P3-04-02: Bevacizumab Treatment Alters Intrinsic Subtypes in a VEGF-Reinforced Xenograft Model of ER-Positive Breast Cancer.

Abstract Background: Anti-vascular endothelial growth factor (anti-VEGF) therapy improves disease-free but not overall survival in metastatic breast cancer. To seek further insight on resistance to anti-VEGF antibody bevacizumab (BEV) at the molecular level, we developed breast cancer xenograft models allowing comparison of tumor response at different time-points. Here we report the gene expression and miRNA analyses of response and non-response to BEV in these models. Methods: MCF-7 cells transfected with control vector (ML20) or VEGF (MV165) were implanted into the mammary fat pads of athymic mice. Tumors from short-term treatment with BEV (3 weeks; Responders to BEV, R) or long-term treatment (8 weeks; Non-Responders, NR) or with vehicle control group (V) were subjected to whole-genome gene expression analysis (Human WG-6v2 Expression Beadchips, Illumina) and miRNA profiling (TaqMan ArrayHuman MicroRNA A+B Cards Set v3.0, Applied Biosystems).Validation of the chosen genes was performed using quantitative real-time RT-PCR (qRT-PCR) and Immunohistochemistry (IHC). Results: Short-term treatment to BEV (3 weeks; 5 mg/kg, i.p./twice weekly) inhibited primary tumor growth significantly in MV165 xenografts compared with vehicle control, whereas BEV treatment did not affect the tumor growth in the ML20 model. MV165 xenografts progressed after 8 weeks of BEV treatment. Gene set enrichment analysis (GSEA) revealed that luminal A-related gene sets were enriched in MV165-R compared to MV165-NR group including DESMEDT (ESR1), SMID_Breast_Cancer_Luminal_A_up, and MASSARWEH_ Tamoxifen_Resistance_ Down. Myoepithelial-specific gene sets were upregulated in both the R and NR groups compared with the vehicle group. qRT-PCR analysis showed that estrogen receptor alpha (ESR1) representative for luminal A decreased significantly in the MV-165-NR group (P=0.001) compared to vehicle. In contrast, Cytokeratin 5 (KRT5) levels increased significantly in both R (P=0.02) and NR (P=0.03) groups. In addition, KRT14 was upregulated in R (P= 0.04) and in NR (P=0.14) group in comparison with the vehicle group, suggesting the upregulation of myoepithelial phenotype specific to BEV treated MV165 model, but not ML20 model. Similar results were obtained by IHC. Consistent with mRNA changes, ESR1 regulated miRNA such as miR-107 (P=0.007) and miRNA important in tamoxifen resistance such as mir-451 (P= 0.0003) were also altered in MV165-NR group compared to vehicle. Conclusion: These results suggest that treatment with BEV may alter the intrinsic subtypes in the presence of VEGF expression. These data may help to explain the variable results to anti-VEGF therapy based on the duration of BEV treatment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-04-02.