The proteasome inhibitor bortezomib, registered for Multiple Myeloma treatment, 
is currently explored for activity in solid tumors including non-small cell lung 
cancer (NSCLC). Here we studied the proteasome-based mechanisms underlying 
intrinsic and acquired bortezomib resistance in NSCLC cells. Various NSCLC cell 
lines displayed differential intrinsic sensitivities to bortezomib. High basal 
chymotrypsin- and caspase-like proteasome activities correlated with bortezomib 
resistance in these cells. Next, via stepwise selection, acquired bortezomib 
resistant cells were obtained with 8-70-fold increased resistance. 
Cross-resistance was found to proteasome inhibitors specifically targeting 
β-subunits, but not to the novel α-subunit-specific proteasome inhibitor (5AHQ). 
Consistently, bortezomib-resistant cells required higher bortezomib 
concentrations to induce G2/M arrest and apoptosis. Interestingly, bortezomib 
concentration-dependent caspase cleavage, Mcl-1 and NOXA accumulation remained 
intact in resistant H460 and SW1573 cells, while A549 resistant cells displayed 
different expression profiles suggesting additional and more protein specific 
adaptations. Furthermore, bortezomib-resistant cells exhibited increased levels 
of both constitutive and immuno-β-subunits. Sequence analysis of the 
bortezomib-binding pocket in the β5-subunit revealed Ala49Thr, Met45Val and 
Cys52Phe substitutions that were not previously described in solid tumors. 
Bortezomib-resistant cells displayed reduced catalytic proteasome activities and 
required higher bortezomib concentrations to achieve comparable inhibition of 
proteasome activity. Taken together, these findings establish that high basal 
levels of proteasome activity correlate with intrinsic bortezomib resistance. 
Furthermore, acquired bortezomib resistance in NSCLC is associated with 
proteasome subunit overexpression and emergence of mutant β5-subunits that 
likely compromise bortezomib binding. α-Subunit-specific proteasome inhibitors, 
however, can efficiently bypass this resistance modality.