Expression of an engrailed-LMO4 fusion protein in mammary epithelial cells inhibits mammary gland development in mice

Abstract

domain factors and associated cofactors are important developmental regulators in pattern formation and organogenesis. In addition, overexpression of two LIM-only factors (LMOs) causes acute lymphocytic leukemia. The more recently discovered LMO factor LMO4 is highly expressed in proliferating epithelial cells, and frequently overexpressed in breast carcinoma. Here we show that while LMO4 is expressed throughout mammary gland development, it is dramatically upregulated in mammary epithelial cells during midpregnancy. The LMO coactivator Clim2/Ldb1/NLI showed a similar expression pattern, consistent with the idea that LMO4 and Clim2 act as a complex in mammary epithelial cells. In MCF-7 cells, LMO4 transcripts were upregulated by heregulin, an activator of ErbB receptors that are known to be important in mammary gland development and breast cancer. To test the hypothesis that LMO4 plays roles in mammary gland development, we created an engrailed-LMO4 fusion protein. This fusion protein maintains the ability to interact with Clim2, but acts as a dominant repressor of both basal and activated transcription when recruited to a DNA-regulatory region. When the engrailed-LMO4 fusion protein was expressed under control of the MMTV promoter in transgenic mice, both ductular development in virgin mice and alveolar development in pregnant mice were inhibited. These results suggest that LMO4 plays a role in promoting mammary gland development.

The LIM-only factor LMO4 regulates expression of the BMP7 gene through an HDAC2-dependent mechanism, and controls cell proliferation and apoptosis of mammary epithelial cells

“…The present study extends previous studies (Wang et al, 2004;Sum et al, 2005c) by showing, in two distinct types of Cre-mediated mammary gland knockouts of LMO4, that there is decreased proliferation of mammary epithelial cells starting in early pregnancy, ultimately resulting in impaired lobuloalveolar development and mammary gland function at the end of pregnancy. Therefore, one of the roles of LMO4 is to maintain proliferation of mammary epithelial cells during early pregnancy.…”

“…While we found an increase in apoptosis of mammary epithelial cells at day 13.5 in WAP-Cre-LMO4 fl/fl mice (data not shown), apoptosis was not increased in the MMTV-Cre-LMO4 fl/fl mice, leading us to conclude that decreased epithelial cell proliferation is the major cause of decreased lobuloalveolar development in LMO4 knockout mice. These data add to previous studies (Wang et al, 2004;Sum et al, 2005c) implicating the LMO4 gene in lobuloalveolar development, by demonstrating consistent phenotypes in two types of mammary gland knockouts and by showing that LMO4 acts as early as day 5.5 in the pregnant mammary gland.…”

“…In addition, overexpression of LMO4 in the mammary gland of mice leads to hyperplasia and intraepithelial neoplasia (Sum et al, 2005b). Furthermore, the LMO4 gene is activated by heregulin (Wang et al, 2004) and is overexpressed in Her2-mediated tumors (Landis et al, 2005). Together, these observations demonstrate that LMO4 has critical functions in mammary epithelial cells.…”

“…To start investigating the effect of LMO4 in breast cancer, we selected the human MCF-7 breast cancer cell line for establishing an inducible LMO4 expression system; MCF-7 cells have low LMO4 levels (Wang et al, 2004). We used the Tet-off system to establish several distinct MCF-7 cell clones, referred to as MCF7-LMO4-TetOff cells, in which the expression of Myctagged LMO4 was repressed by the presence of doxycycline in the medium (Figure 2a).…”

“…LMO4 has been implicated in the cause or progression of breast cancers (Sum et al, 2005b), squamous cell carcinomas of oral cavity (Mizunuma et al, 2003) and primary prostate cancers (Mousses et al, 2002). The LMO4 gene is most highly expressed in mammary epithelial cells during midpregnancy (Wang et al, 2004), a stage of active proliferation and invasion; interference with the protein (Wang et al, 2004) or deletion of the gene (Sum et al, 2005c) leads to impaired lobuloalveolar development of the mammary gland. LMO4 is overexpressed in over half of primary breast cancers and its expression is associated with a worse prognosis (Visvader et al, 2001;Sum et al, 2005b).…”