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

Thuy Phan; Vu H Nguyen; Rui Su; Yangchan Li; Ying Qing; Hanjun Qin; Hyejin Cho; Lei Jiang; Xiwei Wu; Jianjun Chen; Marwan Fakih; Don J Diamond; Ajay Goel; Laleh G Melstrom

doi:10.3389/fonc.2023.1087644

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

Front Oncol. 2023 Feb 17:13:1087644. doi: 10.3389/fonc.2023.1087644. eCollection 2023.

Authors

Thuy Phan¹, Vu H Nguyen², Rui Su³, Yangchan Li³, Ying Qing³, Hanjun Qin⁴, Hyejin Cho⁴, Lei Jiang⁵, Xiwei Wu⁴, Jianjun Chen³, Marwan Fakih⁶, Don J Diamond², Ajay Goel⁷, Laleh G Melstrom¹

Affiliations

¹ Department of Surgery, City of Hope National Medical Center, Duarte, CA, United States.
² Department of Hematology, City of Hope National Medical Center, Duarte, CA, United States.
³ Beckman Research Institute, Department of Systems Biology, City of Hope National Medical Center, Monrovia, CA, United States.
⁴ Beckman Research Institute, The Integrative Genomics Core, City of Hope National Medical Center, Duarte, CA, United States.
⁵ Department of Molecular and Cellular Endocrinology, City of Hope National Medical Center, Duarte, CA, United States.
⁶ Department of Medical Oncology, City of Hope National Medical Center, Duarte, CA, United States.
⁷ Department of Molecular Diagnostics and Experimental Therapeutics, City of Hope National Medical Center, Monrovia, CA, United States.

Abstract

Introduction: Colorectal 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.

Methods: Cell 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.

Results: We 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.

Discussion: Further work exploring the targeted pathways will elucidate precise downstream mechanisms that can potentially translate these findings to clinical trials.

Keywords: FTO; FTO inhibitor; RNA-seq; colorectal cancer; signaling pathways.

Grants and funding

This study was supported in part by Legacy Heritage Fund, the Norman and Sadie Lee Foundation and the City of Hope Chancellors’ Fund.