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 as a sanctuary site, particuarly among women with Her-2 positive tumors, as systemic control of breast cancer metastases improves. We conducted microarray analysis of surgically resected brain metastases of breast cancer, using laser capture microdissection, amplification and 30K cDNA arrays. These data are being 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. Among the genes differentially expressed are overexpression of Her-2, Hexokinase2 and artemis, and underexpression of several invasion, motility and growth suppression genes. Transfections into a brain metastatic cell line are underway to determine functional contributions.We are validating an in vivo assay for brain metastasis using a EGFP labeled brain metastatic variant of the MDA-MB-231 cell line. These cells form imagable brain metastases after intracardiac or intravenous injection into Balb/c/Nu mice, the latter subsequent to a heavy pulmonary metastatic load. Experiments are optimizing injection protocol and quantiation. Targets of translational interest include overexpression of Her-2, and histone deacetylases. Her-2 was amplified in 37% of brain metastatic specimens, larger than historic estimates of amplification in primary tumors. Her-2 transfectants have been made and characterized in vitro. Her-2 overexpressing cells exhibited increased growth in reduced serum in vitro, but equivalent motility to control transfectants. Increased Akt activation is observed in response to EGF, and the effects of trastuzumab and lapatinin are under investigation. In vivo assays are planned. Several genes participatory in HDAC regulation of gene expression were altered in the brain metastasis heatmap including BHC80, LOXL1. The effect of two HDAC inhibitors, SAHA and depsipeptide, have been investigated in vitro. Both inhibitors decrease both proliferation and motility in the brain metastatic cell line. A gene expression profile of HDAC inhibitors has been obtained.