CXCR4 Protein Expression Research Articles

Significance of CXCL12/CXCR4 expression in T-lymphoblastic lymphoma/leukemia

Objective: To investigate the significance of CXCL12/CXCR4 expression in T lymphoblastic lymphoma/leukemia (T-LBL/ALL) and its prognostic significance. Methods: Using immunohistochemical EnVision method, CXCL12, CXCR4 and Ki-67 expression were evaluated in 72 cases of T-LBL/ALL and 30 selected cases of lymph node reactive hyperplasia (LH) as control. In addition, CXCL12 and CXCR4 mRNA expression levels were examined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method. Results: Immunohistochemical results showed that the expression rates of CXCL12 and CXCR4 in T-LBL/ALL were 84.7%(61/72) and 91.6%(66/72), respectively, and these were not different from the expression in the LH control group. The expression indexes of Ki-67 <80% and ≥80% were 25 cases (34.7%, 25/72) and 47 cases (65.3%, 47/72), respectively. Real-time quantitative PCR demonstrated that CXCL12 and CXCR4 mRNA expression in T-LBL/ALL was 62.4% and 71.5%, respectively, and was statistically different (P<0.05) from that of the control group. Single factor analysis found that CXCL12 mRNA expression in T-LBL/ALL was positively correlated with Ann Arbor staging and KPS score (P<0.05); CXCL12 protein expression was positively correlated with splenomegaly (P<0.05); CXCR4 mRNA expression was positively correlated with the IPI score, clinical symptoms, mediastinal widening and bone marrow involvement (P<0.05); CXCR4 protein expression was positively correlated with mediastinal widening (P<0.05); CXCL12 mRNA expression was positively correlated with CXCL12 protein and CXCR4 protein expression (P<0.05), but not the CXCR4 mRNA and protein levels. There was no correlation between CXCL12 and CXCR4 protein expression and CXCR4 mRNA expression. Multivariate COX regression analysis showed that high expression of CXCR4 protein, hepatosplenomegaly and bone marrow involvement were risk factors for T-LBL/ALL outcome. Conclusions: CXCL12/CXCR4 expression is associated with disease progress, mediastinal widening, bone marrow involvement and adverse outcome in T-LBL/ALL. CXCL12/CXCR4 axis plays an essential role in the occurrence and development of T-LBL/ALL. However, CXCL12 and CXCR4 protein expression are not entirely reflected by mRNA transcription levels, and there may be other molecules involved in CXCL12/CXCR4 expression and regulation. With CXCR4 antagonists undergoing clinical trials, targeting the CXCL12/CXCR4 axis may be a promising treatment strategy for T-LBL/ALL.

Modulation of stromal cell-derived factor 1 alpha (SDF-1\u03b1) and its receptor CXCR4 in Porphyromonas gingivalis-induced periodontal inflammation

BackgroundThe production of chemokines by tissue resident cells during inflammation is considered one of the main mechanisms involved in the formation of inflammatory infiltrates. Fibroblasts are the main resident cell type in gingival and periodontal ligament tissues, and their ability to produce chemokine stromal cell-derived factor 1 alpha (SDF-1α) and its receptor CXCR4 under stimulation by gram negative bacteria, Porphyromonas gingivalis, commonly found in periodontal infections was investigated.MethodsWestern blots were used to assess SDF-1α and CXCR4 protein expression levels in human gingival fibroblast cells (HGF-1) induced by Lipopolysaccharide (LPS) from P. gingivalis in the presence or absence of LY294002, a highly selective inhibitor of PI-3K/Akt. RT-PCR and quantitative Real-time PCR was performed using gingival mRNAs from periodontitis patients. Immunohistochemistry was performed to analyze the expression and subcellular localization of SDF-1α and CXCR4, together with NF-kβ phosphorylation, in specimens from patients with periodontitis and in an experimental rat periodontitis model.ResultsWe found that P. gingivalis LPS up-regulated SDF-1α and CXCR4 protein levels and elevated phosphorylation of the SDF-1α-responsive NF-kβ and Akt at 24 h in HGF-1 cells. SDF-1α and CXCR4 mRNA and protein expression levels were high in all patients with periodontitis. In the P. gingivalis-induced rat experimental periodontitis model, SDF-1α and CXCR4 immunoreactivity was higher in gingival and periodontal ligament tissues compared to the control.ConclusionOur data showed that PI-3K/Akt is an upstream participant in the P. gingivalis LPS-mediated induction of SDF-1α. Taken together, these results suggest that the chemokine SDF-1α and its receptor CXCR4 contribute to P. gingivalis-induced periodontal inflammation.

Enhancement of the CXCL12/CXCR4 axis due to acquisition of gemcitabine resistance in pancreatic cancer: effect of CXCR4 antagonists.

BackgroundThe CXCL12-CXCR4 signaling axis in malignant tumor biology has increased in importance, and these peptides are implicated in tumor growth, invasion and metastasis. The aim of our study was to examine the important role of the axis in pancreatic cancer (PaCa) cells’ relationship with stromal cells in gemcitabine-resistant (GEM-R) tumors and to confirm the effectiveness of CXCR4 antagonists for the treatment of GEM-R PaCa cells.MethodsWe established two GEM-R PaCa cell lines using MIA PaCa-2 and AsPC-1 cells. The expression of CXCR4 mRNA in PaCa cells and the expression of CXCL12 mRNA in fibroblasts were examined by reverse transcription polymerase chain reaction (RT-PCR). The expression of CXCR4 protein in PaCa cells was examined by immunosorbent assay (ELISA) and immunocytochemistry. Using Matrigel invasion assays and animal studies, we then examined the effects of two CXCR4 antagonists, AMD11070 and KRH3955, on the invasiveness and tumorigenicity of GEM-R PaCa cells stimulated by CXCL12.ResultsWe found that the expression of CXCR4 in GEM-R PaCa cells was significantly enhanced by GEM but not in normal GEM-sensitive (GEM-S) PaCa cells. In RT-PCR and ELISA assays, the production and secretion of CXCL12 from fibroblasts was significantly enhanced by co-culturing with GEM-R PaCa cells treated with GEM. In Matrigel invasion assays, the invasiveness of GEM-R PaCa cells treated with GEM was significantly activated by fibroblast-derived CXCL12 and was significantly inhibited by CXCR4 antagonists, AMD11070 and KRH3955. In vivo, the tumorigenicity of GEM-R PaCa cells was activated by GEM, and it was significantly inhibited by the addition with CXCR4 antagonists.ConclusionsOur findings demonstrate that the CXCL12-CXCR4 signaling axis plays an important role in PaCa cells’ resistance to GEM. CXCR4 expression was significantly enhanced by the exposure to GEM in GEM-R PaCa cells but not in GEM-S PaCa cells. Furthermore, CXCR4 antagonists can inhibit the growth and invasion of GEM-R PaCa cells. These agents may be useful as second-line chemotherapy for GEM-R PaCa in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2340-z) contains supplementary material, which is available to authorized users.

Difference in CXCR4 expression between sporadic and VHL-related hemangioblastoma.

Central nervous system hemangioblastomas occur sporadically and in patients with von Hippel–Lindau (VHL) disease due to a VHL germline mutation. This mutation leads to enhanced transcription of chemokine receptor 4 (CXCR4), its ligand (CXCL12) and vascular endothelial growth factor A (VEGFA). We aimed to determine in VHL-related and sporadic hemangioblastomas CXCR4, CXCL12, and VEGFA protein expression and to correlate this to hemangioblastoma size and expression in normal surrounding tissue. 27 patients with a hemangioblastoma were included for analysis of immunohistochemistry of tissue, MRI and DNA. Hemangioblastomas overexpress CXCR4, CXCL12, and VEGFA compared to normal surrounding tissue. In sporadic hemangioblastomas the mean percentage of CXCR4 positive hemangioblastoma cells was 16 %, SD 8.4, in VHL-related hemangioblastomas 8 %, SD 4.4 (P = 0.002). There was no relation between preoperative tumor size and CXCR4 or CXCL12 expression. Compared to normal surrounding tissue CXCR4, CXCL12, and VEGFA were overexpressed in hemangioblastomas. Most interestingly, sporadic hemangioblastomas overexpress CXCR4 compared to VHL-related hemangioblastoma.Electronic supplementary materialThe online version of this article (doi:10.1007/s10689-016-9879-3) contains supplementary material, which is available to authorized users.

BK K+ channel blockade inhibits radiation-induced migration/brain infiltration of glioblastoma cells.

Infiltration of the brain by glioblastoma cells reportedly requires Ca2+ signals and BK K+ channels that program and drive glioblastoma cell migration, respectively. Ionizing radiation (IR) has been shown to induce expression of the chemokine SDF-1, to alter the Ca2+ signaling, and to stimulate cell migration of glioblastoma cells. Here, we quantified fractionated IR-induced migration/brain infiltration of human glioblastoma cells in vitro and in an orthotopic mouse model and analyzed the role of SDF-1/CXCR4 signaling and BK channels. To this end, the radiation-induced migratory phenotypes of human T98G and far-red fluorescent U-87MG-Katushka glioblastoma cells were characterized by mRNA and protein expression, fura-2 Ca2+ imaging, BK patch-clamp recording and transfilter migration assay. In addition, U-87MG-Katushka cells were grown to solid glioblastomas in the right hemispheres of immunocompromised mice, fractionated irradiated (6 MV photons) with 5 × 0 or 5 × 2 Gy, and SDF-1, CXCR4, and BK protein expression by the tumor as well as glioblastoma brain infiltration was analyzed in dependence on BK channel targeting by systemic paxilline application concomitant to IR. As a result, IR stimulated SDF-1 signaling and induced migration of glioblastoma cells in vitro and in vivo. Importantly, paxilline blocked IR-induced migration in vivo. Collectively, our data demonstrate that fractionated IR of glioblastoma stimulates and BK K+ channel targeting mitigates migration and brain infiltration of glioblastoma cells in vivo. This suggests that BK channel targeting might represent a novel approach to overcome radiation-induced spreading of malignant brain tumors during radiotherapy.

Macrophages promote vasculogenesis of retinal neovascularization in an oxygen-induced retinopathy model in mice.

To investigate the role of macrophages in oxygen-induced retinal neovascularization (NV) in mice, particularly the involvement of bone marrow-derived cells (BMCs) and the underlying mechanisms, BMCs from green fluorescent protein (GFP) transgenic mice were transplanted into postnatal day (P) 1 mice after irradiation. The mice were exposed to 75% oxygen from P7 to P12 to initiate oxygen-induced retinopathy (OIR). The macrophages were depleted by injection of clodronate-liposomes (lip) intraperitoneally. The eyes were collected at P12 and P17. Retinal flatmounts and histopathological cross-sections were performed to analyze the severity of retinal NV and BMC recruitment. BMCs immunopositive for CD31 (PECAM-1; endothelial cell marker) and α-SMA (smooth muscle cell marker) antigens were detected using a confocal microscope. Expression of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) mRNA was detected by RT-PCR. The VEGF, SDF-1, CXCR4 and CD45 protein expression was detected by western blot examination. The retinal avascular area in OIR mice at P12 was unaffected after macrophage depletion carried out twice (38.27 ± 1.92% reduction) using clodronate-lip. The retinal avascular area and the NV area at P17 were reduced after macrophage depletion four times (79.53 ± 1.02% reduction); these findings were supported by retinal flatmounts and histopathological cross-sections. Macrophage depletion led to significant inhibition of BMC recruitment into the NV tufts at P17, with decreased expression of retinal VEGF, SDF-1, CXCR4 and CD45. The recruited BMCs differentiated primarily into CD31-positive endothelial cells (ECs) and α-SMA-positive smooth muscle cells (SMCs). This study suggested that macrophages promoted the vasculogenesis of retinal NV, particularly the contribution of BMCs in the mouse OIR model, which might be triggered by VEGF and SDF-1 production.

SiRNA-induced TRAF6 knockdown promotes the apoptosis and inhibits the invasion of human lung cancer SPC-A1 cells.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been found to be involved in multiple cancers. However, the effect of small interfering RNA (siRNA)-induced knockdown of TRAF6 on the biological behaviors of cancer cells remains unknown. Thus, the present study aimed to investigate the effect of siRNA-induced knockdown of TRAF6 on the biological behaviors of human lung cancer SPC-A1 cells. The expression of TRAF6 was determined in human lung adenocarcinoma A549, non-small cell lung cancer H1650, human airway epithelial Calu-3 and human lung cancer SPC-A1 cell lines using quantitative RT-PCR (qRT-PCR) and western blotting at the transcriptional and translational levels. TRAF6 expression was knocked down in the SPC-A1 cells using an siRNA technique, and the effects of TRAF6 knockdown on NF-κB activity, cell proliferation, apoptosis, cell cycle, invasion and migration of the SPC-A1 cells were determined using electrophoretic mobility shift assay (EMSA), cell proliferation assay, flow cytometry, Transwell invasion assay and scratch wound assay. In addition, the protein expression of CD24, CXCR4, MMP1, MMP2, MMP9, TWIST, TIMP-2 and Slug was quantified using western blotting assay. Western blotting and qRT-PCR assays showed upregulation of TRAF6 at both the translational and transcriptional levels in the Calu-3 and SPC-A1 cells, and K63-linked ubiquitination of TRAF6 and constitutive NF-κB activation were detected in the SPC-A1 cells. Knockdown of TRAF6 inhibited the migration and invasion and promoted the apoptosis of the SPC-A1 cells, but had little effect on cell proliferation and the cell cycle. In addition, siRNA-induced TRAF6 knockdown caused a marked reduction in the protein expression of CD24 and CXCR4, but had little effect on MMP-1, MMP-2, MMP-9, Twist, TIMP-2 or Slug expression. The present study demonstrated that TRAF6 is upregulated in human lung cancer cells, and siRNA-induced TRAF6 knockdown inhibits the invasion of lung cancer cells and promotes apoptosis. It is suggested that TRAF6 may be a promising target for the therapy of lung cancer.

Role of CXCR4/STAT3 in mesenchymal stromal cell-mediated drug resistance of acute leukemia cells

目的探讨CXCR4/STAT3在骨髓基质细胞介导的急性髓系白血病(AML)细胞耐药中的作用。方法将AML细胞系U937、KG1a和原代AML细胞与健康供者来源的骨髓基质细胞共培养，单独培养的细胞作为对照组。通过膜联蛋白Ⅴ (Annexin Ⅴ)／碘化丙锭(PI)双染色法比较共培养前后AML细胞对米托蒽醌诱导的凋亡的差异，并通过流式细胞术、实时定量PCR及Western blot法检测共培养前后AML细胞CXCR4、磷酸化STAT3(p-STAT3)蛋白的表达；在培养体系中加入STAT3的特异性抑制剂Cucurbitacin Ⅰ或CXCR4拮抗剂AMD3100，检测AML细胞对米托蒽醌诱导的凋亡的变化，并观察AMD3100对p-STAT3表达的影响。结果U937、KG1a细胞与骨髓基质细胞共培养后，对米托蒽醌诱导的凋亡率均低于对照组[U937：(20.08±1.53)%对(45.33±1.03)%，P=0.004; KG1a：(25.60 ± 1.82)%对(40.33±3.29)%，P=0.003]。共培养后，Western blot方法检测发现AML细胞p-STAT3的蛋白表达明显上调，流式细胞术和Western blot检测结果显示CXCR4蛋白表达显著上调，实时定量PCR检测显示CXCR4 mRNA水平明显增高；STAT3的特异性抑制剂Cucurbitacin Ⅰ或CXCR4拮抗剂AMD3100加入共培养体系后，原代AML细胞对米托蒽醌诱导的凋亡率较对照组显著升高，差异均有统计学意义(P值均<0.05)，且加入AMD3100后AML细胞p-STAT3蛋白的表达显著下降。结论AML细胞与骨髓基质细胞共培养可导致p-STAT3、CXCR4蛋白表达的上调，从而导致AML细胞对化疗药物的耐药；针对STAT3、CXCR4的联合靶向治疗可能为AML的治疗提供新思路。

Progesterone modulates endothelial progenitor cell (EPC) viability through the CXCL12/CXCR4/PI3K/Akt signalling pathway.

Progesterone treatment can effectively increase levels of circulating endothelial progenitor cells (EPCs) and improve neurological functional outcome in a traumatic brain injury (TBI) rat model. However, the mechanisms of progesterone's effects on EPC viability remain elusive. The CXCL12/CXCR4 (CXC chemokine ligand 12/CXC chemokine receptor 4) signalling pathway regulates cell proliferation; we hypothesize that it mediates progesterone-induced EPC viability. EPCs were isolated from bone marrow-derived mononuclear cells (BM-MNCs) and treated with progesterone (5, 10 and 100 nm). MTS assay was used to investigate EPC viability. Protein expression was examined by Western blotting, ELISA assay and flow cytometry. Cell membrane and cytoplasm proteins were extracted with membrane and cytoplasm protein extraction kits. CXCR4 antagonist (AMD3100) and phosphatidylinositol 3-kinases (PI3K) antagonist (LY294002) were used to characterize underlying mechanisms. Progesterone-induced EPC viability was time- and dose-dependent. Administration of progesterone facilitated EPC viability and increased expression of CXCL12 and phosphorylated Akt (also known as protein kinase B, pAkt) activity (P < 0.05). Progesterone did not regulate CXCR4 protein expression in cultured EPC membranes or cytoplasm. However, progesterone-induced EPC viability was significantly attenuated by AMD3100 or LY294002. Inhibition of the signalling pathway with AMD3100 and LY294002 subsequently reduced progesterone-induced CXCL12/CXCR4/PI3K/pAkt signalling activity. The CXCL12/CXCR4/PI3K/pAkt signalling pathway increased progesterone-induced EPC viability.

Angiogenesis-Related Markers and Prognosis After Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy for Metastatic Colorectal Cancer.

BackgroundPatients presenting with peritoneal metastases (PM) of colorectal cancer (CRC) can be curatively treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). Angiogenesis is under control of multiple molecules of which HIF1a, SDF1, CXCR4, and VEGF are key players. We investigated these angiogenesis-related markers and their prognostic value in patients with PM arising from CRC treated with CRS and HIPEC.Patients and MethodsClinicopathological data and tissue specimens were collected in 2 tertiary referral centers from 52 patients who underwent treatment for isolated PM of CRC. Whole tissue specimens were subsequently analyzed for protein expression of HIF1a, SDF1, CXCR4, and VEGF by immunohistochemistry. Microvessel density (MVD) was analyzed by CD31 immunohistochemistry. The relationship between overall survival (OS) and protein expression as well as other clinicopathological characteristics was analyzed.ResultsUnivariate analysis showed that high peritoneal cancer index (PCI), resection with residual disease and high expression of VEGF were negatively correlated with OS after treatment with CRS and HIPEC (P < 0.01, P < 0.01, and P = 0.02, respectively). However, no association was found between the other markers and OS (P > 0.05). Multivariate analysis showed an independent association between OS and PCI, resection outcome and VEGF expression (multivariate HR: 6.1, 7.8 and 3.8, respectively, P ≤ 0.05).ConclusionsAn independent association was found between high VEGF expression levels and worse OS after CRS and HIPEC. The addition of VEGF expression to the routine clinicopathological workup could help to identify patients at risk for early treatment failure. Furthermore, VEGF may be a potential target for adjuvant treatment in these patients.Electronic supplementary materialThe online version of this article (doi:10.1245/s10434-015-5023-0) contains supplementary material, which is available to authorized users.

Effects of mesenchymal stem cells surface CXC chemokine receptor 4 over-expression on repair of ischemia reperfusion injured kidney

Objective To observe the effects of mesenchymal stem cells (MSCs) surface CXC chemokine receptor 4 over-expression on the repair of kidney after ischemia reperfusion (I/R) injury. Methods The MSCs were co-cultured with I/R injured renal cell homogenate supernatant. The MSCs surface CXCR4 and stromal cells derived factor-1 (SDF-1α) protein levels were detected by Western blot, chemotactic ability of MSCs to SDF-1 was investigated by transwell test. The I/R injured renal model was made and pathological changes were observed in control group, I/R group, MSCs injection group and CXCR4 neutralize antibody group. Renal CXCR4 protein expression was measured by immunofluorescence histochemistry, SDF-1α、CXCR4、hepatocyte growth factor (HGF) and epidermal growth factor (EGF) mRNA were detected by Real-time quantitative polymerase chain reaction (RT-PCR). Comparisons among multiple groups were performed using One-way analysis of variance, and comparisons between groups were carried out using independent-sample t-test. Results In vitro, the SDF-1α protein expression markedly increased in I/R injured renal tissue homogenate, but the difference was not significant between I/R group and CXCR4 antibody group (t=0.862, P=0.403). MSCs surface CXCR4 protein expression increased significantly after co-cultured with I/R injured renal tissue homogenate (F=95.957, t=10.166, P <0.01) , and the chemotactic ability of MSCs to SDF-1 increased at the same time (F=82.459, t=6.826, P <0.01) , the CXCR4 protein expression (t=13.657, P <0.01) and the chemotactic ability (t=12.662, P <0.01) could be decreased by CXCR4 neutralize antibody. In vivo, renal tubular structure was destroyed in I/R group. After MSCs injection, the renal pathological injury improved rapidly, but the improvement could be inhibited by CXCR4 antibody. The expression of SDF-1α mRNA and level of SDF-1a protein increased in I/R group, but there was no significant difference among different groups (F=1.909, P=0.173). MSCs injection markedly up-regulated the CXCR4 protein and mRNA expression (F=6.663, P=0.006). Following the increase in CXCR4 expression, the expressions of HGF mRNA (F=11.898, P <0.01) and EGF mRNA (F=5.309, P <0.05) increased gradually which could be restrained by CXCR4 antibody (t=5.312, t=4.310, P <0.01). Conclusions I/R injured renal microenvironment markedly increased the mesenchymal stem cells surface CXCR4 expression, and increased CXCR4 expression can induce MSCs chemotaxis and stimulate the secretion of renal protective growth factors paracrine promoting the repair of the kidney. Key words: Mesenchymal stem cells; CXC chemokine receptor 4; Ischemia reperfusion injury; Rats; Renal injury; Chemotaxis; Paracrine; Recovery

Abstract A106: Mechanisms underlying extra-medullary myeloma development

Abstract Extramedullary disease (EMD) involvement in Multiple Myeloma (MM) is associated with poor prognosis and resistance to therapeutic agents. However, molecular alterations that lead to extramedullary metastasis of malignant plasma cells have not been identified. In this study, we developed MM subclones that have tropism to liver EMD sites by performing repetitive in vivo selection in xenograft mice using 2 MM cell lines. Whole body imaging showed that liver-prone clones infiltrated into various EMD site including liver, developing macroscopic EMD tumors, whereas control BM-prone subclones developed little or no macroscopic EMD. Interestingly no significant differences were observed in BM involvement between these subclones. In vitro characterization study showed that cell proliferation rates of these sub-clones were almost identical, while the transwell migration assay revealed that EMD-prone cells had higher chemotaxis index towards serum containing media compared to BM-prone clones. Flow-cytemetry analysis was performed, showing several chemokine receptors were upregulated in EMD-clones compared to BM clones. Among them, CXCR4 was remarkably upregulated in both two MM cell lines. Immunohistochemistry of clinical samples further demonstrated a significant difference in protein expression of CXCR4 in EMD sites compared to BM sites of involvement in the same patients, suggesting that CXCR4 upregulation in mice model is clinical relevant. Gain-of function studies using stable cell lines with high or low CXCR4 expression in a competitive bone metastasis model confirmed that high CXCR4 expressing cell lines were more “fit” for dissemination and engraftment in the liver. For further understanding of the participation of CXCR4 in EMD development, we investigated characteristics of these subclones in hypoxic condition. CXCR4 expression of both EMD and BM-prone subclones were upregulated in hypoxic condition (0.5% O2), becoming almost same level, despite EMD-prone clones had higher CXCR4 expression in normoxia. In normoxic condition, EMD-prone cells exhibited higher chemotaxis toward primary stellate cells, which is thought to be a major source of SDF-1 in liver, compared to BM-prone clones, while no difference was observed in chemotaxis towards BM mesenchymal stem cells at hypoxic condition. These results may suggest possible explanation of EMD tropism of the cells constitutively expressing CXCR4. As BM is highly hypoxic milieu, myeloma cells, in general express higher levels of CXCR4 in BM, but once they migrate outside the BM, the cells decrease CXCR4 expression. The EMD-prone cells remain expressing higher levels of CXCR4 even outside BM and may infiltrate into extrmedullary tissues in response to SDF-1 density gradient. Together, these studies identify novel molecular alterations that are present in MM subclones that metastasize to EMD sites indicating a potential therapeutic role for CXCR4 inhibitors in the treatment of patients with extramedullary myeloma. Citation Format: Yuji Mishima, Aldo Roccaro, Michele Moschetta, Yasuhito Terui, Kiyohiko Hatake, Irene M. Ghobrial. Mechanisms underlying extra-medullary myeloma development. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A106.

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.

HuR-targeted nanotherapy in combination with AMD3100 suppresses CXCR4 expression, cell growth, migration and invasion in lung cancer.

The CXCR4 chemokine receptor plays an important role in cancer cell metastasis. The CXCR4 antagonist, AMD3100, has limited efficacy in controlling metastasis. HuR, an RNA-binding protein, regulates CXCR4 in cancer cells. We therefore investigated whether targeting HuR using a siRNA-based nanoparticle plus AMD3100 would suppress CXCR4 and inhibit lung cancer metastasis. We treated human H1299 lung cancer cell with HuR-specific siRNA contained in a folate-targeted lipid nanoparticle (HuR-FNP) plus AMD3100, and compared this with AMD3100 alone, HuR-FNP alone and no treatment. HuR-FNP plus AMD3100 treatment produced a G1 phase cell-cycle arrest and reduced cell viability above and beyond the effects of AMD3100 alone. HuR and CXCR4 mRNA and protein expression levels were markedly reduced in all treatment groups. Phosphorylated (p) AKTS473 protein was also reduced. P27 protein expression increased with HuR-FNP and combination treatment. Promoter-based reporter studies showed that the combination inhibited CXCR4 promoter activity more than did either treatment alone. Cell migration and invasion was significantly reduced with all treatment; the combination provided the most inhibition. Reduced matrix metalloprotease (MMP) -2 and -9 expression was associated with reduced invasion in all treatment groups. Thus, we found that combined HuR and CXCR4 targeting effectively controlled lung cancer metastasis.

The association of CXCR4 expression with clinicopathological significance and potential drug target in prostate cancer: a meta-analysis and literature review.

CXCR4/CXCL12 axis plays an important role in tumor growth, angiogenesis, metastasis, and therapeutic resistance. The aim of this study is to perform a meta-analysis and literature review to evaluate the association of CXCR4 expression with clinicopathological significance and prognosis in patients with prostate cancer (PCa). A detailed literature search was made in Medline, EMBASE, Web of Science, and Google Scholar for related research publications. The data were extracted and assessed independently. Analysis of pooled data was performed using Review Manager 5.2. Odds ratio (OR) with corresponding confidence intervals were calculated and summarized. The meta-analysis included a total of eleven studies and 630 patients. The rate of CXCR4 protein expression in PCa was significantly higher than in nonmalignant prostate tissues (OR =35.71, P<0.00001). The expression of CXCR4 protein was not significantly associated with Gleason score (P=0.73). However, the frequency of CXCR4 protein expression was significantly higher in T3–4 stage than in T1–2 stage of PCa (OR =2.35, P=0.001). The expression of CXCR4 protein was significantly associated with the presence of lymph node and bone metastasis of PCa: for lymph node metastasis positive versus negative, OR was 5.07 and P=0.0003, and for bone metastasis positive versus negative, OR was 7.03 and P=0.003. Cancer-specific survival of patients with PCa was significantly associated with CXCR4 protein expression, and the pooled Hazard ratio was 0.24 and P=0.002. In conclusion, the high expression of CXCR4 protein is a diagnostic biomarker of PCa, and it is significantly associated with T stages. The increased expression of CXCR4 protein is significantly associated with lymph nodes or bone metastasis, and CXCR4 is a poor prognosis predictor for patients with PCa. Taken together, our findings indicate that CXCR4 could be a target not only for the development of therapeutic intervention but also for the noninvasive monitoring of PCa progression.

The presence of CXCR4+ CD1a+ cells at onset of Langerhans cell histiocytosis is associated with a less favorable outcome

abstractPurpose: Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder characterized by tissue accumulating CD1a+ histiocytes which frequently carry somatic mutations. Irrespective of mutation status, these LCH-cells display constitutively active kinases belonging to the MAPK pathway. We evaluated, in retrospect, the contribution of individual components of the MAPK-activating and chemotaxis-promoting TNF-CXCR4-CXCL12 axis to LCH manifestation and outcome. Experimental design: CXCR4, CXCL12 and TNF protein expression was immunohistochemically analyzed in 70 LCH-affected biopsies. The presence of CXCR4+CD1a+ cells in peripheral blood (PB) and/or bone marrow (BM) samples was evaluated by flowcytometry in 13 therapy-naive LCH-patients. Results: CXCL12 was detected in 68/70 (97%) biopsies. CXCR4+LCH-cells were present in 50/70 (71%) biopsies; their presence was associated with higher levels of intralesional TNF. Circulating CD1a+CXCR4+ cells were detected in 4/13 (31%) therapy-naïve LCH-patients which displayed BRAFV600E (2/4), MAP2K1 (1/4) or no (1/4) mutations in their tissues. These CD11c co-expressing CD1a+CXCR4+cells migrated to CXCL12 in chemotaxis assays. Lesional CXCR4+LCH-cells were detected in 18/20 cases who presented with LCH manifestation at multiple sites and in 5/23 (22%) patients who developed additional lesions after initially presenting with a single lesion. The CXCR4 status at onset proved to be an independent risk factor for LCH reactivation in multivariate analysis (odds ratio 10.4, p = 0.034). Conclusions: This study provides the first evidence that CXCR4 is involved in the homing and retention of LCH-cells in CXCL12-expressing tissues and qualifies CXCR4 as a candidate prognostic marker for less favorable disease outcome.

Abstract 4830: The correlation between CXCR7 expression and EGFR mutation in non-small cell lung cancer

Abstract Objective: To explore the correlation between CXCR7 expression and EGFR mutation in NSCLC. Materials and Methods: Forty-nine paired cases of NSCLC carcinoma and para-carcinoma tissues were analyzed for the expression of CXCR7 mRNA by RT-PCR and CXCR7 protein by IHC. EGFR mutations were detected in carcinoma tissues by ARMS. T-test, one-way ANOVA and correlation analysis were used to analyze the data with SPSS software version 18.0. Results: In NSCLC, CXCR7 protein expression in carcinoma tissues was higher than in para-carcinoma tissues (14450±2049 vs 5206±1066, p&amp;lt;0.0001), while CXCR7 mRNA was not. In NSCLC, CXCR7 mRNA and CXCR7 protein expression levels were both related to the TNM stage (p = 0.007, p = 0.008) and metastatic status (p&amp;lt;0.0001; p&amp;lt;0.0001), but not to gender, age, smoking index and pathology. EGFR and CXCR7 protein were positively correlated (r = 0.289,p = 0.044) Conclusion: Like EGFR, CXCR7 can be used as a marker to predict the prognosis and guide clinical therapy in NSCLC. Keywords: CXCR7 RDC1 EGFR NSCLC Note: This abstract was not presented at the meeting. Citation Format: Yiping Han. The correlation between CXCR7 expression and EGFR mutation in non-small cell lung cancer. [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 4830. doi:10.1158/1538-7445.AM2015-4830

Abstract 1713: IL-24 inhibits lung cancer cell migration and invasion by disrupting the SDF-1/CXCR4 signaling axis

Abstract Introduction The stromal cell derived factor (SDF)-1/chemokine receptor (CXCR)-4 signaling pathway plays a key role in lung cancer metastasis. Therefore, disrupting the SDF-1/CXCR4 signaling axis will reduce the incidence of lung metastasis. In the present study we investigated whether interleukin (IL)-24 can inhibit the SDF-1/CXCR4 axis and suppress lung cancer cell migration and invasion in vitro. Further, the efficacy of IL-24 in combination with CXCR4 antagonists was investigated. Methods Human H1299 lung tumor cell line was stably transfected with a tetracycline-inducible plasmid vector carrying the IL-24. Upon addition of doxycycline (Dox; 1μg/ml), cells were induced to express IL-24 protein. The expression levels of CXCR4 and its downstream molecular mechanisms in H1299 cells were analyzed. The inhibitory effect of IL-24 on SDF-1/CXCR4 axis is determined by RT-qPCR, western blot, luciferase reporter assay, flow-cytometry and immuno-cytochemistry and the consequence of its inhibition on cell migration, and invasion. Results Endogenous CXCR4 protein expression levels varied among four human lung cancer cell lines with H1299 cells showing the highest expression. Doxycycline-induced IL-24 expression in the H1299-IL-24 cell line resulted in reduced CXCR4 mRNA and protein expression. IL-24 post-transcriptionally regulated CXCR4 mRNA expression by decreasing the half-life of CXCR4 mRNA (&amp;gt;40%). Associated with CXCR4 inhibition was the reduced protein expression of pAKTS473, pmTORS2448, pPRAS40T246 and HIF-1α. IL-24 inhibited tumor cell migration and invasion both in the presence and absence of the CXCR4 agonist, SDF-1. However, the combinatorial effect of either IL-24 combined with CXCR4 inhibitors (AMD3100, SJA5) or with CXCR4 siRNA demonstrated enhanced inhibitory activity on tumor cell migration. Conclusions Our study results demonstrate that IL-24 inhibits lung tumor cell migration and invasion by disrupting the SDF-1/CXCR4 signaling pathway and exhibits enhanced anti-metastatic activity when combined with CXCR4 inhibitors. Citation Format: Janani Panneerselvam, Jiankang Jin, Manish Shanker, Jason Lauderdale, Jonathan Bates, Qi Wang, Daniel Zhao, Stephen Archibald, Timothy Hubin, Rajagopal Ramesh. IL-24 inhibits lung cancer cell migration and invasion by disrupting the SDF-1/CXCR4 signaling axis. [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 1713. doi:10.1158/1538-7445.AM2015-1713

Effect of CXCR4 pretreated with ultrasound-exposed microbubbles on accelerating homing of bone marrow mesenchymal stem cells to ischemic myocardium in AMI rats.

To investigate the role and potential mechanism of CXCR4 in promoting targeted homing of bone marrow mesenchymal stem cells (BMSCs) with ultrasound-exposed microbubbles (UM) pretreatment. Third generation BMSCs were divided into four groups control group, ultrasound (US) group, UM group and ultrasound-exposed microbubbles plus catalase group. RT-PCR and western blot were performed to determine the levels of CXCR4 mRNA transcription and protein expression, respectively. Third generation BMSCs were labeled with Fluo-α/AM and divided into three groups: control group, US group and UM group, and fluorescence intensities in the cells were observed immediately, 5min and 15min after intervention under fluorescence microscope. The calcium iron levels in the cells were analyzed. BMSCs were divided into five group: group A without calcium in the medium, group B, group C, group D and group E containing calcium chloride with concentration of l mol, 2mol, 4mol, anti-calcium-sensing receptor antibody, respectively. RT-PCR and western blot were performed to determine the levels of CXCR4 mRNA transcription and proteins expression of the third generation BMSCs of each group, respectively. The levels of CXCR4 mRNA transcription and protein expression between US group and control group had no statistically significant difference (P>0.05) shown by RT-PCR and western blot; the transcription level in the UM group was significantly higher than that in US group and control group (P<0.05); and in the ultrasound-exposed microbubbles plus catalase group, the transcription level was much lower than that in UM group. Fluorescence intensify in the cells of US group had no significant difference compared with that in the cells of the control group (P>0.05), which in the cells of UM group was significantly higher than that in the cells of both US group and control group (P<0.05). Compared to group A, expressions of CXCR4 of group B to D were significantly increased in concentration-dependent manner showed by RT-PCR and western blot (P<0.05). Compared to group C, expressions of CXCR4 of group E were significantly decreased (P<0.05). UM can promote the influx of calcium in BMSCs and increase mRNA transcription and protein expression of CXCR4. The latter may partly be caused by influx of calcium.

GSK3β mediates pancreatic cancer cell invasion in vitro via the CXCR4/MMP-2 Pathway.

BackgroundGlycogen synthase kinase-3β (GSK3β) expression and activity are upregulated in pancreatic cancer tissues. In our previous study, we found that stromal cell-derived factor-1/ chemokine receptor C-X-C motif chemokine receptor 4 (SDF-1α/CXCR4) upregulated matrix metalloproteinase 2 (MMP-2) and promoted invasion in PANC1 and SW-1990 pancreatic cancer cells by activating p38 mitogen-activated protein kinase (p38 MAPK). Additionally, inhibition of GSK3β reduced MMP-2 secretion.MethodsTo investigate the molecular mechanism of GSK3β in pancreatic cancer tissues, we created stable PANC1 cells up-regulation of GSK3β by transfecting GSK3β overexpression plasmid, and down-regulation of GSK3β using two different types of RNA interference.ResultsWestern blotting showed that overexpression of GSK3β up-regulated CXCR4 and MMP-2 expression; suppression of GSK3β down-regulated CXCR4 and MMP-2 protein expression. Up-regulation of MMP2 induced by overexpression of GSK3β was blocked by inhibition of CXCR4. Overexpression of GSK3β promoted PANC1 cell invasion, and down-regulation of GSK3β suppressed PANC1 cell invasion in the transwell invasion assays. However, inhibition of CXCR4 using shRNA attenuated the ability of GSK3β to promote PANC1 cell invasion.ConclusionsThis study demonstrated that GSK3β promotes PANC1 cell invasion via the CXCR4/MMP-2 pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0216-y) contains supplementary material, which is available to authorized users.