Alan S. Mak

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.

Caldesmon is an integral component of podosomes in smooth muscle cells

Podosomes are highly dynamic actin-based structures commonly found in motile and invasive cells such as macrophages, osteoclasts and vascular smooth muscle cells. Here, we have investigated the role of caldesmon, an actin-binding protein, in the formation of podosomes in aortic smooth muscle A7r5 cells induced by the phorbol ester PDBu. We found that endogenous low molecular weight caldesmon (l-caldesmon), which was normally localised to actin-stress fibres and membrane ruffles, was recruited to the actin cores of PDBu-induced podosomes. Overexpression of l-caldesmon in A7r5 cells caused dissociation of actin-stress fibres and disruption of focal adhesion complexes, and significantly reduced the ability of PDBu to induce podosome formation. By contrast, siRNA interference of caldesmon expression enhanced PDBu-induced formation of podosomes. The N-terminal fragment of l-caldesmon, CaD40, which contains the myosin-binding site, did not label stress fibres and was not translocated to PDBu-induced podosomes. Cad39, the C-terminal fragment housing the binding sites for actin, tropomyosin and calmodulin, was localised to stress fibres and was translocated to podosomes induced by PDBu. The caldesmon mutant, CadCamAB, which does not interact with Ca2+/calmodulin, was not recruited to PDBu-induced podosomes. These results show that (1) l-caldesmon is an integral part of the actin-rich core of the podosome; (2) overexpression of l-caldesmon suppresses podosome formation, whereas siRNA knock-down of l-caldesmon facilitates its formation; and (3) the actin-binding and calmodulin-binding sites on l-caldesmon are essential for the translocation of l-caldesmon to the podosomes. In summary, this data suggests that caldesmon may play a role in the regulation of the dynamics of podosome assembly and that Ca2+/calmodulin may be part of a regulatory mechanism in podosome formation.

The Protein Kinase C Cascade Regulates Recruitment of Matrix Metalloprotease 9 to Podosomes and Its Release and Activation

ABSTRACT Podosomes are transient cell surface structures essential for degradation of extracellular matrix during cell invasion. Protein kinase C (PKC) is involved in the regulation of podosome formation; however, the roles of individual PKC isoforms in podosome formation and proteolytic function are largely unknown. Recently, we reported that PDBu, a PKC activator, induced podosome formation in normal human bronchial epithelial cells. Here, we demonstrate that phorbol-12,13-dibutyrate (PDBu)-induced podosome formation is mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes. Interestingly, although blocking atypical PKCζ did not affect PDBu-induced podosome formation, it significantly reduced matrix degradation at podosomes. Inhibition of PKCζ reduced recruitment of matrix metalloprotease 9 (MMP-9) to podosomes and its release and activation. Downregulation of MMP-9 by small interfering RNA (siRNA) or neutralization antibody also significantly reduced matrix degradation. The regulatory effects of PKCζ on matrix degradation and recruitment of MMP-9 to podosomes were PKCζ kinase activity dependent. PDBu-induced recruitment of PKCζ and MMP-9 to podosomes was blocked by inhibition of novel PKC with rottlerin or PKCδ siRNA. Our data suggest that multiple PKC isozymes form a signaling cascade that controls podosome formation and dynamics and MMP-9 recruitment, release, and activation in a coordinated fashion.

Cdc42-interacting protein 4 is a Src substrate that regulates invadopodia and invasiveness of breast tumors by promoting MT1-MMP endocytosis.

Invadopodia are actin-rich membrane protrusions that promote extracellular matrix degradation and invasiveness of tumor cells. Src protein-tyrosine kinase is a potent inducer of invadopodia and tumor metastases. Cdc42-interacting protein 4 (CIP4) adaptor protein interacts with actin regulatory proteins and regulates endocytosis. Here, we show that CIP4 is a Src substrate that localizes to invadopodia in MDA-MB-231 breast tumor cells expressing activated Src (MDA-SrcYF). To probe the function of CIP4 in invadopodia, we established stable CIP4 knockdown in MDA-SrcYF cell lines by RNA interference. Compared with control cells, CIP4 knockdown cells degrade more extracellular matrix (ECM), have increased numbers of mature invadopodia and are more invasive through matrigel. Similar results are observed with knockdown of CIP4 in EGF-treated MDA-MB-231 cells. This inhibitory role of CIP4 is explained by our finding that CIP4 limits surface expression of transmembrane type I matrix metalloprotease (MT1-MMP), by promoting MT1-MMP internalization. Ectopic expression of CIP4 reduces ECM digestion by MDA-SrcYF cells, and this activity is enhanced by mutation of the major Src phosphorylation site in CIP4 (Y471). Overall, our results identify CIP4 as a suppressor of Src-induced invadopodia and invasion in breast tumor cells by promoting endocytosis of MT1-MMP.

Doubles Game: Src-Stat3 versus p53-PTEN in Cellular Migration and Invasion

ABSTRACT We have recently shown that Src induces the formation of podosomes and cell invasion by suppressing endogenous p53, while enhanced p53 strongly represses the Src-induced invasive phenotype. However, the mechanism by which Src and p53 play antagonistic roles in cell invasion is unknown. Here we show that the Stat3 oncogene is a required downstream effector of Src in inducing podosome structures and related invasive phenotypes. Stat3 promotes Src phenotypes through the suppression of p53 and the p53-inducible protein caldesmon, a known podosome antagonist. In contrast, enhanced p53 attenuates Stat3 function and Src-induced podosome formation by upregulating the tumor suppressor PTEN. PTEN, through the inactivation of Src/Stat3 function, also stabilizes the podosome-antagonizing p53/caldesmon axis, thereby further enhancing the anti-invasive potential of the cell. Furthermore, the protein phosphatase activity of PTEN plays a major role in the negative regulation of the Src/Stat3 pathway and represses podosome formation. Our data suggest that cellular invasiveness is dependent on the balance between two opposing forces: the proinvasive oncogenes Src-Stat3 and the anti-invasive tumor suppressors p53-PTEN.

Both lipid- and protein-phosphatase activities of PTEN contribute to the p53-PTEN anti-invasion pathway

We have recently identified mutually antagonizing signaling pathways that regulate podosome formation and invasive phenotypes in Src-transformed vascular smooth muscle cells and fibroblasts. Cross-talks between the anti-invasion p53-PTEN, and the pro-invasion Src-Stat3 and Src-PI3K-Akt pathways serve as a check and balance that dictates the outcome of either an invasive or non-invasive phenotype. Using a retrovirus vector encoding PTEN phosphatase mutants that retain either protein- or lipid-phosphatase activity on a Src(Y527F)background, we report here that both lipid- and protein-phosphatase activities of PTEN contribute to the suppression of Src-induced podosome formation and associated invasive phenotypes in rat aortic smooth muscle cells. This data suggests that p53 up-regulation of PTEN inhibits cell invasion via a two-prong mechanism: inactivating podosome agonists by its protein-phosphatase activity on the one hand, and antagonising the PI3K-Akt pathway by its lipid-phosphatase activity on the other.

Phorbol ester-induced podosomes in normal human bronchial epithelial cells

Spreading and migration of the basal cells neighboring a wound is essential for airway epithelial repair. To gain insight into the molecular mechanisms that govern these cellular processes, we asked whether normal human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls migration and invasion. Herein, we report that phorbol‐12, 13‐dibutyrate (PDBu), a protein kinase C activator, induced reorganization of cytoskeletal structure in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. Z‐stack scanning confocal microscopy showed that PDBu‐induced podosome‐like structures contain actin‐rich columns that arise from the ventral surface of the cell, and also revealed the presence of circular ruffles/waves at the dorsal cell surface. The molecular components of these cytoskeletal structures were determined with immunofluorescent staining. Using in situ zymography, we demonstrated that PDBu‐induced podosomes were capable of degrading fibronectin‐gelatin‐sucrose matrix. PDBu also increased epithelial cell invasion across Transwell chamber. Podosomes and circular dorsal ruffles may be important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration. J. Cell. Physiol. 218: 366–375, 2009.

MEK/ERK pathway mediates PKC activation-induced recruitment of PKCζ and MMP-9 to podosomes

Podosomes are adhesive structures on the ventral surface of cells that invade and degrade the extracellular matrix. Recently, we reported that phorbol 12,13‐dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in normal human bronchial epithelial (NHBE) cells, and atypical PKCζ regulated MMP‐9 recruitment to podosomes for its release and activation. The objective of this study was to explore signaling pathways that are involved in PKC activation‐induced podosome formation and matrix degradation. Herein, we found that PDBu increased phosphorylation of PI3K p85, Akt, Src, ERK1/2, and JNK. Inhibitors for PI3K, Akt, and Src suppressed PDBu‐induced podosome formation and matrix degradation. In contrast, blockers for MEK/ERK or JNK did not inhibit podosome formation but reduced proteolytic activity of podosomes. Inhibition of PKCζ activity with its pseudosubstrate peptide (PS)‐inhibited PDBu‐induced phosphorylation of MEK/ERK and JNK. On the other hand, inhibition of MEK/ERK or JNK pathway did not affect PKCζ phosphorylation, but reduced the recruitment of PKCζ and MMP‐9 to podosomes. We conclude that PKCζ may regulate MEK/ERK and JNK phosphorylation and in turn activated MEK/ERK and JNK may regulate the proteolytic activity of PDBu‐induced podosomes by influencing the recruitment of PKCζ and MMP‐9 to podosomes. J. Cell. Physiol. 228: 416–427, 2013.

p53 regulation of podosome formation and cellular invasion in vascular smooth muscle cells

The p53 transcription factor, discovered in 19791;2, is well known as a potent suppressor of tumor development by inhibiting cell cycle progression, and promoting senescence or apoptosis, when the genome is compromised or under oncogenic stress3. Accumulating evidence has pointed to an alternative role of p53 in the curtailment of tumor progression and colonization of secondary sites by negatively regulating tumor cell metastasis4;5. Recently, we have found that p53 suppresses Src-induced formation of podosomes and associated invasive phenotypes in fibroblasts and vascular smooth muscle cells (VSMC)6;7. In this review, I will focus on some recent studies that have identified p53 as a suppressor of cell migration and invasion in general, and VSMC podosome formation and ECM degradation in particular.

The roles of akt isoforms in the regulation of podosome formation in fibroblasts and extracellular matrix invasion.

Mesenchymal cells employ actin-based membrane protrusions called podosomes and invadopodia for cross-tissue migration during normal human development such as embryogenesis and angiogenesis, and in diseases such as atherosclerosis plaque formation and cancer cell metastasis. The Akt isoforms, downstream effectors of phosphatidylinositol 3 kinase (PI3K), play crucial roles in cell migration and invasion, but their involvement in podosome formation and cell invasion is not known. In this study, we have used Akt1 and/or Akt2 knockout mouse embryonic fibroblasts and Akt3-targeted shRNA to determine the roles of the three Akt isoforms in Src and phorbol ester-induced podosome formation, and extracellular matrix (ECM) digestion. We found that deletion or knockdown of Akt1 significantly reduces Src-induced formation of podosomes and rosettes, and ECM digestion, while suppression of Akt2 has little effect. In contrast, Akt3 knockdown by shRNA increases Src-induced podosome/rosette formation and ECM invasion. These data suggest that Akt1 promotes, while Akt3 suppresses, podosome formation induced by Src, and Akt2 appears to play an insignificant role. Interestingly, both Akt1 and Akt3 suppress, while Akt2 enhances, phorbol ester-induced podosome formation. These data show that Akt1, Akt2 and Akt3 play different roles in podosome formation and ECM invasion induced by Src or phorbol ester, thus underscoring the importance of cell context in the roles of Akt isoforms in cell invasion.