Yongzhen Qian

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)

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, PDGFRβ, 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. Clin Cancer Res; 17(1); 142–53. ©2010 AACR.

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

BackgroundBrain 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.MethodsTwo 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.ResultsShort 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.ConclusionsOur results show that elevated expression of MMP-1 can promote the local growth and the formation of brain metastases by breast cancer cells.

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. Mol Cancer Ther; 11(9); 1959–67. ©2012 AACR.

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.

Pazopanib Inhibits the Activation of PDGFRβ-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 β (at tyrosine 751; p751-PDGFRβ) was identified around perivascular brain micrometastases. p751-PDGFRβ(+) 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-PDGFRβ(+) 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.

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 O6-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. Clin Cancer Res; 20(10); 2727–39. ©2014 AACR.

Nm23-H1 Binds to Gelsolin and Inactivates Its Actin-Severing Capacity to Promote Tumor Cell Motility and Metastasis

Nm23-H1 has been identified as a metastasis suppressor gene, but its protein interactions have yet to be understood with any mechanistic clarity. In this study, we evaluated the proteomic spectrum of interactions made by Nm23-H1 in 4T1 murine breast cancer cells derived from tissue culture, primary mammary tumors, and pulmonary metastases. By this approach, we identified the actin-severing protein Gelsolin as binding partner for Nm23-H1, verifying their interaction by coimmunoprecipitation in 4T1 cells as well as in human MCF7, MDA-MB-231T, and MDA-MB-435 breast cancer cells. In Gelsolin-transfected cells, coexpression of Nm23-H1 abrogated the actin-severing activity of Gelsolin. Conversely, actin severing by Gelsolin was abrogated by RNA interference-mediated silencing of endogenous Nm23-H1. Tumor cell motility was negatively affected in parallel with Gelsolin activity, suggesting that Nm23-H1 binding inactivated the actin-depolymerizing function of Gelsolin to inhibit cell motility. Using indirect immunoflourescence to monitor complexes formed by Gelsolin and Nm23-H1 in living cells, we observed their colocalization in a perinuclear cytoplasmic compartment that was associated with the presence of disrupted actin stress fibers. In vivo analyses revealed that Gelsolin overexpression increased the metastasis of orthotopically implanted 4T1 or tail vein-injected MDA-MB-231T cells (P = 0.001 and 0.04, respectively), along with the proportion of mice with diffuse liver metastases, an effect ablated by coexpression of Nm23-H1. We observed no variation in proliferation among lung metastases. Our findings suggest a new actin-based mechanism that can suppress tumor metastasis.

Abstract P6-11-04: Profound prevention of experimental brain metastases of breast cancer by temozolomide in a MGMT-dependent manner

Purpose: Brain metastases of breast cancer cause neurocognitive damage and are incurable. We evaluated in experimental brain metastasis model a role of temozolomide, an oral brain permeable alkylating agent characterized by significant uptake in the central nervous system, in the prevention of brain metastases of breast cancer. Material and methods: To assess preventive role of temozolomide, mice were inoculated with 175,000 triple-negative 231-BR-EGFP cells in 0.1 mL PBS in the left ventricle of the heart. Three days after tumor cell inoculation, mice were randomized to temozolomide at the dose of 50 mg/kg delivered by oral gavage in saline, 5 days a week for 4 weeks, or vehicle. Subsequent experiments used temozolomide doses of 25, 10, 5, 1 and 0.5 mg/kg. To evaluate the efficacy of temozolomide in treating established BM, mice received temozolomide (50 mg/kg) beginning on either day 18 or day 24 post-injection of 231-BR-EGFR cells, 5 days a week for two and one week, respectively. To investigate the impact of temozolomide on survival, mice injected with 231-BR-EGFP cells were randomized to vehicle, temozolomide on days 3-14, or temozolomide on days 17-28 post-injection, per the schedule described above. To determine the functional contribution of MGMT expression in the BM preventive model, similar experiments were performed using 231-BR-EGFP cells with induced MGMT expression, and MGMT-positive Jimt-1 cells. Metastases were counted in step sections of one hemisphere of each brain. Additionally, 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/week, beginning 3 days after inoculation, completely prevented the formation of experimental brain metastases from MGMT-negative 231-BR-EGFP cell line. 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. Both schedules of temozolomide (days 3-14 and days 17-28) significantly increased survival (P = .0003 by long-rank test). Earlier administration of temozolomide resulted in long term survival of 6 and 2 out of 10 mice, respectively; a significant difference compared to vehicle ( P Conclusions: Temozolomide profoundly prevents the outgrowth of experimental brain metastases of breast cancer in a MGMT-dependent manner. The majority of patients had low MGMT expressing brain metastases. These data provide a compelling rationale for investigating preventive efficacy of temozolomide in high-risk advanced breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-11-04.

Abstract 2846: Pigment epithelium-derived factor (PEDF) functions as a brain metastasis suppressor of breast cancer

Brain metastases occur in 10-20% of metastatic breast cancer patients. The one year survival rate is approximately 20%. Brain metastasis is typically diagnosed late in breast cancer progression; however, studies suggest that the incidence of brain metastasis may be increasing under current chemotherapeutic regimens. We have investigated changes in gene expression associated with the acquisition of brain metastatic potential by breast cancer. A DNA microarray analysis comparing human brain metastases and a cohort of unlinked primary breast carcinomas identified Pigment Epithelium-Derived Factor (PEDF) as down-regulated in the brain metastases relative to the primary tumors (mean ∼14X lower by QPCR validation). The data suggest the hypothesis that PEDF may have a negative impact on brain metastatic progression. PEDF, a secreted factor, has been shown to act as a tumor suppressor, has strong anti-angiogenic activity, but supports the proliferation/viability/ differentiation of neural tissue. We hypothesize that the multiple roles of PEDF make it a potent agent which targets not only tumor cells, but also the microenvironment of the brain. In vitro experiments demonstrated that PEDF can promote death of breast cancer cells. Conversely, PEDF promoted neuronal survival, in vitro. In a xenograft model system (231BR, a brain-tropic derivative of human MDA-MB-231 breast cancer cells) PEDF overexpression inhibited formation of large brain metastases (∼2-fold, p=0.001) and reduced proliferation of PEDF-expressing breast cancer cells in mouse brain metastases (11% Ki67-positive, PEDF-positive vs. >56% Ki67-positive, PEDF-negative). In an intracranial implantation model, expression of PEDF by breast cancer cells lead to a rapid reduction in brain metastatic tumor volume. Histological examination suggests that PEDF reduces gliosis and protects neurons concomitant to suppression of metastatic growth. The data suggest the intriguing possibility that PEDF overexpression may concurrently inhibit brain metastasis of breast cancer cells but preserve neuronal function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2846. doi:10.1158/1538-7445.AM2011-2846