Abstract 2627: Mechanisms of bortezomib resistance in human NSCLC cell lines; potential role of proteasome beta5-subunits and autophagy

Abstract

Abstract Non-small cell lung cancer (NSCLC) is a disease with poor prognosis and novel treatments are warranted. The proteasome inhibitor bortezomib (BTZ, Velcade), which is successfully applied for the treatment of primary and refractory Multiple Myeloma, may also elicit activity against solid tumors, including lung cancer. To further explore this, we investigated mechanisms underlying a 12-fold difference in BTZ sensitivity in a panel of 3 human NSCLC cells : H460 (IC50:69 nM), A549 (IC50:22 nM) and SW1573 (IC50:6 nM) after 72 hours drug exposure. We also evaluated the effects of long term BTZ exposure to identify whether BTZ resistance may emerge in these NSCLC cells. For this purpose, H460, A549 and SW1573 cells were exposed to stepwise increasing concentrations of BTZ over a period of 5 months resulting in BTZ-resistance levels of 2.6-, 6.9- and 4.9-fold, respectively, compared to their parental counterparts. Since BTZ primarily targets the beta-5 subunit of the proteasome, we examined cross-resistance to other proteasome inhibitors; 4A6, a 6-mer peptide that specifically targets the beta-5 subunit of the proteasome, and 5-amino-8-hydroxyquinoline (5AHQ), which primarily targets the non-catalytic alpha-7 subunit of the proteasome. Moreover, proteasome catalytic activities were measured by means of an intact cell-based fluorogenic assay. Finally, the potential role of autophagy as a possible cell survival or cell death mechanism after BTZ exposure was examined utilizing immunohistochemistry staining with LC3B as a marker for authophagy. The generated BTZ-resistant NSCLC cells displayed up to 10-fold cross-resistance to 4A6, but retained full sensitivity to 5AHQ, suggesting that quantitative or qualitative alterations of the beta-5 subunit are implicated in BTZ resistance. The basal proteasome catalytic activity demonstrated an increase of 2.9- and 1.4-fold for the chymotrypsin like activity and a 4.5- and 1.5-fold for the caspase-like activity in H460 compared to SW1573 and A549, respectively, indicating a relationship between basal proteasome activity and BTZ sensitivity. Of note, no marked differences in basal proteasome activity levels were observed between parental and BTZ-resistant cells. Nevertheless, to inhibit proteasome activity by > 80%, 2-fold higher BTZ concentrations were required in BTZ-resistant cells. Finally, the role of autophagy as a mechanism contributing to BTZ resistance was examined. We observed a marked induction of BTZ-induced autophagy in H460 and SW1573 cells upon exposure to 100 nM and 10 nM BTZ, respectively. Strikingly, no signs of autophagy were noted in the generated BTZ-resistant H460 and SW1573 cells. In conclusion, these data support the notion that alterations in proteasome beta-5 subunit targeting and loss of autophagy may contribute to a reduced efficacy of BTZ in NSCLC cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2627.