Abstract
Dynamic reciprocal interactions between a tumor and its microenvironment impact both the establishment and progression of metastases. These interactions are mediated, in part, through proteolytic sculpting of the microenvironment, particularly by the matrix metalloproteinases, with both tumors and stroma contributing to the proteolytic milieu. Because bone is one of the predominant sites of breast cancer metastases, we used a co-culture system in which a subpopulation of the highly invasive human breast cancer cell line MDA-MB-231, with increased propensity to metastasize to bone, was overlaid onto a monolayer of differentiated osteoblast MC3T3-E1 cells in a mineralized osteoid matrix. CLIP-CHIP® microarrays identified changes in the complete protease and inhibitor expression profile of the breast cancer and osteoblast cells that were induced upon co-culture. A large increase in osteoblast-derived MMP-13 mRNA and protein was observed. Affymetrix analysis and validation showed induction of MMP-13 was initiated by soluble factors produced by the breast tumor cells, including oncostatin M and the acute response apolipoprotein SAA3. Significant changes in the osteoblast secretomes upon addition of MMP-13 were identified by degradomics from which six novel MMP-13 substrates with the potential to functionally impact breast cancer metastasis to bone were identified and validated. These included inactivation of the chemokines CCL2 and CCL7, activation of platelet-derived growth factor-C, and cleavage of SAA3, osteoprotegerin, CutA, and antithrombin III. Hence, the influence of breast cancer metastases on the bone microenvironment that is executed via the induction of osteoblast MMP-13 with the potential to enhance metastases growth by generating a microenvironmental amplifying feedback loop is revealed.
Highlights
Dynamic reciprocal interactions between a tumor and its microenvironment impact both the establishment and progression of metastases
This ensured that the two cell types were physically separated from one another such that we could identify the effects exerted by soluble paracrine factors produced by both the osteoblasts and tumor cells
We have demonstrated the influence that breast cancer cells can exert on stroma to alter the proteolytic potential within the tumor microenvironment
Summary
Dynamic reciprocal interactions between a tumor and its microenvironment impact both the establishment and progression of metastases. The combined overexpression of three of these molecules (osteopontin, IL11, and CXCR4 or connective tissue growth factor or MMP-1) in the parental MDA-231 was required to achieve the same level of bone metastasis, indicating that multiple interactions are involved in promoting bone metastasis These approaches only consider the contribution of the tumor cells and not the cells of the bone microenvironment. The RANK/RANKL pathway is a focus of considerable research as a potential therapeutic target (5, 6), it is clear that other molecules and pathways are involved in promoting the establishment of bone metastasis, and identifying these pathways may lead to alternative therapeutic targets Such pathways are in part mediated or regulated through the expression of proteases and proteolytic modulation of the microenvironment, with both tumors and stroma contributing to the proteolytic milieu. Global approaches are needed, as individual proteases do not act alone but function as part of a network, the “protease web” (7), and are regulated by a myriad of other proteins such as activators, inhibitors, co-factors, receptors, substrates, and cleavage products
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