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A bivalent promoter contributes to stress-induced plasticity of CXCR4 in Ewing sarcoma.

ABSTRACT: Tumor heterogeneity is a major impediment to cancer cures. Tumor cell heterogeneity can arise by irreversible genetic mutation, as well as by non-mutational mechanisms, which can be reversibly modulated by the tumor microenvironment and the epigenome. We recently reported that the chemokine receptor CXCR4 is induced in Ewing sarcoma cells in response to microenvironmental stress. In the current study, we investigated plasticity of CXCR4 expression in vivo and assessed whether CXCR4 impacts on tumor growth. Our studies showed that Ewing sarcoma cells convert between CXCR4 negative and CXCR4 positive states in vivo and that positive cells are most abundant adjacent to areas of necrosis. In addition, tumor volumes directly correlated with CXCR4 expression supporting a role for CXCR4 in growth promotion. Mechanistically, our results show that, in ambient conditions where CXCR4 expression is low, the CXCR4 promoter exists in a poised, bivalent state with simultaneous enrichment of both activating (H3K4me3) and repressive (H3K27me3) post-translational histone modifications. In contrast, when exposed to stress, CXCR4 negative cells lose the H3K27me3 mark. This loss of promoter bivalency is associated with CXCR4 upregulation. These studies demonstrate that stress-dependent plasticity of CXCR4 is, in part, mediated by epigenetic plasticity and a bivalent promoter.

SUBMITTER: Krook MA

PROVIDER: S-EPMC5308690 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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A bivalent promoter contributes to stress-induced plasticity of CXCR4 in Ewing sarcoma.

Krook Melanie A MA Hawkins Allegra G AG Patel Rajiv M RM Lucas David R DR Van Noord Raelene R Chugh Rashmi R Lawlor Elizabeth R ER

Oncotarget 20160901 38

Tumor heterogeneity is a major impediment to cancer cures. Tumor cell heterogeneity can arise by irreversible genetic mutation, as well as by non-mutational mechanisms, which can be reversibly modulated by the tumor microenvironment and the epigenome. We recently reported that the chemokine receptor CXCR4 is induced in Ewing sarcoma cells in response to microenvironmental stress. In the current study, we investigated plasticity of CXCR4 expression in vivo and assessed whether CXCR4 impacts on tu ...[more]

PMID: 27528222

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Project description:BackgroundEwing sarcoma is an aggressive, highly metastatic primary bone and soft tissue tumor most frequently occurring in the bone of young adolescents. Patients, especially those diagnosed with a metastatic disease, have a poor overall survival. Chemokine receptor CXCR4 has a key pro-tumorigenic role in the tumor microenvironment of Ewing sarcoma and has been suggested to be involved in the increased metastatic propensity. Earlier studies on CXCR4 protein expression in Ewing sarcoma yielded contradictory results when compared to CXCR4 RNA expression studies. Previously, we demonstrated that CXCR4 expression could be detected in vivo using the fluorescently tagged CXCR4-specific peptide MSAP-Ac-TZ14011. Therefore, we studied the membranous CXCR4 expression in Ewing sarcoma cell lines using MSAP-Ac-TZ14011.MethodsThe CXCR4 membrane expression levels were studied in EWS cell lines by flow cytometry using the hybrid peptide MSAP-Ac-TZ14011 and were correlated to CXCR4 RNA expression levels. The measurements were compared to levels detected using the CXCR4 antibody ab2074 under various cell preparation conditions. In addition, the staining patterns were analyzed by confocal fluorescence microscopy over time.ResultsThe hybrid peptide MSAP-Ac-TZ14011 levels showed a strong and better correlation of CXCR4 membrane expression with the CXCR4 RNA expression levels than observed with the anti-CXCR4 antibody ab2074. With the hybrid peptide MSAP-Ac-TZ14011 using live cell confocal microscopy CXCR4 membrane staining and internalization was detected and the signal intensity correlated well with CXCR4 mRNA expression levels.ConclusionsThe fluorescently labeled CXCR4 targeting peptide-based method provides a reliable alternative to antibody staining to study the CXCR4 membrane expression in live cells using either flow cytometry or live cell fluorescence microscopy. The fluorescently tagged CXCR4 targeting peptide could enable in vivo detection of CXCR4 expression in Ewing sarcoma which may help to stratify cases for anti-CXCR4 therapy.

Project description:PurposePropagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity.Experimental designExisting tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays.ResultsWe found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis.ConclusionsEwing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13. See related commentary by Weiss and Bailey, p. 4360.

Project description:UnlabelledBackgroundChemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a sarcoma with poor prognosis despite current therapies, particularly for patients with advanced-stage disease. Lungs and bone (marrow), organs of predilection for (primary/metastatic) Ewing sarcoma, represent predominant CXCL12 sources.MethodsTo gain insight into the role of the CXCR4-CXCL12 axis in Ewing sarcoma, CXCR4, CXCL12 and hypoxia-inducible factor-1? protein expression was studied in therapy-naïve and metastatic tumors by immunohistochemistry. CXCR4 function was assessed in vitro, by flow cytometry and proliferation/ cell viability assays, in the presence of recombinant CXCL12 and/or CXCR4-antagonist AMD3100 or under hypoxic conditions.ResultsWhereas CXCR4 was predominantly expressed by tumor cells, CXCL12 was observed in both tumor and stromal areas. Survival analysis revealed an (expression level-dependent) negative impact of CXCR4 expression (p?<?0.04). A role for the CXCR4-CXCL12 axis in Ewing sarcoma growth was suggested by our observations that i) CXCR4 expression correlated positively with tumor volume at diagnosis (p?=?0.013), ii) CXCL12 was present within the microenvironment of virtually all cases, iii) CXCL12 induced proliferation of CXCR4-positive Ewing sarcoma cell lines, which could be abrogated by AMD3100. CXCR4 expression was not correlated with occurrence of metastatic disease. Also, therapy-naïve tumors demonstrated higher CXCR4 expression as compared to metastases (p?=?0.027). Evaluation of in vivo hypoxia-inducible factor-1? expression and culture of cells under hypoxic conditions revealed no role for hypoxia in CXCR4 expression.ConclusionsTogether, our results imply a crucial role for the CXCR4-CXCL12 axis in auto- and/or paracrine growth stimulation. Integration of CXCR4-targeting strategies into first- and/or second-line treatment regimens may represent a promising treatment option for Ewing sarcoma.

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A bivalent promoter contributes to stress-induced plasticity of CXCR4 in Ewing sarcoma.

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A bivalent promoter contributes to stress-induced plasticity of CXCR4 in Ewing sarcoma.

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