Notch as a Diagnostic Marker and Therapeutic Target in Human Breast Cancer

PUBLIC ABSTRACT

In the United States, breast cancer is the second most common cancer and contributes to 40,000 deaths a year. Early detection is key to improving survival as more than 90% of women will live longer than 10 years after the diagnosis if tumors are 10 millimeters or smaller in size. Current diagnostic tools include mammography and biopsy, but the former technique is nonspecific (cannot distinguish between malignant and nonmalignant tumors) and is not infallible, as a certain proportion (11%) of tumors are not detected. Thus, all women would benefit by the development of a more specific and sensitive method of screening. Fortunately, progress has been made in our understanding of the biochemical and molecular mechanisms of breast cancer that permit us to better delineate cellular and local microenvironment changes that occur during the initial phase of tumor growth. Unfortunately, technologies capable of exploiting this knowledge for the early detection and treatment of this devastating disease have been poor at best. The goal of this proposal is to develop enhanced diagnostic methods and to explore a potential novel therapy for the treatment of breast cancer by exploiting the molecular signatures that best define this disease state.

To accomplish these goals, we will utilize molecular markers that help identify patients with breast cancer in the early stages of their disease and/or a cancer that needs aggressive treatment. Our focus is on the cell surface proteins Notch and its ligands, which are highly expressed in human breast cancer and the vasculature that supplies it. Moreover, this cell signaling system is known to promote breast cancer growth and as well as the formation of tumor blood vessels. We will focus specifically on the Notch receptor, Notch1, and the Notch ligands known as Jagged-1 and Delta-4 (Delta-like 4). There are several compelling reasons to develop diagnostic tools to detect this class of cell surface proteins. First, Delta-4 is overexpressed on the vasculature supplying human breast cancer in contrast to nontumor vessels, thus making it an excellent candidate marker for diagnosis by imaging. Second, Jagged-1 and Notch1 are highly produced in breast cancer patients with poor prognosis. In fact, preliminary data suggests that breast cancer undergoing metastasis expresses high levels of the activated form of Notch1. Thus, it would of great benefit to be able to detect elevated expression of these proteins in such individuals, as they may need a more aggressive course of therapy. Moreover, preliminary in vivo observations also suggest that blockade of the Notch signaling pathway by use of a decoy receptor significantly retards tumor growth.

The first aims of this proposal are to develop update diagnostic strategies for assessing serum levels as well as tissue and tumor vessel expression of Notch and its ligands. We propose to generate antibodies that specifically recognize these proteins to ultimately develop an ELISA-based detection system for Jagged-1 and Notch1 in blood that can be used in clinics. In addition, such antibodies will be used to target nanoparticles to tumor vessels that can be detected by commercially available imaging modalities such as ultrasound and MRI. Xenografts of human breast cancer cell lines that overexpress Jagged-1 will be grown in mice. Serum will be collected and analyzed for the extracellular domains of Jagged-1 that may be shed into the blood stream from breast tumors. We will also incorporate antibodies directed against Notch ligands, such as Delta-4, into a nanoparticle-based detection system and evaluate their ability to preferentially localize on blood vessels supplying breast cancer tumors using state-of-the-art imaging technology, confocal intravital microscopy. This imaging system permits direct observation of nanoparticle localization on the vessel wall in real time in a living animal. Information gleaned from such experiments include the (1) selective nanoparticle binding to Delta-like 4, and (2) the rate of accumulation and duration of binding, events that are critical for determining optimal viewing time and signal strength for future clinical imaging modalities. These combined studies may lead to a series of noninvasive assays that can be applied in the clinic in a timely fashion. US Food and Drug Administration approval is currently being sought for the in vivo use of the nanoparticle imaging system and alliances with industry have already been formed, which will also expedite clinical development.

Ultimately, the successful treatment of breast cancer will depend on a pipeline of novel and safe inhibitors of tumor growth and angiogenesis. This proposal will also explore novel therapeutics that target the Notch signaling pathway. To date, no published work has described specific inhibitors of ligand-dependent Notch. We will make neutralizing antibodies to its ligands, Jagged-1 and Delta-4, and compare their utility and efficacy to an inhibitor we have already developed, Notch1 decoy, that shows promise in retarding tumor growth in vivo. Exploring multiple inhibitory paradigms (decoys versus neutralizing antibodies) is merited if one is to define the most efficacious and least toxic reagent to pursue in the clinic. Thus, we present a update, and hopefully powerful, series of approaches to address the need for novel Notch inhibitors for the treatment of breast cancer.