The Potential Role of N6-Methyladenosine (m6A) Demethylase Fat Mass and Obesity-Associated Gene (FTO) in Human Cancers.

N6-methyladenosine (m6A) demethylase fat mass and obesity-associated gene(FTO), previously recognized to be related with obesity and diabetes, was gradually discovered to be dysregulated in multiple cancers and plays an oncogenic or tumor-suppressive role. However, the specific expression and pro- or anti-cancer role of FTO in various cancers remained controversial. In this review, through summarizing the available literature, we found that FTO single nucleotide polymorphisms (SNPs) were closely related with cancer risk. Additionally, the dysregulation of FTO was implicated in multiple biological processes, such as cancer cell apoptosis, proliferation, migration, invasion, metastasis, cell-cycle, differentiation, stem cell self-renewal and so on. These modulations mostly relied on the communications between FTO and specific signaling pathways, including PI3K/AKT, MAPK and mTOR signaling pathways. Furthermore, FTO had great potential for clinical application by serving as a prognostic biomarker.

Introduction

Fat mass and obesity-associated gene (FTO) was previously recognized to be associated with the occurrence and development of childhood and adult obesity and type 2 diabetes (T2D).1–3 Later, researchers found that the FTO A allele was not only associated with increased body mass index (BMI), but also associated with decreased risk of lung cancer and increased risk of kidney cancer.4 Accumulating studies revealed that there was a close connection between FTO and the risk of various human cancers, including breast cancer,5,6 colon cancer,7 gastric cancer,8 pancreatic cancer,9 prostate cancer10 and so on. Further studies discovered that the regulatory role of FTO in cancers might rely on FTO-mediated N6-methyladenosine (m6A) demethylation.11,12

It was widely known that there were over 100 post-transcriptional modifications of RNA identified in living organisms, and these post-transcriptional modifications provided a functional diversity that allowed basic ribonucleotide residues to obtain various functions.13 M6A modification was first identified in mRNA-enriched RNA fractions from novikoff hepatoma cells in 1974.14 It was the most prevalent internal RNA modification and was a dynamic and reversible modification process in eukaryotic cells.15 With time going on, Dominissini et al16 presented in 2012 that m6A-seq, dependent on antibody-mediated capture and massively parallel sequencing, was able to landscape the human and mouse m6A modification in a transcriptome-wide manner. With the application of the technology for monitoring m6A, insights into the potential mechanisms had been explored in recent decades. For example, existing evidence revealed that m6A modification was maintained by methyltransferase (MTase) complex and demethylase, and functionally regulated the eukaryotic transcriptome to affect mRNA splicing, export, localization, translation, and stability.17 MTase complex termed as “writers”, including methyltransferase-like 3/14/16 (METTL3/14/16), KIAA1429, wilms tumor 1 (WT1)-associated protein(WTAP) and RBM15, and acted to add m6A-modified sites.18 However, FTO belonged to demethylase. Demethylase, termed as “erasers”, functioned to reverse the methylation and affect biological functions accordingly (Figure 1).19 In detail, FTO had been found to modify multiple RNAs, such as microRNAs (miRNAs)20 and messenger RNAs (mRNAs).21 It was also significantly related with multiple biological functions of cancers, including cell cycle,11 tumor growth,12 proliferation,22 survival,23 migration,24 invasion,25 stem cell maintenance26 and self-renewal.27

Figure 1

FTO, belonged to demethylase, termed as “erasers” and functioned to reverse the methylation.

In order to better describe the role of FTO in various cancers, we first searched the literature about FTO and multiple cancers in PubMed () and classified them according to the types of cancers. After obtaining the literature, we focused on the relationship between FTO and cancer risks and the underlying mechanisms of FTO in various cancers.

FTO Single Nucleotide Polymorphism (SNP) with Cancer Risk

In 2010, Gaudet et al28 first explored the relationship between FTO and cancer risk. Unfortunately, the results of genome-wide association scans indicated that there was no association between FTO and endometrial cancer risk. Later, FTO variants, including rs9939609, rs17817449, rs8050136, rs1477196, rs6499640, rs16953002, rs11075995 and rs1121980 were found to be related with cancer risk.29 Lewis et al10 carried out a genetic association study of the connection between FTO and prostate cancer risk. Interestingly, FTO rs9939609, which was a SNP known to be associated with obesity, was inversely related with low-grade prostate cancer risk, but positively related with high-grade prostate cancer. Salgado-Montilla et al30 also discovered the same correlation between FTO rs9939609 and prostate cancer risk; nonetheless, the result was not statistically significant upon adjustment.

As for breast cancer, one research revealed that FTO might have a close connection with the risk of breast cancer.31 Kaklamani et al5 evaluated the role of four FTO SNPs, including rs7206790, rs8047395, rs9939609 and rs1477196 in breast cancer patients from Northwestern University in Chicago, Illinois. The results showed that all SNPs, especially FTO rs1477196, were significantly associated with breast cancer risk. Likewise, FTO rs1477196 significantly depressed breast cancer risk, and FTO rs16953002 significantly increased breast cancer risk in Chinese population.32 Additionally, FTO rs11075995 was closely connected with breast cancer risk, but this connection was eliminated with further adjustment for BMI.33 However, in Iranian population, neither FTO rs1477196 nor FTO rs9939609 was statistically significantly related with the risk of breast cancer.34 Similarly, FTO rs1121980 and rs9939609 did not show any significant association with breast cancer development.6,35 Another research genotyped two polymorphic sites located in FTO gene (rs993909 and rs9930506), and did not find any association between FTO and breast cancer risk in patients from Copernicus Memorial Hospital in Lodz, Poland.36 What is more, FTO rs3751812 was not significantly connected with breast cancer risk in Chinese population.37 In conclusion, up to date, FTO rs7206790, rs8047395, rs9939609, rs1477196 and rs16953002 might be associated with breast cancer risk.

As for colorectal cancer, Yang et al38 examined 677 FTO SNPs in patients from the Colon Cancer Family Registry, and did not find any evidence that FTO SNPs were related with colorectal cancer risk. Whereas, another research found that FTO rs1558902, rs8050136, rs3751812, and rs9939609 showed a positive association with colorectal cancer in Japanese population.7

Furthermore, FTO rs9939609 polymorphism might be associated with the susceptibility of pancreatic cancer and endometrial cancer, especially in Asian populations, while no statistical significance was found in other cancers.39 A meta-analysis suggested that FTO rs9939609 was not significantly related with the increased risk of cancers, with the exception of pancreatic cancer.33,40 A case-control study in Japan revealed that FTO rs9939609 was correlated with pancreatic cancer risk and possibly independent of obesity.9 Additionally, although FTO rs9939609 was associated with increased risk of endometrial carcinoma, this association was eliminated after adjusting for BMI in white non-Hispanic women.41

In other cancers, researches revealed that FTO was not only associated with a decreased risk of lung cancer but also associated with a weak increased risk of kidney cancer.4 And FTO rs8047395 was closely associated with papillary thyroid cancer in German population.42 All associations between FTO SNPs and cancer risk are listed in Table 1.

Table 1

The Associations Between FTO SNPs and Various Cancers

Cancer	SNPs	Population	Association	Ref
Prostate cancer	rs9939609	/	+	10
	rs9939609	/	/	30
Breast cancer	rs7206790rs8047395rs9939609rs1477196	Patients from Northwestern University in Chicago	+	5
	rs1477196rs16953002	Chinese population	+	32
	rs11075995	/	/	33
	rs1477196rs9939609	Iranian population	/	34
	rs1121980rs9939609	/	/	6
	rs993909rs9930506	Patients from Copernicus Memorial Hospital in Poland	/	36
	rs3751812	Chinese population	/	37
Colorectal cancer	rs1558902rs8050136rs3751812rs9939609	Japanese population	+	7
Pancreatic cancer	rs9939609	Asian population	+	39
		/	+	9
Endometrial cancer	rs9939609	Asian population	+	39
		White non-Hispanic population	/	41
Papillary thyroid cancer	rs8047395	German population	+	42

Notes: +: indicated significantly related. /: indicated not significantly related.

The Biological Functions and the Underlying Mechanisms of FTO in Multiple Cancers

According to the existing researches, the association between FTO SNPs and the risk of various cancers might rely on the molecular mechanisms of FTO, which played a critical role in cancer tumorigenesis.43 For instance, the expression of FTO was dramatically dysregulated in cancers and took a great part in the growth of cancer cells through modulating cellular metabolic pathways, including phosphoinositide 3-kinases/protein kinase B (PI3K/AKT) and adenosine monophosphate-activated protein kinase(AMPK) signaling pathways.23 Next, we would further explore the detailed molecular mechanisms of FTO in the occurrence and progression of cancers. The expression, clinical significance and biological functions of FTO in various cancers are shown in Table 2.

Table 2

Expression, Clinical Significance and Biological Functions of FTO in Various Cancers

Cancer	Expression	Role	Biological Function	Target	Ref
Bladder Cancer	Down-regulated	Tumor suppressor	Proliferation, migration, cytotoxicity	/	61
Breast Cancer	Up-regulated	Oncogene	Survival, colony formation	IRX3	45,46
	Up-regulated	Oncogene	Cell energy metabolism	PI3K/AKT	48
	/	Oncogene	Proliferation	PI3K/AKT	31
	Up-regulated	Oncogene	Proliferation, colony formation, metastasis	BNIP3	47
Cervical Cancer	Up-regulated	Oncogene	Chemo-radiotherapy resistance	β-catenin, ERCC1	21
	Up-regulated	Oncogene	Proliferation, migration	E2F1, Myc	67
Clear Cell Renal Cell Carcinoma	Down-regulated	Tumor suppressor	Cell growth, apoptosis, mitochondrial biogenesis, oxidative phosphorylation, oxidative stress	PGC-1α	62
Colorectal Cancer	/	Oncogene	Proliferation	/	20
Endometrial Carcinoma	Up-regulated	Oncogene	Proliferation, invasion	PI3K/AKTMPAK	25
	Up-regulated	Oncogene	Proliferation	mTOR	78
Esophageal Squamous Cell Carcinoma	Up-regulated	Oncogene	Cell growth, migration, tumorigenicity	MMP13	24
Gastric Cancer	Up-regulated	Oncogene	Proliferation, migration, invasion	/	8
	Down-regulated	Tumor suppressor	/	/	57
Hepatocellular Carcinoma	Up-regulated	Oncogene	Proliferation, tumor growth, cell cycle	PKM2	11
	Down-regulated	Tumor suppressor	/	/	60
Lung Cancer	Up-regulated	Oncogene	Proliferation, colony formation, tumor growth	USP7	12
	Up-regulated	Oncogene	Proliferation, invasion, apoptosis	MZF1	52
Ovarian Cancer	Down-regulated	Tumor suppressor	Stemness	cAMP	27
Pancreatic Cancer	Up-regulated	Oncogene	Proliferation apoptosis	cMYC	22
Leukemia	Up-regulated	Oncogene	Proliferation, viability, cell-cycle arrest, apoptosis	MYC/CEBPA	87
	Up-regulated	Oncogene	Differentiation	ASB2,RARA	81

FTO in Breast Cancer

In 2015, Tan et al44 first explored the association between FTO and breast cancer. The results showed that the expression of FTO was significantly higher in breast cancer tissues, especially HER2-overexpressed breast cancer. Furthermore, FTO inhibitor obviously suppressed the survival and colony formation of panresistant triple-negative inflammatory breast cancer cells, this regulation might depend on obesity-associated cis-acting elements in non-coding region of FTO, which acted to modulate the expression of IRX3 gene and activate obesity networks.45,46 Niu et al47 also found that FTO was elevated in breast cancer cell lines and tissues, and enhanced cancer cell proliferation, colony formation and metastasis through regulating m6A demethylation in the 3ʹUTR of BNIP3mRNA, which was a tumor suppressor, and inducing its degradation by a YTHDF2independent mechanism.

Due to the role of FTO in metabolism, Liu et al48 assessed the effect of FTO on the energy metabolism of breast cancer cells. Mechanism researches found out that FTO inhibitor restrained pyruvate kinase and hexokinase activity and suppressed breast cancer cell glycolysis, partly through lowering the levels of PI3K, p-PI3K, Akt and p-Akt, which were members of PI3K/AKT signaling pathway. It was also disclosed by Gholamalizadeh et al31 that FTO functioned to activate the PI3K/Akt signaling pathway and promote breast cancer cell proliferation in estrogen receptor positive breast cancer patients. Additionally, the association of FTO and breast cancer was affected by the status of estrogen receptors and estrogen might exert its influence on breast cancer through FTO. The specific mechanisms of FTO in breast cancer are displayed in Figure 2.

Figure 2

The specific mechanisms of FTO in breast cancer. FTO greatly participated in the survival, colony formation, metastasis, glycolysis and proliferation of breast cancer through targeting IRX3, BNIP3 and PI3K/AKT.

FTO in Lung Cancer

FTOwas up-regulated in non-small cell lung cancer (NSCLC) tissues and cell lines, and FTO knockdown decreased the proliferation rate, inhibited the colony formation ability of cancer cells and retained tumor growth in vivo via increasing mRNA stability of ubiquitin-specific protease(USP7).12 USP7 was recognized to regulate the activities of numerous proteins and known as tumor suppressors, DNA repair proteins, immune responders, viral proteins, and epigenetic modulators.49–51 FTO was also drastically overexpressed in lung squamous cell carcinoma (LUSC), and loss-of-function assays indicated that the knockdown of FTO effectively retained the proliferation and invasion of cancer cells, while enhanced the apoptosis, via targeting myeloid zinc finger protein 1 (MZF1).52 MZF1 was a member of the SCAN-Zinc finger transcription factor family and had been proved to facilitate lung adenocarcinoma (LUAD) progression.53 The specific mechanisms of FTO in breast cancer are displayed in Figure 3.

Figure 3

The specific mechanisms of FTO in lung cancer. FTO obviously promoted lung cancer cell colony formation, proliferation, invasion and tumor growth and inhibited apoptosis via promoting the expression of USP7 and MZF1.

FTO in Gastrointestinal Cancer

FTO in Esophageal Squamous Cell Carcinoma (ESCC)

FTO was obviously up-regulated in ESCC tissues and functional assays revealed that FTO silence retained ESCC cell growth, migration and tumorigenicity through regulating matrix metalloproteinases 13 (MMP13), and FTO overexpression exhibited the opposite results.24 MMP13 was an important member of MMPs, who were a family of Zn2+-dependent endopeptidases, mainly existed in connective tissue and had a significant influence on tumor genesis and biological behavior.54–56

FTO in Gastric Cancer (GC)

The mRNA and protein expression of FTO was up-regulated in GC tissues and contributed to cancer cell proliferation, migration and invasion.8 However, Li et al57 disclosed that as opposed to the mRNA level, FTO protein level was significantly down-regulated in signet ring cells and GC tissues.

FTO in Hepatocellular Carcinoma (HCC)

Overexpressed FTO in the HCC tissues and cells modulated cancer cell proliferation, cell cycle and in vivo tumor growth, mechanically through triggering the demethylation of pyruvate kinase (PKM2) mRNA and accelerating the translation.11 PKM2 was one of the key glycolysis pyruvate kinase isoenzyme and transformed the glucose metabolism from the normal respiratory chain to lactate production in tumor cells, thus contributing to tumorigenesis.58,59 However, it was claimed by Zhao et al60 that FTO mRNA and protein levels were significantly down-regulated in HCC tissues.

FTO in Pancreatic Cancer and Colorectal Cancer

It was proved that FTO was overexpressed in pancreatic cancer cell lines, and FTO knockdown promoted cancer cell apoptosis and inhibited proliferation partly via communicating with cMYC proto-oncogene, which was a critical mediator in regulating cell entry into S phase of cell cycle.22 FTO, targeted by microRNA-1266, promoted proliferation of colorectal cancer cell lines.20 The specific mechanisms of FTO in gastrointestinal cancer are displayed in Figure 4.

Figure 4

The detailed mechanisms of FTO in gastrointestinal cancer. FTO promoted colorectal and gastric cancer cell proliferation, migration and tumor growth. FTO also advanced cancer cell growth, migration and tumorigenicity through up-regulating MMP13 in esophageal squamous cell carcinoma, and enhanced cancer cell proliferation and tumor growth via up-regulating PKM2 in hepatocellular carcinoma. Finally, FTO promoted pancreatic cancer cell proliferation and inhibited apoptosis by regulating the expression of cMYC.

FTO in Urological Cancer

FTO in Bladder Cancer

The mRNA and protein expression level of FTO were decreased in bladder cancer cell lines and bladder urothelial carcinoma tissues compared with the normal control.61 Further cell counting kit-8 and wound healing assays revealed that FTO knockdown enhanced cancer cell proliferation and migration, and cisplatin-induced cytotoxicity of bladder cancer cells could be rescued by a highly selective inhibitor of FTO. However, further mechanism explorations had not been conducted.

FTO in Clear Cell Renal Cell Carcinoma (ccRCC)

The expression of FTO is suppressed in ccRCC tissues and FTO severed to modulate mitochondrial activity, oxidative phosphorylation and cancer cell growth and apoptosis through demethylating the PPARg coactivators (PGC) −1αmRNA.62 PGC −1α was a member of transcriptional coactivators, which acted to be a central regulator of mitochondrial biogenesis and oxidative phosphorylation, and played a tumor-suppressive and pro-tumorigenic role in variant cancers.63–66 The specific mechanisms of FTO in urological cancer are shown in Figure 5.

Figure 5

The specific mechanisms of FTO in urological cancer. FTO advanced bladder cancer cell proliferation and migration and suppressed cytotoxicity. Additionally, FTO greatly participated in cancer cell apoptosis, mitochondrial activity, oxidative stress, ROS production and tumor growth through modulating PGC-1α in clear cell renal cell carcinoma.

FTO in Gynecological Cancer

FTO in Cervical Cancer

Up-regulated FTO enhanced the chemo-radiotherapy resistance of cervical squamous cell carcinoma (CSCC) partly through reducing m6A level of β-catenin mRNA transcripts and in turn increasing excision repair cross-complementation group 1(ERCC1) activity.21 ERCC1 was a significant regulator of nucleotide excision repair and positively associated with chemo-radiotherapy resistance of CSCC. Likely, FTO served as a positive modulator of cervical cancer cell proliferation and migration through affecting the translation efficiency of E2F1 and Myc.67

FTO in Endometrial Cancer

In FTO in Breast Cancer, we found that there was a mutual relationship between FTO and estrogen in breast cancer. As far as we know, aberrant estrogen metabolism was also greatly involved in endometrial cancer growth and metastasis.68–70 Zhang et al25 first explored the association between FTO and estrogen in endometrial cancer, they found that β-estradiol (E2) up-regulated FTO expression, thus enhancing endometrial cancer cell proliferation, migration and invasion via activating phosphatidylinositol-3-kinase (PI3K)/protein kinase b (AKT) and mitogen-activated protein kinase (MAPK) signal pathways. PI3Ks were key regulators of intracellular signaling in response to the extracellular stimulators. The activation of PI3K/AKT signaling pathway was one of the most common events in human cancers.71–73 MAPK pathway was also a pivotal bridge in the switch from extracellular signals to intracellular responses and frequently involved in oncogenesis, tumor progression and drug resistance.74–77 It was also proved by Zhu et al78 that estrogen promoted FTO nuclear localization and advanced mammalian target of rapamycin (mTOR) signaling pathway in endometrial carcinoma, thus promoting proliferative activity of cancer cells. mTOR signaling pathway was often activated in tumors, and acted to modulate cell proliferation, immune cell differentiation, tumor metabolism through affecting gene transcription and protein synthesis.79,80

FTO in Ovarian Cancer

However, FTO was down-regulated in ovarian tumors and inhibited the self-renewal of ovarian cancer stem cells (CSC) and suppressed tumorigenesis in vivo via blocking cAMPsignaling.27 The detailed mechanisms of FTO in gynecological cancer are shown in Figure 6.

Figure 6

The specific mechanisms of FTO in gynecological cancer. FTO greatly influenced ovarian cancer cell stemness and tumorigenesis via suppressing the expression of cAMP. FTO also affected endometrial carcinoma cell proliferation and invasion by modulating PI3K/AKT and MAPK signaling pathways and nuclear localization. Finally, FTO played an important role in the chemo-radiotherapy resistance of cervical squamous cell carcinoma and cancer cell proliferation and migration through up-regulating β-catenin, E2F1 and Myc.

FTO in Leukemia

Similarly, FTO was extremely overexpressed in acute myeloid leukemia (AML) with t (11q23)/MLL rearrangements, t (15;17)/PML-RARA, FLT3-ITD, and/or NPM1 mutations. Mechanically, FTO enhanced leukemogenesis, cell proliferation and transformation, and suppressed apoptosis through modulating m6A level in ASB2 and RARA mRNA transcripts.81 ASB2 and RARA had an anti-leukemic effect via degrading MLL during hematopoietic differentiation via ubiquitination.82–86 Furthermore, R-2-hydroxyglutarate (R-2HG) inhibited leukemia cell proliferation/viability and promoted cell-cycle arrest and apoptosis via increasing m6A RNA modification in the sensitive cells, modulating the stability of MYC/CEBPA transcripts and thus suppressing relevant pathways.87 CEBPA was a vital hematopoiesis-related transcription factor which was essential for leukemogenesis.88–90 The specific mechanisms of FTO in leukemia are displayed in Figure 7.

Figure 7

The detailed mechanisms of FTO in leukemia. FTO up-regulated MYC/CEBPA and down-regulatedASB2and RARA, thus promoting leukemia cell viability, proliferation, transformation and leukemogenesis and inhibiting cell-cycle arrest and apoptosis.

The Role of FTO Inhibitors

As the promoting role of FTO in leukemogenesis, Huang et al19,91 developed a promising FTO inhibitor, termed as FB23-2, which directly bound to FTO and selectively retained m6A demethylase activity. Further functional assays revealed that FB23-2 could remarkably inhibit the proliferation and advance the differentiation/apoptosis of AML cells, thus inhibiting the progression of AML. Considering the oncogenic roles of FTO in kinds of cancers, Su et al26 also developed two potent small-molecule FTO inhibitors, which proclaimed prominent anti-tumor effects in multiple cancers. In detail, inhibitors of FTO took a great part in cancer stem cell self-renewal and immune evasion by mediating the expression of immune checkpoint genes, especially LILRB4. In conclusion, the discovery of FTO inhibitors highlighted the broad potential of targeting FTO for cancer therapy.

FTO in Cancer Prognosis

High level of FTO was significantly correlated with lower survival rates in patients with advanced stage of breast cancer and patients with ER negative breast cancer.47 In addition, high expression of FTO was associated with poor prognosis and early relapse of endometrial carcinoma.78 Overexpression of FTO predicted the lower survival rate in HCC patients.11 High expression of FTO was also positively correlated with low differentiation, lymph node metastasis, high TNM stage and poor prognosis in gastric cancer patients.8 It was also observed in lung cancer that higher expression of FTO was significantly related with poor prognosis.52 Li et al57 revealed that although higher mRNA level of FTO was associated with poor overall survival (OS), further immunohistochemistry (IHC) staining and evaluation found that lower FTO protein expression was associated with shorter OS in GC patients. ESCC patients with high FTO expression had shorter OS, despite the statistical significance was absent.24 The prognostic value of FTO in OSCC patients for OS is dependent on the expression of β-catenin.21

However, in ccRCC patients, low expression of FTO was significantly associated with poor survival, such as shortened OS and disease-free survival (DFS).62,92,93 Additionally, HCC patients with decreased FTO expression had shorter OS and progression-free survival (PFS).60

Discussion

FTOwas dysregulated and played a tumor-suppressive or oncogenic role in human cancers, including breast cancer, bladder cancer, cervical cancer, renal cell carcinoma, endometrial cancer, esophageal carcinoma, gastric cancer, hepatocellular carcinoma, lung cancer, leukemia and so on. Through m6A modification, FTO regulated cancer cell apoptosis, proliferation, viability, migration, invasion, metastasis, cell-cycle, differentiation, stem cell self-renewal, colony formation, chemo-radiotherapy resistance and so on. These effects were achieved by regulating various pathways, such as mTOR signaling pathway, PI3K/AKT and MPAK signal pathways. In addition, miRNAs and estrogen could modulate the expression of FTO. Given that FTO patterns in RNA transcripts play important roles in multiple cancers, researchers focused on the rational design of potent and specific FTO inhibitors in medicine use and several FTO inhibitors had been developed, which might have extensive application for cancer therapy. What is more, FTO had great potential for clinical application by serving as prognostic targets. However, further studies are still needed to clarify FTO patterns in human cancers and pave the way for research into the discovery and development of FTO-specific drugs.