The oncogenic m6A demethylase FTO promotes tumorigenesis and immune escape by upregulating GPNMB in hepatocellular carcinoma

Abstract

<p>Background and Aims: Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer-related <br>death worldwide. Immune checkpoint inhibitor (ICI) targeting PD-L1 combined with VEGF is the first-<br>line therapy for advanced HCC. However, the efficiency of Immune checkpoint inhibitor (ICI) is not <br>satisfactory in HCC patients. FTO (Fat mass and obesity-associated protein) is the first identified N6-<br>methyladenosine (m6A) demethylase and has been reported to play an oncogenic or tumor-<br>suppressive role in liver cancer. A better understanding of the functional role and mechanistic basis of <br>FTO in HCC is needed. In this study, we aimed to functionally characterize the roles of m6A <br>demethylase FTO in HCC. </p><p>Method: To investigate the tumorigenic functions of FTO, colony formation, proliferation, migration <br>and invasion assays were conducted in vitro. Co-culture system of HCC cells and CD8 T cells or THP-<br>1 cells was used to examine the activation of effector memory T cells and polarization and recruitment <br>of macrophages upon stable knockdown of FTO (FTO-KD). Multiple xenograft and spontaneous HCC <br>forming mouse models were used to evaluate the effect of FTO on self-renewal, tumor growth, and <br>response to ICI treatment. RNA-sequencing and m6A-sequincing were done to identify the gene <br>targets related to m6A modification by FTO. </p><p>Results: FTO expression was up-regulated in patients’ HCC tumors, and high FTO expression (2-fold <br>cutoff) was significantly associated with poorer survival rates. Depletion of FTO resulted in reduced <br>pro-oncogenic functions in vitro, and suppressed tumor formation, progression, metastasis in vivo. <br>Stable FTO-KD significantly increased tumor-infiltrating immune cells and cytotoxic CD8+ T cells in <br>mouse tumor tissues and was accompanied by a significant reduction of PD1+ T cell exhaustion. We <br>also observed a trend of increase in anti-tumoral M1 macrophages in mouse tumors. Consistently, in <br>vitro co-culture assays of human HCC cells with T cells isolated from PBMC of healthy human <br>subjects showed that FTO KD increased the proportion of effector memory T cells and enhanced the <br>proliferation of CD8+ T cells. FTO silencing increased the anti-tumoral polarization and migration of <br>M1 macrophages. We identified GPNMB as a novel downstream target of FTO, which reduced the <br>m6A abundance of GPNMB, hence stabilizing it from degradation by YTHDF2. GPNMB was able to <br>inhibit CD8+ T cell activation. Furthermore, targeting FTO by its potential inhibitor CS2 promoted the <br>therapeutic effect of anti-PD1 in a well-established ‘cold-tumor’ mouse model (Trp53KO/C-MycOE), <br>resulting in significantly smaller tumors and higher tumor-infiltrating CD8 T cells in the liver tumor <br>tissues. </p><p>Conclusion: We demonstrated that FTO exerts oncogenic function and promotes immune escape in <br>HCC by regulating the m6A demethylation of GPNMB mRNA. Targeting FTO may provide a new <br>therapeutic approach for treating HCC.</p>