Abstract 3316: The role of high mobility group box 1 in the combination therapy of gemcitabine and radiation in muscle invasive bladder cancer
Authors: Mansure Jose J. , Almajed Wael S. , Shrivastava Sanhita , Cury Fabio , Ferbeyre Gerardo , Popovic Marija , Seuntjens Jan , Kassouf Wassim 
Published: August 2015

Purpose: Radical cystectomy is the gold standard treatment for muscle invasive bladder cancer. However, it causes a serious impact in the quality of life of the patients. Radiation therapy when combined with radio-sensitizing chemotherapy is an attractive alternative as it offers bladder preservation allowing for normal urinary and sexual functions. However, lack of local control and dose dependent toxicity are the main drawbacks of the combination therapy. As HMGB1 has been found to be associated with DNA damage response and chronic inflammation pathways of radiation resistance, we sought to investigate the role of HMGB1 in the response to radiation in bladder cancer.

Methods: The expression of HMGB1 among different bladder cancer cell lines was analyzed by mRNA and Protein quantification. The mean growth inhibition to Gemcitabine was assessed by colorimetric assays of cellular viability. Knock down of HMGB1 was done using lentiviral shRNA system. To assess the effect of the combination therapy, Clonogenic assay was done. Cells were exposed to the mean growth inhibition dose of gemcitabine for 6 hours followed by varying doses of radiation (2-8 Gy). Analysis of cell cycle was done by Flow Cytometry. Western blot was utilized to assess pathways involved in cellular survival. In vivo, athymic mice were subcutaneously injected with two bladder cancer cell lines. Treatment consisted of either placebo or combined gemcitabine (1 g/m2, 6 hours prior to radiation) and fractionated IR (total 6Gy = 3Gy×2) in cells with scramble vs knockdown of HMGB1. Mice were followed over 3 weeks. Tumor weight was measured at experimental endpoint.

Results: HMGB1 expression was analyzed in eight urothelial carcinoma cell lines. Amongst all, UM-UC3 and UM-UC5 had the highest expression while 253J-BV had the lowest expression of HMGB1. There was a significant correlation between the levels of HMGB1 and resistance to radiation (P value &lt;0.0001). Our results from clonogenic assays post combined treatment shows that knockdown of HMGB1 resulted in a dose modifying factor (DMF) of 2.95 for a survival fraction of 0.5. Moreover, western blot analysis revealed that high expression of HMGB1 was associated with increased and sustained activation of ERK pathway. Also, cell cycle analysis showed that upon HMGB1 knockdown, cell cycle arrest was induced at S phase post gemcitabine treatment and at G2 phase post radiation and combination therapy. To validate these results, subcutaneous tumors were induced in athymic mice and underwent two fractions of the combined treatment. Significant sensitization effect upon HMGB1 knockdown was observed, evident by tumor volume (P value &lt;0.0001) and weight (P value &lt;0.05).

Conclusion: Our in vitro and in vivo results strongly suggest that investigating HMGB1 increases efficacy of combination therapy of gemcitabine and radiation. Baseline of HMGB1 could be a good marker to predict response to such therapy.

Citation Format: Jose J. Mansure, Wael S. Almajed, Sanhita Shrivastava, Fabio Cury, Gerardo Ferbeyre, Marija Popovic, Jan Seuntjens, Wassim Kassouf. The role of high mobility group box 1 in the combination therapy of gemcitabine and radiation in muscle invasive bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3316. doi:10.1158/1538-7445.AM2015-3316