C-X-C Motif) Receptor 4 Research Articles

CXCR4-Targeted Therapy Inhibits VEGF Expression and Chondrosarcoma Angiogenesis and Metastasis

Chondrosarcoma is notable for its lack of response to conventional cytotoxic chemotherapy, propensity for developing lung metastases, and poor survival. Therefore, a better understanding of angiogenic and metastatic pathways is needed. Multiple pathways regulate angiogenesis and metastasis, including chemokines and their receptors. In this study, we investigated chemokine (C-X-C motif) receptor 4 (CXCR4) signaling in chondrosarcoma and tested the hypotheses that CXCR4 inhibition suppresses tumor angiogenesis and metastasis. CXCR4 expression, analyzed by real-time PCR and Western blot, was increased in human chondrosarcoma cell line JJ compared with normal chondrocytes and was further increased in JJ by hypoxia (2% O2), vascular endothelial growth factor A (VEGFA; 10 ng/mL), and in xenograft tumors in nude mice. The CXCR4 ligand CXCL12 (10 ng/mL) doubled secreted VEGFA, measured with ELISA, under hypoxic conditions and this conditioned media increased human umbilical vein endothelial cell tube formation. These effects were inhibited by CXCR4 siRNA or AMD3100 (5 μg/mL). In a xenograft mouse model, four weeks of AMD3100 treatment (1.25 mg/kg, intraperitoneally twice daily) inhibited tumor angiogenesis, tumor growth, and metastasis. VEGFA content in tumor extracts was decreased (7.19 ± 0.52 ng/mL control vs. 3.96 ± 0.66 treatment) and bioimaging of angiogenesis was decreased by 56%. Tumor volumes averaged 4.44 ± 0.68 cm(3) in control compared with 2.48 ± 0.61 cm(3) in the treatment group. The number of lung metastatic nodules was 23 ± 9 in control compared with 10 ± 6 in the treatment group (N = 8/group). Therefore, CXCR4-targeted therapy may be a treatment strategy for chondrosarcoma.

Natriuretic Peptide Receptor A Signaling Regulates Stem Cell Recruitment and Angiogenesis: A Model to Study Linkage Between Inflammation and Tumorigenesis

Natriuretic peptide receptor A (NPRA), the signaling receptor for the cardiac hormone, atrial natriuretic peptide (ANP), is expressed abundantly in inflamed/injured tissues and tumors. NPRA deficiency substantially decreases tissue inflammation and inhibits tumor growth. However, the precise mechanism of NPRA function and whether it links inflammation and tumorigenesis remains unknown. Since both injury repair and tumor growth require stem cell recruitment and angiogenesis, we examined the role of NPRA signaling in tumor angiogenesis as a model of tissue injury repair in this study. In in vitro cultures, aortas from NPRA-KO mice show significantly lower angiogenic response compared to wild-type counterparts. The NPRA antagonist that decreases NPRA expression, inhibits lipopolysaccharide-induced angiogenesis. The reduction in angiogenesis correlates with decreased expression of vascular endothelial growth factor and chemokine (C-X-C motif) receptor 4 (CXCR4) implicating a cell recruitment defect. To test whether NPRA regulates migration of cells to tumors, mesenchymal stem cells (MSCs) were administered i.v., and the results showed that MSCs fail to migrate to the tumor microenvironment in NPRA-KO mice. However, coimplanting tumor cells with MSCs increases angiogenesis and tumorigenesis in NPRA-KO mice, in part by promoting expression of CXCR4 and its ligand, stromal cell-derived factor 1α. Taken together, these results demonstrate that NPRA signaling regulates stem cell recruitment and angiogenesis leading to tumor growth. Thus, NPRA signaling provides a key linkage between inflammation and tumorigenesis, and NPRA may be a target for drug development against cancers and tissue injury repair.

SDF‐1 promotes ox‐LDL induced vascular smooth muscle cell proliferation

The mechanism of the regulatory roles of stromal cell derived factor-1 (SDF-1)/C-X-C motif receptor 4 (CXCR4) on cell proliferation and apoptosis in vascular smooth muscle cells (VSMCs) via the protein kinase C (PKC) and nuclear factor-kappa B (NF-κB) signalling pathways have been investigated. Rat aortic VSMCs were treated with control or an oxidised low-density lipoprotein (ox-LDL) atherosclerosis (AS) model. Cells exposed to the AS model were treated with SDF-1 plus inhibitors specific for PKC (Ro31-8220), CXCR4 (12G5) or NF-κB (pyrrolidine dithiocarbamate, PDTC). Cell proliferation was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis by flow cytometry. NF-κB protein expression was analysed using Western blotting. The proliferation rate in the AS model group was significantly higher than the control group, but lower than the SDF-1 group (P < 0.05). Apoptosis in the AS model group (ox-LDL) was significantly higher than the normal control group (P < 0.05). In addition, the apoptosis rate in the SDF-1 group was significantly lower than the normal control group (P < 0.05); however, there was no difference from the Ro31-8220 group. NF-κB protein expression in the SDF-1 group was significantly higher than the AS model (ox-LDL) group (P < 0.05). In conclusion, SDF-1 can promote the proliferation of VSMCs induced by ox-LDL and inhibit cell apoptosis, via the SDF-1/CXCR4 axis.

Reduced CXCR4 expression is associated with extramedullary disease in a mouse model of myeloma and predicts poor survival in multiple myeloma patients treated with bortezomib

Reduced CXCR4 expression is associated with extramedullary disease in a mouse model of myeloma and predicts poor survival in multiple myeloma patients treated with bortezomib

Maraviroc treatment in non-R5-HIV-1-infected patients results in the selection of extreme CXCR4-using variants with limited effect on the total viral setpoint

Using deep sequencing methods, we intensively investigated the selective pressure of maraviroc on the viral population in four patients with dual/mixed HIV-1 experiencing treatment failure. Patients received maraviroc add-on therapy (n = 4). Tropism was determined by Monogram's Trofile assay and/or 'deep' sequencing. Longitudinal 'deep' sequence analysis used triplicate HIV V3 RT-PCR on plasma samples. Sequences were interpreted using the geno2phenocoreceptor algorithm with a 3.5% false-positive rate (FPR) cut-off. Patients had a median viral load of 4.7 log10 HIV RNA copies/mL with a median of 24% chemokine (C-X-C motif) receptor 4 (CXCR4)-using virus at baseline. Following maraviroc exposure, the chemokine (C-C motif) receptor 5 (CCR5)-using virus (R5) plasma viral load decreased by at least 1 log10, and only non-R5 variants with extremely low FPR values predominated after 21 days. Virus with an FPR ≤1.8% accounted for more than 90% of the circulating virus, having expanded to occupy the 'space' left by the suppression of R5 variants. Population genetic estimates of viral fitness in the presence of maraviroc showed a steep rise around an FPR value of 2%. Longitudinal analysis of independent R5 and non-R5 HIV populations shows that maraviroc selects viruses with an extremely low FPR, implying that the antiviral activity of maraviroc may extend to a broader range of HIV variants than previously suspected.

Abstract 1419: A phase I study of the CXCR4 peptide antagonist LY2510924 in patients with advanced cancer.

Abstract Background: The over-expression of C-X-C motif receptor 4 (CXCR4), a chemokine receptor implicated in tumor progression, tumor cell growth, migration, and invasion, correlates with a worse prognosis. LY2510924 is a selective peptide antagonist to CXCR4. This peptide blocks stromal cell-derived factor-1 (SDF-1), the only known ligand to CXCR4, from binding to the receptor. Disruption of the CXCR4/SDF-1 axis with CXCR4 antagonists is known to mobilize WBC, neutrophils, and CD34+ cells. The primary objective of this study was to determine the maximum tolerated dose (MTD). Secondary objectives were to determine safety, pharmacokinetics, efficacy, and pharmacodynamic response, which included mobilization of CD34+ hematopoietic stem cells into the peripheral blood. Material &amp; Methods: This phase I study in patients (pts) with advanced cancer included two Parts: a 3+3 dose escalation (Part A) and dose confirmation (Part B). LY2510924 was administered as a daily injection subcutaneously (s.c.) on a 28-day cycle. Results: Forty-five pts, 98% with solid tumors, were enrolled onto study, 25 in Part A and 20 in Part B. The median age was 67.0 years (range: 39-86), 51.1% were male, and 80.0% were Caucasian. The Eastern Cooperative Oncology Group performance status was 1 for 73.3% of pts. In Part A, pts in 6 cohorts were treated with doses of LY2510924: 1.0 (n=3), 2.5 (n=5), 5.0 (n=3), 10 (n=3), 20 (n=4), and 30 mg/day (n=7). Two pts in the 30 mg/day cohort experienced dose-limiting toxicities, grade 3 neutrophil count increase. The MTD was determined to be 20 mg/day. Pts in Part B were treated with either 2.5 (n=10) or 20 mg/day (n=10). For Parts A and B combined, the most frequently reported (≥15%) treatment emergent adverse events (TEAE), regardless of causality, included fatigue, nausea, constipation, anemia, and anorexia. The most common grade 3 TEAE were increased neutrophil count, anemia, asthenia, and dypsnea (all occurring in 4.4% of pts). There were no grade 4 TEAE. The best response was stable disease for 9 pts (20.0%). At the end of Cycle 1, mean peak LY2510924 plasma concentration (Cmax) and the 24-hour area under the plasma concentration vs. time curve (AUC) increased slightly more than dose proportionally. Mean Cmax and AUC ranged from 18.8 ng/mL to 1250 ng/mL, and 61.5 ng•h/mL to 5720 ng•h/mL, respectively, in the 1-30 mg dose range. Median time to Cmax was 0.5 hours independent of dose. Receptor occupancy was consistently high with median values of 96.9-100% for 2.5 mg, 20 mg and 30 mg, between 0.5 hours through 24 hours after dosing. There was an up to 18-fold mean increase from baseline in CD34+ cell count in peripheral blood, with an apparent dose-response relationship between 1mg and 10 mg, and little additional response with 20 mg or 30 mg. The increase persisted to the end of Cycle 1, but was somewhat blunted. Conclusions: LY2510924 demonstrated CD34+ cell mobilization with an acceptable safety profile up to an MTD of 20 mg/day (s.c.). Citation Format: Nicholas Vogelzang, Mansoor Saleh, Paul Conkling, Eyas J. Abu-Raddad, John P. Polzer, Stephanie Roberson, John R. Stille, Donald E. Thornton. A phase I study of the CXCR4 peptide antagonist LY2510924 in patients with advanced cancer. [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 1419. doi:10.1158/1538-7445.AM2013-1419

Airway smooth muscle CXCR3 ligand production: regulation by JAK-STAT1 and intracellular Ca2+

In asthma, airway smooth muscle (ASM) chemokine (C-X-C motif) receptor 3 (CXCR3) ligand production may attract mast cells or T lymphocytes to the ASM, where they can modulate ASM functions. In ASM cells (ASMCs) from people with or without asthma, we aimed to investigate JAK-STAT1, JNK, and Ca²⁺ involvement in chemokine (C-X-C motif) ligand (CXCL)10 and CXCL11 production stimulated by interferon-γ, IL-1β, and TNF-α combined (cytomix). Confluent, growth-arrested ASMC were treated with inhibitors for pan-JAK (pyridone-6), JAK2 (AG-490), JNK (SP-600125), or the sarco(endo)plasmic reticulum Ca²⁺ATPase (SERCA) pump (thapsigargin), Ca²⁺ chelator (BAPTA-AM), or vehicle before and during cytomix stimulation for up to 24 h. Signaling protein activation as well as CXCL10/CXCL11 mRNA and protein production were examined using immunoblot analysis, real-time PCR, and ELISA, respectively. Cytomix-induced STAT1 activation was lower and CXCR3 ligand mRNA production was more sensitive to pyridone-6 and AG-490 in asthmatic than nonasthmatic ASMCs, but CXCL10/CXCL11 release was inhibited by the same proportion. Neither agent caused additional inhibition of release when used in combination with the JNK inhibitor SP-600125. Conversely, p65 NF-κB activation was higher in asthmatic than nonasthmatic ASMCs. BAPTA-AM abolished early CXCL10/CXCL11 mRNA production, whereas thapsigargin reduced it in asthmatic cells and inhibited CXCL10/CXCL11 release by both ASMC types. Despite these inhibitory effects, neither Ca²⁺ agent affected early activation of STAT1, JNK, or p65 NF-κB. In conclusion, intracellular Ca²⁺ regulated CXCL10/CXCL11 production but not early activation of the signaling molecules involved. In asthma, reduced ASM STAT1-JNK activation, increased NF-κB activation, and altered Ca²⁺ handling may contribute to rapid CXCR3 ligand production and enhanced inflammatory cell recruitment.

Abstract C32: The critical roles of tumor-initiating cell and lymph node stromal microenvironment in human colorectal cancer extranodal metastasis

Abstract In the U.S., colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer-related mortality, with an estimated incidence of 143,000 cases and 51,000 deaths per year. The outcome of CRC patients depends on multiple factors, such as, staging (depth of tumor invasion into colon or rectum wall), lymph node (LN) involvement, local recurrence, and extra-nodal metastasis. Despite optimal oncologic treatment including surgery, chemotherapy and/or radiation therapy, up to 50% of the American Joint Committee on Cancer stage II and III patients will develop extra-nodal metastasis. The most common metastatic sites are the liver or lung. These metastatic cancer cells have acquired a chemoresistant phenotype and developed genetic alterations. Recent studies from our group and others have proposed that metastasis or recurrence is closely associated with the presence of CRC-cancer stem cells or -tumor initiating cells (Co-TIC) expressing CD133 and the chemokine (C-X-C motif) receptor 4 (CXCR4), and their interaction with the LN microenvironment. Our hypothesis is that LN stromal cells prime, support, educate, and stimulate Co-TIC through soluble factors such as the chemokine (C-X-C motif) ligand 12 (CXCL12, ligand to CXCR4), and CD133+CXCR4+ Co-TIC are responsible for drug resistance and extra-nodal metastasis. In this study, we investigated the critical roles of Co-TIC and LN stromal microenvironment in human CRC extra-nodal metastasis using our unique humanized orthortopic xenograft model. The luciferase-tagged colon cancer cell lines and CRC cancer cells isolated from consented patients were injected intra-rectal (IR) to NOD/SCID mice. The LN stromal cell lines HK and CXCL12 knockdown HK (HK-KD-A3) cells were co-inoculated with CRC cells. In addition, Co-TIC were FACS sorted based on positivity of cell surface markers CD326, CD133 and CXCR4. Tumor growth and extra-nodal metastasis were followed by luciferase activity as detected by the IVIS imaging system and quantitative PCR for human DNA in mice livers and lungs. The IR model closely mimicked the expected pattern of CRC spread by showing progressive CRC tumor growth under rectum mucosa with liver and lung metastasis in NOD/SCID mice both injected with CRC cell lines and patient cancer cells. The presence of HK cells or pre-incubation of cancer cells with HK cell conditioned media promoted CRC cell distant metastasis. CXCL12 knockdown HK-KD-A3 cells showed significantly impaired HK cell support of CRC primary tumor formation and more importantly distant organ metastasis. In the presence of HK cells, CD133+CXCR4+ Co-TIC showed increased tumor formation and extra-nodal metastasis in comparison to un-separated and double negative cancer cell populations. Our results indicate both Co-TIC and LN stromal factors play crucial roles in CRC metastasis through CXCL12/CXCR4 axis. Blocking the signaling in Co-TIC/LN stromal interaction may lead to an effective therapy to prevent CRC extra-nodal metastasis, a major cause of cancer morbidity and mortality. In addition, the IR model would serve as a launching pad for evaluating the mechanism of CRC metastasis and testing novel targeted therapies. Citation Format: Tamara Myers, Izi Obokhare, Chelsea Grimes, Xin Zhang, Jennifer Silinsky, Jose Cordova, Heather Green, David Margolin, Li Li. The critical roles of tumor-initiating cell and lymph node stromal microenvironment in human colorectal cancer extranodal metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C32.

Astrocyte-Derived CXCL10 Drives Accumulation of Antibody-Secreting Cells in the Central Nervous System during Viral Encephalomyelitis

Microbial infections of the central nervous system (CNS) are often associated with local accumulation of antibody (Ab)-secreting cells (ASC). By providing a source of Ab at the site of infection, CNS-localized ASC play a critical role in acute viral control and in preventing viral recrudescence. Following coronavirus-induced encephalomyelitis, the CNS accumulation of ASC is chemokine (C-X-C motif) receptor 3 (CXCR3) dependent. This study demonstrates that CNS-expressed CXCR3 ligand CXCL10 is the critical chemokine regulating ASC accumulation. Impaired ASC recruitment in CXCL10(-/-) but not CXCL9(-/-) mice was consistent with reduced CNS IgG and κ-light chain mRNA and virus-specific Ab. Moreover, the few ASC recruited to the CNS in CXCL10(-/-) mice were confined to the vasculature, distinct from the parenchymal localization in wild-type and CXCL9(-/-) mice. However, neither CXCL9 nor CXCL10 deficiency diminished neutralizing serum Ab, supporting a direct role for CXCL10 in ASC migration. T cell accumulation, localization, and effector functions were also not affected in either CXCL9(-/-) or CXCL10(-/-) mice, consistent with similar control of infectious virus. There was also no evidence for dysregulation of chemokines or cytokines involved in ASC regulation. The distinct roles of CXCL9 and CXCL10 in ASC accumulation rather coincided with their differential localization. While CXCL10 was predominantly expressed by astrocytes, CXCL9 expression was confined to the vasculature/perivascular spaces. These results suggest that CXCL10 is critical for two phases: recruitment of ASC to the CNS vasculature and ASC entry into the CNS parenchyma.

Heparin Oligosaccharides Inhibit Chemokine (CXC Motif) Ligand 12 (CXCL12) Cardioprotection by Binding Orthogonal to the Dimerization Interface, Promoting Oligomerization, and Competing with the Chemokine (CXC Motif) Receptor 4 (CXCR4) N Terminus

The ability to interact with cell surface glycosaminoglycans (GAGs) is essential to the cell migration properties of chemokines, but association with soluble GAGs induces the oligomerization of most chemokines including CXCL12. Monomeric CXCL12, but not dimeric CXCL12, is cardioprotective in a number of experimental models of cardiac ischemia. We found that co-administration of heparin, a common treatment for myocardial infarction, abrogated the protective effect of CXCL12 in an ex vivo rat heart model for myocardial infarction. The interaction between CXCL12 and heparin oligosaccharides has previously been analyzed through mutagenesis, in vitro binding assays, and molecular modeling. However, complications from heparin-induced CXCL12 oligomerization and studies using very short oligosaccharides have led to inconsistent conclusions as to the residues involved, the orientation of the binding site, and whether it overlaps with the CXCR4 N-terminal site. We used a constitutively dimeric variant to simplify the NMR analysis of CXCL12-binding heparin oligosaccharides of varying length. Biophysical and mutagenic analyses reveal a CXCL12/heparin interaction surface that lies perpendicular to the dimer interface, does not involve the chemokine N terminus, and partially overlaps with the CXCR4-binding site. We further demonstrate that heparin-mediated enzymatic protection results from the promotion of dimerization rather than direct heparin binding to the CXCL12 N terminus. These results clarify the structural basis for GAG recognition by CXCL12 and lend insight into the development of CXCL12-based therapeutics.

The Role of CXCL12-CXCR4 Signaling Pathway in Pancreatic Development

Chemokine (C-X-C motif) receptor 4 (CXCR4) is the receptor for chemokine (C-X-C motif) ligand 12 (CXCL12, also known as stromal derived factor-1, Sdf1). CXCR4, a protein consisting 352 amino acids, is known to transduce various signals such as cell differentiation, cell survival, cell proliferation, cell chemotaxis and apoptosis [1, 2]. The expression of CXCR4 is observed in embryonic stem cells, blood cells, haematopoietic stem cells, endothelial cells, angioblasts and smooth muscle cells [3-9]. The CXCL12-CXCR4 signaling pathway has very important roles in the embryonic development. Mutant mice for CXCL12 or CXCR4 genes showed lethality due to defects in neurogenesis, angiogenesis, cardiogenesis, myelopoiesis, lymphopoiesis and germ cell development [10-13]. Recently, we reported that CXCL12-CXCR4 signaling pathway has a crucial role in regional specification of the gut endoderm during early development [14]. Here, we would like to focus on the role of CXCL12-CXCR4 signaling pathway in pancreatic development and summarize recent findings of its role in the induction of the pancreatic progenitor cells.

Differential Cytokine Release from Brain Microvascular Endothelial Cells Treated with Dexamethasone and Multiple Sclerosis Patient Sera

Objective: Multiple sclerosis (MS) is a neurodegenerative disorder of the central nervous system (CNS). Damage of the blood-brain barrier integrity is a key pathogenic event leading to the migration of lymphocytes into the CNS and subsequent demyelination. This process is tightly regulated by chemokines and cytokines which are target of therapeutic strategies in MS, such as anti-inflammatory glucocorticosteroid treatment. Here, we examine the effects of dexamethasone-treatment and MS patient sera on the expression of cytokines and chemokines in brain microvascular cell line, cEND in vitro. Methods: We conducted 96-well Mouse Cytokines and Receptors qPCR arrays to quantitatively compare the cytokine and chemokine expression profiles after treatment with dexamethasone. For selected cytokines, we studied the effects of pre-treatment with MS patient sera from active phase of disease (exacerbation) or in relapse (remission) in combination with dexamethasone. Results: After dexamethasone treatment, colony stimulating factor 3 (Csf3) and interleukin 17F (IL17f) were significantly up-regulated, whereas the chemokine (C-C motif) ligand 12 (CCL12/MCP-5), chemokine (C-X-C motif) receptor 3 (CXCR3) and kit oncogene (Kit) were significantly down-regulated. These results were confirmed in qRT-PCR using gene-specific primers. For Csf3 and CCL12 we analyzed the dexamethasone-mediated changes in protein levels secreted into the cell culture medium. Dexamethasone treatment increased the release of Csf3 into the culture medium and decreased the release of CCL12 by cEND. Additionally, we examined the effects of MS-patient sera on dexamethasone-induced cytokine secretion. Pretreatment with MS-patient serum from exacerbation phase augmented dexamethasone effects on Csf3 and CCL12 release in cEND cells. The expression of Csf3- and CCL12-receptors was demonstrated on protein and mRNA level in cEND cells. Conclusion: We identified Csf3 (G-Csf) and CCL12 as cytokines differentially regulated by dexamethasone on mRNA and protein level. This effect was even more pronounced after pretreatment with MS patient serum, especially from patients with acute relapses.

Dual Inhibition of Plasminogen Kringle 5 on Angiogenesis and Chemotaxis Suppresses Tumor Metastasis by Targeting HIF-1α Pathway

We had demonstrated that plasminogen kringle 5 (K5), a potent angiogenic inhibitor, inhibited retinal neovascularization and hepatocellular carcinoma growth by anti-angiogenesis. The current study investigated the effects and the underlying mechanisms of K5 on both tumor growth and spontaneous pulmonary metastasis in Lewis lung carcinoma (LLC) implanted mouse model. Similarly, K5 could decrease expression of VEGF in LLC cells and grafted tissues and suppress tumor angiogenesis and growth. K5 had no direct effect on proliferation and apoptosis of LLC. However, K5 could significantly inhibit SDF-1α-induced chemotaxis movement of LLC cells and resulted in a great reduction of surface metastatic nodules and micrometastases in the lungs of LLC tumor-bearing mice. K5 also decreased expression of chemokine (C-X-C motif) receptor 4 (CXCR4) in LLC cells and grafted tissues. Furthermore, K5 down-regulated SDF-1α expression in metastatic lung tissues of LLC-bearing mice. Therefore, K5 may suppress tumor pulmonary metastasis through inhibiting SDF-1α-CXCR4 chemotaxis movement and down-regulation of VEGF. Moreover, the role of hypoxia inducible factor-1α (HIF-1α), a crucial transcriptional factor for both VEGF and CXCR4 expression, was evaluated. The siRNA of HIF-1α attenuated expression of VEGF and CXCR4 and inhibited LLC migration. K5 decreased HIF-1α protein level and impaired nuclear HIF-1α accumulation. These results showed for the first time that K5 inhibits LLC growth and metastasis via the dual effects of anti-angiogenesis and suppression of tumor cell motility by targeting the pivotal molecule, HIF-1α.

Epigenetic expansion of VHL-HIF signal output drives multiorgan metastasis in renal cancer

Inactivation of the von Hippel-Lindau tumor suppressor (VHL) is an archetypical tumor-initiating event in clear cell renal carcinoma (ccRCC), leading to the activation of hypoxia-inducible transcription factors (HIFs). However, VHL mutation status in ccRCC is not correlated with clinical outcome. Here we show that during ccRCC progression, cancer cells exploit diverse epigenetic alterations to empower a branch of the VHL-HIF pathway for metastasis, and the strength of this activation is associated with poor clinical outcome. By analyzing metastatic subpopulations of VHL-deficient ccRCC cells, we discovered an epigenetically altered VHL-HIF response specific to metastatic ccRCC. Focusing on the two most prominent pro-metastatic VHL-HIF target genes, we show that liberation from PRC2-dependent repressive histone methylation (H3K27me3) activates HIF-driven CXCR4 expression in support of chemotactic cell invasion, whereas loss of DNA methylation enables HIF-driven CYTIP expression to protect cancer cells from death cytokine signals. Thus, metastasis in ccRCC is based on an epigenetically expanded output of the tumor-initiating pathway.

Cordyceps sinensis protects against renal ischemia/reperfusion injury in rats

Cordyceps sinensis (CS) is an entomogenous fungus used as a tonic food and Chinese medicine to replenish health. This study investigated the protective effects of CS in rats post-renal ischemia-reperfusion (I/R) sequence by analyzing the influence on stromal cell-derived factor-1α (SDF-1α and chemokine (C-X-C motif) receptor 4 (CXCR4) expressions and senescence during recovery. Chemokine SDF-1 [now called chemokine C-X-C motif ligand 12 (CXCL12)] and its receptor CXCR4 are crucial in kidney repair after ischemic acute renal failure. CS treatment significantly alleviated I/R-induced renal damage assessed by creatinine levels (p < 0.05) and abated renal tubular damages assessed by periodic acid-Schiff with diastase (PASD) staining. CS induced early SDF-1α expression and increased CXCR4 expression 1-6 h post-reperfusion. Histology studies have revealed that CS induced SDF-1α in squamous cells of Bowman's capsule, mesangial cells, distal convoluted tubules (DCT), and proximal convoluted tubules (PCT). CS also improved renal repair in I/R-induced injury by increasing Ki-67 staining. I/R induced renal senescence after 3 and 6 h of reperfusion. However, CS alleviated I/R-induced senescence at early stage (1 and 3 h). We conclude that CS protects against I/R injury via the SDF-1/CXCR4-signaling axis and alleviates senescence.

HIV‐1 co‐receptor expression and epithelial immune cells of the cervix in asymptomatic women attending a genitourinary medicine clinic

The aim of the study was to qualitatively and semiquantitatively characterize the expression of the principal HIV co-receptors chemokine (C-C motif) receptor 5 (CCR5) and chemokine (C-X-C motif) receptor 4 (CXCR4) on susceptible CD4 T-helper cell, monocyte/macrophage and Langerhans dendritic cell populations within the cervical epithelia of asymptomatic women attending a genitourinary medicine clinic. Of 77 asymptomatic women recruited, 35 were excluded: 21 because they were found to have bacterial vaginosis, eight because they were found to have candida and six for other reasons. Cervical cytobrush samples from 11 women with Chlamydia trachomatis infection and 31 women without any detectable genital infection were stained with fluorescently labelled antibodies specific for cell surface CCR5, CXCR4, CD4, CD3, CD1a and CD19 expression, then analysed by flow cytometry. CD4/CD3 T-helper cells (84%), CD1a Langerhans dendritic cells (75%) and CD4/CD14 monocytes/macrophages (59%) were detected in the samples. CCR5 and CXCR4 HIV co-receptor expression was observed on 46-86% of the above subsets. CD1a cells exhibited significantly higher CCR5 and CXCR4 positivity and median fluorescence than CD4 cells and higher CXCR4 positivity and median fluorescence than CD14 cells (P < 0.05 or less). Increased detection of CCR5 over CXCR4 was seen in CD14 cells (P < 0.05). No significant differences in CCR5 or CXCR4 expression were found in samples from asymptomatic women with or without chlamydial infection. Co-receptor expression confirms the potential for CD1a Langerhans cells, monocytes/macrophages and T-helper cells in the cervix as primary targets for HIV infection. Previously observed selective transmission of CCR5-tropic isolates cannot be accounted for by a lack of CXCR4-expressing CD4 cervical immune cells. We were unable to identify any specific impact of chlamydial infection on co-receptor expression in this study.

TGF-β polymorphism and its expression correlated with CXCR4 expression in human breast cancer

The role of chemokines and the growth factors has been extensively analyzed both in cancer risk and tumor progression. The transforming growth factor beta (TGF-β) and chemokine (C-X-C motif) receptor 4 (CXCR4) genes are implicated in several diseases, including breast cancer. Genomic DNA was obtained from 21 samples of peripheral blood or from normal tissue, previously fixed in formalin and embedded in paraffin for TGF-β T869C polymorphism analyses. Total cellular RNA was extracted from the same 21 patients, but from fresh tissue (tumor and adjacent healthy from the same breast) for expression analysis by Real Time PCR. No significant differences were observed in genotype distribution according to clinicopathological characteristics. Transforming growth factor beta (TGF-β) mRNA expression was assessed according to T869C polymorphism and CC patients presented a higher TGF-β expression but not significant when compared to other genotypes (p = 0.064). A positive correlation was observed in relative mRNA expressions of CXCR4 and TGF-β (p = 0.020). It is known that overexpression of TGF-β by both tumor and stromal tissue can facilitate the development of metastases, mainly by TGF-β stimulated angiogenesis and increased tumor cell motility. Our findings suggested a role of these genes as progression markers for breast carcinoma.

The Inflammatory Chemokine CXC Motif Ligand 16 Triggers Platelet Activation and Adhesion Via CXC Motif Receptor 6–Dependent Phosphatidylinositide 3-Kinase/Akt Signaling

The recently discovered chemokine CXC motif ligand 16 (CXCL16) is highly expressed in atherosclerotic lesions and is a potential pathogenic mediator in coronary artery disease. The aim of this study was to test the role of CXCL16 on platelet activation and vascular adhesion, as well as the underlying mechanism and signaling pathway. Reverse-transcriptase polymerase chain reaction, Western blotting, confocal microscopy, and flow cytometry revealed that CXCL16-specific receptor, CXC motif receptor 6, is highly expressed in platelets. According to flow cytometry and confocal microscopy, stimulation of platelets with CXCL16 induced platelet degranulation, integrin α(IIb)β(3) activation, and shape change. CXCL16 increased Akt phosphorylation (Thr(308)/Ser(473)), an effect abrogated by phosphatidylinositide 3-kinase inhibitors wortmannin (100 nmol/L) and LY294002 (25 µmol/L). The phosphatidylinositide 3-kinase inhibitors and Akt inhibitor SH-6 (20 µmol/L) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin α(IIb)β(3) activation, and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXC motif receptor 6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro after high arterial shear stress (2000(-s)) and to injured vascular wall in vivo after carotid ligation. CXCL16-induced stimulation of platelet adhesion again was prevented by phosphatidylinositide 3-kinase and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P(2)Y(1) (MRS2179, 100 µmol/L) and especially P(2)Y(12) (Cangrelor, 10 µmol/L) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotid ligation in vivo. The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXC motif receptor 6-dependent phosphatidylinositide 3-kinase/Akt signaling and paracrine activation, suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.

Potentiation of Inflammatory CXCL8 Signalling Sustains Cell Survival in PTEN-deficient Prostate Carcinoma

BackgroundInflammation and genetic instability are enabling characteristics of prostate carcinoma (PCa). Inactivation of the tumour suppressor gene phosphatase and tensin homolog (PTEN) is prevalent in early PCa. The relationship of PTEN deficiency to inflammatory signalling remains to be characterised. ObjectiveTo determine how loss of PTEN functionality modulates expression and efficacy of clinically relevant, proinflammatory chemokines in PCa. Design, setting, and participantsExperiments were performed in established cell-based PCa models, supported by pathologic analysis of chemokine expression in prostate tissue harvested from PTEN heterozygous (Pten+/−) mice harbouring inactivation of one PTEN allele. InterventionsSmall interfering RNA (siRNA)– or small hairpin RNA (shRNA)–directed strategies were used to repress PTEN expression and resultant interleukin-8 (CXCL8) signalling, determined under normal and hypoxic culture conditions. Outcome measurements and statistical analysisChanges in chemokine expression in PCa cells and tissue were analysed by real-time polymerase chain reaction (PCR), immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry; effects of chemokine signalling on cell function were assessed by cell cycle analysis, apoptosis, and survival assays. Results and limitationsTransient (siRNA) or prolonged (shRNA) PTEN repression increased expression of CXCL8 and its receptors, chemokine (C-X-C motif) receptor (CXCR) 1 and CXCR2, in PCa cells. Hypoxia-induced increases in CXCL8, CXCR1, and CXCR2 expression were greater in magnitude and duration in PTEN-depleted cells. Autocrine CXCL8 signalling was more efficacious in PTEN-depleted cells, inducing hypoxia-inducible factor-1 (HIF-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription and regulating genes involved in survival and angiogenesis. Increased expression of the orthologous chemokine KC was observed in regions displaying atypical cytologic features in Pten+/− murine prostate tissue relative to normal epithelium in wild-type PTEN (PtenWT) glands. Attenuation of CXCL8 signalling decreased viability of PCa cells harbouring partial or complete PTEN loss through promotion of G1 cell cycle arrest and apoptosis. The current absence of clinical validation is a limitation of the study. ConclusionsPTEN loss induces a selective upregulation of CXCL8 signalling that sustains the growth and survival of PTEN-deficient prostate epithelium.

Cellular and subcellular localization of CXCL12 and CXCR4 in rat nociceptive structures: physiological relevance

Initial studies implicated the chemokine CXC motif ligand 12 (CXCL12) and its cognate CXC motif receptor 4 (CXCR4) in pain modulation. However, there has been no description of the distribution, transport and axonal sorting of CXCL12 and CXCR4 in rat nociceptive structures, and their direct participation in nociception modulation has not been demonstrated. Here, we report that acute intrathecal administration of CXCL12 induced mechanical hypersensitivity in naive rats. This effect was prevented by a CXCR4-neutralizing antibody. To determine the morphological basis of this behavioural response, we used light and electron microscopic immunohistochemistry to map CXCL12- and CXCR4-immunoreactive elements in dorsal root ganglia, lumbar spinal cord, sciatic nerve and skin. Light microscopy analysis revealed CXCL12 and CXCR4 immunoreactivity in calcitonin gene related peptide-containing peptidergic primary sensory neurons, which were both conveyed to central and peripheral sensory nerve terminals. Electron microscopy clearly demonstrated CXCL12 and CXCR4 immunoreactivity in primary sensory nerve terminals in the dorsal horn; both were sorted into small clear vesicles and large dense-core vesicles. This suggests that CXCL12 and CXCR4 are trafficked from nerve cell bodies to the dorsal horn. Double immunogold labelling for CXCL12 and calcitonin gene related peptide revealed partial vesicular colocalization in axonal terminals. We report, for the first time, that CXCR4 receptors are mainly located on the neuronal plasma membrane, where they are present at pre-synaptic and post-synaptic sites of central terminals. Receptor inactivation experiments, behavioural studies and morphological analyses provide strong evidence that the CXCL12/CXCR4 system is involved in modulation of nociceptive signalling.