Alterations in the tumor stroma contribute significantly to the malignant characteristics of human cancers. Still, little is known about the specific signaling pathways utilized in the tumor stroma to support malignancy. In this grant we will focus on a new biological paradigm i.e. the role of the nuclear receptor coactivator AIB1 (amplified in breast cancer 1 or NCoA3 or SRC3) in the control of stromal function during mammary tumorigenesis. We will also evaluate a more potent isoform of AIB1 that lacks the N-terminal domain due to alternative slicing of exon 3 (AIB1-?3). AIB1 is an oncogene that is amplified and/or overexpressed in many human epithelial tumors, including breast cancer. Recent data suggest a new role for AIB1 in the control of stromal function: We have reported that MMTV-HER2 induced mammary tumorigenesis and tumor angiogenesis are reduced in AIB1 knockout mice. Our recent studies in AIB1 knockout mice reveal poor healing of full-thickness skin wounds, defective neo-angiogenesis of wounds, macrophage invasion and extracellular matrix remodeling that was also observed in subcutaneous Matrigel plug colonization assays. In addition, depletion of AIB1 impacts on endothelial monolayer formation in vitro. These data suggest that AIB1 plays a significant role in tumor stromal response and damage repair of normal tissues. In this proposal we will examine the hypothesis that AIB1 plays a significant role in controlling stromal function during mammary tumorigenesis. We propose that epithelial/stromal crosstalk are impacted by AIB1 effects on signaling in the tumor epithelial as well as the tumor stromal cells. We also propose that the ?3 splice isoform of AIB1 plays a distinct role in epithelial control of stromal function because it escapes an intracellular AIB1 repression mechanism.
The Specific Aims of the proposal are:
Aim 1 : To define the role of AIB1 and ?3 in mammary tumor progression and epithelial/stromal cross talk in vivo. We will use mammary transplantation models to determine if continuous expression of AIB1 or ?3 in the A) epithelium or B) stroma is required for maintenance of mammary tumorigenesis and the tumor stromal response that includes recruitment of bone marrow derived cells. This will extend to studies of signaling pathways utilized.
Aim 2 : To analyze the effects of AIB1 and ?3 on stromal cell phenotype and identify critical AIB1-dependent signaling pathways in stromal/epithelial crosstalk in cultured cells in vitro. We will establish the conditions in which endothelial cells co cultured with epithelium, fibroblasts and monocytes, are influenced by changes in AIB1 expression. We will identify the AIB1-controlled signaling pathways activated in endothelial cells by by biochemical and proteomics approaches as well as gene expression pattern analysis.
Aim 3 : To examine the mechanism of increased potency of the ?3 splice isoform of AIB1 on cell signaling and transcriptional activation. We will Identify and characterize the repressor(s) interacting with the N-terminal domain of AIB1 using high sensitivity pull down approaches followed by identification with proteomics. We will undertake a series of functional assays to validate that the identified protein(s) has coactivator repressor function and determine its impact on mammary tumorigenesis. We will use data mining of human breast cancer array sets to determine if there are changes in the levels of the repressor associated with tumorigenesis in human breast epithelium and stroma.
Understanding the role tumor stroma in the development of cancer is critical for detection, prevention and therapeutic stratagies. AIB1 is a protein highly expressed in human breast cancer that predicts poor prognosis and reduced survival. We have recently discovered that AIB1 is required for stromal function including the healing of wounds as well as in tumor angiogenesis. In addition, we observed that AIB1 is overexpressed in the stroma of clinical breast cancers relative to stromal tissues in normal breast. In this grant we aim to uncover the contribution of AIB1 and mechanisms that control the cross talk between cancer epithelia and stroma including the recruitment of bone marrow derived cells from the circulation. Identification of novel pathways can be used to understand drivers of malignant progression and design novel therapeutic strategies.
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