Abstract P6-15-13: Biologic Activity of a Novel Heat Shock Protein 90 Inhibitor, PF-4928473, in Human Breast Cancer Cell Lines
Authors: Tsang RY , Conklin D , Kalous O , Desai A , Dering J , Ginther C , Christensen J , Slamon DJ , Finn RS. 
Published: April 2011

Abstract
Background: Heat shock protein 90 (HSP90) is a key molecular chaperone involved with the post-translational folding and maturation of various cellular proteins, including Raf, Src, ER, EGFR, HER2/ErbB2, IGFR, and VEGFR. Therefore, targeting HSP90 represents an attractive strategy for modulating aberrant oncogenic signal transduction in human malignancies. In the present study, we investigate the anti-cancer activity of PF-4928473, a novel small molecule inhibitor of HSP90, in a diverse panel of human breast cancer cell lines in vitro.
Methods: 49 breast cancer cell lines (representing 10 luminal, 18 HER2- amplified, 18 non-luminal, 3 immortalized subtypes), including two HER2- amplified lines with conditioned trastuzumab and lapatinib resistance (SKBR3 parental), were assessed. Cell proliferation assays with two-fold drug dilutions over 9 concentrations were performed over 6 days to determine growth-adjusted IC50 and IC100 values, the latter as a surrogate of in vitro drug lethality. Flow-cytometry was performed in a selected subset of 21 cell lines to assess effects on cell cycle and apoptosis after 48 hours and 5 days of drug exposure, respectively. Western blot analysis evaluated the biochemical effects of HSP90 drug inhibition. Results: In our panel, IC50s ranged from 8 (±1) nM to 525 (±2) nM, with 45 cell lines exhibiting IC50s ≥50 nM. Differential drug sensitivity was observed with HER2-amplified lines, with 15 of 18 demonstrating IC50s ≥28 nM. For SKBR3-trastuzumab and -lapatinib-resistant lines, IC50s were 13 (±1) nM and 21 (±2) nM, respectively. IC100s ranged from 9 (±3) nM to &gt; 1000 nM. 17 of 18 HER2-amplified lines had IC100s between 9 (±3) nM and 69 (±1) nM, including SKBR3-trastuzumab and -lapatinib-resistant lines at 31 (±2) nM and 66 (±2) nM, respectively. Flow cytometry with 50 nM of PF-4928473 revealed moderate early apoptotic effects in cell lines with IC50s &lt; 30 nM, encompassing the majority of HER2-amplified lines. Mild effects on cell cycle, primarily G0/G1 arrest, were observed in 8 lines at 50 and/or 100 nM. Western blots of representative cell lines at 50 and 100 nM of PF-4928473 identified downregulation of p-AKT and p-ERK in the HER2-amplified SKBR3 line (IC50: 19 ±4 nM), vs. relative lack of effect for the non-luminal MDA-MB-468 (IC50: 24 ±5 nM) and luminal KPL-1 (IC50: 68 ±2 nM) lines. Upregulation of HSP70, a known biomarker associated with HSP90 inhibition, was observed at 12 hours post-drug exposure regardless of cell sensitivity. Further biochemical studies are ongoing.
Conclusions: PF-4928473 demonstrated low nanomolar in vitro anti-proliferative activity, lethality, and apoptosis in a wide panel of human breast cancer cell lines, including trastuzumab and lapatinib-resistant lines. Biological effects were most pronounced in HER2-amplified lines, in keeping with HER2 as a known protein client of HSP90. Based on these results, further translational evaluation of HSP90 inhibitors in this population merits consideration.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-13.