Protein or amino acid (AA) limitation activates an AA response (AAR) comprised of several partially characterized signaling pathways. The best studied of these involves AA sensing by GCN2 kinase and culminates in ATF4-dependent transcription via genomic C/EBP-ATF response element (CARE) enhancer sites that exhibit AAR element (AARE) function. Using HepG2 human hepatocellular carcinoma (HCC) and U87 human glioblastoma cells as models, our recent research indicates that there are GCN2/ATF4-independent pathways that exhibit little or no contribution in non-transformed cells. One of these pathways is mediated through the RAS/RAF/MEK/ERK arm of MAPK signaling and terminates with ELK1 transcription factor binding to ETS enhancer sequences not previously known to exhibit AARE function. We have discovered that the ERK pathway makes a significant contribution to the regulation of hundreds of AA-responsive genes in human HCC cells, but not in normal hepatocytes. These novel observations underscore the significant gaps in our knowledge about the impact of AA availability on basic cell functions. Our global hypothesis is that in some tumor cells the mammalian AAR extends beyond GCN2/ATF4 and that AA signaling via MAPK pathways is a significant component of the AAR, which permits tumor cells to proliferate despite AA limitation. The proposed experiments will test the following specific hypotheses in cultured cells and tumor-bearing mice. (Hypothesis I) An ERK pathway contributes to novel AA signaling independent of GCN2-ATF4. (Hypothesis II) The ERK AAR pathway includes transcription factors not previously known to be AA-responsive and terminates in novel genomic sequences that have functional AARE activity. (Hypothesis III) Induction of ERK-dependent signaling contributes to continued tumor growth despite AA limitation. The proposed studies are conceptually innovative for the following reasons. 1) The current dogma that the GCN2-ATF4 pathway induces most/all AA- responsive genes must be expanded to include an unknown AA sensor, MAPK signaling, and enhancer sequences previously unknown to have AARE activity. 2) AA-dependent transcription via ELK1 reveals an entirely new family of transcription factors, the ETS family, within the AAR. 3) The ERK-driven induction of genes, such as EGR1, FOS, JUN, and MYC, in AA-deprived tumor cells suggests a link between protein/AA nutrition and tumor proliferation.
Dietary protein/amino acid availability is a critical component in the maintenance of health, particularly during fetal/neonatal development, post-surgical recovery, and in the progression of a wide range of diseases, including cancer. Despite these important relationships, we know relatively little about the impact of fluctuation of dietary amino acids on the proliferation of cancer cells versus non-cancerous cells. The proposed studies will compare normal and cancerous human liver cells, as well as mouse tumor models, to investigate newly discovered amino acid regulated signaling mechanisms.
