Diane Palmieri

Breast Cancer Metastasis to the Central Nervous System

Clinically symptomatic metastases to the central nervous system (CNS) occur in approximately 10 to 15% of patients with metastatic beast cancer. CNS metastases are traditionally viewed as a late complication of systemic disease, for which few effective treatment options exist. Recently, patients with Her-2-positive breast tumors who were treated with trastuzumab have been reported to develop CNS metastases at higher rates, often while responding favorably to treatment. The blood:brain barrier and the unique brain microenvironment are hypothesized to promote distinct molecular features in CNS metastases that may require tailored therapeutic approaches. New research approaches using cell lines that reliably and preferentially metastasize in vivo to the brain have been reported. Using such model systems, as well as in vitro analogs of blood-brain barrier penetration and tissue-based studies, new molecular leads into this disease are unfolding.

Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer

Purpose: Brain metastases of breast cancer appear to be increasing in incidence, confer significant morbidity, and threaten to compromise gains made in systemic chemotherapy. The blood–tumor barrier (BTB) is compromised in many brain metastases; however, the extent to which this influences chemotherapeutic delivery and efficacy is unknown. Herein, we answer this question by measuring BTB passive integrity, chemotherapeutic drug uptake, and anticancer efficacy in vivo in two breast cancer models that metastasize preferentially to brain. Experimental Design:Experimental brain metastasis drug uptake and BTB permeability were simultaneously measured using novel fluorescent and phosphorescent imaging techniques in immune-compromised mice. Drug-induced apoptosis and vascular characteristics were assessed using immunofluorescent microscopy. Results: Analysis of over 2,000 brain metastases from two models (human 231-BR-Her2 and murine 4T1-BR5) showed partial BTB permeability compromise in greater than 89% of lesions, varying in magnitude within and between metastases. Brain metastasis uptake of 14C-paclitaxel and 14C-doxorubicin was generally greater than normal brain but less than 15% of that of other tissues or peripheral metastases, and only reached cytotoxic concentrations in a small subset (∼10%) of the most permeable metastases. Neither drug significantly decreased the experimental brain metastatic ability of 231-BR-Her2 tumor cells. BTB permeability was associated with vascular remodeling and correlated with overexpression of the pericyte protein desmin. Conclusions: This work shows that the BTB remains a significant impediment to standard chemotherapeutic delivery and efficacy in experimental brain metastases of breast cancer. New brain permeable drugs will be needed. Evidence is presented for vascular remodeling in BTB permeability alterations. Clin Cancer Res; 16(23); 5664–78. ©2010 AACR.

Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth

The development of life-threatening cancer metastases at distant organs requires disseminated tumour cells’ adaptation to, and co-evolution with, the drastically different microenvironments of metastatic sites. Cancer cells of common origin manifest distinct gene expression patterns after metastasizing to different organs. Clearly, the dynamic interaction between metastatic tumour cells and extrinsic signals at individual metastatic organ sites critically effects the subsequent metastatic outgrowth. Yet, it is unclear when and how disseminated tumour cells acquire the essential traits from the microenvironment of metastatic organs that prime their subsequent outgrowth. Here we show that both human and mouse tumour cells with normal expression of PTEN, an important tumour suppressor, lose PTEN expression after dissemination to the brain, but not to other organs. The PTEN level in PTEN-loss brain metastatic tumour cells is restored after leaving the brain microenvironment. This brain microenvironment-dependent, reversible PTEN messenger RNA and protein downregulation is epigenetically regulated by microRNAs from brain astrocytes. Mechanistically, astrocyte-derived exosomes mediate an intercellular transfer of PTEN-targeting microRNAs to metastatic tumour cells, while astrocyte-specific depletion of PTEN-targeting microRNAs or blockade of astrocyte exosome secretion rescues the PTEN loss and suppresses brain metastasis in vivo. Furthermore, this adaptive PTEN loss in brain metastatic tumour cells leads to an increased secretion of the chemokine CCL2, which recruits IBA1-expressing myeloid cells that reciprocally enhance the outgrowth of brain metastatic tumour cells via enhanced proliferation and reduced apoptosis. Our findings demonstrate a remarkable plasticity of PTEN expression in metastatic tumour cells in response to different organ microenvironments, underpinning an essential role of co-evolution between the metastatic cells and their microenvironment during the adaptive metastatic outgrowth. Our findings signify the dynamic and reciprocal cross-talk between tumour cells and the metastatic niche; importantly, they provide new opportunities for effective anti-metastasis therapies, especially of consequence for brain metastasis patients.

Her-2 Overexpression Increases the Metastatic Outgrowth of Breast Cancer Cells in the Brain

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 microm(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.

In vivo MRI of cancer cell fate at the single-cell level in a mouse model of breast cancer metastasis to the brain.

Metastasis (the spread of cancer from a primary tumor to secondary organs) is responsible for most cancer deaths. The ability to follow the fate of a population of tumor cells over time in an experimental animal would provide a powerful new way to monitor the metastatic process. Here we describe a magnetic resonance imaging (MRI) technique that permits the tracking of breast cancer cells in a mouse model of brain metastasis at the single‐cell level. Cancer cells that were injected into the left ventricle of the mouse heart and then delivered to the brain were detectable on MR images. This allowed the visualization of the initial delivery and distribution of cells, as well as the growth of tumors from a subset of these cells within the whole intact brain volume. The ability to follow the metastatic process from the single‐cell stage through metastatic growth, and to quantify and monitor the presence of solitary undivided cells will facilitate progress in understanding the mechanisms of brain metastasis and tumor dormancy, and the development of therapeutics to treat this disease. Magn Reson Med, 2006. Published 2006 Wiley‐Liss, Inc.

Effect of Lapatinib on the Outgrowth of Metastatic Breast Cancer Cells to the Brain

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 microm(2)) 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.

Nm23-H1 Metastasis Suppressor Phosphorylation of Kinase Suppressor of Ras via a Histidine Protein Kinase Pathway

The metastasis-suppressive activity of Nm23-H1 was previously correlated with its in vitro histidine protein kinase activity, but physiological substrates have not been identified. We hypothesized that proteins that interact with histidine kinases throughout evolution may represent partners for Nm23-H1 and focused on the interaction of Arabidopsis“two-component” histidine kinase ERS with CTR1. A mammalian homolog of CTR1 was previously reported to be c-Raf; we now report that CTR1 also exhibits homology to the kinase suppressor of Ras (KSR), a scaffold protein for the mitogen-activated protein kinase (MAPK) cascade. Nm23-H1 co-immunoprecipitated KSR from lysates of transiently transfected 293T cells and at endogenous protein expression levels in MDA-MB-435 breast carcinoma cells. Autophosphorylated recombinant Nm23-H1 phosphorylated KSR in vitro. Phosphoamino acid analysis identified serine as the major target, and two peaks of Nm23-H1 phosphorylation were identified upon high performance liquid chromatography analysis of KSR tryptic peptides. Using site-directed mutagenesis, we found that Nm23-H1 phosphorylated KSR serine 392, a 14-3-3-binding site, as well as serine 434 when serine 392 was mutated. Phosphorylated MAPK but not total MAPK levels were reduced in annm23-H1 transfectant of MDA-MB-435 cells. The data identify a complex in vitro histidine-to-serine protein kinase pathway, which may contribute to signal transduction and metastasis.

Analyses of Resected Human Brain Metastases of Breast Cancer Reveal the Association between Up-Regulation of Hexokinase 2 and Poor Prognosis

Brain metastases of breast cancer seem to be increasingin 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 glucose-limiting 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)

Vorinostat Inhibits Brain Metastatic Colonization in a Model of Triple-Negative Breast Cancer and Induces DNA Double-Strand Breaks

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 [14C]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)

The Biology of Metastasis to a Sanctuary Site

Metastasis to the brain is prevalent in solid tumors and lymphomas, and is associated with shortened survival. The brain is regarded as a sanctuary site for metastatic tumor cells where they exist partially protected from drugs by the blood-tumor barrier. Model systems for brain metastasis have been developed and are now yielding mechanistic insights into the roles of angiogenesis, energy metabolism, the Her-2 and Stat3 signaling pathways, and dormancy. Specific, new approaches to combat brain metastatic disease are needed.