CXCR4 Protein Research Articles

CXCR4 Protein Epitope Mimetic Antagonist POL5551 Disrupts Metastasis and Enhances Chemotherapy Effect in Triple-Negative Breast Cancer.

The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow-disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a "chemotherapy framing" dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases.

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.

Imaging CXCR4 Expression with (99m)Tc-Radiolabeled Small-Interference RNA in Experimental Human Breast Cancer Xenografts.

Noninvasive quantification of chemokine receptor 4 (CXCR4) expression could serve as a prognostic indicator and may be of value for the design of personalized therapies and posttreatment monitoring. The objective of the present study was to assess the use of (99m)Tc-radiolabeled small-interference RNA (siRNA) targeting CXCR4 to detect CXCR4 expression in vivo. CXCR4 siRNAs were radiolabeled with (99m)Tc using the bifunctional chelator hydrazinonicotinamide (HYNIC), and the labeling efficiency, specific activity and radiochemical purity were determined. The stability of the probe in serum was assessed by measuring its radiochemical purity and inhibitory activity by RT-PCR and western blotting. Biodistribution studies and static imaging were performed in MDA-MB-231 tumor-bearing mice. Radiochemical purity remained highly stable in PBS and fresh human serum at room temperature and at 37°C. Radiolabeled siRNA1 showed strong inhibitory effects similar to those of unlabeled siRNA1 on both CXCR4 messenger RNA (mRNA) and protein in vitro. The excretion of the probe occurred mainly through the liver and kidneys. Tumors were clearly visualized at 1-10h after injection of the probe, but not after injection of the control probe. (99m)Tc-labeled CXCR4 siRNA1 shows tumor-specific accumulation and could be a promising strategy for the visualization of CXCR4 expression in human breast cancer.

Cigarette smoke exposure triggers the autophagic cascade via activation of the AMPK pathway in mice.

We previously demonstrated that cigarette smoke (CS) exposure decreases primordial follicle counts and induces autophagy in ovarian granulosa cells in preference to apoptosis. Therefore, the objective of this study was to investigate molecular targets underlying smoke-induced activation of the reparative autophagy pathway in the ovary. Briefly, ovarian homogenates were prepared from adult female mice exposed to mainstream CS twice daily for 8 wk, using a whole-body exposure system. A gene array revealed that CS exposure induced a greater than 2-fold significant increase in the expression of proautophagic genes Cdkn1b, Map1lc3a, Bad, and Sqstm1/p62. A significant increase in Prkaa2, Pik3c3, and Maplc31b expression, as well as a significant decrease in Akt1 and Mtor expression, was detected by quantitative PCR. The 5'-AMP-activated protein kinase catalytic subunit (AMPK) alpha1 + alpha2 and ATG7 protein expression was significantly increased, whereas AKT1, mTOR, CDKN1B/p27, and CXCR4 proteins were significantly decreased in CS exposed versus control ovaries. Up-regulation of AMPK alpha1 + alpha2, a known initiator of autophagic signaling, and ATG7 further suggests activation of the autophagy cascade. Two prosurvival factors, AKT and mTOR, were decreased in expression, an outcome that favors induction of the autophagy pathway, whereas decreased levels of CDKN1B is suggestive of cell cycle dysregulation. In summary, our data suggest that CS exposure induces ovarian follicle loss through induction of the autophagic cascade via the AMPK pathway together with inhibition of antiautophagic markers AKT and mTOR. We further postulate that toxicant-induced dysregulation of reparative autophagy is a novel pathway central to impaired follicle development and subfertility.

GLI1 orchestrates CXCR4/CXCR7 signaling to enhance migration and metastasis of breast cancer cells.

The up-regulation of chemokine receptors CXCR4 and CXCR7 impacts on the distant metastasis and prognosis of breast cancer, though knowledge about the regulatory mechanism of their expressions is limited. Meanwhile, the GLI transcription factors of Hedgehog signaling have been reported to play a pivotal role in the development and progression of many types of human cancer. In breast cancer, the increased expression of GLI1 correlated with metastasis and unfavorable overall prognosis, though its molecular mechanism is also not fully understood. Based on our findings that GLI1 enhanced the lung metastasis of breast cancer cells in a mouse model system, we comprehensively screened for genes up-regulated by GLI1 in breast cancer cells, and as such identified CXCR4, CXCR7/ACKR3, and actin-binding protein LCP1/L-PLASTIN, all of which have been reported to be involved in CXCL12-stimulating signaling. In breast cancer cells, we found that GLI1 and GLI2 up-regulated these expressions, while treatment with GLI-specific inhibitor GANT61 reduced the expressions. As for CXCR4, we confirmed it as a direct target of GLI1 through the reporter assay and the chromatin immunoprecipitation assay. We also found that GLI1 enhanced CXCL12-induced ERK phosphorylation and cell migration, both of which were blocked by either CXCR4-specific inhibitor or knockdown of CXCR7 or LCP1. These evidences suggest an indispensable role of GLI1 in the migration and metastasis of breast cancer cells through CXCL12/CXCR4 signaling enhancement.

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.

CXCR3 chemokine receptor signaling mediates itch in experimental allergic contact dermatitis.

Persistent itch is a common symptom of allergic contact dermatitis (ACD) and represents a significant health burden. The chemokine CXCL10 is predominantly produced by epithelial cells during ACD. Although the chemokine CXCL10 and its receptor CXCR3 are implicated in the pathophysiology of ACD, it is largely unexplored for itch and pain accompanying this disorder. Here, we showed that CXCL10 and CXCR3 mRNA, protein, and signaling activity were upregulated in the dorsal root ganglion after contact hypersensitivity (CHS), a murine model of ACD, induced by squaric acid dibutylester. CXCL10 directly activated a subset of cutaneous dorsal root ganglion neurons innervating the area of CHS through neuronal CXCR3. In behavioral tests, a CXCR3 antagonist attenuated spontaneous itch- but not pain-like behaviors directed to the site of CHS. Injection of CXCL10 into the site of CHS elicited site-directed itch- but not pain-like behaviors, but neither type of CXCL10-evoked behaviors was observed in control mice. These results suggest that CXCL10/CXCR3 signaling mediates allergic itch but not inflammatory pain in the context of skin inflammation. Thus, upregulation of CXCL10/CXCR3 signaling in sensory neurons may contribute to itch associated with ACD. Targeting the CXCL10/CXCR3 signaling might be beneficial for the treatment of allergic itch.

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.

Prolonged ischemia elicits acute allograft rejection involved in CXCR3 activation in rat kidney transplants

BackgroundAcute rejection is a major obstacle in patients with prolonged ischemia in deceased-donor renal transplantation. Chemokines and their receptors play a critical role in leukocyte trafficking, resulting in allograft rejection; therefore, the role of chemokine receptor CXCR3 in acute rejection induced by prolonged ischemia in rat kidney transplantation models was evaluated. MethodsSyngeneic and allogeneic renal transplantations were performed. For cold ischemia, grafts were stored in 4.0°C University of Wisconsin solution for 12 or 16h. Serum and renal tissues were harvested 7.0d after surgery and serum TNF-α, IL-6, and renal function were measured. Graft histology was stained with periodic acid–Schiff and immunohistochemical staining and further evaluated for signs of acute rejection. CXCR3 proteins were quantified by Western blot. The transplanted rats were divided into 4 groups as follows: iso-12-h=isogeneic transplant with 12-h CIT graft; iso-16-h=isogeneic kidney transplant with 16-h CIT graft; allo-12-h=allogeneic renal transplant with 12-h CIT graft; allo-16h=allogeneic renal transplant with 16-h CIT graft; and 16h+T=allogeneic 16-h CIT graft received tacrolimus. ResultsProlonged cold ischemia time (CIT; 16h) enhanced acute glomerular damage, interstitial inflammation, and tubulointerstitial cellular infiltration in allografts with and without immunosuppressant tacrolimus; but it was not apparent in the isografts. The expression of CXCR3 protein and the proportion of CXCR3-positive cells were significantly higher in the allo-16h and 16h+T groups than that in the allo-12h group 7d post-surgery. ConclusionsCIT triggered acute rejection in allogeneic, but not in isogeneic, kidney transplants, accompanied by an elevation of leukocyte recruitment and damaged graft function. The upregulated expression of chemokine receptor CXCR3 promoted inflammatory infiltration and acute allograft rejection.

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

Abstract 1906: CXCR4 is a novel target of cancer chemopreventative isothiocyanates in prostate cancer cells

Abstract Isothiocyanates (ITCs) derived from cruciferous vegetables, including phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit in vivo activity against prostate cancer in xenograft and transgenic mouse models, and thus are appealing for chemoprevention of this disease. Watercress constituent PEITC and SFN-rich broccoli sprout extract are under clinical investigation but the molecular mechanisms underlying their cancer chemopreventive effects are not fully understood. The present study demonstrates that chemokine receptor CXCR4 is a novel target of ITCs in prostate cancer cells. Exposure of prostate cancer cells (LNCaP, 22Rv1, C4-2, and PC-3) to pharmacologically applicable concentrations of PEITC, benzyl isothiocyanate (BITC), and SFN (2.5 and 5 μmol/L) resulted in downregulation of CXCR4 expression. None of the isothiocyanates affected secretion of CXCR4 ligand (stromal-derived factor-1). In vivo inhibition of PC-3 xenograft growth upon PEITC treatment was associated with a significant decrease in CXCR4 protein level. A similar trend was discernible in the tumors from SFN-treated TRAMP mice compared with those of control mice, but the difference was not significant. Stable overexpression of CXCR4 in PC-3 cells conferred significant protection against wound healing, cell migration, and cell viability inhibition by ITCs. Inhibition of cell migration resulting from PEITC and BITC exposure was significantly augmented by RNA interference of CXCR4. This study demonstrates, for the first time, that cancer chemopreventive ITCs suppress CXCR4 expression in prostate cancer cells in vitro as well as in vivo. These results suggest that CXCR4 downregulation may be an important pharmacodynamic biomarker of cancer chemopreventative ITCs in prostate adenocarcinoma. Citation Format: Ali I. Amjad, Kozue Sakao, Avani R. Vyas, Sreenivasa R. Chinni, Rahul Parikh, Shivendra V. Singh. CXCR4 is a novel target of cancer chemopreventative isothiocyanates in prostate cancer cells. [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 1906. doi:10.1158/1538-7445.AM2015-1906

Type 1 Diabetes Prone NOD Mice Have Diminished Cxcr1 mRNA Expression in Polymorphonuclear Neutrophils and CD4+ T Lymphocytes.

In humans, CXCR1 and CXCR2 are two homologous proteins that bind ELR+ chemokines. Both receptors play fundamental roles in neutrophil functions such as migration and reactive oxygen species production. Mouse Cxcr1 and Cxcr2 genes are located in an insulin-dependent diabetes genetic susceptibility locus. The non obese diabetic (NOD) mouse is a spontaneous well-described animal model for insulin-dependent type 1 diabetes. In this disease, insulin deficiency results from the destruction of insulin-producing beta cells by autoreactive T lymphocytes. This slow-progressing disease is dependent on both environmental and genetic factors. Here, we report descriptive data about the Cxcr1 gene in NOD mice. We demonstrate decreased expression of mRNA for Cxcr1 in neutrophils and CD4+ lymphocytes isolated from NOD mice compared to other strains, related to reduced NOD Cxcr1 gene promoter activity. Looking for Cxcr1 protein, we next analyze the membrane proteome of murine neutrophils by mass spectrometry. Although Cxcr2 protein is clearly found in murine neutrophils, we did not find evidence of Cxcr1 peptides using this method. Nevertheless, in view of recently-published experimental data obtained in NOD mice, we argue for possible Cxcr1 involvement in type 1 diabetes pathogenesis.

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.

Al[18F]NOTA-T140 Peptide for Noninvasive Visualization of CXCR4 Expression.

Chemokine receptor CXCR4 plays an important role in tumor aggressiveness, invasiveness, and metastasis formation. Quantification of CXCR4 expression by tumors may have an impact on prediction and evaluation of tumor response to therapies. In this study, we developed a robust and straightforward F-18 labeling route of T140, a CXCR4 peptide-based antagonist. T140 derivative was conjugated to 1,4,7-triazacyclononane-triacetic acid (NOTA) and labeled with Al[(18)F]. Al[(18)F]NOTA-T140 was evaluated in vitro in cell-based assay and stability in mouse serum and in vivo using CXCR4 positive and negative tumor xenograft models. Labeling of Al[(18)F]NOTA-T140 was completed within 30 min with a radiochemical yield of 58 ± 5.3 % at the end of synthesis, based on fluoride-18 activity. Al[(18)F]NOTA-T140 accumulated in CHO-CXCR4 positive but not negative tumors. Al[(18)F]NOTA-T140 uptake in the tumors correlated with CXCR4 protein expression. Moreover, Al[(18)F]NOTA-T140 had high accumulation in CXCR4-positive metastatic tumors. The simplicity of Al[(18)F]NOTA-T140 labeling along with its properties to specifically image CXCR4 expression by tumors warrant further clinical application for the diagnosis of CXCR4 clinically.

CXCR4 Regulates Extra-Medullary Myeloma through Epithelial-Mesenchymal-Transition-like Transcriptional Activation

Extra-medullary disease (EMD) in multiple myeloma (MM) is associated with poor prognosis and resistance to chemotherapy. However, molecular alterations that lead to EMD have not been well defined. We developed bone marrow (BM)- and EMD-prone MM syngeneic cell lines; identified that epithelial-to-mesenchymal transition (EMT) transcriptional patterns were significantly enriched in both clones compared to parental cells, together with higher levels of CXCR4 protein; and demonstrated that CXCR4 enhanced the acquisition of an EMT-like phenotype in MM cells with a phenotypic conversion for invasion, leading to higher bone metastasis and EMD dissemination in vivo. In contrast, CXCR4 silencing led to inhibited tumor growth and reduced survival. Ulocuplumab, a monoclonal anti-CXCR4 antibody, inhibited MM cell dissemination, supported by suppression of the CXCR4-driven EMT-like phenotype. These results suggest that targeting CXCR4 may act as a regulator of EMD through EMT-like transcriptional modulation, thus representing a potential therapeutic strategy to prevent MM disease progression.

Co-expression of CXCR4 and CD133 in gastric neoplastic tissue and their correlation with clinicopathological factors and prognosis in gastric cancer

Purpose: Worldwide Gastric carcinoma considered as the second most common cause of cancer related death. Cancer stem cell plays significant role in prognosis and invasion of gastric cancer. CXCR4 is a chemokine receptor and plays an important role in self renewal, differentiation potential, and cell adhesion of cancer stem cell (CSC). On the other hand CD133 is a cell surface glycoprotein and could serve as a prognostic indicator for tumor re-growth, malignant progression, and patient survival. The aim of this study was to establish the expression pattern of CXCR4 and CD133 in gastric cancer tissues; and their correlation with clinicopathological factors like age and gender of patients, position, size and depth of tumor, lymphatic invasion and node metastasis. Methods: Expression of CXCR4 and CD133 proteins were assessed by immunohistochemical and immunofluorescence staining of paraffin–embedded tissues, and followed by RT-PCR in 90 tumors (observed group) and 30 normal gastric samples. The clinical pathological data was statistically analyzed by chi-square methods. Results: The positive rate of CXCR4 and CD133 expression in the observed group was 94.44 (85/90) and 95.55 (86/90) respectively. The expression of CXCR4 and CD133 were correlated with age and gender of patients, and position, size, & depth of the tumor, lymphatic invasion and node metastasis ( p < 0.05). While CXCR4 was positive, CD133 had a positive rate of 92.22% but the positive rate was 2.22% when CXCR4 expression was negative (χ 2 = 58.657; p < 0.001). Conclusion: Overall this data suggests that increased expression of CXCR4 and CD133 might be attributed with disease progression and malignant transformation of gastric epithelium cells. A significant correlation was found in between CXCR4 and CD133 expression and their co-expression may play significant role in invasiveness of gastric cancer.

SDF-1/CXCR4 promotes F5M2 osteosarcoma cell migration by activating the Wnt/β-catenin signaling pathway.

Osteosarcoma (OS), the most common primary malignant bone tumor in children and adolescents, lacks an effective therapy. Stromal cell-derived factor (SDF-1) and its receptor, CXCR4, play multiple roles in migration, proliferation, and survival of different tumor cells. This study aimed to investigate whether the functional SDF-1/CXCR4 signaling mediates chemotaxis in F5M2 OS cells as well as the underlying mechanisms. Immunohistochemistry and immunofluorescence microscopy were used. RNA expression was detected by real-time quantitative polymerase chain reaction, and protein expression was examined by Western blotting. Migration assays were carried out in F5M2 cells. The results showed that the expression of CXCR4 and β-catenin mRNA and protein was significantly higher in OS tissues compared to the surrounding non-neoplastic tissues. SDF-1 promoted F5M2 cell migration by activating the AKT and Wnt/β-catenin signaling pathway, which was abrogated by preincubation with AMD3100 and LY294002. In conclusion, SDF-1/CXCR4 axis-promoted F5M2 cell migration was regulated by the Wnt/β-catenin signaling pathway.

Studying the binding interactions of allosteric agonists and antagonists of the CXCR4 receptor

Several examples of allosteric modulators of GPCRs have been reported recently in the literature, but understanding their molecular mechanism presents a new challenge for medicinal chemistry. For the specific case of the cellular receptor CXCR4, it is known that pepducins (lipidated fragments of intracellular GPCR loops) such as ATI-2341 modulate CXCR4 activity agonistically via an allosteric mechanism. Moreover, there are also examples of small organic molecules such as AMD11070 and GSK812397 which may also act as allosteric antagonists. However, incomplete knowledge of the ligand-binding sites has hampered a detailed molecular understanding of how these inhibitors work. Here, we attempt to answer this question by analysing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose two different allosteric binding sites, one located in the intracellular loops 1, 2 and 3 (ICL1, ICL2 and ICL3) which binds the pepducin agonist ATI-2341, and the other at a subsite of the main extracellular orthosteric binding pocket between extracellular loops 1 and 2 and the N-terminus, which binds the antagonists AMD11070 and GSK812397. Allosteric interactions between the CXCR4 and ATI-2341 were predicted by combining different modeling approaches. First, a rotational blind docking search was applied and the best poses were subsequently refined using flexible docking methods and molecular dynamic simulations. For the AMD11070 and GSK812397 antagonists, the entire CXCR4 protein surface was explored by blind docking in order to define the binding region. A second docking analysis by subsites was then performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 allosteric modulators.