FTO demethylates m6A modifications in HOXB13 mRNA and promotes endometrial cancer metastasis by activating the WNT signalling pathway

Abstract

Although many studies have confirmed the relationship between obesity and endometrial cancer (EC), the molecular mechanism between obesity and EC progression has not been elucidated. Overexpression of fat mass and the obesity associated protein FTO leads to weight gain, although recently it has been discovered that FTO can serve as a demethylase which erases N6-methyladenosine (m6A) modification and regulates the metabolization of mRNAs. In this study, we found high expression of FTO in metastatic EC and that this action promote both metastasis and invasion in vivo and in vitro. Mechanistically, FTO can catalyse demethylation modification in 3'UTR region of HOXB13 mRNA, thereby abolishing m6A modification recognition with the YTHDF2 protein. Decreasing HOXB13 mRNA decay and increasing HOXB13 protein expression was accompanied by WNT signalling pathway activation and the expression of downstream proteins, leading to tumour metastasis and invasion. We also found the WNT signalling pathway inhibitor ICG-001 can block HOXB13 gene-induced tumour metastasis, therefore ICG-001 may be a promising molecular intervention. This study provides insight into the relationship between obesity and the pathogenesis of endometrial cancer while highlighting future areas of research.

Keywords: Endometrial cancer; cancer metastasis; fto; hoxb13; m6A.

Figure 1.

FTO is overexpressed in metastatic EC cancer. (A) FTO mRNA expression in metastatic EC (N = 30) and nonmetastatic EC tissues (N = 66). (B) Expression of the FTO protein in nonmetastatic EC tissue and different types of metastatic EC tissues. (C) The IHC score of the FTO protein in different EC samples, (No metastasis, N = 66), (With metastasis, N = 30), (Abdominal metastasis, N = 24), (Lymph node metastasis, N = 22). **P < 0.01, n.s indicates not significant

Figure 2.

FTO promotes EC cell metastasis and invasion. (A) FTO overexpression and knockdown regulate cell migratory abilities in KLE cells according to wound-healing assays. (B) Wound-healing assays demonstrate that FTO regulates cell metastasis of AN3CA cells. (C) Effects of FTO overexpression and knockdown on cell invasive capacities by Transwell assays. (D) Weight of xenografts derived from AN3CA cells (n = 5 mice/group). (E) IHC staining of FTO and Ki-67 in tumour samples. FTO (+) NC: Negative control lentiviral vector. FTO shRNA: knockdown of FTO by shRNA lentiviruses. FTO shRNA NC: Negative control shRNA lentiviruses. Error bars indicate means ± SDs, **P < 0.01

Figure 3.

FTO recognizes and regulates m⁶A in HOXB13 mRNA. (A) KEGG enrichment map of genes specifically enriched by RNA-seq after FTO knockdown. (B) MeRIP-seq detects changes in m6A modifications in mRNA after silencing FTO expression. (C) Top motif identified by HOMER with m⁶A-seq peaks. (D) Distribution of new m⁶A peaks in mRNA detected by MeRIP-seq after knocking down FTO expression. (E) Venn diagram shows the genes enriched by MeRIP-seq, RNA-seq, and RIP-seq. (F, G) qPCR and WB confirmed decreased HOXB13 mRNA after FTO knockdown in AN3CA and KLE cells. (H) RIP-PCR validates exogenous FTO binding to HOXB13 mRNA. (I) MeRIP-PCR confirmed that the m⁶A peak in the 3ʹ untranslated region of HOXB13 mRNA was regulated by FTO. (J) The m⁶A peak in HOXB13 mRNA transcripts in FTO knockdown samples (IP and input) and the negative control (IP and input). Error bars indicate means ± SDs, **P < 0.01

Figure 4.

YTHDF2 regulates HOXB13 expression in an m⁶A-dependent manner. (A, B) qPCR and WB confirmed elevated HOXB13 mRNA and protein expression after YTHDF2 knockdown. (C) RIP-PCR validates exogenous YTHDF2 binding to HOXB13 mRNA. (D) Prolonged RNA lifetime of HOXB13 mRNA after knockdown of YTHDF2 expression. (E) Shortened RNA lifetime of HOXB13 mRNA after knockdown of FTO expression. (F) Relative activity of the wild-type or mutant HOXB13 3′ UTR luciferase reporter in AN3CA cells expressing YTHDF2 shRNA or control. (G) Position of the m⁶A peak in HOXB13 mRNA (top). RNA probe sequences for RNA pulldowns (bottom). (H) YTHDF2 recognizes the m⁶A site in the 3′ UTR of HOXB13 mRNA as shown by RNA pull-down assays. Error bars indicate means ± SDs, **P < 0.01, n.s indicates no significance

Figure 5.

HOXB13 promotes EC cell metastasis and invasion. (A, B) HOXB13 overexpression and knockdown regulate cell migratory abilities in KLE and AN3CA cells by wound-healing assays. (C) Effects of HOXB13 overexpression and knockdown on cell invasive capacities by Transwell assays. (D) Expression of HOXB13 protein in the tissue array validated by IHC, (No metastasis, N = 60), (With metastasis, N = 30), (Abdominal metastasis, N = 24), (Lymph node metastasis, N = 22). Error bars indicate means ± SDs, **P < 0.01

Figure 6.

HOXB13 promotes EC cell metastasis and invasion (A, B) WNT signalling pathway inhibitor (ICG-001) inhibits metastasis and invasion of KLE and AN3CA cells. (C, D) WNT signalling pathway inhibitor (ICG-001) blocked the effect of promoting tumour cell invasion and metastasis mediated by HOXB13. Error bars indicate means ± SDs, *P < 0.05, **P < 0.01

Figure 7.

HOXB13 activates the WNT signalling pathway and promotes the expression of downstream genes