Alterations in Pericyte Subpopulations Are Associated with Elevated Blood-Tumor Barrier Permeability in Experimental Brain Metastasis of Breast Cancer.

PURPOSE: The blood-brain barrier (BBB) is modified to a blood-tumor barrier (BTB) as a brain metastasis develops from breast or other cancers. We (i) quantified the permeability of experimental brain metastases, (ii) determined the composition of the BTB, and (iii) identified which elements of the BTB distinguished metastases of lower permeability from those with higher permeability. EXPERIMENTAL DESIGN: A SUM190-BR3 experimental inflammatory breast cancer brain metastasis subline was established. Experimental brain metastases from this model system and two previously reported models (triple-negative MDA-231-BR6, HER2+ JIMT-1-BR3) were serially sectioned; low- and high-permeability lesions were identified with systemic 3-kDa Texas Red dextran dye. Adjoining sections were used for quantitative immunofluorescence to known BBB and neuroinflammatory components. One-sample comparisons against a hypothesized value of one were performed with the Wilcoxon signed-rank test. RESULTS: When uninvolved brain was compared with any brain metastasis, alterations in endothelial, pericytic, astrocytic, and microglial components were observed. When metastases with relatively low and high permeability were compared, increased expression of a desmin+ subpopulation of pericytes was associated with higher permeability (231-BR6 P = 0.0002; JIMT-1-BR3 P = 0.004; SUM190-BR3 P = 0.008); desmin+ pericytes were also identified in human craniotomy specimens. Trends of reduced CD13+ pericytes (231-BR6 P = 0.014; JIMT-1-BR3 P = 0.002, SUM190-BR3, NS) and laminin α2 (231-BR6 P = 0.001; JIMT-1-BR3 P = 0.049; SUM190-BR3 P = 0.023) were also observed with increased permeability. CONCLUSIONS: We provide the first account of the composition of the BTB in experimental brain metastasis. Desmin+ pericytes and laminin α2 are potential targets for the development of novel approaches to increase chemotherapeutic efficacy. Clin Cancer Res; 22(21); 5287-99. ©2016 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.

Analysis of radiation therapy in a model of triple-negative breast cancer brain metastasis.

Most cancer patients with brain metastases are treated with radiation therapy, yet this modality has not yet been meaningfully incorporated into preclinical experimental brain metastasis models. We applied two forms of whole brain radiation therapy (WBRT) to the brain-tropic 231-BR experimental brain metastasis model of triple-negative breast cancer. When compared to sham controls, WBRT as 3 Gy × 10 fractions (3 × 10) reduced the number of micrometastases and large metastases by 87.7 and 54.5 %, respectively (both p < 0.01); whereas a single radiation dose of 15 Gy × 1 (15 × 1) was less effective, reducing metastases by 58.4 % (p < 0.01) and 47.1 % (p = 0.41), respectively. Neuroinflammation in the adjacent brain parenchyma was due solely to a reaction from metastases, and not radiotherapy, while adult neurogenesis in brains was adversely affected following both radiation regimens. The nature of radiation resistance was investigated by ex vivo culture of tumor cells that survived initial WBRT ("Surviving" cultures). The Surviving cultures surprisingly demonstrated increased radiosensitivity ex vivo. In contrast, re-injection of Surviving cultures and re-treatment with a 3 × 10 WBRT regimen significantly reduced the number of large and micrometastases that developed in vivo, suggesting a role for the microenvironment. Micrometastases derived from tumor cells surviving initial 3 × 10 WBRT demonstrated a trend toward radioresistance upon repeat treatment (p = 0.09). The data confirm the potency of a fractionated 3 × 10 WBRT regimen and identify the brain microenvironment as a potential determinant of radiation efficacy. The data also nominate the Surviving cultures as a potential new translational model for radiotherapy.

DNA double-strand break repair genes and oxidative damage in brain metastasis of breast cancer.

BACKGROUND: Breast cancer frequently metastasizes to the brain, colonizing a neuro-inflammatory microenvironment. The molecular pathways facilitating this colonization remain poorly understood. METHODS: Expression profiling of 23 matched sets of human resected brain metastases and primary breast tumors by two-sided paired t test was performed to identify brain metastasis-specific genes. The implicated DNA repair genes BARD1 and RAD51 were modulated in human (MDA-MB-231-BR) and murine (4T1-BR) brain-tropic breast cancer cell lines by lentiviral transduction of cDNA or short hairpin RNA (shRNA) coding sequences. Their functional contribution to brain metastasis development was evaluated in mouse xenograft models (n = 10 mice per group). RESULTS: Human brain metastases overexpressed BARD1 and RAD51 compared with either matched primary tumors (1.74-fold, P < .001; 1.46-fold, P < .001, respectively) or unlinked systemic metastases (1.49-fold, P = .01; 1.44-fold, P = .008, respectively). Overexpression of either gene in MDA-MB-231-BR cells increased brain metastases by threefold to fourfold after intracardiac injections, but not lung metastases upon tail-vein injections. In 4T1-BR cells, shRNA-mediated RAD51 knockdown reduced brain metastases by 2.5-fold without affecting lung metastasis development. In vitro, BARD1- and RAD51-overexpressing cells showed reduced genomic instability but only exhibited growth and colonization phenotypes upon DNA damage induction. Reactive oxygen species were present in tumor cells and elevated in the metastatic neuro-inflammatory microenvironment and could provide an endogenous source of genotoxic stress. Tempol, a brain-permeable oxygen radical scavenger suppressed brain metastasis promotion induced by BARD1 and RAD51 overexpression. CONCLUSIONS: BARD1 and RAD51 are frequently overexpressed in brain metastases from breast cancer and may constitute a mechanism to overcome reactive oxygen species-mediated genotoxic stress in the metastatic brain.

Profound prevention of experimental brain metastases of breast cancer by temozolomide in an MGMT-dependent manner.

PURPOSE: Brain metastases of breast cancer cause neurocognitive damage and are incurable. We evaluated a role for temozolomide in the prevention of brain metastases of breast cancer in experimental brain metastasis models. EXPERIMENTAL DESIGN: Temozolomide was administered in mice following earlier injection of brain-tropic HER2-positive JIMT-1-BR3 and triple-negative 231-BR-EGFP sublines, the latter with and without expression of O(6)-methylguanine-DNA methyltransferase (MGMT). In addition, the percentage of MGMT-positive tumor cells in 62 patient-matched sets of breast cancer primary tumors and resected brain metastases was determined immunohistochemically. RESULTS: Temozolomide, when dosed at 50, 25, 10, or 5 mg/kg, 5 days per week, beginning 3 days after inoculation, completely prevented the formation of experimental brain metastases from MGMT-negative 231-BR-EGFP cells. At a 1 mg/kg dose, temozolomide prevented 68% of large brain metastases, and was ineffective at a dose of 0.5 mg/kg. When the 50 mg/kg dose was administered beginning on days 18 or 24, temozolomide efficacy was reduced or absent. Temozolomide was ineffective at preventing brain metastases in MGMT-transduced 231-BR-EGFP and MGMT-expressing JIMT-1-BR3 sublines. In 62 patient-matched sets of primary breast tumors and resected brain metastases, 43.5% of the specimens had concordant low MGMT expression, whereas in another 14.5% of sets high MGMT staining in the primary tumor corresponded with low staining in the brain metastasis. CONCLUSIONS: Temozolomide profoundly prevented the outgrowth of experimental brain metastases of breast cancer in an MGMT-dependent manner. These data provide compelling rationale for investigating the preventive efficacy of temozolomide in a clinical setting.

CNS metastases in breast cancer: old challenge, new frontiers.

Despite major therapeutic advances in the management of patients with breast cancer, central nervous system (CNS) metastases remain an intractable problem, particularly in patients with metastatic HER2-positive and triple-negative breast cancer. As systemic therapies to treat extracranial disease improve, some patients are surviving longer, and the frequency of CNS involvement seems to be increasing. Furthermore, in the early-stage setting, the CNS remains a potential sanctuary site for relapse. This review highlights advances in the development of biologically relevant preclinical models, including the development of brain-tropic cell lines for testing of agents to prevent and treat brain metastases, and summarizes our current understanding of the biology of CNS relapse. From a clinical perspective, a variety of therapeutic approaches are discussed, including methods to improve drug delivery, novel cytotoxic agents, and targeted therapies. Challenges in current trial design and endpoints are reviewed. Finally, we discuss promising new directions, including novel trial designs, correlative imaging techniques, and enhanced translational opportunities.

Paclitaxel-hyaluronic nanoconjugates prolong overall survival in a preclinical brain metastases of breast cancer model.

Brain (central nervous system; CNS) metastases pose a life-threatening problem for women with advanced metastatic breast cancer. It has recently been shown that the vasculature within preclinical brain metastasis model markedly restricts paclitaxel delivery in approximately 90% of CNS lesions. Therefore to improve efficacy, we have developed an ultra-small hyaluronic acid (HA) paclitaxel nanoconjugate (∼5 kDa) that can passively diffuse across the leaky blood-tumor barrier and then be taken up into cancer cells (MDA-MB-231Br) via CD44 receptor-mediated endocytocis. Using CD44 receptor-mediated endocytosis as an uptake mechanism, HA-paclitaxel was able to bypass p-glycoprotein-mediated efflux on the surface of the cancer cells. In vitro cytoxicity of the conjugate and free paclitaxel were similar in that they (i) both caused cell-cycle arrest in the G2-M phase, (ii) showed similar degrees of apoptosis induction (cleaved caspase), and (iii) had similar IC50 values when compared with paclitaxel in MTT assay. A preclinical model of brain metastases of breast cancer using intracardiac injections of Luc-2 transfected MDA-MB-231Br cells was used to evaluate in vivo efficacy of the nanoconjugate. The animals administered with HA-paclitaxel nanoconjugate had significantly longer overall survival compared with the control and the paclitaxel-treated group (P < 0.05). This study suggests that the small molecular weight HA-paclitaxel nanoconjugates can improve standard chemotherapeutic drug efficacy in a preclinical model of brain metastases of breast cancer.

Pazopanib inhibits the activation of PDGFRß-expressing astrocytes in the brain metastatic microenvironment of breast cancer cells.

Brain metastases occur in more than one-third of metastatic breast cancer patients whose tumors overexpress HER2 or are triple negative. Brain colonization of cancer cells occurs in a unique environment, containing microglia, oligodendrocytes, astrocytes, and neurons. Although a neuroinflammatory response has been documented in brain metastasis, its contribution to cancer progression and therapy remains poorly understood. Using an experimental brain metastasis model, we characterized the brain metastatic microenvironment of brain tropic, HER2-transfected MDA-MB-231 human breast carcinoma cells (231-BR-HER2). A previously unidentified subpopulation of metastasis-associated astrocytes expressing phosphorylated platelet-derived growth factor receptor ß (at tyrosine 751; p751-PDGFRß) was identified around perivascular brain micrometastases. p751-PDGFRß(+) astrocytes were also identified in human brain metastases from eight craniotomy specimens and in primary cultures of astrocyte-enriched glial cells. Previously, we reported that pazopanib, a multispecific tyrosine kinase inhibitor, prevented the outgrowth of 231-BR-HER2 large brain metastases by 73%. Here, we evaluated the effect of pazopanib on the brain neuroinflammatory microenvironment. Pazopanib treatment resulted in 70% (P = 0.023) decrease of the p751-PDGFRß(+) astrocyte population, at the lowest dose of 30 mg/kg, twice daily. Collectively, the data identify a subpopulation of activated astrocytes in the subclinical perivascular stage of brain metastases and show that they are inhibitable by pazopanib, suggesting its potential to prevent the development of brain micrometastases in breast cancer patients.

The role of MMP-1 in breast cancer growth and metastasis to the brain in a xenograft model.

BACKGROUND: Brain metastasis is an increasingly common complication for breast cancer patients; approximately 15- 30% of breast cancer patients develop brain metastasis. However, relatively little is known about how these metastases form, and what phenotypes are characteristic of cells with brain metastasizing potential. In this study, we show that the targeted knockdown of MMP-1 in breast cancer cells with enhanced brain metastatic ability not only reduced primary tumor growth, but also significantly inhibited brain metastasis. METHODS: Two variants of the MDA-MB-231 human breast cancer cell line selected for enhanced ability to form brain metastases in nude mice (231-BR and 231-BR3 cells) were found to express high levels of matrix metalloproteinase-1 (MMP-1). Short hairpin RNA-mediated stable knockdown of MMP-1 in 231-BR and 231-BR3 cells were established to analyze tumorigenic ability and metastatic ability. RESULTS: Short hairpin RNA-mediated stable knockdown of MMP-1 inhibited the invasive ability of MDA-MB 231 variant cells in vitro, and inhibited breast cancer growth when the cells were injected into the mammary fat pad of nude mice. Reduction of MMP-1 expression significantly attenuated brain metastasis and lung metastasis formation following injection of cells into the left ventricle of the heart and tail vein, respectively. There were significantly fewer proliferating cells in brain metastases of cells with reduced MMP-1 expression. Furthermore, reduced MMP-1 expression was associated with decreased TGFα release and phospho-EGFR expression in 231-BR and BR3 cells. CONCLUSIONS: Our results show that elevated expression of MMP-1 can promote the local growth and the formation of brain metastases by breast cancer cells.

Effect of inhibition of the lysophosphatidic acid receptor 1 on metastasis and metastatic dormancy in breast cancer.

BACKGROUND: Previous studies identified the human nonmetastatic gene 23 (NME1, hereafter Nm23-H1) as the first metastasis suppressor gene. An inverse relationship between Nm23-H1 and expression of lysophosphatidic acid receptor 1 gene (LPAR1, also known as EDG2 or hereafter LPA1) has also been reported. However, the effects of LPA1 inhibition on primary tumor size, metastasis, and metastatic dormancy have not been investigated. METHODS: The LPA1 inhibitor Debio-0719 or LPA1 short hairpinned RNA (shRNA) was used. Primary tumor size and metastasis were investigated using the 4T1 spontaneous metastasis mouse model and the MDA-MB-231T experimental metastasis mouse model (n = 13 mice per group). Proliferation and p38 intracellular signaling in tumors and cell lines were determined by immunohistochemistry and western blot to investigate the effects of LPA1 inhibition on metastatic dormancy. An analysis of variance-based two-tailed t test was used to determine a statistically significant difference between treatment groups. RESULTS: In the 4T1 spontaneous metastasis mouse model, Debio-0719 inhibited the metastasis of 4T1 cells to the liver (mean = 25.2 liver metastases per histologic section for vehicle-treated mice vs 6.8 for Debio-0719-treated mice, 73.0% reduction, P < .001) and lungs (mean = 6.37 lesions per histologic section for vehicle-treated mice vs 0.73 for Debio-0719-treated mice, 88.5% reduction, P < .001), with no effect on primary tumor size. Similar results were observed using the MDA-MB-231T experimental pulmonary metastasis mouse model. LPA1 shRNA also inhibited metastasis but did not affect primary tumor size. In 4T1 metastases, but not primary tumors, expression of the proliferative markers Ki67 and pErk was reduced by Debio-0719, and phosphorylation of the p38 stress kinase was increased, indicative of metastatic dormancy. CONCLUSION: The data identify Debio-0719 as a drug candidate with metastasis suppressor activity, inducing dormancy at secondary tumor sites.

In Vivo Magnetic Resonance Imaging for Investigating the Development and Distribution of Experimental Brain Metastases due to Breast Cancer.

INTRODUCTION: The overall goal of this study was to assess the utility of three-dimensional magnetic resonance imaging (MRI) for monitoring the temporal and spatial development of experimental brain metastasis in mice. MATERIALS AND METHODS: Brain metastatic human breast cancer cells (231-BR or 231-BR-HER2) were injected intracardially in nude mice for delivery to the brain. Mouse brains were imaged in vivo at different time points using a balanced steady-state-free precession (bSSFP) pulse sequence at 1.5 T. Brains were categorized into four regions: cortex, central brain, olfactory, and posterior. The number of metastases and their volumes were quantified for both cell lines. RESULTS: There was no difference in the mean number of metastases for either cell line. The volumes of metastases in mice injected with 231-BR-HER2 cells were significantly larger than those for mice injected with 231-BR cells. The growth rate for 231-BR-HER2 metastases was 67.5% compared with 54.4% for the 231-BR metastases. More than 50% of metastases were located in the cortex and 25% to 30% of metastases were identified in the central brain for each time point and for mice injected with either cell line. The volumes of metastases were significantly larger in mice with fewer metastases at end point. SIGNIFICANT CONCLUSIONS: MRI provided a comprehensive accounting of the number and size of experimental brain metastases in the whole mouse brain at multiple time points. This approach has provided new information about the temporal and spatial development of metastases in the brain not possible by other histopathologic or imaging methods.

TPI-287, a new taxane family member, reduces the brain metastatic colonization of breast cancer cells.

Brain metastases of breast and other cancers remain resistant to chemotherapeutic regimens that are effective systemically, in part due to the blood-brain barrier. We report that TPI-287, a new microtubule-stabilizing agent, displays in vitro cytotoxic activity similar to taxanes and epothilones. Unlike the taxanes, TPI-287 is permeable through the blood-brain barrier. Brain-to-plasma ratios of TPI-287 after a single injection typically exceeded one and were as high as 63.8 in the rat and 14.1 in the mouse. A brain-tropic derivative of the MDA-MB-231 triple-negative breast cancer cell line, 231-BR, was used to test whether TPI-287 may be efficacious at preventing or treating brain metastases. TPI-287 had growth inhibitory effects comparable with paclitaxel when 231-BR tumor cells were injected into the mammary fat pad. Brain metastatic colonization was determined by intracardiac injection of 231-BR cells, with treatment beginning on day 3 to 4 postinjection, culminating in a histologic count of brain metastases in brains necropsied days 25 to 28 postinjection. In this assay, paclitaxel, ixabepilone, and nab paclitaxel did not have significant inhibitory activity. TPI-287 was ineffective in the same assay using a 6 mg/kg every week schedule; however an 18 mg/kg dose delivered on days 3, 7, and 11 significantly reduced the outgrowth of brain metastases (55% reduction, P = 0.028) and reduced proliferation in brain metastases (16% reduction, P = 0.008). When TPI-287 treatment was delayed until days 18, 22, and 26 postinjection, efficacy was reduced (17% reduction, not significant). These data suggest that TPI-287 may have efficacy when administered early in the course of the disease.

Opposing effects of pigment epithelium-derived factor on breast cancer cell versus neuronal survival: implication for brain metastasis and metastasis-induced brain damage.

Brain metastases are a significant cause of morbidity and mortality for patients with cancer, yet preventative and therapeutic options remain an unmet need. The cytokine pigment epithelium-derived factor (PEDF) is downregulated in resected human brain metastases of breast cancer compared with primary breast tumors, suggesting that restoring its expression might limit metastatic spread. Here, we show that outgrowth of large experimental brain metastases from human 231-BR or murine 4T1-BR breast cancer cells was suppressed by PEDF expression, as supported by in vitro analyses as well as direct intracranial implantation. Notably, the suppressive effects of PEDF were not only rapid but independent of the effects of this factor on angiogenesis. Paralleling its cytotoxic effects on breast cancer cells, PEDF also exerted a prosurvival effect on neurons that shielded the brain from tumor-induced damage, as indicated by a relative 3.5-fold reduction in the number of dying neurons adjacent to tumors expressing PEDF. Our findings establish PEDF as both a metastatic suppressor and a neuroprotectant in the brain, highlighting its role as a double agent in limiting brain metastasis and its local consequences.

Lapatinib distribution in HER2 overexpressing experimental brain metastases of breast cancer.

PURPOSE: Lapatinib, a small molecule EGFR/HER2 inhibitor, partially inhibits the outgrowth of HER2+ brain metastases in preclinical models and in a subset of CNS lesions in clinical trials of HER2+ breast cancer. We investigated the ability of lapatinib to reach therapeutic concentrations in the CNS following (14)C-lapatinib administration (100 mg/kg p.o. or 10 mg/kg, i.v.) to mice with MDA-MD-231-BR-HER2 brain metastases of breast cancer. METHODS: Drug concentrations were determined at differing times after administration by quantitative autoradiography and chromatography. RESULTS: (14)C-Lapatinib concentration varied among brain metastases and correlated with altered blood-tumor barrier permeability. On average, brain metastasis concentration was 7-9-fold greater than surrounding brain tissue at 2 and 12 h after oral administration. However, average lapatinib concentration in brain metastases was still only 10-20% of those in peripheral metastases. Only in a subset of brain lesions (17%) did lapatinib concentration approach that of systemic metastases. No evidence was found of lapatinib resistance in tumor cells cultured ex vivo from treated brains. CONCLUSIONS: Results show that lapatinib distribution to brain metastases of breast cancer is partially restricted and blood-tumor barrier permeability is a key component of lapatinib therapeutic efficacy which varies between tumors.

The B-Raf status of tumor cells may be a significant determinant of both antitumor and anti-angiogenic effects of pazopanib in xenograft tumor models.

Pazopanib is an FDA approved Vascular Endothelial Growth Factor Receptor inhibitor. We previously reported that it also inhibits tumor cell B-Raf activity in an experimental brain metastatic setting. Here, we determine the effects of different B-Raf genotypes on pazopanib efficacy, in terms of primary tumor growth and anti-angiogenesis. A panel of seven human breast cancer and melanoma cell lines harboring different mutations in the Ras-Raf pathway was implanted orthotopically in mice, and tumor growth, ERK1/2, MEK1/2 and AKT activation, and blood vessel density and permeability were analyzed. Pazopanib was significantly inhibitory to xenografts expressing either exon 11 mutations of B-Raf, or HER2 activated wild type B-Raf; no significant inhibition of a xenograft expressing the common V600E B-Raf mutation was observed. Decreased pMEK staining in the responsive tumors confirmed that B-Raf was targeted by pazopanib. Interestingly, pazopanib inhibition of tumor cell B-Raf also correlated with its anti-angiogenic activity, as quantified by vessel density and area. In conclusion, using pazopanib, tumor B-Raf status was identified as a significant determinant of both tumor growth and angiogenesis.

Inhibition of Polo-like kinase 1 prevents the growth of metastatic breast cancer cells in the brain.

Few therapeutic strategies exist for the treatment of metastatic tumor cells in the brain because the blood-brain barrier (BBB) limits drug access. Thus the identification of molecular targets and accompanying BBB permeable drugs will significantly benefit brain metastasis patients. Polo-like kinase 1 (Plk1) is an attractive molecular target because it is only expressed in dividing cells and its expression is upregulated in many tumors. Analysis of a publicly available database of human breast cancer metastases revealed Plk1 mRNA expression was significantly increased in brain metastases compared to systemic metastases (P = 0.0018). The selective Plk1 inhibitor, GSK461364A, showed substantial uptake in normal rodent brain. Using a breast cancer brain metastatic xenograft model (231-BR), we tested the efficacy of GSK461364A to prevent brain metastatic colonization. When treatment was started 3 days post-injection, GSK461364A at 50 mg/kg inhibited the development of large brain metastases 62% (P = 0.0001) and prolonged survival by 17%. GSK461364A sensitized tumor cells to radiation induced cell death in vitro. Previously, it was reported that mutations in p53 might render tumor cells more sensitive to Plk1 inhibition; however, p53 mutations are uncommon in breast cancer. In a cohort of 41 primary breast tumors and matched brain metastases, p53 immunostaining was increased in 61% of metastases; 44% of which were associated with primary tumors with low p53. The data suggest that p53 overexpression occurs frequently in brain metastases and may facilitate sensitivity to Plk1 inhibition. These data indicate Plk1 may be a new druggable target for the prevention of breast cancer brain metastases.

Notch1 inhibition alters the CD44hi/CD24lo population and reduces the formation of brain metastases from breast cancer.

Brain metastasis from breast cancer is an increasingly important clinical problem. Here we assessed the role of CD44(hi)/CD24(lo) cells and pathways that regulate them, in an experimental model of brain metastasis. Notch signaling (mediated by γ-secretase) has been shown to contribute to maintenance of the cancer stem cell (CSC) phenotype. Cells sorted for a reduced stem-like phenotype had a reduced ability to form brain metastases compared with unsorted or CD44(hi)/CD24(lo) cells (P < 0.05; Kruskal-Wallis). To assess the effect of γ-secretase inhibition, cells were cultured with DAPT and the CD44/CD24 phenotypes quantified. 231-BR cells with a CD44(hi)/CD24(lo) phenotype was reduced by about 15% in cells treated with DAPT compared with DMSO-treated or untreated cells (P = 0.001, ANOVA). In vivo, mice treated with DAPT developed significantly fewer micro- and macrometastases compared with vehicle treated or untreated mice (P = 0.011, Kruskal-Wallis). Notch1 knockdown reduced the expression of CD44(hi)/CD24(lo) phenotype by about 20%. In vitro, Notch1 shRNA resulted in a reduction in cellular growth at 24, 48, and 72 hours time points (P = 0.033, P = 0.002, and P = 0.009, ANOVA) and about 60% reduction in Matrigel invasion was observed (P < 0.001, ANOVA). Cells transfected with shNotch1 formed significantly fewer macrometastases and micrometastases compared with scrambled shRNA or untransfected cells (P < 0.001; Kruskal-Wallis). These data suggest that the CSC phenotype contributes to the development of brain metastases from breast cancer, and this may arise in part from increased Notch activity.

Pazopanib reveals a role for tumor cell B-Raf in the prevention of HER2+ breast cancer brain metastasis.

PURPOSE: Brain metastases of breast cancer contribute significantly to patient morbidity and mortality. We have tested pazopanib, a recently approved antiangiogenic drug that targets VEGFR1, VEGFR2, VEGFR3, PDGFRß, PDGFRα, and c-kit, for prevention of experimental brain metastases and mechanism of action. EXPERIMENTAL DESIGN: In vitro assays included B-Raf enzymatic assays, Western blots, and angiogenesis assays. For in vivo assays, HER2 transfectants of the brain seeking sublines of MDA-MB-231 cells (231-BR-HER2) and MCF7 cells (MCF7-HER2-BR3, derived herein) were injected into the left cardiac ventricle of mice and treated with vehicle or pazopanib beginning on day 3 postinjection. Brain metastases were counted histologically, imaged, and immunostained. RESULTS: Treatment with 100 mg/kg of pazopanib resulted in a 73% decline in large 231-BR-HER2 metastases (P < 0.0001) and a 39% decline in micrometastases (P = 0.004). In vitro, pazopanib was directly antiproliferative to 231-BR-HER2 breast cancer cells and inhibited MEK and ERK activation in vitro despite B-Raf and Ras mutations. Enzymatic assays demonstrated that pazopanib directly inhibited the wild type and exon 11 oncogenic mutant, but not the V600E mutant forms of B-Raf. Activation of the B-Raf targets pERK1/2 and pMEK1/2 was decreased in pazopanib-treated brain metastases whereas blood vessel density was unaltered. In the MCF7-HER2-BR3 experimental brain metastasis model, pazopanib reduced overall brain metastasis volume upon magnetic resonance imaging (MRI) by 55% (P = 0.067), without affecting brain metastasis vascular density. CONCLUSIONS: The data identify a new activity for pazopanib directly on tumor cells as a pan-Raf inhibitor and suggest its potential for prevention of brain metastatic colonization of HER2(+) breast cancer.

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.

Translational research in brain metastasis is identifying molecular pathways that may lead to the development of new therapeutic strategies.

Central nervous system (CNS) or brain metastasis is an emerging area of interest in organ-specific metastasis research. Lung and breast cancers are the most common types of primary tumors to develop brain metastases. This disease complication contributes significantly to the morbidity and mortality of both of these common cancers; as such, brain metastasis is designated an unmet medical need by the US Food and Drug Administration (FDA). Recently, an increase in incidence of CNS disease has been noted in the literature for breast cancer, while it has been an ongoing major complication from lung cancer. Progress in treating brain metastases has been hampered by a lack of model systems, a lack of human tissue samples, and the exclusion of brain metastatic patients from many clinical trials. While each of those is significant, the major impediment to effectively treating brain metastatic disease is the blood-brain barrier (BBB). This barrier excludes most chemotherapeutics from the brain and creates a sanctuary site for metastatic tumors. Recent findings on the biology of this disease and translational leads identified by molecular studies are discussed in this article.