Figure 4

(A) The BD BioCoatTM MatrigelTM Invasion Chamber was used to assess cell invasion/migration of T47D cells stably transduced with empty MLV-backbone (T47D-BB) or MLV-HA-RASSF1C (T47D-1C). T47D-1C or T47D-BB cells treated with doxycycline at 1ug/ml were co-incubated with Hs27a human stromal cells. Hs27a cells express the stromal derived factor-1 (SDF-1), the ligand for the CXCR4 receptor. After 24 hr incubation period the lower sides of the filters were fixed and stained and cells in four microscopic fields were counted. The average cell number count was plotted. T47D cells over-expression RASSF1C showed a higher number of cells invading the Matrigel chamber and migrating to the other side of the filter compared to T47D-cells, p <0.003. (B) T47D-lenti-shRNA control (Mission, Sigma), T47D-lenti-shRNA to silence RASSF1C (T47D-siRNA-1C) (Mission, Sigma), and T47D-1C cells were incubated with either non-conditioned serum-free medium or SDF-1-conditioned serum-free medium for 24 hr. Lower number T47D-siRNA-1C cells invaded the Matrigel chamber and migrating to the other side of the filter compared to T47D-siRNA control. (C) MDA-MB231-BB (MDA-BB) and MDA-MB231-1C (MDA-1C) cells were plated in the chamber inserts and placed in wells containing either non-conditioned serum-free medium or SDF-1-conditioned serum-free medium, and incubated for 24 hr. MDA-1C cells showed a higher number of cells invading the Matrigel chamber and migrating to the other side of the filter compared to MDA-BB cells, p < 0.001. The data suggests a potential role for RASSF1C in cancer cell metastasis, perhaps through the up-regulation of CXCR gene expression.