CXC Receptor 4 Expression Research Articles

Prognostic role of the stromal cell derived factor-1 in patients with hepatitis B virus-related acute-on-chronic liver failure.

Stromal cell derived factor-1 (SDF-1) plays a pivotal role in the recruitment of stem cells to injured livers. However, the changes of SDF-l in patients with hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) have yet to be elucidated. To study the SDF-1 changes in patients with HBV-related ACLF. 30 patients with HBV-related ACLF, 27 patients with chronic hepatitis B and 20 healthy individuals are involved in our study. The SDF-l mRNA expression in liver tissue was detected by quantitative real-time polymerase chain reaction. Immunohistochemical staining was performed to illustrate the expression of SDF-l, CXC receptor 4 (CXCR4) and Ki67. The serum SDF-l concentrations were also detected by enzyme-linked immunosorbent assays. The expression of SDF-1 mRNA from ACLF patients was remarkably higher than that from other patients (both P < 0.05). The expression of SDF-l, CXCR4 and Ki67 from ACLF were the highest among the three groups (all P < 0.01). The serum SDF-l levels in ACLF patients were significantly lower than that in other patients (both P < 0.01). Moreover, in ACLF patients, the serum SDF-1 Levels were positively correlated with serum total bilirubin and international normalized ratio. In addition, the serum SDF-l levels in survival were significantly lower compared with the non-survivals (P < 0.05). The area under the curve for the serum SDF-1 level in predicting 28-d mortality was 0.722 (P < 0.05). This study provides the SDF-1 changes in patients with HBV-related ACLF. The SDF-1 Level at admission may serve as a promising prognostic marker for predicting short-term prognosis.

SDF-1/CXCR4 axis participants in the pathophysiology of adult patients with moyamoya disease

BackgroundMoyamoya disease (MMD) is characterized by an abundance of moyamoya vessels; however, the precise mechanism driving the spontaneous angiogenesis of these compensatory vessels remains unclear. Previous research has established a link between the stromal cell-derived factor-1 (SDF-1)/ CXC receptor 4 (CXCR4) axis and angiogenesis under hypoxic conditions. Nevertheless, the alterations in this axis within the cerebrospinal fluid, arachnoid membranes and vascular tissue of MMD patients have not been fully investigated. MethodsOur study enrolled 66 adult MMD patients and 61 patients with atherosclerotic vascular disease (ACVD). We investigated the SDF-1 concentration in cerebrospinal fluid (CSF) and CXCR4 expression level on the arachnoid membranes and vascular tissue. We utilized enzyme-linked immunosorbent assay and immunohistochemistr. Additionally, we cultured and stimulated human brain microvascular endothelial cells (HBMECs) and smooth muscle cells (SMCs) under oxygen and glucose deprivation (OGD) conditions followed by reoxygenation, to examine any changes in the SDF-1/CXCR4 axis. ResultsThe results demonstrated an elevation in the level of SDF-1 in CSF among MMD patients compared to those with ACVD. Moreover, the expression of CXCR4 in arachnoid membranes and vascular tissue showed a similar trend. Furthermore, the content of CXCR4 in HBMECs and SMCs increased with the duration of ischemia and hypoxia. However, it was observed that the expression of CXCR4 decreased at OGD/R 24h compared to OGD 24h. The temporal pattern of SDF-1 expression in HBMECs and SMCs mirrored that of CXCR4 expression. ConclusionThese findings indicate a critical role for the SDF-1/CXCR4 axis in the angiogenesis of moyamoya disease.

P1253: MAVORIXAFOR DISRUPTS THE CROSS TALK BETWEEN WALDENSTRÖM’S MACROGLOBULINEMIA CELLS AND THE BONE MARROW MICROENVIRONMENT RESULTING IN ENHANCED SENSITIVITY TO B-CELL TARGETED THERAPIES

Background: Waldenström’s macroglobulinemia (WM) is a rare B-cell malignancy characterized by infiltration of bone marrow (BM) and lymphoid tissues with lymphoplasmacytic cells along with hypersecretion of monoclonal immunoglobulin M (IgM) by progenitor B cells. Studies have shown that more than 90% of patients with WM have an L265P mutation in the myeloid differentiation primary response gene 88 (MYD88), and 30% to 40% of those patients show mutations in the carboxyl terminus of C-X-C receptor 4 (CXCR4). The CXCR4/C-X-C motif ligand 12 (CXCL12) axis plays an important role in the homing and retention of WM cells in the BM. The interaction of WM cells and the BM microenvironment, via cell-to-cell contacts or indirectly through secreted factors (e.g. interleukin 6 [IL-6], C-C motif ligand 5 [CCL5]), is thought to be essential for the pathogenesis of WM and to confer drug resistance. A clinical trial evaluating the efficacy of the CXCR4 antagonist, mavorixafor, in combination with the BTK inhibitor, ibrutinib in patients with MYD88L265P and CXCR4WHIM WM is currently ongoing (NCT04274738). However, the effects of mavorixafor with ibrutinib and other B-cell targeted therapies on WM cells harboring only the single mutation in MYD88L265P and without a CXCR4 mutation have not been evaluated. Aims: This study was designed to elucidate the crosstalk between WM cells and the BM stromal cell (BMSC)-based microenvironment and to explore the effect of mavorixafor in combination with different B-cell targeted therapies in a WM/BMSC co-culture model. Methods: WM cells (MWCL-1 cell line, MYD88L265PCXCR4WT) pretreated with mavorixafor and B-cell targeted therapies (BTK antagonists: ibrutinib, zanubrutinib, evobrutinib, pirtobrutinib, nemtabrutinib; or BCL2 inhibitor venetoclax) were co-cultured with established BMSCs (HS27a cells). Apoptosis, IL-6 production, and IgM release were measured after 48 or 72 hours. The effects of mavorixafor on calcium mobilization, cell migration, and adhesion to BMSCs were also measured. Results: Co-culture of WM cells with established BMSCs led to increased IgM secretion and enhanced surface expression of CXCR4 in WM cells by activation of the IL-6 receptor (IL-6R)/ Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Co-culture with established BMSCs protected WM cells from the effects of B-cell targeted therapies, reducing apoptosis of tumor cells and decreasing the IgM-reducing efficacy of the targeted therapies. Mavorixafor alone reduced IgM secretion, blocked CXCL12-induced cell migration and Ca2+ mobilization and disrupted the adhesion of WM cells to established BMSCs. Mavorixafor in combination with B-cell targeted therapies synergistically enhanced antitumor activity in WM cells, inhibited BMSC-induced IgM hypersecretion, and overcame the protective effects of BMSCs on tumor cells. Summary/Conclusion: Our studies provide preliminary evidence for the potential use of mavorixafor in disrupting the interaction of WM cells with the BMSC-based microenvironment, enhancing the efficacy of B-cell targeted therapies in the treatment of WM and potentially other lymphomas. Further studies using additional WM cell lines and/or primary patient cells are warranted to support these findings.

Association of SDF-1 Gene Polymorphism with Increased Risk of Acute Myeloid Leukemia Patients.

Background:Acute myeloid leukemia (AML) is a heterogenous group of disorders that emerge from the malignant transformation of hematopoietic stem cells. Chemokine stromal cell-derived factor 1(SDF-1) and its receptor CXC receptor 4 (CXCR4) has an essential role in dissemination of blast cells. Study aimed to detect CXCR4 expression and the SDF-1 (rs1801157) gene polymorphisms and correlate them with prognosis and outcome in AML patients. Subjects and Methods:The study was conducted on 60 de-novo AML patients, and 60 healthy controls. SDF-1 (rs1801157) gene polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and CXCR4 expression was done using flow cytometry analysis. Results:SDF-1 dominant model (AG+AA) had higher risk AML (p 0.002). CXCR4 positive cases were associated significantly with toxic manifestations (p 0.019), lower CR rates (p 0.004), and unfavorable cytogenetics (p 0.027). Multivariate analysis showed that combined CXCR4positive with dominant SDF-1 considered as independent prognostic factor for shorter overall survival (OS) in AML patients (p 0.031). Conclusion:SDF-1 dominant model had a higher risk to develop AML, and CXCR4 positive expression predicts poor prognosis in AML patients and it could represent a targeted therapy in AML. In addition, CXCR4 could be easily integrated into the initial routine diagnostic work up of AML.

Targeting CXCR4 in AML and ALL.

The interaction of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts with the bone marrow microenvironment regulates self-renewal, growth signaling, as well as chemotherapy resistance. The chemokine receptor, CXC receptor 4 (CXCR4), with its ligand chemokine ligand 12 (CXCL12), plays a key role in the survival and migration of normal and malignant stem cells to the bone marrow. High expression of CXCR4 on AML and ALL blasts has been shown to be a predictor of poor prognosis for these diseases. Several small molecule inhibitors, short peptides, antibodies, and antibody drug conjugates have been developed for the purposes of more effective targeting and killing of malignant cells expressing CXCR4. In this review we will discuss recent results and strategies in targeting CXCR4 with these agents in patients with AML or ALL.

High Mobility Group Box 1 Neutralization in the Brain Prevents Inflammation, Sympathoexcitation and Hypertension

High mobility group box 1 (HMGB1) is a nuclear protein, that once translocated to cytoplasm, can act as a chemoattractant and a proinflammatory mediator. Post‐translationally modified HMGB1 can interact with different receptors, such as toll‐like receptor 4 (TLR4) and chemokine receptor CXC receptor 4 (CXCR4), to induce acute and chronic inflammation. We have previously showed that central TLR4 blockade delayed progression of hypertension and improved cardiac function. However, the involvement of HMGB1 and its interaction with TLR4 in the pathogenesis of hypertension has not been fully explored. Therefore, in this study, we investigated the role of HMGB1 in mediating hypertension, inflammation and sympathoexcitation. We hypothesized that blockade of HMGB1 prevents the development of hypertension. Chronic angiotensin II (AngII) infusion (200 ng/kg/min, SC, 14 days) significantly increased mean arterial pressure (telemetry) in male Sprague‐Dawley rats compared to saline‐infused controls (163 ±5 vs. 105 ±4 mmHg, n=7, p&lt;0.01). AngII‐infusion significantly increased HMGB1 levels in the plasma (21 ±3 vs.15 ±2 ng/ml, p&lt;0.01) and cerebrospinal fluid (34±3 vs. 21±1 ng/ml, p&lt;0.01), and elevated HMGB1 protein expression (2.5 fold, n=6, p&lt;0.01 vs. Control) in the brain hypothalamic paraventricular nucleus (PVN). Intracerebroventricular infusion of neutralizing monoclonal anti‐HMGB1 antibody (10 μg/kg/day) attenuated AngII‐induced hypertension (120 ±2 mmHg, p&lt;0.01 vs. AngII). Furthermore, AngII‐infusion significantly increased expression of TNF, IL‐1, IL‐6, TLR4, CXCR4, and NF‐kB in the PVN, which was prevented by HMGB1 neutralization. AngII infusion resulted in sympathoexcitation, as indicated by increased plasma norepinephrine levels (1602 ±53 vs. 549 ±25 pg/ml, P&lt;0.01 vs. Control), but this increase was blunted by HMGB1 neutralization (720 ±25 pg/ml, P&lt;0.05 vs. AngII). Taken together, these data suggest that neutralization of HMGB1 in the brain reduces inflammation and decreases sympathoexcitation, thereby preventing the development of AngII‐induced hypertension.Support or Funding InformationThe American Heart Association, 15SDG25720021 (SS), 16GRNT30510012 (JF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of CXC-motif-chemokine 12 and the receptor C-X-C receptor 4 in glioma and theeffect on peritumoral brain edema.

The present study aimed to evaluate the association between CXC-motif-chemokine 12 (CXCL12)/C-X-C receptor 4 (CXCR4) expression and peritumoral brain edema (PTBE) in glioma patients. Immunohistochemical techniques were used to detect the expression of CXCR4 and CXCL12 in 58 glioma tissues. Magnetic resonance imaging was used to evaluate the extent and type of brain edema in preoperative glioma patients. The association between edema and CXCL12/CXCR4 expression was examined by χ2 analysis. The prognostic significance of CXCL12 or CXCR4 was determined by log-rank tests and Cox's proportional hazards model. Expression of CXCL12 and CXCR4 was observed in vascular endothelial cells and tumor cells. The degree (P=0.033) and morphology (P=0.033) of PTBE were significantly associated with the level of CXCL12 expression in vascular endothelial cells. The degree (P=0.001) and morphology (P=0.001) of PTBE were associated with the level of CXCR4 expression in tumor cells. CXCR4-positive vascular endothelial cells were significantly associated only with the degree of edema (P=0.030). Therefore, the present study indicated that levels of CXCL12 expression in vascular endothelial cells and CXCR4 expression in tumor cells are associated with PTBE.

Overexpression of CXCR4 promotes invasion and migration of non-small cell lung cancer via EGFR and MMP-9.

The aim of the present study was to verify whether overexpression of CXC receptor 4 (CXCR4) promotes the invasion and migration of non-small cell lung cancer (NSCLC) via epidermal growth factor receptor (EGFR) and matrix metallopeptidase-9 (MMP-9), and to detect the association between CXCR4, EGFR and MMP-9. The effects of overexpression of CXCR4 on lung cancer cell functions were investigated by migration and invasion assays. Western blotting and zymograph assays were used to analyze the protein expression levels of EGFR and the production of MMP-9, respectively. Immunohistochemistry was applied to analyze the expression of EGFR, CXCR4 and MMP-9 in NSCLC. Statistical analyses were used to detect the associations among EGFR, CXCR4 and MMP-9 in NSCLC. Finally, survival analyses were performed. CXCR4 overexpression enhanced cell motility and invasion. CXCR4 also promoted expression of EGFR and elevated MMP-9 production. CXCR4, EGFR and MMP-9 were highly expressed in NSCLC, and were not identified as associated with age and sex (P>0.05). However, they were associated with tumor differentiation and lymph node metastasis (P<0.05). CXCR4, EGFR and CXCR4 expression were positively associated with one another in NSCLC (P<0.05). In addition, patients with positive expression of CXCR4, EGFR or MMP-9 in tumors exhibited significantly shorter overall survival compared with those with negative expression (P<0.05). In conclusion, CXCR4 overexpression enhanced cell motility and invasion via EGFR and MMP-9. CXCR4, EGFR and MMP-9 were identified as highly expressed in NSCLC, and there was positive correlation among them.

Nuclear Respiratory Factor-1 (NRF-1) Regulates Transcription of the CXC Receptor 4 (CXCR4) in the Rat Retina.

The CXC receptor 4 (CXCR4) is required for various physiologic and pathologic processes in the eye, including stem cell trafficking, neuronal development, immune responses, and ocular neovascularization. Here, we used the rat retina models to determine the mechanisms driving CXCR4 transcription. The expression pattern of CXCR4 and nuclear respiratory factor-1 (NRF-1) were profiled in the rat retina during the course of development. Chromatin immunoprecipitation (CHiP) assay determined the transcriptional mechanism of CXCR4 in rat retina. A rat model of oxygen-induced retinopathy (OIR) that mimics retinal ischemia-reperfusion injury was established. Under either normoxic or hypoxic conditions, CXCR4 and NRF-1 expression in rat retinas was tracked by RT-PCR and Western analysis. Immunofluorescence staining localized CXCR4 and NRF-1. Both CXCR4 and NRF-1 were highly expressed in the neonatal rat retina, down-regulated in parallel, and silenced in fully developed retinas (1 month of age). ChIP assays revealed that NRF-1 was required for CXCR4 promoter activity in rat retinas. In the OIR rat model, retinal hypoxia induced up-regulation of CXCR4 and NRF-1 concurrently. Our findings suggest that NRF-1 regulates the expression of CXCR4 in normal retinal development and in pathologic processes of retinal hypoxia and neovascularization.

Anti-Leukemic Activation of Immune Cells in a Leukemic-Induced Microenvironment By CXCR4 Inhibition

The bone marrow microenvironment (BMM) provides a protective niche that supports growth and survival of leukemic stem cells. Stromal-derived factor-1 (SDF-1)/CXC receptor 4 (CXCR4) plays pivotal roles in the cross-interactions between blasts and the BMM to prevent retention and mobilization of leukemic cells, as well as in normal hematopoiesis including the development of immune cells. Here, we show that the CXCR4 antagonist, Plerixafor, decreased the level of CXCR4 expression and inhibited SDF-1-induced migration of leukemic cells. Further, the inhibition of the interaction between leukemic cells and the BMM by the CXCR4 antagonist enhanced cytotoxic activity of immune cells as a result of increased susceptibility of leukemic cells to chemotherapeutic agents such as cytosine arabinoside (Ara-C) in a mouse model of acute myeloid leukemia (AML), suggesting biological effects of the BMM through immune cell activation. To examine the level of CXCR4 expressed by primary AML blasts and murine leukemic cells and the role of CXCR4 in migration at various concentration, 19 AML samples and murine C1498 and human Jurkat cells were subjected to FACS analysis and migration assay. CXCR4 expression in C1498 cells was about 31.29%, compared to 99.7% in the CXCR4+ Jurkat cell line and RNA expression was also confirmed. C57Bl/6J mice were used to construct a syngeneic AML animal model by infusion of 2x106 C1498 cells, and peripheral blood from a facial vein and samples from multiple organs from the sacrificed mice were obtained until day 30 post-injection. Total RNA was extracted from BM cells, liver, and spleen of mice at day 15 and 1ug RNA was reverse transcribed into cDNA using a Transcriptor First Strand cDNA Synthesis Kit. All data were normalized to the amount of GAPDH expression, with samples run in triplicate. The quantitative real-time PCR assay for IFN-γ, perforin, and granzyme B, main cytokines produced by cytotoxic T and NK cells, was performed to examine the functional capacity of subsets. Data revealed that both normal human samples and AML mononuclear cells highly expressed CXCR4. CXCR4 expression in both groups was significantly decreased by CXCR4 inhibition, when treated with 5 µM Plerixafor. Our migration assay clearly showed the inhibitory effect of Plerixafor on SDF-1α-induced migration of primary AML and C1498 cells. Under SDF-1α conditions (100 ng/ml), C1498 cells were co-cultured with the CXCR4 antagonist at various concentrations. Migrations of both C1498 and primary AML cells were similarly inhibited. To test the direct role of the CXCR4 antagonist in apoptosis, C1498 cells were cultured with or without Ara-C. The Ara-C and Plerixafor dual-treated group (termed P+A group) displayed no significant difference in apoptosis when compared to the Ara-C only group, suggesting that apoptosis is exclusively controlled by Ara-C, but not Plerixafor, in vitro. However, leukemic blasts synergistically and significantly decreased in the P+A group, compared to those of the other groups, in a syngeneic leukemic mouse model experiments, suggesting an unexpected role for Plerixafor in leukemic blast suppression specifically in the AML niche. The frequency of CD4 and CD8 cells was maintained in the P+A group, compared to the Ara-C-only, Plerixafor treated only, and C1498 injected groups, implying that Plerixafor cannot independently alter the frequency of immune cells. Most of all, the expression levels of IFN-γ, perforin, and granzyme B in the spleens of the P+A group were significantly increased, compared to those of the control groups including the Plerixafor only group. This study demonstrates that the effects of CXCR4 inhibition on blast suppression and immune cell function in the tumor microenvironment and chemotherapy with Plerixafor represents an advanced therapeutic strategy of targeting the leukemic niche. DisclosuresNo relevant conflicts of interest to declare.

High glucose microenvironments inhibit the proliferation and migration of bone mesenchymal stem cells by activating GSK3β.

Diabetes mellitus involves metabolic changes that can impair bone repair. Bone mesenchymal stem cells (BMSCs) play an important role in bone regeneration. However, the bone regeneration ability of BMSCs is inhibited in high glucose microenvironments. It can be speculated that this effect is due to changes in BMSCs' proliferation and migration ability, because the recruitment of factors with an adequate number of MSCs and the microenvironment around the site of bone injury are required for effective bone repair. Recent genetic evidence has shown that the Cyclin D1 and the CXC receptor 4 (CXCR-4) play important roles in the proliferation and migration of BMSCs. In this study we determined the specific role of glycogen synthase kinase-3β (GSK3β) in the proliferation and migration of BMSCs in high glucose microenvironments. The proliferation and migration ability of BMSCs were suppressed under high glucose conditions. We showed that high glucose activates GSK3β but suppresses CXCR-4, β-catenin, LEF-1, and cyclin D1. Inhibition of GSK3β by LiCl led to increased levels of β-catenin, LEF-1, cyclin D1, and CXCR-4 expression. Our data indicate that GSK3β plays an important role in regulating the proliferation and migration of BMSCs by inhibiting cyclin D1 and CXCR-4 under high glucose conditions.

Effect of the LPA-mediated CXCL12-CXCR4 axis in the tumor proliferation, migration and invasion of ovarian cancer cell lines.

Ovarian cancer is the most fatal gynecological cancer, with a 5-year survival rate of only 30%. Lysophosphatidic acid (LPA), which possesses growth factor-like functions, is a major regulatory factor in the peritoneal metastasis of ovarian cancer. LPA stimulates the expression of numerous genes that are associated with angiogenesis and metastasis. Ovarian epithelial carcinoma specifically expresses chemotactic factor C-X-C motif chemokine ligand 12 (CXCL12) and its receptor, CXC receptor 4 (CXCR4). The CXCL12-CXCR4 axis directly contributes to ovarian cancer cell proliferation, migration and invasion. The present study investigated the regulation of LPA on the CXCL12-CXCR4 axis and the effect of the LPA-mediated CXCL12-CXCR4 axis on the tumor proliferation, migration and invasion of ovarian cancer cell lines. The CXCR4 proteins expressed in the cell membrane and the cytoplasm of ovarian cancer cells, CAOV3 and SKOV3, were detected by immunocytochemistry. The expression of CXCR4 and CXCL12 was increased in the ovarian cancer cells in a dose- and time-dependent manner when treated with LPA compared with the control groups (P<0.05), as determined by reverse transcription polymerase chain reaction and flow cytometry. LPA (20 μM) and CXCL12 (100 ng/ml) enhanced the proliferation, migration and invasion of the ovarian cancer cells, CAOV3 and SKOV3, as identified by MTT, Transwell and Matrigel assays following co-treatment for 24 h. LPA promoted invasiveness of ovarian cancer by upregulating CXCL12-CXCR4 axis expression.

Effect of CO2 pneumoperitoneum on the expression of the chemokine receptors CXCR4 and CCR7 in colorectal carcinoma cells in vitro

Background The ability of pneumoperitoneum in laparoscopic surgery to promote proliferation and metastasis of colorectal cancer has become a focus of research in the field of minimally invasive surgery. The aim of this research was to investigate the effect of CO2 pneumoperitoneum under different pressures and exposed times on the expression of chemokine receptors in colorectal carcinoma cells. Methods We constructed an in vitro pneumoperitoneum model. SW480 colon carcinoma cells were exposed to CO2 pneumoperitoneum under different pressures (6, 9, 12, and 15 mmHg) for 1, 2, and 4 hours. These cells were then cultivated under the same conditions as normal SW480 colon carcinoma cells without CO2 pneumoperitoneum (control group), treated at 37°C, and 5% CO2. The expression of the chemokine receptors CXC receptor 4 (CXCR4) and chemokine C receptor 7 (CCR7) was detected by immunocytochemistry and reverse transcriptase polymerase chain reaction after being cultivated for 0, 24, 48, and 72 hours. Results Immunocytochemistry showed that CXCR4 expression in SW480 cells was significantly decreased in the 6, 9, 12, and 15 mmHg CO2 pneumoperitoneum-treated groups for the same exposure times compared with controls (P &lt;0.05). CCR7 expression in SW480 cells was significantly decreased in the 12 and 15 mmHg CO2 pneumoperitoneum-treated groups compared with controls (P &lt;0.05). CXCR4 and CCR7 expression increased up to the level of the control group after 24 and 48 hours (P &gt;0.05). If the CO2 pneumoperitoneum pressure increased, CXCR4 and CCR7 expression decreased at all exposure times. If the CO2 pneumoperitoneum exposure time prolonged, there were no significant differences in CXCR4 and CCR7 expression under the same pressure. Under all exposure times, CXCR4 and CCR7 mRNA expression was significantly decreased in the 6, 9, 12, and 15 mmHg CO2 pneumoperitoneum-treated groups (P &lt;0.05) compared with controls, and it increased up to the level of controls after being cultivated for 48 hours (P &gt;0.05). If the CO2 pneumoperitoneum pressure increased (with all exposure times) and exposure time prolonged (under the same pressure), there were no significant differences in CXCR4 and CCR7 expression. Conclusions CXCR4 and CCR7 expression is temporarily affected after continuous CO2 pneumoperitoneum treatment. The high pressure of CO2 pneumoperitoneum plays an important role in suppressing the expression of these chemokine receptors. Different lengths of time of exposure to a CO2 pneumoperitoneum-like environment do not change CXCR4 and CCR7 expression.

CXC receptor 4 and stromal cell–derived factor 1 in primary tumors and liver metastases of colorectal cancer

BackgroundIt has been determined that the chemokine receptor CXC receptor 4 (CXCR4) and its ligand, stromal cell-derived factor 1 (SDF-1), regulate several key processes in a wide variety of cancers. In this study, we investigate the possible role of SDF-1 (noncancerous liver tissue) and CXCR4 in liver metastases of colorectal cancer (CRC). Materials and methodsReverse transcription–polymerase chain reaction was performed to examine the expression of SDF-1 in noncancerous liver tissues of 16 CRC patients with liver metastasis and in normal liver tissues of six patients with benign liver disease. We also examined the expression of CXCR4 in cancerous tissues from primary and metastatic tumors. ResultsUsing reverse transcription–polymerase chain reaction, CXCR4 expression in metastatic tumors tended to be higher than that in primary tumors (P = 0.16). High CXCR4 expression in a primary tumor was found to be related to an increased lymphatic invasion (P = 0.01), an advanced depth of tumor invasion (P = 0.07), and a decrease in the overall survival rate. The SDF-1 expression observed in noncancerous liver tissues of CRC with liver metastasis was significantly higher than that observed in normal liver tissues of benign liver disease (P < 0.05). ConclusionsIn CRC with liver metastasis, CXCR4 expression demonstrated associations with local progression, liver metastasis, and poor overall survival.

Abstract 5118: Cysteine (C)-X-C receptor 4 undergoes transportin 1 dependent nuclear localization and is functional at the nucleus of metastatic prostate cancer cells.

Abstract The G-Protein coupled receptor (GPCR) Cysteine (C)-X-C Receptor 4 (CXCR4) plays an important role in prostate cancer (PCa) metastasis. To date, considerable evidence support the presence of GPCRs at the plasma membrane (PM), or within the perinuclear/nuclear compartments of cells and have been suggested to regulate a number of physiological processes including cell proliferation, survival, inflammatory responses and tumorigenesis. In accordance with previous clinical observations in diverse tumor tissues, we observed ubiquitous expression of CXCR4 in metastatic PCa tissues and several PCa cells lines. Additionally, Gαi immunoprecipitation and calcium mobilization studies elucidated that nuclear CXCR4 was functional and participated in G-protein signaling. However, a mechanism by which CXCR4 is translocated to the nucleus is poorly understood.An initial search of the CXCR4 sequence via the PSORT II NLS prediction software program revealed a putative, nontraditional nuclear localization sequence (NLS), “RPRK” in CXCR4.Additionally,the members of karyopherin beta (β) family, Karyopherin-β2 or Transportin 1 (TRN1) were reported to be involved in transportation of various cargos into the nucleus, including the chemokine GPCRs. Collectively, based on our results and previous studies, we suggest that the NLS, “RPRK”and TRN1 play a role in translocation of CXCR4 into the nucleus of advanced PCa. To determine the involvement of “RPRK," manipulations of the NLS revealed that single point mutations of basic resides within the NLS was insufficient to eliminate to abrogate nuclear translocation of CXCR4. However, when the NLS was deleted, nuclear CXCR4 was not detected. Additionally, TRN1 immunoprecipitation and siRNA-TRN1 studies delineated an association between CXCR4 and TRN1,and that nuclear translocation of CXCR4 depended on TRN1. Little information is available about nuclear GPCRs and nuclear CXCR4 in cancer. Thus, the significance of the study is that since CXCR4 is a major signaling receptor involved in PCa metastasis, the dualistic localization pattern and function at the nucleus may encourage recurrence,metastasis and poorer prognosis after tumors have been treated with therapy that targets CXCR4.In conclusion, our studies will provide insight to the development of therapeutics towards CXCR4 and transport mechanisms, as current therapy targets membrane receptors, which may be ineffective if CXCR4 is translocated and functional inside of the cell. Citation Format: Ayesha S. Don-Salu-Hewage, Siu Y. Chan, Kathleen McAndrews, Mahandranauth A. Chetram, Michelle R. Dawson, Cimona V. Hinton. Cysteine (C)-X-C receptor 4 undergoes transportin 1 dependent nuclear localization and is functional at the nucleus of metastatic prostate cancer cells. [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 5118. doi:10.1158/1538-7445.AM2013-5118

Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), ‘RPRK’, within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinβ1 and Transportinβ1-binding to CXCR4 promoted its nuclear translocation. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

ROS enhances CXCR4-mediated functions through inactivation of PTEN in prostate cancer cells

Inactivation of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is heavily implicated in the tumorigenesis of prostate cancer. Conversely, the upregulation of the chemokine (CXC) receptor 4 (CXCR4) is associated with prostate cancer progression and metastasis. Studies have shown that loss of PTEN permits CXCR4-mediated functions in prostate cancer cells. Loss of PTEN function is typically due to genetic and epigenetic modulations, as well as active site oxidation by reactive oxygen species (ROS); likewise ROS upregulates CXCR4 expression. Herein, we show that ROS accumulation permitted CXCR4-mediated functions through PTEN catalytic inactivation. ROS increased p-AKT and CXCR4 expression, which were abrogated by a ROS scavenger in prostate cancer cells. ROS mediated PTEN inactivation but did not affect expression, yet enhanced cell migration and invasion in a CXCR4-dependent manner. Collectively, our studies add to the body of knowledge on the regulatory role of PTEN in CXCR4-mediated cancer progression, and hopefully, will contribute to the development of therapies that target the tumor microenvironment, which have great potential for the better management of a metastatic disease.

Ginsenoside Rg3 inhibits CXCR4 expression and related migrations in a breast cancer cell line

Ginsenoside Rg3 is an extract from the natural product ginseng. Previous studies have linked Rg3 with anti-metastasis of cancer in vivo and in vitro. CXC receptor 4 (CXCR4) is a vital molecule in migration and homing of cancer to the docking regions. In this study, the effects of Rg3 on CXCR4 expression were investigated in a breast cancer cell line. Immunohistochemistry, chemotaxis and wound healing mobility assays were performed in cultured MDA-MB-231 cells. At a dosage without obvious cytotoxicity, Rg3 treatment elicits a weak CXCR4 stain color, decreases the number of migrated cells in CXCL12-elicited chemotaxis and reduces the width of the scar in wound healing. This work suggests that Rg3 is a new CXCR4 inhibitor from a natural product.

Hyperglycemia and Oxidized-LDL exert a deleterious effect on Endothelial Progenitor Cell migration in Type 2 Diabetes Mellitus

Introduction Type 2 diabetes mellitus (DM) patients with coronary artery disease (CAD) have elevated plasma oxidized-LDL (OxLDL) levels and impaired neovascularization. Hyperglycemia and hyperlipidemia impair endothelial progenitor cell (EPC) migration, and endothelial nitric oxide (NO) bioavailability and NO synthase (NOS) activity are essential for EPC migration. Stromal-derived factor-1α (SDF1α) contributes to EPC mobilization and homing by stimulating the CXC receptor-4 (CXCR4) on the EPC plasmalemma to activate the Pi3K/Akt/eNOS signaling pathway. Therefore, we investigated the effect of high glucose (HG) and OxLDL on the migration and NO bioavailability of EPCs from healthy individuals, and then correlated the findings with those of EPCs from type 2 DM patients with and without CAD. Materials and Methods EPCs from 15 healthy and 55 patients were exposed to HG, OxLDL, or both before evaluating EPC count, migration and NO production, and expression of CXCR4 and members of Pi3K/Akt/eNOS signaling cascade. Results Counts, migration, CXCR4 expression, and NO production were significantly reduced in EPCs from DM and CAD patients compared with that obtained in EPCs from healthy, and were further reduced in DM patients with CAD. The expression of CXCR4 and activation of Pi3K/Akt/eNOS signaling cascade were suppressed in OxLDL- and HG-treated EPCs, and this suppression was exacerbated when EPCs were treated simultaneously with HG and OxLDL. Conclusions Hyperglycemia and elevated circulating OxLDL in DM patients with CAD severely impair EPC migration. These results suggest that the underlying mechanism for this impaired EPC migration is linked to the CXCR4/Pi3K/Akt/eNOS signaling pathway.

Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1α(CXCL12)/CXCR4 axis

AbstractFlt3 ligand (FL) enhances hematopoietic cell proliferation and facilitates hematopoietic stem cell mobilization in vivo, while the stromal-derived factor 1α (SDF-1α, CXC ligand 12 [CXCL12])/CXC receptor 4 (CXCR4) axis is critical for their homing and trafficking. We investigated if FL and its receptor, Flt3, functionally interact with CXCL12/CXCR4 to regulate hematopoietic cell migration. FL stimulated chemokinetic activity when used alone, but synergistically enhanced short-term migration of CD34+ cells, Ba/F3 cells expressing human Flt3 (Ba/F3-Flt3), and human RS4;11 acute leukemia cells, induced by CXCL12. Moreover, overexpression of constitutively activated internal tandem duplication (ITD)–Flt3 mutants in Ba/F3 cells dramatically enhanced migration toward CXCL12. In Ba/F3-Flt3 cells, synergistic cell migration to FL plus CXCL12 was associated with enhanced phosphorylation of mitogen-activated protein kinase p42/p44 (MAPKp42/p44), cyclic adenosine monophosphate response element binding protein (CREB), and Akt, and was partially inhibited by pretreatment of cells with selective inhibitors for MAPKp42/p44, protein kinase A (PKA), or phosphatidylinositol 3–kinase (PI3-kinase), implicating these pathways in migration to FL plus CXCL12. In contrast, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulated CXCR4 expression, inhibited CXCL12-mediated phosphorylation of MAPKp42/p44, CREB, and Akt, and impaired migration toward CXCL12. These findings suggest that FL/Flt3 may facilitate hematopoietic cell migration/homing and mobilization by enhancing or inhibiting CXCL12/CXCR4 signaling pathways and that the FL/Flt3 axis participates in trafficking of normal and transformed hematopoietic cells.