05-185 Sigma-AldrichAnti-avian Src Antibody, clone EC10

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.

Nontransformed cells can force tumor cells to assume a normal morphology and phenotype by the process of contact normalization. Transformed cells must escape this process to become invasive and malignant. However, mechanisms underlying contact normalization have not been elucidated. Here, we have identified genes that are affected by contact normalization of Src-transformed cells. Tumor cells must migrate to become invasive and malignant. Src must phosphorylate the adaptor protein Cas (Crk-associated substrate) to promote tumor cell motility. We report here that Src utilizes Cas to induce podoplanin (Pdpn) expression to promote tumor cell migration. Pdpn is a membrane-bound extracellular glycoprotein that associates with endogenous ligands to promote tumor cell migration leading to cancer invasion and metastasis. In fact, Pdpn expression accounted for a major part of the increased migration seen in Src-transformed cells. Moreover, nontransformed cells suppressed Pdpn expression in adjacent Src-transformed cells. Of greater than 39,000 genes, Pdpn was one of only 23 genes found to be induced by transforming Src activity and suppressed by contact normalization of Src-transformed cells. In addition, we found 16 genes suppressed by Src and induced by contact normalization. These genes encode growth factor receptors, adaptor proteins, and products that have not yet been annotated and may play important roles in tumor cell growth and migration.

Differentiation and transformation of untransformed and ts-Src-transformed canine kidney MDCK cells in 2D and 3D environment were investigated using microarray technique, RT-PCR, confocal microscopy and functional assays. Activated Src induced epithelial-mesenchymal transition (EMT) in 2D environment followed by translocation of junctional proteins to the cytoplasm, without significant changes in protein expression. In 3D environment untransformed MDCK cells formed cell cysts with apical domain facing a lumen, E-cadherin delineating the lateral membranes, ZO-1 at tight junctions and caspase-3 in apoptotic cells captured within the lumen. This was accompanied by reduced expression of an apoptosis inhibitor, survivin and vesicle transport effectors, rab 7 and 8, whereas rab 5 expression increased. In 3D environment activated Src induced changes in expression of over 100 genes as revealed by microarray analysis, mostly involved in cell signaling, division and energy metabolism. Only response in cytoskeletal components was decreased expression of actin and Arp2/3 by v-Src, whereas two p120catenin binding proteins Kaiso and Nanos increased their expression. Concomitantly, apoptosis was inhibited by v-Src resulting in formation of a sphere with epitheloid cells facing extracellular matrix and undifferentiated cells captured within the cluster. This was accompanied by increased expression of apoptosis inhibitor survivin, as revealed by western blotting. Mitochondrial membrane potential in untransformed MDCK cells was lower than in ts-Src-MDCK cells in early days of cluster formation correlating with the induction of apoptosis. Hence, v-Src activation in 3D environment did not induce EMT, but brought about inhibition of apoptosis and increased proliferation where increased expression of survivin and inhibition of the mitochondrial permeability have a role.

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 pageing 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.

The Src tyrosine kinase associates with the focal adhesion adaptor protein Cas (Crk-associated substrate) to suppress the expression of potential tumor suppressor genes. For example, Src utilizes Cas to suppress the expression of the LIM-only protein Fhl1 (four and a half LIM domains 1), in order to promote non-anchored tumor-cell growth and migration. Here, we report that the promoter region of the Fhl1 gene was methylated more in Src-transformed cells than non-transformed cells. In addition, global expression analysis indicates that Fhl1 induced expression of serum deprivation response factor (Sdpr) in Src-transformed cells. Moreover, Fhl1 and Sdpr was expressed in approximately 87% and 40% of samples obtained from non-transformed breast, 100% of samples obtained from non-transformed kidney, and over 60% of samples obtained from non-transformed prostate. In contrast, Fhl1 and Sdpr was detected in approximately 40% and 7% of matched samples from mammary carcinoma, less than 11% of matched samples from kidney carcinoma, and in less than 22% of matched samples from prostate carcinoma. These data indicate that Fhl1 and Sdpr expression was significantly reduced in tumors of the breast (P < 0.02 and P < 0.001), kidney (P < 0.01), and prostate (P < 0.05). In addition, although Src can activate mitogen-activated protein kinase (MAPK) to promote tumor-cell growth, our data indicate that Src did not rely on MAPK activity to suppress the expression of Fhl1 and Sdpr in transformed cells. Thus, Src induced methylation of the promoter region of the Fhl1 gene; Src suppressed Fhl1 and Sdpr expression independent of mitogen-activated protein kinase (MAPK) activity; Fhl1 induced the expression of Sdpr in Src-transformed cells; and Fhl1 and Sdpr expression was suppressed in tumors of the breast, kidney, and prostate.

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.

The growth of many types of cancer cells can be controlled by surrounding normal cells. However, mechanisms underlying this phenomenon have not been defined. We used a layered culture system to investigate how nontransformed cells suppress the growth of neighboring transformed cells. Direct physical contact between transformed and nontransformed cells was required for growth suppression of transformed cells in this system; communication by diffusible factors was not sufficient. However, significant gap junctional communication was not required, indicating that other intercellular junctions mediated this growth regulatory response. We also report that the Src kinase activity in transformed cells was not directly inhibited by contact with nontransformed cells. Instead, nontransformed cells increased the expression of serum deprivation-response protein and the transcription factor four and a half LIM domain 1 in tumor cells. In addition, these results suggest mechanisms by which normal cells may block Wnt signaling, inhibit insulin-like growth factor activity, and promote host recognition of neighboring tumor cells.

Cas is a member of the focal adhesion complex. Phosphorylation of Cas by Src is an important event leading to cell transformation. Using mass spectrometry, we have mapped 11 sites in Cas that are phosphorylated by Src. These sites are all located between residues 132 and 414 of Cas, in a region that is required for binding to a number of other proteins including Crk. We tested synthetic peptides modeled on Cas phosphorylation sites, and found that the sequence containing tyrosine 253 was phosphorylated by Src most efficiently. Using cells derived from Cas-deficient mice, we confirmed that Cas greatly enhanced the ability of Src to transform cells. Phosphorylation of Cas on tyrosine 253 was not required for Src to increase growth rate, suppress contact inhibition, or suppress anchorage dependence. Yet, in contrast to these growth characteristics, phosphorylation of Cas on tyrosine 253 was required for Src to promote cell migration. Thus, a single phosphorylation site on this focal adhesion adaptor protein can effectively separate cell migration from other transformed growth characteristics.

Cellular transformation by oncogenic retroviruses encoding protein tyrosine kinases coincides with the tyrosine-specific phosphorylation of multiple protein substrates. Previous studies have shown that tyrosine phosphorylation of a protein of 120 kDa, p120, correlated with src transformation in chicken embryo fibroblasts. Additionally, we previously identified two phosphotyrosine-containing cellular proteins, p130 and p110, that formed stable complexes with activated variants of pp60src, the src-encoded tyrosine kinase. To study transformation-relevant tyrosine kinase substrates, we have generated monoclonal antibodies to individual tyrosine phosphoproteins, including p130, p120, p110, and five additional phosphoproteins (p210, p125, p118, p85, and p185/p64). These antibodies detected several of the same tyrosine phosphoproteins in chicken embryo fibroblasts transformed by avian retroviruses Y73 and CT10, encoding the yes and crk oncogenes, respectively. Protein substrates in mouse, rat, hamster, and human cells overexpressing activated variants of chicken pp60src were also detected by several of the monoclonal antibodies.