Our laboratory has had a long standing interest in identifying the molecules and understanding the mechanisms that regulate tumor growth. We have recently identified the presence of NGAL (Neutrophil Gelatinase- Associated Lipocalin) both alone, and complexed to the gelatinase MMP-9, in the urine of women with breast cancer. NGAL is a member of the lipocalin family of proteins, a group of small, secreted proteins that can transport and present ligands, bind to cell-surface receptors, and form macromolecular complexes. The presence of NGAL has been correlated with breast cancer development and progression and its potential as a biomarker of human breast cancer has been suggested. We have recently demonstrated that the overexpression of NGAL in human breast cancer cells induces an EMT (Epithelial to Mesenchymal Transformation), a hallmark of cancer progression. We have found that NGAL induces the down-regulation of epithelial markers such as E-cadherin, along with a concomitant upregulation of the mesenchymal markers vimentin and fibronectin as well as inducing the development or a scattering phenotype consistent with the loss of E-cadherin- mediated cell-cell adhesion. We have also determined that the expression level of the transcription factor Slug, known to induce EMT, is increased in NGAL-expressing cells. In addition, the expression of the estrogen receptor alpha (ERalpha) was significantly downregulated by NGAL over-expression. Finally, we have demonstrated that NGAL overexpression leads to significantly increased human breast cancer cell motility and invasivity. Taken together, these data demonstrate that NGAL can induce EMT and may regulate breast cancer aggressiveness. Within the context of the Specific Aims of our proposal, we will determine the mechanism(s) by which NGAL induces EMT in breast cancer cells. These mechanistic studies will be complemented by a series of in vivo ones in which we will determine the effects of NGAL on human breast cancer progression in vivo using three complementary in vivo models. These studies will also evaluate the potential role of NGAL as a therapeutic target in the treatment of breast cancer. In the third aim of this study, we will determine whether NGAL, alone or in combination with other cancer biomarkers, may be a diagnostic and/or prognostic biomarker for breast cancer. These studies are proposed within the context of the following Specific Aims: 1. To determine the mechanism(s) by which NGAL induces an epithelial to mesenchymal transition in breast cancer cells; 2. To determine whether NGAL can promote human breast cancer progression in vivo; 3. To determine whether the presence of NGAL, alone or multiplexed with other urinary cancer biomarkers, predicts tumor presence and therapeutic efficacy in animal models of breast cancer and in human patients with breast cancer. By systematically identifying novel molecules that may be playing a role in the development of aggressive breast cancer, and by dissecting the mechanisms by which such molecules may be exerting their effects, we will have the opportunity to develop new and improved therapeutic, diagnostic and prognostic strategies that could result in significantly improved breast cancer patient survival. ? ? ? ? ?
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