Cisplatin response of NSCLC cells irresponsive to knocking down VRAC subunits LRRC8A or LRRC8D

Abstract Platinum compounds represent the backbone of combination chemotherapy protocols for advanced lung cancer. Their efficacies are limited chiefly by either primal or chemotherapeutically acquired cellular platinum resistance whose complex mechanisms became more accessible through recent discoveries (Voets et al., 2015, EMBO J 34(24): 2985-2987). By stepwise increasing the cisplatin concentration in the culture medium, we generated cisplatin-resistant sublines of the wild type A240286S cell strain which derived from the suprarenal metastasis of a lung adenocarcinoma (Heuser et al., 2005, Cancer Lett 223(1): 57-66). All IC50 values were determined under flavin-protecting conditions (Granzow et al., 1995, Cancer Res 55(21): 4837-4843). For cisplatin, IC50 values were determined of 0.46±0.05 µM in the wild type A240286S cells, 6.03±1.39 µM in the Pt2.0 A240286S subline (growing in the presence of 2 µM cisplatin), 9.09±0.56 µM in the Pt4.0 A240286S subline (growing in the presence of 4 µM cisplatin), and 18.49±1.96 µM in the Pt8.0 A240286S subline (growing in the presence of 8 µM cisplatin). Testing for cross resistance to oxaliplatin of these tumor cell lines yielded respective IC50 values of 0.19±0.01 µM, 0.50±0.06 µM, 0.58±0.18 µM, and 0.78±0.11 µM oxaliplatin. Cultivation in the absence of cisplatin for seven months reduced the respective IC50 values for cisplatin to 0.99±0.07 µM in the Pt2.0 A240286S subline and to 4.02±0.23 µM in the Pt4.0 A240286S subline. As described by Planells-Cases et al., 2015, EMBO J 34(24): 2993-3008, the subunit composition of VRAC may determine the cellular resistance to platinum-based anti-cancer drugs in haploid KBM7 leukemia cells. In our study, the LRRC8D expression decreased with increasing cisplatin levels. In the Pt8.0 A240286S subline, LRRC8D expression was nearly abolished. However, knockdown of the LRRC8A or LRRC8D subunits of VRAC showed no effect on the cisplatin response in A240286S wild type cells. The respective IC50 values determined were 0.47±0.03 µM in the LRRC8A knockdown cells and 0.61±0.01 µM in the LRRC8D knockdown cells. In conclusion, in A240286S NSCLC cells, knockdown of the subunit composition of VRAC had no effect on cisplatin response. In addition, and in agreement with clinical experience (Stordal et al., 2007, Cancer Treat Rev 33(4): 347-357), the induction of cisplatin resistance in these cells was accompanied by cross resistance to oxaliplatin. Acknowledgments: We thank Rolf Jaggi and Irene Keller for contributing excellent technical support. Citation Format: Nico O. Ruprecht, Martin Hungerbühler, Christof Granzow, Christoph M. Kempf, Johannes T. Heverhagen. Cisplatin response of NSCLC cells irresponsive to knocking down VRAC subunits LRRC8A or LRRC8D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2844.