C. Subramanian1, N. Zhang1, B. B. Blagg2, M. S. Cohen1 1University of Michigan,General Surgery,Ann Arbor, MI, USA 2University of Notre Dame,Department Of Chemistry And Biochemistry,Notre Dame, INDIANA, USA
Introduction: Heat shock protein 90 (Hsp90) is a molecular chaperone that is critical for several cellular processes including the folding, maturation and degradation of client proteins involved in the development of cancers (including the breast cancer) and their metastatic spread. Current Hsp90 inhibitors in clinical trials non-selectively target all four isoforms of Hsp90 and have shown detrimental toxicities. Through an innovative structure guided design, our collaborative group has recently developed the first Hsp90β isoform specific inhibitors. During the initial screens with our lead β specific Hsp90 inhibitor, KUNB105, we noted that cancer cells with wild type (wt) p53 were more sensitive to the drug than p53 mutant (mt) cells. Therefore, we have hypothesized that it is the differential effect in p53 pathway proteins to Hsp90β selective inhibition that leads to this selective effect on p53 wt breast cancer (BC) cells compared to mt p53 cell lines.
Methods: Validated BC cell lines MCF7 (p53 wt) and MDA-MB-231 (p53 mt) were treated with KUNB105 and viability of cells were measured using cell TiterGlo. Changes in expression levels of Hsp90 clients were analyzed by western. Apoptosis was measured using RealTime-Glo Annexin V Apoptosis and Necrosis Assay. Changes in cancer pathway genes was analyzed after treatment of cells with KUNB105 using Qiagen Cancer Pathway Profiler assay. Prism software was used for statistical analysis and all experiments were repeated in triplicate.
Results: Viability of MDA-MB-231 and MCF7 cells after treatment with KUNB105 showed an IC50 value of 20.5 & 8.2 μ M respectively. Analysis of p53-pathway by Western showed dose dependent increase in p53 (3-5 fold) and mdm2 (2-3 fold) starting from 10 μ M KUNB105 treatment for MCF7 cells whereas the levels of p53 did not show observable change even at the highest concentration of 30 μ M KUNB105 (p<0.001) for MDA-MB-231. By contrast, the cell cycle dependent protein p21 showed dose dependent upregulation starting at 1μ M KUNB105 (2- fold, p<0.01) in both cell lines indicating that KUNB105 treatment leads to cell cycle arrest. Evaluation of the effect of KUNB105 treatment on apoptosis showed increase in luminescence starting from 2 h (30000-33000 compared to around 12000 for the control, p<0.001) and peaked at 10 h (50,000-54,000, p<0.001) for both the cells indicating significant induction of apoptosis. Evaluation of cancer pathway after KUNB105 treatment of BC cells showed differential expression of genes in cell cycle (AURKA, CCND2, E2F4, MK167, WEE1, STM1), DNA damage and repair (ERCC5 and GADD45G) as well as apoptosis (BCL2L11).
Conclusions: Our results indicate that KUNB105 has selectivity to p53 wt BC and induces apoptosis through p53 pathway-specific mechanistic differences between wt and mt BC cell lines. Further studies related to this unique targeting effect are needed for translational applications of this promising and novel Hsp90β inhibitor.