Oncogene MYCN regulates localization of NKT cells to the site of disease in neuroblastoma
Liping Song, Tasnim Ara, Hong-Wei Wu, Chan-Wook Woo, C. Patrick Reynolds, Robert C. Seeger, Yves A. DeClerck, Carol J. Thiele, Richard Sposto, Leonid S. Metelitsa
Liping Song, Tasnim Ara, Hong-Wei Wu, Chan-Wook Woo, C. Patrick Reynolds, Robert C. Seeger, Yves A. DeClerck, Carol J. Thiele, Richard Sposto, Leonid S. Metelitsa
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Research Article Oncology

Oncogene MYCN regulates localization of NKT cells to the site of disease in neuroblastoma

Abstract

Vα24-invariant natural killer T (NKT) cells are potentially important for antitumor immunity. We and others have previously demonstrated positive associations between NKT cell presence in primary tumors and long-term survival in distinct human cancers. However, the mechanism by which aggressive tumors avoid infiltration with NKT and other T cells remains poorly understood. Here, we report that the v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN), the hallmark of aggressive neuroblastoma, repressed expression of monocyte chemoattractant protein–1/CC chemokine ligand 2 (MCP-1/CCL2), a chemokine required for NKT cell chemoattraction. MYCN knockdown in MYCN-amplified neuroblastoma cell lines restored CCL2 production and NKT cell chemoattraction. Unlike other oncogenes, MYCN repressed chemokine expression in a STAT3-independent manner, requiring an E-box element in the CCL2 promoter to mediate transcriptional repression. MYCN overexpression in neuroblastoma xenografts in NOD/SCID mice severely inhibited their ability to attract human NKT cells, T cells, and monocytes. Patients with MYCN-amplified neuroblastoma metastatic to bone marrow had 4-fold fewer NKT cells in their bone marrow than did their nonamplified counterparts, indicating that the MYCN-mediated immune escape mechanism, which we believe to be novel, is operative in metastatic cancer and should be considered in tumor immunobiology and for the development of new therapeutic strategies.

Authors

Liping Song, Tasnim Ara, Hong-Wei Wu, Chan-Wook Woo, C. Patrick Reynolds, Robert C. Seeger, Yves A. DeClerck, Carol J. Thiele, Richard Sposto, Leonid S. Metelitsa

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Figure 1

MYCN represses CCL2 expression and inhibits the chemoattraction of NKT and T cells.

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MYCN represses CCL2 expression and inhibits the chemoattraction of NKT a...
CHLA-255 neuroblastoma cells were stably transduced with MYCN cDNA or empty vector control. (A) Representative Western blot analysis of MYCN protein in the nuclear extracts from indicated cells. (B) CCL2 RNA expression was quantified by TaqMan RT-PCR; values are relative to expression in parental cells. Results are mean ± SD from 3 experiments. (C) CBA analysis. Five indicated chemokines were measured in supernatants collected after 24 hours’ culture of 80% confluent cells. Chemokine concentration is proportional to the fluorescence intensity on the x axis and was calculated by standard curve analysis. Concentrations above 20 pg/ml are shown. (D) The same supernatants as in C were placed in lower chambers of dual-chamber plates with 5-μm pore membrane. Freshly isolated human monocyte-depleted PBLs were added in upper chambers and allowed to migrate for 4 hours. Absolute numbers of NKT and T cells were determined by flow cytometry using CD3/6B11 staining and TruCount tubes with a known number of beads (see Methods). Percent migration reflects the proportion of migrated cells relative to input cells. Specific migration was calculated by subtracting the number of cells that migrated to control medium. Results are mean ± SD from 4 experiments.