CXCR4 Activation Research Articles

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.

Mouse Modeling Dissecting Macrophage-Breast Cancer Communication Uncovered Roles of PYK2 in Macrophage Recruitment and Breast Tumorigenesis.

Macrophage infiltration in mammary tumors is associated with enhanced tumor progression, metastasis, and poor clinical outcome, and considered as target for therapeutic intervention. By using different genetic mouse models, the authors show that ablation of the tyrosine kinase PYK2, either in breast cancer cells, only in the tumor microenvironment, or in both, markedly reduces the number of infiltrating tumor macrophages and concomitantly inhibits tumor angiogenesis and tumor growth. Strikingly, PYK2 ablation only in macrophages is sufficient to induce similar effects. These phenotypic changes are associated with reduced monocyte recruitment and a substantial decrease in tumor‐associated macrophages (TAMs). Mechanistically, the authors show that PYK2 mediates mutual communication between breast cancer cells and macrophages through critical effects on key receptor signaling. Specifically, PYK2 ablation inhibits Notch1 signaling and consequently reduces CCL2 secretion by breast cancer cells, and concurrently reduces the levels of CCR2, CXCR4, IL‐4Rα, and Stat6 activation in macrophages. These bidirectional effects modulate monocyte recruitment, macrophage polarization, and tumor angiogenesis. The expression of PYK2 is correlated with infiltrated macrophages in breast cancer patients, and its effects on macrophage infiltration and pro‐tumorigenic phenotype suggest that PYK2 targeting can be utilized as an effective strategy to modulate TAMs and possibly sensitize breast cancer to immunotherapy.

CXCR2 Mediates Distinct Neutrophil Behavior in Brain Metastatic Breast Tumor.

Simple SummaryBrain metastasis is one of the common complications associated with breast cancers. Neutrophils form the predominant type of circulating white blood cells and play an important role in tumor pathogenesis. However, the role of neutrophils in the evolution of brain metastasis of breast cancers has not been clearly understood. Using simple neutrophil-tumor cell-culture models, we studied the possible biomolecular mechanisms by which the brain-metastatic breast cancer cells could potentially re-program the neutrophils as a tumor-promoting strategy, and how drug-induced inhibition of certain key neutrophil functionalities could help reverse this behavior. Brain metastasis is one of the main causes of mortality among breast cancer patients, but the origins and the mechanisms that drive this process remain poorly understood. Here, we report that the upregulation of certain CXCR2-associated ligands in the brain metastatic variants of the breast cancer cells (BrM) dynamically activate the corresponding CXCR2 receptors on the neutrophils, thereby resulting in the modulation of certain key functional neutrophil responses towards the BrM. Using established neutrophil-tumor biomimetic co-culture models, we show that the upregulation of CXCR2 increases the recruitment of Tumor-Associated Neutrophils (TANs) towards the BrM, to enable location-favored formation of Neutrophil Extracellular Traps (NETs). Inhibition of CXCR2 using small molecule antagonist AZD5069 reversed this behavior, limiting the neutrophil responses to the BrM and retarding the reciprocal tumor development. We further demonstrate that abrogation of NETs formation using Neutrophil Elastase Inhibitor (NEI) significantly decreases the influx of neutrophils towards BrM but not to their parental tumor, suggesting that CXCR2 activation could be used by the brain metastatic tumors as a mechanism to program the tumor-infiltrating TANs into a pro-NETotic state, so as to assume a unique spatial distribution that assists in the subsequent migration and invasion of the metastatic tumor cells. This new perspective indicates that CXCR2 is a critical target for suppressing neutrophilic inflammation in brain metastasis.

Stromal cell-derived factor-1 exerts opposing roles through CXCR4 and CXCR7 in angiotensin II-induced adventitial remodeling

Recent studies have emphasized the role of vascular adventitia inflammation and immune response in hypertension. It has been reported that stromal cell-derived factor-1 (SDF-1) plays various biological functions through its receptors C-X-C motif chemokine receptor 4 (CXCR4) and CXCR7 in tumor growth and tissue repair. However, it is unclear that whether SDF-1/CXCR4/CXCR7 axis is involved in hypertensive vascular remodeling. In the present study, the involvement of SDF-1/CXCR4/CXCR7 axis was evaluated with lentivirus-mediated shRNA of SDF-1 and CXCR7, CXCR4 antagonist AMD3100 and CXCR7 agonist VUF11207 in angiotensin II (AngII)-induced hypertensive mice and in cultured adventitial fibroblasts (AFs). Results showed that AngII infusion markedly increased SDF-1 expressed in vascular adventitia, but not in media and endothelium. Importantly, blockade of SDF-1/CXCR4 axis strikingly potentiated AngII-induced adventitial thickening and fibrosis, as indicated by enhanced collagen I deposition. In contrast, CXCR7 shRNA largely attenuated AngII-induced adventitial thickness and fibrosis, whereas CXCR7 activation with VUF11207 significantly potentiated AngII-induced adventitial thickening and fibrosis. In consistent with these in vivo study, CXCR4 inhibition with AMD3100 and CXCR7 activation with VUF11207 aggravated AngII-induced inflammation, proliferation and migration in cultured AFs. In summary, these results suggested that SDF-1 exerted opposing effects through CXCR4 and CXCR7 in AngII-induced vascular adventitial remodeling.

ENTREP/FAM189A2 encodes a new ITCH ubiquitin ligase activator that is downregulated in breast cancer

The HECT‐type ubiquitin E3 ligases including ITCH regulate many aspects of cellular function through ubiquitinating various substrates. These ligases are known to be allosterically autoinhibited and to require an activator protein to fully achieve the ubiquitination of their substrates. Here we demonstrate that FAM189A2, a downregulated gene in breast cancer, encodes a new type of ITCH activator. FAM189A2 is a transmembrane protein harboring PPxY motifs, and the motifs mediate its association with and ubiquitination by ITCH. FAM189A2 also associates with Epsin and accumulates in early and late endosomes along with ITCH. Intriguingly, FAM189A2 facilitates the association of a chemokine receptor CXCR4 with ITCH and enhances ITCH‐mediated ubiquitination of CXCR4. FAM189A2‐knockout prohibits CXCL12‐induced endocytosis of CXCR4, thereby enhancing the effects of CXCL12 on the chemotaxis and mammosphere formation of breast cancer cells. In comparison to other activators or adaptors known in the previous studies, FAM189A2 is a unique activator for ITCH to desensitize CXCR4 activity, and we here propose that FAM189A2 be renamed as ENdosomal TRansmembrane binding with EPsin (ENTREP).

Paradoxical anxiolytic effect of the ‘bath salt’ synthetic cathinone MDPV during early abstinence is inhibited by a chemokine CXCR4 or CCR5 receptor antagonist

Chemokine CXCR4 and CCR5 receptors are best known as HIV co-entry receptors, but evidence that CXCR4 or CCR5 blockade reduces rewarding and locomotor-stimulant effects of psychostimulants in rats suggests a role in psychostimulant use disorders. We investigated the impact of CXCR4 or CCR5 receptor antagonism on anxiety-related effects of the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) in the elevated zero-maze (EZM) assay. Rats exposed to a 4-day MDPV binge dosing paradigm and tested 24 or 72 h post-treatment spent more time in the open compartment at the 24-h time point but less time at the 72-h post-binge time point. Daily administration of AMD 3100, a CXCR4 antagonist (10 mg/kg), or maraviroc, a CCR5 antagonist (2.5 mg/kg), during MDPV treatment inhibited the MDPV-induced increase in time spent in the open compartment. Neither antagonist affected the MDPV-induced reduction in time spent in the open compartment at the 72-h post-binge time point. Cocaine, administered in the same paradigm as MDPV, did not increase time spent in the open compartment 24-h post-binge, suggesting specificity to MDPV. The present results identify a surprising anxiolytic-like effect of MDPV 24 h after cessation of repeated exposure that is sensitive to chemokine CXCR4 and CCR5 receptor activity.

Bone Marrow Adipocyte Promotes Epithelial-Mesenchymal-Transition-like Activation in Multiple Myeloma Via CXCL-12/CXCR4 Axis

Extra-medullary disease (EMD) in multiple myeloma (MM) is associated with poor prognosis and resistance to chemotherapy. The bone marrow microenvironment is a key regulator of myeloma metastatic progression through the secretion of growth factors that activate epithelial-mesenchymal transition (EMT)-like characters. Bone marrow adipocyte (BMA) accumulates in obese patients and displays distinct immune regulatory properties rather than provides energy substrates, forming a bulk portion of the bone marrow microenvironment. Emerging evidence indicates the potential for adipocytes to influence cancer cell progression through the secretion of adipokines that can induce EMT activation. The molecular mechanisms underlying how adipocytes enhance multiple myeloma progression is largely unknown. The present study aims to investigate the precise mechanism of BMA promoting myeloma EMT-like activation and new potential therapeutic strategies targeting bone marrow adipocytes.Newly diagnosed MM patients in our cancer center were recruited and divided into lean or obese group according to their body mass index (BMI). The bioinformatics-based analysis of transcriptome sequencing data for bone marrow stromal cells (BMSCs) derived from lean and obese MM patients identified that obese patients showed more obvious EMT-like transcriptional regulation characteristics and altered the expression of genes regulating EMT (Fig A), which was further confirmed by the immunohistochemistry staining of bone marrow biopsy (Fig B). Co-culture bone marrow adipocytes with myeloma cells significantly enhanced the proliferation and migration (Fig C-D). The transcriptome sequencing data of BMA further demonstrated that CXCL12 was enriched in BMA derived from obese MM patients (Fig E). We further confirmed that CXCL12 secreted by adipocytes induce EMT-like activation in myeloma cells, and the effect of CXCL12 expression on EMT is mediated through activation of CXCR4 (Fig F-G). Blocking of CXCR4 signaling in myeloma cells, decreased proliferation and invasion capabilities of myeloma cells cultured with CXCL12 (Fig H-I). Together, our results suggest that bone marrow adipocytes can enhance the aggressive behavior of myeloma cells and induce an EMT-like activation through paracrine CXCL12/CXCR4 signaling (Fig J).In conclusion, paracrine signaling by BMAs can induce EMT-like activation and results in increased proliferation and invasion characteristics of myeloma cells. These results suggested that targeting CXCL12/CXCR4 may act as a regulator of EMD through EMT-like transcriptional modulation, thus representing a potential therapeutic strategy to prevent MM disease progression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Role of CXCR4 and MMP-9 Expressions with Regional Cervical Lymph Nodes Metastatic Status in Papillary Thyroid Carcinoma

Background: Papillary thyroid carcinoma (PTC) that has metastasized has a higher risk because of the poor prognosis, ranging from decreased quality of life of the patient to death. There is a need for markers that can understand the image of the tumor so that it can predict earlier the level of aggressiveness and the ability of tumor cells to metastasize. Tumor cells that proliferate excessively will cause CXCR4 expression. Activation of CXCR4 will increase MMP-9 via the MAPK/ ERK line. MMP-9 will degrade the extracellular matrix causing tumor cell metastasis. This study is aimed to analyze the correlation of CXCR4 and MMP-9 expressions with lymph node metastasis (LNM) in PTC.Methods: This cross-sectional analytic observational study was conducted in 43 PTC samples taken from the Anatomical Pathology Laboratory of Dr. Soetomo Hospital from January 2011 to December 2018. The samples were divided into 2 groups based on the regional cervical LNM status. Immunohistochemical staining was used to assess the expression of CXCR4 and MMP-9. Spearman test was used to analyze the correlation of CXCR4 and MMP expression with cervical LNM status.Results: The expression of CXCR4 and MMP-9 was higher in the papillary thyroid carcinoma group with cervical regional LNM (p = 0.007 and p = 0.030). There was a significant positive relationship between CXCR4 and MMP-9 expressions with regional LNM status with rs = 0.468 and p = 0.001 (p < 0.05). Conclusions: Expression of CXCR4 and MMP-9 was proven to correlate with regional cervical LNM status in papillary thyroid carcinoma. Both can be used as predictive markers for metastasis in papillary thyroid carcinoma.

Intravesical CD74 and CXCR4, macrophage migration inhibitory factor (MIF) receptors, mediate bladder pain

Activation of intravesical protease activated receptor 4 (PAR4) leads to release of urothelial macrophage migration inhibitory factor (MIF). MIF then binds to urothelial MIF receptors to release urothelial high mobility group box-1 (HMGB1) and elicit bladder hyperalgesia. Since MIF binds to multiple receptors, we investigated the contribution of individual urothelial MIF receptors to PAR4-induced HMGB1 release in vivo and in vitro and bladder pain in vivo. We tested the effect of intravesical pre-treatment with individual MIF or MIF receptor (CD74, CXCR4, CXCR2) antagonists on PAR4-induced HMGB1 release in vivo (female C57/BL6 mice) and in vitro (primary human urothelial cells) and on PAR4-induced bladder hyperalgesia in vivo (mice). In mice, PAR4 induced HMGB1 release and bladder hyperalgesia through activation of intravesical MIF receptors, CD74 and CXCR4. CXCR2 was not involved in these effects. In primary urothelial cells, PAR4-induced HMGB1 release through activation of CD74 receptors. Micturition parameters in mice were not changed by any of the treatments. Urothelial MIF receptors CD74 and CXCR4 mediate bladder pain through release of urothelial HMGB1. This mechanism may set up persistent pain loops in the bladder and warrants further investigation. Urothelial CD74 and CXCR4 may provide novel targets for interrupting bladder pain.

CXCR4 hyperactivation cooperates with TCL1 in CLL development and aggressiveness

Aberrant CXCR4 activity has been implicated in lymphoma pathogenesis, disease progression, and resistance to therapies. Using a mouse model with a gain-of-function CXCR4 mutation (CXCR4C1013G) that hyperactivates CXCR4 signaling, we identified CXCR4 as a crucial activator of multiple key oncogenic pathways. CXCR4 hyperactivation resulted in an expansion of transitional B1 lymphocytes, which represent the precursors of chronic lymphocytic leukemia (CLL). Indeed, CXCR4 hyperactivation led to a significant acceleration of disease onset and a more aggressive phenotype in the murine Eµ-TCL1 CLL model. Hyperactivated CXCR4 signaling cooperated with TCL1 to cause a distinct oncogenic transcriptional program in B cells, characterized by PLK1/FOXM1-associated pathways. In accordance, Eµ-TCL1;CXCR4C1013G B cells enriched a transcriptional signature from patients with Richter’s syndrome, an aggressive transformation of CLL. Notably, MYC activation in aggressive lymphoma was associated with increased CXCR4 expression. In line with this finding, additional hyperactive CXCR4 signaling in the Eµ-Myc mouse, a model of aggressive B-cell cancer, did not impact survival. In summary, we here identify CXCR4 hyperactivation as a co-driver of an aggressive lymphoma phenotype.

Abstract 670: CXCR4 expression in tumor associated circulating cells of patients with pancreatic cancer is prognostic for progression and overall survival

Abstract Background: CXCR4 is a chemotaxis receptor implicated in the progression of Pancreatic Cancer (PC) via the activation and invasion of tumor cells. To better elucidate its role in the PC metastasis, we examined the role of CXCR4 expression on a variety of tumor associated circulating cells (TACs) [i.e. circulating tumor cells (CTCs), epithelial to mesenchymal cells (EMTs), and cancer associated macrophage-like cells (CAMLs)] as it relates to patient outcomes. A PC cell line PANC-1 was used to model the activation and expression pathway of CXCR4 in cells. Then blood samples were procured from 30 PC patients prior to the start of therapeutic intent to evaluate CXCR4 expression in TACs. CAMLs, EMTs, and CTCs were found in 93%, 63%, and 30% of PC patients respectively, with high CXCR4 expression found to significantly relate with higher TAC populations in circulation. Further, in a 2-year univariate analysis it was found that high expression of CXCR4 in CAMLs or EMTs was significantly related to both progression free survival (PFS) and overall survival (OS). These data suggest that CXCR4 expression in circulating cells in PC is strongly related to the progression and metastatic spread of the disease. Methods: PANC-1 cells were used to examine the expression and upregulation of CXCR4 by staining with an anti-CXCR4 antibody after stimulation with Isoproterenol. A prospective pilot study was initiated using anonymized peripheral blood samples from (n=30) PC patients, obtained in accordance with local IRB regulations with informed consent. TACs were isolated from the blood using a low-flow CellSieve Microfiltration system and stained for cytokeratin, CXCR4, and CD45. Wilcoxon ranked sum test and univariate analysis were used to analyze the association of CXCR4 expression on all TACs and to evaluate 24-month PFS/OS. Results: Within the PANC-1 cell line, we observed a median expression of CXCR4 intensity of roughly 210 after activation and upregulation with Isoproterenol. Within TACs in the circulation, higher numbers of CAMLs (p=0.017), EMTs (p=0.001), and CTCs (p<0.001) were all significantly related to cells with ≥210 CXCR4 expression. Further, higher expression of CXCR4 on CAMLs correlated with worse PFS (HR=4.0, 95%CI 1.5-10.5, p=0.012) and worse OS (HR=4.8, 95%CI 1.7-13.1, p=0.006), with higher CXCR4 expression in EMTs also correlated with worse PFS (HR=4.6, 95%CI 1.2-15.4, p=0.033) and worse OS (HR=5.4, 95%CI 1.5-19.2, p=0.021). Expression of CXCR4 in CTCs was not significant for PFS or OS. Conclusion: Expression of chemotaxis related receptor CXCR4 appears to have a strong relationship to higher numbers of TACs in circulation. This higher CXCR4 expression in CAMLs and EMTs also appears to correlate with rapid progression and death, indicating a relationship CXCR4 in the spread of PC and its potential use as a target for therapy. Citation Format: Kirby P. Gardner, Susan Tsai, Mohammed Aldakkak, Stephen Gironda, Cha-Mei Tang, Daniel L. Adams. CXCR4 expression in tumor associated circulating cells of patients with pancreatic cancer is prognostic for progression and overall survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 670.

Abstract 2104: 3D spheroid: A rapid drug screening model for epigenetic clinical targets against heterogenic cancer stem cells

Abstract Recent scientific studies showed that transcript start sequences (TSS) are regulated by epigenetic bivalent histone modifications (H3K4me3 and H3K27me3) of target genes for heterogenic cancer stem cells (CSCs) reprogramming. Most of these studies are based on monolayer or 2D culture grew cancer cells. Studies of 3D spheroid models showed a rapid screening of anticancer drugs against CSCs. The 3D-spheroid models closely mimic in vivo tumor microenvironment (TME). Hence, 3D spheroid in vitro model development is a standout amongst other portrayed cancer models regarding 3D cell culture based on its similitude to physiological tumor tissues. Our previous studies of 3D spheroid models showed the effectiveness of small molecules targeted to the bivalent histone modifications against the CSCs in pancreatic cancer. The current study is aimed to explore the epigenetic clinical targets of bivalent histone modifications with the expression of heterogenic CSCs (CD133, CD15 CD44, CD49f, CD24, ALDH1A3, CXCR4) of 3D spheroids in glioblastomas, oral, and colorectal cancers (CRC). The Cancer Stem Cells Therapeutic Target Database Version 2.0 (CSCTTv2.0) reported the highest expression of CSCs in glioblastomas, oral and CRCs that showed resistance to anticancer drugs (such as Bevacizumab, 5-FU, cisplatin, cetuximab, oxaliplatin). The biological informatics methods (String; Cytoscape, BioGrid, Reactome, MatrixDB, Pubchem database) were used in our current study. The NCBI Gene Expression Omnibus (GEO) database showed only 4 studies of 3D spheroids of CSCs which revealed drug-resistant heterogenic CSCs, in 7 samples of glioblastoma spheroids and 4 samples of CSCs in MCF-7 breast cancer spheroids from 6 samples; drug-resistant CSCs of MDA-MB 231 spheroids from 6 samples; CSCs of colorectal cancer (CRC) spheroids from 6 samples, but the clinical the target of bivalent histone modifications with heterogenic CSCs in the glioblastomas, oral and CRCs have not been explored earlier using 3D spheroids for rapid drug screening. The protein-protein interaction (PPI) network of the current study showed a relationship of closely related 17 significant genes. Among these 17 genes, a total of 6 CSCs was closely associated with epigenetic bivalent histone modifications through KDM7A, PHF1and EZH2. We also found from Cytoscape analysis of Reactome network that UniProtKB - P61073 (CXCR4_HUMAN) has significant interaction with other target network genes from 106 network data and it has been mentioned that Plerixafor, an antagonist inhibits CXCR4 activity. Therefore, it is important in designing any small molecules or drugs against heterogenic CSCs of glioblastomas, oral, and CRC with epigenetic target of histone bivalent modifications using a 3D spheroid model which could be rapid screening model for anticancer drug discovery and our current study highlighted this important relationship. Citation Format: Madhumita Das, Maylet Cortes Santana, Stephanie Barraque, Jhoselyn Cardenas, Johnny Amaya Galindo, Misael Cortes, Jose Ramos, Alberto Sanchez Prado, Melissa Tejeda Castillo, Victor Villar, Claudy Vincent, Elizabeth Justo, Mariano Mendez, Indira Mera, Juan Pachon, Kamila Perez, Adara Marin, Nabendu Murmu, Maharaj Biswas, Marco Ruiz, Jayanta Kumar Das. 3D spheroid: A rapid drug screening model for epigenetic clinical targets against heterogenic cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2104.

G-CSF Signaling Counteracts TGF-β—PAI-1-Dependent Retention of Hematopoietic Stem Cells in the Niche

Hematopoietic stem cells (HSCs) reside in the supportive stromal niche in bone marrow (BM); when needed, however, they are rapidly mobilized into the circulation, suggesting that HSPCs are intrinsically highly motile but are usually stayed in the niche. Recently, we have reported that niche cell-producing TGF-β induces intracellular PAI-1 expression in HSCs, which inhibits the proteolytic activity of proprotein convertase Furin-dependent maturation of MT1-MMP, a protease involved in the mobilization of HSPCs, thereby keeping HSCs retaining in the BM niche. Pharmacological inhibition or genetic disruption of the TGF-β−PAI-1 signal increased MT1-MMP-dependent cellular motility, causing a detachment of HSCs from the TGF-β-expressing niche cells, demonstrated a crucial role for TGF-β−PAI-1 signal in the retention of HSCs in the BM. On the other hand, HSCs frequently egress from the BM to circulation and replenish blood supply in order to maintain the homeostasis of blood system. At this moment, however, how environmental stimuli, such as a blood G-CSF concentration rise, counteract the TGF-β-PAI-1 signaling and releases the motility restriction of HSCs remains unknown.To identify the molecular mechanism that govern the dynamic motion of HSCs in the niche, we chose to investigate the cellular activities of HSCs in response to G-CSF. Here, we provide evidence that G-CSF inhibits Smad-dependent signaling by regulating cAMP-PKA-dependent signaling pathway in HSCs. G-CSF-induced activation of this pathway inhibits Smad-dependent expression of PAI-1 through an increase in phosphorylation levels of CREB protein. Mechanistically, increased pCREB competitively inhibited the Smad from binding to the p300/CBP, which ultimately resulting in a transcriptional suppression of Smad-p300/CBP transcription regulatory complex-dependent PAI-1 gene expression. In line with this, administration of G-CSF to mice strongly downregulated intracellular PAI-1 expression in HSCs, which resulted in enhanced Furin-dependent MT1-MMP maturation. The enhanced expression of MT1-MMP results in MT1-MMP-mediated CD44 cleavage as well as CXCR4 and VLA-4 activation, which in turn stimulates detachment of HSPCs from the niche and active trafficking into the blood stream. Expectedly, pharmacological inhibition of intracellular PAI-1 activity synergized with G-CSF-induced HSC mobilization.Our study reveals a novel role for G-CSF signaling in controlling TGF-β−PAI-1-dependent retention of HSCs to recruit them into the circulation, which potentially improve clinical HSC mobilization and transplantation protocols. DisclosuresAndo:CHUGAI PHARMACEUTICAL: Other: Donation to institute; Meiji Seika Pharma: Other: Donation to institute; NOVARTIS: Other: Donation to institute; TOYAMA CHEMICAL: Other: Donation to institute; MOCHIDA PHARMACEUTICAL: Other: Donation to institute; Sumitomo Dainippon Pharma: Other: Donation to institute; Eisai: Other: Donation to institute; Bristol-Myers Squibb: Other: Donation to institute; MSD: Other: Donation to institute; Kyowa Hakko Kirin: Other: Donation to institute; ALEXION: Other: Donation to institute; Takeda: Other: Donation to institute; Japan Blood Products Organization: Other: Donation to institute; Nippon Shinyaku: Other: Donation to institute; NIHON PHARMACEUTICAL: Other: Donation to institute; TAIHO: Other: Donation to institute; Asahi KASEI: Other: Donation to institute.

Retinoblastoma tumor suppressor gene 1 enhances 5-Fluorouracil chemosensitivity through SDF-1/CXCR4 axis by regulating autophagy in gastric cancer

Due to lack of effective biomarkers for early diagnosis, most patients are diagnosed with advanced gastric cancer and have lower survival rates. 5-Fluorouracil (5-FU) is one of commonly used drugs for chemotherapy of gastric cancer, but drug resistance limits the wide application of agents. Retinoblastoma tumor suppressor gene 1 (RB1) is a key regulator in the progression of various human cancers, including gastric cancer. However, the effects of RB1 on chemosensitivity and the underlying mechanisms in gastric cancer (GC) are not clear. In this study, expressions of RB1 in GC cell lines were evaluated by RT-qPCR and western blot assay. CCK-8 was applied to examine the effect of 5-FU on cell viability. Meanwhile, IC50 values were calculated. The drug-resistance protein MDR1 and autophagy-related proteins were detected by western blot assay. Flow cytometry was used to detect cell cycle. The results showed that RB1 expressions were downregulated in GC cell lines and had significant differences between 5-FU resistance cell lines (SNU-620/5-FU and NUGC-3/5-FU) and non-resistance cell lines (SNU-620 and NUGC-3). Overexpression of RB1 enhanced 5-FU sensitivity of GC cells and caused cell cycle arrest in the S phase. Meanwhile, autophagy-related proteins were downregulated. Mechanistically, SDF-1/CXCR4 participated in the regulation of RB1 on cell autophagy. Autophagy activator, SDF-1 treatment and CXCR4 activation reversed the promoted effects of RB1 on 5-FU sensitivity in GC cells. In conclusion, our data revealed that RB1 was downregulated in GC cell lines. RB1 overexpression enhanced 5-FU chemosensitivity in GC cells by regulating cell autophagy via SDF-1/CXCR4 pathway. RB1 might serve as a promising therapeutic target of GC.

Asthmatic bronchial smooth muscle migration towards rhinovirus-infected epithelium through a CXCR3 isoform-dependant pathway

Asthma is the most common chronic respiratory disease. In severe patients, one of the main feature of the physiopathology is the increased bronchial smooth muscle (BSM) mass, which is related to frequency and severity of asthma exacerbations. Exacerbations are mainly triggered by rhinovirus infection of the epithelium. In addition, histology analysis has shown that distance between BSM cells and epithelium decrease proportionally to the severity of the disease. We thus hypothesis that rhinovirus infection of the epithelium increase the migration of asthmatic BSM cells. Primary BSM cells from control and asthmatic patients were obtained from bronchial biopsies. Epithelial cells were cultivated in an air-liquid Interface. After complete differentiation, epithelium were exposed to RVA-16 (MOI 0,1) and epithelium supernatant were collected 24 h post infection. BSM migration was assessed in response to epithelium supernatant in a chemotaxis assay for 24 h using live microscopy. Transcriptomics and ELISA were used to analysed chemokine content within the epithelial supernatant. Immunohistochemistry and flow cytometry were used to quantify CXCR3 isoforms distribution ( i.e CXCR3 A and B). Finally, western blot, cAMP assay and Ca2+ fluorometric probes were used to assess CXCR3 activation pathway. BSM cells from asthmatics migrates towards RV-infected epithelium whereas such a chemotactic effect was not observed in control BSM cells. Analysis of epithelium supernatant show that CXCL9 and CXCL10 were both induced by RVA-16. Interestingly, blocking of CXCR3 receptor, which is activated, by both CXCL9 and 10 abolished asthmatic BSM migration. They are two main isoforms of CXCR3 (A and B) which are described as activator and inhibitor of cell migration reciprocally. Interestingly, CXCR3-B expression and activation pathway through PKC and cAMP are increased in control BSM cells whereas CXCR3-A signalling pathway through calcium and Raf-1 activation are increase in asthmatic BSM cells. Asthmatic BSM cells specifically migrate toward RV-infected epithelium through the activation of CXCR3-A. This new mechanism could be a new therapeutic target against BSM remodelling in asthma.

Antitumor and Radiosensitization Effects of a CXCR2 Inhibitor in Nasopharyngeal Carcinoma.

CXCR2, a member of the G-protein-coupled cell surface chemokine receptor family, is commonly found on leukocytes, endothelial cells and tumor cells including nasopharyngeal carcinoma cells. However, how the activity of CXCR2 and its ligand CXCL8 affects the development of nasopharyngeal carcinoma (NPC) remains unknown. Here, we found that CXCR2 and CXCL8 were both predicted poor prognosis in NPC patients. Furthermore, we identified that treatment with CXCR2 antagonist SB225002 of nasopharyngeal carcinoma cell lines resulted tumorigenesis inhibition in vitro and in vivo. In addition, we found that SB225002 could enhance NPC cells radiosensitivity through regulating cell circle distribution and interfering with cellular DNA damage repair. SB225002 also exhibited an efficient radiosensitization effect in C666-1 and HONE-1 bearing mice. Functionally, we showed that SB225002 reduced microvessel density and proliferation and induced tumor apoptosis. Furthermore, changes in the tumor microenvironment were also observed in this study. We observed that SB225002 reduced tumor-associated neutrophils (TANs) in the tumors tissue which were recruited especially after irradiation. Taken together, our results suggested that targeting the CXCL8-CXCR2 pathway is a promising therapeutic strategy for comprehensive NPC treatment.

Tumor-Associated Macrophage Promotes the Survival of Cancer Cells upon Docetaxel Chemotherapy via the CSF1/CSF1R-CXCL12/CXCR4 Axis in Castration-Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is an advanced stage of prostate cancer that can progress rapidly even in patients treated with castration. Previously, we found that tumor-associated macrophages (TAM) can be recruited by CSF-1 secreted by docetaxel-treated prostate cancer cells and promote the survival of cancer cells in response to chemotherapy. The inhibition of CSF-1R can impede this effect and significantly prolong survival in xenograft mice. However, the actual mechanism of how TAM improves cancer cell survival still remains elusive and controversial. Here, for the first time, we found that the enhanced survival of cancer cells achieved by TAM was mainly mediated by CXCR4 activation from the increased secretion of CXCL12 from CSF-1 activated TAM. This finding helps to clarify the mechanism of chemoresistance for second-line chemotherapy using docetaxel, facilitating the development of novel drugs to overcome immune tolerance in castration-resistant prostate cancer.

CXCR4 engagement triggers CD47 internalization and antitumor immunization in a mouse model of mesothelioma

Boosting antitumor immunity has emerged as a powerful strategy in cancer treatment. While releasing T‐cell brakes has received most attention, tumor recognition by T cells is a pre‐requisite. Radiotherapy and certain cytotoxic drugs induce the release of damage‐associated molecular patterns, which promote tumor antigen cross‐presentation and T‐cell priming. Antibodies against the “do not eat me” signal CD47 cause macrophage phagocytosis of live tumor cells and drive the emergence of antitumor T cells. Here we show that CXCR4 activation, so far associated only with tumor progression and metastasis, also flags tumor cells to immune recognition. Both CXCL12, the natural CXCR4 ligand, and BoxA, a fragment of HMGB1, promote the release of DAMPs and the internalization of CD47, leading to protective antitumor immunity. We designate as Immunogenic Surrender the process by which CXCR4 turns in tumor cells to macrophages, thereby subjecting a rapidly growing tissue to immunological scrutiny. Importantly, while CXCL12 promotes tumor cell proliferation, BoxA reduces it, and might be exploited for the treatment of malignant mesothelioma and a variety of other tumors.

Conformational Dynamics of Classical and Atypical GPCRs, CXCR4 and ACKR3, by Single‐Molecule FRET

The G protein-coupled receptors (GPCRs) CXCR4 and ACKR3 both bind the same natural agonist CXCL12, yet CXCR4 signals through G proteins and arrestins, while ACKR3 only couples to the latter. This naturally occurring receptor-mediated signaling bias also manifests in mutations to CXCL12 that cause the agonist to no longer activate CXCR4, but still acts as an ACKR3 agonist. How does CXCL12 binding lead to the different activity of these related receptors? Here, we have investigated the conformational dynamics of the two receptors by single-molecule Förster resonance energy transfer (smFRET) using probes to detect translocation of transmembrane helix 6 (TM6) of purified receptors reconstituted in nanodiscs. Our results show a consistent picture of both CXCR4 and ACKR3 occupying multiple conformational states in the absence of ligands, which collapse to mostly a single FRET state with the addition of agonist. Additionally, the system responds as expected to antagonists and mutants of CXCL12 induce opposite conformational changes for CXCR4 and ACKR3. Additionally, our experimental setup is amenable to the addition of ancillary binders, such as G proteins and arrestins, and we observe further population shifts when these are included. In this way we are able to observe differences in activation of ACKR3 and CXCR4 by the same ligand. The optimization of our single-molecule system and investigations into the activation differences of CXCR4 and ACKR3 will be discussed along with potential caveats.

Structural insights into the activation of chemokine receptor CXCR2

The C-X-C motif chemokine CXCL8 (interleukin-8, IL-8) and its receptor chemokine receptor 2 (CXCR2) mediate neutrophil migration during cell development and inflammatory responses and thus are related to numerous inflammatory diseases and cancers. We have determined the cryo-electron microscopy structure of CXCL8 bound CXCR2 coupled to Gi protein, as well as the crystal structure of inactive CXCR2 in complex with a designed allosteric antagonist. These results reveal the binding modes between CXCL8 and CXCR2, CXCR2 and G protein, and the detailed binding pattern of the allosteric antagonist, 00767013. Further structural analysis of the inactive- and active- states of CXCR2 reveals the unique shallow-pocket activation mechanism of C-X-C chemokine receptors and promotes our understanding on how a G protein-coupled receptor (GPCR) is activated by an endogenous protein molecule. In addition, the cholesterol molecule is observed in the activated CXCR2 structure, providing the structural basis of the potential allosteric modulation role of cholesterol in chemokine receptors.