SKA 1 induces de novo MTX ‐resistance in osteosarcoma through inhibiting FPGS transcription

: De novo methotrexate (MTX)-resistance, whose underlying mechanism remains largely unknown, usually leads to very poor prognosis in patients with osteosarcoma (OS). In this study, we established the de novo MTX-resistant OS cell line SF-86 and identified the candidate gene spindle and kinetochore associated complex subunit 1 (SKA1) as potentially related to de novo MTX-resistance. Analysis of a cohort of 95 OS patients demonstrated that SKA1 overexpression significantly correlated with de novo MTX-resistance and poor 5-year survival. Mechanistically, SKA1 overexpression lead to a downregulation of folylpoly-γ-glutamate synthetase (FPGS), a key enzyme that converts MTX into its active form, MTX-PG. We further demonstrated that SKA1 interacts with DNA-directed RNA polymerase II subunit RPB3. ChIP analysis revealed that RPB3 binds the promoter region of the FPGS gene and triggers FPGS transcription upon MTX treatment in SW1353, a MTX-sensitive OS cell line lacking endogenous SKA1 expression. On the contrary, this process is blocked in SF-86 cells due to the formation of an inhibitory SKA1-RPB3 complex. Furthermore, downregulation of SKA1 levels restores MTX sensitivity in SF-86. Collectively, our study has established the de novo MTX-resistant cell line SF-86 and identified SKA1 as a novel regulator of FPGS, playing a key role in the development of de novo MTX-resistance in OS.