SDF-1alpha promotes invasion of head and neck squamous cell carcinoma by activating NF-kappaB.
ABSTRACT: CXCL12/stromal cell-derived factor-1alpha (SDF-1alpha), a chemokine ligand for the G protein-coupled receptor CXCR4, plays an important role in the directed movement of cells. Many studies have documented the importance of CXCR4 in tumor progression and organ-specific metastasis. Recently, several studies have implicated a role for SDF-1alpha in head and neck squamous cell carcinoma (HNSCC) metastasis, but currently there is little information about how SDF-1alpha promotes HNSCC metastasis. In this report we show that the NF-kappaB signaling pathway is activated in response to SDF-1alpha in HNSCC while primary and immortalized keratinocytes show no SDF-1alpha-mediated NF-kappaB activity. We found that SDF-1alpha-mediated NF-kappaB signaling is independent of phosphoinositide 3-kinase/Akt and ERK/MAPK pathways. We observed that SDF-1alpha induces IkappaBalpha phosphorylation and degradation and the nuclear translocation of NF-kappaB in HNSCC cell lines, suggesting that SDF-1alpha activates the classical NF-kappaB signaling pathway. Contrary to previous reports, SDF-1alpha-induced NF-kappaB activation is not mediated by tumor necrosis factor alpha. Furthermore, blocking the NF-kappaB signaling pathway with an IKKbeta inhibitor significantly reduces SDF-1alpha-mediated HNSCC invasion. Taken together, our data suggest SDF-1alpha/CXCR4 may promote HNSCC invasion and metastasis by activating NF-kappaB and that targeting NF-kappaB may provide therapeutic opportunities in preventing HNSCC metastasis mediated by SDF-1alpha.
Project description:Cervical cancer is frequently associated with HPV infection. The expression of E6 and E7 HPV oncoproteins is a key factor in its carcinogenicity and might also influence its virulence, including metastatic conversion. The cellular mechanisms involved in metastatic spread remain elusive, but pro-adhesive receptors and their ligands, such as SDF-1alpha and CXCR4 are implicated. In the present study, we assessed the possible relationship between SDF-1alpha/CXCR4 signaling, E6/E7 status and the metastatic process. We found that SDF-1alpha stimulated the invasion of E6/E7-positive cancer cell lines (HeLa and TC-1) in Matrigel though CXCR4 and subsequent Rho/ROCK activation. In pulmonary metastatic foci generated by TC-1 cells IV injection a high proportion of cells expressed membrane-associated CXCR4. In both cases models (in vitro and in vivo) cell adhesion and invasion was abrogated by CXCR4 immunological blockade supporting a contribution of SDF-1alpha/CXCR4 to the metastatic process. E6 and E7 silencing using stable knock-down and the approved anti-viral agent, Cidofovir decreased CXCR4 gene expression as well as both, constitutive and SDF-1alpha-induced cell invasion. In addition, Cidofovir inhibited lung metastasis (both adhesion and invasion) supporting contribution of E6 and E7 oncoproteins to the metastatic process. Finally, potential signals activated downstream SDF-1alpha/CXCR4 and involved in lung homing of E6/E7-expressing tumor cells were investigated. The contribution of the Rho/ROCK pathway was suggested by the inhibitory effect triggered by Cidofovir and further confirmed using Y-27632 (a small molecule ROCK inhibitor). These data suggest a novel and highly translatable therapeutic approach to cervix cancer, by inhibition of adhesion and invasion of circulating HPV-positive tumor cells, using Cidofovir and/or ROCK inhibition.
Project description:Stromal cell-derived factor-1alpha (SDF-1alpha) binding to its cognate receptor, CXCR4, regulates a variety of cellular functions such as stem cell homing, trafficking, and differentiation. However, the role of the SDF-1alpha-CXCR4 axis in modulating myocardial ischemia/reperfusion injury is unknown.In mice subjected to ischemic preconditioning, myocardial SDF-1alpha mRNA was found to be increased 3 hours later (P<0.05). Myocardial SDF-1alpha and CXCR4 mRNA and protein were found to be expressed in both cardiac myocytes and fibroblasts. SDF-1alpha production increased significantly after 1 or 4 hours of hypoxia and 18 hours of reoxygenation in cultured myocytes (P<0.05) but did not change in fibroblast cultures. In isolated myocytes, CXCR4 activation by SDF-1alpha resulted in increased phosphorylation of both ERK 1/2 and AKT and decreased phosphorylation of JNK and p38. Cultured myocytes pretreated with SDF-1alpha were resistant to hypoxia/reoxygenation damage, exhibiting less lactate dehydrogenase release, trypan blue uptake, and apoptotic cell death (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay) (P<0.05). This protective effect was blocked by the CXCR4 selective antagonist AMD3100. In vivo, administration of SDF-1alpha before 30 minutes of coronary occlusion followed by 4 hours of reperfusion decreased infarct size (P<0.05). The decrease in infarct size with SDF-1alpha administration also was blocked by AMD3100.We conclude that SDF-1alpha and its receptor, CXCR4, constitute a paracrine or autocrine axis in cardiac myocytes that is activated in response to preconditioning and hypoxic stimuli, recruiting the antiapoptotic kinases ERK and AKT and promoting an antiapoptotic program that confers protection against ischemia/reperfusion damage.
Project description:Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4-mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4-mediated phosphoproteome, including construction of kinase-substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, ?-catenin, and PPP1C? in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy.
Project description:SDF-1alpha/CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions. We previously reported that bone marrow-derived stromal cells inhibit chemotherapy-induced apoptosis in acute myeloid leukemia (AML). Here we demonstrate that the CXCR4 inhibitor AMD3465 antagonized stromal-derived factor 1alpha (SDF-1alpha)-induced and stroma-induced chemotaxis and inhibited SDF-1alpha-induced activation of prosurvival signaling pathways in leukemic cells. Further, CXCR4 inhibition partially abrogated the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. Fetal liver tyrosine kinase-3 (FLT3) gene mutations activate CXCR4 signaling, and coculture with stromal cells significantly diminished antileukemia effects of FLT3 inhibitors in cells with mutated FLT3. Notably, CXCR4 inhibition increased the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of the FLT3 inhibitor sorafenib. In vivo studies demonstrated that AMD3465, alone or in combination with granulocyte colony-stimulating factor, induced mobilization of AML cells and progenitor cells into circulation and enhanced antileukemic effects of chemotherapy and sorafenib, resulting in markedly reduced leukemia burden and prolonged survival of the animals. These findings indicate that SDF-1alpha/CXCR4 interactions contribute to the resistance of leukemic cells to signal transduction inhibitor- and chemotherapy-induced apoptosis in systems mimicking the physiologic microenvironment. Disruption of these interactions with CXCR4 inhibitors represents a novel strategy of sensitizing leukemic cells by targeting their protective bone marrow microenvironment.
Project description:Tyrosine sulfation of the chemokine receptor CXCR4 enhances its interaction with the chemokine SDF-1alpha. Given similar post-translational modification of other receptors, including CCR5, CX3CR1 and CCR2b, tyrosine sulfation may be of universal importance in chemokine signaling. N-terminal domains from seven transmembrane chemokine receptors have been employed for structural studies of chemokine-receptor interactions, but never in the context of proper post-translational modifications known to affect function. A CXCR4 peptide modified at position 21 by expressed tyrosylprotein sulfotransferase-1 and unmodified peptide are both disordered in solution, but bind SDF-1alpha with low micromolar affinities. NMR and fluorescence polarization measurements showed that the CXCR4 peptide stabilizes dimeric SDF-1alpha, and that sulfotyrosine 21 binds a specific site on the chemokine that includes arginine 47. We conclude that the SDF-1alpha dimer preferentially interacts with receptor peptide, and residues beyond the extreme N-terminal region of CXCR4, including sulfotyrosine 21, make specific contacts with the chemokine ligand.
Project description:Interleukin-18 (IL-18) plays pivotal roles in linking inflammatory immune responses and tumor progression and metastasis, yet the manner in which this occurs remains to be sufficiently clarified. Here we report that hypoxia induces the transcription and secretion of IL-18, which subsequently induces the expression of hypoxia-inducible factor-1alpha (HIF-1alpha). Mechanistically, IL-18 induces HIF-1alpha through the activity of the GTPase Rac1, which inducibly associates with the IL-18 receptor beta (IL-18Rbeta) subunit, via a PI3K-AKT-NF-kappaB-dependent pathway. Importantly, the knockdown of the IL-18Rbeta subunit inhibited IL-18-driven tumor cell metastasis. Collectively, these findings demonstrate a feed-forward pathway in HIF-1alpha-mediated tumor progression, in which the induction of IL-18 by hypoxia or inflammatory cells augments the expression of both HIF-1alpha and tumor cell metastasis.
Project description:Ischemia exists in many diseased tissues, including arthritic joints, atherosclerotic plaques, and malignant tumors. Macrophages accumulate in these sites and up-regulate hypoxia-inducible transcription factors (HIFs) 1 and 2 in response to the hypoxia present. Here we show that the gene expression profile in primary human and murine macrophages changes markedly when they are exposed to hypoxia for 18 hours. For example, they were seen to up-regulate the cell surface receptors, CXCR4 and GLUT1, and the potent, tumor-promoting cytokines, vascular endothelial growth factor A, interleukin (IL)-1beta and IL-8, adrenomedullin, CXCR4, and angiopoietin-2. Hypoxia also stimulated their expression and/or phosphorylation of various proteins in the nuclear factor-kappaB (NF-kappaB) signaling pathway. We then used both genetic and pharmacologic methods to manipulate the levels of HIFs-1alpha and 2alpha or NF-kappaB in primary macrophages to elucidate their role in the hypoxic induction of many of these key genes. These studies showed that both HIF-1 and -2, but not NF-kappaB, are important transcriptional effectors regulating the responses of macrophages to such a period of hypoxia. Further studies using experimental mouse models are now warranted to investigate the role of such macrophage responses in the progression of various diseased tissues, such as malignant tumors.
Project description:The NL4.3 T-cell-line-tropic human immunodeficiency virus type 1 strain is sensitive to the CXC chemokine stromal cell-derived factor 1alpha (SDF-1alpha), the natural ligand for CXC chemokine receptor 4 (CXCR4); the 50% inhibitory concentration (IC50) in MT-4 cells is 130 ng/ml. We generated resistant virus through passaging of the virus in the presence of increasing concentrations of SDF-1alpha. After 24 passages, the virus was no longer sensitive to SDF-1alpha (SDF-1alpha(res) virus) (IC50, >2 microg/ml) and became resistant to SDF-1beta (IC50, >2 microg/ml) and to a specific CXCR4 monoclonal antibody (IC50, >20 microg/ml). The SDF-1alpha(res) virus was about 10-fold less sensitive than the wild-type virus to the bicyclam AMD3100, a specific CXCR4 antagonist. The SDF-1alpha(res) virus contained the following mutations in the gp120 molecule: N106K in the V1 loop; S134N and F145L in the V2 loop; F245I in the C2 loop; K269E, Q278H, I288V, and N293D in the V3 loop; a deletion of 5 amino acids (FNSTW) at positions 364 to 368 in the V4 loop; and R378T in the CD4 binding domain. Replication of the NL4.3 wild-type virus and the SDF-1alpha(res) virus was demonstrated in U87 cells that coexpressed CD4 and CXCR4 (U87.CD4.CXCR4) but not in U87.CD4.CCR5 cells. Thus, the resistant virus was not able to switch to the CC chemokine receptor 5 (CCR5) coreceptor (the main coreceptor for macrophage-tropic viruses). The SDF-1alpha(res) virus replicated in HOS.CD4 cells expressing CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4 but also in HOS.CD4.pBABE cells. However, all HOS transfectant cells expressed a low level of CXCR4. Neither of the two virus strains was able to infect HOS.CXCR4 or HOS.CCR5 transfectants, demonstrating the necessity of the CD4 receptor. The T-cell-line-tropic SDF-1alpha(res) virus was thus able to overcome the inhibitory effect of SDF-1alpha through mutations in gp120 but still needed CXCR4 to enter the cells.
Project description:Insights into the interacting mode of CXCR4 with SDF-1alpha are crucial in understanding the structural and functional characteristics of CXCR4 receptor. In this paper a computational pipeline, integrating protein structure prediction, molecular dynamics simulations, automated molecular docking, and Brownian dynamics simulations were employed to investigate the dynamic and energetic aspects of CXCR4 associating with SDF-1alpha. The entire simulation revealed the surface distribution feature of electrostatic potentials and conformational "open-close" process of the receptor. The possible binding conformation of CXCR4 was identified, and the CXCR4-SDF-1alpha binding complex was generated. Arg188-Glu277 salt bridge plays an important role for both the extracellular domain conformational change and SDF-1alpha binding. Two binding sites were mapped at the extracellular domain (Site 1) and inside the transmembrane domain (Site 2), which are composed of conserved residues. Sites 1 and 2 contribute approximately 60% and 40% to the binding affinity with SDF-1alpha, respectively. The binding model is in agreement with most of the experimental data. Transmembrane VI has more significant motion in the harmonious conformational transition of CXCR4 during SDF-1alpha binding, which may be possibly associated with signal transduction. Based on the modeling and simulation, a binding mechanism hypothesis between CXCR4 and SDF-1alpha and its relationship to the signal transduction has been proposed.
Project description:SDF-1alpha is a member of the chemokine family implicated in various reactions in the immune system. The interaction of SDF-1alpha with its receptor, CXCR4, is responsible for metastasis of a variety of cancers. SDF-1alpha is also known to play a role in HIV-1 pathogenesis. The structures of SDF-1alpha determined by NMR spectroscopy have been shown to be monomeric while X-ray structures are dimeric. Biochemical data and in vivo studies suggest that dimerization is likely to be important for the function of chemokines. We report here the dynamics of SDF-1alpha determined through measurement of main chain (15)N NMR relaxation data. The data were obtained at several concentrations of SDF-1alpha and used to determine a dimerization constant of approximately 5 mM for a monomer-dimer equilibrium. The dimerization constant was subsequently used to extrapolate values for the relaxation data corresponding to monomeric SDF-1alpha. The experimental relaxation data and the extrapolated data for monomeric SDF-1alpha were analyzed using the model free approach. The model free analysis indicated that SDF-1alpha is rigid on the nano- to picosecond timescale with flexible termini. Several residues involved in the dimer interface display slow micro- to millisecond timescale motions attributable to chemical exchange such as monomer-dimer equilibrium. NMR relaxation measurements are shown to be applicable for studying oligomerization processes such as the dimerization of SDF-1alpha.