Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer.

ABSTRACT

MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) orchestrates diverse environmental signals to facilitate cell growth and is frequently activated in cancer. Translocation of MTORC1 from the cytosol to the lysosomal surface by the RRAG GTPases is the key step in MTORC1 activation. Here, we demonstrated that transcription factors MEF2A and MEF2D synergistically regulated MTORC1 activation via modulating its cyto-lysosome shutting. Mechanically, MEF2A and MEF2D controlled the transcription of FNIP1 and FNIP2, the components of the FLCN-FNIP1 or FNIP2 complex that acts as a RRAGC-RRAGD GTPase-activating element to promote the recruitment of MTORC1 to lysosome and its activation. Furthermore, we determined that the pro-oncogenic protein kinase SRC/c-Src directly phosphorylated MEF2D at three conserved tyrosine residues. The tyrosine phosphorylation enhanced MEF2D transcriptional activity and was indispensable for MTORC1 activation. Finally, both the protein and tyrosine phosphorylation levels of MEF2D are elevated in human pancreatic cancers, positively correlating with MTORC1 activity. Depletion of both MEF2A and MEF2D or expressing the unphosphorylatable MEF2D mutant suppressed tumor cell growth. Thus, our study revealed a transcriptional regulatory mechanism of MTORC1 that promoted cell anabolism and proliferation and uncovered its critical role in pancreatic cancer progression.Abbreviation ACTB actin beta; ChIP chromatin immunoprecipitation; EGF epidermal growth factor; EIF4EBP1 eukaryotic translation initiation factor 4E binding protein 1; FLCN folliculin; FNIP1 folliculin interacting protein 1; FNIP2 folliculin interacting protein 2; GAP GTPase activator protein; GEF guanine nucleotide exchange factors; GTPase guanosine triphosphatase; LAMP2 lysosomal associated membrane protein 2; MAP1LC3B/LC3B microtubule associated protein 1 light chain 3 beta; MEF2 myocyte enhancer factor 2; MEF2A myocyte enhancer factor 2A; MEF2D myocyte enhancer factor 2D; MEF2D-3YF Y131F, Y333F, Y337F mutant; MTOR mechanistic target of rapamycin kinase; MTORC1 MTOR complex 1; NR4A1 nuclear receptor subfamily 4 group A member 1; RPTOR regulatory associated protein of MTOR complex 1; RHEB Ras homolog, mTORC1 binding; RPS6KB1 ribosomal protein S6 kinase B1; RRAG Ras related GTP binding; RT-qPCR real time-quantitative PCR; SRC SRC proto-oncogene, non-receptor tyrosine kinase; TMEM192 transmembrane protein 192; WT wild-type.