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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. Cancer Res; 72(1); 144–53. ©2012 AACR.

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)

Interactions between tumor cells and the microenvironment are crucial to tumor formation and metastasis. The central nervous system serves as a "sanctuary" site for metastasis, resulting in poor prognosis in diagnosed patients. The incidence of brain metastasis is increasing; however, little is known about interactions between the brain and metastatic cells. Brain pathology was examined in an experimental model system of brain metastasis, using a subline of MDA-MB-231 human breast cancer cells. The results were compared with an analysis of sixteen resected human brain metastases of breast cancer. Experimental metastases formed preferentially in specific brain regions, with a distribution similar to clinical cases. In both the 231-BR model, and in human specimens, Ki67 expression indicated that metastases were highly proliferative (approximately 50%). Little apoptosis was observed in either set of tumors. In the model system, metastases elicited a brain inflammatory response, with extensive reactive gliosis surrounding metastases. Similarly, large numbers of glial cells were found within the inner tumor mass of human brain metastases. In vitro co-cultures demonstrated that glia induced a approximately 5-fold increase in metastatic cell proliferation (P0.001), suggesting that brain tissue secretes factors conducive to tumor cell growth. Molecules used to signal between tumor cells and the surrounding glia could provide a new avenue of therapeutic targets for brain metastases.

The Netrin/RGMa receptor, Neogenin, has recently been identified on neuronal and gliogenic progenitors, including radial glia in the embryonic mouse cortex and ganglionic eminences, respectively [Fitzgerald, D.P., Cole, S.J., Hammond, A., Seaman, C., Cooper, H.M., 2006a. Characterization of Neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain. Neuroscience 142, 703-716]. Here we have undertaken a detailed analysis of Neogenin expression in the embryonic mouse central nervous system at key developmental time points. We demonstrate that Neogenin protein is present on actively dividing neurogenic precursors during peak phases of neurogenesis (embryonic days 12.5-14.5) in the forebrain, midbrain and hindbrain. Furthermore, we show that Neogenin protein is localized to the cell bodies and glial processes of neurogenic radial glial populations in all these regions. We have also observed Neogenin on gliogenic precursors within the subventricular zones of the forebrain late in development (embryonic day 17.5). Adult neural stem cells found in the subventricular zone of the lateral ventricle of the rodent forebrain are direct descendants of the embryonic striatal radial glial population. Here we show that Neogenin expression is maintained in the neural stem cell population of the adult mouse forebrain. In summary, this study demonstrates that Neogenin expression is a hallmark of many neural precursor populations (neurogenic and gliogenic) in both the embryonic and adult mammalian central nervous system.

Two cases of severe low cardiac output and right ventricular failure after coronary artery bypass grafting necessitated pulmonary artery balloon counterpulsation after intraaortic balloon pumping and maximal inotropic/ pressor support were unsuccessful in maintaining a satisfactory cardiac output. Hemodynamic improvement was sufficient to allow removal of the device 2 and 3 days postoperatively, with survival in 1 patient. Pulmonary artery counterpulsation is less morbid in comparison with other mechanical methods of right ventricular support and is applicable in right ventricular failure of intermediate severity.

To investigate the molecular mechanisms responsible for breast cancer metastasis to the brain, a gene expression analysis was performed using formalin-fixed, paraffin embedded sections of matched primary breast tumors and resected brain metastases. The neuronal cell adhesion molecule L1CAM was upregulated by approximately 1.57 fold in the brain metastases relative to the primary tumors (p=0.037). This trend was most apparent in Her-2+ tumors (p=0.044) and did not extend to ER+ patients (p=0.42). L1CAM has been correlated to decreased patient survival in breast cancer. MDA-MB-231 breast cancer cells selected to grow in the brain (231-BR) expressed 6.4 fold greater L1CAM on western blots than the parental cell line (231-P). Using three shRNA constructs, L1CAM expression was decreased by 1.76, 3.82, and 63.3 fold in the 231-BR cell line. Decreased L1CAM expression inhibited the clonogenic formation of 231-BR cells by 40-50% (p 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 2667. doi:10.1158/1538-7445.AM2011-2667

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