P5-06-01: Gene Expression Analysis of Resistance to Bevacizumab in a VEGF-Reinforced Xenograft Model of ER-Positive Breast Cancer.

Abstract Background: Bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor (VEGF), had promising therapeutic efficacy in breast cancer. However, intrinsic or acquired resistance is common in the clinic. To improve our understanding of the underlying mechanisms of resistance to bevacizumab (BEV), we report the gene expression analysis of resistance to bevacizumab in a VEGF-overexpressing xenograft model of ER-positive breast cancer. Methods: We developed a nude mouse xenograft model of resistance to anti-VEGF therapy with BEV in which MCF-7 control (ML20) or MCF-7 VEGF (MV165) transfectants were implanted in mammary fat pads, allowed to grow, then treated with BEV, with collection of tumor at early or late time points (while responding (R) to or progressing (NR) on anti-VEGF therapy). To elucidate differentially expressed gene profiling associated with tumor resistance to BEV, we performed whole-genome gene expression analysis (Human WG-6v2 Expression Beadchips, Illumina) and miRNA profiling (TaqMan ***ArrayHuman MicroRNAA+B Cards Set v3.0, Applied Biosystems). Validation of the chosen genes was performed using quantitative real-time RT-PCR (qRT-PCR). Results: Gene expression analysis revealed differentially regulated genes in the MV165-NR group compared with the MV165-R group. Among the significant genes, Follistatin (FST) and HEY2 were the top genes upregulated in NR compared to R by ANOVA. Expression of HEY2 is induced by the Notch signaling pathway. Using qRT-PCR, we validated the expression of FST and Notch in our system. FST was significantly decreased (Fold change= −3.2; P=0.03) in the R group compared with vehicle in MV165 xenografts. In contrast to R group, FST was upregulated significantly (Fold change= 9.3; P=0.05) in the NR group. Notch4 displayed increased levels of expression in NR group, but it did not reach significance (P=0.23). In addition, correlation of mRNA and miRNA profiles showed that miRNAs targeting FST and Notch4 were differentially regulated in NR group compared to R group in MV165 xenograft tumors. Among the miRNAs, TGF-β-induced oncomiR miR-181a is up-regulated in NR and targets both FST and Notch4. Other miRNAs that target both Notch4 and FST include miR-1, miR-133a, miR-133b, and mir-449b. Conclusion: Our data serve as a potential mechanistic explanation for acquired resistance to bevacizumab. These data may shed light on the transitory effect of BEV observed in the E2100 firstline metastatic breast cancer trial, where VEGF-targeted therapy prolongs progression-free survival in metastatic breast cancer without improving overall survival. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-01.