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. 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. 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. Her-2 overexpression conferred a three fold increase in the number of large brain metastases, proportional to MRI detectable metastases in a human brain. No change was observed in the number of micrometastases. We determined the efficacy of the dual EGFR/Her-2 tyrosine kinase inhibitor, lapatinib, on the brain metastatic colonization of a human breast carcinoma cell line.). To determine the effect of lapatinib on brain metastatic colonization in vivo, mice received intracardiac injections of either the control or the Her-2 transfected 231BR cells. Five days later mice were randomized to vehicle, 30 or 100 mg/kg lapatinib by twice daily oral gavage. Histologic analysis was conducted to quantify both micrometastases and large metastases from 10 step sagittal sections in one hemisphere of the brain. For the control transfectant mean (95% CI) large metastases were 3.36 (2.73-3.98) per section in the vehicle controls, were unaffected by 30 mg/kg lapatinib but declined 54% to 1.56 (0.94-2.17) when treated with 100 mg/kg lapatinib (p=0.0001). The Her-2 transfectant produced 6.83 (5.86-7.79) large metastases per section. Treatment with 30 mg/kg resulted in a 53% decline to 3.21 (2.31-4.11) large metastases (p<0.0001), while treatment with 100 mg/kg resulted in a 50% decline to 3.44 (2.55-4.32) large metastases (P<0.0001). The data indicate that lapatinib can inhibit the brain colonization of dual EGFR-Her-2+ breast cancer cells. Inhibition of activated (phospho) Her-2 but not phospho-EGFR was observed upon IHC of brain sections. These data compare favorably to a recently published update of the registration latatinib/capecitabine vs capecitabine metastatic trial, where the combination arm now significantly prevented brain as a first site of replapse. 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. Of the genes validated, experiments are ongoing for hexokinase 2 (HK2 and pigment epithelium derived factor (PEDF). A cohort study was conducted of HK2 expression by immunohistochemistry. High expression of HK2 significantly correlated with poor patient survival after neurosurgery. Expression of a shRNA to HK2 has been achieved, with significant reductions in tumor cell HK2 expression in indepedent clones. Other hexokinases were not affected. Cellular ATP levels were reduced in the shRNA knockdown clones. 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. Exploration in an additional model system is underway. 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. Vorinostat was selected from the multiple HDAC inhibitors since its structure appeared favorable for brain penetration. Treatment of mice with 100 mg/kg vorinostat qd, beginning on day five postinjection, significantly reduced the number of large brain metastases. In contrast to the literature, we find no effect of vorinostat on brain histone acetylation as determined by IHC, nor on apoptotic rates. We did uncover a novel mechanism of action, the induction of DNA double stand breaks. These data have been extended by Dr. Kevin Camphausen to look at interactions with radiation. A clinical trial of Vorinostat and radiation has opened at Thomas Jefferson University.
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