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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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Acute over-expression of MAF in MPNST cells transiently increases cell death and reduces anchorage independent growth, while sustained lower MAF expressing cells proliferate and maintain increased TORC1 signaling through DEPTOR(A) Short term MTS assay on S462-TY cells transduced with mCherry tagged MAF for 48 hours. (B) Flow cytometry on PI and Annexin stained S462-TY cells transduced with inducible MAF 48 hours after doxycycline induction. (C) Anchorage independent growth of S462-TY cells assessed by colony formation in soft agar after overexpression of MAF using mCherry tagged MAF (mCh-MAF) or inducible MAF (iMAF). (D) Longer time course MTS assay on S462-TY cells transduced with inducible MAF showing recovery of cell growth by 6 days. (E) Flow analysis of BrdU incorporation in doxycycline induced pLVX (empty vector control) and iMAF transduced MPNST cells. (F) Western blot analysis of pLVX (empty vector control) or iMAF transduced S462-TY cells comparing uninduced, low (0.2 ug/ml) and high (2 ug/ml) doxycycline induced cells for 2 or 6 days, showing cell death (cleaved caspase 3) is transient and correlates with high levels of MAF expression.
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Figure 5: Acute over-expression of MAF in MPNST cells transiently increases cell death and reduces anchorage independent growth, while sustained lower MAF expressing cells proliferate and maintain increased TORC1 signaling through DEPTOR(A) Short term MTS assay on S462-TY cells transduced with mCherry tagged MAF for 48 hours. (B) Flow cytometry on PI and Annexin stained S462-TY cells transduced with inducible MAF 48 hours after doxycycline induction. (C) Anchorage independent growth of S462-TY cells assessed by colony formation in soft agar after overexpression of MAF using mCherry tagged MAF (mCh-MAF) or inducible MAF (iMAF). (D) Longer time course MTS assay on S462-TY cells transduced with inducible MAF showing recovery of cell growth by 6 days. (E) Flow analysis of BrdU incorporation in doxycycline induced pLVX (empty vector control) and iMAF transduced MPNST cells. (F) Western blot analysis of pLVX (empty vector control) or iMAF transduced S462-TY cells comparing uninduced, low (0.2 ug/ml) and high (2 ug/ml) doxycycline induced cells for 2 or 6 days, showing cell death (cleaved caspase 3) is transient and correlates with high levels of MAF expression.

Mentions: If MAF promotes cell differentiation, then restoring MAF expression in MPNST cells might affect tumorigenic potential. S462-TY cells transduced with MAF for 48 hours significantly decreased metabolic activity as measured by MTS assay (Figure 5A). Flow cytometric analysis of PI and Annexin stained S462-TY cells transduced with MAF for 48 hours revealed increased apoptotic cell death (Figure 5B). Importantly, these cytostatic and cytotoxic effects were accompanied by decreased anchorage independent growth in soft agar in S462-TY cells overexpressing MAF constitutively or inducibly (Figure 5C). However, after these transient effects, MAF expressing cells were able to proliferate in culture, as confirmed by increased metabolic activity in MTS assays (Figure 5D) and BrdU incorporation (Figure 5E). Relative levels of MAF protein expression were higher 2 days after induction than at day 6, correlating with increased apoptosis evidenced by cleaved caspase 3 (Figure 5F). Low MAF levels by day 6 corresponded with increased proliferation. Thus, effects of MAF expression on MPNST cells are dose dependent.


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)

Acute over-expression of MAF in MPNST cells transiently increases cell death and reduces anchorage independent growth, while sustained lower MAF expressing cells proliferate and maintain increased TORC1 signaling through DEPTOR(A) Short term MTS assay on S462-TY cells transduced with mCherry tagged MAF for 48 hours. (B) Flow cytometry on PI and Annexin stained S462-TY cells transduced with inducible MAF 48 hours after doxycycline induction. (C) Anchorage independent growth of S462-TY cells assessed by colony formation in soft agar after overexpression of MAF using mCherry tagged MAF (mCh-MAF) or inducible MAF (iMAF). (D) Longer time course MTS assay on S462-TY cells transduced with inducible MAF showing recovery of cell growth by 6 days. (E) Flow analysis of BrdU incorporation in doxycycline induced pLVX (empty vector control) and iMAF transduced MPNST cells. (F) Western blot analysis of pLVX (empty vector control) or iMAF transduced S462-TY cells comparing uninduced, low (0.2 ug/ml) and high (2 ug/ml) doxycycline induced cells for 2 or 6 days, showing cell death (cleaved caspase 3) is transient and correlates with high levels of MAF expression.
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Related In: Results  -  Collection

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Figure 5: Acute over-expression of MAF in MPNST cells transiently increases cell death and reduces anchorage independent growth, while sustained lower MAF expressing cells proliferate and maintain increased TORC1 signaling through DEPTOR(A) Short term MTS assay on S462-TY cells transduced with mCherry tagged MAF for 48 hours. (B) Flow cytometry on PI and Annexin stained S462-TY cells transduced with inducible MAF 48 hours after doxycycline induction. (C) Anchorage independent growth of S462-TY cells assessed by colony formation in soft agar after overexpression of MAF using mCherry tagged MAF (mCh-MAF) or inducible MAF (iMAF). (D) Longer time course MTS assay on S462-TY cells transduced with inducible MAF showing recovery of cell growth by 6 days. (E) Flow analysis of BrdU incorporation in doxycycline induced pLVX (empty vector control) and iMAF transduced MPNST cells. (F) Western blot analysis of pLVX (empty vector control) or iMAF transduced S462-TY cells comparing uninduced, low (0.2 ug/ml) and high (2 ug/ml) doxycycline induced cells for 2 or 6 days, showing cell death (cleaved caspase 3) is transient and correlates with high levels of MAF expression.
Mentions: If MAF promotes cell differentiation, then restoring MAF expression in MPNST cells might affect tumorigenic potential. S462-TY cells transduced with MAF for 48 hours significantly decreased metabolic activity as measured by MTS assay (Figure 5A). Flow cytometric analysis of PI and Annexin stained S462-TY cells transduced with MAF for 48 hours revealed increased apoptotic cell death (Figure 5B). Importantly, these cytostatic and cytotoxic effects were accompanied by decreased anchorage independent growth in soft agar in S462-TY cells overexpressing MAF constitutively or inducibly (Figure 5C). However, after these transient effects, MAF expressing cells were able to proliferate in culture, as confirmed by increased metabolic activity in MTS assays (Figure 5D) and BrdU incorporation (Figure 5E). Relative levels of MAF protein expression were higher 2 days after induction than at day 6, correlating with increased apoptosis evidenced by cleaved caspase 3 (Figure 5F). Low MAF levels by day 6 corresponded with increased proliferation. Thus, effects of MAF expression on MPNST cells are dose dependent.

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