PurposeThe 26S proteasome plays important roles in many intracellular processes and is therefore a critical intracellular cellular target for anticancer treatments. The primary aim of the current study was to identify critical proteins that may play roles in opposing the antisurvival effect of the proteasome inhibitor bortezomib together with the calcium-chelator BAPTA-AM in cancer cells using label-free LC-MS/MS. In addition, based on the results of the proteomic technique, a novel and more effective inhibitor combination involving bortezomib as well as OTSSP167 was developed for breast cancer cells.Methods and resultsUsing label-free LC-MS/MS, it was found that expressions of 1266 proteins were significantly changed between the experimental groups. Among these proteins were cell division cycle 5-like (Cdc5L) and drebrin-like (DBNL). We then hypothesized that inhibition of the activities of these two proteins may lead to more effective anticancer inhibitor combinations in the presence of proteasomal inhibition. In fact, as presented in the current study, Cdc5L phosphorylation inhibitor CVT-313 and DBNL phosphorylation inhibitor OTSSP167 were highly cytotoxic in 4T1 breast cancer cells and their IC50 values were 20.1 and 43nM, respectively. Under the same experimental conditions, the IC50 value of BAPTA-AM was found 19.9M. Using WST 1 cytotoxicity assay, it was determined that 10nM bortezomib+10nM CVT-313 was more effective than the control, the single treatments, or than 5nM bortezomib+5nM CVT-313. Similarly, 10nM bortezomib+10nM OTSSP167 was more cytotoxic than the control, the monotherapies, 5nM bortezomib+5nM OTSSP167, or than 5nM bortezomib+10nM OTSSP167, indicating that bortezomib+OTSSP167 was also more effective than bortezomib+CVT-313 in a dose-dependent manner. Furthermore, the 3D spheroid model proved that bortezomib+OTSSP167 was more effective than the monotherapies as well as bortezomib+CVT-313 and bortezomib+BAPTA-AM combinations. Finally, the effect of bortezomib+OTSSP167 combination was tested on MDA-MB-231 breast cancercells, and it similarly determined that 20nM bortezomib +40nM OTSSP167 combination completely blocked the formation of 3D spheroids.ConclusionsAltogether, the results presented here indicate that bortezomib+OTSSP167 is a novel and effective combination and may be tested further for cancer treatment in vivo and in clinical settings.