Whole mount of adult ear skin as a model to study vascular malformations.

BACKGROUND: Genetic analysis in human patients has linked mutations in PIK3CA, the catalytic subunit of PI-3'Kinase, to sporadic incidences of vascular malformations. METHODS: We have developed a mouse model with inducible and endothelial-specific expression of PIK3CAH1047R , resulting in the development of vascular malformations. Systemic induction of this mutation in adult mice results in rapid lethality, limiting our ability to track and study these lesions; therefore, we developed a topical and local induction protocol using the active metabolite of tamoxifen, 4OH-T, on the ear skin of adults. RESULTS: This approach allows us to successfully model the human disease in a mature and established vascular bed and track the development of vascular malformations. To validate the utility of this model, we applied a topical rapamycin ointment, as rapamycin is therapeutically beneficial to patients in clinical trials. We found that the induced ear lesions showed significant attenuation after treatment, which was easily quantified. CONCLUSIONS: These data collectively provide evidence of a new model to study vascular malformations in adult tissues, which should be particularly useful in environments lacking specialized small-animal imaging facilities.

Laminin-511 and α6 integrins regulate the expression of CXCR4 to promote endothelial morphogenesis.

During angiogenesis, endothelial cells engage components of the extracellular matrix through integrin-mediated adhesion. Endothelial expression of laminin-411 and laminin-511 is known to promote vessel stability. However, little is known about the contribution of these laminins to endothelial morphogenesis. We used two organotypic cell culture angiogenesis assays, in conjunction with RNAi approaches, to demonstrate that depletion of either the α4 chain of laminin-411 (LAMA4) or the α5 chain of laminin-511 (LAMA5) from endothelial cells inhibits sprouting and tube formation. Depletion of α6 (ITGA6) integrins resulted in similar phenotypes. Gene expression analysis indicated that loss of either laminin-511 or α6 integrins inhibited the expression of CXCR4, a gene previously associated with angiogenic endothelial cells. Pharmacological or RNAi-dependent inhibition of CXCR4 suppressed endothelial sprouting and morphogenesis. Importantly, expression of recombinant CXCR4 rescued endothelial morphogenesis when α6 integrin expression was inhibited. Additionally, the depletion of α6 integrins from established tubes resulted in the loss of tube integrity and laminin-511. Taken together, our results indicate that α6 integrins and laminin-511 can promote endothelial morphogenesis by regulating the expression of CXCR4 and suggest that the α6-dependent deposition of laminin-511 protects the integrity of established endothelial tubes.

Activation of Ras in the Vascular Endothelium Induces Brain Vascular Malformations and Hemorrhagic Stroke.

Cerebrovascular malformations (CVMs) affect approximately 3% of the population, risking hemorrhagic stroke, seizures, and neurological deficits. Recently Ras mutations have been identified in a majority of brain arterio-venous malformations. We generated an endothelial-specific, inducible HRASV12 mouse model, which results in dilated, proliferative blood vessels in the brain, blood-brain barrier breakdown, intracerebral hemorrhage, and rapid lethality. Organoid morphogenesis models revealed abnormal cessation of proliferation, abnormalities in expression of tip and stalk genes, and a failure to properly form elongating tubes. These defects were influenced by both hyperactive PI-3' kinase signaling and altered TGF-ß signaling. Several phenotypic changes predicted by the in vitro morphogenesis analysis were validated in the mouse model. These data provide a model of brain vascular malformations induced by mutant Ras and reveal insights into intersecting molecular mechanisms in the pathogenesis of brain vascular malformations.

mTORc1 activity is necessary and sufficient for phosphorylation of eNOSS1177.

Nitric oxide, produced by eNOS, plays critical roles in the regulation of vascular function and maintenance. Chronic PI3K signaling has recently been associated with vascular malformations. A well described substrate downstream of PI3K signaling is eNOS. Another critical downstream target of PI3K is the metabolic regulator, mTORc1. The relationship between mTORc1 and eNOS regulation, has not been determined. We generated cells with manipulated PI3K signaling by expressing the activating mutation, PIK3CAH1047R , or knocking down PTEN expression. We investigated eNOSS1177 phosphorylation, a major activating regulatory site, following mTORC1 inhibition. We also tested the sufficiency of mTORc1 activation to stimulate eNOSS1177 phosphorylation. Our data indicate mTORc1 activity is required for the phosphorylation of eNOSS1177 , even in the presence of robust AKT activation. Moreover, we found that expression of RHEB, which functions in the absence of AKT activation to activate mTORc1, is sufficient to phosphorylate this site. Our data indicate that mTORc1, rather than AKT, may be the critical determinant of eNOSS1177 phosphorylation. As mTORc1 is a central regulator of cellular metabolism, the finding that this regulatory complex can directly participate in the regulation of eNOS provides new insights into metabolic uncoupling and vascular disease that often accompanies diabetes, high fat diets, and aging.

BranchAnalysis2D/3D automates morphometry analyses of branching structures.

BACKGROUND: Morphometric analyses of biological features have become increasingly common in recent years with such analyses being subject to a large degree of observer bias, variability, and time consumption. While commercial software packages exist to perform these analyses, they are expensive, require extensive user training, and are usually dependent on the observer tracing the morphology. NEW METHOD: To address these issues, we have developed a broadly applicable, no-cost ImageJ plugin we call 'BranchAnalysis2D/3D', to perform morphometric analyses of structures with branching morphologies, such as neuronal dendritic spines, vascular morphology, and primary cilia. RESULTS: Our BranchAnalysis2D/3D algorithm allows for rapid quantification of the length and thickness of branching morphologies, independent of user tracing, in both 2D and 3D data sets. COMPARISON WITH EXISTING METHODS: We validated the performance of BranchAnalysis2D/3D against pre-existing software packages using trained human observers and images from brain and retina. We found that the BranchAnalysis2D/3D algorithm outputs results similar to available software (i.e., Metamorph, AngioTool, Neurolucida), while allowing faster analysis times and unbiased quantification. CONCLUSIONS: BranchAnalysis2D/3D allows inexperienced observers to output results like a trained observer but more efficiently, thereby increasing the consistency, speed, and reliability of morphometric analyses.

Suppression of integrin α3ß1 by α9ß1 in the epidermis controls the paracrine resolution of wound angiogenesis.

Development of wound therapies is hindered by poor understanding of combinatorial integrin function in the epidermis. In this study, we generated mice with epidermis-specific deletion of α3ß1, α9ß1, or both integrins as well as keratinocyte lines expressing these integrin combinations. Consistent with proangiogenic roles for α3ß1, α3-null keratinocytes showed reduced paracrine stimulation of endothelial cell migration and survival, and wounds of epidermis-specific α3 knockout mice displayed impaired angiogenesis. Interestingly, α9ß1 in keratinocytes suppressed α3ß1-mediated stimulation of endothelial cells, and wounds of epidermis-specific α9 knockout mice displayed delayed vascular normalization and reduced endothelial apoptosis, indicating that α9ß1 cross-suppresses α3ß1 proangiogenic functions. Moreover, α9ß1 inhibited α3ß1 signaling downstream of focal adhesion kinase (FAK) autoactivation at the point of Src-mediated phosphorylation of FAK Y861/Y925. Finally, α9ß1 cross-suppressed many α3ß1-dependent genes, including the gene that encodes MMP-9, which we implicated as a regulator of integrin-dependent cross talk to endothelial cells. Our findings identify a novel physiological context for combinatorial integrin signaling, laying the foundation for therapeutic strategies that manipulate α9ß1 and/or α3ß1 during wound healing.

Integrins promote cytokinesis through the RSK signaling axis.

Cytokinesis is the final stage in cell division. Although integrins can regulate cytokinesis, the mechanisms involved are not fully understood. In this study, we demonstrate that integrin-regulated ERK (extracellular signal-related kinase) and RSK (p90 ribosomal S6 kinase) signaling promotes successful cytokinesis. Inhibiting the activation of ERK and RSK in CHO cells by a mutation in the integrin ß1 cytoplasmic tail or with pharmacological inhibitors results in the accumulation of cells with midbodies and the formation of binucleated cells. Activation of ERK and RSK signaling by the expression of constitutively active RAF1 suppresses the mutant phenotype in a RSK-dependent manner. Constitutively active RSK2 also restores cytokinesis inhibited by the mutant integrin. Importantly, the regulatory role of the RSK pathway is not specific to CHO cells. MCF-10A human mammary epithelial cells and HPNE human pancreatic ductal epithelial cells exhibit a similar dependence on RSK for successful cytokinesis. In addition, depriving mitotic MCF10A cells of integrin-mediated adhesion by incubating them in suspension suppressed ERK and RSK activation and resulted in a failure of cytokinesis. Furthermore, inhibition of RSK or integrins within the 3D context of a developing salivary gland organ explant also leads to an accumulation of epithelial cells with midbodies, suggesting a similar defect in cytokinesis. Interestingly, neither ERK nor RSK regulates cytokinesis in human fibroblasts, suggesting cell-type specificity. Taken together, our results identify the integrin-RSK signaling axis as an important regulator of cytokinesis in epithelial cells. We propose that the proper interaction of cells with their microenvironment through integrins contributes to the maintenance of genomic stability by promoting the successful completion of cytokinesis.

Focal adhesion kinase is a phospho-regulated repressor of Rac and proliferation in human endothelial cells.

Focal adhesion kinase (FAK) is critically positioned to integrate signals from the extracellular matrix and cellular adhesion. It is essential for normal vascular development and has been implicated in a wide range of cellular functions including the regulation of cell proliferation, migration, differentiation, and survival. It is currently being actively targeted therapeutically using different approaches. We have used human endothelial cells as a model system to compare the effects of inhibiting FAK through several different approaches including dominant negatives, kinase inhibitors and shRNA. We find that manipulations of FAK signaling that result in inhibition of FAK 397 phosphorylation inhibit proliferation and migration. However, abolition of FAK expression using stable (shRNA) or transient (siRNA) approaches does not interfere with these cellular functions. The ability to regulate cell proliferation by FAK manipulation is correlated with the activation status of Rac, an essential signal for the regulation of cyclin-dependent kinase inhibitors. The knockdown of FAK, while not affecting cellular proliferation or migration, dramatically interferes with vascular morphogenesis and survival, mirroring in vivo findings. We propose a novel model of FAK signaling whereby one of the multifunctional roles of FAK as a signaling protein includes FAK as a phospho-regulated repressor of Rac activation, with important implications on interpretation of research experiments and therapeutic development.

Loss of NF1 expression in human endothelial cells promotes autonomous proliferation and altered vascular morphogenesis.

Neurofibromatosis is a well known familial tumor syndrome, however these patients also suffer from a number of vascular anomalies. The loss of NFl from the endothelium is embryonically lethal in mouse developmental models, however little is known regarding the molecular regulation by NF1 in endothelium. We investigated the consequences of losing NF1 expression on the function of endothelial cells using shRNA. The loss of NF1 was sufficient to elevate levels of active Ras under non-stimulated conditions. These elevations in Ras activity were associated with activation of downstream signaling including activation of ERK, AKT and mTOR. Cells knocked down in NF1 expression exhibited no cellular senescence. Rather, they demonstrated augmented proliferation and autonomous entry into the cell cycle. These proliferative changes were accompanied by enhanced expression of cyclin D, phosphorylation of p27(KIP), and decreases in total p27(KIP) levels, even under growth factor free conditions. In addition, NF1-deficient cells failed to undergo normal branching morphogenesis in a co-culture assay, instead forming planar islands with few tubules and branches. We find the changes induced by the loss of NF1 could be mitigated by co-expression of the GAP-related domain of NF1 implicating Ras regulation in these effects. Using doxycycline-inducible shRNA, targeting NF1, we find that the morphogenic changes are reversible. Similarly, in fully differentiated and stable vascular-like structures, the silencing of NF1 results in the appearance of abnormal vascular structures. Finally, the proliferative changes and the abnormal vascular morphogenesis are normalized by low-dose rapamycin treatment. These data provide a detailed analysis of the molecular and functional consequences of NF1 loss in human endothelial cells. These insights may provide new approaches to therapeutically addressing vascular abnormalities in these patients while underscoring a critical role for normal Ras regulation in maintaining the health and function of the vasculature.

Integrins regulate microtubule nucleating activity of centrosome through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling.

Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and γ-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin ß1 tail and show that this mutant substantially decreases microtubule nucleation and γ-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of γ-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.

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.

Activation of endothelial ras signaling bypasses senescence and causes abnormal vascular morphogenesis.

Angiogenesis is crucial for embryogenesis, reproduction, and wound healing and is a critical determinant of tumor growth and metastasis. The multifunctional signal transducer Ras is a proto-oncogene and frequently becomes mutated in a variety of human cancers, including angiosarcomas. Regulation of Ras is important for endothelial cell function and angiogenesis. Hyperactivation of Ras is linked with oncogene-induced senescence in many cell types. Given links between vascular malformations and angiosarcoma with activated Ras signaling, we sought to determine the consequence of sustained Ras activation on endothelial cell function. We find that sustained Ras activation in primary endothelial cells leads to prolonged activation of progrowth signaling, accompanied by a senescence bypass, enhanced proliferation, autonomous growth, and increased survival. Moreover, Ras severely compromises the ability of these cells to organize into vascular structures, instead promoting formation of planar endothelial sheets. This abnormal phenotype is regulated by phosphoinositide 3-kinase signaling, highlighting the therapeutic potential of agents targeting this axis in dealing with vascular morphogenic disorders and vascular normalization of tumors.

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 beta-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.

Endostatin and anastellin inhibit distinct aspects of the angiogenic process.

BACKGROUND: Endostatin and anastellin, fragments of collagen type XVIII and fibronectin, respectively, belong to a family of endogenous inhibitors of angiogenesis which inhibit tumor growth and metastasis in a number of mouse models of human cancer. The mechanism of action of these inhibitors is not well understood, but they have great potential usefulness as non-toxic long-term therapy for cancer treatment. METHODS: In this study, we compare the anti-angiogenic properties of endostatin and anastellin using cell proliferation and transwell migration assays. RESULTS: Anastellin but not endostatin completely inhibited human dermal microvessel endothelial cell proliferation in response to serum stimulation. Both anastellin and endostatin additively inhibited endothelial cell migration in response to VEGF. Anastellin but not endostatin lowered basal levels of active ERK. CONCLUSION: These data indicate that anastellin and endostatin exert their anti-angiogenic effects by modulating distinct steps in the angiogenic pathway and suggest that matrix-derived inhibitors of angiogenesis may exhibit higher efficacy when used in combination.

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.

Endothelial beta2 adrenergic signaling to AKT: role of Gi and SRC.

We have recently demonstrated that endothelial beta(2) adrenergic receptors (beta(2)AR) regulate eNOS activity and consequently vascular tone, through means of PKB/AKT. In this work we explored the signal transduction pathway leading to AKT/eNOS activation in endothelial cells (EC). Using pharmacological and molecular inhibitors both in cultured EC cells and in ex vivo rat carotid preparations, we found that G(i) coupling of the beta(2)AR is needed for AKT activation and vasorelaxation. Since endothelial activation is sensitive to pertussis toxin but not to G(ibetagamma) inhibition by betaARKct, we conclude that G(alphai) mediates betaAR induced AKT activation. Downstream, betaAR signalling requires the soluble tyrosine kinase SRC, as both in cultured EC and rat carotid, the mutant dominant negative of SRC prevent beta(2)AR induced endothelial activation and vasodilation. In EC, G(alphai) directly interacts with SRC and this interaction leads to SRC activation and phosphorylation in a manner that is regulated by beta(2)AR stimulation. We propose a novel signal transduction pathway for beta(2)AR stimulation trough G(alphai) and SRC, leading to activation of AKT.

Integrin alpha3beta1-dependent activation of FAK/Src regulates Rac1-mediated keratinocyte polarization on laminin-5.

Upon epidermal wounding, keratinocytes at the wound edge become activated, deposit newly synthesized laminin-5 into the extracellular matrix, and migrate into the wound bed. The interaction between integrin alpha3beta1 and laminin-5 is essential for establishment of a stable, leading lamellipodium and persistent keratinocyte migration. We previously showed that integrin alpha3beta1 activates the Rho family GTPase Rac1 and regulates Rac1-dependent formation of polarized, leading lamellipodia in migrating keratinocytes. In the present study, we explored the role of focal adhesion kinase (FAK) and src signaling in this process. We show that overexpression of the FAK inhibitor FAK-related non-kinase or of the FAK(Y397F) auto-phosphorylation mutant, induced abnormal, non-polarized spreading of keratinocytes on laminin-5. Integrin alpha3beta1 was required for full FAK auto-phosphorylation at Y397, and subsequent src kinase-dependent phosphorylation of FAK at residues Y861 and Y925, sites responsible for promoting signal transduction downstream of FAK, indicating that alpha3beta1 regulates the coordination of FAK/src signal transduction. Inhibiting either src kinase activity or FAK signaling interfered with alpha3beta1-mediated Rac1 activation and polarized cell spreading. These findings reveal a novel pathway in migratory keratinocytes wherein alpha3beta1-laminin-5 interactions regulate src kinase signaling through FAK, promoting Rac1 activation and polarized lamellipodium extension.

Focal adhesion kinase controls cellular levels of p27/Kip1 and p21/Cip1 through Skp2-dependent and -independent mechanisms.

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.

CXCL-12/stromal cell-derived factor-1alpha 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.