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MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1.

Brundage ME, Tandon P, Eaves DW, Williams JP, Miller SJ, Hennigan RH, Jegga A, Cripe TP, Ratner N - Oncogene (2014)

Bottom Line: Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene are common in cancer and can cause resistance to therapy.RAD001 blocked MAF-mediated tumor growth, and MAF regulated the mTOR pathway through DEPTOR.MAPK inhibition with NF1 loss of function is predicted to show limited efficacy due to reactivation of mTOR signaling via MAF.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology and Cancer Biology, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.

ABSTRACT
Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene are common in cancer and can cause resistance to therapy. Using transcriptome analysis we identified MAF as an NF1- regulated transcription factor and verified MAF regulation through RAS/MAPK/AP-1 signaling in malignant peripheral nerve sheath tumor (MPNST) cell lines. MAF was also downregulated in human MPNST. Acute re-expression of MAF promoted expression of glial differentiation markers in MPNST cells in vitro, decreased self-renewal of embryonic precursors and transiently affected tumor cell phenotypes in vitro by increasing MPNST cell death and reducing metabolic activity and anchorage-independent growth. Paradoxically, chronic MAF overexpression enhanced MPNST cell tumor growth in vivo, correlating with elevated pS6 in vitro and in vivo. RAD001 blocked MAF-mediated tumor growth, and MAF regulated the mTOR pathway through DEPTOR. MAPK inhibition with NF1 loss of function is predicted to show limited efficacy due to reactivation of mTOR signaling via MAF.

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The NF1-GRD normalizes MAF expression in MPNST cells(A) Heatmap of gene expression microarray data comparing normal human Schwann cells (NHSC), ST8814 NF1 patient-derived malignant peripheral nerve sheath tumor (MPNST) cells (UN), ST8814 cells infected with an adenoviral vector expressing green fluorescent protein (Vector) or the NF1-GRD (GRD). Samples were analyzed in triplicate, and expression is shown relative to the mean gene expression in 6 NHSC samples. Yellow = normal; red = increased expression; blue = reduced expression. A list of 45 genes brought closer to levels in normal human Schwann cells (NHSC) by the NF1-GRD is displayed to the right of the heatmap. The top cluster includes 36 genes with expression downregulated at least 2.3-fold in MPNST relative to NHSC and increased at least 3-fold in MPNST cells expressing NF1-GRD relative to MPNST cells expressing GFP control, shown as positive GRD fold-change. The bottom cluster includes 9 genes with expression upregulated at least 2.3-fold in MPNST relative to NHSC and decreased at least 3-fold in MPNST cells expressing NF1-GRD relative to MPNST cells expressing GFP control, shown as negative GRD fold-change. * GRD regulation data confirmed by RT-PCR; ** GRD regulation not confirmed by RT-PCR. (B) Quantification of microarray expression data relative to NHSC, showing a trend toward normalization of MAF expression in MPNST cells expressing the NF1-GRD. Expression levels in MPNST cells infected with GFP control are not statistically different than uninfected MPNST cells; expression level differences in MPNST cells infected with NF1-GRD versus GFP control are statistically different (***; p = 0.001). (C) Time course RT-PCR analysis in MPNST cells at 18, 22, and 24 hours post-infection with NF1-GRD relative to NHSC, showing rapid induction of MAF expression by NF1-GRD. (D) Heatmap of gene expression microarray data comparing normal human Schwann cells (NHSCs), dermal (dNFSC) and plexiform (pNFSC) neurofibroma derived Schwann cells, and patient-derived malignant peripheral nerve sheath tumor cell lines (MPNST). Red = overexpression; blue = under-expression. (E) Heatmap of gene expression microarray data comparing normal human nerve, dermal (dNF) and plexiform (pNF) neurofibromas, and malignant peripheral nerve sheath tumors (MPNST). Red = overexpression; blue = under-expression.
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Figure 1: The NF1-GRD normalizes MAF expression in MPNST cells(A) Heatmap of gene expression microarray data comparing normal human Schwann cells (NHSC), ST8814 NF1 patient-derived malignant peripheral nerve sheath tumor (MPNST) cells (UN), ST8814 cells infected with an adenoviral vector expressing green fluorescent protein (Vector) or the NF1-GRD (GRD). Samples were analyzed in triplicate, and expression is shown relative to the mean gene expression in 6 NHSC samples. Yellow = normal; red = increased expression; blue = reduced expression. A list of 45 genes brought closer to levels in normal human Schwann cells (NHSC) by the NF1-GRD is displayed to the right of the heatmap. The top cluster includes 36 genes with expression downregulated at least 2.3-fold in MPNST relative to NHSC and increased at least 3-fold in MPNST cells expressing NF1-GRD relative to MPNST cells expressing GFP control, shown as positive GRD fold-change. The bottom cluster includes 9 genes with expression upregulated at least 2.3-fold in MPNST relative to NHSC and decreased at least 3-fold in MPNST cells expressing NF1-GRD relative to MPNST cells expressing GFP control, shown as negative GRD fold-change. * GRD regulation data confirmed by RT-PCR; ** GRD regulation not confirmed by RT-PCR. (B) Quantification of microarray expression data relative to NHSC, showing a trend toward normalization of MAF expression in MPNST cells expressing the NF1-GRD. Expression levels in MPNST cells infected with GFP control are not statistically different than uninfected MPNST cells; expression level differences in MPNST cells infected with NF1-GRD versus GFP control are statistically different (***; p = 0.001). (C) Time course RT-PCR analysis in MPNST cells at 18, 22, and 24 hours post-infection with NF1-GRD relative to NHSC, showing rapid induction of MAF expression by NF1-GRD. (D) Heatmap of gene expression microarray data comparing normal human Schwann cells (NHSCs), dermal (dNFSC) and plexiform (pNFSC) neurofibroma derived Schwann cells, and patient-derived malignant peripheral nerve sheath tumor cell lines (MPNST). Red = overexpression; blue = under-expression. (E) Heatmap of gene expression microarray data comparing normal human nerve, dermal (dNF) and plexiform (pNF) neurofibromas, and malignant peripheral nerve sheath tumors (MPNST). Red = overexpression; blue = under-expression.

Mentions: To identify genes with expression dysregulated in MPNST compared to human Schwann cells (NHSC) and normalized by the NF1-GRD, we first normalized gene expression in MPNST cells infected with NF1-GRD or control to gene expression in NHSC. Second, we identified 152 probe sets with differential expression ≥ 2.3-fold in MPNST cells expressing the NF1-GRD and not differentially expressed in GFP-expressing cells (Figure 1A). Third, we identified 128 of 152 probe sets with gene expression brought closer to levels in NHSC by NF1-GRD. Finally, we identified 45 known genes with expression restored to ≥ 3-fold levels of NHSC in MPNST cells infected with NF1-GRD relative to control. Microarray differential gene expression induced by GRD was confirmed by RT-PCR analysis for DACH1 (24), EGFL5, MAF, PCDH20, and FOXD1; SOX9 and SPRY4 were tested but failed to confirm, perhaps due to timing of transcriptional regulation.


MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1.

Brundage ME, Tandon P, Eaves DW, Williams JP, Miller SJ, Hennigan RH, Jegga A, Cripe TP, Ratner N - Oncogene (2014)

The NF1-GRD normalizes MAF expression in MPNST cells(A) Heatmap of gene expression microarray data comparing normal human Schwann cells (NHSC), ST8814 NF1 patient-derived malignant peripheral nerve sheath tumor (MPNST) cells (UN), ST8814 cells infected with an adenoviral vector expressing green fluorescent protein (Vector) or the NF1-GRD (GRD). Samples were analyzed in triplicate, and expression is shown relative to the mean gene expression in 6 NHSC samples. Yellow = normal; red = increased expression; blue = reduced expression. A list of 45 genes brought closer to levels in normal human Schwann cells (NHSC) by the NF1-GRD is displayed to the right of the heatmap. The top cluster includes 36 genes with expression downregulated at least 2.3-fold in MPNST relative to NHSC and increased at least 3-fold in MPNST cells expressing NF1-GRD relative to MPNST cells expressing GFP control, shown as positive GRD fold-change. The bottom cluster includes 9 genes with expression upregulated at least 2.3-fold in MPNST relative to NHSC and decreased at least 3-fold in MPNST cells expressing NF1-GRD relative to MPNST cells expressing GFP control, shown as negative GRD fold-change. * GRD regulation data confirmed by RT-PCR; ** GRD regulation not confirmed by RT-PCR. (B) Quantification of microarray expression data relative to NHSC, showing a trend toward normalization of MAF expression in MPNST cells expressing the NF1-GRD. Expression levels in MPNST cells infected with GFP control are not statistically different than uninfected MPNST cells; expression level differences in MPNST cells infected with NF1-GRD versus GFP control are statistically different (***; p = 0.001). (C) Time course RT-PCR analysis in MPNST cells at 18, 22, and 24 hours post-infection with NF1-GRD relative to NHSC, showing rapid induction of MAF expression by NF1-GRD. (D) Heatmap of gene expression microarray data comparing normal human Schwann cells (NHSCs), dermal (dNFSC) and plexiform (pNFSC) neurofibroma derived Schwann cells, and patient-derived malignant peripheral nerve sheath tumor cell lines (MPNST). Red = overexpression; blue = under-expression. (E) Heatmap of gene expression microarray data comparing normal human nerve, dermal (dNF) and plexiform (pNF) neurofibromas, and malignant peripheral nerve sheath tumors (MPNST). Red = overexpression; blue = under-expression.
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Related In: Results  -  Collection

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Figure 1: The NF1-GRD normalizes MAF expression in MPNST cells(A) Heatmap of gene expression microarray data comparing normal human Schwann cells (NHSC), ST8814 NF1 patient-derived malignant peripheral nerve sheath tumor (MPNST) cells (UN), ST8814 cells infected with an adenoviral vector expressing green fluorescent protein (Vector) or the NF1-GRD (GRD). Samples were analyzed in triplicate, and expression is shown relative to the mean gene expression in 6 NHSC samples. Yellow = normal; red = increased expression; blue = reduced expression. A list of 45 genes brought closer to levels in normal human Schwann cells (NHSC) by the NF1-GRD is displayed to the right of the heatmap. The top cluster includes 36 genes with expression downregulated at least 2.3-fold in MPNST relative to NHSC and increased at least 3-fold in MPNST cells expressing NF1-GRD relative to MPNST cells expressing GFP control, shown as positive GRD fold-change. The bottom cluster includes 9 genes with expression upregulated at least 2.3-fold in MPNST relative to NHSC and decreased at least 3-fold in MPNST cells expressing NF1-GRD relative to MPNST cells expressing GFP control, shown as negative GRD fold-change. * GRD regulation data confirmed by RT-PCR; ** GRD regulation not confirmed by RT-PCR. (B) Quantification of microarray expression data relative to NHSC, showing a trend toward normalization of MAF expression in MPNST cells expressing the NF1-GRD. Expression levels in MPNST cells infected with GFP control are not statistically different than uninfected MPNST cells; expression level differences in MPNST cells infected with NF1-GRD versus GFP control are statistically different (***; p = 0.001). (C) Time course RT-PCR analysis in MPNST cells at 18, 22, and 24 hours post-infection with NF1-GRD relative to NHSC, showing rapid induction of MAF expression by NF1-GRD. (D) Heatmap of gene expression microarray data comparing normal human Schwann cells (NHSCs), dermal (dNFSC) and plexiform (pNFSC) neurofibroma derived Schwann cells, and patient-derived malignant peripheral nerve sheath tumor cell lines (MPNST). Red = overexpression; blue = under-expression. (E) Heatmap of gene expression microarray data comparing normal human nerve, dermal (dNF) and plexiform (pNF) neurofibromas, and malignant peripheral nerve sheath tumors (MPNST). Red = overexpression; blue = under-expression.
Mentions: To identify genes with expression dysregulated in MPNST compared to human Schwann cells (NHSC) and normalized by the NF1-GRD, we first normalized gene expression in MPNST cells infected with NF1-GRD or control to gene expression in NHSC. Second, we identified 152 probe sets with differential expression ≥ 2.3-fold in MPNST cells expressing the NF1-GRD and not differentially expressed in GFP-expressing cells (Figure 1A). Third, we identified 128 of 152 probe sets with gene expression brought closer to levels in NHSC by NF1-GRD. Finally, we identified 45 known genes with expression restored to ≥ 3-fold levels of NHSC in MPNST cells infected with NF1-GRD relative to control. Microarray differential gene expression induced by GRD was confirmed by RT-PCR analysis for DACH1 (24), EGFL5, MAF, PCDH20, and FOXD1; SOX9 and SPRY4 were tested but failed to confirm, perhaps due to timing of transcriptional regulation.

Bottom Line: Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene are common in cancer and can cause resistance to therapy.RAD001 blocked MAF-mediated tumor growth, and MAF regulated the mTOR pathway through DEPTOR.MAPK inhibition with NF1 loss of function is predicted to show limited efficacy due to reactivation of mTOR signaling via MAF.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology and Cancer Biology, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.

ABSTRACT
Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene are common in cancer and can cause resistance to therapy. Using transcriptome analysis we identified MAF as an NF1- regulated transcription factor and verified MAF regulation through RAS/MAPK/AP-1 signaling in malignant peripheral nerve sheath tumor (MPNST) cell lines. MAF was also downregulated in human MPNST. Acute re-expression of MAF promoted expression of glial differentiation markers in MPNST cells in vitro, decreased self-renewal of embryonic precursors and transiently affected tumor cell phenotypes in vitro by increasing MPNST cell death and reducing metabolic activity and anchorage-independent growth. Paradoxically, chronic MAF overexpression enhanced MPNST cell tumor growth in vivo, correlating with elevated pS6 in vitro and in vivo. RAD001 blocked MAF-mediated tumor growth, and MAF regulated the mTOR pathway through DEPTOR. MAPK inhibition with NF1 loss of function is predicted to show limited efficacy due to reactivation of mTOR signaling via MAF.

Show MeSH
Related in: MedlinePlus