Molecular Analysis of Breast Cancer Metastasis to the Br

Basic Sciences

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Abstract

Brain metastases occur in approximately 15% of metastatic breast cancer patients and confer a dismal prognosis. Brain metastases are thought to increasing, particuarly among women with Her-2 positive tumors. We conducted microarray analysis of surgically resected brain metastases of breast cancer, using laser capture microdissection, amplification and 30K cDNA arrays. These data were compared to a cohort of unmatched primary breast tumors, matched for histopathology, TNM and grade. A heat map comparing gene expression differences between brain metastses and unmatched primary tumors has been compiled and expression trends validated by QRT-PCR using an independent cohort. Analysis of the gene expression data indicated that 80% of the differentially expressed genes were down-regulated in the brain metastases. We asked whether a HDAC inhibitor could restore gene expression using a MDA-MB-231 human breast carcinoma cell subline selected for brain tropism. SAHA was selected from the multiple HDAC inhibitors since its structure appeared favorable for brain penetration. At an IC50, SAHA restored the expression of multiple down-regulated genes, and inhibited colonization and motility. We have developed a quantitative in vivo model for brain metastasis using the 231-BR cell subline. Treatment of mice with 100 mg/kg SAHA qd, beginning on day five postinjection, significantly reduced the number of large brain metastases. We are preparing these data for publication and Merck is planning a clinical trial of SAHA. Future plans will test SAHA in a treatment model and examine interactions with radiation. In collaboration with Ken Aldape, Her-2 was overexpressed in 37% of brain metastatic specimens, larger than historic estimates of amplification in primary tumors. At least three factors could explain the increased incidence of brain metastases in Her-2+ patients: their increased lifespan, the inability of Herceptin to cross the blood-brain barrier, and a brain metastasis promoting effect of Her-2 overexpression. We tested the hypothesis that Her-2 overexpression alters the natural history of breast cells to render them more brain metastatic. Her-2 transfectants of the 231-BR cells have been engineered and characterized in vitro and in vivo. In vitro, the Her-2 transfectants exhibited increased colonization; in vivo, Her-2 overexpression conferred a three fold increase in the number of large brain metastases. Experiments are ongoing using lapatinib, a Her-2/EGFR inhibitor.