ALKBH5 activates FAK signaling through m6A demethylation in ITGB1 mRNA and enhances tumor-associated lymphangiogenesis and lymph node metastasis in ovarian cancer

Theranostics. 2023 Jan 5;13(2):833-848. doi: 10.7150/thno.77441. eCollection 2023.

Abstract

Background: Lymph node (LN) metastasis is common in patients with epithelial ovarian cancer (EOC) and is associated with poor prognosis. Tumor-associated lymphangiogenesis is the first stage of LN metastasis. Research on lymphangiogenesis and lymph node metastases can help develop new anti-LN-targeted therapies. Aberrant N6-methyladenosine (m6A) modifications have been reported to be linked to LN metastasis in several cancers, however, their role in EOC lymphangiogenesis and LN metastasis remains unclear. Methods: m6A levels in EOC tissues with or without LN metastases were evaluated by dot blot analysis. Real-time polymerase chain reaction (PCR) and immunofluorescence were used to examine the expression of m6A-related enzymes. Additionally, in vitro and in vivo functional studies were performed to discover the importance of the AlkB homolog 5 (ALKBH5) gene in EOC lymphatic metastasis. To identify the downstream target genes regulated by ALKBH5, we performed RNA pulldown, RNA-binding protein immunoprecipitation-quantitative PCR, co-immunoprecipitation, m6A-modified RNA immunoprecipitation-quantitative PCR, and luciferase reporter assays. Results: m6A modification was reduced in ovarian cancers with LN metastases. ALKBH5 overexpression increased tumor-associated lymphangiogenesis and LN metastasis both in vitro and in vivo. ALKBH5 overexpression also reversed the m6A modification in ITGB1 mRNA and suppressed the YTHDF2 protein-mediated m6A-dependent ITGB1 mRNA degradation, which resulted in increased expression of ITGB1 and phosphorylation of the focal adhesion kinase (FAK) and Src proto-oncogene proteins, thereby increasing LN metastasis. Furthermore, hypoxia induced the expression of hypoxia inducible factor 1 subunit alpha, which increased ALKBH5 expression and enhanced LN metastasis in EOC. Conclusions: The ALKBH5/m6A-ITGB1/FAK signalling axis is important in ovarian cancer lymphangiogenesis and LN metastasis. Antibodies that block ITGB1 and FAK kinase-inhibitors are promising anti-metastatic agents.

Keywords: ALKBH5; ITGB1; N6-methyladenosine; epithelial ovarian cancer; lymphatic metastasis.

MeSH terms

  • AlkB Homolog 5, RNA Demethylase / genetics
  • Carcinoma, Ovarian Epithelial / genetics
  • Demethylation
  • Female
  • Focal Adhesion Protein-Tyrosine Kinases / metabolism
  • Humans
  • Lymphangiogenesis* / genetics
  • Lymphatic Metastasis
  • Ovarian Neoplasms* / genetics
  • RNA / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism

Substances

  • RNA, Messenger
  • Focal Adhesion Protein-Tyrosine Kinases
  • RNA
  • ALKBH5 protein, human
  • AlkB Homolog 5, RNA Demethylase