Kevin Pumiglia

CXCL-12/Stromal Cell–Derived Factor-1α Transactivates HER2-neu in Breast Cancer Cells by a Novel Pathway Involving Src Kinase Activation

Experimental evidence suggests that CXCR4, a Gi protein-coupled receptor for the ligand CXCL12/stromal cell-derived factor-1alpha (SDF-1alpha), plays a role in breast cancer metastasis. Transactivation of HER2-neu by G protein-coupled receptor activation has been reported as a ligand-independent mechanism of activating tyrosine kinase receptors. We found that SDF-1alpha transactivated HER2-neu in the breast cancer cell lines MDA-MB-361 and SKBR3, which express both CXCR4 and HER2-neu. AMD3100, a CXCR4 inhibitor, PKI 166, an epidermal growth factor receptor/HER2-neu tyrosine kinase inhibitor, and PP2, a Src kinase inhibitor, each blocked SDF-1alpha-induced HER2-neu phosphorylation. Blocking Src kinase, with PP2 or using a kinase-inactive Src construct, and inhibiting epidermal growth factor receptor/HER2-neu signaling with PKI 166 each inhibited SDF-1alpha-stimulated cell migration. We report a novel mechanism of HER2-neu transactivation through SDF-1alpha stimulation of CXCR4 that involves Src kinase activation.

Integrin α3β1 directs the stabilization of a polarized lamellipodium in epithelial cells through activation of Rac1

Epithelial cell migration is a crucial event in wound healing, yet little is known about mechanisms whereby integrins regulate epithelial cell polarization and migration. In the present work, we demonstrate the importance of adhesion through the α3β1 integrin in promoting the stabilization of leading lamellipodia in migrating keratinocytes. We demonstrate that this integrin is found at the leading edge of migrating keratinocytes and that inhibition of α3β1 binding to laminin-5 prevents the formation of stable leading lamellipodia. Consistent with this observation, keratinocytes derived from α3β1-deficient mice fail to form stable leading lamellipodia but retain the ability to form actin-containing protrusions that rapidly extend and retract from the cell membrane. Formation of a leading lamellipodium also requires α3β1-dependent activation of Rac1, because α3β1-deficient keratinocytes show decreased activation of Rac1 compared with α3β1-expressing cells, and formation of stable leading lamellipodia can be inhibited in the latter cells by expression of the dominant negative Rac1 mutant Rac1N17. Furthermore, α3β1-deficient keratinocytes expressing constitutively active Rac1L61 failed to form stable lamellipodia when plated onto laminin-5, demonstrating that α3β1 is required for Rac1-mediated formation of a stable lamellipodium. These observations identify a crucial role for integrin-mediated adhesion and signaling in the formation of large, polarized, stable lamellipodia by migrating epithelial cells. To our knowledge, this study is the first to demonstrate that signal transduction through a specific integrin is required to direct the development of a lamellipodium from an initial protrusion and promote persistent epithelial cell migration.

α3β1 integrin promotes keratinocyte cell survival through activation of a MEK/ERK signaling pathway

Inadequate or inappropriate adhesion of epithelial cells to extracellular matrix leads to a form of apoptosis known as anoikis. During various tissue remodelling events, such as wound healing or carcinoma invasion, changes in the physical properties, and/or composition of the extracellular matrix, can lead to anoikis of epithelial cells that lack appropriate receptor-matrix interactions. Laminin-5 is the major ligand for keratinocyte adhesion in the epidermis, and it also promotes keratinocyte survival in vivo and in vitro. Integrins α3β1 and α6β4 are the major receptors for laminin-5; however, specific roles for these integrins in keratinocyte survival have not been determined. In the current study, we exploited keratinocyte cell lines derived from wild-type or α3 integrin knockout mice to reveal a critical role for α3β1 in protecting keratinocytes from apoptosis upon serum withdrawal. We show that α3β1-mediated adhesion to laminin-5 extracellular matrix inhibits proteolytic activation of caspase-3 and TUNEL-staining, both hallmarks of apoptosis. We also show that α3β1-mediated adhesion activates focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), and that inhibition of either FAK or ERK signaling leads to apoptosis of keratinocytes attached to laminin-5. α6β4-mediated adhesion to laminin-5 only partially protects cells from apoptosis in the absence of α3β1, and α6β4 is not necessary for cell survival in the presence of α3β1. These results suggest that α3β1 is necessary and sufficient for maximal keratinocyte survival on laminin-5. We propose a model to address the potential importance of α3β1-mediated survival for migrating keratinocytes at the leading edge of a cutaneous wound.

SRC-induced tyrosine phosphorylation of VE-cadherin is not sufficient to decrease barrier function of endothelial monolayers

Activation of Src family kinases (SFK) and the subsequent phosphorylation of VE-cadherin have been proposed as major regulatory steps leading to increases in vascular permeability in response to inflammatory mediators and growth factors. To investigate Src signaling in the absence of parallel signaling pathways initiated by growth factors or inflammatory mediators, we activated Src and SFKs by expression of dominant negative Csk, expression of constitutively active Src, or knockdown of Csk. Activation of SFK by overexpression of dominant negative Csk induced VE-cadherin phosphorylation at tyrosines 658, 685, and 731. However, dominant negative Csk expression was unable to induce changes in the monolayer permeability. In contrast, expression of constitutively active Src decreased barrier function and promoted VE-cadherin phosphorylation on tyrosines 658 and 731, although the increase in VE-cadherin phosphorylation preceded the increase in permeability by 4–6 h. Csk knockdown induced VE-cadherin phosphorylation at sites 658 and 731 but did not induce a loss in barrier function. Co-immunoprecipitation and immunofluorescence studies suggest that phosphorylation of those sites did not impair VE-cadherin ability to bind p120 and β-catenin or the ability of these proteins to localize at the plasma membrane. Taken together, our data show that Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to promote an increase in endothelial cell monolayer permeability and suggest that signaling leading to changes in vascular permeability in response to inflammatory mediators or growth factors may require VE-cadherin tyrosine phosphorylation concurrently with other signaling pathways to promote loss of barrier function.

α3β1 integrin regulates MMP-9 mRNA stability in immortalized keratinocytes: a novel mechanism of integrin-mediated MMP gene expression

Matrix metalloproteinases facilitate cell migration and tumor invasion through their ability to proteolyse the extracellular matrix. The laminin-binding integrin α3β1 is expressed at high levels in squamous cell carcinomas and in normal keratinocytes during cutaneous wound healing. We showed previously that α3β1 is required for MMP-9/gelatinase B secretion in immortalized mouse keratinocytes (MK cells) and that this regulation was acquired as part of the immortalized phenotype, suggesting a possible role for α3β1 during malignant conversion. In the current study, we identify a novel mechanism whereby α3β1 regulates the induction of MMP-9 expression that occurs in response to activation of a MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Inhibition of MEK/ERK signaling in wild-type MK cells with a pharmacological inhibitor, U0126, showed that ERK activation was necessary for high levels of endogenous MMP-9 gene expression and activity of a transfected MMP-9 promoter. Furthermore, activation of MEK/ERK signaling in these cells with an oncogenic mutant of Ras, RasV12, increased both endogenous MMP-9 gene expression and MMP-9 promoter activity. Experiments with α3β1-deficient MK cells revealed that α3β1 was required for both baseline levels and RasV12-induced levels of MMP-9 mRNA expression. However, α3β1 was not required for RasV12-mediated activation of ERK or for ERK-dependent MMP-9 promoter activity. Direct comparison of mRNA turnover in the wild type and α3-null MK cells identified a requirement for α3β1 in stabilization of MMP-9 mRNA transcripts. These results identify a novel function for integrins in promoting mRNA stability as a mechanism to potentiate MAPK-mediated gene expression. They also suggest a role for α3β1 in maintaining high levels of MMP-9 mRNA expression in response to oncogenic activation of MEK/ERK signaling pathways.

Activated Ras induces a proangiogenic phenotype in primary endothelial cells.

Angiogenic factors alter endothelial cell phenotype to promote the formation of new blood vessels, a process critical for a number of normal and pathological conditions. Ras is required for the induction of the angiogenic phenotype in response to vascular endothelial growth factor (VEGF). However, VEGF generates many signals, several of which are not dependent upon Ras activation. Our current study investigates the sufficiency of Ras activation for driving angiogenic responses. An activated RasV12 mutant induces prominent membrane ruffling, branching morphogenesis on three-dimensional collagen, DNA synthesis, and cell migration in primary endothelial cells. An upregulation of PI3K/AKT, Erk, and Jnk signaling pathways accompany these phenotypic changes. The inhibition of Erk blocked cell proliferation, but only partially attenuated migration. Blocking PI3K had no effect on DNA synthesis, but caused a modest reduction in cell migration. Lastly, Jnk played a significant role in both the proliferation and migration response. These effects of RasV12 are not the result of increased autocrine secretion of VEGF. These data suggest that the acquisition of activating Ras mutations can lead to a proangiogenic conversion in the phenotype of primary endothelial cells. Furthermore, these data raise the possibility that chronic Ras activation in endothelial cells may be sufficient to promote angiogenesis and the development of vascular anomalies.

Vascular endothelial growth factor-mediated activation of p38 is dependent upon Src and RAFTK/Pyk2

Vascular endothelial growth factor (VEGF) induces activation of p38 mitogen-activated protein kinase (MAPK) in primary endothelial cells and may be critical for VEGF-induced angiogenesis. We investigated the molecular basis for p38 activation in response to VEGF. The expression of a C-terminal splice variant of FAK, FRNK, had no affect on VEGF-induced activation of p38; however, expression of a dominant-negative RAFTK/Pyk2 mutant led to a decrease in the activation of p38, but had no affect on extracellular signal-regulated kinase (ERK). Since calcium regulates RAFTK/Pyk2, we investigated its role in p38 activity. Preincubation with EGTA suppressed p38 activation, while calcium ionophore induced p38 activity. Inhibition of phospholipase C (PLC) resulted in complete inhibition of ERK, while having no affect on p38 activity. These data suggested a bifurcation in the regulation of MAPKs that occurs at the level of PLC and RAFTK/Pyk2 activation. Src family kinases interact with RAFTK/Pyk2. Inhibition of Src by either pharmacological or genetic means decreased p38 activity. Finally, we found that both Src and RAFTK/Pyk2 were essential for endothelial cell migration. These data identified a novel regulatory network involving extracellular calcium, RAFTK/Pyk2, Src and p38. This signaling network appears to be critical for VEGF-induced endothelial cell migration.

Focal Adhesion Kinase Controls Cellular Levels of p27/Kip1 and p21/Cip1 through Skp2-Dependent and -Independent Mechanisms

ABSTRACT Endothelial cell proliferation is a critical step in angiogenesis and requires a coordinated response to soluble growth factors and the extracellular matrix. As focal adhesion kinase (FAK) integrates signals from both adhesion events and growth factor stimulation, we investigated its role in endothelial cell proliferation. Expression of a dominant-negative FAK protein, FAK-related nonkinase (FRNK), impaired phosphorylation of FAK and blocked DNA synthesis in response to multiple angiogenic stimuli. These results coincided with elevated cyclin-dependent kinase inhibitors (CDKIs) p21/Cip and p27/Kip, as a consequence of impaired degradation. FRNK inhibited the expression of Skp2, an F-box protein that targets CDKIs, by inhibiting mitogen-induced mRNA. The FAK-regulated degradation of p27/Kip was Skp2 dependent, while levels of p21/Cip were regulated independent of Skp2. Skp2 is required for endothelial cell proliferation as a consequence of degrading p27. Finally, knockdown of both p21 and p27 in FRNK-expressing cells completely restored mitogen-induced endothelial cell proliferation. These data demonstrate a critical role for FAK in the regulation of CDKIs through two independent mechanisms: Skp2 dependent and Skp2 independent. They also provide important insights into the requirement of focal adhesion kinase for normal vascular development and reveal novel regulatory control points for angiogenesis.

An immortalization-dependent switch in integrin function up-regulates MMP-9 to enhance tumor cell invasion.

Integrins, the major receptors for cell adhesion to the extracellular matrix, play important roles during tumor progression. However, it is still unclear whether genetic lesions that occur during carcinoma development can lead to altered integrin function, and how changes in integrin function contribute to subsequent carcinoma progression. Loss-of-function mutations in p53 and activating mutations in H-Ras, which immortalize and transform epithelial cells, respectively, are common causal events in squamous cell carcinoma (SCC). Phenotypes resulting from these two genetic lesions promote SCC progression and are, therefore, potential targets for anticancer therapies. We developed a model system of keratinocyte transformation that has allowed us to investigate the individual roles of p53 mutation and oncogenic Ras mutation in the acquisition of integrin alpha3beta1-regulated phenotypes that promote SCC progression. Using this model, we show that keratinocyte immortalization by p53-null mutation causes a switch in alpha3beta1 function that induces matrix metalloproteinase (MMP)-9 gene expression in tumorigenic cells. This acquired alpha3beta1-dependent regulation of MMP-9 was maintained during subsequent transformation by oncogenic Ras, and it promoted invasion of tumorigenic keratinocytes. Our results show that loss of p53 function leads to changes in integrin-mediated gene regulation that occur during SCC progression and play a critical role in tumor cell invasion.

Pro-inflammatory angiogenesis is mediated by p38 MAP kinase

Chronic inflammation is tightly linked to diseases associated with endothelial dysfunction including aberrant angiogenesis. To better understand the endothelial role in pro‐inflammatory angiogenesis, we analyzed signaling pathways in continuously activated endothelial cells, which were either chronically exposed to soluble TNF or the reactive oxygen species (ROS) generating H2O2, or express active transmembrane TNF. Testing in an in vitro capillary sprout formation assay, continuous endothelial activation increased angiogenesis dependent on activation of p38 MAP kinase, NADPH oxidase, and matrix metalloproteinases (MMP). p38 MAP kinase‐ and MMP‐9‐dependent angiogenesis in our assay system may be part of a positive feed forward autocrine loop because continuously activated endothelial cells displayed up‐regulated ROS production and subsequent endothelial TNF expression. The pro‐angiogenic role of the p38 MAP kinase in continuously activated endothelial cells was in stark contrast to the anti‐angiogenic activity of the p38 MAP kinase in unstimulated control endothelial cells. In vivo, using an experimental prostate tumor, pharmacological inhibition of p38 MAP kinase demonstrated a significant reduction in tumor growth and in vessel density, suggesting a pro‐angiogenic role of the p38 MAP kinase in pathological angiogenesis in vivo. In conclusion, our results suggest that continuous activation of endothelial cells can cause a switch of the p38 MAP kinase from anti‐angiogenic to pro‐angiogenic activities in conditions which link oxidative stress and autocrine TNF production. J. Cell. Physiol. 226: 800–808, 2011.