Research Grants Awarded
Novel Regulators of EGFR Signaling in Breast Cancer
Tumor Cell Biology I
The epidermal growth factor receptor (EGFR) is frequently upregulated in breast cancer and is a drug target for treating cancers including breast cancer. The molecular mechanism for EGFR upregulation in breast cancer is not completely understood. Defects in receptor degradation have been suggested to play a role, in addition to gene amplification. Resistance to anti-EGFR therapies may be caused by redundant signaling pathways or constitutively active down-stream signaling components, as well as mutations in the EGFR that renders this receptor tyrosine kinase itself constitutively active. Prostasin is a glycosylphosphatidylinositol (GPI)-anchored extracellular serine protease, an epithelial differentiation marker which plays a role in maintaining epithelial integrity via regulating tight junction structure and function. Prostasin is expressed in normal breast epithelial cells, but down-regulated by promoter DNA methylation in invasive breast cancer cells. Re-expression of prostasin inhibited tumor invasion in vitro. We have new preliminary data to show activation of membrane prostasin by the transmembrane extracellular serine protease matriptase, and cleavage of the EGFR extracellular domain (ECD) as a result of the coordinated expression and actions of matriptase and prostasin. The novel EGFR ECD cleavage is predicted to abolish EGF-binding with an expected functional impact on EGFR signal modulation. We proposed three specific aims to test the hypothesis that a matriptase-prostasin extracellular proteolytic cascade is a key regulator of EGFR signaling in breast cancer. Specific Aim 1, Determination of the cleavage sites in the EGFR ECD for membrane serine proteases: we will determine the prostasin cleavage site of the EGFR molecule by site-directed mutagenesis and biochemical analysis. The novel cleavage of the EGFR ECD is expected to have two specific potential outcomes, a reduction of the cell surface receptor number, and changes of receptor function for both the truncated and the wild-type EGFR. Specific Aim 2, Characterization of the novel EGFR C-terminal fragments: we will determine the effect of cleavage on EGFR phosphorylation, ligand binding, and kinase activities using the CHO cell line as the host. Specific Aim 3, Determination of cell signaling impact of the novel EGFR ECD cleavage in vitro and in vivo: we will use a human breast cancer cell line with high EGFR expression but no prostasin or matriptase expression, MDA-MB-231; and another with prostasin as well as matriptase expression but low EGFR, MCF-7, as the experimental models. We will determine if the novel cleavage or the truncated EGFR molecule itself affects tumor cell morphology, and growth in vitro and in nude mice. The outcome of the project will positively impact the state-of-the-art in anti-EGFR therapies for breast cancer.
Each year in the US more than 200,000 new cases of invasive breast cancer and about 60,000 new cases of in situ breast cancer are diagnosed, and more than 40,000 women die of this disease (from XXX). Early detection and improved treatment have contributed to a 2.3% decline in breast cancer death rate from 1990 to 2001. Growth factors and growth factor receptors are essential molecules for normal tissue development in the body, but are often present at abnormal quantities and with abnormal activities in cancer. The epidermal growth factor receptor (EGFR) has been investigated for its role in cancer biology and for its applicability as a target for cancer treatment over the past two decades. Currently, inhibitors of the EGFR kinase and monoclonal antibodies against the EGFR protein are in clinical use to treat lung, head and neck, and colorectal cancers, and in trials for breast cancer. The understanding of exactly how EGFR is involved in cancer development and progression, however, still remains limited. Due to such limitations in the understanding of EGFR biology, it remains difficult to predict patient response toward anti-EGFR therapies, which also have significant side effects. Much effort in the state-of-the-art is focused on how to improve the ability to predict patient responses to anti-EGFR therapies, and to design new combination therapies more efficacious for treating cancer. A membrane serine protease, prostasin, abundantly expressed in normal breast epithelial cells but down-regulated in invasive breast cancer cells, was shown to be an invasion suppressor. We have new preliminary data to propose an extracellular proteolytic cleavage cascade model involving prostasin and another membrane serine protease, matriptase, to serve as a key regulator of EGFR signaling in breast cancer. Novel extracellular cleavage of the EGFR by this extracellular serine protease cascade will be investigated in this project for its impact on the EGFR molecule¡¯s cellular function, and on the breast cancer cells expressing EGFR, in tissue culture, and in animal xenograft model. The outcome of this study will enhance our knowledge on EGFR signal regulation in breast cancer and provide new strategies for EGFR-targeting to treat breast cancer.