LMO4 is an essential mediator of ErbB2/HER2/Neu-induced breast cancer cell cycle progression.

ErbB2/HER2/Neu-overexpressing breast cancers are characterized by poor survival due to high proliferation and metastasis rates and identifying downstream targets of ErbB2 should facilitate developing novel therapies for this disease. Gene expression profiling revealed the transcriptional regulator LIM-only protein 4 (LMO4) is upregulated during ErbB2-induced mouse mammary gland tumorigenesis. Although LMO4 is frequently overexpressed in breast cancer and LMO4-overexpressing mice develop mammary epithelial tumors, the mechanisms involved are unknown. In this study, we report that LMO4 is a downstream target of ErbB2 and PI3K in ErbB2-dependent breast cancer cells. Furthermore, LMO4 silencing reduces proliferation of these cells, inducing a G2/M arrest that was associated with decreased cullin-3, an E3-ubiquitin ligase component important for mitosis. Loss of LMO4 subsequently results in reduced Cyclin D1 and Cyclin E. Further supporting a role for LMO4 in modulating proliferation by regulating cullin-3 expression, we found that LMO4 expression oscillates throughout the cell cycle with maximum expression occurring during G2/M and these changes precede oscillations in cullin-3 levels. LMO4 levels are also highest in high-grade/less differentiated breast cancers, which are characteristically highly proliferative. We conclude that LMO4 is a novel cell cycle regulator with a key role in mediating ErbB2-induced proliferation, a hallmark of ErbB2-positive disease.

Ovarian hyperstimulation induces centrosome amplification and aneuploid mammary tumors independently of alterations in p53 in a transgenic mouse model of breast cancer.

Aneuploidy and genomic instability are common features of human cancers, including breast cancer; however, mechanisms by which such abnormalities develop are not understood. The exquisite dependence of the mammary gland on hormones for growth and development as well as hormonal contributions to breast cancer risk and progression suggest that tumorigenic mechanisms in the breast should be considered in the context of hormonal stimulation. We used transgenic mice that overexpress luteinizing hormone with subsequent ovarian hyperstimulation as a model to identify mechanisms involved in hormone-induced mammary cancer. Tumor pathology in these mice is highly variable, suggesting individual tumors undergo distinct initiating or promoting events. Supporting this notion, hormone-induced tumors display considerable chromosomal instability and aneuploidy, despite the presence of functional p53. The presence of extensive centrosome amplification in tumors and hyperplastic glands prior to tumor formation suggests that alterations in the ovarian hormonal milieu dysregulate the centrosome cycle in mammary epithelial cells, leading to aneuploidy and cancer.

Sustained trophism of the mammary gland is sufficient to accelerate and synchronize development of ErbB2/Neu-induced tumors.

Epidemiological studies indicate that parity enhances HER2/ErbB2/Neu-induced breast tumorigenesis. Furthermore, recent studies using multiparous, ErbB2/Neu-overexpressing mouse mammary tumor virus (MMTV-Neu) mice have shown that parity induces a population of cells that are targeted for ErbB2/Neu-induced transformation. Although parity accelerates mammary tumorigenesis, the pattern of tumor development in multiparous MMTV-Neu mice remains stochastic, suggesting that additional events are required for ErbB2/Neu to cause mammary tumors. Whether such events are genetic in nature or reflective of the dynamic hormonal control of the gland that occurs with pregnancy remains unclear. We postulated that young age at pregnancy initiation or chronic trophic maintenance of mammary epithelial cells might provide a cellular environment that significantly increases susceptibility to ErbB2/Neu-induced tumorigenesis. MMTV-Neu mice that were maintained pregnant or lactating beginning at 3 weeks of age demonstrated accelerated tumorigenesis, but this process was still stochastic, indicating that early pregnancy does not provide the requisite events of tumorigenesis. However, bitransgenic mice that were generated by breeding MMTV-Neu mice with a luteinizing hormone-overexpressing mouse model of ovarian hyperstimulation developed multifocal mammary tumors in an accelerated, synchronous manner compared to virgin MMTV-Neu animals. This synchrony of tumor development in the bitransgenic mice suggests that trophic maintenance of the mammary gland provides the additional events required for tumor formation and maintains the population of cells that are targeted by ErbB2/Neu for transformation. Both the synchrony of tumor appearance and the ability to characterize a window of commitment by ovariectomy/palpation studies permitted microarray analysis to evaluate changes in gene expression over a defined timeline that spans the progression from normal to preneoplastic mammary tissue. These approaches led to identification of several candidate genes whose expression changes in the mammary gland with commitment to ErbB2/Neu-induced tumorigenesis, suggesting that they may either be regulated by ErbB2/Neu and/or contribute to tumor formation.