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Widespread metastatic disease causes >90% deaths in breast cancer patients. BRMS1, a metastasis suppressor gene, suppresses lung metastasis in breast, bladder and melanoma animal models, without significantly blocking primary tumor growth. This study tested the hypothesis that BRMS1 suppresses metastasis to multiple organs. MDA-MB-231 and -435 cells were transduced to constitutively express green fluorescent protein (GFP) (designated 231GFP and 435GFP). These were then stably transduced to express myc epitope-tagged BRMS1 (designated 231GFP-BRMS1 and 435GFP-BRMS1). 231GFP and 435GFP and 3 clones each of BRMS1 expressors (2×105 cells/mouse) were injected into the left ventricle of the heart into female athymic mice, in order to avoid first-pass clearance in the lung. At 5 wk, mice were euthanized and all bones and major organs were examined for the presence of metastatic foci by fluorescence stereomicroscopy. In both the 231 and 435 experiments, the mean number of bone metastases was significantly suppressed in all BRMS1 expressing clones as compared to controls (p<0.01). Metastasis to brain, pancreas, adrenals, and kidneys were similarly suppressed. To determine which step(s) in the metastatic cascade BRMS1 blocks, kinetic studies were done using 231GFP and 231GFP-BRMS1 clone 9 at times from 1 hr to 5 wk. Both the femurs were collected and the numbers of fluorescent foci at the distal end of the femurs were calculated by fluorescence stereomicroscopy. Significantly fewer BRMS1-expressing cells (mean - 3.5) reached the distal end of the femur compared to the controls (mean - 15) at 1 hr. Similar differences were observed in lungs following intravenous injection. To determine whether decreased ‘seeding’ might be due to differential anoikis, parental (231GFP and 435GFP) and BRMS1-expressing cells were plated onto poly-HEMA plates and assessed for caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. While no difference in anoikis was observed in unstimulated cells, after stimulation with 50 ng/mL EGF, BRMS1-expressing cells showed significantly higher caspase-3 activation and PARP cleavage. These differential effects were seen as early as 5 min, suggesting that BRMS1-expressing cells are more prone to anoikis compared to control cells. Thus, BRMS1 can suppress metastasis of two different human breast carcinoma cell lines to multiple organs. In addition, GFP is an excellent reporter for dissecting the steps in the metastatic cascade at which metastasis suppressor genes, like BRMS1 act. Support: DAMD17-02-1-0541, CA87728, NFCR Center for Metastasis Research,

[Proc Amer Assoc Cancer Res, Volume 47, 2006]