Chief of Surgical Oncology, Associate Professor of Surgery and Immuno Oncology City of Hope Medical Center La Verne, California, United States
Disclosure: Disclosure information not submitted.
Participants should be aware of the following financial/non-financial relationships:
Laleh Melstrom, MD MSCI: Disclosure information not submitted.
Introduction: Colorectal cancer (CRC) remains a significant cause of cancer related mortality. FTO is m6A mRNA demethylase that plays an oncogenic role in various type of cancer. In this study we evaluate the role of FTO in CRC tumorigenesis.
Methods: MTS assays were conducted in 6 CRC cell lines with the FTO inhibitor CS-1 (50-3200nM)(+/- 5-FU 5-80μM) and after lentivirus mediated FTO knockdown. Cell cycle and apoptosis assays were conducted in HCT116 cells (24, 48hr, 290nM CS1). Western blot and m6A dot blot assays were performed to assess CS1 inhibition cell cycle proteins and FT0 demethylase activity. Migration and invasion assays of sh-FTO cells and CS-1 treated cells were performed. An in vivo heterotopic model of HCT116 cells treated with CS-1 or with FTO knockdown cells was performed. RNAseq was performed on sh-FTO cells to assess which pathways were impacted. RT-PCR was conducted on select genes down-regulated by FTO knockdown.
Results: The FTO inhibitor ,CS-1 suppressed CRC cell proliferation in HT-29, COLO, HCT116, LoVo, SW480, SW620 cells and in the 5FU resistant HCT116-5FUR cell line (p< 0.05). CS1(290 nM) induced cell cycle arrest in the G2/M phase by down regulation of CDC25c and promoted apoptosis of HCT116 cells (24h 13.8 vs 9.9% , P< .0.001). CS1 suppressed in vivo tumor growth by day 21 post treatment in the HCT116 heterotopic model (Figure A, P< 0.05). Lentivirus knockdown of FTO in HCT116 cells (shFTO) mitigated in vitro demethylase activity, cell growth, migration and invasion compared to shScr controls. (P< 0.01). RNA-seq of sh-FTO cells compared to sh-Scr demonstrated these top 4 down-regulated genes (EREG, KRAP, PDE48 and SLC38A2). These genes were markedly down-regulated in sh-FTO cells as confirmed by RT-PCR as well as in CS-1 treated cells (P< 0.05).
Conclusions: Inhibition of FTO by the small molecule CS1 or by lentivirus mediated knockdown mitigates proliferation, invasion and migration in vitro and in vivo in human CRC. Further work exploring the targeted pathways will elucidate precise downstream mechanisms that can potentially translate these findings to clinical trials.
Learning Objectives:
1. Upon completion, the participant will be able to understand the mechanism of action of FTO.
2. Upon completion the participant will learn that FTO inhibition mitigates CRC growth.
3. Upon completion the participant will understand the pathways impacted with FTO knockdown.
