T. A. Phan1, R. A. Su4, A. A. Goel3, D. A. Diamond2, L. A. Melstrom1 1City Of Hope National Medical Center, Surgery, Duarte, CA, USA 2City Of Hope National Medical Center, Hematology, Duarte, CA, USA 3City Of Hope National Medical Center, Experimental Therapeutics, Duarte, CA, USA 4City Of Hope National Medical Center, Systems Biology, Duarte, CA, USA
Introduction: Fat mass and obesity-associated protein (FTO) is a m6A mRNA demethylase that plays an oncogenic role in various malignancies. Our previous study showed that knockdown (KD) of FTO in HCT116 colorectal cancer cells mitigates tumor proliferation and most significantly down regulates epigregulin (EREG). EREG is a ligand of the epidermal growth factor receptor (EGFR) which is important colorectal cancer growth. In this study we hypothesize that EREG down-regulation is a mechanism of action of FTO mediated growth inhibition in colorectal cancer cells.
Methods: The plasmid for EREG overexpression was constructed by inserting EREG ORF (pcDNA3.1, Genscript) into pCDH-puro. Overexpression of EREG in HCT116 colorectal cancer cells was performed using Lentivirus transduction. EREG overexpression in selected clones was confirmed by Western blot analysis. MTS proliferation assays were conducted to assess the growth inhibition by the FTO inhibitor CS1 in control, pCDH and EREG overexpression HCT116 cell lines. Those 3 cell lines were treated with CS1 (50-3200nM) for 48hrs, and RT-PCR with an EGFR signaling primer library was conducted to assess which down -stream EREG related pathways are impacted.
Results:EREG overexpression was demonstrated by western blot analysis after transduction of cells with the EREG transcript. Overexpressed EREG in HCT116 mitigated in part the growth inhibition of the FTO inhibitor CS1 (88% vs 70% vs 75% at 100nM, 68% vs 44% vs 49% at 200nM, and 50% vs 33% vs 36% at 400nM, 48hrs) compared to parental cell lines or pCDH (p<0.0001), respectively. This demonstrates that in FTO mediated growth inhibition is in part due to down regulation of EREG. In order to identify downstream pathways impacted by EREG overexpression in CS1 treated cells, RT-PCR using the EGFR signaling primer library identified 3 top up-regulated genes FASLG, MAP3K2 and MAPK12 in EREG overexpressed HCT116 cells after CS1 treatment comparing to controls.
Conclusion:In this study, we demonstrate that overexpressed EREG can overcome in part colorectal cancer cell growth inhibition induced by the FTO inhibitor CS1. We have identified three potential down-stream mechanisms including tumor expression of FASLG, MAPS3K2, MAPK12. Further work is necessary to assess the role of EREG as a therapeutic target in colorectal cancer.
