Death from cancer is usually the result of dissemination of cancer cells from a primary tumor to secondary vital organs, and the formation of metastases. This process involves a series of steps, each of which have become targets of anticancer therapies such as intravasation of cancer cells into the bloodstream or lymphatics, delivery to organs (e.g., liver, lung, bone, brain, and lymph nodes), extravasation of cells into the organ parenchyma, cell proliferation to form secondary tumors, and development of new blood vessels to sustain continued growth (1). Importantly, single metastatic cells (2,3) or prevascular micrometastases (4) may also remain dormant within an organ, persisting until conditions are suitable for proliferation. Therefore, while surgical treatment of the primary tumor may be successful, undetectable dormant single metastatic cells or prevascular micrometastases can remain clinically silent for long periods and may eventually result in tumor formation and patient relapse (3,4).Metastasis to the brain can occur with many tumor types, including breast cancer, lung cancer, and melanoma. For breast cancer patients, the prevalence of brain metastases was historically estimated at 10 -16% with a 1-year survival rate of 20% (5). More recent studies, however, have demonstrated the prevalence of brain metastases in breast cancer patients to be closer to 22-30% (6), suggesting that its incidence may be increasing as a sanctuary site as systemic control improves. Brain metastases are typically treated with stereotactic radiosurgery or surgery with whole-brain radiation, supplemented with corticosteroid therapy for symptomatic relief. Patchell et al. (7) reported that surgery and whole-brain radiation can cure up to 90% of solitary brain metastases, which suggests that undiagnosed micrometastases or dormant cells are responsible for treatment failure. Thus, identification of micrometastatic and dormant brain metastatic tumor cells may facilitate an understanding of their biology and development of therapeutic interventions.For brain metastases of breast cancer, only a handful of experimental model systems have been reported. Yoneda et al. (8) performed six rounds of selection of human MDA-MB-231 breast carcinoma cells for brain metastasis in mice, followed by excision of the lesion and establishment of a cell culture. The resulting MDA-MB-231BR "brain-seeking" clone metastasized to the brain following intracardiac injection in 100% of the mice. Metastasis was identified histologically, which provided only one time point per animal. Clearly, studies of the metastatic process would greatly benefit from techniques that could dynamically monitor metastases from their earliest stage to endstage growth throughout entire organs or animals. This

Clinically symptomatic metastases to the central nervous system (CNS) occur in Natural History of CNS MetastasisOf the nearly 1.3 million people diagnosed with cancer in the United States each year, ϳ100,000 to 170,000 will develop brain metastases, for an annual incidence of ϳ4.1 to 11.1 per 100,000 population (American Cancer Society Cancer Facts and Figures 2005, available at http://www.cancer.org). 1 Large autopsy studies suggest that between 20% and 40% of all patients with metastatic cancer will have brain metastases (http://www.cancer. org). [1][2][3][4] Given their overall greater frequency, lung and breast cancer are by far the most common tumors to present with brain metastases. 2,4 The incidence of symptomatic brain metastases among women with metastatic breast cancer ranges from 10 to 16%. 5 On average, the median latency between the initial diagnosis of breast cancer and the onset of brain metastasis is ϳ2 to 3 years. 1,2 In most cases, breast cancer patients develop brain metastases after metastases have appeared systemically in the lung, liver, and/or bone. 6 For the purposes of this review, central nervous system (CNS) and brain are used interchangeably.Several risk factors for brain metastases have been reported. Young age appears to correlate with elevated risk. 5,7 In a study of 1015 women with metastatic breast cancer, brain metastases occurred in 9% of women with estrogen receptor-negative (ERϪ) primary tumors, compared to 5% of patients with ERϩ primary tumors. 8 Many human breast cancers (25 to 33%) express Her-2, also known as the epidermal growth factor receptor erbB2 or the neu oncogene [Online Mendelian Inheritance in Man (OMIM) accession number 164870; http://www.ncbi. nlm.nih.gov/entrez/dispomim.cgi?id ϭ 164870, accessed 2.25.05]. Amplification or overexpression of Her-2 correlates with a shorter disease-free and overall survival time 9 and also appears to associate with a higher incidence of brain metastases. 10 -12 The metastasis of breast cancer to the CNS, either the brain parenchyma or the leptomeninges, is generally a late feature of metastatic disease. Metastases to the brain parenchyma are thought to be hematogenous in origin. In a retrospective survey of breast cancer patients with brain metastases, 78% had multiple intracerebral metastases, 14% had a solitary intracerebral metastasis, and the remaining 8% had leptomeningeal metastases. 7 Breast cancer is the most common solid tumor to exhibit leptomeningeal colonization. 13 Within the three membranous coverings, or meninges, that surround the brain, leptomeningeal metastases arise on the innermost covering (pia) and the middle membrane (arachnoid) or in the cerebral spinal fluid (CSF)-filled space between the arachnoid and the pia (subarachnoid space). 4 Spread to the leptomeninges may occur via multiple routes including hematogenous, direct extension, transport through

Retrospective studies of breast cancer patients suggest that primary tumor Her-2 overexpression or trastuzumab therapy is associated with a devastating complication: the development of central nervous system (brain) metastases. Herein, we present Her-2 expression trends from resected human brain metastases and data from an experimental brain metastasis assay, both indicative of a functional contribution of Her-2 to brain metastatic colonization. Of 124 archival resected brain metastases from breast cancer patients, 36.2% overexpressed Her-2, indicating an enrichment in the frequency of tumor Her-2 overexpression at this metastatic site. Using quantitative real-time PCR of laser capture microdissected epithelial cells, Her-2 and epidermal growth factor receptor (EGFR) mRNA levels in a cohort of 12 frozen brain metastases were increased up to 5-and 9-fold, respectively, over those of Her-2-amplified primary tumors. Co-overexpression of Her-2 and EGFR was also observed in a subset of brain metastases. We then tested the hypothesis that overexpression of Her-2 increases the colonization of breast cancer cells in the brain in vivo. A subclone of MDA-MB-231 human breast carcinoma cells that selectively metastasizes to brain (231-BR) overexpressed EGFR; 231-BR cells were transfected with low (4-to 8-fold) or high (22-to 28-fold) levels of Her-2. In vivo, in a model of brain metastasis, low or high Her-2-overexpressing 231-BR clones produced comparable numbers of micrometastases in the brain as control transfectants; however, the Her-2 transfectants yielded 3-fold greater large metastases (>50 Mm 2 ; P < 0.001). Our data indicate that Her-2 overexpression increases the outgrowth of metastatic tumor cells in the brain in this model system. [Cancer Res 2007;67(9):4190-8]

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.

Brain metastases of breast cancer seem to be increasing in incidence as systemic therapy improves. Metastatic disease in the brain is associated with high morbidity and mortality. We present the first gene expression analysis of laser-captured epithelial cells from resected human brain metastases of breast cancer compared with unlinked primary breast tumors. The tumors were matched for histology, tumor-node-metastasis stage, and hormone receptor status. Most differentially expressed genes were down-regulated in the brain metastases, which included, surprisingly, many genes associated with metastasis. Quantitative real-time PCR analysis confirmed statistically significant differences or strong trends in the expression of six genes: BMP1, PEDF, LAMγ3, SIAH, STHMN3, and TSPD2. Hexokinase 2 (HK2) was also of interest because of its increased expression in brain metastases. HK2 is important in glucose metabolism and apoptosis. In agreement with our microarray results, HK2 levels (both mRNA and protein) were elevated in a brain metastatic derivative (231-BR) of the human breast carcinoma cell line MDA-MB-231 relative to the parental cell line (231-P) in vitro. Knockdown of HK2 expression in 231-BR cells using short hairpin RNA reduced cell proliferation when cultures were maintained in glucoselimiting conditions. Finally, HK2 expression was analyzed in a cohort of 123 resected brain metastases of breast cancer. High HK2 expression was significantly associated with poor patient survival after craniotomy (P = 0.028). The data suggest that HK2 overexpression is associated with metastasis to the brain in breast cancer and it may be a therapeutic target. (Mol Cancer Res 2009;7(9):1438-45)

Purpose: As chemotherapy and molecular therapy improve the systemic survival of breast cancer patients, the incidence of brain metastases increases. Few therapeutic strategies exist for the treatment of brain metastases because the blood-brain barrier severely limits drug access. We report the pharmacokinetic, efficacy, and mechanism of action studies for the histone deactylase inhibitor vorinostat (suberoylanilide hydroxamic acid) in a preclinical model of brain metastasis of triple-negative breast cancer. Experimental Design: The 231-BR brain trophic subline of the MDA-MB-231 human breast cancer cell line was injected into immunocompromised mice for pharmacokinetic and metastasis studies. Pharmacodynamic studies compared histone acetylation, apoptosis, proliferation, and DNA damage in vitro and in vivo. Results: Following systemic administration, uptake of [ 14 C]vorinostat was significant into normal rodent brain and accumulation was up to 3-fold higher in a proportion of metastases formed by 231-BR cells. Vorinostat prevented the development of 231-BR micrometastases by 28% (P = 0.017) and large metastases by 62% (P < 0.0001) compared with vehicle-treated mice when treatment was initiated on day 3 post-injection. The inhibitory activity of vorinostat as a single agent was linked to a novel function in vivo: induction of DNA double-strand breaks associated with the down-regulation of the DNA repair gene Rad52. Conclusions:We report the first preclinical data for the prevention of brain metastasis of triple-negative breast cancer. Vorinostat is brain permeable and can prevent the formation of brain metastases by 62%. Its mechanism of action involves the induction of DNA double-strand breaks, suggesting rational combinations with DNA active drugs or radiation. (Clin Cancer Res 2009;15(19):6148-57) Significant advances have been made in the treatment of primary breast cancer; one of the unfortunate complications of this progress is an increase in the incidence of brain metastases (reviewed in refs. 1, 2). Combinations of cytotoxic and targeted therapies have afforded metastatic breast cancer patients' clinical responses or stable disease, but the poor penetration of these drugs into the brain and leptomeninges creates a "sanctuary site" for recurrence. With an increased number of metastatic breast cancer patients having stable disease or responding to treatment systemically when they develop brain metastases,

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p53 is believed to sense cellular ribonucleotide depletion in the absence of DNA strand breaks and to respond by imposition of a p21-dependent G 1 cell cycle arrest. We now report that the p53-dependent G 1 checkpoint is blocked in human carcinoma cell lines after inhibition of de novo purine synthesis by folate analogs inhibitory to glycinamide ribonucleotide formyltransferase (GART). p53 accumulated in HCT116, MCF7, or A549 carcinoma cells upon GART inhibition, but, surprisingly, transcription of several p53 targets, including p21 cip1/waf1 , was impaired. The mechanism of this defect was examined. The p53 accumulating in these cells was nuclear but was not phosphorylated at serines 6, 15, and 20, nor was it acetylated at lysines 373 or 382. The DDATHF-stabilized p53 bound to the p21 promoter in vitro and in vivo but did not activate histone acetylation over the p53 binding sites in the p21 promoter that is an integral part of the transcriptional response mediated by the DNA damage pathway. We concluded that the robust initial response of the p53 pathway to GART inhibitors is not transcriptionally propagated to target genes due to a defect in p53 post-translational modifications and a failure to open chromatin structure despite promoter binding of this unmodified p53.