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

Abstract

The zinc finger protein LMO4 is overexpressed in a high proportion of breast carcinomas. Here, we report that overexpression of a mouse mammary tumor virus (MMTV)-Lmo4 transgene in the mouse mammary gland elicits hyperplasia and mammary intraepithelial neoplasia or adenosquamous carcinoma in two transgenic strains with a tumor latency of 13-18 months. To investigate cellular processes controlled by LMO4 and those that may be deregulated during oncogenesis, we used RNA interference. Down-regulation of LMO4 expression reduced proliferation of human breast cancer cells and increased differentiation of mouse mammary epithelial cells. Furthermore, small-interfering-RNA-transfected breast cancer cells (MDA-MB-231) had a reduced capacity to migrate and invade an extracellular matrix. Conversely, overexpression of LMO4 in noninvasive, immortalized human MCF10A cells promoted cell motility and invasion. Significantly, in a cohort of 159 primary breast cancers, high nuclear levels of LMO4 were an independent predictor of death from breast cancer. Together, these findings suggest that deregulation of LMO4 in breast epithelium contributes directly to breast neoplasia by altering the rate of cellular proliferation and promoting cell invasion.

Fig. 1.

Overexpression of Lmo4 in transgenic mice leads to mammary hyperplasia and tumors. (A) Schematic representation of the MMTV-Lmo4 transgene. Lmo4 cDNA was cloned into the MMTV-RG-SV40 vector. (B) Northern blot analysis of total RNA (15 μg) from the mammary glands of transgenic strains 34 and 36. Filters were hybridized with a transgene-specific SV40 probe followed by a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe. dP, day pregnant. (C–E) Hematoxylin and eosin sections of mammary glands and lung from strain 34, showing high grade MIN (C) and acinar hyperplasia (D), and alveolar bronchocarcinoma (E). (F–H) Hematoxylin and eosin sections from strain 36 showing mammary glands from multiparous mice. (F) Littermate control. (G) Diffuse acinar hyperplasia. (H) Adenosquamous carcinoma. [Original magnification: ×400 (C–E and H) and ×200 (F and G).]

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

Overexpression of LMO4 in breast cancer is a predictor of poor clinical outcome. (A) LMO4 immunostaining was performed on tissue microarrays containing archival breast tumor specimens using α-LMO4 (20F8) mAb. Representative images of tumor specimens displaying low (Left) and high (Right) levels of LMO4 expression are shown. (B) Kaplan–Meier curves for overall survival (OS) of 159 breast cancer patients according to LMO4 nuclear staining. High LMO4 expression was significantly associated with decreased OS in univariate (P = 0.023) analysis.