Novel splice variants of CXCR4 identified by transcriptome sequencing

Sand, Laurens G.L.; Jochemsen, Aart G.; Beletkaia, Elena; Schmidt, Thomas; Hogendoorn, Pancras C.W.; Szuhai, Károly

doi:10.1016/j.bbrc.2015.08.113

Cited by 10 publications

(10 citation statements)

References 28 publications

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“…Splicing isoforms and post-translational modifications such as glycosylation and ubiquitination can affect CXCR4 surface expression and function [ 27 , 29 ]. Because such CXCR4 splicing variants have been reported in Ewing sarcoma [ 30 , 31 ], we first examined CXCR4 protein expression in Western blots of whole cell lysates. This revealed multiple bands as previously shown in other cancers [ 27 , 29 ] (Fig.…”

Section: Resultsmentioning

confidence: 99%

The CXCR4 antagonist plerixafor (AMD3100) promotes proliferation of Ewing sarcoma cell lines in vitro and activates receptor tyrosine kinase signaling

et al. 2018

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BackgroundThe CXCR4 receptor antagonist plerixafor (AMD3100) is raising interest as an anti-cancer agent that disrupts the CXCL12-CXCR4 chemokine – receptor interaction between neoplastic cells and their microenvironment in tumor progression and metastasis. Here, we investigated plerixafor for anti-cancer activity in Ewing sarcoma, a rare and aggressive cancer of bone and soft tissues.MethodsWe used a variety of methods such as cell viability and migration assays, flow cytometry, phospho-tyrosine arrays and western blotting to determine plerixafor effects on five characterized Ewing sarcoma cell lines and a low-passage culture in vitro.ResultsUnexpectedly, plerixafor led to an increase in cell viability and proliferation in standard cell growth conditions, and to chemotactic migration towards plerixafor. Exploring potential molecular mechanisms underlying this effect, we found that Ewing sarcoma cell lines divided into classes of high- and low-level CXCR4 surface expression. Proliferative plerixafor responses were observed in both groups, maintained despite significant CXCR4 down-regulation or inhibition of Gαi-protein signal transduction, and involved activation of multiple receptor tyrosine kinases (DDR2, MERTK, MST1R, NTRK1, RET), the most prominent being platelet-derived growth factor receptor beta (PDGFRB). PDGFRB was activated in response to inhibition of the CXCL12-CXCR4 axis by plerixafor and/or pertussis toxin (Gαi-protein inhibitor). Dasatinib, a multi-kinase inhibitor of both PDGFRB and the CXCR4 downstream kinase SRC, counteracted this activation in some but not all cell lines.ConclusionThese data suggest a feedback interaction between the CXCR4 chemokine receptor and RTK signaling cascades that elicits compensatory cell survival signaling and can shift the net effect of plerixafor towards proliferation. PDGFRB was identified as a candidate driver RTK and potential therapeutic co-target for CXCR4 in Ewing sarcoma. Although as yet limited to in vitro studies, these findings call for further investigation in the cancer – microenvironment interplay in vivo.Electronic supplementary materialThe online version of this article (10.1186/s12964-018-0233-2) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

The CXCR4 antagonist plerixafor (AMD3100) promotes proliferation of Ewing sarcoma cell lines in vitro and activates receptor tyrosine kinase signaling

et al. 2018

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“…In EWS, however, although RNA expression has been reported, protein expression in metastases was absent in paraffin embedded material using immunohistochemistry [8, 9]. CXCR4 consists of multiple isoforms with varying N-terminal ends of which one, CXCR4–2, is dominantly expressed [34]. Both the N- and C-terminal ends of CXCR4 contain many potential post-translational modification sites and changes at these sites may influence antibody recognition, potentially explaining the various staining patterns observed in earlier studies [35–37].…”

Section: Discussionmentioning

confidence: 99%

Fluorescent CXCR4 targeting peptide as alternative for antibody staining in Ewing sarcoma

Sand

Buckle

Leeuwen³

et al. 2017

BMC Cancer

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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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3352-z) contains supplementary material, which is available to authorized users.

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“…32,33 Two additional splice variants are reported from Ewing sarcoma cells, although the functional significance of these is uncertain, with only one responding to ligand treatment. 34 Posttranslational modifications can also modulate CXCR4 activity, sulfation of Tyr, 7 Tyr, 12 and Tyr 20 has been shown to increase binding to a subset of ligands. 35 In particular, sulfation of Tyr 20 increases CXCL12 binding and dimerization.…”

Section: Cxcr4 Isoforms and Posttranslational Modificationmentioning

confidence: 99%

“…CXCR4‐B but not CXCR4‐A mediates efficient HIV‐1 X4 binding and infection, although the chemotactic activity affected through each isoform was the same 32,33 . Two additional splice variants are reported from Ewing sarcoma cells, although the functional significance of these is uncertain, with only one responding to ligand treatment 34 . Posttranslational modifications can also modulate CXCR4 activity, sulfation of Tyr, 7 Tyr, 12 and Tyr 20 has been shown to increase binding to a subset of ligands 35 .…”

Section: Cxcr4/cxcl12 Molecular Pathwaymentioning

confidence: 99%

Polyfunctionality of the CXCR4/CXCL12 axis in health and disease: Implications for therapeutic interventions in cancer and immune‐mediated diseases

Britton¹,

Poznansky²,

Reeves³

2021

The FASEB Journal

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Historically the chemokine receptor CXCR4 and its canonical ligand CXCL12 are associated with the bone marrow niche and hematopoiesis. However, CXCL12 exhibits broad tissue expression including brain, thymus, heart, lung, liver, kidney, spleen, and bone marrow. CXCR4 can be considered as a node which is integrating and transducing inputs from a range of ligand-receptor interactions into a responsive and divergent network of intracellular signaling pathways that impact multiple cellular processes such as proliferation, migration, and stress resistance. Dysregulation of the CXCR4/CXCL12 axis and consequent fundamental cellular processes, are associated with a panoply of disease. This review frames the polyfunctionality of the receptor at a molecular, physiological, and pathophysiological levels. Transitioning our perspective of this axis from a single gene/protein:single function model to a polyfunctional signaling cascade highlights the potential for finer therapeutic intervention and cautions against a reductionist approach.

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Novel splice variants of CXCR4 identified by transcriptome sequencing

Cited by 10 publications

References 28 publications

The CXCR4 antagonist plerixafor (AMD3100) promotes proliferation of Ewing sarcoma cell lines in vitro and activates receptor tyrosine kinase signaling

The CXCR4 antagonist plerixafor (AMD3100) promotes proliferation of Ewing sarcoma cell lines in vitro and activates receptor tyrosine kinase signaling

Fluorescent CXCR4 targeting peptide as alternative for antibody staining in Ewing sarcoma

Polyfunctionality of the CXCR4/CXCL12 axis in health and disease: Implications for therapeutic interventions in cancer and immune‐mediated diseases

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