AMD1 upregulates hepatocellular carcinoma cells stemness by FTO mediated mRNA demethylation

Abstract

Background: S-adenosylmethionine decarboxylase proenzyme (AMD1) is a key enzyme involved in the synthesis of spermine (SPM) and spermidine (SPD), which are associated with multifarious cellular processes. It is also found to be an oncogene in multiple cancers and a potential target for tumor therapy. Nevertheless, the role AMD1 plays in hepatocellular carcinoma (HCC) is still unknown.

Methods: HCC samples were applied to detect AMD1 expression and evaluate its associations with clinicopathological features and prognosis. Subcutaneous and orthotopic tumor mouse models were constructed to analyze the proliferation and metastasis of HCC cells after AMD1 knockdown or overexpression. Drug sensitive and tumor sphere assay were performed to investigate the effect of AMD1 on HCC cells stemness. Real-time quantitative PCR (qRT-PCR), western blot, immunohistochemical (IHC) and m6A-RNA immunoprecipitation (Me-RIP) sequencing/qPCR were applied to explore the potential mechanisms of AMD1 in HCC. Furthermore, immunofluorescence, co-IP (Co-IP) assays, and mass spectrometric (MS) analyses were performed to verify the proteins interacting with AMD1.

Results: AMD1 was enriched in human HCC tissues and suggested a poor prognosis. High AMD1 level could promote SRY-box transcription factor 2 (SOX2), Kruppel like factor 4 (KLF4), and NANOG expression of HCC cells through obesity-associated protein (FTO)-mediated mRNA demethylation. Mechanistically, high AMD1 expression increased the levels of SPD in HCC cells, which could modify the scaffold protein, Ras GTPase-activating-like protein 1 (IQGAP1) and enhance the interaction between IQGAP1 and FTO. This interaction could enhance the phosphorylation and decrease the ubiquitination of FTO.

Conclusions: AMD1 could stabilize the interaction of IQGAP1 with FTO, which then promotes FTO expression and increases HCC stemness. AMD1 shows prospects as a prognostic predictor and a therapeutic target for HCC.

Keywords: AMD1; FTO; IQGAP1; N6-methyladenosine; hepatocellular carcinoma; polyamination; stemness.

FIGURE 1

AMD1 expression is upregulated in HCC and predicts poor prognosis. (A‐D) AMD1 mRNA levels in different cancers were analyzed by UALCAN, ***p < 0.001. (E) The mRNA expression of AMD1 of 233 paired tumors and para‐tumor tissues in GSE14520 cohorts. (F) The mRNA level of AMD1 in HCC tissues compared with tumor tissues (T) and para‐tumor tissues (PT) in our study. (G) AMD1 expression in 85 paired HCC tissues and para‐tumor tissues from the immunohistochemistry results, ***p < 0.001. (H) Representative images of AMD1 expression in HCC tissues and adjacent para‐tumor liver tissues. (I and J) Kaplan–Meier analysis indicates high expression of AMD1 is correlated with poor overall survival and disease‐free survival (n = 85). (K‐M) Overall survival analysis of HCC patients according to AMD1 expression in GSE14520, ICGC‐LIRI‐JP, and TCGA cohorts performed by HCCDB Abbreviations: CHOL, cholangio carcinoma; COAD, colon adenocarcinoma; HCC, hepatocellular carcinoma; PT, para‐tumor tissues; STAD, stomach adenocarcinoma; T, tumor tissues.

FIGURE 2

Downregulation of AMD1 inhibits HCC growth and metastasis in vivo. (A) Real‐time PCR and western blot and analysis of the AMD1 mRNA and protein expression in different HCC cells. *p < 0.05. (B) The mRNA levels of transcription factors in HCC cells infected with AMD1 overexpression or knockdown and negative control lentivirus. *p < 0.05, **p < 0.005. (C and D) Macrograph and absolute volume of subcutaneous xenograft tumors. ***p < 0.001. (E) Macrograph volume of orthotopic xenograft tumors. (F) The percentage of mice with metastasis in the lungs. (G and H) Representative pictures for lung metastasis of each mouse

FIGURE 3

AMD1 promotes the stemness of HCC cells. (A and B) The effects of AMD1 overexpression or knockdown on sphere‐forming ability detected by sphere‐forming assays. *p < 0.05, **p < 0.005. (C and D) The effects of AMD1 overexpression or knockdown on glycogen content in HCC cells examined by PAS stain. *p < 0.05, **p < 0.005. (E and F) Representative images and statistical results of PLC, MHCC97H and MHCCLM3 cells clone formation assay after transfected with corresponding vectors. **p < 0.01 (G) The IC50 and growth‐inhibitory curves of AMD1 overexpression or knockdown in HCC cells were assayed by ic50‐calculator. (H) CCK8 assay was performed to detect cell proliferation after treatment with sorafenib for 72 h. **p < 0.01, ***p < 0.001. (I) Subgroup analysis of TCGA cohort from Kaplan‐Meier plotter revealed that patients with low AMD1 expression treated with sorafenib had better clinical outcomes. (J) Flow cytometry using antibodies specifically targeting CD44 and CD90 detected an enrichment of the CD90+CD44+ cells. (K) Flow cytometry detected an enrichment of the CD90+ cells after sphere‐forming assay

FIGURE 4

AMD1 regulates pluripotency factors expression of HCC cells. (A and B) The mRNA and protein levels of transcription factors in HCC cells infected with AMD1 overexpression or knockdown and negative control lentivirus. *p < 0.05, **p < 0.005, ***p < 0.001. (C) AMD1 increased NANOG, SOX2, and KLF4 mRNA stability in HCC cells. *p < 0.05, **p < 0.005

FIGURE 5

AMD1 regulates m6A methyladenosine modification of HCC cells. (A) Overexpression of AMD1 decreased m6A content in PLC cells, while knockdown of AMD1 increased m6A content in MHCC97H cells. *p < 0.05. (B) The significantly increased (red) or decreased (green) m6A peaks (p < 0.05) in MHCC97HAMKD cells compared with MHCC97HAMNC cells. (C) m6A abundance in modification sites of SOX2 and KLF4 mRNA identified with m6A‐Seq in MHCC97HAMKD cells compared with MHCC97HAMNC cells. (D) m6A sites were predicted in NANOG, SOX2, KLF4, and OCT4 with SRAMP. (E) Gene‐specific m6A qPCR analysis of alterations in the m6A level in NANOG CDs and OCT4 3′UTR regions in HCC cells with different AMD1 levels. *p < 0.05. (F) The m6A methyltransferase/demethylase expression in PLC and MHCC97H cells transfected with AMD1 overexpression or knockdown and the negative control vectors

FIGURE 6

AMD1 promotes HCC stemness through FTO. (A) FTO expression in 85 paired HCC tissuesand para‐tumor tissuesfrom the immunohistochemistry results. ***p < 0.001. (B) Positive correlation between AMD1 and FTO in HCC tissues from TMA (n = 85). (C and D) Representative images of IHC staining of FTO in tumor tissues of HCC patients with high or low level of AMD1 expression. (E) Representative immunohistochemical staining of AMD1 and FTO of subcutaneous xenograft tumors (magnification 200×, Scale bar: 50 μm). (F and G) Kaplan–Meier analysis indicates high expression of FTO is correlated with poor overall survival and disease‐free survival (n = 85)

FIGURE 7

FTO acted as a functional downstream target of AMD1. (A and B) FTO knockdown in PLC^AMOE cells decreased the sizes and formation rate of tumor spheres, while FTO restoration in MHCC97H^AMKD cells increased the sizes and formation rate of tumor spheres. *p < 0.05. (C and D) FTO knockdown in PLC^AMOE cells decrease transcription factors expression while FTO restoration in MHCC97H^AMKD cells promotes transcription factors expression. *p < 0.05, **p < 0.005. (E) R‐2HG decreased FTO and transcription factors expression in PLC^AMOE cells

FIGURE 8

Spermidine promotes FTO expression via IQGAP1. (A and B) Co‐IP and immunofluorescence assays were used to detect the direct interaction of AMD1 and FTO. (C) Quantification of the spermidine level in MHCC97H cells transfected with AMD1 knockdown and the negative control vectors. **p < 0.005 (D) Western blot analysis was used to detect the expression change of FTO, NANOG, SOX2, and KLF4 on spermidine treatment. (E) Immunofluorescence assays were used to detect the direct interaction of FTO and IQGAP1. (F and G) Co‐IP and western blot analysis were used to evaluate the associativity of FTO and IQGAP1 and the phosphorylation of FTO on spermidine treatment. IQCAP1 was used as a loading control. (H) Ubiquitination assay for the effects of spermidine on FTO ubiquitination. (I) Western blot analysis was used to detect the expression change of FTO, NANOG, SOX2, and KLF4 on IQGAP1 deficient condition

FIGURE 9

Mechanisms by which AMD1 promotes hepatocellular carcinoma cell stemness