Abstract 498: A bivalent promoter regulates stress dependent induction of CXCR4 in Ewing sarcoma

Abstract

Abstract CXCR4 is the most commonly expressed chemokine receptor in human cancer and high expression contributes to metastasis. We recently showed that CXCR4 expression is dynamic in Ewing sarcoma (ES) and that its expression is induced by cell stress. In the current study we investigated the potential contribution of epigenetic regulation to this stress response. Chromatin Immunoprecipitation (ChIP) was used to analyze histone modifications at the CXCR4 locus. CXCR4 mRNA levels were quantified using qRT-PCR. Chemotactic migration to CXCL12 was evaluated using transwell assays in the presence and absence of the bromodomain inhibitor JQ1. ChIP showed enrichment of both H3K4me3 and H3K27me3 marks at the CXCR4 promoter in unstressed ES cells. ChIP-re-ChIP of the same chromatin confirmed that the promoter exists in a bivalent state in ambient conditions. Levels of CXCR4 directly correlated with H3K4me3 and inversely correlated with H3K27me3 marks suggesting that transcription of CXCR4 is, at least in part, regulated by the competing actions of these polycomb- and trithorax-dependent histone modifications. This was confirmed using studies of CXCR4+ and CXCR4- cells that had been isolated by FACS prior to ChIP. In these sorted populations we detected preferential enrichment of H3K4me3 in CXCR4+ and H3K27me3 in CXCR4- cells. Significantly, exposure of ES cells to stress, either serum-deprivation or hypoxia, resulted in loss of bivalency as demonstrated by diminished enrichment of H3K27me3 in the continued presence of high-levels of H3K4me3. Loss of bivalency was accompanied by upregulation of CXCR4 thereby confirming the key contribution of the promoter's chromatin state to transcriptional regulation. Finally, we noted that an active enhancer exists upstream of the CXCR4 locus in HeLa cells, which express constitutively high levels of CXCR4. Therefore, we hypothesized that this enhancer may also contribute to CXCR4 regulation in ES. In support of this, JQ1 treatment of ES cells resulted in marked down-regulation of CXCR4 expression in ambient conditions. In addition, JQ1 treated cells failed to up-regulate CXCR4 in response to both serum-deprivation and hypoxia. Significantly, chemotactic migration of ES cells to CXCL12 was impeded by JQ1. The effects of JQ1 on basal and stress-dependent induction of CXCR4 were reproduced in both HeLa and neuroblastoma cells indicating that epigenetic regulation of CXCR4 extends to other cancer types. In summary, we have demonstrated that stress-dependent induction of CXCR4 in ES is, in part, epigenetically regulated. In particular, the CXCR4 promoter in ES cells resides in a bivalent state which permits rapid and reversible transitions between CXCR4- and CXCR4+ states. Further, enhancer-dependent regulation of CXCR4 transcription opens the door to the potential of bromodomain inhibitors as novel therapeutics that could be used to antagonize metastasis-promoting cell state transitions. Citation Format: Melanie A. Krook, Elizabeth R. Lawlor. A bivalent promoter regulates stress dependent induction of CXCR4 in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 498. doi:10.1158/1538-7445.AM2015-498