p53 Suppresses Src-Induced Podosome and Rosette Formation and Cellular Invasiveness through the Upregulation of Caldesmon

Abstract

The tumor-suppressive role of p53 at the level of tumor initiation is well documented. It has also been shown previously that p53 acts against tumor progression/metastasis. However, its role in modulating cell migration and invasion leading to metastasis is poorly understood. In this study, using vascular smooth muscle cells and NIH 3T3 fibroblast cells, we have shown that p53 potently suppresses Src-induced podosome/rosette formation, extracellular matrix digestion, cell migration, and invasion. The overexpression of exogenous wild-type p53 or the activation of the endogenous p53 function suppresses, while the short hairpin RNA-mediated knockdown of p53 expression or the blocking of its function exacerbates, Src-induced migratory and invasive phenotypes. We have also found that p53 expression and function are downregulated in cells stably transformed with constitutively active Src that exhibit aggressive invasive properties. Lastly, p53 upregulates the expression of caldesmon, an actin-binding protein that has been shown to be an inhibitor of podosome/invadopodium formation. The ability of p53 to suppress Src phenotypes in transformed cells was largely abolished by knocking down caldesmon. This study reports a novel molecular mechanism (caldesmon), as well as a structural basis (podosomes/rosettes), to show how p53 can act as an anti-motility/invasion/metastasis agent.

Sox4-mediated caldesmon expression facilitates skeletal myoblast differentiation

“…There is evidence that h-CaD expression enhances stress fiber stability and reduces cell motility in non-muscle cells (Castellino et al, 1992;Castellino et al, 1995;Eppinga et al, 2006;Li et al, 2004;Mayanagi et al, 2008;Mirzapoiazova et al, 2005). Moreover, CaD expression induced by p53 suppresses Src-kinase-dependent podosome formation, which inhibits fibroblast and rat aortic SMC migration (Mukhopadhyay et al, 2009). By contrast, HeLa-l-CaD expression in endothelial cells and endothelial progenitor cells in the vasculature of various human tumors promotes cell migration for vasculogenesis and angiogenesis during tumor development (Zheng et al, 2004;Zheng et al, 2007).…”

Src-mediated phosphorylation of mammalian Abp1 (DBNL) regulates podosome rosette formation in transformed fibroblasts

“…Formation of podosomes is generally correlated with invasive migration (Mukhopadhyay et al, 2009;Linder, 2007;Seals et al, 2005). However, the relationship between podosome rosettes and invasion has not been defined in Src-transformed cells.…”

“…Both podosome dots and rosettes are capable of degrading the ECM (Mukhopadhyay et al, 2009;Linder, 2007;Seals et al, 2005). To determine whether mAbp1-deficient cells retain their capacity to degrade the ECM, we plated cells on coverslips coated with a mixed substrate of fibronectin and Alexa-Fluor-568-conjugated gelatin.…”

Automated Podosome Identification and Characterization in Fluorescence Microscopy Images

“…With lifetimes between 2-12 min, podosomes are very dynamic and continuously change size, shape, and composition~Destaing et al, 2003;Evans et al, 2003!. They have initially been identified in Src-transformed fibroblasts, and although previously thought to be restricted to only a few cell types, they have now been observed in smooth muscle cells, activated endothelial cells, and cells of the myeloid lineage such as osteoclasts, macrophages, and dendritic cells DCs!~Murphy & Courtneidge, 2011!. More recently, podosome-associated structures named invadopodia have been found on the ventral membranes of tumor cells where they are thought to play a central role in tumor metastasis Blouw et al, 2008;Mukhopadhyay et al, 2009!. Most of our current understanding of podosome formation, turnover, and distribution is based on studies that exploit conventional and confocal microscopy techniques. Although these techniques are potentially quantitative, largescale image analysis is required to obtain enough statistical power to be able to detect subtle and transient variations in podosome composition.…”