A New Gene Regulation Factor, LMO-4, in Breast Cancer

Institution: University of California, Irvine
Investigator(s): Bogi Andersen, M.D. - Bogi Andersen, M.D. -
Award Cycle: 1999 (Cycle V) Grant #: 5JB-0119A Award: $54,823
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

This is a collaboration with: 5JB-0119 -

Initial Award Abstract (1999)
The etiology of sporadic breast cancers is multifactorial and is thought to involve stepwise mutations in several known oncogenes (tumor promoting) and tumor suppressor genes. The elucidation of novel pathways involved in regulation of proliferation and differentiation of breast epithelial cells has important implications for pathogenesis, prognosis and the identification of targets for therapeutic agents in breast cancer. We have recently cloned a novel gene regulation co-factor, LMO-4, which exhibits prominent expression in breast epithelial cells. Previously characterized members of this gene family have been shown to be required for normal blood cell differentiation and cause lymphocyte-type tumors. Consistent with a role in proliferation and/or early differentiation, the LMO-4 gene is highly expressed in proliferating epithelial cells of the breast. Data suggests that LMO-4 functions as a component of multiprotein complexes that regulate transcription. We hypothesize that LMO-4 participates in regulation of proliferation and differentiation in normal breast epithelial cells, and this may lead to altered patterns of gene expression that contribute to breast cancer.

To test this hypothesis, we plan to study in more detail the expression pattern of LMO-4 both during normal breast development and in breast cancers particularly with respect to cellular proliferation and differentiation. We plan to introduce both wild-type (i.e., the normal, non-mutated form) LMO-4 and an artificial LMO-4 repressor in the mammary glands of mice. Using mice that are genetically engineered (transgenic) we will be able to examine the effects of LMO-4 on mammary development, differentiation, and tumor processes. As a final goal, we will use the yeast two-hybrid technology to search for proteins that interact with LMO-4 and work with it in gene regulation. Specifically, we suspect that LMO-4 interacts with previously described transcription factors, either nuclear oncoproteins or tumor suppressors, known to be important for the pathogenesis of breast cancer.

These experiments should provide insights into how LMO-4 regulates cellular proliferation and differentiation in normal and neoplastic breast.

Final Report (2002)
Note: This project was extended one year, and Dr. Andersen relocated to the University of California, Irvine.

Understanding the mechanisms involved in regulating proliferation and differentiation of breast epithelial cells is important for further understanding the causes, diagnosis and treatment of breast cancer. We have recently identified a new protein called LMO-4, a member of a family of proteins that participate in gene regulation. Proteins belonging to this group have been shown to cause leukemia. We were therefore intrigued to find that LMO-4 is highly abundant in breast epithelial cells when these cells are proliferating. Our hypothesis is therefore that LMO-4 is involved in regulation of proliferation of breast epithelial cells in normal breast and in breast cancer. To test this hypothesis, we pursued the following Specific Aims:

#1. Define the expression pattern of LMO-4 during normal mouse breast development and in human breast cancer. We have completed the generation of LMO-4 antisera both in rabbits and guinea pigs. Our experiments show that LMO-4 and its co-factor Clim are most highly expressed in breast epithelial cells during mid-pregnancy, suggesting a role in promoting proliferation and/or invasion of breast epithelial cells. Consistent with this idea, other investigators have shown that LMO-4 is overexpressed in a large portion of invasive human breast cancer.

#2. Define the role of LMO-4 in normal breast development and in breast cancer, using a mouse transgenic approach. We have created transgenic mice expressing wild type LMO-4, LMO-4 fused to a transcriptional repressor domain, LMO-4 fused to a transactivation domain and a dominant negative CLIM under the MMTV promoter. We have obtained founder mice for 3 out of 4 of the lines. Our analyses indicates that ductal growth and branching is inhibited in mice expressing LMO-4 fused to a transcriptional repressor domain, indicating that LMO-4 plays a role in normal mammary gland development.

#3. Identify and characterize protein partners for LMO-4 in human breast tissue. With the yeast-two hybrid system we have identified at least three proteins that potentially act as LMO-4 partners in human breast. The gene encoding one of these molecules, which is thought to be involved in processing of mRNA, was recently found to be amplified in human breast cancer cell lines. We are currently trying to characterize this interaction further.

#4. Define the effect of LMO-4 on gene expression in breast cancer cell lines. We have generated stable MCF-7 breast cancer cell lines in which the expression of LMO-4 and Clim can be conditionally induced. We are in the process of using these cell lines and microarray analyses to identify the downstream targets of LMO-4/Clim that may be involved in breast cancer.

This work has identified novel genes, providing insights into the causes of breast cancer and thus impacting on reducing the human/economic cost of breast cancer in California.

Symposium Abstract (2003)
Many properties of breast cancer cells, including increased proliferation and invasion, are common to epithelial cells of the developing mammary gland. This suggests that understanding of developmental control in normal mammary glands may provide important insights into the biology of breast cancer. This notion is supported by work in many organ systems, demonstrating that subversion of developmental control genes plays role in carcinogenesis. LIM domain factors, and associated co-regulators, are important developmental regulators involved in pattern formation and organogenesis in a wide spectrum of organisms, including mammals. We isolated a LIM only factor, LMO-4, which is highly expressed in epithelium cells, including mammary epithelium. Interestingly, LMO factors are known to be oncogenic in lymphocytes where their overexpression causes acute lymphocytic leukemia.

We have studied expression of LMO-4 in mammary glands of mice and found that it is most highly expressed in proliferating mammary epithelial cells during pregnancy. This suggests that the LMO-4 gene may play a role in proliferation. Since LMOs do not bind to DNA it is likely that they regulate transcription by interacting with DNA-binding proteins and other transcriptional co-regulators. To search for such factors, we have screened a human breast cancer cDNA library with LMO-4 as ”bait” in the yeast two hybrid technique. We found several potential interacting partners, including DNA-binding protein, Clim/Nli/ldb co-regulators, and splicing factors previously shown to be amplified in breast cancer cell lines. To test the role of LMO-4 in mammary gland biology, we have generated three lines of transgenic mice expressing it under control of the MMTV promoter a) wild-type LMO-4, b) LMO-4 fused to the VP-16 transactivation domain and c) LMO-4 fused to the engrailed repression domain. Whole mount mammary gland analyses of these transgenic mice are in progress and preliminary results will be presented. Analyses of the EST databases indicate that LMO-4 is highly expressed in mammary carcinomas, and we are in the process of evaluating its expression in breast cancer.

We conclude that LMO-4 may be an important regulator of mammary epithelial cells and propose a hypothesis that its high level expression in mammary tumors may play a role in mammary carcino-genesis.