Epigenetic therapy inhibits metastases by disrupting premetastatic niches.

Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.

Targeting ESE3/EHF With Nifurtimox Inhibits CXCR2+ Neutrophil Infiltration and Overcomes Pancreatic Cancer Resistance to Chemotherapy and Immunotherapy.

BACKGROUND & AIMS: Because pancreatic cancer responds poorly to chemotherapy and immunotherapy, it is necessary to identify novel targets and compounds to overcome resistance to treatment. METHODS: This study analyzed genomic single nucleotide polymorphism sequencing, single-cell RNA sequencing, and spatial transcriptomics. Ehf-knockout mice, KPC (LSL-KrasG12D/+, LSL-Trp53R172H/+ and Pdx1-Cre) mice, CD45.1+ BALB/C nude mice, and CD34+ humanized mice were also used as subjects. Multiplexed immunohistochemistry and flow cytometry were performed to investigate the proportion of tumor-infiltrated C-X-C motif chemokine receptor 2 (CXCR2)+ neutrophils. In addition, multiplexed cytokines assays and chromatin immunoprecipitation assays were used to examine the mechanism. RESULTS: The TP53 mutation-mediated loss of tumoral EHF increased the recruitment of CXCR2+ neutrophils, modulated their spatial distribution, and further induced chemo- and immunotherapy resistance in clinical cohorts and preclinical syngeneic mice models. Mechanistically, EHF deficiency induced C-X-C motif chemokine ligand 1 (CXCL1) transcription to enhance in vitro and in vivo CXCR2+ neutrophils migration. Moreover, CXCL1 or CXCR2 blockade completely abolished the effect, indicating that EHF regulated CXCR2+ neutrophils migration in a CXCL1-CXCR2-dependent manner. The depletion of CXCR2+ neutrophils also blocked the in vivo effects of EHF deficiency on chemotherapy and immunotherapy resistance. The single-cell RNA-sequencing results of PDAC treated with Nifurtimox highlighted the therapeutic significance of Nifurtimox by elevating the expression of tumoral EHF and decreasing the weightage of CXCL1-CXCR2 pathway within the microenvironment. Importantly, by simultaneously inhibiting the JAK1/STAT1 pathway, it could significantly suppress the recruitment and function of CXCR2+ neutrophils, further sensitizing PDAC to chemotherapy and immunotherapies. CONCLUSIONS: The study demonstrated the role of EHF in the recruitment of CXCR2+ neutrophils and the promising role of Nifurtimox in sensitizing pancreatic cancer to chemotherapy and immunotherapy.

Synergistic antitumor activity by dual blockade of CCR1 and CXCR2 expressed on myeloid cells within the tumor microenvironment.

BACKGROUND: Chemokine signaling within the tumor microenvironment can promote tumor progression. Although CCR1 and CXCR2 on myeloid cells could be involved in tumor progression, it remains elusive what effect would be observed if both of those are blocked. METHODS: We employed two syngeneic colorectal cancer mouse models: a transplanted tumor model and a liver metastasis model. We generated double-knockout mice for CCR1 and CXCR2, and performed bone marrow (BM) transfer experiments in which sub-lethally irradiated wild-type mice were reconstituted with BM from either wild-type, Ccr1-/-, Cxcr2-/- or Ccr1-/-Cxcr2-/- mice. RESULTS: Myeloid cells that express MMP2, MMP9 and VEGF were accumulated around both types of tumors through CCR1- and CXCR2-mediated pathways. Mice reconstituted with Ccr1-/-Cxcr2-/- BM exhibited the strongest suppression of tumor growth and liver metastasis compared with other three groups. Depletion of CCR1+CXCR2+ myeloid cells led to a higher frequency of CD8+ T cells, whereas the numbers of Ly6G+ neutrophils, FOXP3+ Treg cells and CD31+ endothelial cells were significantly decreased. Furthermore, treatment with a neutralizing anti-CCR1 mAb to mice reconstituted with Cxcr2-/- BM significantly suppressed tumor growth and liver metastasis. CONCLUSION: Dual blockade of CCR1 and CXCR2 pathways in myeloid cells could be an effective therapy against colorectal cancer.

The non-ELR CXC chemokine encoded by human cytomegalovirus UL146 genotype 5 contains a C-terminal ß-hairpin and induces neutrophil migration as a selective CXCR2 agonist.

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. The UL146 gene exists as 14 diverse genotypes among clinical isolates, which encode 14 different CXC chemokines. One genotype (vCXCL1GT1) is a known agonist for CXCR1 and CXCR2, while two others (vCXCL1GT5 and vCXCL1GT6) lack the ELR motif considered crucial for CXCR1 and CXCR2 binding, thus suggesting another receptor targeting profile. To determine the receptor target for vCXCL1GT5, the chemokine was probed in a G protein signaling assay on all 18 classical human chemokine receptors, where CXCR2 was the only receptor being activated. In addition, vCXCL1GT5 recruited ß-arrestin in a BRET-based assay and induced migration in a chemotaxis assay through CXCR2, but not CXCR1. In contrast, vCXCL1GT1 stimulated G protein signaling, recruited ß-arrestin and induced migration through both CXCR1 and CXCR2. Both vCXCL1GT1 and vCXCL1GT5 induced equally potent and efficacious migration of neutrophils, and ELR vCXCL1GT4 and non-ELR vCXCL1GT6 activated only CXCR2. In contrast to most human chemokines, the 14 UL146 genotypes have remarkably long C-termini. Comparative modeling using Rosetta showed that each genotype could adopt the classic chemokine core structure, and predicted that the extended C-terminal tail of several genotypes (including vCXCL1GT1, vCXCL1GT4, vCXCL1GT5, and vCXCL1GT6) forms a novel ß-hairpin not found in human chemokines. Secondary NMR shift and TALOS+ analysis of vCXCL1GT1 supported the existence of two stable ß-strands. C-terminal deletion of vCXCL1GT1 resulted in a non-functional protein and in a shift to solvent exposure for tryptophan residues likely due to destabilization of the chemokine fold. The results demonstrate that non-ELR chemokines can activate CXCR2 and suggest that the UL146 chemokines have unique C-terminal structures that stabilize the chemokine fold. Increased knowledge of the structure and interaction partners of the chemokine variants encoded by UL146 is key to understanding why circulating HCMV strains sustain 14 stable genotypes.

CXCL8 and its cognate receptors CXCR1/CXCR2 in primary myelofibrosis.

BCR::ABL1 negative myeloproliferative neoplasms (MPN) form a distinct group of hematologic malignancies characterized by sustained proliferation of cells from multiple myeloid lineages. With a median survival of 16-35 months in patients with high-risk disease, primary myelofibrosis (PMF) is considered the most aggressive entity amongst all BCR::ABL1 MPN. Additionally, for a significant subset of patients, MPN evolve into secondary acute myeloid leukemia (AML), which has an even poorer prognosis compared to de novo AML. As the exact mechanisms of disease development and progression remain to be elucidated, current therapeutic approaches fail to prevent disease progression or transformation into secondary AML. As each MPN entity is characterized by sustained activation of various immune cells and raised cytokine concentrations within bone marrow (BM) and peripheral blood (PB), MPN may be considered to be typical inflammation-related malignancies. However, the exact role and consequences of increased cytokine concentrations within BM and PB plasma has still not been completely established. Up-regulated cytokines can stimulate cellular proliferation, or contribute to the development of an inflammation-related BM niche resulting in genotoxicity and thereby supporting mutagenesis. The neutrophil chemoattractant CXCL8 is of specific interest as its concentration is increased within PB and BM plasma of patients with PMF. Increased concentration of CXCL8 negatively correlates with overall survival. Furthermore, blockage of the CXCR1/2 axis appears to be able to reduce BM fibrosis and megakaryocyte dysmorphia in murine models. In this review, we summarize available evidence on the role of the CXCL8-CXCR1/2 axis within the pathogenesis of PMF, and discuss potential therapeutic modalities targeting either CXCL8 or its cognate receptors CXCR1/2.

Endothelial CXCR2 deficiency attenuates renal inflammation and glycocalyx shedding through NF-κB signaling in diabetic kidney disease.

BACKGROUND: The incidence of diabetic kidney disease (DKD) continues to rapidly increase, with limited available treatment options. One of the hallmarks of DKD is persistent inflammation, but the underlying molecular mechanisms of early diabetic kidney injury remain poorly understood. C-X-C chemokine receptor 2 (CXCR2), plays an important role in the progression of inflammation-related vascular diseases and may bridge between glomerular endothelium and persistent inflammation in DKD. METHODS: Multiple methods were employed to assess the expression levels of CXCR2 and its ligands, as well as renal inflammatory response and endothelial glycocalyx shedding in patients with DKD. The effects of CXCR2 on glycocalyx shedding, and persistent renal inflammation was examined in a type 2 diabetic mouse model with Cxcr2 knockout specifically in endothelial cells (DKD-Cxcr2 eCKO mice), as well as in glomerular endothelial cells (GECs), cultured in high glucose conditions. RESULTS: CXCR2 was associated with early renal decline in DKD patients, and endothelial-specific knockout of CXCR2 significantly improved renal function in DKD mice, reduced inflammatory cell infiltration, and simultaneously decreased the expression of proinflammatory factors and chemokines in renal tissue. In DKD conditions, glycocalyx shedding was suppressed in endothelial Cxcr2 knockout mice compared to Cxcr2 L/L mice. Modulating CXCR2 expression also affected high glucose-induced inflammation and glycocalyx shedding in GECs. Mechanistically, CXCR2 deficiency inhibited the activation of NF-κB signaling, thereby regulating inflammation, restoring the endothelial glycocalyx, and alleviating DKD. CONCLUSIONS: Taken together, under DKD conditions, activation of CXCR2 exacerbates inflammation through regulation of the NF-κB pathway, leading to endothelial glycocalyx shedding and deteriorating renal function. Endothelial CXCR2 deficiency has a protective role in inflammation and glycocalyx dysfunction, suggesting its potential as a promising therapeutic target for DKD treatment.

CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth.

BACKGROUND: Though the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. METHODS: To characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven BrafV600E/Pten-/-/Cxcr2-/- and NRasQ61R/INK4a-/-/Cxcr2-/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in BrafV600E/Pten-/- and NRasQ61R/INK4a-/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). RESULTS: Genetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1, a key tumor suppressive transcription factor, was the only gene significantly induced with a log2 fold-change greater than 2 in these three different melanoma models. CONCLUSIONS: Here, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

METTL3 Inhibits Antitumor Immunity by Targeting m6A-BHLHE41-CXCL1/CXCR2 Axis to Promote Colorectal Cancer.

BACKGROUND & AIMS: N6-Methyladenosine (m6A) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic m6A modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC. METHODS: Mettl3 knockout mice, CD34+ humanized mice, and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay, respectively. M6A sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n = 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration. RESULTS: We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4+ and CD8+ T cells, and eventually suppressed CRC in ApcMin/+Mettl3+/- mice, CD34+ humanized mice, and syngeneic mice models. Mechanistically, METTL3 activated the m6A-BHLHE41-CXCL1 axis by analysis of m6A sequencing, RNA sequencing, and cytokine arrays. METTL3 promoted BHLHE41 expression in an m6A-dependent manner, which subsequently induced CXCL1 transcription to enhance MDSC migration in vitro. However, the effect was negligible on BHLHE41 depletion, CXCL1 protein or CXCR2 inhibitor SB265610 administration, inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently, depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 in vivo. Importantly, targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment. CONCLUSIONS: Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the m6A-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC.

Viral genomic, metagenomic and human transcriptomic characterization and prediction of the clinical forms of COVID-19.

COVID-19 is characterized by respiratory symptoms of various severities, ranging from mild upper respiratory signs to acute respiratory failure/acute respiratory distress syndrome associated with a high mortality rate. However, the pathophysiology of the disease is largely unknown. Shotgun metagenomics from nasopharyngeal swabs were used to characterize the genomic, metagenomic and transcriptomic features of patients from the first pandemic wave with various forms of COVID-19, including outpatients, patients hospitalized not requiring intensive care, and patients in the intensive care unit, to identify viral and/or host factors associated with the most severe forms of the disease. Neither the genetic characteristics of SARS-CoV-2, nor the detection of bacteria, viruses, fungi or parasites were associated with the severity of pulmonary disease. Severe pneumonia was associated with overexpression of cytokine transcripts activating the CXCR2 pathway, whereas patients with benign disease presented with a T helper "Th1-Th17" profile. The latter profile was associated with female gender and a lower mortality rate. Our findings indicate that the most severe cases of COVID-19 are characterized by the presence of overactive immune cells resulting in neutrophil pulmonary infiltration which, in turn, could enhance the inflammatory response and prolong tissue damage. These findings make CXCR2 antagonists, in particular IL-8 antagonists, promising candidates for the treatment of patients with severe COVID-19.

A developmentally prometastatic niche to hepatoblastoma in neonatal liver mediated by the Cxcl1/Cxcr2 axis.

BACKGROUND AND AIMS: Hepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigate whether the neonatal liver provides a protumorigenic niche to HB development. APPROACH AND RESULTS: HB development was compared between orthotopic transplantation models established in postnatal day 5 (P5) and 60 (P60) mice (P5Tx and P60Tx models). Single-cell RNA-sequencing (sc-RNAseq) was performed using tumor and liver tissues from both models and the top candidate cell types and genes identified are investigated for their roles in HB cell growth, migration, and survival. CONCLUSIONS: We found that various HB cell lines including HepG2 cells were consistently and considerably more tumorigenic and metastatic in the P5Tx model than in the P60Tx models. Sc-RNAseq of the P5Tx and P60Tx HepG2 models revealed that the P5Tx tumor was more hypoxic and had a larger number of activated hepatic stellate cells (aHSCs) in the tumor-surrounding liver that express significantly higher levels of Cxcl1 than those from the P60Tx model. We found these differences were developmentally present in normal P5 and P60 liver. We showed that the Cxcl1/Cxcr2 axis mediated HB cell migration and was critical to HB cell survival under hypoxia. Treating HepG2 P60Tx model with recombinant CXCL1 protein induced intrahepatic and pulmonary metastasis and CXCR2 knockout (KO) in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in tumors from patients with metastatic HB, there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with prognosis of patients with HB among pediatric cancers. We demonstrated that the neonatal liver provides a prometastatic niche to HB development through the Cxcl1/Cxcr2 axis.

Chemokine signaling via the CXCR2 receptor reinforces senescence.

Cells enter senescence, a state of stable proliferative arrest, in response to a variety of cellular stresses, including telomere erosion, DNA damage, and oncogenic signaling, which acts as a barrier against malignant transformation in vivo. To identify genes controlling senescence, we conducted an unbiased screen for small hairpin RNAs that extend the life span of primary human fibroblasts. Here, we report that knocking down the chemokine receptor CXCR2 (IL8RB) alleviates both replicative and oncogene-induced senescence (OIS) and diminishes the DNA-damage response. Conversely, ectopic expression of CXCR2 results in premature senescence via a p53-dependent mechanism. Cells undergoing OIS secrete multiple CXCR2-binding chemokines in a program that is regulated by the NF-kappaB and C/EBPbeta transcription factors and coordinately induce CXCR2 expression. CXCR2 upregulation is also observed in preneoplastic lesions in vivo. These results suggest that senescent cells activate a self-amplifying secretory network in which CXCR2-binding chemokines reinforce growth arrest.

Heterocomplexes between the atypical chemokine MIF and the CXC-motif chemokine CXCL4L1 regulate inflammation and thrombus formation.

To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine-chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. Platelet chemokine CXCL4L1 (but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12) was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures that can be exploited to modulate inflammation and thrombosis.

Circ_0002295 facilitated myocardial fibrosis progression through the miR-1287/CXCR2 axis.

Myocardial fibrosis (MF) is involved in hypertension, myocardial infarction and heart failure. It has been reported that circular RNA (circRNA) is a key regulatory factor of MF progression. In this study, we revealed that circ_0002295 and CXCR2 were elevated, and miR-1287 was reduced in MF patients. Knockdown of circ_0002295 effectively suppressed the proliferation, migration and MF progression. Circ_0002295 was the molecular sponge of miR-12878, and miR-1287 inhibitor reversed the biological functions of circ_0002295 on the myocardial fibrosis. CXCR2 was a target gene of miR-1287, and CXCR2 silencing relieved the impacts of miR-1287 inhibitor on cardiac myofibroblasts. Circ_0002295 promoted MF progression by regulating the miR-1287/CXCR2 axis, providing a possible circRNA-targeted therapy for MF.

CXC- receptor 2 promotes extracellular matrix production and attenuates migration in peripapillary human scleral fibroblasts under mechanical strain.

As the main loading-bearing tissue of eye, sclera exerts an important role in the pathophysiology of glaucoma. Intraocular pressure (IOP) generates mechanical strain on sclera. Recent studies have demonstrated that sclera, especially the peripapillary sclera, undergoes complicated remodelling under the mechanical strain. However, the mechanisms of the hypertensive scleral remodelling in human eyes remained uncertain. In this study, peripapillary human scleral fibroblasts (ppHSFs) were applied cyclic mechanical strain by Flexcell-5000™ tension system. We found that CXC- ligands and CXCR2 were differentially expressed after strain. Increased cell proliferation and inhibited cell motility were observed when CXCR2 was upregulated under the strain, whereas cell proliferation and motility did not have a significant change when CXCR2 was knocked down. CXCR2 could facilitate cell proliferation ability, modulate the mRNA and protein expressions of type I collagen and matrix metalloproteinase 2 via JAK1/2-STAT3 signalling pathway. In addition, CXCR2 might inhibit cell migration via FAK/MLC2 pathway. Taken together, CXCR2 regulated protein production and affected cell behaviours of ppHSFs. It might be a potential therapeutic target for the hypertensive scleral remodelling.

Interleukin-8: An evolving chemokine.

Early in the 1980s several laboratories mistakenly reported that partially purified interleukin-1 (IL-1) was chemotactic for neutrophils. However, further investigations by us, revealed that our purified IL-1 did not have neutrophil chemotactic activity and this activity in the LPS-stimulated human monocyte conditioned media could clearly be separated from IL-1 activity on HPLC gel filtration. This motivated Teizo Yoshimura and Kouji Matsushima to purify the monocyte-derived neutrophil chemotactic factor (MDNCF), present in LPS conditioned media and molecularly clone the cDNA for MDNCF. They found that MDNCF protein (later renamed IL-8, and finally termed CXCL8) is first translated as a precursor form consisting of 99 amino acid residues and the signal peptide is then removed, leading to the secretion and processing of biologically active IL-8 of 72 amino acid form (residues 28-99). There are four cysteine residues forming two disulfide linkage and 14 basic amino acid residues which result in a very basic property for the binding of IL-8 to heparan sulfate-proteoglycan. The IL-8 gene consists of 4 exons and 3 introns. IL-8 is produced by various types of cells in inflammation. The 5'-flanking region of IL-8 gene contains several nuclear factor binding sites, and NF-κB in combination with AP-1 or C/EBP synergistically activates IL-8 gene in response to IL-1 and TNFα. Two receptors exist for IL-8, CXCR1 and CXCR2 in humans, which belong to γ subfamily of GTP binding protein (G-protein) coupled rhodopsin-like 7 transmembrane domain receptors. Rodents express CXCR2 and do not produce IL-8, but produce numerous homologues instead. Once IL-8 binds to the receptor, ß and γ subunits of G-protein are released from Gα (Gαi2 in neutrophils) and activate PI3Kγ, PLCß2/ß3, PLA2 and PLD. Gαi2 inhibits adenyl cyclase to decrease cAMP levels. Small GTPases Ras/Rac/Rho/cdc42/Rap1, PKC and AKT (PKB) exist down-stream of ß and γ subunits and regulate cell adhesion, actin polymerization, membrane protrusion, and eventually cell migration. PLCß activation generates IP3 and induces Ca++ mobilization, DAG generation to activate protein kinase C to lead granule exocytosis and respiratory burst. MDNCF was renamed interleukin 8 (IL-8) at the International Symposium on Novel Neutrophil Chemotactic Activating Polypeptides, London, UK in 1989. The discovery of IL-8 prompted us to also purify and molecularly clone the cDNA of MCAF/MCP-1 responsible for monocyte chemotaxis, and other groups to identify a large family of chemotactic cytokines capable of attracting other types of leukocytes. In 1992, most of the investigators contributing to the discovery of this new family of chemotactic cytokines gathered in Baden, Austria and agreed to name this family "chemokines" and subsequently established the CXCL/CCL and CXCR/CCR nomenclature. The discovery of chemokines resulted in solving the long-time enigma concerning the mechanism of cell type specific leukocyte infiltration into inflamed tissues and provided a molecular basis for immune and hematopoietic cell migration and interactions under physiological as well as pathological conditions. To our surprise based on its recently identified multifunctional activities, IL-8 has evolved from a neutrophil chemoattractant to a promising therapeutic target for a wide range of inflammatory and neoplastic diseases. IL-8 was initially characterized as a chemoattractant of neutrophils engaged in acute inflammation and then discovered to also be chemotactic for endothelial cells with a major role in angiogenesis. These two activities of IL-8 foster its stimulatory effect on tumor growth. This is abetted by recent additional discoveries showing that IL-8 has stimulatory effects on stem cells and can therefore directly promote the growth of receptor expressing cancer stem cells. IL-8 by interacting with bone marrow stem/progenitor cells has also the capacity to mobilize and release hematopoietic cells into the peripheral circulation. This includes the mobilization of neutrophilic myeloid-derived suppressor cells (N-MDSC) to infiltrate into tumors and thus further promotes the immune escape of tumors. Finally, the capacity of IL-8 to induce trans-differentiation of epithelial cancer cells into mesenchymal phenotype (EMT) increases the malignancy of tumors by promoting their metastatic spread and resistance to chemotherapeutics and cytotoxic immune cells. These observations have stimulated considerable current efforts to develop receptor antagonists for IL-8 and humanized anti-IL-8 antibody for the therapy of cancer, particularly in combination with immune checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies.

Crosstalk between head and neck cancer cells and lymphatic endothelial cells promotes tumor metastasis via CXCL5-CXCR2 signaling.

Head and neck squamous cell carcinoma (HNSCC) metastasizes to the locoregional lymph nodes at high rates and is related to poor clinical outcomes. However, the mechanism by which cancer cells migrate to the lymph nodes is unclear. To address this, we established a conditioned medium culture system for HNSCC cells and lymphatic endothelial cells (LECs) and investigated their crosstalk. Stimulation with tumor-conditioned medium (TCM) activated LECs, resulting in a robust increase in cell proliferation to induce lymphatic hyperplasia. Further, stimulation of HNSCC cells with activated LEC Conditioned media (TCM-LEC CM) induced cell invasion. Among various chemokines, CXCL5 promoted the invasion of TCM-LEC CM-treated HNSCC cells. The level of CXCL5 protein was higher in cancer tissues than those in normal tissues from HNSCC patients. Furthermore, treatment with SB225002, a CXCR2 (CXCL5 receptor) inhibitor, resulted in decreased lymph node metastasis in vivo. In conclusion, inhibition of CXCL5-CXCR2 signaling between cancer cells and LECs suppresses cancer cell invasion and metastasis in vitro and in vivo. This novel therapeutic strategy might be a practical approach to the clinical management of HNSCC.

Tumor-associated macrophages promote resistance of hepatocellular carcinoma cells against sorafenib by activating CXCR2 signaling.

BACKGROUND: Sorafenib (SOR) is the first line treatment for advanced hepatocellular carcinoma (HCC), but resistance develops frequently. Tumor-associated macrophages (TAMs) have been reported to affect the progression of HCC. We therefore aimed to study the role of TAMs in promoting SOR resistance. METHODS: Immunofluorescence staining for the M2 marker CD204 and the cancer stem cell (CSC) markers CD44 and CD133 was performed in paired HCC and adjacent noncancerous tissues and HCC tissues stratified by response of SOR treatment. HCC/U937 coculture system and cytokines were used to induce M2 polarization for studying the effects of M2 TAMs on CSC properties and apoptotic death of HCC cells after SOR treatment. RESULTS: Higher expression of CD204, CD44, and CD133 was observed in patients with SOR nonresponse (SNR) than in those with SOR response (SR), suggesting that SNR is positively correlated to levels of CSCs and M2 TAMs. After coculture, M2 TAMs could increase the level of CSCs but decrease SOR-induced apoptosis. Incubation of HCC cells with coculture conditioned medium increased the formation of spheres that were resistant to SOR. Furthermore, CXCL1 and CXCL2 were found to be the potential paracrine factors released by M2 TAMs to upregulate SOR resistance in HCC cells. Treatment with CXCL1 and CXCL2 could increase HCC CSC activity but decrease SOR-induced apoptosis by affecting BCL-2 family gene expression. Using pharmacological inhibitors, CXCR2/ERK signaling was found to be critical to CXCL1- and CXCL2-mediated SOR resistance. CONCLUSION: This study identified CXCL1, CXCL2, and their downstream CXCR2/ERK signaling as potential therapeutic targets to overcome SOR resistance in HCC.

Pharmacological characterization and biological function of the interleukin-8 receptor, CXCR2, in largemouth bass (Micropterus salmoides).

Interleukin-8 (IL-8 or C-X-C motif chemokine ligand 8, CXCL8) is a cytokine secreted by numerous cell types and is best known for its functional roles in inflammatory response by binding to specific receptors (the interleukin-8 receptors, IL-8Rs). From the transcriptomic data of largemouth bass (Micropterus salmoides), we identified an IL-8R that is highly homologous to the functionally validated teleost IL-8Rs. The M. salmoides IL-8 receptor (MsCXCR2) was further compared with the C-X-C motif chemokine receptor 2 subfamily by phylogenetic analysis. Briefly, the full-length CDS sequence of MsCXCR2 was cloned into the pEGFP-N1 plasmid, and the membrane localization of fusion expressing MsCXCR2-EGFP was revealed in HEK293 cells. To determine the functional interaction between IL-8 and MsCXCR2, secretory expressed Larimichthys crocea IL-8 (LcIL-8) was used to stimulate MsCXCR2 expressing cells. MsCXCR2 was demonstrated to be activated by LcIL-8, leading to receptor internalization, which was further revealed by the detection of extracellular regulated protein kinase (ERK) phosphorylation. Quantitative real-time PCR was used to evaluate the expressional distribution and variation of MsCXCR2 in healthy and Nocardia seriolae infected fish. Based on our findings, MsCXCR2 was constitutively expressed in all examined tissues, despite at different levels. Furthermore, gene expression was found to be significantly upregulated in the liver and head kidney of diseased fish. Collectively, our findings reveal the molecular activity of MsCXCR2 and indicate the functional involvement of this IL-8R in the immune response induced by N. seriolae in M. salmoides.

Senescent Human Pancreatic Stellate Cells Secrete CXCR2 Agonist CXCLs to Promote Proliferation and Migration of Human Pancreatic Cancer AsPC-1 and MIAPaCa-2 Cell Lines.

Interactions between pancreatic cancer cells and pancreatic stellate cells (PSCs) play an important role in the progression of pancreatic cancer. Recent studies have shown that cellular senescence and senescence-associated secretory phenotype factors play roles in the progression of cancer. This study aimed to clarify the effects of senescence-induced PSCs on pancreatic cancer cells. Senescence was induced in primary-cultured human PSCs (hPSCs) through treatment with hydrogen peroxide or gemcitabine. Microarray and Gene Ontology analyses showed the alterations in genes and pathways related to cellular senescence and senescence-associated secretory phenotype factors, including the upregulation of C-X-C motif chemokine ligand (CXCL)-1, CXCL2, and CXCL3 through the induction of senescence in hPSCs. Conditioned media of senescent hPSCs increased the proliferation-as found in an assessment with a BrdU incorporation assay-and migration-as found in an assessment with wound-healing and two-chamber assays-of pancreatic cancer AsPC-1 and MIAPaca-2 cell lines. SB225002, a selective CXCR2 antagonist, and SCH-527123, a CXCR1/CXCR2 antagonist, attenuated the effects of conditioned media of senescent hPSCs on the proliferation and migration of pancreatic cancer cells. These results suggest a role of CXCLs as senescence-associated secretory phenotype factors in the interaction between senescent hPSCs and pancreatic cancer cells. Senescent PSCs might be novel therapeutic targets for pancreatic cancer.

CXCR2 Receptor: Regulation of Expression, Signal Transduction, and Involvement in Cancer.

Chemokines are a group of about 50 chemotactic cytokines crucial for the migration of immune system cells and tumor cells, as well as for metastasis. One of the 20 chemokine receptors identified to date is CXCR2, a G-protein-coupled receptor (GPCR) whose most known ligands are CXCL8 (IL-8) and CXCL1 (GRO-α). In this article we present a comprehensive review of literature concerning the role of CXCR2 in cancer. We start with regulation of its expression at the transcriptional level and how this regulation involves microRNAs. We show the mechanism of CXCR2 signal transduction, in particular the action of heterotrimeric G proteins, phosphorylation, internalization, intracellular trafficking, sequestration, recycling, and degradation of CXCR2. We discuss in detail the mechanism of the effects of activated CXCR2 on the actin cytoskeleton. Finally, we describe the involvement of CXCR2 in cancer. We focused on the importance of CXCR2 in tumor processes such as proliferation, migration, and invasion of tumor cells as well as the effects of CXCR2 activation on angiogenesis, lymphangiogenesis, and cellular senescence. We also discuss the importance of CXCR2 in cell recruitment to the tumor niche including tumor-associated neutrophils (TAN), tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), and regulatory T (Treg) cells.