GALNT6, transcriptionally inhibited by KLF9, promotes osteosarcoma progression by increasing EFEMP1 expression via O-glycosylation modification.

Osteosarcoma (OS) is one of the deadliest malignancies in adolescents and its treatment status and prognosis remain unsatisfactory. N-acetylgalactosamine transferase 6 (GALNT6), one of the key enzymes regulating O-glycosylation, functions vary in different types of cancer. Currently, the function of GALNT6 in OS is unclear. Our results showed that GALNT6 was highly expressed in OS tissues, and the patients with higher GALNT6 expression exhibited a lower overall survival rate than patients with lower GALNT6 expression. We constructed the GALNT6-knockdown and GALNT6-overexpression vectors based on Tet-on system and packaged lentiviral particles to modulate GALNT6 expression. GALNT6 silencing impaired OC cell growth and metastasis both in vivo and vitro. Kruppel-like factor 9 (KLF9), a transcription factor known to suppress OS progression, was found to block GALNT6 transcription by binding to its promoter. Meanwhile, GALNT6 overexpression restored the effects caused by KLF9 upregulation. GALNT6 was known to affect protein stability by O-glycosylation regulation, thus the label-free proteomics combined with co-immunoprecipitation/mass-spectrum (MS) analysis were conducted to identify the potential mechanism of GALNT6 in promoting OS progression. EGF-containing fibulin extracellular matrix protein 1 (EFEMP1), contained several O-glycosylation sites and was upregulated in GALNT6 overexpressing cells (Log₂FC = 1.3195, p = 0.0160), attracted our attention. We demonstrated that GALNT6 interacted with EFEMP1 at protein level. The O-glycosylation of EFEMP1 was increased by GALNT6 overexpression, which slowed the degradation rate of EFEMP1. EFEMP1 knockdown reversed the effects of GALNT6 overexpression. Collectively, our observations demonstrate that KLF9/GALNT6/EFEMP1 may be a promising direction for OS treatment.