Overexpression of LMO4 induces mammary hyperplasia, promotes cell invasion, and is a predictor of poor outcome in breast cancer

Down-regulation of LMO4 expression in breast epithelial cells by RNAi inhibits proliferation and augments differentiation. (A) Western blot analysis of protein lysates from MCF-7 and BT-549 breast cancer cells transiently transfected with LMO4-specific siRNA276 and siRNA376, compared with control siRNA and mock-transfected cells, 2 and 6 days after transfection by using α-LMO4 (20F8) mAb. Anti-tubulin was used to verify protein loading. (B) The proliferation rate of siRNA-transfected MCF-7 and BT-549 cells, and mock-transfected cells, was determined from days 0–5, in three independent experiments. Error bars indicate SEM; n = 3. (C) Cell cycle defect in LMO4-deficient MCF7 cells. siRNA and mock-transfected cells were fixed in ice-cold 70% ethanol for 24 h before staining with 0.5 μg/ml propidium iodide (PI). FACScan analysis revealed an increase in G₁/G₀ cells and a decrease in S-phase cells in LMO4-deficient MCF-7 cells, compared with control cells, in three independent experiments. Error bars indicate SEM; n = 3. *, P < 0.05. (D) Down-regulation of LMO4 expression in HC11 cells transiently transfected with siRNA376 (or a control siRNA) was confirmed by Western blot analysis of cells harvested 3 days after transfection by using α-LMO4 (20F8) mAb. Anti-tubulin immunoblotting provided a control. (E) RT-PCR analysis was performed by using total RNA derived from RNAi-treated HC11 cells that were stimulated with (+) prolactin, insulin, and dexamethasone or unstimulated (–) for 48 h. β-casein and Hprt were used as markers of differentiation and loading, respectively. At least three independent experiments were performed.

Reduced LMO4 expression impedes the migration and invasion of breast cancer cells. (A) Western blot analysis of protein lysates from MDA-MB-231 breast cancer cells transfected with siRNA276, siRNA376, control siRNA, or mock-transfected cells, 2 and 6 days after transfection by using α-LMO4 (20F8) mAb. Anti-tubulin provided a control. (B) Focal adhesions in MDA-MB-231 cells transfected with siRNA376 or a control siRNA were visualized by indirect immunofluorescence by using α-vinculin mAb. Costaining with α-LMO4 (20F8) mAb confirmed the reduction in LMO4 expression in siRNA376-transfected cell nuclei. [Scale bars, 20 (i and ii) and 8 (iii and iv) μm.] (C) The number of migrating MDA-MB-231 cells transfected with either LMO4-specific siRNA276 or siRNA376, or a mutant siRNA, was determined by counting 10 random fields in each of three independent experiments. Error bars indicate SEM; n = 3. (D) The number of MDA-MB-231 cells, transfected with siRNA276, siRNA376, or a mutant siRNA, capable of invading Matrigel was determined for three independent experiments, as in C. Error bars indicate SEM; n = 3.

LMO4 overexpression augments mammary epithelial cell migration and invasion. (A) The motility of MCF10A(EcoR) cells, stably transduced with a FLAG-LMO4 retroviral construct or vector alone (pBabe-puro), was determined by counting the number of migrated cells in 10 random fields from each of four experiments. Error bars indicate SEM; n = 4. (B) The number of MCF10A(EcoR) cells expressing either FLAG-LMO4 or vector alone (pBabepuro), capable of invading through Matrigel, was determined in four experiments, as described for A. Error bars indicate SEM; n = 4. (C) Western blot analysis confirmed the expression of FLAG-LMO4 in the transductants. Lysates from cells expressing FLAG-LMO4 or empty vector were subjected to SDS/PAGE and immunoblotted with mouse α-FLAG mAb. Anti-tubulin was used to control for protein loading.