CXC Chemokine Receptor 4 Signaling Research Articles

Berberine-Promoted CXCR4 Expression Accelerates Endothelial Repair Capacity of Early Endothelial Progenitor Cells in Persons with Prehypertension.

To evaluate whether the berberine treatment can improve endothelial repair capacity of early endothelial progenitor cells (EPCs) from prehypertensive subjects through increasing CXC chemokine receptor 4 (CXCR4) signaling. EPCs were isolated from prehypertensive and healthy subjects and cultured. In vivo reendothelialization capacity of EPCs from prehypertensive patients with or without in vitro berberine treatment was examined in a nude mouse model of carotid artery injury. The protein expressions of CXCR4/Janus kinase-2 (JAK-2) signaling of in vitro EPCs were detected by Western blot analysis. CXCR4 signaling and alteration in migration and adhesion functions of EPCs were evaluated. Basal CXCR4 expression was significantly reduced in EPCs from prehypertensive patients compared with normal subjects (P<0.01). Also, the phosphorylation of JAK-2 of EPCs, a CXCR4 downstream signaling, was significantly decreased (P<0.01). Berberine promoted CXCR4/JAK-2 signaling expression of in vitro EPCs (P<0.01). Transplantation of EPCs pretreated with berberine markedly accelerated in vivo reendothelialization (P<0.01). The increased in vitro function and in vivo reendothelialization capacity of EPCs were inhibited by CXCR4 neutralizing antibody or pretreatment with JAK-2 inhibitor AG490, respectively (P<0.01). Berberinemodified EPCs via up-regulation of CXCR4 signaling contributes to enhanced endothelial repair capacity in prehypertension, indicating that berberine may be used as a novel potential primary prevention means against prehypertension-related atherosclerotic cardiovascular disease.

Suppression of the SDF‑1/CXCR4/β‑catenin axis contributes to bladder cancer cell growth inhibition invitro and invivo.

Previous studies have found that the activation of stromal cell‑derived factor‑1 (SDF‑1)/CXC chemokine receptor‑4 (CXCR4)/β‑catenin signaling is associated with biological malignant potential in cancers. However, its function has been rarely reported in the progression of bladder cancer (BCa). The aim of the present study was to investigate the association of SDF‑1/CXCR4 signaling and β‑catenin in regards to BCa cell proliferation, colony formation, migration and invasion. The methods used were MTS, colony formation, and Transwell migration and invasion assays which were performed in SW780 cells following treatment with the CXCR4 antagonist AMD3465, SDF‑1, the β‑catenin antagonist FH535, AMD3465+SDF‑1 or FH535+SDF‑1. The mRNA and protein levels were assayed by RT‑qPCR and western blotting, respectively. The effect of AMD3465 on SW780 cell xenograft growth invivo was evaluated using a nude mouse model. According to our results, human BCa SW780 cells were identified as having high expression of CXCR4 and β‑catenin. Subsequently, we found that both CXCR4 and β‑catenin antagonists could significantly inhibit the proliferation, colony formation, migration and invasion of SW780 cells. Notably, SDF‑1 could reverse the inhibitory effects of AMD3465 and FH535 on proliferation, colony formation, migration and invasion in SW780 cells. In AMD3465‑treated SW780 cells, the expression of c‑myc was significantly upregulated, and E‑cadherin was downregulated in the presence of SDF‑1. Furthermore, the tumor volume and average weight in the AMD3465‑treated group were evidently less than these parameters in the control group, indicating that AMD3465 can inhibit SW780 cell growth invivo. In conclusion, targeting the SDF‑1/CXCR4/β‑catenin axis may be a potential therapeutic target for suppressing BCa progression.

Inhibition of HIV Fusion by Small Molecule Agonists through Efficacy-Engineering of CXCR4.

CXC chemokine receptor 4 (CXCR4) is involved in multiple physiological and pathological processes, notably as a coreceptor for human immunodeficiency virus (HIV) cell entry. Its broad expression pattern and vital biological importance make CXCR4 a troublesome drug target, as disruption of the interaction with its endogenous ligand, CXC chemokine ligand 12 (CXCL12), has severe consequences. In fact, only one CXCR4 drug, the bicyclam antagonist and HIV entry inhibitor AMD3100 (Plerixafor/Mozobil), has been approved for clinical use, however only for stem cell mobilization-a consequence of CXCR4 antagonism. Here, we report the engineering of an efficacy switch mutation in CXCR4-F292A7.43 in the middle of transmembrane helix 7-that converted the antagonists AMD3100 and AMD11070 into partial agonists. As agonists on F292A CXCR4, AMD3100 and AMD11070 were less disruptive to CXCR4 signaling while they remained efficient inhibitors of HIV fusion. This demonstrates that small molecule CXCR4 agonists can have a therapeutic potential as HIV entry inhibitors.

Critical role of SDF-1/CXCR4 signaling pathway in stem cell homing in the deafened rat cochlea after acoustic trauma.

Previous animal studies have shown that stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor-4 (CXCR4) signaling pathway plays an important role in the targeted migration of bone marrow-derived mesenchymal stem cells (BMSCs) to the injured area. In the present study, we aimed to investigate the potential role of chemotactic SDF-1/CXCR4 signaling pathway in the homing of transplanted BMSCs to the injured cochlea after noise-induced hearing loss (NIHL) in a rat model. White noise exposure (110 dB) paradigm was used for hearing loss induction in male rats for 6 hours in 5 days. Distortion-product otoacoustic emission (DPOAE) responses were recorded before the experiment and post noise exposure. Hoechst 33342-labeled BMSCs and CXCR4 antagonist (AMD3100)-treated BMSCs were injected into the rat cochlea through the round window. SDF-1 protein expression in the cochlear tissue was assayed using western blot assay. The number of labeled BMSCs reaching the endolymph was determined after 24 hours. SDF-1 was significantly increased in the cochlear tissue of rats in the noise exposure group than in the control group. The number of Hoechst 33342-labeled BMSCs reaching the endolymph of the cochlea was significantly smaller in the AMD3100-treated BMSCs group than in the normal BMSCs group. Our present findings suggest that the SDF-1/CXCR4 signaling pathway has a critical role in BMSCs migration to the injured cochlea in a rat model of noise-induced hearing loss.

Targeting CXCR4-dependent immunosuppressive Ly6Clow monocytes improves antiangiogenic therapy in colorectal cancer

Antiangiogenic therapy with antibodies against VEGF (bevacizumab) or VEGFR2 (ramucirumab) has been proven efficacious in colorectal cancer (CRC) patients. However, the improvement in overall survival is modest and only in combination with chemotherapy. Thus, there is an urgent need to identify potential underlying mechanisms of resistance specific to antiangiogenic therapy and develop strategies to overcome them. Here we found that anti-VEGFR2 therapy up-regulates both C-X-C chemokine ligand 12 (CXCL12) and C-X-C chemokine receptor 4 (CXCR4) in orthotopic murine CRC models, including SL4 and CT26. Blockade of CXCR4 signaling significantly enhanced treatment efficacy of anti-VEGFR2 treatment in both CRC models. CXCR4 was predominantly expressed in immunosuppressive innate immune cells, which are recruited to CRCs upon anti-VEGFR2 treatment. Blockade of CXCR4 abrogated the recruitment of these innate immune cells. Importantly, these myeloid cells were mostly Ly6Clow monocytes and not Ly6Chigh monocytes. To selectively deplete individual innate immune cell populations, we targeted key pathways in Ly6Clow monocytes (Cx3cr1-/- mice), Ly6Chigh monocytes (CCR2-/- mice), and neutrophils (anti-Ly6G antibody) in combination with CXCR4 blockade in SL4 CRCs. Depletion of Ly6Clow monocytes or neutrophils improved anti-VEGFR2-induced SL4 tumor growth delay similar to the CXCR4 blockade. In CT26 CRCs, highly resistant to anti-VEGFR2 therapy, CXCR4 blockade enhanced anti-VEGFR2-induced tumor growth delay but specific depletion of Ly6G+ neutrophils did not. The discovery of CXCR4-dependent recruitment of Ly6Clow monocytes in tumors unveiled a heretofore unknown mechanism of resistance to anti-VEGF therapies. Our findings also provide a rapidly translatable strategy to enhance the outcome of anti-VEGF cancer therapies.

Role of C-X-C chemokine ligand 12/C-X-C chemokine receptor 4 in the progression of hepatocellular carcinoma.

The efficacy of the current non-surgical treatments for advanced hepatocellular carcinoma (HCC) remains limited and novel treatments are required to improve patient outcomes. The majority of HCCs develop from chronically damaged tissue that contains a high degree of inflammation and fibrosis, which promotes tumor progression and resistance to therapy. Understanding the interaction between stromal components and cancer cells (and the signaling pathways involved in this interaction) could aid the identification of novel therapeutic targets. Numerous studies have demonstrated a marked association between high C-X-C chemokine receptor 4 (CXCR4) expression and the invasiveness, progression and metastasis of HCC. The present review will investigate the different roles of CXCR4 in the progression of HCC and discuss possible future treatments. Through the C-X-C chemokine ligand 12 (CXCL12)/CXCR4 signaling pathway, ephrin A1 activation enhances the migration of endothelial progenitor cells to HCC to enable the neovascularization of tumors. There is an association between nuclear CXCR4 expression and the lymph node metastasis of HCC to distant areas. CXCR4 enhances cell migration in vitro and cell homing in vivo. CXCR4 levels are concentrated at the border of a tumor and in perivascular areas, inducing invasive behavior. The binding of CXCL12 to CXCR4 activates intracellular signaling pathways and induces crosstalk with transforming growth factor-β signaling, which enhances the migration of cancer cells. The CXCL12/CXCR4 axis also activates expression of matrix metalloproteinase 10, which further stimulates migration. CXCR4 is likely to crosstalk with the sonic hedgehog signaling pathway, contributing to tumor invasiveness and supporting the cancer stem-cell population; as a result, CXCR4 can be regarded as a cancer stem-cell marker. CXCR4 influences interstitial fluid flow-induced invasion. CXCR4 expression and HCC cell migration are promoted by α-fetoprotein, which activates AKT/mechanistic target of rapamycin signaling. CXCR4 also has the potential to affect sorafenib treatment for HCC. Targeting the CXCL12/CXCR4 signaling pathway may, therefore, be a promising strategy in HCC treatment.

The Function of SDF-1-CXCR4 Axis in SP Cells-Mediated Protective Role for Renal Ischemia/Reperfusion Injury by SHH/GLI1-ABCG2 Pathway.

Renal ischemia-reperfusion (I/R) injury ranks as the primary cause of acute renal injury with severe morbidity and mortality. Side population (SP) cells have recently drawn increasing attention due to their critical role in injury repair and regeneration. Unfortunately, the underlying mechanism involved in renal I/R remains poorly elucidated. Here, pronounced increases of stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) were substantiated in I/R kidneys from C57BL/6 mice subjected to clamp the bilateral renal pedicles to mimic renal ischemia. Similar up-regulation of them was also determined in SP cells upon simulated ischemia/reperfusion (SI/R). In contrast to non-SP cells, SP cells exhibited higher viability, apoptosis resistance, chemotaxis, and paracrine actions following SI/R treatment, and these were further enhanced after SDF-1 stimulation. Interestingly, blocking CXCR4 signaling with AMD3100 notably ameliorated the above effects. Mechanism analysis corroborated that SDF-1/CXCR4 further induced the expression of ATP-binding cassette transporter ABCG2, an essential element for SP-mediated kidney regeneration after renal I/R injury. Moreover, AMD3100 pretreatment strikingly attenuated ABCG2 elevation in SP cells. Additionally, sonic hedgehog (SHH)-Gli 1 signaling was involved in SDF-1/CXCR4-mediated ABCG2 expression. When SP cells pretreated with AMD3100 were intravenously injected into I/R mice, SP cell-mediated decreases in blood urea nitrogen, serum creatinine, and histological score of kidney were noticeably attenuated, indicating that blocking CXCR4 pathway mitigated the therapeutic function of SP cells in renal I/R injury. Together, this research suggests that SDF-1/CXCR4 axis might act, via Shh-Gli1-ABCG2 signaling, as a positive regulator of SP cell-based therapies for renal I/R by Shh-Gli 1-ABCG2 signaling.

CXCR4 signaling contributes to alveolar bone resorption in Porphyromonas gingivalis-induced periodontitis in mice.

Periodontitis caused by bacterial infection gradually progresses accompanied by periodontal tissue destruction. As a result, teeth lose their supporting structures, and this leads to tooth exfoliation. CXC-chemokine receptor 4 (CXCR4) is known to be expressed in lymphocytes, fibroblasts and osteoclasts in periodontal tissues, suggesting that periodontal CXCR4 signaling contributes to alveolar bone resorption in the milieu of periodontitis. However, the role of CXCR4 signaling in the pathogenesis of periodontitis has remained unknown. We established a mouse model of periodontitis by inoculation of Porphyromonas gingivalis (P.g.) into a silk ligature placed around the maxillary molar. Although there was no significant difference in the mechanical sensitivity in the periodontal tissue between P.g. treatment and sham treatment during the experimental period, mechanical allodynia in the periodontal tissue was induced after gingival injection of complete Freund's adjuvant compared with that resulting from sham and P.g. treatment alone. Moreover, CXCR4 neutralization in the periodontal tissue following P.g. treatment enhanced periodontal inflammatory cell infiltration and depressed alveolar bone resorption. These findings suggest that periodontal CXCR4 signaling in several cell types in P.g.-induced periodontal inflammation depresses alveolar bone resorption in periodontitis. CXCR4 signaling might be a target for therapeutic intervention to prevent alveolar bone resorption in periodontitis.

Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices

The atomic-level mechanisms by which G protein-coupled receptors (GPCRs) transmit extracellular ligand binding events through their transmembrane helices to activate intracellular G proteins remain unclear. Using a comprehensive library of mutations covering all 352 residues of the GPCR CXC chemokine receptor 4 (CXCR4), we identified 41 amino acids that are required for signaling induced by the chemokine ligand CXCL12 (stromal cell-derived factor 1). CXCR4 variants with each of these mutations do not signal properly but remain folded, based on receptor surface trafficking, reactivity to conformationally sensitive monoclonal antibodies, and ligand binding. When visualized on the structure of CXCR4, the majority of these residues form a continuous intramolecular signaling chain through the transmembrane helices; this chain connects chemokine binding residues on the extracellular side of CXCR4 to G protein-coupling residues on its intracellular side. Integrated into a cohesive model of signal transmission, these CXCR4 residues cluster into five functional groups that mediate (i) chemokine engagement, (ii) signal initiation, (iii) signal propagation, (iv) microswitch activation, and (v) G protein coupling. Propagation of the signal passes through a "hydrophobic bridge" on helix VI that coordinates with nearly every known GPCR signaling motif. Our results agree with known conserved mechanisms of GPCR activation and significantly expand on understanding the structural principles of CXCR4 signaling.

Abstract 4579: Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased cell signaling and tumor cell intravasation

Abstract G-protein-coupled receptor (GPCR) heterodimerization has emerged as a means by which new signaling entities can be created with respect to agonist binding. Preliminary data from our lab indicates that agonist-bound GPCR C-X-C Chemokine Receptor 4 (CXCR4) and agonist-bound cannabinoid receptor 2 (CB2R) interact at the cell membrane and that receptor interaction mitigates the effects of CXCL12-mediated CXCR4 signaling. RhoA, Rac1 and Cdc42 are small GTP-binding proteins of the Rho family that are activated downstream of CXCR4 signaling. The effects of RhoA, Rac1 and Cdc42 on the actin cytoskeleton have led to established roles for Rho proteins in cancer cell motility, metastasis and intravasation. Here we report the novel finding that CXCR4/CB2R heterodimerization decreases Rho signaling, resulting in decreased intravasation. To test the effect of receptor heterodimerization on RhoA, Rac1, and Cdc42 activation, PC3 cells were treated with the CXCR4 agonist SDF1α, the CB2R agonist AM1241 or with a combination of SDF1α and AM1241 for 1 minute. Cell lysates were collected and immunoprecipitation experiments were performed to detect RhoA, Rac1 and Cdc42. To test the effects of CXCR4/CB2R heterodimerization on intravsation, we performed a zymography assay for the detection of metalloproteinase-2 (MMP2) and metalloproteinase-9 (MMP9), which are commonly secreted by CXCR4 signaling. Immunopreciptation results indicated an increase in RhoA and Rac1 protein levels after 1 minute in PC3 cells compared to the untreated control. However, when treated with SDF1α and AM1241 for 1 minute, RhoA and Rac1 levels decreased compared to SDF1α independent treatment; there was no observable change in Cdc42 protein levels. The results from the zymography experiments demonstrated that co-treatment with SDF1α and AM1241, which induces CXCR4/CB2R heterodimerization, led to decreased MMP-2 and MMP-9 activity, as compared to the SDF1α-independent treatment. Preliminary results provide novel evidence that CXCR4 and CB2R heterodimerize at the cell membrane, which may result in decreased signaling through RhoA and Rac1, but not through Cdc42. Furthermore, CXCR4/CB2R heterodimerization results in decreased MMP activation, thus abrogating intravsation. Citation Format: Imani N. Fennell, Kisha A. Scarlett, Christopher J. Coke. Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased cell signaling and tumor cell intravasation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4579.

Abstract 4574: C-X-C chemokine receptor 4 and cannabinoid receptor 2 interact to abrogate CXCL12-mediated cellular response

Abstract Expression of C-X-C Chemokine Receptor 4 (CXCR4) encourages tumors cells to migrate to distal organs expressing its cognate ligand, CXCL12, thus facilitating metastasis. Thus, targeting the CXCR4/CXCL12 signaling axis is a good strategy to inhibit the metastatic spread and progression of cancer. It has been suggested that homo- or heterodimerization of GPCRs causes decreased signaling through receptor complexes, representing functional desensitization. In the context of cancer treatment, it is plausible that CXCR4 signaling can be silenced through heterodimeric association with other receptors, thereby inhibiting functions that would lead to metastasis. Various studies suggest that cannabis, signaling via cannabinoid receptors, have anti-proliferative and anti-metastatic properties; though a biochemical mechanism describing how this phenomenon has not been described. We’ve confirmed that agonist-bound CXCR4 and agonist-bound Cannabinoid Receptor 2 (CB2) form a heterodimer that decreases cancer cell migration. Simultaneous treatment of the breast cancer cell line, MDA-MB-231, with CXCL12 and AM1241 (synthetic CB2 agonist), desensitized the intrinsic cellular CXCR4 response of cells to migrate towards CXCL12. Furthermore, through co-immunoprecipitation and proximity ligation assays (PLA), we determined increased interaction between the two receptors upon co-stimulation of respective agonists. To further delineate whether co-stimulation of both receptors led to subsequent heterodimerization, CXCR4 receptor activity was inhibited by an antagonist, AMD3100, or expression of endogenous CXCL12 was downregulated by siRNA. An interaction was observed in simultaneously treated AM1241/CXCL12 cells between CXCR4 and CB2, and surprisingly, in AM1241-treated cells. However, upon pretreatment with AMD3100, and CXCL12-siRNA, receptor association was no longer observed in AM1241-treated cells, supporting the therapeutic notion that treating tumors that endogenously secreted CXCL12 with an exogenous AM1241 can induce dimerization. Moreover, when CXCR4 and CB2 were activated simultaneously with various agonists, decreases in migration were observed, confirming that the regulatory activity was receptor-based, not agonist-based. Further, cells pre-treated with CB2 antagonist (AM630) and combinations of CXCL12 and AM1241 the anticipated decrease in migration was no longer seen. Finally, to determine whether simultaneously-treated, dimerized receptors inhibited activity of respective receptors, calcium mobilization assays showed that transiently activated calcium levels were significantly lower in response to simultaneous treated cells when compared to cells treated with their individual ligands. Therefore, a physical interaction between CXCR4 and CB2 may inhibit the response to CXCL12, leading to a loss in metastatic potential. Citation Format: Christopher Coke, Kisha Scarlett, Kia Jones, Cimona V. Hinton. C-X-C chemokine receptor 4 and cannabinoid receptor 2 interact to abrogate CXCL12-mediated cellular response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4574.

AMD3100 Attenuates Matrix Metalloprotease-3 and -9 Expressions and Prevents Cartilage Degradation in a Monosodium Iodo-Acetate–Induced Rat Model of Temporomandibular Osteoarthritis

Temporomandibular joint osteoarthritis (TMJOA) is an important subtype of temporomandibular disorder. This study investigated the inflammatory role of the stromal cell-derived factor-1 (SDF-1) and C-X-C chemokine receptor-4 (CXCR4) axis and the probable signaling pathway involved in matrix metalloprotease (MMP)-3 and MMP-9 productions stimulated by the SDF-1-CXCR4 axis in an experimental rat model of TMJOA. Rats were randomly divided into a control group, a pathologic model group, and an AMD3100 group. Effects of the bicyclam derivative AMD3100 (the specific antagonist of SDF-1-CXCR4 axis) were studied in TMJOA experimentally induced by monosodium iodo-acetate. Productions of SDF-1 and CXCR4 were compared in the normal and pathologic model groups, and cartilage changes and expressions of MMP-3, MMP-9, and phosphorylated extracellular signal-regulated kinase (p-ERK) were compared in the control, pathologic model, and AMD3100 groups. Expressions of SDF-1 and CXCR4 in the pathologic model group were increased compared with the control group (P < .05). Releases of MMP-3, MMP-9, and p-ERK and cartilage changes were downregulated in the AMD3100 group compared with the pathologic model group (P < .05), and these changes occurred in a dose-dependent manner with AMD3100 concentrations. Moreover, there were strong predictive relations between the expression of p-ERK with MMP-3 (r(2)= 0.419; P < .001) and with MMP-9 (r(2)= 0.542; P < .001). The SDF-1-CXCR4 signaling pathway plays a proinflammatory role in experimental TMJOA, the bicyclam derivative AMD3100 can alleviate the severity of experimental TMJOA, and there might be a potential relation between the SDF-1-CXCR4 axis and the ERK signaling pathway.

MiR-181a Targets RGS16 to Promote Chondrosarcoma Growth, Angiogenesis, and Metastasis

Chondrosarcoma is the most common primary malignant bone tumor in adults, has no effective systemic treatment, and patients with this disease have poor survival. Altered expression of microRNA (miR) is involved in tumorigenesis; however, its role in chondrosarcoma is undetermined. miR-181a is overexpressed in high-grade chondrosarcoma, is upregulated by hypoxia, and increases VEGF expression. Here, the purpose was to determine the mechanism of miR-181a regulation of VEGF, determine whether miR-181a overexpression promotes tumor progression, and to evaluate an antagomir-based approach for chondrosarcoma treatment. Therapeutic inhibition of miR-181a decreased expression of VEGF and MMP1 in vitro, and angiogenesis, MMP1 activity, tumor growth, and lung metastasis, all by more than 50%, in a xenograft mouse model. A target of miR-181a is a regulator of G-protein signaling 16 (RGS16), a negative regulator of CXC chemokine receptor 4 (CXCR4) signaling. CXCR4 signaling is increased in chondrosarcoma, its expression is also increased by hypoxia, and is associated with angiogenesis and metastasis; however, receptor blockade is only partially effective. RGS16 expression is restored after miR-181a inhibition and partially accounts for the antiangiogenic and antimetastatic effects of miR-181a inhibition. These data establish miR-181a as an oncomiR that promotes chondrosarcoma progression through a new mechanism involving enhancement of CXCR4 signaling by inhibition of RGS16. Targeting miR-181a can inhibit tumor angiogenesis, growth, and metastasis, thus suggesting the possibility of antagomir-based therapy in chondrosarcoma.

Abstract 4177: Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased angiogenesis

Abstract Tumor angiogenesis is a complex process modulated by numerous signaling molecules and pathways that influence cell migration, invasion, survival and tube formation. The expression of the G-protein-coupled receptor (GPCR) C-X-C Chemokine Receptor 4 (CXCR4) has been correlated with the increased angiogenic potential of cancer cells. The ligand-specific binding of CXCL12 to CXCR4 increases tumor angiogenesis by upregulating the expression of vascular endothelial growth factor (VEGF) at both mRNA and protein levels. Thus, modulating the CXCR4/CXCL12 signaling axis provides an attractive target for the inhibition of tumor angiogenesis and cancer progression. Recent studies have suggested that in addition to its palliative properties, cannabis may have anti-proliferative and anti-metastatic effects by acting through the GPCR, cannabinoid receptor 2 (CB2R). GPCR heterodimerization has emerged as a means by which new signaling entities can be created with respect to agonist binding. Preliminary co-immunoprecipitation data from our lab indicates that agonist-bound CXCR4 and agonist-bound CB2R interact at the cell membrane and that receptor interaction mitigates the effects of CXCL12-mediated CXCR4 signaling. Here we show that co-stimulation of prostate cancer cells with CXCR4 and CB2R-specific agonists results in decreased VEGF expression in vitro. Furthermore, treatment of human endothelial cells with conditioned medium collected from co-stimulated prostate cancer cells decreased endothelial cell migration in wound healing and invasion assays, and decreased endothelial cell tubulogenesis. Taken together, these results demonstrate that desensitization of CXCR4 signaling through association with CB2R is a novel anti-angiogenic strategy and reinforces the clinical relevance of cannabinoids in cancer treatment. Note: This abstract was not presented at the meeting. Citation Format: Kisha A. Scarlett, Brittney Sandifer, Christopher J. Coke, Cimona V. Hinton. Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased angiogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4177. doi:10.1158/1538-7445.AM2015-4177

Abstract 4467: Targeting of chemokine receptor CXCR4 inhibits invasion and sensitizes melanoma cells to chemotherapeutic agents

Abstract Malignant melanoma is highly metastatic and resistant to conventional therapies. Although numerous clinical approaches for treating melanoma have been adopted, none led to a remarkable therapeutic effect. Recently new drugs have been developed for treatment of metastatic melanoma, and hold great promise; however complete effects are still distant dream because of resistance. We have learnt that the most aggressive melanomas are generally resistant to strategies which target one signaling pathway. Thus an agent targeting multiple signaling pathways would maximize therapeutic outcome. We have identified one such agent named as gambogic acid (GA) that is natural and derived from medicinal plant Garcinia hanburyi (also called mangosteen or kokum). Moreover this agent has a potential to target several pathways responsible for survival and chemoresistance of several cancers including melanoma. Stromal cell-derived factor 1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4), has been associated with inferior survival, poor prognosis, chemoresistance and metastasis of malignant melanoma. We, therefore, investigated whether GA could inhibit CXCR4 signaling and suppress invasion of malignant melanoma cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on melanoma cells in a dose- and time-dependent manner. Suppression of CXCR4 expression by GA correlated with both inhibition of SDF-1α-induced invasion of melanoma cells. GA also inhibited expression of MMP-9, VEGF, c-Myc and phosphorylation of Erk1/2 in melanoma cells. Importantly, GA markedly enhances the efficacy of PLX-4032 and dacarbazine against melanoma cells. Citation Format: Manoj K. Pandey, Deepkamal Karelia, Vijay P. Kale, Krishne Gowda, Arun K. Sharma, Rogerio I. Neves, Shantu G. Amin. Targeting of chemokine receptor CXCR4 inhibits invasion and sensitizes melanoma cells to chemotherapeutic agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4467. doi:10.1158/1538-7445.AM2015-4467

Abstract 3786: Gambogic acid inhibits chemokine receptor CXCR4 signaling pathways and osteoclastogenesis in multiple myeloma

Abstract Bone disease, characterized by the presence of lytic lesions and osteoporosis is the hallmark of multiple myeloma (MM). Stromal cell-derived factor 1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4), has been implicated as a regulator of bone resorption, suggesting that agents that can suppress SDF1α/CXCR4 signaling might inhibit osteoclastogenesis, a process closely linked to bone resorption. We, therefore, investigated whether gambogic acid (GA), a xanthone, could inhibit CXCR4 signaling and suppress osteoclastogenesis induced by MM cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on MM cells in a dose- and time-dependent manner. The down-regulation of CXCR4 was not due to proteolytic degradation but rather to transcriptional regulation, as indicated by down-regulation of CXCR4 mRNA expression, inhibition of nuclear factor-kappa B (NF-κB) activity, and suppression of p65 binding at CXCR4 promoter analyzed by quantitative chromatin immunoprecipitation (qChIP) assay. Suppression of CXCR4 expression by GA correlated with both inhibition of SDF-1α-induced invasion of MM cells. GA also inhibited phosphorylation of Akt, p38, and Erk1/2 in MM cells. GA suppressed the RANKL-induced differentiation of macrophages to osteoclasts in a dose- and time-dependent manner. Finally, we found that MM cells, induced differentiation of macrophages to osteoclasts, and that GA suppressed this process. Overall, our results show that GA is a novel inhibitor of CXCR4 expression and thus has a strong potential to suppress osteoclastogenesis mediated by MM cells. Citation Format: Manoj K. Pandey, Vijay P. Kale, Chunhua Song, Shen-shu Sung, Arun K. Sharma, Sinisa Dovat, Shantu G. Amin. Gambogic acid inhibits chemokine receptor CXCR4 signaling pathways and osteoclastogenesis in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3786. doi:10.1158/1538-7445.AM2014-3786

Gambogic acid inhibits multiple myeloma mediated osteoclastogenesis through suppression of chemokine receptor CXCR4 signaling pathways

Bone disease, characterized by the presence of lytic lesions and osteoporosis is the hallmark of multiple myeloma (MM). Stromal cell-derived factor 1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4), has been implicated as a regulator of bone resorption, suggesting that agents that can suppress SDF1α/CXCR4 signaling might inhibit osteoclastogenesis, a process closely linked to bone resorption. We, therefore, investigated whether gambogic acid (GA), a xanthone, could inhibit CXCR4 signaling and suppress osteoclastogenesis induced by MM cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on MMcells in a dose- and time-dependent manner. The down-regulation of CXCR4 was not due to proteolytic degradation, but rather GA suppresses CXCR4 mRNA expression by inhibiting nuclear factor-kappa B (NF-κB) DNA binding. This was further confirmed by quantitative chromatin immunoprecipitation assay, as GA inhibits p65 binding at the CXCR4 promoter. GA suppressed SDF-1α-induced chemotaxis of MMcells and downstream signaling of CXCR4 by inhibiting phosphorylation of Akt, p38, and Erk1/2 in MMcells. GA abrogated the RANKL-induced differentiation of macrophages to osteoclasts in a dose- and time-dependent manner. In addition, we found that MMcells induced differentiation of macrophages to osteoclasts, and that GA suppressed this process. Importantly, suppression of osteoclastogenesis by GA was mediated through IL-6 inhibition. Overall, our results show that GA is a novel inhibitor of CXCR4 expression and has a strong potential to suppress osteoclastogenesis mediated by MMcells.

Matrix metalloproteinase-1-mediated mesenchymal stem cell tumor tropism is dependent on crosstalk with stromal derived growth factor 1/C-X-C chemokine receptor 4 axis.

Human bone marrow-derived mesenchymal stem cells (MSCs) have the unique ability to home toward injuries or tumor sites. We have previously shown that the tumor-tropic property is dependent on the intrinsic expression and activity of the matrix remodeling gene, matrix metalloproteinase 1 (MMP-1). Herein, crosstalk between MMP-1/protease activated receptor 1 (PAR-1) and the G-protein coupled receptor stromal-derived growth factor 1 (SDF-1)/C-X-C chemokine receptor 4 (CXCR-4) in facilitating cell migration was investigated. Gain-of-function and RNA interference (RNAi) technology were used to evaluate the interplay between the key players. The downstream effect on the tumor-tropic migration of MSCs was investigated using modified Boyden chamber assay. Neutralizing PAR-1 activation using monoclonal antibody and targeted knockdown of MMP-1 using RNAi resulted in decreased expression of SDF-1, which was not observed in control-RNAi-transfected cells. Overexpression of CXCR-4 failed to promote MSC migration; the percentage of migrated cells toward tumor cell conditioned medium was similar to the vector-transduced and the CXCR-4-transduced MSCs. Furthermore, inhibition of SDF-1/CXCR-4 signaling using AMD3100 reduced MSC migration through the deregulation of MMP-1 promoter activities, protein expression, and metalloproteinase activity. Collectively, our results showed that MMP-1-mediated MSC tumor tropism is dependent on crosstalk with the SDF-1/CXCR-4 axis.

Anti-invasive effects of celastrol in hypoxia-induced fibroblast-like synoviocyte through suppressing of HIF-1α/CXCR4 signaling pathway.

Rheumatoid arthritis (RA) joints are in a hypoxic condition. Hypoxia-induced migration and invasion of fibroblast-like synoviocytes (FLSs) are considered to play a critical role in the pathogenesis of RA. Among the key genes upregulated by hypoxia-inducible factor-1α (HIF-1α), CXC chemokine receptor 4 (CXCR4) plays an important role in FLS migration and invasion. Our previous studies have shown that celastrol exerts anti-arthritic effects by inhibiting FLS migration and invasion under normoxic conditions. However, the effect and molecular mechanisms underlying the effect of celastrol on hypoxia-induced FLS migration and invasion are poorly understood. In the present study, we assessed the effect of celastrol on hypoxia-induced FLS migration and invasion. Results showed that celastrol suppressed hypoxia-induced FLS migration and invasion. In addition, we also found that celastrol inhibited hypoxia-induced CXCR4 expression at both the mRNA and the protein levels in RA-FLSs. Meanwhile, it is revealed that celastrol inhibited the transcriptional activity of CXCR4 under hypoxic conditions by suppressing the binding activity of HIF-1α in the CXCR4 promoter, and blocked hypoxia-induced accumulation of nuclear HIF-1α. Furthermore, treatment with HIF-1α inhibitor reduced the hypoxia-induced expression and transcriptional activity of CXCR4. In conclusion, our results indicate that celastrol inhibits hypoxia-induced migration and invasion via suppression of HIF-1α mediated CXCR4 expression in FLSs under hypoxic conditions. These results provide a strong rationale for further testing and validation of the use of celastrol as a new alternative for using in the treatment of RA.

CXC Chemokine Receptor 4 is Essential for Maintenance of Renal cell Carcinoma-Initiating Cells and Predicts Metastasis

In many solid tumors, cancer stem cells (CSC) represent a population with tumor-initiating, self-renewal, and differentiation potential, which can be identified by surface protein markers. No generally applicable markers are yet known for renal cell carcinoma (RCC). Two RCC cell lines (RCC-26, RCC-53) were found to differ widely in their capacity to form spheres in vitro and to establish tumors in mice, potentially reflecting differences in CSC content. A subpopulation expressing the CXC chemokine receptor 4 (CXCR4) was present only in the more tumorigenic cell line RCC-53. When grown as spheres, most of the RCC-53 cells were CXCR4-positive, expressed stem cell-associated transcription factor genes at elevated levels, and were more resistant toward the tyrosine kinase inhibitors sunitinib, sorafenib, and pazopanib. Sorted CXCR4-positive cells exhibited greater capacity for sphere formation and tumor growth-inducing potential in vivo than CXCR4-negative cells. Significantly, higher CXCR4 mRNA levels in primary RCC tumors from patients with localized but not disseminated disease predicted shorter survival. Downregulation of CXCR4 expression by small interfering RNA (siRNA) or pharmacological inhibition by AMD3100 compromised tumor sphere formation, viability of CXCR4-positive cells, and increased their responsiveness toward tyrosine kinase inhibitors. In conclusion, CXCR4 identifies a subpopulation of tumor-initiating cells in RCC cell lines and plays a role in their maintenance. The relative insensitivity of such cells to tyrosine kinase inhibitors might contribute to the development of therapy resistance in RCC patients. Future therapies therefore could combine blockade of the CXCR4 signaling pathway with standard therapies for more effective treatments of metastatic RCC.