Upregulation Of CXCR4 Expression Research Articles

HIV glycoprotein gp120 enhances mesenchymal stem cell migration by upregulating CXCR4 expression

BackgroundHIV infection and/or the direct pathogenic effects of circulating HIV proteins impairs the physiological function of mesenchymal stem cells (MSCs), and contribute to the pathogenesis of age-related clinical comorbidities in people living with HIV. The SDF-1/CXCR4 pathway is vital for modulating MSC proliferation, migration and differentiation. HIV glycoprotein gp120 inhibits SDF-1 induced chemotaxis by downregulating the expression and function of CXCR4 in monocytes, B and T cells. The influence of gp120 on CXCR4 expression and migration in MSCs is unknown. MethodsWe investigated CXCR4 expression and SDF-1/CXCR4-mediated MSC migration in response to gp120, and its effect on downstream signaling pathways: focal adhesion kinase (FAK)/Paxillin and extracellular signal-regulated kinase (ERK). ResultsGp120 upregulated MSC CXCR4 expression. This potentiated the effects of SDF-1 in inducing chemotaxis; FAK/Paxillin and ERK pathways were over-activated, thereby facilitating actin stress fiber reorganization. CXCR4 blockage or depletion abrogated the observed effects. ConclusionGp120 from both T- and M- tropic HIV strains upregulated CXCR4 expression in MSCs, resulting in enhanced MSC chemotaxis in response to SDF-1. General significanceHIV infection and its proteins are known to disrupt physiological differentiation of MSC; increased gp120-driven migration amplifies the total MSC population destined for ineffective and inappropriate differentiation, thus contributing to the pathogenesis of HIV-related comorbidities. Additionally, given that MSCs are permissive to HIV infection, initial cellular priming by gp120 results in increased expression of CXCR4 and could lead to co-receptor switching and cell tropism changes in chronic HIV infection and may have implications against CCR5-knockout based HIV cure strategies.

Cyclam-Modified PEI for Combined VEGF siRNA Silencing and CXCR4 Inhibition To Treat Metastatic Breast Cancer.

Chemokine receptor CXCR4 plays an important role in cancer cell invasion and metastasis. Recent findings suggest that anti-VEGF therapies upregulate CXCR4 expression, which contributes to resistance to antiangiogenic therapies. Here, we report the development of novel derivatives of polyethylenimine (PEI) that effectively inhibit CXCR4 while delivering anti-VEGF siRNA. PEI was alkylated with different amounts of a CXCR4-binding cyclam derivative to prepare PEI-C. Modification with the cyclam derivatives resulted in a considerable decrease in cytotoxicity when compared with unmodified PEI. All the PEI-C showed significant CXCR4 antagonism and the ability to inhibit cancer cell invasion. Polyplexes of PEI-C prepared with siVEGF showed effective silencing of the VEGF expression in vitro. In vivo testing in a syngeneic breast cancer model showed promising antitumor and antimetastatic activity of the PEI-C/siVEGF polyplexes. Our data demonstrate the feasibility of using PEI-C as a carrier for simultaneous VEGF silencing and CXCR4 inhibition for enhanced antiangiogenic cancer therapies.

NFκB-Mediated Invasiveness in CD133+ Pancreatic TICs Is Regulated by Autocrine and Paracrine Activation of IL1 Signaling.

Tumor-initiating cells (TIC) have been implicated in pancreatic tumor initiation, progression, and metastasis. Among different markers that define this cell population within the tumor, the CD133+ cancer stem cell (CSC) population has reliably been described in these processes. CD133 expression has also been shown to functionally promote metastasis through NF-κB activation in this population, but the mechanism is unclear. In the current study, overexpression of CD133 increased expression and secretion of IL1β (IL1B), which activates an autocrine signaling loop that upregulates NF-κB signaling, epithelial-mesenchymal transition (EMT), and cellular invasion. This signaling pathway also induces CXCR4 expression, which in turn is instrumental in imparting an invasive phenotype to these cells. In addition to the autocrine signaling of the CD133 secreted IL1β, the tumor-associated macrophages (TAM) also produced IL1β, which further activated this pathway in TICs. The functional significance of the TIC marker CD133 has remained elusive for a very long time; the current study takes us one step closer to understanding how the downstream signaling pathways in these cells regulate the functional properties of TICs.Implications: This study demonstrates the important role of tumor- and macrophage-derived IL1β stimulation in pancreatic cancer. IL1 signaling is increased in cells with CD133 expression, leading to increased NF-kB activity, EMT induction, and invasion. Increased invasiveness via IL1β stimulation is mediated by the upregulation of CXCR4 expression. The study highlights the importance of IL1-mediated signaling in TICs. Mol Cancer Res; 16(1); 162-72. ©2017 AACR.

Monocytes/Macrophages promote vascular CXCR4 expression via the ERK pathway in hepatocellular carcinoma

ABSTRACTWe recently identified CXCR4 as a novel vascular marker for vessel sprouting in hepatocellular carcinoma (HCC) tissues. Thus, CXCR4+ endothelial cells (ECs) could serve as a potential predictor for patients who may benefit from sorafenib treatment; however, the mechanism that regulates vascular CXCR4 expression in HCC remains largely unknown. Here, we revealed a large number of monocytes/macrophages (Mo/Mφ) to be selectively enriched in the perivascular areas of CXCR4+ vessels in HCC samples. The depletion of Mo/Mφ with gadolinium chloride (GdCl3) or zoledronic acid (ZA) treatment significantly reduced vascular CXCR4 expression in HCC tumors. This phenomenon was also confirmed in CCR2-KO mice, which exhibited reduced infiltration of inflammatory Mo/Mφ in tumor tissues. Mechanistic studies revealed that inflammatory cytokines derived from tumor conditioned Mo/Mφ, especially TNF-α, could up-regulate CXCR4 expression on ECs. TNF-α-induced activation of the Raf-ERK pathway, but not Notch signaling, was responsible for the expression of CXCR4. Moreover, the combination treatment of sorafenib with ZA was associated with improved anti-tumor efficacy by significantly reducing vascular CXCR4 expression. These findings revealed that Mo/Mφ could regulate CXCR4 expression in the tumor vasculature. Thus, the inhibition of Mo/Mφ inflammation might enhance the treatment efficacy of sorafenib in HCC.

BAG3 promotes stem cell-like phenotype in breast cancer by upregulation of CXCR4 via interaction with its transcript

BAG3 is an evolutionarily conserved co-chaperone expressed at high levels and has a prosurvival role in many tumor types. The current study reported that BAG3 was induced under specific floating culture conditions that enrich breast cancer stem cell (BCSC)-like cells in spheres. Ectopic BAG3 overexpression increased CD44+/CD24− CSC subpopulations, first-generation and second-generation mammosphere formation, indicating that BAG3 promotes CSC self-renewal and maintenance in breast cancer. We further demonstrated that mechanically, BAG3 upregulated CXCR4 expression at the post-transcriptional level. Further studies showed that BAG3 interacted with CXCR4 mRNA and promoted its expression via its coding and 3′-untranslational regions. BAG3 was also found to be positively correlated with CXCR4 expression and unfavorable prognosis in patients with breast cancer. Taken together, our data demonstrate that BAG3 promotes BCSC-like phenotype through CXCR4 via interaction with its transcript. Therefore, this study establishes BAG3 as a potential adverse prognostic factor and a therapeutic target of breast cancer.

Platelet activating factor-induced expression of p21 is correlated with histone acetylation

Ultraviolet (UV)-irradiated keratinocytes secrete the lipid mediator of inflammation, platelet-activating factor (PAF). PAF plays an essential role in UV-induced immune suppression and skin cancer induction. Dermal mast cell migration from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced PAF activates mast cell migration by up-regulating mast cell CXCR4 surface expression. Recent findings indicate that PAF up-regulates CXCR4 expression via histone acetylation. UV-induced PAF also activates cell cycle arrest and disrupts DNA repair, in part by increasing p21 expression. Do epigenetic alterations play a role in p21 up-regulation? Here we show that PAF increases Acetyl-CREB-binding protein (CBP/p300) histone acetyltransferase expression in a time and dose-dependent fashion. Partial deletion of the HAT domain in the CBP gene, blocked these effects. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the p21 promoter. PAF-treatment had no effect on other acetylating enzymes (GCN5L2, PCAF) indicating it is not a global activator of histone acetylation. This study provides further evidence that PAF activates epigenetic mechanisms to affect important cellular processes, and we suggest this bioactive lipid can serve as a link between the environment and the epigenome.

Transmembrane tumor necrosis factor-α promotes the recruitment of MDSCs to tumor tissue by upregulating CXCR4 expression via TNFR2

Myeloid-derived suppressor cells (MDSCs) accumulated in tumor sites promote immune evasion. We found that TNFR deficiency-induced rejection of transplanted tumor was accompanied with markedly decreased accumulation of MDSCs. However, the mechanism(s) behind this phenomenon is not completely understood. Here, we demonstrated that TNFR deficiency did not affect the amount of MDSCs in bone marrow (BM), but decreased accumulation of Gr-1+CD11b+ MDSCs in the spleen and tumor tissues. The chemotaxis of Tnfr−/− MDSCs was prominently decreased in response to both tumor cell culture supernatants and tumor tissue homogenates from Tnfr−/− and wild-type mice, indicating an effect of TNFR signaling on chemokine receptor expression in MDSCs. We used real-time PCR to detect gene expression for several chemokine receptors in MDSCs from BM and found that CXCR4 was the most affected molecule at the transcriptional level in Tnfr−/− MDSCs. Neutralizing CXCR4 in wild-type MDSCs by a specific antibody blocked their chemotactic migration. Interestingly, it was tmTNF-α, but not sTNF-α, that induced CXCR4 expression in MDSCs. This effect of tmTNF-α was totally blocked in TNFR2−/− but not in TNFR1−/− MDSCs, and partially inhibited by PDTC or SB203580, an inhibitor of NF-κB or p38 MAPK pathway, respectively. Adoptive transfer of wild-type MDSCs restored MDSCs accumulation in tumors of Tnfr−/− mice, but this could be partially blocked by treatment with a CXCR4 inhibitor AMD3100. Our data suggest that tmTNF-α upregulates CXCR4 expression that promotes chemotaxis of MDSCs to tumor, and give a new insight into a novel mechanism by which tmTNF-α facilitates tumor immune evasion.

Androgen Triggers the Pro-Migratory CXCL12/CXCR4 Axis in AR-Positive Breast Cancer Cell Lines: Underlying Mechanism and Possible Implications for the Use of Aromatase Inhibitors in Breast Cancer

Background/Aims: Reports regarding the role of androgen in breast cancer (BC) are conflicting. Some studies suggest that androgen could lead to undesirable responses in the presence of certain BC tumor characteristics. We have shown that androgen induces C-X-C motif chemokine 12 (CXCL12) in BC cell lines. Our aim was to identify the mechanisms regulating the phenotypic effects of androgen-induced CXCL12 on Androgen Receptor (AR) positive BC cell lines. Methods: We analyzed the expression of CXCL12 and its receptors with qPCR and ELISA and the role of Nuclear Receptor Coactivator 1 (NCOA1) in this effect. AR effects on the CXCL12 promoter was studied via Chromatin-immunoprecipitation. We also analyzed publically available data from The Cancer Genome Atlas to verify AR-CXCL12 interactions and to identify the effect or Aromatase Inhibitors (AI) therapy on CXCL12 expression and disease progression in AR positive cases. Results: CXCL12 induction occurs only in AR-positive BC cell lines, possibly via an Androgen Response Element, upstream of the CXCL12 promoter. The steroid receptor co-regulator NCOA1 is critical for this effect. Androgen only induced the motility of p53-mutant BC cells T47D cells via upregulation of CXCR4 expression while they had no effect on wild-type p53 MCF-7 cells. Loss of CXCR4 expression and depletion of CXCL12 abolished the effect of androgen in T47D cells while inhibition of p53 expression in MCF-7 cells made them responsive to androgen and increased their motility in the presence to androgen. Patients with estrogen receptor positive (ER+)/AR+ BC treated with AIs were at increased risk of disease progression compared to ER+/AR+ non-AI treated and ER+/AR- AI treated cases. Conclusion: AIs may lead to unfavorable responses in some ER/AR positive BC cases, especially in patients with AR+, p53 mutant tumors.

Hypoxia and low-dose inflammatory stimulus synergistically enhance bone marrow mesenchymal stem cell migration.

Cell migration is necessary for numerous physiological cell processes. Although either inflammatory or hypoxic stimuli of certain dose and duration have positive influence on cell migration, their combination has not been shown to result in a synergistic effect. In this study, we investigated combined effects of hypoxia and low-dose inflammatory stimulus (one-tenth of that of a previously used concentration) on migration of human bone marrow-derived mesenchymal stem cells (BMMSCs). Our results from real-time PCR, Western blot analysis and an immunofluorescence assay, showed that dual stimulation up-regulated CXCR4 expression. Based on tablet scratch experimentation and transwell assay, the dual stimuli exhibited greater positive effects on cell migration than a single inflammatory or hypoxic stimulus. When effects of various pre-treatments on cell proliferation, differentiation and immunosuppression were screened, cells subjected to the hypoxic stimulus or dual stimuli had increased cell proliferation, while short-term inflammatory stimulus and/or hypoxic stimulus had no negative effect on cell differentiation and immunosuppression. These findings suggest that the combination of hypoxia and low-dose inflammatory stimuli enhances the potential of BMMSCs to migrate, thus identifying cell pre-treatment conditions that could enhance future stem cell-based therapeutics.

Stromal cell-derived factor-1-directed bone marrow mesenchymal stem cell migration in response to inflammatory and/or hypoxic stimuli

ABSTRACTDirecting cell trafficking toward a target site of interest is critical for advancing stem cell therapy in clinical theranostic applications. In this study, we investigated the effects of inflammatory and/or hypoxic stimuli on the migration of bone marrow mesenchymal stem cells (BMMSCs) during in vitro culture and after in vivo implantation. Using tablet scratch experiments and observations from a transwell system, we found that both inflammatory and hypoxic stimuli significantly enhanced cell migration. However, the combination of inflammatory and hypoxic stimuli did not result in a synergistic effect. The presence of stromal cell-derived factor-1 (SDF-1) significantly enhanced cell migration irrespective of the incubation conditions, and these positive effects could be blocked by treatment with AMD3100. Based on a time course experiment, we found that preconditioning cells with either inflammatory or hypoxic stimuli for 24 h or with both stimuli for 12 h led to high levels of chemokine receptor type 4 (CXCR4) expression. In vivo studies further demonstrated that pretreatment of BMMSCs with inflammatory and/or hypoxic stimuli resulted in an increased number of systemically injected cells migrating toward skin injuries, and local SDF-1 administration significantly increased cell migration. These findings suggest that in vitro control of either inflammatory or hypoxic stimuli has significant potential to enhance SDF-1-directed BMMSC migration via the upregulation of CXCR4 expression. Although combining the stimuli did not necessarily lead to a synergistic effect, the potential to reduce the dose and time required for cell preconditioning indicates that combinations of various strategies warrant further exploration.

MP84-16 PHOSPHATIDYLINOSITOL 4-KINASE TYPE III? (PI4KA) AND CXCR4 CROSSTALK IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-16 PHOSPHATIDYLINOSITOL 4-KINASE TYPE III? (PI4KA) AND CXCR4 CROSSTALK IN PROSTATE CANCER CELLS Michael Cher, Diego Sbrissa, Louie Semaan, Yanfeng Li, Assia Shisheva, and Srinivasa Chinni Michael CherMichael Cher More articles by this author , Diego SbrissaDiego Sbrissa More articles by this author , Louie SemaanLouie Semaan More articles by this author , Yanfeng LiYanfeng Li More articles by this author , Assia ShishevaAssia Shisheva More articles by this author , and Srinivasa ChinniSrinivasa Chinni More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2240AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The CXCR4 receptor directs homing of hematopoietic stem cells to CXCL12 ligand-rich bone marrow. Previous studies demonstrated that the CXCL12/CXCR4 axis promotes prostate cancer (PC) invasion and bone metastasis. We showed that CXCR4 expression is transcriptionally upregulated by TMPRSS2-ERG gene fusions in PC tumors, and its localization in lipid raft membrane microdomains promotes expression of invasive proteases. To identify novel lipid raft-associated CXCR4 regulators supporting invasion and metastasis, we performed proteomics analysis of lipid raft microdomains from stable CXCR4-overexpressing (CXCR4) and CXCR4-knockdown (shCXCR4) PC3 cell lines generated by lentiviral infection. METHODS We used stable isotope labeling by amino acids in cell culture (SILAC) proteomics, sucrose gradient buoyant density ultracentrifugation, fluorescence activated cell sorting (FACS), knockdown by siRNA silencing, cell invasion/growth assays, [2-3H]inositol cell labeling, SDS PAGE, Western blotting (WB), and inositol-lipid HPLC profiling. RESULTS SILAC identified phosphatidylinositol 4-kinase IIIα (PI4KA) to be highly associated with CXCR4 cells, along with slightly elevated SAC1 phosphatase. WB confirmed that expression of these phosphoinositide-regulating enzymes is associated with CXCR4 overexpression, thus anticipating their promotion of invasion and metastasis. Several lines of data demonstrated a link between PI4KA and CXCR4 signaling: PI4KA knockdown (i) reduced surface CXCR4 expression and CXCL12-stimulated invasion of CXCR4 cells; (ii) blocked CXCR4/AKT signaling lowering pAKT(Ser473) and pCXCR4(Ser339); and (iii) dropped global cellular PI4P levels. Unexpectedly, PI4KA knockdown resulted in a rise in PI4,5P2, suggesting a homeostatic response. Conversely, CXCR4 knockdown showed an expected increase in PI4,5P2 but an unexpected decrease in PI4P. Importantly, human PC tumor microarray data showed higher PI4KA expression in metastatic vs. localized PC or normal adjacent tissue. CONCLUSIONS These data confirm that PI4KA upregulates CXCR4 expression by altering inositol lipid levels thereby promoting CXCR4/AKT signaling, and that inhibition of CXCR4 signaling via PI4KA knockdown can suppress PC growth/invasion. Thus, PI4KA inhibitors may beneficial in patients with prostate cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1095 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Michael Cher More articles by this author Diego Sbrissa More articles by this author Louie Semaan More articles by this author Yanfeng Li More articles by this author Assia Shisheva More articles by this author Srinivasa Chinni More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Impact of a CXCL12/CXCR4 Antagonist in Bleomycin (BLM) Induced Pulmonary Fibrosis and Carbon Tetrachloride (CCl4) Induced Hepatic Fibrosis in Mice.

Modulation of chemokine CXCL12 and its receptor CXCR4 has been implicated in attenuation of bleomycin (BLM)-induced pulmonary fibrosis and carbon tetrachloride (CCl4)-induced hepatic injury. In pulmonary fibrosis, published reports suggest that collagen production in the injured lung is derived from fibrocytes recruited from the circulation in response to release of pulmonary CXCL12. Conversely, in hepatic fibrosis, resident hepatic stellate cells (HSC), the key cell type in progression of fibrosis, upregulate CXCR4 expression in response to activation. Further, CXCL12 induces HSC proliferation and subsequent production of collagen I. In the current study, we evaluated AMD070, an orally bioavailable inhibitor of CXCL12/CXCR4 in alleviating BLM-induced pulmonary and CCl4-induced hepatic fibrosis in mice. Similar to other CXCR4 antagonists, treatment with AMD070 significantly increased leukocyte mobilization. However, in these two models of fibrosis, AMD070 had a negligible impact on extracellular matrix deposition. Interestingly, our results indicated that CXCL12/CXCR4 signaling has a role in improving mortality associated with BLM induced pulmonary injury, likely through dampening an early inflammatory response and/or vascular leakage. Together, these findings indicate that the CXCL12-CXCR4 signaling axis is not an effective target for reducing fibrosis.

Pim-1, Beyond an Oncogene, in Acute Myeloid Leukemia

Background and Objective:There are 3 members in Pim family, Pim-1, Pim-2 and Pim-3. They are serine/threonine kinase coding proto-oncogene. It is reported that Pim-1 plays a role in solid tumor, leukemia and polycythemia vera, et al. Pim-3, as a newly cloned oncogene has discovered to functioning in hepatic carcinoma, pancreatic cancer, et al. We are trying to find out the roles of Pim1 in acute myeloid leukemia.Methods:1 Evaluate the expressions of Pim family genes in acute myeloid leukemia patients and analysis the profile of Pim expressions with clinical characteristics.2 Up-regulating the expression of Pim1 in AML cell lines by transient transfection or long-term infection through GFP-expressing plasmids and lenti-virus system and analyze the proliferation, apoptosis and chemotaxis features of the transfected AML cell lines.Results:1 Our investigation showed that Pim-1 is up-regulated in around 15% of acute myeloid leukemia (AML) patients.2 As shown in growth curves and apoptosis assays, over expression of Pim-1induces growth up-regulation and anti-apoptosis. We hypothesized that phenomenon could be originated from the enhanced expression of c-myc, cyclin D1 and Bad phosphorylation shown in western blotting analysis.3 Our chemotaxis assay and bone marrow stromal cell co-culture model demonstrated that overexpression of Pim-1can enhance the AML cells chemotactic movement toward SDF-1¦Á, with calcium influx increment and phosphorylation of CXCR4.4 Flow cytometry analysis and confocal immunofluorescence observation demonstrated the up-regulated CXCR4 expression and internalization induced by SDF-1¦Á.5 We also checked the angiogenesis and adhesion molecule during the process but did not show any contribution.Conclusion:Pim-1, beyond as an oncogene, can promote growth or anti-apoptosis of AML cells, can interact with microenviroment through SDF-1¦Á-CXCR4 axis. The interplay between AML cells and microenviroment mediated by Pim-1 can make sense in AML target therapy and make a good adjunctive for transplantation conditioning regimen. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effect of silencing HIF-1α by RNA interference on adhesion and invasion of the human nasopharyngeal carcinoma cell line CNE-1

To investigate the effects of HIF-1α on adhesion and invasion of human nasopharyngeal carcinoma CNE-1 cells under hypoxia and underlying molecular mechanisms. CoCl₂was used to mimic tumor hypoxic microenvironment. mRNA and protein expressions of HIF-1α, E-cadherin and CXCR4 in CNE-1 cells at different hypoxic time phases were detected by RT-PCR and ELISA respectively. The influences of silencing HIF-1α using RNA interference on E-cadherin and CXCR4 expressions were evaluated. Adhesion test Transwell invasion test were used to evaluate the effects of HIF-1α gene silencing on cell adhesion and invasion. Under hypoxia, HIF-1α mRNA expression in CNE-1 cells was stable, but its protein expression increased obviously (P<0.05). Both mRNA and protein expressions of E-cadherin were decreased significantly with prolonged hypoxia, while mRNA and protein expressions of CXCR4 increased significantly (P<0.05). After silencing HIF-1α gene, expression of E-cadherin protein was up-regulated, but with down-regulated expression of CXCR4 protein, with a decrease significantly in adhesion rate or invasive cell number of CNE-1 cells (P<0.05). Hypoxia can increase HIF-1α protein expression in nasopharyngeal carcinoma cell line CNE-1. Silencing HIF-1α by RNA interference can reduce inhesion and invasion abilities of CNE-1 cells, which may be mediated by down-regulating E-cadherin expression and up-regulating CXCR4 expression.

Tetramethylpyrazine (TMP), an Active Ingredient of Chinese Herb Medicine Chuanxiong, Attenuates the Degeneration of Trabecular Meshwork through SDF-1/CXCR4 Axis.

BackgroundA traditional Chinese medicine, Tetramethylpyrazine (TMP), has been prescribed as a complementary treatment for glaucoma to improve patient prognosis. However, the pharmacological mechanism of action of TMP is poorly understood. In previous studies, we demonstrated that TMP exerts potent inhibitory effects on neovascularization, suppresses the tumorigenic behavior of glioma cells, and protects neural cells by regulating CXCR4 expression. Here, we further investigated whether the SDF-1/CXCR4 pathway is also involved in the TMP-mediated activity in trabecular meshwork cells.Methodology/Principal FindingsCXCR4 expression was examined by quantitative real-time PCR in trabecular and iris specimens from 54 primary open-angle glaucoma (POAG) patients who required surgery and 19 non-glaucomatous donors. Our data revealed markedly elevated CXCR4 expression in the trabecular meshwork of POAG patients compared with that of controls. Consistently, CXCR4 expression was much higher in glaucomatous trabecular meshwork cells than in normal trabecular meshwork cells. Using RT-PCR and western blot assays, we determined that glaucoma-related cytokines and dexamethasone (DEX) also significantly up-regulated CXCR4 expression in primary human trabecular meshwork (PHTM) cells. Moreover, the TGF-β1-mediated induction of CXCR4 expression in PHTM cells was markedly down-regulated by TMP compared with control treatment (PBS) and the CXCR4 antagonist AMD3100. In addition, TMP could counteract the TGF-β1-induced effects on stress fiber accumulation and expansion of PHTM cells. TMP markedly suppressed the migration of PHTM cells stimulated by TGF-β1 in transwell and scratch wound assays. TMP also suppressed the extracellular matrix (ECM) accumulation induced by TGF-β2.ConclusionsOur findings demonstrate that CXCR4 might be involved in the pathogenetic changes in the trabecular meshwork of patients with POAG. Additionally, TMP might exert its beneficial effects in POAG patients by down-regulating CXCR4 expression.

Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma.

Chronic inflammation contributes to tumor development through the induction of oncogenic mutations, genomic instability, early tumor promotion, and enhanced angiogenesis. Here, we report that IL-1 receptor 1 (IL-1R1) was expressed in 40 of 41 human tongue squamous cell carcinomas (TSCC). IL-1β up-regulated the expression of CXCR4, a CXC chemokine receptor that mediates cancer growth and metastasis, at both mRNA and protein levels in Tca8113 TSCC cells. IL-1β treatment of Tca8113 cells promoted migration in response to CXCR4 ligand stromal-derived factor α (SDF-1α). The inhibition of IL-1R1 by its antagonist IL-1Ra or RNA interference significantly reversed the up-regulation of CXCR4 induced by IL-1β. IL-1R1 activation also up-regulated the expression of IL-1β itself, suggesting a positive feedback regulation of CXCR4 expression. Furthermore, IL-1β induced the activation of Notch, which was originally considered a stem cell regulator. Pharmacological inhibition of Notch signaling reversed the up-regulation of CXCR4 induced by IL-1β, suggesting that Notch signaling may be involved in the growth and metastasis of cancers via up-regulation of CXCR4. In addition, IL-1β induced the activation of extracellular signal regulated kinase (ERK) and ERK inhibition decreased the up-regulation of CXCR4 induced by IL-1β, suggesting the involvement of ERK signaling in cancer metastasis. Taken together these data suggest that IL-1β and IL-1R1 promote cancer growth and metastasis by up-regulating CXCR4 expression and that CXCR4 may be a link between inflammation and cancer.

Targeting survival and cell trafficking in multiple myeloma and Waldenstrom macroglobulinemia using pan-class I PI3K inhibitor, buparlisib.

The phosphatidylinositol-3 kinase (PI3K) pathway is activated in multiple myeloma (MM) and Waldenstrom Macroglobulenima (WM), and plays a crucial role in tumor progression and drug resistance. In this study, we characterized the role of pan-class I PI3K inhibition on cell trafficking and survival of MM and WM cells. We tested the effect of pan-class I PI3K inhibition by siRNA silencing or pharmacologic inhibition with buparlisib on MM cell survival, apoptosis and cell cycle in vitro and tumor growth and mobilization of MM cells in vivo. We then evaluated buparlisib-dependent mechanisms of induced MM cell mobilization. Moreover, the effect of buparlisib on cell survival, apoptosis, and adhesion of WM cells to bone marrow stromal cells (BMSCs) has been evaluated. We showed that buparlisib induced toxicity in MM cells, supported by induction of apoptosis and cell cycle arrest. Buparlisib was also found to reduce tumor progression in vivo. Importantly, buparlisib enhanced MM cell mobilization in vivo which was driven by decreased adhesion of MM cells to BMSCs and increased chemotaxis via up-regulation of CXCR4 expression. Similar to its effects on MM cells, buparlisib also induced cell survival and apoptosis, and decreased adhesion in WM cells. These data highlight the critical contribution of class I PI3K signaling to the regulation of survival and cell dissemination in B-cell malignancies.

Abstract 245: Plasminogen Augments Cxcl12/cxcr4-regulated Stem Cell Homing And Contributes To Cardiac Repair After Myocardial Infarction.

Myocardial infarction (MI) is a leading cause of morbidity and mortality worldwide. Bone marrow (BM)-derived stem cells promote tissue repair and regeneration after MI. Thrombolytic treatment with plasminogen (Plg) activators significantly improves the clinical outcome in MI by restoration of cardiac perfusion. In addition to its canonical function, Plg is critical for cardiac repair, wound healing and liver injury, however, the mechanism for Plg-regulated tissue repair remains unclear. Here, we show a novel role of Plg in stem cell-mediated neovascularization and cardiac repair after MI. Our data show that Granulocyte colony-stimulating factor (G-CSF), a stem cell mobilizer, significantly increased neovascularization and decreased infarct size in the infarct area, and improved ejection fraction and LV internal diameter by echocardiogram in wild-type mice. No improvement in tissue repair and heart function was observed in Plg deficient (Plg-/-) mice indicating that Plg is required for stem cell-regulated cardiac repair after MI. In vivo tracking of GFP-expressing BM cells after BM transplantation revealed that in Plg-/- mice, recruitment of BM-derived stem cells (GFP+c-kit+ cells) to the infarcted heart and stem cell-derived vessels and arteries are dramatically decreased (by 11 fold) suggesting that Plg may regulate stem cell homing to the lesion sites and subsequently contribute stem cell-mediated tissue regeneration. Mechanistic studies show that Plg up-regulated CXCR4 expression on stem cell in vivo and in vitro, suggesting Plg may promotes stem cell homing by induction of CXCR4 expression in stem cells. Stem cell migration was enhanced by endogenous Plg in vitro, however, AMD3100, a CXCR4 antagonist, significantly inhibited Plg-regulated stem cell migration. Furthermore, lentiviral reconstitution of CXCR4 expression in BM cells rescued stem cell homing to the infarcted heart in Plg-/- mice, indicating that Plg mediates stem cell homing through regulating CXCR4 expression. These findings identified a novel role of Plg in cardiac repair by promoting stem cell homing to the injured heart after MI. Thus, targeting Plg may offer a new therapeutic strategy for strengthening stem cell-mediated cardiac repair and regeneration after MI.

Effect of Helicobacter pylori infection on stromal-derived factor-1/CXCR4 axis in bone marrow-derived mesenchymal stem cells

Background:Recent studies have demonstrated that during chronic Helicobacter pylori (H. pylori) infection bone marrow-derived-mesenchymal stem cells (BMD-MSCs) migrate to the gastric tissue and could be also the origin of gastric adenocarcinoma. The chemokine CXCR4 through binding to its ligand stromal-derived factor (SDF-1) plays a crucial role in migration of inflammatory and stem cells. However, the possible effect of H. pylori infection on the SDF-1/CXCR4 axis has not yet been elucidated.Materials and Methods:Gastric epithelial cell line, AGS, and BMD-MSCs were cocultured with H. pylori for 24 h. The expression of CXCR4 was examined in BMD-MSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry, and SDF-1 expression in AGS cells was detected by qRT-PCR and enzyme-linked immunosorbent assay. Further, migration of BMD-MSCs toward SDF-1 was evaluated by chemotaxis assay.Results:We found that coculture of H. pylori with BMD-MSCs or AGS: (i) enhanced CXCR4 expression on the cell surface of BMD-MSCs and (ii) increased SDF-1 secretion by AGS cells. Consistently, we observed that H. pylori-treated BMD-MSCs showed a higher capability to migrate toward SDF-1 gradient compared with untreated cells.Conclusion:We found that H. pylori upregulates CXCR4 expression in BMD-MSCs and enhance their migration toward SDF-1. This study provides the first evidence that H. pylori infection may enhance BMD-MSC migration through acting on the SDF-1/CXCR4 axis.

Abstract 4794: An undesired effect of chemotherapy: gemcitabine promotes pancreatic cancer cell invasiveness through upregulation of CXCR4.

Abstract Pancreatic cancer (PC) remains a therapeutic challenge with a rising incidence and unabated mortality. Gemcitabine (GEM), the current standard of care drug, is largely ineffective due to chemoresistance, thus underscoring the need for a refined understanding of the molecular mechanisms that drive drug-resistant nature of this malignancy. Recently, we have shown that CXCL12/CXCR4 signaling axis is involved in conferring chemoresistance to PC cells. Here, we explored the effect of GEM on this resistance mechanism and its functional consequences. Our data demonstrated that GEM upregulated CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time- dependent manner. GEM-induced CXCR4 expression was dependent on generation of reactive oxygen species (ROS) as it was abrogated upon pre-treatment of PC cells with radical scavenger N-acetyl-L-cysteine (NAC). CXCR4 upregulation by GEM correlated with time-dependent accumulation of p65/NF-κB and HIF-1α in the nucleus. An enhanced binding of p65/NF-κB and HIF-1α to the CXCR4 promoter was observed in chromatin immunoprecipitation assay in GEM-treated PC cells, while their silencing by RNA interference suppressed GEM-induced CXCR4 expression. ROS induction upon GEM treatment preceded the nuclear accumulation of p65/NF-κB and HIF-1α and suppression of ROS diminished these effects. Effect of ROS on p65/NF-κB and HIF-1α was mediated through activation of ERK1/2 and Akt and their pharmacological inhibition also suppressed GEM-induced CXCR4 upregulation. Lastly, our data demonstrated that GEM-treated PC cells were more motile and exhibited significantly greater invasiveness against a CXCL12 gradient. Altogether, these findings reinforces the role of CXCL12/CXCR4 signaling in GEM resistance and point toward an undesired effect of chemotherapy that is potentiation of invasion and spread of resistant tumor cells. Citation Format: Sumit Arora, Arun Bhardwaj, Seema Singh, Sanjeev K. Srivastava, Steven McClellan, William E. Grizzle, Laurie B. Owen, Ajay P. Singh. An undesired effect of chemotherapy: gemcitabine promotes pancreatic cancer cell invasiveness through upregulation of CXCR4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4794. doi:10.1158/1538-7445.AM2013-4794