Brain metastases occur in approximately 15% of metastatic breast cancer patients and confer a dismal prognosis. Brain metastases are thought to increasing, particuarly among metastatic patients with Her-2+ or triple negative tumors. Our goals are to identify genes that functionally contribute to brain metastatic progression and to identify and validate preclinical leads. A murine preclinical model of brain metastasis was developed using a derivative of the MDA-MB-231 breast carcinoma cell line (231-BR cells). The proliferative and apoptotic rates exhibited by this cell line, as well as the presence of a neuro-inflammatory response, closely correlate with data from 16 resected brain metastases of human breast cancer, suggesting that the model is relevant. Additional models of brain metastasis of breast cancer have been estalblished to provide a measure of heterogeneity including the murine 4T1 mammary cell line (4T1-BR5), Her-2 transfected human MCF-7 (ER+) cells (MCF-7-Her-2-BR3), and naturally Her-2+ Jimt-1 and SUM190 cells. In collaboration with Drs. Quentin Smith and Paul Lockman, Texas Tech University, the permeability of experimental 231-BR and 4T1-BR5 metastases was quantified. Published collaborative research showed that experimental brain metastases of breast cancer were heterogeneous in their permeability, both within and between metastases in the same brain. While most metastases were permeable as compared to the normal brain blood-brain barrier, 10% exhibited sufficient permeability to enable a cytotoxic response to a systemic drug. The data argue that a new class of inherently brain-permeable drugs will be needed for this complication. Ongoing experiments in the lab investigate the hypothesis that alterations in tumor or brain microenvironmental gene expression underlie altered permeability of metastases. Permeable and impermeable brain lesions are being identified in frozen sections of mouse brains;using the adjacent section the material was laser-capture microdissected. RNA extracted from these lesions was hybridized to both human (tumor cell genes) and mouse (brain microenvironment genes) microarrays and leads are now under investigation.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 produced three fold greater large brain metastases, proportional to MRI detectable metastases in a human brain. The efficacy of the dual EGFR/Her-2 tyrosine kinase inhibitor, lapatinib, was tested on the brain metastatic colonization of a human breast carcinoma cell line.). Lapatinib prevented the brain colonization of 231-BR-Her-2 cells by 53% and inhibited the phospho-Her-2 staining of treated brain metastases in vivo, demonstrating that the drug hit its target. A pharmacokinetic analysis of lapatinib uptake in experimental brain metastases was conducted. In these experiments, lapatinib resistant tumor cells were cultured from the brains of treated animals. The lapatinib resistant cells were sensitive to lapatinib inhibition of proliferation in vitro, suggesting that lapatinib resistance in the brain is mostly due to inadequate drug delivery. Rational combinations with lapatinib were investigated to improve its preventive efficacy. Pazopanib, a VEGFR, PDGFR and c-kit inhibitor, was investigated. Pazopanib as a single agent signficantly prevented the formation of 231-Her2 brain metastases in mice, and was found to exert B-Raf inhibitory activity. Lapatinib/pazopanib combination experiments are under consideration. Pazopanib was also found to affect the neuroinflammatory response to brain metastases. In collaboration with Dr. George Sledge, Indiana University, and Drs. Jacek Jassem and Renata Duchnowska, Poland, a 13-gene signature was developed from gene expression profiling of primary tumor material from metastatic Her-2+ breast cancer patients, that predicts short (3 years) time to the development of brain metastases. Validation experiments have revised the signature to four of the original 13 genes, which is being prepared for publication. One of the genes in the four gene signature is the DNA double strand break repair gene Rad51.Comparison of matched primary tumors and resected brain metastases revealed that Rad51 and another DNA double strand break repair protein, Bard1, were overexpressed in the brain lesions. We have found that transfection of either gene into 231-BR or 4T1-BR cells increases brain metastsis formation by approximately four fold. The mechanism of action of this observation is under evaluation.The role of Pigment-Epithelium derived factor was determined in experimental models of brain metastasis. PEDF is a secreted protein with anti-angiogenic, tumor suppressor and neuronal viability properties, possibly mediated through multiple receptors. Overexpression of PEDF in the 231-BR cell line reduced the number of large parenchymal metastases significantly. Due to the instability of the PEDF transfection construct in vivo, intracranial injections were used for analysis of an interaction with the microenvironment. PEDF overexpressing tumor cells formed smaller intracranial tumors then control transfectants. Interestingly, neuronal damage, assessed by two independent neuropathologists, was reduced in the zone surrounding the tumor cells overexpressing PEDF. Neuronal damage was assessed by traditional staining and a new Flourojade technology that the lab has adapted to cancer studies. PEDF therefore stands as a potential strategy to reduce the damage to surrounding nerves caused by developing metastases. In preclinical experiments, 17 potential therapeutics have been tested for prevention of 231-BR brain metastatic colonization. Of these, only four have demonstrated efficacy, highlighting the prohibitive role of the blood-brain barrier. Where tested, the active drugs prevented brain metastases, but statistically failed to treat (shrink) an established brain metastasis. Considering that all clinical trials are conducted with the primary endpoint of responses in established lesions, the data indicate that we are missing some potent activities with preventive potential. New clinical trial designs are needed.
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