The Role of LMO4 in Breast Cancer

Institution: University of California, Irvine
Investigator(s): Zhengquan Yu, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11FB-0142 Award: $135,000
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2005)
Cancer cells are able to proliferate uncontrollably because of genetic alterations or abnormal regulation of genes that promote or inhibit cell proliferation. LM04 is potentially responsible for activating proliferation-promoting genes in breast cancer. It is a member of the family of LIM only proteins (LMOs); two other members of the LMO family, LMO1 and LM02, are oncogenes in leukemia. Normally, LM04 is expressed in breast epithelial cells and its expression is dramatically increased during mid-pregnancy. This genetic expression pattern is consistent with a role in the regulation of cell proliferation and therefore it is not surprising that interference with LM04 in mouse mammary glands leads to inhibition of mammary gland development. There is also strong evidence that LM04 may play a role in promoting breast cancer. LM04 was first found in breast cancer tissues and was referred to as Human Breast Tumor Autoantigen. Later studies found that LM04 is overexpressed in about 50% of invasive breast cancers. In recent experiments, it was found that LM04 expression level is greatly increased in ErbB2-induced breast cancer in mice. Moreover, when the LM04 activity was inhibited, cell proliferation was inhibited and programmed cell death was induced in breast cancer cells.

We are using state-of the art methods in mouse genetics to understand breast cancer. Our preliminary results showed that LM04 regulates several key genes implicated in tumorigenesis. . Specifically we propose that LM04 plays its role in breast cancer by functioning in the well known breast cancer oncogene ErbB2 pathway. To study the role of LM04 in mammary gland development and breast cancer, we will generate mice in which the LM04 gene is specifically deleted in the mammary gland. To link the role of LM04 in ErbB2/Her2/Neu-induced breast cancer, we plan to use transgenic mice overexpressing ErbB2 with deleted LM04 gene in the mammary glands. In order to further study the mechanisms of action for LM04, we plan to lower LM04 levels in breast cancer cell lines by RNA-mediated interference (RNAi). We will study the effect of LM04 on the cell cycle and programmed cell death, and determine how LM04 regulates cancer-causing genes to affect these processes.

The long-term goal of our laboratory is to understand transcriptional control mechanisms that underlie normal development of epidermis, hair follicles and mammary glands, and to use this knowledge to gain insights into carcinogenesis in epithelial tissues. The project will investigate new biological mechanism of breast cancer, in which a small nuclear protein, LM04, might potentially take part in the well known breast cancer ErbB2 pathway.


Final Report (2008)
LMO4 is highly upregulated (increased) in breast cancer, especially estrogen receptor negative tumors, and its overexpression in mice leads to hyperplasia and tumor formation. LMO4 (LIM domain only 4) may play a role as a transcriptional regulator in embryogenesis or possibly act as an oncogene. Our goals were to investigate whether LMO4 plays roles in normal mammary gland development by regulating proliferation and apoptosis of mammary epithelial cells and whether LMO4 plays its role as a downstream target gene in ErbB2-induced breast cancer.

During the 3-yr funding period, we essentially completed most of the research project aims. We did not have time to complete role LMO4 in ErbB2 (Her-2) tumorigenesis, but we plan to continue this work. The major accomplishments of the research project are:
  1. Whole mount and histological analyses of Wap-Cre-LMO4fl/fl and MMTV-Cre-LMO4fl/fl mice mammary glands showed decreased lobuloalveolar structures.
  2. We have investigated proliferation and apoptosis of mammary epithelial cell in LMO4 knockout mice. We found that mammary epithelial cell proliferation was reduced and, that apoptosis of mammary epithelial cells was significantly increased in mid-pregnancy in LMO4 knockout mice
  3. We generated MCF7-LMO4-TetOff cells and identified LMO4 responsive genes, such as BMP7, GASS, and LIN7A, in MCF7-LMO4-TetOff cell clones and the MCF7-DN-Clim-TetOff cells by microarray
  4. We have further found that BMP7 and LMO4 are highly correlated in human breast cancers, and that BMP7 expression is regulated by LMO4 in breast cancer cells. These results strongly suggested BMP7 is a direct target of LMO4
  5. The reporter assay showed that LMO4 regulates the BMP7 promoter
  6. Chromatin immunoprecipitation (ChiP) studies showed that LMO4 and its co-factor Clim2 are recruited to the BMP7 promoter.
  7. We demonstrated that HDAC2 recruitment to the BMP7 promoter is inhibited by upregulation of LMO4 and that HDAC2 knockdown upregulates the promoter.

Future plans: We will continue to study the role of BMP7 (a member of the TGF-beta superfamily) in mammary gland development and breast cancer.


Symposium Abstract (2005)
Cancer cells are able to proliferate uncontrollably because of genetic alterations or abnormal regulation of genes that promote or inhibit cell proliferation. LM04 is potentially responsible for activating proliferation-promoting genes in breast cancer. It is a member of the family of LIM only proteins (LMOs); two other members of the LMO family, LMO1 and LM02, are oncogenes in leukemia. Normally, LM04 is expressed in breast epithelial cells and its expression is dramatically increased during mid-pregnancy. This genetic expression pattern is consistent with a role in the regulation of cell proliferation and therefore it is not surprising that interference with LM04 in mouse mammary glands leads to inhibition of mammary gland development. There is also strong evidence that LM04 may play a role in promoting breast cancer. LM04 was first found in breast cancer tissues and was referred to as Human Breast Tumor Autoantigen. Later studies found that LM04 is overexpressed in about 50% of invasive breast cancers. In recent experiments, it was found that LM04 expression level is greatly increased in ErbB2-induced breast cancer in mice. Moreover, when the LM04 activity was inhibited, cell proliferation was inhibited and programmed cell death was induced in breast cancer cells.

We are using state-of the art methods in mouse genetics to understand breast cancer. Our preliminary results showed that LM04 regulates several key genes implicated in tumorigenesis. . Specifically we propose that LM04 plays its role in breast cancer by functioning in the well known breast cancer oncogene ErbB2 pathway. To study the role of LM04 in mammary gland development and breast cancer, we will generate mice in which the LM04 gene is specifically deleted in the mammary gland. To link the role of LM04 in ErbB2/Her2/Neu-induced breast cancer, we plan to use transgenic mice overexpressing ErbB2 with deleted LM04 gene in the mammary glands. In order to further study the mechanisms of action for LM04, we plan to lower LM04 levels in breast cancer cell lines by RNA-mediated interference (RNAi). We will study the effect of LM04 on the cell cycle and programmed cell death, and determine how LM04 regulates cancer-causing genes to affect these processes.

The long-term goal of our laboratory is to understand transcriptional control mechanisms that underlie normal development of epidermis, hair follicles and mammary glands, and to use this knowledge to gain insights into carcinogenesis in epithelial tissues. The project will investigate new biological mechanism of breast cancer, in which a small nuclear protein, LM04, might potentially take part in the well known breast cancer ErbB2 pathway.


Symposium Abstract (2007)
The nuclear LIM-only protein LMO4 is a transcriptional regulator that becomes upregulated in breast cancer, especially estrogen receptor negative tumors, and its overexpression in mice leads to hyperplasia and tumor formation. Here, we show that deletion of LMO4 in the mammary glands of mice leads to impaired lobuloalveolar development due to decreased epithelial cell proliferation. With the goal of discovering potential LMO4-target genes, we also developed a conditional expression system in MCF-7 cells for both LMO4 and a dominant negative (DN) form of its co-regulator, Co-factor of LIM domains (Clim/Ldb/Nli). We then used DNA microarrays to identify genes responsive to LMO4 and DN-Clim upregulation. One of the genes common to both datasets was BMP7, whose expression is also significantly correlated with LMO4 transcript levels in a large dataset of human breast cancers, suggesting that BMP7 is a bona fide target gene of LMO4 in breast cancer. Inhibition of BMP7 partially blocks the effects of LMO4 on apoptosis, indicating that BMP7 mediates at least some functions of LMO4. Gene transfer studies show that LMO4 regulates the BMP7 promoter, and chromatin immunoprecipitation studies show that LMO4 and its co-factor Clim2 are recruited to the BMP7 promoter. Furthermore, we demonstrate that HDAC (histone deacetylase)-2 recruitment to the BMP7 promoter is inhibited by upregulation of LMO4 and that HDAC2 knockdown upregulates the promoter. These studies suggest a novel mechanism of action for LMO4: LMO4, Clim2 and HDAC2 are part of a transcriptional complex, and increased LMO4 levels can disrupt the complex, leading to decreased HDAC2 recruitment and increased promoter activity.

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.
Periodical:Oncogene
Index Medicus: Oncogene
Authors: Wang N, Lin KK, Lu Z, Lam KS, Newton R, Xu X, Yu Z, Gill GN, Andersen B
Yr: 2007 Vol: 26 Nbr: 44 Abs: Pg:6431-41