A major research goal of this project is to understand the role of the extracellular matrix in tumor invasion and metastasis, focusing currently on identifying integrin-mediated signaling pathways that regulate tumor invadopodia assembly, their function in proteolytic degradation, and tumor cell invasion. A fundamental question in cancer biology is the relationship of the local matrix environment to cancer progression. For example, a common feature of advanced carcinomas is the induction of a dense collagenous matrix surrounding tumors during the process of desmoplasia. However, whether this increase in collagen is protective against tumor expansion or actually contributes to cancer pathogenesis was not well characterized. Micro-invasive structures termed invadopodia can mediate local proteolytic degradation of matrix to permit tumor cell invasion. We hypothesized that the dense accumulations of extracellular matrix found in advanced human tumors during the desmosplastic response could trigger formation of invadopodia. We generated thin, cell-free 3D sections of human tumor tissue extracellular matrix for comparisons with adjacent normal tissue matrix. Desmoplastic matrix induced large numbers of invadopodia. We discovered that this induction could be mimicked by a novel high-density fibrillar collagen (HDFC) matrix, which is a potent inducer of invadopodia even in the absence of serum factors. In carcinoma cells, HDFC matrix induces invadopodia via a specific integrin signaling pathway independent of growth factors or even any alterations in mRNA or protein expression. Instead, phospho-proteomics analyses identified major changes in a complex phosphosignaling network initiated by the collagen receptor integrin α2β1, which was both necessary and sufficient for potent induction of invadopodia. Immediately downstream of this integrin, kindlin2 serine phosphorylation proved to be a novel regulatory element according to phospho-null and -mimetic analyses. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. Surprisingly, HDFC was equally effective for induction of matrix-degrading invadopodia in non-transformed primary human dermal fibroblasts in the absence of serum. Contrary to findings in three tumor cell lines, this invadopodial induction in non-malignant fibroblasts was suppressed completely by serum factors, identifying a distinct difference in the regulation of invadopodia induction. Thus, a novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the matrix. We are currently evaluating how changes in matrix rigidity alone can affect tumor cell migration compared to non-malignant cells. Interactions at the cell surface are likely to play important roles in many diseases. We have been involved in a long-term collaboration with Dr. Subhash Dhawan in CBER, FDA to characterize cell-surface and extracellular interactions involved in the pathogenesis of infectious diseases. Some molecules are found to enhance infectivity, while others can suppress this process. For example, HIV-1 infectivity for human macrophages was found to be markedly increased by co-exposure to the PrP106-126a peptide mimetic of pathogenic prion protein PrP(sc), which enhanced cytokine secretion and resulted in macrophage clustering indicative of cellular activation. These findings underscore the significance of pathogenic crosstalk of host cellular responses to extracellular stimuli. In contrast, the host cellular response to extracellularly provided hemin, an inducer of heme oxygenase-1 (HO-1), involves host protective responses against viral infections including HIV and poxvirus. Human macrophages infected by vaccinia virus show selective up-regulation of a variety of small nuclear RNAs (snRNAs). Treatment with hemin induced expression of HO-1, completely reversed virus-induced snRNA induction, and suppressed vaccinia virus infection. HIV-1 infection of macrophages induces cell fusion in a process that closely resembles osteoclastogenesis. Hemin treatment also suppresses HIV-induced osteoclastogenesis. It also inhibits classical osteoclastogenesis, suggesting promise for possible application in ameliorating osteopenia. We suggest that HO-1 induction via hemin, an FDA approved drug, might provide a potential host-defense therapeutic strategy against a variety of pathogens.
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