothelial carcinoma cell line MB49 was engineered to express a shRNA construct targeting HMGB1. The shRNA expressing cell line underwent characterization to ensure its comparability to the parental MB49 cell line. An orthotopic tumor model was used to compare the in vivo antitumor efficacy of bacillus Calmette-Guerin in the parental cell line (24 control and 24 bacillus Calmette-Guerin treated) vs the HMGB1 knockdown line (23 control and 21 treated). Results: Expression of the shRNA construct decreased HMGB1 expression and its release in response to bacillus Calmette-Guerin. The parental and shRNA cell lines showed similar in vitro doubling time and cytotoxicity in response to bacillus Calmette-Guerin. Treatment significantly decreased tumor volume vs controls in parental MB49 tumor bearing mice (p = 0.036). Tumor volume in treated mice inoculated with the shRNA cell line was higher than that in sham treated shRNA controls (p = 0.12). Of the bacillus Calmette-Guerin treated mice tumor volume was significantly lower in parental tumor bearing mice vs the shRNA group (p < 0.00001). ANOVA revealed a significant interaction between the cell line (shRNA vs parental) and the bacillus Calmette-Guerin effect (p = 0.0076). Conclusions: The direct tumor response to bacillus Calmette-Guerin, culminating in HMGB1 release, may be an important contributor to the clinical efficacy of bacillus Calmette-Guerin.