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Interfering Breast Cancer Metastasis By Blocking Ngal Function
Investigator Initiated Research
Breast cancer is the most common cancer in women worldwide and accounts for one-sixth of cancer deaths in the United States. Although the majority of early stage breast cancer can be cured by surgical resection, most of the cancer deaths are caused by advanced disease and distal metastasis. Therefore, blocking the tumor metastasis is the key to reduce breast cancer mortality. By expression profiling studies of 133 primary breast cancers, we identified that NGAL (Neutrophil Gelatinase Associated Lipocalin) is associated with a high stage of breast cancer. In addition, we found that NGAL is also associated with two aggressive forms of breast cancers, those having the so called triple negative basal type and HER2/neu over-expressing breast cancers. Both types are typically associated with poor clinical outcome. Consistent with its name (NGAL), NGAL is reported to stabilize MMP9, which we have confirmed in our mouse studies. To further confirm the role of NGAL in breast cancer metastasis, we first used the mouse breast cancer 4T1 cells as a model system. 4T1 mouse breast cancer cell line is an extremely aggressive tumor cell line which causes extensive tumor metastasis and early mouse death. The 4T1 cell line expressed and secreted a very high level of 24p3 protein (mouse counterpart of human NGAL protein). 4T1 cells and 24p3 protein are detected in the blood plasma of tumor-bearing mice. Knocking down 24p3 expression in the 4T1 cell line reduced tumor metastasis. Treating tumor-bearing mice with neutralizing antibody against 24p3 also blocked the tumor metastasis to lung. These preliminary mouse data support the role of 24p3/NGAL in breast cancer metastasis in mouse model. The current proposed studies are to further investigate the role of NGAL in human breast cancer metastasis (Aim 1), and to use a genetic approach to study the function of NGAL/24p3 in breast tumor development and progression/metastasis in a mouse model (Aim2), and to generate a neutralizing monoclonal antibody against NGAL to inhibit human breast cancer metastasis (Aim 3). STUDY DESIGN. Aim 1: we will investigate the role of NGAL in human breast cancer metastasis in mouse xenograft model. Several cell lines of low metastatic potential with low NGAL secretion and high metastatic potential with high NGAL secretion will be used in these experiments. We will knockdown (loss-of-function) NGAL in high metastatic cell lines, and over-express (gain-of-function) NGAL in low metastatic cell lines to investigate the role of NGAL in breast cancer metastasis by establishing mouse xenograft model. Aim 2: We will assess the role of 24p3 in breast cancer metastasis using a HER2tg/24p3-/-mouse model. We will generate two HER2tg/24p3-/- mice models. One is to cross HER2 (wild type) transgenic mice with 24p3 null mice to generate HER2 (wild type)tg/24p3-/- mice. The other is to cross activated HER2 (V664E) transgenic mice with 24p3 null mice to generate HER2 (V664E)tg/24p3-/- mice. Using these mice, we will be able to evaluate whether loss of NGAL/24p3 function will affect breast cancer formation and metastasis in (a) HER2 (wild type)tg background, a condition closely mimicking the human HER2 positive breast cancer development; and (b) HER2 (V664E)tg ,a much more aggressive form of breast cancer. Aim 3: we will generate monoclonal antibody against human NGAL to test whether anti-human NGAL antibody will reduce human breast cancer cell metastases, as seen in our antibody experiments using affinity-purified polyclonal antibody made against mouse 24p3 to reduce tumor metastases caused by mouse 4T1 breast cancer cells. POTENTIAL OUTCOMES AND BENEFITS OF THE RESEARCH. Our preliminary studies in human primary breast cancers have demonstrated a correlation between high NGAL expression and high tumor stage, and aggressive tumor types with poor clinical outcome. Our preliminary studies in a mouse breast cancer model have further confirmed the role of NGAL in tumor metastasis. We believe the proposed studies will likely validate the role of NGAL in human breast cancer metastasis. The neutralizing antibody against NGAL will likely to inhibit metastasis of human breast cancer cells. If the proposed studies are successfully completed, a humanized neutralizing monoclonal antibody against NGAL will be developed as a therapeutic agent to reduce tumor metastasis in human breast cancer. Patients with aggressive breast cancers, such as triple negative basal type and HER2/neu over-expressing breast cancers, who account for up to 50% of total breast cancer patients and have high chance of distal metastasis and high mortality, should benefit most from this treatment modality.
Breast cancer is curable if it is in an early stage. The mortality is high if the disease progresses to an advanced stage with metastasis. The poor clinical outcome is also associated with particular aggressive types of breast cancers, i.e. the HER2/neu over-expressing and basal type (HER2-, ER-) of breast cancers. Although Herceptin (Trastuzumab) prolongs the survival of Her2/neu positive breast cancer patients, it is only effective in up to 50% of the patients. Most of these Her2 positive patients eventually die of metastatic disease. In terms of Her2- and ER- basal type of breast cancers, there is currently no specific therapy against them. Therefore, there is a need to develop a therapeutic strategy to fight these aggressive forms of breast cancers, and to block these tumors from developing distal metastasis. Our preliminary studies have indicated the association of NGAL/24p3 expression and secretion with aggressive phenotype of human breast cancer and aggressive forms of breast cancer, specifically the HER2 positive and basal types. The elevated expression of NGAL/24p3 increased tumor cell migration and invasion in vitro. Our mouse experiments further support the role of NGAL/24p3 in tumor metastasis. Neutralizing antibody against 24p3 strongly inhibited lung metastasis in mouse breast cancer experiments, indicating the potential of neutralizing antibody in treating metastatic breast cancer. Therefore, we hypothesize that NGAL expression and secretion by human breast cancer cells will facilitate tumor metastasis; and that neutralizing antibody against human NGAL will strongly inhibit breast cancer metastasis. The objectives of this study is to validate the role of NGAL in human breast cancer metastasis, and to generate a monoclonal antibody against NGAL to reduce human breast cancer metastasis. Our long term goal is to generate a humanized inhibitory monoclonal antibody against NGAL to use in the therapy (e.g. i.v. delivery) of breast cancer in combination with other treatment regimens.