Abstract 1828: A genomic-transcriptomic-phosphoproteomic study to interrogate lapatinib resistance in an HER2 over expressing SKBR3 human breast cancer cell line model
Authors: Thavasu Parames , Poele Robert T. , Hughes Mark , Stewart Adam , Workman Paul , Banerji Udai 
Published: May 2015

Abstract
Background: Lapatinib is a tyrosine kinase inhibitor that inhibits EGFR and HER2 and is licensed to treat HER2-amplified breast cancer. Understanding mechanisms of acquired resistance is an unmet clinical need.
Methods: SKBR3 cells were continuously exposed to increasing concentrations of lapatinib over a 12-month period. The parent cell line (SKBR3) and resulting lapatinib-resistant cell line (SKBR3LR) then underwent a set of studies including: a) western blot analysis for PGP, b) whole exome sequencing, c) whole genome expression microarray, d) quantification of 587 phosphoproteins. All data were integrated using Metacore software. Sensitivity of SKBR3 and SKBR3LR to a panel of drugs based on the findings was carried out by 96-hr sulforhodamine assays.
Results: The GI50 values for lapatinib in SKBR3LR and SKBR3 cells were 3582 nM (SD 241.6) and 10.3 nM (SD 1.1). There was no induction of PGP protein or mRNA expression levels in SKBR3LR compared to SKBR3. Somatic mutations in KIT and MEK3 which were not present in SKBR3 were detected in SKBR3LR. There was a reduction in the levels of p-HER2 and p-EGFR in SKBR3LR; however, no differences in the levels of p-HER3, p-HER4, and p-KIT between the cell lines were observed. Analysis of proteins downstream in the RAS-RAF-MEK and PI3K-AKT-m-TOR showed increased levels of p-MEK but no significant changes in p38MAPK and no differences in p-AKT levels, but an increase in p-P70S6K in SKBR3LR compared to SKBR3. The phosphorylation network changes were integrated with transcription factors that showed significant enrichment for binding sites in the genes that were differentially expressed in SKBR3LR. Deregulation of the MAPK signalling network was one of the proposed mechanisms of resistance of SKBR3LR cells to lapatinib. Differential sensitivity to small-molecule agents was used to interrogate the mechanism of lapatinib resistance further. There was no significant differential sensitivity of SKBR3 and SKBR3LR to imatinib, GI50 values 16.2 uM (SD 1.2) and 13.64 uM (SD1.1), respectively. SKBR3LR was more resistant than SKBR3 to an AKT inhibitor AKT1/2i; GI50 values 8 uM (SD 0.45) vs 17.3 uM (SD 1.1) respectively. Interestingly, SKBR3LR was more sensitive to the HSP90 inhibitor, AUY922; GI50 values 3.2 nM (SD 1.1) and 11.2 nM (SD 0.42) in SKBR3LR and SKBR3 cells, respectively. Experiments are ongoing using targeted agents to understand the mechanism of lapatinib resistance and suggest potential therapeutic strategies to overcome these, including novel combinations, particularly the use of HSP90 inhibitors.
Conclusions: Integrating genomic-transcriptomic and phosphoproteomic data provides useful insights into the mechanism of resistance to lapatinib. The lapatinib-resistant cell line model was sensitive to the HSP90 inhibitor, AUY922, suggesting HSP90 inhibitors could be clinically effective in this setting.
Citation Format: Parames Thavasu, Robert Te Poele, Mark Hughes, Adam Stewart, Paul Workman, Udai Banerji. A genomic-transcriptomic-phosphoproteomic study to interrogate lapatinib resistance in an HER2 over expressing SKBR3 human breast cancer cell line model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1828. doi:10.1158/1538-7445.AM2014-1828