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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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Inducing MAF expression modulates DEPTOR(A) Relative expression of DEPTOR from gene expression array analysis comparing MPNST and NHSC. (B) RT-PCR analysis of DEPTOR expression in S462-TY cells after overexpression of MAF. (C) Western blot analysis (reprobes of blots from 5F) of pLVX empty vector (control) or iMAF transduced S462-TY cells comparing uninduced, low (0.2 ug/ml) and high (hi; 2 ug/ml) doxycycline induced cells for 2 or 6 days. Downstream of MAF expression, phosphorylation of the mTOR substrates S6 and 4EBP1 are elevated while P-AKT is reduced. (D) Western blot analysis of S462-TY cells showing induction of MAF with and without DEPTOR overexpression. Overexpression of MAF attenuated DEPTOR levels and DEPTOR reduced pS6. (E) MTS assay showing that the decreased cell numbers caused by MAF are rescued by co-expression of DEPTOR.
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Figure 6: Inducing MAF expression modulates DEPTOR(A) Relative expression of DEPTOR from gene expression array analysis comparing MPNST and NHSC. (B) RT-PCR analysis of DEPTOR expression in S462-TY cells after overexpression of MAF. (C) Western blot analysis (reprobes of blots from 5F) of pLVX empty vector (control) or iMAF transduced S462-TY cells comparing uninduced, low (0.2 ug/ml) and high (hi; 2 ug/ml) doxycycline induced cells for 2 or 6 days. Downstream of MAF expression, phosphorylation of the mTOR substrates S6 and 4EBP1 are elevated while P-AKT is reduced. (D) Western blot analysis of S462-TY cells showing induction of MAF with and without DEPTOR overexpression. Overexpression of MAF attenuated DEPTOR levels and DEPTOR reduced pS6. (E) MTS assay showing that the decreased cell numbers caused by MAF are rescued by co-expression of DEPTOR.

Mentions: As MAF can act as an oncogene in some cell types, we studied what survival mechanism(s) the MAF expressing population might exploit after initial growth suppression. We examined gene expression changes in NF1 tumors and cell lines compared to human Schwann cells from our Affymetrix array analysis (24). DEPTOR mRNA was 15-fold up-regulated in MPNST tumors and cell lines versus normal human Schwann cells (Figure 6A). DEPTOR is a MAF target and negative regulator of the AKT/mTOR pathway (31). Restoring MAF to S462-TY cells reduced DEPTOR mRNA expression (Figure 6B). Western blot analysis of S462-TY cells transduced with inducible MAF showed increased phosphorylation of S6 and 4E-BP1 and decreased phosphorylation of AKT (Figure 6C), readouts of TORC1 activity and its negative feedback onto AKT (39). Overexpression of MAF did not change levels of pERK, consistent with MAF acting downstream of MAPK signaling. Importantly, overexpression of MAF, plus simultaneous DEPTOR overexpression, attenuated DEPTOR levels and pS6 (Figure 6D), and DEPTOR blocked MAF-induced decreased cell survival in MTS assay (Figure 6E). These data suggest that MAF regulates AKT/mTOR signaling through DEPTOR.


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)

Inducing MAF expression modulates DEPTOR(A) Relative expression of DEPTOR from gene expression array analysis comparing MPNST and NHSC. (B) RT-PCR analysis of DEPTOR expression in S462-TY cells after overexpression of MAF. (C) Western blot analysis (reprobes of blots from 5F) of pLVX empty vector (control) or iMAF transduced S462-TY cells comparing uninduced, low (0.2 ug/ml) and high (hi; 2 ug/ml) doxycycline induced cells for 2 or 6 days. Downstream of MAF expression, phosphorylation of the mTOR substrates S6 and 4EBP1 are elevated while P-AKT is reduced. (D) Western blot analysis of S462-TY cells showing induction of MAF with and without DEPTOR overexpression. Overexpression of MAF attenuated DEPTOR levels and DEPTOR reduced pS6. (E) MTS assay showing that the decreased cell numbers caused by MAF are rescued by co-expression of DEPTOR.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4127377&req=5

Figure 6: Inducing MAF expression modulates DEPTOR(A) Relative expression of DEPTOR from gene expression array analysis comparing MPNST and NHSC. (B) RT-PCR analysis of DEPTOR expression in S462-TY cells after overexpression of MAF. (C) Western blot analysis (reprobes of blots from 5F) of pLVX empty vector (control) or iMAF transduced S462-TY cells comparing uninduced, low (0.2 ug/ml) and high (hi; 2 ug/ml) doxycycline induced cells for 2 or 6 days. Downstream of MAF expression, phosphorylation of the mTOR substrates S6 and 4EBP1 are elevated while P-AKT is reduced. (D) Western blot analysis of S462-TY cells showing induction of MAF with and without DEPTOR overexpression. Overexpression of MAF attenuated DEPTOR levels and DEPTOR reduced pS6. (E) MTS assay showing that the decreased cell numbers caused by MAF are rescued by co-expression of DEPTOR.
Mentions: As MAF can act as an oncogene in some cell types, we studied what survival mechanism(s) the MAF expressing population might exploit after initial growth suppression. We examined gene expression changes in NF1 tumors and cell lines compared to human Schwann cells from our Affymetrix array analysis (24). DEPTOR mRNA was 15-fold up-regulated in MPNST tumors and cell lines versus normal human Schwann cells (Figure 6A). DEPTOR is a MAF target and negative regulator of the AKT/mTOR pathway (31). Restoring MAF to S462-TY cells reduced DEPTOR mRNA expression (Figure 6B). Western blot analysis of S462-TY cells transduced with inducible MAF showed increased phosphorylation of S6 and 4E-BP1 and decreased phosphorylation of AKT (Figure 6C), readouts of TORC1 activity and its negative feedback onto AKT (39). Overexpression of MAF did not change levels of pERK, consistent with MAF acting downstream of MAPK signaling. Importantly, overexpression of MAF, plus simultaneous DEPTOR overexpression, attenuated DEPTOR levels and pS6 (Figure 6D), and DEPTOR blocked MAF-induced decreased cell survival in MTS assay (Figure 6E). These data suggest that MAF regulates AKT/mTOR signaling through DEPTOR.

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