Abstract 746: Acquired bortezomib resistance in non-small cell lung cancer is associated with overexpression of the mutated β5 proteasome subunit

Abstract

Abstract The proteasome inhibitor bortezomib (Velcade®) is registered for the treatment of multiple myeloma, but has limited activity in solid tumors such as non-small cell lung cancer (NSCLC). Bortezomib predominantly inhibits proteasome subunit β5 and mutations in this subunit were recently associated with bortezomib resistance in leukemic cell line models. Herein we studied the molecular mechanisms underlying intrinsic and acquired bortezomib resistance in NSCLC cells. H460, A549 and SW1573 NSCLC cells demonstrated differential intrinsic sensitivities towards bortezomib with IC50 values of 12.6, 9.0 and 2.1 nM, respectively. Basal proteasome activities in these cell lines measured by an intact cell proteasome activity assay revealed that high basal levels of chymotrypsin- and caspase-like proteasome activities strongly correlate with intrinsic bortezomib resistance (R2=0.99, P<0.05 and R2=0.99, P<0.06, respectively).We established a model for acquired bortezomib resistance in H460, A549 and SW1573 cells by exposing cells to gradually increasing concentrations of bortezomib starting from 5nM to 80 or 200 nM for H460, 40 or 100 nM for A549 as well as 30 or 150 nM for SW1573. These bortezomib-resistant cell lines, named H460BTZR80, H460BTZR200, A549BTZR40, A549BTZR100, SW1573BTZR30 and SW1573BTZR150 displayed 14 to 57-fold bortezomib resistance. Consistantly, bortezomib-resistant cells required higher bortezomib concentrations to induce a G2/M cell cycle arrest and activation of apoptosis. Overall activation of apoptosis was reduced even at higher bortezomib concentrations when compared to parental cells treated with equitoxic drug doses. Furthermore, bortezomib-resistant cells exhibited increased levels of both constitutive β-subunits and immuno-β-subunits. Moreover, sequence analyses of the bortezomib binding pocket encoded by exon 2 of the PSMB5 gene revealed a previously described Ala49Thr mutation and two newly identified Met45Val and Cys52Phe mutations. Nevertheless, alterations in the proteasome subunits did not affect basal catalytic proteasome activities, although higher bortezomib concentrations were required in bortezomib-resistant cells to achieve comparable inhibition levels of proteasome activity. Cross-resistance was also observed to other proteasome inhibitors that specifically target the β-subunits of the proteasome. Interestingly, no cross-resistance was found to 5AHQ, a novel non-competitive proteasome inhibitor that targets the α7-subunit. Taken together, these findings establish a correlation between bortezomib-sensitivity and low basal levels of proteasome activity. In addition, acquired resistance to bortezomib in NSCLC was associated with expression of mutant β5-subunits that may compromise bortezomib binding. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 746. doi:10.1158/1538-7445.AM2011-746