Inactivation of chemokine (C-C motif) receptor 1 (CCR1) suppresses colon cancer liver metastasis by blocking accumulation of immature myeloid cells in a mouse model.
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ABSTRACT:
SUBMITTER: Kitamura T
PROVIDER: S-EPMC2919974 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:To understand colon cancer metastasis, we earlier analyzed a mouse model that developed liver metastasis of cancer cells disseminated from the spleen. We suggested that CCR1(+) bone marrow (BM)-derived cells are recruited to the microenvironment of disseminated colon cancer cells, and produce metalloproteinases MMP9 and MMP2, helping metastatic colonization. In the present study, we have examined these myeloid cells expressing CCR1 and/or MMPs in detail. To this end, we have established bacterial artificial chromosome (BAC)-based transgenic mouse lines in which membrane-targeted Venus fluorescent protein (mVenus) was expressed under the control of Ccr1 gene promoter. Then, myeloid cells obtained from the BM and liver metastatic foci were analyzed by the combination of flow cytometry and cytology/immunohistochemistry, in situ RNA hybridization, or quantitative RT-PCR. We have found four distinct types of myeloid cells recruited to the metastatic foci; neutrophils, eosinophils, monocytes and fibrocytes. These cell types exhibited distinct expression patterns for CCR1, MMP2 and MMP9. Namely, neutrophils found in the early phase of cancer cell dissemination expressed CCR1 exclusively and MMP9 preferentially, whereas fibrocytes accumulated in later phase expressed MMP2 exclusively. Either genetic inactivation of Ccr1 or antibody-mediated neutrophil depletion reduced subsequent recruitment of fibrocytes. The recruitment of CCR1(+) neutrophils in early phase of colon cancer dissemination appears to cause that of fibrocytes in late phase. These results implicate the key role of CCR1 in colon cancer metastasis in this mouse model, and explain why both MMP9 and MMP2 are essential as genetically demonstrated previously. The results also suggest relevant mechanisms in humans.
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Project description:BackgroundCancer associated fibroblasts (CAFs) communicate metabolically with tumor genesis and development. Rocuronium bromide (RB) is reported to exert certain inhibitory effect on tumor. Here, we investigate the role of RB in esophageal cancer (EC) malignant progression.MethodsTumor xenograft models with EC cells were locally and systemically administrated with RB to detect the influence of different administrations on tumor progression. Mouse CAFs PDGFRα+/F4/80- were sorted by Flow cytometry with specific antibodies. CAFs were treated with RB and co-cultured with EC cells. The proliferation, invasion and apoptosis assays of EC cells were performed to detect the influences of RB targeting CAFs on EC cell malignant progression. Human fibroblasts were employed to perform these detections to confirm RB indirect effect on EC cells. The gene expression changes of CAFs response to RB treatment were detected using RNA sequencing and verified by Western blot, immunohistochemistry and ELISA.ResultsTumors in xenograft mice were observed significantly inhibited by local RB administration, but not by systemic administration. Moreover EC cells did not show obvious change in viability when direct stimulated with RB in vitro. However, when CAFs treated with RB were co-cultured with EC cells, obvious suppressions were observed in EC cell malignancy, including proliferation, invasion and apoptosis. Human fibroblasts were employed to perform these assays and similar results were obtained. RNA sequencing data of human fibroblast treated with RB, and Western blot, immunohistochemistry and ELISA results all showed that CXCL12 expression was significantly diminished in vivo and in vitro by RB. EC cells direct treated with CXCL12 showed much higher malignancy. Moreover cell autophagy and PI3K/AKT/mTOR signaling pathway in CAFs were both suppressed by RB which can be reversed by Rapamycin pretreatment.ConclusionsOur data suggest that RB could repress PI3K/AKT/mTOR signaling pathway and autophagy to block the CXCL12 expression in CAFs, thereby weakening the CXCL12-mediated EC tumor progression. Our data provide a novel insight into the underlying mechanism of RB inhibiting EC, and emphasize the importance of tumor microenvironment (cytokines from CAFs) in modulating cancer malignant progression.
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Project description:Chemotactic cytokines (chemokines) can help regulate tumor cell invasion and metastasis. Here, we show that chemokine 25 (CCL25) and its cognate receptor chemokine receptor 9 (CCR9) inhibit colorectal cancer (CRC) invasion and metastasis. We found that CCR9 protein expression levels were highest in colon adenomas and progressively decreased in invasive and metastatic CRCs. CCR9 was expressed in both primary tumor cell cultures and colon-cancer-initiating cell (CCIC) lines derived from early-stage CRCs but not from metastatic CRC. CCL25 stimulated cell proliferation by activating AKT signaling. In vivo, systemically injected CCR9+ early-stage CCICs led to the formation of orthotopic gastrointestinal xenograft tumors. Blocking CCR9 signaling inhibited CRC tumor formation in the native gastrointestinal CCL25+ microenvironment, while increasing extraintestinal tumor incidence. NOTCH signaling, which promotes CRC metastasis, increased extraintestinal tumor frequency by stimulating CCR9 proteasomal degradation. Overall, these data indicate that CCL25 and CCR9 regulate CRC progression and invasion and further demonstrate an appropriate in vivo experimental system to study CRC progression in the native colon microenvironment.
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Project description:Leukocyte migration, a hallmark of the inflammatory response, is stimulated by the interactions between chemokines, which are expressed in injured or infected tissues, and chemokine receptors, which are G protein-coupled receptors (GPCRs) expressed in the leukocyte plasma membrane. One mechanism for the regulation of chemokine receptor signaling is biased agonism, the ability of different chemokine ligands to preferentially activate different intracellular signaling pathways via the same receptor. To identify features of chemokines that give rise to biased agonism, we studied the activation of the receptor CCR1 by the chemokines CCL7, CCL8, and CCL15(Δ26). We found that, compared to CCL15(Δ26), CCL7 and CCL8 exhibited biased agonism towards cAMP inhibition and away from β-Arrestin 2 recruitment. Moreover, N-terminal substitution of the CCL15(Δ26) N-terminus with that of CCL7 resulted in a chimera with similar biased agonism to CCL7. Similarly, N-terminal truncation of CCL15(Δ26) also resulted in signaling bias between cAMP inhibition and β-Arrestin 2 recruitment signals. These results show that the interactions of the chemokine N-terminal region with the receptor transmembrane region play a key role in selecting receptor conformations coupled to specific signaling pathways.
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Project description:Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b(+)Ly6G(+) immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b(+)Ly6G(+) cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b(+)Ly6G(+) IMCs in early cancer development.
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Project description:Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses.
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Project description:The CC motif chemokine receptor 1 (CCR1) has been implicated in tumor invasion and metastasis in numerous cancers. However, the detailed mechanism of CCR1 upregulation in metastatic tumor cells is poorly understood. The aim of this study was to clarify the regulatory mechanism underlying transcriptional activation of the CCR1 gene in response to epidermal growth factor (EGF) stimulation in breast cancer cells. CCR1 was highly expressed in human breast invasive ductal carcinoma (IDC) compared to adjacent normal tissues. Upon EGF stimulation, CCR1 expression was upregulated at the transcriptional level. Promoter analysis showed that signal transducer and activator of transcription 3 (STAT3) is necessary for EGF-induced CCR1 promoter activation, and STAT3 silencing abrogated EGF-induced CCR1 transcription. Pharmacological inhibition and short hairpin RNA-mediated knockdown experiments showed that AKT-dependent mammalian target of rapamycin (mTOR) activation was involved in the phosphorylation of serine-727 of STAT3, which in turn stimulated the transcription of the CCR1 gene. In conclusion, the AKT-mTOR-STAT3 signaling axis contributes to EGF-induced CCR1 expression, which promotes invasion and metastasis in breast cancer cells. We propose that the AKT-mTOR-STAT3 axis is a potential therapeutic target for blocking the invasion and metastasis of breast cancers.
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Project description:Chemokine receptors are a specific class of G-protein-coupled receptors (GPCRs) that control cell migration associated with routine immune surveillance, inflammation and development. In addition to their roles in normal physiology, these receptors and their ligands are involved in a large number of inflammatory diseases, cancer and AIDS, making them prime therapeutic targets in the pharmaceutical industry. Like other GPCRs, a significant obstacle in determining structures and characterizing mechanisms of activation has been the difficulty in obtaining high levels of pure, functional receptor. Here we describe a systematic effort to express the chemokine receptor CCR1 in mammalian cells, and to purify and reconstitute it in functional form. The highest expression levels were obtained using an inducible HEK293 system. The receptor was purified using a combination of N- (StrepII or Hemagglutinin) and C-terminal (His8) affinity tags. Function was assessed by ligand binding using a novel fluorescence polarization assay with fluorescein-labeled chemokine. A strict dependence of function on the detergent composition was observed, as solubilization of CCR1 in n-dodecyl-beta-D-maltopyranoside/cholesteryl hemisuccinate yielded functional receptor with a K(d) of 21 nM for the chemokine CCL14, whereas it was non-functional in phosphocholine detergents. Differences in function were observed despite the fact that both these detergent types maintained the receptor in a state characterized by monomers and small oligomers, but not large aggregates. While optimization is still warranted, yields of approximately 0.1-0.2mg of pure functional receptor per 10(9) cells will permit biophysical studies of this medically important receptor.
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