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Annexin 1 released by necrotic human glioblastoma cells stimulates tumor cell growth through the formyl peptide receptor 1.

ABSTRACT: Highly malignant human gliomas overexpress the G-protein-coupled chemoattractant receptor formyl peptide receptor (FPR1), which promotes tumor progression when activated. Our previous studies demonstrated that necrotic glioblastoma cells release chemotactic agonist(s) that activate FPR1 on viable tumor cells. In the present study, we identified an FPR1 agonist released by necrotic human glioblastoma cells. Necrotic tumor cell supernatant (NecSup) contained Annexin 1 (Anx A1), a chemotatic polypeptide agonist for FPR1. Immunoabsorption of Anx A1 with a specific antibody markedly reduced the chemotactic activity of NecSup for tumor cells and diminished its capacity to promote tumor cell growth, invasion, and colony formation on soft agar. In addition, Anx A1 was present in tumor xenografts formed by human glioblastoma cells in nude mice. Anx A1 knockdown significantly reduced the tumorigenicity of glioblastoma cells in nude mice, but FPR1/Anx A1 double knockdown diminished tumor growth even further. The clinical relevance of Anx A1 in gliomas was supported by the observation that Anx A1 was more highly expressed in poorly differentiated human primary gliomas compared with lower grade tumors. Our study implicates Anx A1 as a major component in necrotic tumor cell-derived stimulants of the growth of glioblastoma via the activation of FPR1.

SUBMITTER: Yang Y

PROVIDER: S-EPMC3157278 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Annexin 1 released by necrotic human glioblastoma cells stimulates tumor cell growth through the formyl peptide receptor 1.

Yang Yan Y Liu Ying Y Yao Xiaohong X Ping Yifang Y Jiang Tao T Liu Qin Q Xu Senlin S Huang Jian J Mou Haiwei H Gong Wanghua W Chen Keqiang K Bian Xiuwu X Wang Ji Ming JM

The American journal of pathology 20110722 3

Highly malignant human gliomas overexpress the G-protein-coupled chemoattractant receptor formyl peptide receptor (FPR1), which promotes tumor progression when activated. Our previous studies demonstrated that necrotic glioblastoma cells release chemotactic agonist(s) that activate FPR1 on viable tumor cells. In the present study, we identified an FPR1 agonist released by necrotic human glioblastoma cells. Necrotic tumor cell supernatant (NecSup) contained Annexin 1 (Anx A1), a chemotatic polype ...[more]

PMID: 21782780

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Project description:BackgroundDespite advancements in therapies, brain metastasis in patients with triple negative subtype of breast cancer remains a therapeutic challenge. Activated microglia are often observed in close proximity to, or within, malignant tumor masses, suggesting a critical role that microglia play in brain tumor progression. Annexin-A1 (ANXA1), a glucocorticoid-regulated protein with immune-regulatory properties, has been implicated in the growth and metastasis of many cancers. Its role in breast cancer-microglia signaling crosstalk is not known.MethodsThe importance of microglia proliferation and activation in breast cancer to brain metastasis was evaluated in MMTV-Wnt1 spontaneous mammary tumor mice and BALBc mice injected with 4T1 murine breast cancer cells into the carotid artery using flow cytometry. 4T1 induced-proliferation and migration of primary microglia and BV2 microglia cells were evaluated using 2D and coculture transwell assays. The requirement of ANXA1 in these functions was examined using a Crispr/Cas9 deletion mutant of ANXA1 in 4T1 breast cancer cells as well as BV2 microglia. Small molecule inhibition of the ANXA1 receptor FPR1 and FPR2 were also examined. The signaling pathways involved in these interactions were assessed using western blotting. The association between lymph node positive recurrence-free patient survival and distant metastasis-free patient survival and ANXA1 and FPR1 and FPR2 expression was examined using TCGA datasets.ResultsMicroglia activation is observed prior to brain metastasis in MMTV-Wnt1 mice with primary and secondary metastasis in the periphery. Metastatic 4T1 mammary cancer cells secrete ANXA1 to promote microglial migration, which in turn, enhances tumor cell migration. Silencing of ANXA1 in 4T1 cells by Crispr/Cas9 deletion, or using inhibitors of FPR1 or FPR2 inhibits microglia migration and leads to reduced activation of STAT3. Finally, elevated ANXA1, FPR1 and FPR2 is significantly associated with poor outcome in lymph node positive patients, particularly, for distant metastasis free patient survival.ConclusionsThe present study uncovered a network encompassing autocrine/paracrine ANXA1 signaling between metastatic mammary cancer cells and microglia that drives microglial recruitment and activation. Inhibition of ANXA1 and/or its receptor may be therapeutically rewarding in the treatment of breast cancer and secondary metastasis to the brain.

Project description:ObjectiveNecrotic cells evoke potent innate immune responses through unclear mechanisms. The mitochondrial fraction of the cell retains constituents of its bacterial ancestors, including N-formyl peptides, which are potentially immunogenic. Thus, we hypothesized that the mitochondrial fraction of the cell, particularly N-formyl peptides, contributes significantly to the activation of monocytes by necrotic cells.DesignHuman peripheral blood monocytes were incubated with necrotic cell fractions and mitochondrial proteins to investigate their potential for immune cell activation.SettingUniversity Medical Center Research Laboratory.SubjectsHealthy human adults served as blood donors.Measurements and main resultsHuman blood monocyte activation was measured after treatment with cytosolic, nuclear and mitochondrial fractions of necrotic HepG2 cells or necrotic HepG2 cells depleted of N-formyl peptides [Rho(0) cells]. The specific role of the high affinity formyl peptide receptor (FPR) was then tested using specific pharmacologic inhibitors and RNA silencing. The capacity of mitochondrial N-formyl peptides to activate monocytes was confirmed using a synthetic peptide conforming to the N-terminus of mitochondrial nicotinamide adenine dinucleotide subunit 6. The results demonstrated that mitochondrial cell fractions most potently activated monocytes, and interleukin (IL)-8 was selectively released at low-protein concentrations. Mitochondria from Rho(0) cells induced minimal monocyte IL-8 release, and specific pharmacologic inhibitors and RNA-silencing confirmed that FPR contributes significantly to monocyte IL-8 responses to both necrotic cells and mitochondrial proteins. N-formyl peptides alone did not induce monocyte IL-8 release; whereas, the combination of mitochondrial N-formyl peptides and mitochondrial transcription factor A (TFAM) dramatically increased IL-8 release from monocytes. Likewise, high mobility group box 1, the nuclear homolog of TFAM, did not induce monocyte IL-8 release unless combined with mitochondrial N-formyl peptides.ConclusionsInteractions between mitochondrial N-formyl peptides and FPR in the presence of other mitochondrial antigens (e.g., TFAM) contributes significantly to the activation of monocytes by necrotic cells.

Project description:The N-formyl peptide receptors (FPRs) are a family of G-protein coupled receptors that respond to proinflammatory N-formylated bacterial peptides (e.g., formyl-Met-Leu-Phe, fMLF) and, thus, contribute to the host response to bacterial infection. Paradoxically, a growing body of evidence suggests that some members of this receptor family may also be targets for certain anti-inflammatory molecules, including annexin A1 (ANXA1), which is an important mediator of glucocorticoid (GC) action. To explore further the potential role of FPRs in mediating ANXA1 actions, we have focused on the pituitary gland, where ANXA1 has a well-defined role as a cell-cell mediator of the inhibitory effects of GCs on the secretion of corticotrophin (ACTH), and used molecular, genetic, and pharmacological approaches to address the question in well-established rodent models. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis identified mRNAs for four FPR family members in the mouse anterior pituitary gland, Fpr-rs1, Fpr-rs2, Fpr-rs6, and Fpr-rs7. Functional studies confirmed that, like dexamethasone, ANXA1 and two ANXA1-derived peptides (ANXA1(1-188) and ANXA1(Ac2-26)) inhibit the evoked release of ACTH from rodent anterior pituitary tissue in vitro. Fpr1 gene deletion failed to modify the pituitary responses to dexamethasone or ANXA1(Ac2-26). However, lipoxin A4 (LXA4, 0.02-2 microM, a lipid mediator with high affinity for Fpr-rs1) mimicked the inhibitory effects of ANXA1 on ACTH release as also did fMLF in high (1-100 microM) but not lower (10-100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 microM) overcame the effects of dexamethasone, ANXA1(1-188), ANXA1(Ac2-26), fMLF, and LXA4 on ACTH release, although at a lower concentration (50 microM), it was without effect. Together, the results suggest that the actions of ANXA1 in the pituitary gland are independent of Fpr1 but may involve other FPR family members, in particular, Fpr-rs1 or a closely related receptor. They thus provide the first evidence for a role of the FPR family in the regulation of neuroendocrine function.

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Annexin 1 released by necrotic human glioblastoma cells stimulates tumor cell growth through the formyl peptide receptor 1.

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Annexin 1 released by necrotic human glioblastoma cells stimulates tumor cell growth through the formyl peptide receptor 1.

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