Darren Seals Lab
My lab uses cellular and molecular biology approaches to address the basic mechanisms of cell migration and invasion, particularly as it relates to tumor growth and metastasis. Our efforts are concentrated on the study of podosomes/invadopodia, cytoskeletal structures that both engage and remodel the extracellular matrix. Our study of the Src tyrosine kinase substrate and adaptor protein Tks5 serves as a conduit into defining podosome formation and function and the role of podosomes in cancer progression.
Podosomes/invadopodia are actin-based adhesion structures that protrude from the ventral cell surface into the underlying extracellular matrix. Though they share the same cytoskeletal regulatory machinery (integrins, tyrosine kinases, Arp2/3, WASp) as other adhesions, they are distinguished by marker proteins (cortactin, dynamin2, Tks5) and proteases (MMPs, ADAMs, serine proteases) that regulate their highly dynamic and proteolytic capabilities. Originally described in Src-transformed fibroblasts (Figure 1), podosomes are found in normal cells with an invasive character like osteoclasts, macrophages, dendritic cells, and the endothelial and mural cells of the vasculature. By convention, structures called invadopodia are present in an ever-growing list of cancer cell lines.
c-src was the first identified proto-oncogene. It encodes a nonreceptor tyrosine kinase that, when activated, transforms cells to a neoplastic phenotype. It has long been known that Src promotes adhesion-based cell motility and tissue invasion, processes that may be driven by the podosomes that are enriched in Src-activated cells. Tks5 is a substrate of Src, and is localized to podosomes of Src-transformed fibroblasts and the invadopodia of several cancer cell lines. Our past work indicates that Tks5 is a controlling factor for invadopodia development in cancer cells, augmenting or attenuating their protease-driven invasive behavior depending on its degree of expression. Silencing of Tks5 also limits tumor growth and angiogenesis in vivo. Tks5 is also a scaffolding protein with an amino terminal PX domain followed by five SH3 domains (Figure 2). The PX domain is a binding module for the phosphoinositides.
PtdIns(3,4)P2 and PtdIns(3,4,5)P3. Removal of the PX domain mislocalizes Tks5 in Src-transformed fibroblasts, and this construct fails to support invadopodia formation in T47D breast cancer cells. The SH3 domains support associations with Grb2, N-WASp, and ADAMs family metalloproteinases. Ongoing research suggests that the recruitment of these proteins by Tks5 helps drive focalized actin polymerization (in the case of N-WASp) and extracellular matrix degradation (in the case of ADAMs). This is consistent with the direct correlation between Tks5 expression and invasive behavior in transformed cells. Currently we are looking at the role of Src and Tks5 in invadopodia assembly and invasive function in prostate cancer cell lines.
Another avenue of research is in the role of Tks5 and podosome assembly in normal cell types, particularly those that may support tumor progression. Normal cell types within the tumor stroma (fibroblasts, leukocytes, vascular cells), by releasing growth factors and cytokines or by contributing nutrients and oxygen, help overcome rate-limiting processes like angiogenesis during tumor progression. Moreover, these cell types have invasive capabilities as well the ability to assemble podosomes. We are studying the invasive mural cell of the tumor vasculature as a means of addressing whether Tks5 and podosomes directly influences angiogenesis. We also are studying Tks5 and podosomes in the tumor-associated macrophage, as this cell type has been linked to the intravasation step of the metastatic cascade.
Our current efforts are directed towards the relative contributions of Src and Tks5 to podosome development among breast and prostate cancer cells and among normal cell types (macrophages, mural cells) of the tumor microenvironment. We consider their role in the assembly of scaffolds that permit podosome formation, proteolysis of matrix proteins, cell invasion, and tumor growth and metastasis.