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The Role of the Rho Exchange Factor NET1 in Breast Cancer
Tumor Cell Biology V
Background: RhoA is a small GTP binding protein that may contribute to the development of many cancers, including breast cancer. We have found that mRNA for the RhoA activating protein NET1 is overexpressed in 70 percent of human breast cancer tumors, and by immunohistochemical staining we have demonstrated that NET1 protein is overexpressed and mislocalized in human breast tumor tissue. We have also shown that NET1 transforms cells in vitro by constitutively activating RhoA and by interacting with regulatory proteins through a C-terminal PDZ binding domain . Furthermore, we have shown that this binding site mediates interaction with the tumor suppressor Dlg1. Hypothesis: Our central hypothesis is that overexpression of NET1 contributes to breast cancer by inhibiting the tumor suppressor activity of Dlg1 and promoting the constitutive activation of RhoA . Specific Aims: (1) Analyze NET1 expression levels and the DNA sequence of NET1 isoforms in primary human breast epithelial and breast tumor specimens. (2) Demonstrate the requirement for NET1 activity in the proliferation and transformation of immortal breast epithelial cells and breast cancer cells. (3) Define the interaction of NET1 with proteins that regulate its transforming activity in vitro and in breast cancer cells. Study Design: In Aim 1 we will analyze the expression of wt NET1 and NET1A mRNA and protein in human breast epithelial and breast cancer tissues by quantitative PCR and immunohistochemistry. We will also determine whether the DNA sequence of the wt NET1 and NET1A coding regions is mutated in the breast cancer specimens. In Aim 2 we will test the requirement for NET1 isoform expression in the proliferation and transformation of immortal breast epithelial and breast cancer cells. This will be achieved by overexpression of each protein, as well as by RNAi-mediated knockdown of the expression of endogenous NET1 proteins. In Aim 3 we will define the molecular basis for the interaction of NET1 with a protein complex containing the tumor suppressor Dlg1 in vitro and in breast epithelial cells. Potential Benefits of Research: Completion of these Aims will identify NET1 as a potential biomarker for breast cancer, demonstrate its role in breast epithelial cell proliferation and transformation, and define the basis for interaction between NET1 and regulatory proteins such as the tumor suppressor Dlg1.
Cancer is a genetic disease caused by the permanent activation of genes that promote cell proliferation, as well as the inactivation of genes that normally inhibit this process. Examples in breast cancer are the overexpression of the growth factor receptor gene HER2 and the inactivation of the tumor suppressors BRCA1 and BRCA2. Unfortunately, these genetic lesions account for only a fraction of all breast cancers, so it is imperative to identify other changes associated with the development of this disease. Recently we have found that an enzyme called NET1 is overexpressed in breast tumors as opposed to the surrounding normal tissue. We have shown previously that expression of this gene product causes normal cells to become cancerous in laboratory cultures. Our data shows that the cancer causing activity of NET1 in vitro is due to the constitutive activation of its enzymatic target RhoA, as well as an interaction with the tumor suppressor protein Dlg1. In this study we will test the hypothesis that NET1 overexpression contributes to breast cancer by inhibiting the activity of the growth suppressor protein Dlg1, as well as by continuously activating RhoA. Thus, in Aim 1 we will verify that overexpression of NET1 is a reliable biomarker of breast cancer by contrasting its expression levels in normal and cancerous human breast tissue. We will also sequence the DNA of the NET1 genes in the cancer tissue to determine whether they have been mutated in the cancer cells. In Aim 2 we will determine whether NET1 expression levels affect the proliferation or cancer phenotypes of common model breast cell lines used in the lab. In Aim 3 we will define the molecular basis for the interaction of NET1 with the tumor suppressor protein Dlg1, with the goal of understanding how NET1 may inactivate its growth suppressor properties. Completion of these Aims will identify NET1 as a novel biomarker for breast cancer, define the role of NET1 in the proliferation and transformation of breast epithelial and cancer cell lines commonly used in the lab, and single out new potential targets for therapeutic intervention in this disease.