): The long-term goal of this proposal is to elucidate the mechanisms of hematopoietic disruption caused by breast cancer (BC) metastasis to the bone marrow (BM), their preferred homing site. Despite """"""""successful"""""""" treatments, BM involvement is common. BC metastasis to the BM is associated with poor prognosis. We arbitrarily assign BC metastasis as two stages, early and late. Early being at a time long before the primary tumor is detectable and late being the time when the cancer cells disrupt the BM. This study will focus on early metastasis since understanding early interactions among BC and BM cells will allow preventive and/or novel therapeutic targets. We hypothesize that preprotachykinin-I (PPT-I) and the genes for its receptors, neurokinin (NK)-1 and NK-2, over expressed in BC cells, are important mediators in the early integration of BC cells within BM stromal cells. Preliminary studies from our lab support this hypothesis.
Four specific aims will test this hypothesis. We will use an in vitro co-culture model with BC and BM stromal cells to study their interactions. For this proposed period, we will use twelve defined malignant cell lines and four normal mammary epithelial cell lines. Preliminary studies show that the latter do not integrate and survive among BM stroma.
Aim I will expand on preliminary studies to understand early integration between BC and BM stromal cells using the in vitro co-cultures. We will determine the type of BC-stromal interactions (direct vs. indirect) and determine the relationship between expression of PPT-I, NK-1, and NK-2, and co-culture confluence. Preliminary studies indicate changes in specific gone expression in both the BC and BM stromal cells during co-culture. Furthermore, although the preliminary studies show that PPT-I are involved in the integration of BC cells to the BM, the evidence indicate that it interacts with other factors. To this end, aim 2 will take advantage of vast numbers of commercial DNA arrays to analyze differences in various categories of genes in BC and stromal cells during different levels of confluence.
Aim 3 will study the role of PPT-I in more detail. In the first set of experiments, we will transiently knockout PPT-I with antisense to beta-PPT-I and then study they ability integrate within BM stromal cells. Since multiple genes are dysregulated in cancer cells, the second set of experiments will use a more defined model to study the role of PPT-I. We will use normal mammary epithelial, stably transfected with PPT-I and/or NK-1 expression vectors. Initial experiments will be qualitative with the genes under the control of relatively strong (RSV) and weak (CMV) promoters. The results of these experiments will direct studies with PPT-I quantitatively expressed with the Tet-off system. Since BC metastasis involves two major cell subsets, mesenchymal (stroma) and epithelial (BC), aim 4 will determine if regulation of PPT-I, NK-1 and NK-2 is tissue specific by studying their promoters in these cell subsets.
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