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Triple-negative breast cancer (TNBC) exhibits more traits possessed by cancer stem cells (CSC) than other breast cancer subtypes and is more likely to develop brain metastases. TNBC patients usually have shorter survival time after diagnosis of brain metastasis, suggesting an innate ability of TNBC tumor cells in adapting to the brain. In this study, we establish novel animal models to investigate early tumor adaptation in brain metastases by introducing both patient-derived and cell line–derived CSC-enriched brain metastasis tumorsphere cells into mice. We discovered astrocyte-involved tumor activation of protocadherin 7 (PCDH7)-PLCβ-Ca2+-CaMKII/S100A4 signaling as a mediator of brain metastatic tumor outgrowth. We further identified and evaluated the efficacy of a known drug, the selective PLC inhibitor edelfosine, in suppressing the PCDH7 signaling pathway to prohibit brain metastases in the animal models. The results of this study reveal a novel signaling pathway for brain metastases in TNBC and indicate a promising strategy of metastatic breast cancer prevention and treatment by targeting organ-adaptive cancer stem cells. Significance: These findings identify a compound to block adaptive signaling between cancer stem cells and brain astrocytes. Cancer Res; 78(8); 2052–64. ©2018 AACR.

Purpose: Lazaroid U-74389G (LAZ) is a 21-aminosteroid that has radioprotective effect against radiation-induced lipid peroxidation. In-vitro antiproliferative effect was reported against Glioblastoma primary cell lines. The aim of this study was to use a liposomal formulation in a proof-of-concept efficacy study to evaluate LAZ radioprotective and antiproliferative effects in Glioblastoma mouse model. Methods: LAZ PEGylated liposomes (Lipo G) were developed in our laboratory. Lipo G size was 80-90 nm with Zeta potential of -22mv. Glioblastoma cell line U87-Lexpressing firefly luciferase reporter gene (100,000 cells in 2 μL) was injected intracranially in each male SCID hairless outbred mouse. The mice were randomized among 4 treatment groups (n=8-9, each): brain model (M) without treatment (control), radiation of 2Gy once a week (M+R), Lipo G at 5 mg/kg dose IP twice a week (M+L) and radiation with Lipo G (M+R+L). The treatment lasted three weeks. The tumor size was monitored using non-invasive bioluminescence imaging (BLI), in each mouse, expressed as the relative BLI photon intensity on the second and third week to that of the first measurement. The mice were sacrificed at the end of the third week and the whole brain was harvested for immunohistochemistry examinations. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. The survival was evaluated using Kaplan Meier analysis at P = 0.05. Results: The relative BLI intensity was 4002.03±1737.67, 2034±737.72, 1387.36±684.53 and 2498.89±2521.32 % for M, M+R, M+L and M+R+L, respectively. The tumor size of the M+L group was reduced by 65% compared to the control group. For the radiation treated groups (M+R and M+R+L), there was no significant difference in the tumor size suppression compared to the control group. MDA brain concentration in M+L and M+R+L groups was significantly less than that in M+R group (8.27±0.78 and 10.37±3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M+R, M+R+L and M+L groups, respectively. The mean survival duration of LAZ treated groups (M+L and M+R+L) was significantly longer than that of the control group (27.13 and 25.22 day vs. 22.67 day, respectively). Conclusion: LAZ liposomal formulations administered at 5 mg/kg dose delivered a therapeutic concentration of LAZ to the brain for the efficacy of suppressing Glioblastoma tumor growth (65% reduction). The liposomal LAZ also protected the brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration (lipid peroxidation surrogate) to 50%. 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 5460. doi:10.1158/1538-7445.AM2011-5460

Brain metastasis is the worst stage of breast cancer, as no therapy exists that prevents or eliminates breast cancer spreading to the brain. New therapeutic strategies depend on specific knowledge of tumor cell properties that allow breast cancer cells get through blood-brain barrier and grow in brain tissue. To provide information in this direction, we developed a microfluidic single-cell patch clamp microdevice to study the electrophysiological and biomechanical properties of human breast cancer brain metastatic cells for the first time. The results show that the brain metastatic cells possess higher membrane resistance and greater membrane stiffness compared to non-brain metastatic cells. The over-expression of HER2 and EGFR decrease the cell membrane resistance, while increase the cell stiffness, which correlates with the enhanced cell invasion through the in vitro blood-brain barrier and increased formation of brain metastatic loci in the xenograft mice. Gene expression analysis of the brain metastatic cells identifies cell membrane protein PCDH7 mediates the increased cell mechanics property functionally, and PCDH7 is also an enhancer of blood-brain barrier crossing and brain colonization for the brain metastatic cells, through the cytoskeleton rearrangement mechanism. This cell mechanics-cytoskeleton-PCDH7 protein loop mechanism may prove to be an attraction therapeutic target to block the brain metastasis of breast cancer. 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 5161. doi:10.1158/1538-7445.AM2011-5161

Phase I/II clinical studies with rapamycin analogs in breast and other cancers have demonstrated favorable responses. However, little is known on the effects of the mTOR inhibitor on breast cancer cell metastasis, which is a major cause of morbidity and death. We developed a highly sensitive 3-dimensional (3D) proliferation/invasion assay using quantitative bioluminescence (BL) imaging and applied this assay to evaluate the effects of rapamycin on the triple negative breast cancer cell line MDA-MB231. Without cytotoxicity of rapamycin on this cell line, rapamycin at 10nM inhibited the cell migration/invasion, but not at 1nM and 100nM, which was confirmed by the time-lapse single cell tracking analysis. The quantification of cytoskeleton changes showed most potent effects of 10nM rapamycin on the MDA-MB231 cells, with the formation and rearrangement of specialized cell membrane structures and actin fiber implicated in cell motility. Then, the Panorama Cell Signaling Antibody Microarray, enabling the global comparative analysis of cell signal proteins simultaneously, was exploited to analyze the effects of rapamycin on the cellular signaling network of the MDA-MB231 breast cancer cell line. 100nM rapamycin activated the MAPK pathway obviously, through the attenuated negative feedback of activated S6K1 to PI3K-Raf, which increased the expressions of activated Jun N-terminus kinase (JNK), Erk1/2, MEK-1, Raf-pS621, and MAPK-activated protein kinase 2 (MAPKAPK2) in the cells exposed to 100nM rapamycin. MEK inhibitor U0126 or PD98059 could restore the anti-migration effects of 100nM rapamycin on the MDA-MB231 cells. Furthermore, the combination of MEK inhibitors and rapamycin performed synergism on inhibiting the cell proliferation and migration/invasion. Accordingly, rapamycin at a certain dose suppresses MDA-MB231 cell migration/invasion, and the co-targeting of mTOR and MAPK pathways enhances the inhibition on cell proliferation and migration/invasion, underscoring the potential therapeutic utility of rapamycin, and rapamycin combining with MAPK inhibitors in triple negative breast cancer progression, and the results highlight the cross-talk homeostasis of mTOR and MAPK pathways in cancer treatment. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5080.