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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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MAF is regulated by NF1 through a RAS/MAPK/AP-1 pathway(A) RT-PCR analysis showing mRNA expression of S462TY MPNST cells treated with the MEK inhibitor PD0325901 or vehicle control for 24 hours, relative to β-actin, with corresponding protein levels below. (B) Luciferase reporter assays for AP-1 response after transfecting full length human NF1 (NF1), dominant negative H-Ras (RasN17), the NF1 gap-related domain (GRD), an N-terminal NF1 fragment (NF1 N-term), a C-terminal NF1 fragment (NF1 c-term), or active RasG12V (RasV12) into MPNST cells. (C) Luciferase reporter assay for MAF response to dominant negative aFOS transfected into MPNST cell lines. (D) Model showing that NF1 loss through Ras activation promotes ERK activation and AP-1, thereby suppressing MAF expression.
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Figure 2: MAF is regulated by NF1 through a RAS/MAPK/AP-1 pathway(A) RT-PCR analysis showing mRNA expression of S462TY MPNST cells treated with the MEK inhibitor PD0325901 or vehicle control for 24 hours, relative to β-actin, with corresponding protein levels below. (B) Luciferase reporter assays for AP-1 response after transfecting full length human NF1 (NF1), dominant negative H-Ras (RasN17), the NF1 gap-related domain (GRD), an N-terminal NF1 fragment (NF1 N-term), a C-terminal NF1 fragment (NF1 c-term), or active RasG12V (RasV12) into MPNST cells. (C) Luciferase reporter assay for MAF response to dominant negative aFOS transfected into MPNST cell lines. (D) Model showing that NF1 loss through Ras activation promotes ERK activation and AP-1, thereby suppressing MAF expression.

Mentions: Increased MAPK signaling in MPNST cells is due to loss of NF1 RAS-Gap function (10, 11). Confirming that MAF is downstream of RAS-RAF-MAPK, MEK inhibitors restored MAF mRNA in MPNST cell lines and protein expression in S462TY cells (Figure 2A). AP-1 transcription factors are common effectors of the MAPK pathway (34) and MPNST cells have increased AP-1 activity (35). We tested whether NF1 regulates MAF via AP-1 activity. Transfecting MPNSTs with the GRD, dominant negative RasN17, or full length NF1 constructs decreased AP-1 luciferase reporter activity compared to the empty vector negative control, while constitutively active RasV12 activated AP-1 (Figure 2B). Mutant NF1 constructs lacking the GRD did not repress AP-1 activity, consistent with AP-1 regulation by RAS signaling (Figure 2B). Importantly, a dominant negative aFos (36) transfected into MPNST cell lines along with a MAF promoter luciferase reporter increased MAF activity, indicating that AP-1 represses MAF transcription in MPNST cells (Figure 2C) (37). Thus MAF is a novel NF1 target in MPNST, repressed by the RAS/MAPK/AP-1 pathway (Figure 2D).


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)

MAF is regulated by NF1 through a RAS/MAPK/AP-1 pathway(A) RT-PCR analysis showing mRNA expression of S462TY MPNST cells treated with the MEK inhibitor PD0325901 or vehicle control for 24 hours, relative to β-actin, with corresponding protein levels below. (B) Luciferase reporter assays for AP-1 response after transfecting full length human NF1 (NF1), dominant negative H-Ras (RasN17), the NF1 gap-related domain (GRD), an N-terminal NF1 fragment (NF1 N-term), a C-terminal NF1 fragment (NF1 c-term), or active RasG12V (RasV12) into MPNST cells. (C) Luciferase reporter assay for MAF response to dominant negative aFOS transfected into MPNST cell lines. (D) Model showing that NF1 loss through Ras activation promotes ERK activation and AP-1, thereby suppressing MAF expression.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4127377&req=5

Figure 2: MAF is regulated by NF1 through a RAS/MAPK/AP-1 pathway(A) RT-PCR analysis showing mRNA expression of S462TY MPNST cells treated with the MEK inhibitor PD0325901 or vehicle control for 24 hours, relative to β-actin, with corresponding protein levels below. (B) Luciferase reporter assays for AP-1 response after transfecting full length human NF1 (NF1), dominant negative H-Ras (RasN17), the NF1 gap-related domain (GRD), an N-terminal NF1 fragment (NF1 N-term), a C-terminal NF1 fragment (NF1 c-term), or active RasG12V (RasV12) into MPNST cells. (C) Luciferase reporter assay for MAF response to dominant negative aFOS transfected into MPNST cell lines. (D) Model showing that NF1 loss through Ras activation promotes ERK activation and AP-1, thereby suppressing MAF expression.
Mentions: Increased MAPK signaling in MPNST cells is due to loss of NF1 RAS-Gap function (10, 11). Confirming that MAF is downstream of RAS-RAF-MAPK, MEK inhibitors restored MAF mRNA in MPNST cell lines and protein expression in S462TY cells (Figure 2A). AP-1 transcription factors are common effectors of the MAPK pathway (34) and MPNST cells have increased AP-1 activity (35). We tested whether NF1 regulates MAF via AP-1 activity. Transfecting MPNSTs with the GRD, dominant negative RasN17, or full length NF1 constructs decreased AP-1 luciferase reporter activity compared to the empty vector negative control, while constitutively active RasV12 activated AP-1 (Figure 2B). Mutant NF1 constructs lacking the GRD did not repress AP-1 activity, consistent with AP-1 regulation by RAS signaling (Figure 2B). Importantly, a dominant negative aFos (36) transfected into MPNST cell lines along with a MAF promoter luciferase reporter increased MAF activity, indicating that AP-1 represses MAF transcription in MPNST cells (Figure 2C) (37). Thus MAF is a novel NF1 target in MPNST, repressed by the RAS/MAPK/AP-1 pathway (Figure 2D).

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