Pazopanib reveals a role for tumor cell B-Raf in the prevention of HER2+ breast cancer brain metastasis

Abstract

Purpose: Brain metastases of breast cancer contribute significantly to patient morbidity and mortality. We have tested pazopanib, a recently approved antiangiogenic drug that targets VEGFR1, VEGFR2, VEGFR3, PDGFRβ, PDGFRα, and c-kit, for prevention of experimental brain metastases and mechanism of action.

Experimental design: In vitro assays included B-Raf enzymatic assays, Western blots, and angiogenesis assays. For in vivo assays, HER2 transfectants of the brain seeking sublines of MDA-MB-231 cells (231-BR-HER2) and MCF7 cells (MCF7-HER2-BR3, derived herein) were injected into the left cardiac ventricle of mice and treated with vehicle or pazopanib beginning on day 3 postinjection. Brain metastases were counted histologically, imaged, and immunostained.

Results: Treatment with 100 mg/kg of pazopanib resulted in a 73% decline in large 231-BR-HER2 metastases (P < 0.0001) and a 39% decline in micrometastases (P = 0.004). In vitro, pazopanib was directly antiproliferative to 231-BR-HER2 breast cancer cells and inhibited MEK and ERK activation in vitro despite B-Raf and Ras mutations. Enzymatic assays demonstrated that pazopanib directly inhibited the wild type and exon 11 oncogenic mutant, but not the V600E mutant forms of B-Raf. Activation of the B-Raf targets pERK1/2 and pMEK1/2 was decreased in pazopanib-treated brain metastases whereas blood vessel density was unaltered. In the MCF7-HER2-BR3 experimental brain metastasis model, pazopanib reduced overall brain metastasis volume upon magnetic resonance imaging (MRI) by 55% (P = 0.067), without affecting brain metastasis vascular density.

Conclusions: The data identify a new activity for pazopanib directly on tumor cells as a pan-Raf inhibitor and suggest its potential for prevention of brain metastatic colonization of HER2(+) breast cancer.

Figure 1. Pazopanib directly targets breast tumor cells

A- Effect on cell viability. 231-BR-vector and 231-BR-HER2 transfectants were incubated with increasing concentrations of pazopanib for 96 h. Cells were assayed for viability using MTT. Data are represented as mean ± standard error of the mean (SEM) of three independent experiments. B- Effect on signaling pathways. 231-BR-vector or 231-BR-HER2 cells were serum starved overnight and subsequently treated with pazopanib or DMSO for 24 hours. After treatment, cells were stimulated with 10 ng/mL VEGF for 10 minutes, and effects on signaling proteins assayed. “p-” refers to phosphorylation of the residue(s) in parentheses. The data shown are representative of three separate experiments. C- Cell Cycle analysis. Cells were treated with vehicle (DMSO) or 5μM pazopanib for 72h then analyzed by flow cytometry.

Figure 2. Pazopanib inhibits B-Raf

A- Effect of pazopanib on B-Raf protein. Increasing concentrations (0.022, 0.11, 0.22, 0.44, 2.2 μM) of pazopanib, lapatinib or a non-clinical Raf inhibitor were incubated with B-Raf for 20 min at 30°C. Inactive MEK1 was added for 30 minutes and the level of MEK1 phosphorylation was analyzed with pMEK1 and total MEK1 antibodies. A Raf inhibitor was used as a positive control and lapatinib was used as negative control. The (−) column represents the level of MEK1 phosphorylation without the addition of enzyme. In the (+) column, no inhibitors were added, representing the maximum level of pMEK1. B- Effect of pazopanib on Raf activity in cell lysates. The same protocol was followed substituting MCF7-HER2, 231-BR or SKMEL28 cell lysates for B-Raf protein and using the following concentrations of inhibitors: 0.022, 00.22, 2.2 μM. C- Effect on cell viability. 231-BR-vector cells were incubated with increasing concentrations of pazopanib, Raf inhibitor or MEK inhibitor (U0126) for 96 h. Cells were assayed for viability using MTT. Data are represented as mean ± SEM. D and E- B-Raf siRNA transfection. 231-BR-vector (D) and MCF7-HER2 (E) cell lines were transfected with two different B-Raf siRNA constructs (S1 and S2), with a non targeting siRNA (C), or treated with the transfectant agent alone (T). Cell lysates were collected at 48 and 96h after transfection.

Figure 3. Pazopanib inhibits brain metastatic colonization of 231-BR-HER2 cells targeting Raf-MEK-ERK pathway

Brain sections from five mice per group were stained for pERK1/2, pMEK1/2 and pAKT. The intensity of staining was quantified using the intensity scores 0, 1+, 2+ or 3+. Representative photographs of the staining are shown for each treatment group. P values are reported where it was significant (p<0.01).

Figure 4. Pazopanib does not affect blood vessel density

A- Top- Mean number of blood vessels per high power field. Bottom- Percentage of area occupied by blood vessels, both determined by image analysis of CD31 staining. P values comparing brain metastasis to normal brain are indicated. Raw data are presented but statistical analysis was performed on square root transformed data. B- Representative photographs of CD31 staining in normal brain and metastases.

Figure 5. Pazopanib decreases the brain metastatic outgrowth of MCF7-HER2-BR3 breast carcinoma cells without an effect on the vasculature

A- Mice were injected with MCF7-HER2-BR3 cells and randomized to receive vehicle or 100 mg/kg pazopanib. MRI analyses were performed on 10 mice per group. Each mouse had a single metastasis. Each dot represents a mouse. B- Representative MRI scans of mouse brains. C and D- Vascular changes. C- Blood vessel density. D- Percentage of brain section covered by blood vessels. Raw data are presented but statistical analysis was performed on square root transformed data. E to F- Representative photographs taken at 100x magnification: CD31 staining in (E) normal brain tissue and (F) in metastatic tissue.