Abstract
Pazopanib is an FDA approved Vascular Endothelial Growth Factor Receptor inhibitor. We previously reported that it also inhibits tumor cell B-Raf activity in an experimental brain metastatic setting. Here, we determine the effects of different B-Raf genotypes on pazopanib efficacy, in terms of primary tumor growth and anti-angiogenesis. A panel of seven human breast cancer and melanoma cell lines harboring different mutations in the Ras-Raf pathway was implanted orthotopically in mice, and tumor growth, ERK1/2, MEK1/2 and AKT activation, and blood vessel density and permeability were analyzed. Pazopanib was significantly inhibitory to xenografts expressing either exon 11 mutations of B-Raf, or HER2 activated wild type B-Raf; no significant inhibition of a xenograft expressing the common V600E B-Raf mutation was observed. Decreased pMEK staining in the responsive tumors confirmed that B-Raf was targeted by pazopanib. Interestingly, pazopanib inhibition of tumor cell B-Raf also correlated with its anti-angiogenic activity, as quantified by vessel density and area. In conclusion, using pazopanib, tumor B-Raf status was identified as a significant determinant of both tumor growth and angiogenesis.
Highlights
The validation of drug targets, for multi-kinase inhibitors, will be key to predicting sensitivity and developing rational strategies to address resistance
A panel of seven human breast carcinoma and melanoma cell lines was examined for pazopanib responsiveness in vitro and in vivo
The breast cancer cell lines included the brain seeking variant of the ‘‘triple negative’’ MDA-MB-231 cell line, 231 BR ‘‘brain seeking’’ (231-BR), the estrogen receptor positive MCF7 cell line and the MCF7 line transfected with HER2
Summary
Its antiangiogenic activity was observed using corneal micropocket and matrigel plug assays. We recently identified B-Raf as a new, direct target for pazopanib [2]. Pazopanib altered the in vitro signaling of a brain metastatic derivative of MDA-MB-231 breast carcinoma cells, 231-BR, resulting in a reduction in the activity of the ERK pathway despite the presence of both Ras and B-Raf mutations. Enzymatic assays showed direct inhibition of B-Raf by pazopanib. Pazopanib prevented experimental brain metastases by 231-BR cells or HER2 transfectants of MCF7 breast carcinoma cells (selected for brain tropism, (MCF7-HER2-BR3)) by 73% and 55%, respectively; a concomitant reduction in pERK activity was observed, suggesting that B-Raf was a drug target in vivo. No antiangiogenic response was observed in the brain metastasis models, which may reflect the highly vascular nature of the brain where cooption of existing blood vessels is predicted to occur [3,4,5]
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