Bone Marrow Stroma Influences Transforming Growth Factor-β Production in Breast Cancer Cells to Regulate c-myc Activation of the Preprotachykinin-I Gene in Breast Cancer Cells

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Abstract
Breast cancer cells (BCCs) have preference for the bone marrow (BM). This study used an in vitro coculture of BCCs and BM stroma to represent a model of early breast cancer metastasis to the BM. The overarching hypothesis states that once BCCs are in the BM, microenvironmental factors induce changes in the expression of genes for cytokines and preprotachykinin-I (PPT-I) in both BCCs and stromal cells. Consequently, the expression of both PPT-I and cytokines are altered to facilitate BCC integration within BM stroma. Cytokine and transcription factor arrays strongly suggested that transforming growth factor-β (TGF-β) and c-myc regulate the expression of PPT-I so as to facilitate BCC integration among stroma. Northern analyses and TGF-β bioassays showed that stromal cells and BCCs influence the level of PPT-I and TGF-β in each other. In cocultures, PPT-I and TGF-β expressions were significantly (P < 0.05) increased and decreased, respectively. TGF-β and PPT-I were undetectable in separate stromal cultures but were expressed as cocultures. Two consensus sequences for c-myc in the 5′ flanking region of the PPT-I gene were shown to be functional using gel shift and reporter gene assays. Mutagenesis of c-myc sites, neutralization studies with anti-TGF-β, and transient tranfections all showed that c-myc is required for TGF-β-mediated induction of PPT-I in BCCs. TGF-β was less efficient as a mediator of BCC integration within stroma for c-myc-BCCs. Because the model used in this study represents BCC integration within BM stroma, these studies suggest that TGF-β is important to the regulation of PPT-I in the early events of bone invasion by BCCs.