Targeting fat mass and obesity-associated protein mitigates human colorectal cancer growth in vitro and in a murine model

Abstract

IntroductionColorectal cancer (CRC) remains a significant cause of cancer related mortality. Fat mass and obesity-associated protein (FTO) is a m6A mRNA demethylase that plays an oncogenic role in various malignancies. In this study we evaluated the role of FTO in CRC tumorigenesis.MethodsCell proliferation assays were conducted in 6 CRC cell lines with the FTO inhibitor CS1 (50-3200 nM) (± 5-FU 5-80 mM) and after lentivirus mediated FTO knockdown. Cell cycle and apoptosis assays were conducted in HCT116 cells (24 h and 48 h, 290 nM CS1). Western blot and m6A dot plot assays were performed to assess CS1 inhibition of cell cycle proteins and FTO demethylase activity. Migration and invasion assays of shFTO cells and CS1 treated cells were performed. An in vivo heterotopic model of HCT116 cells treated with CS1 or with FTO knockdown cells was performed. RNA-seq was performed on shFTO cells to assess which molecular and metabolic pathways were impacted. RT-PCR was conducted on select genes down-regulated by FTO knockdown.ResultsWe found that the FTO inhibitor, CS1 suppressed CRC cell proliferation in 6 colorectal cancer cell lines and in the 5-Fluorouracil resistant cell line (HCT116-5FUR). CS1 induced cell cycle arrest in the G2/M phase by down regulation of CDC25C and promoted apoptosis of HCT116 cells. CS1 suppressed in vivo tumor growth in the HCT116 heterotopic model (p< 0.05). Lentivirus knockdown of FTO in HCT116 cells (shFTO) mitigated in vivo tumor proliferation and in vitro demethylase activity, cell growth, migration and invasion compared to shScr controls (p< 0.01). RNA-seq of shFTO cells compared to shScr demonstrated down-regulation of pathways related to oxidative phosphorylation, MYC and Akt/ mTOR signaling pathways.DiscussionFurther work exploring the targeted pathways will elucidate precise downstream mechanisms that can potentially translate these findings to clinical trials.

Targeting RNA modifications with pharmacological agents: New frontiers in cancer therapy

“…CS1 suppresses in vivo tumour growth in the HCT116 heterotopic model. CRC [188,189] Treatment leads to increased apoptosis and cell cycle arrest at the G0 phase in human AML cells. Glioblastoma [191] Ena21 Shows a dose-dependent inhibition of cell growth in cell lines tested.…”

Targeting key RNA methylation enzymes to improve the outcome of colorectal cancer chemotherapy (Review)

“…An inhibitor of FTO called CS1 inhibited the proliferation of six CRC cell lines, including HT-29, COLO, HCT-116 and 5-FU-resistant cell lines (HCT-116/5FU). It also induced G25/M phase cell cycle arrest and promoted apoptosis of HCT-116 cells by downregulating doublecortin domain containing 2C ( 113 ).…”