Journal of the National Cancer Institute Advance Access originally published online on July 29, 2008
JNCI Journal of the National Cancer Institute 2008 100(15):1092-1103; doi:10.1093/jnci/djn216
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© The Author 2008. Published by Oxford University Press.
ARTICLES |
Effect of Lapatinib on the Outgrowth of Metastatic Breast Cancer Cells to the Brain
Affiliations of authors: Women's Cancers Section, Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD (BG, DP, JLB, PSS); Laboratory Animal Sciences Program, SAIC-Frederick, National Cancer Institute, National Institutes of Health, Frederick, MD (JMH, EVV, LF); Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Research, National Institutes of Health, Bethesda, MD (DJL, SMS); Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD (MJM); GlaxoSmithKline, Philadelphia, PA (SDR)
Correspondence to: Patricia S. Steeg, PhD, Women's Cancers Section, Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 37, Room 1122, MSC 4254, Bethesda, MD 20892 (e-mail: steegp{at}mail.nih.gov).
Background: The brain is increasingly being recognized as a sanctuary site for metastatic tumor cells in women with HER2-overexpressing breast cancer who receive trastuzumab therapy. There are no approved or widely accepted treatments for brain metastases other than steroids, cranial radiotherapy, and surgical resection. We examined the efficacy of lapatinib, an inhibitor of the epidermal growth factor receptor (EGFR) and HER2 kinases, for preventing the outgrowth of breast cancer cells in the brain in a mouse xenograft model of brain metastasis.
Methods: EGFR-overexpressing MDA-MB-231-BR (231-BR) brain-seeking breast cancer cells were transfected with an expression vector that contained or lacked the HER2 cDNA and used to examine the effect of lapatinib on the activation (ie, phosphorylation) of cell signaling proteins by immunoblotting, on cell growth by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and on cell migration using a Boyden chamber assay. The outgrowth of large (ie, >50 µm2) and micrometastases was counted in brain sections from nude mice that had been injected into the left cardiac ventricle with 231-BR cells and, beginning 5 days later, treated by oral gavage with lapatinib or vehicle (n = 22–26 mice per treatment group). All statistical tests were two-sided.
Results: In vitro, lapatinib inhibited the phosphorylation of EGFR, HER2, and downstream signaling proteins; cell proliferation; and migration in 231-BR cells (both with and without HER2). Among mice injected with 231-BR-vector cells, those treated with 100 mg lapatinib/kg body weight had 54% fewer large metastases 24 days after starting treatment than those treated with vehicle (mean number of large metastases per brain section: 1.56 vs 3.36, difference = 1.80, 95% confidence interval [CI] = 0.92 to 2.68, P < .001), whereas treatment with 30 mg lapatinib/kg body weight had no effect. Among mice injected with 231-BR-HER2 cells, those treated with either dose of lapatinib had 50%–53% fewer large metastases than those treated with vehicle (mean number of large metastases per brain section, 30 mg/kg vs vehicle: 3.21 vs 6.83, difference = 3.62, 95% CI = 2.30 to 4.94, P < .001; 100 mg/kg vs vehicle: 3.44 vs 6.83, difference = 3.39, 95% CI = 2.08 to 4.70, P < .001). Immunohistochemical analysis revealed reduced phosphorylation of HER2 in 231-BR-HER2 cell–derived brain metastases from mice treated with the higher dose of lapatinib compared with 231-BR-HER2 cell–derived brain metastases from vehicle-treated mice (P < .001).
Conclusions: Lapatinib is the first HER2-directed drug to be validated in a preclinical model for activity against brain metastases of breast cancer.
| CONTEXT AND CAVEATS Prior knowledge The brain is increasingly being recognized as a sanctuary site for metastatic tumor cells in women with HER2-overexpressing breast cancer who receive trastuzumab therapy. Treatment options for brain metastases are currently limited to steroids, cranial radiotherapy, and surgical resection. Study design A preclinical mouse model was used to test the efficacy of lapatinib, an inhibitor of the epidermal growth factor receptor (EGFR) and HER2 kinases, for preventing the outgrowth of breast cancer cells in the brain. EGFR-overexpressing brain-seeking breast cancer cells with or without HER2 overexpression were used to examine the effect of lapatinib on the phosphorylation of cell signaling proteins, on cell growth, and on cell migration. Contribution In vitro, lapatinib inhibited the phosphorylation of EGFR, HER2, and downstream signaling proteins; cell proliferation; and migration in brain-seeking breast cancer cells (both with and without HER2 overexpression). In vivo, lapatinib inhibited the formation of large brain metastases by HER2-overexpressing brain-seeking breast cancer cells by 50%–53% and reduced phosphorylation of HER2 in the brain metastases. Implications Lapatinib is the first HER2-directed drug to be validated in a preclinical prevention model for activity against brain metastases of breast cancer. Limitations Lapatinib did not completely inhibit the formation of large brain metastases, which suggests that some breast cancer cells may be resistant to this drug. From the Editors
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B. Gril and D. Palmieri contributed equally to this work.
Present address: Office of Science Planning and Assessment, NCI, Rockville, MD (J. L. Bronder).
We thank Drs Azize Sahin and Paul Meltzer (NCI) for sequencing EGFR in the 231-BR cell line and Dr Tona Gilmer (GlaxoSmithKline) for her helpful discussions concerning this manuscript. The authors take sole responsibility for the design of the study; the collection, analysis, and interpretation of the data; the writing of the manuscript; and the decision to submit the manuscript for publication.
Manuscript received October 22, 2007; revised May 5, 2008; accepted May 30, 2008.
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