Abstract 1565: Pazopanib reveals B-Raf regulation of anti-angiogenesis pathways and prevents brain metastatic colonization of HER2+ breast cancer cells

Abstract

Abstract Brain metastases occur in approximately 35% of metastatic breast cancer patients whose tumors overexpress HER2. Using a quantitative model system to study breast cancer brain metastasis, HER2 overexpression increased the brain colonization of a brain seeking subline of MDA-MB-231 cells (231-BR-HER2). Since HER2 has been shown to induce overexpression of VEGF, we hypothesized that the anti-angiogenic drug pazopanib (GW786034) would reduce the metastatic outgrowth of breast cancer cells in the brain. Pazopanib is a multispecific tyrosine kinase inhibitor targeting VEGFR-1, −2, and −3, PDGFR-alpha and beta, and c-kit. In vitro, pazopanib not only inhibited proliferation of human brain endothelial cells but also the 231-BR tumor cell line (IC50 = 4microM). Pazopanib disrupted the Erk signaling pathway despite the fact that 231-BR cells have a constitutively activated Ras pathway; expressing mutant K-Ras (codon13) and mutant B-Raf (G464V). Interestingly, using either purified B-Raf or cell lysates, we showed that pazopanib could directly inhibit B-Raf kinase activity as shown by a decreased in pMEK1/2. This data identifies a new mechanism of action of pazopanib as a direct inhibitor of B-Raf. Pazopanib inhibition of B-Raf was greater for the wild type enzyme and the G464V mutant than the V600E mutation. The inhibition of Ras-Raf-ERK pathway by pazopanib was confirmed in vivo in xenograft models using seven different breast carcinoma and melanoma cell lines presenting different genotypes for the Ras-Raf-ERK pathway (mutation in Ras, mutation in B-Raf, overexpression of kinases upstream Ras (HER2) or wild type for the entire pathway). Anti-angiogenic potency in this cell line panel directly correlated with anti-B-Raf activity, suggesting that B-Raf pathways control angiogenic mediator production. Finally we demonstrated that Pazopanib prevented the outgrowth of brain metastasis. In the brain metastasis mice model, 231-BR-HER2 cells produced a mean of 3.92 large metastases (>300microM) and 101.9 micro metastasis per brain section in the vehicle treated mice. Treatment with 30 mg/kg Pazopanib resulted in a 51% decline to 1.93 large metastases (p=0.0002) and 25% decrease to 76.4 micro metastasis (p=0.07), while treatment with 100 mg/kg resulted in a 73% decline to 1.05 large metastases (p<0.0001) and 39% decline in micro metastasis to 61.7 (p=0.004). These experiments contradict previous research claiming that anti-angiogenic agents promote metastasis, and support a clinical trial for the treatment or prevention of HER2+ brain metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1565.