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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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MEK inhibition recapitulates effects of MAF in MPNST cells(A) Western blot analysis of S462-TY cells treated with RAD001, PD0325901, or a combination showing downstream effects of restored MAF expression or drug treatment on DEPTOR and downstream signaling, and SOX9 expression. (B) RT-PCR analysis showing changes in neural crest marker SOX9 and differentiation factor mRNAs on exposure to MEK inhibition. (C) MTS assay of RAD001 or PD0325901 treated MPNST cells showing decreased proliferation with drug treatment, especially in response to the combination. (D) Immunohistochemical staining of vehicle or PD0325901 treated S462-TY xenograft tumors. Staining with anti-pERK and anti-MAF showing that PD0325901 resistant tumors show MAF expression. Reaction product is brown.
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Figure 8: MEK inhibition recapitulates effects of MAF in MPNST cells(A) Western blot analysis of S462-TY cells treated with RAD001, PD0325901, or a combination showing downstream effects of restored MAF expression or drug treatment on DEPTOR and downstream signaling, and SOX9 expression. (B) RT-PCR analysis showing changes in neural crest marker SOX9 and differentiation factor mRNAs on exposure to MEK inhibition. (C) MTS assay of RAD001 or PD0325901 treated MPNST cells showing decreased proliferation with drug treatment, especially in response to the combination. (D) Immunohistochemical staining of vehicle or PD0325901 treated S462-TY xenograft tumors. Staining with anti-pERK and anti-MAF showing that PD0325901 resistant tumors show MAF expression. Reaction product is brown.

Mentions: Since MAF regulated changes in DEPTOR affected TORC1 signaling, we reasoned that overexpressing MAF might enhance sensitivity to mTOR pathway inhibition. Normally, S462TY cells are poorly responsive to RAD001. However, the combination of treatment with 10 or 30 nM RAD001 in addition to MAF overexpression resulted in an enhanced response (Figure 7C). In xenograft models, the S462TY cells are also not significantly responsive to treatment with 10mg/kg RAD001 (Figure 7D), but a significant decrease in tumor volume compared to the vector control tumors was seen in combination with MAF overexpression (Figure 7E). IHC analysis confirmed a decrease in pS6 staining due to RAD001 inhibition of TORC1 activity in vector control and MAF expressing tumors (Figure 7F). Treatment with PD0325901 increased MAF expression (Figure 2A; 8A), decreased DEPTOR, increased pS6 and p4E-BP1 and decreased P-AKT (Figure 8A). The combination of PD0325901 with RAD001 abolished changes in pS6 and p4E-BP1 (Figure 8A). PD0325901 decreased mRNA and protein encoding the neural crest marker SOX9 (Figure 8A,B) and increased mRNA encoding Schwann cell differentiation markers BLBP/FABP7, S100β and MBP; RAD001 had no significant effects on cell differentiation. S462TY cells are resistant to RAD001 in MTS assays (Figure 8C), but responded to PD0325901 at 1uM (13). Combinations of these inhibitors had additive effects on cell viability (Figure 8C). S462-TY cells xenografted into immune-compromised mice delayed tumor growth when mice were exposed to 10mg/kg/day PD0325901 (13). We analyzed sections of tumor xenografts from mice treated with PD0325901 for 11 days. Tumors responding to drug (n=3) had little or no MAF expression, while faster growing tumors (n=2) showed MAF positivity (Figure 8D).


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)

MEK inhibition recapitulates effects of MAF in MPNST cells(A) Western blot analysis of S462-TY cells treated with RAD001, PD0325901, or a combination showing downstream effects of restored MAF expression or drug treatment on DEPTOR and downstream signaling, and SOX9 expression. (B) RT-PCR analysis showing changes in neural crest marker SOX9 and differentiation factor mRNAs on exposure to MEK inhibition. (C) MTS assay of RAD001 or PD0325901 treated MPNST cells showing decreased proliferation with drug treatment, especially in response to the combination. (D) Immunohistochemical staining of vehicle or PD0325901 treated S462-TY xenograft tumors. Staining with anti-pERK and anti-MAF showing that PD0325901 resistant tumors show MAF expression. Reaction product is brown.
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Related In: Results  -  Collection

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Figure 8: MEK inhibition recapitulates effects of MAF in MPNST cells(A) Western blot analysis of S462-TY cells treated with RAD001, PD0325901, or a combination showing downstream effects of restored MAF expression or drug treatment on DEPTOR and downstream signaling, and SOX9 expression. (B) RT-PCR analysis showing changes in neural crest marker SOX9 and differentiation factor mRNAs on exposure to MEK inhibition. (C) MTS assay of RAD001 or PD0325901 treated MPNST cells showing decreased proliferation with drug treatment, especially in response to the combination. (D) Immunohistochemical staining of vehicle or PD0325901 treated S462-TY xenograft tumors. Staining with anti-pERK and anti-MAF showing that PD0325901 resistant tumors show MAF expression. Reaction product is brown.
Mentions: Since MAF regulated changes in DEPTOR affected TORC1 signaling, we reasoned that overexpressing MAF might enhance sensitivity to mTOR pathway inhibition. Normally, S462TY cells are poorly responsive to RAD001. However, the combination of treatment with 10 or 30 nM RAD001 in addition to MAF overexpression resulted in an enhanced response (Figure 7C). In xenograft models, the S462TY cells are also not significantly responsive to treatment with 10mg/kg RAD001 (Figure 7D), but a significant decrease in tumor volume compared to the vector control tumors was seen in combination with MAF overexpression (Figure 7E). IHC analysis confirmed a decrease in pS6 staining due to RAD001 inhibition of TORC1 activity in vector control and MAF expressing tumors (Figure 7F). Treatment with PD0325901 increased MAF expression (Figure 2A; 8A), decreased DEPTOR, increased pS6 and p4E-BP1 and decreased P-AKT (Figure 8A). The combination of PD0325901 with RAD001 abolished changes in pS6 and p4E-BP1 (Figure 8A). PD0325901 decreased mRNA and protein encoding the neural crest marker SOX9 (Figure 8A,B) and increased mRNA encoding Schwann cell differentiation markers BLBP/FABP7, S100β and MBP; RAD001 had no significant effects on cell differentiation. S462TY cells are resistant to RAD001 in MTS assays (Figure 8C), but responded to PD0325901 at 1uM (13). Combinations of these inhibitors had additive effects on cell viability (Figure 8C). S462-TY cells xenografted into immune-compromised mice delayed tumor growth when mice were exposed to 10mg/kg/day PD0325901 (13). We analyzed sections of tumor xenografts from mice treated with PD0325901 for 11 days. Tumors responding to drug (n=3) had little or no MAF expression, while faster growing tumors (n=2) showed MAF positivity (Figure 8D).

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