In vitro assessment of BBI608 in 2D and 3D culture models for drug repositioning in oral squamous cell carcinoma.

STAT3 is abnormally activated in several types of cancer, and elevated nuclear levels of STAT3 are strongly associated with poor prognosis in oral squamous cell carcinoma (OSCC). Despite ongoing progress in developing targeted therapies, there is no Food and Drug Administration‑approved drug currently targeting STAT3 in OSCC. To evaluate the anticancer effects of BBI608, a potent STAT3 inhibitor, in two human OSCC cell lines (HSC‑3 and HSC‑4), various two‑dimensional (2D) or 3D in vitro analyses were performed, including western blot analysis, colony formation assay, DAPI staining, sub‑G₁ population analysis and Annexin V/PI staining. The molecular mechanisms of BBI608 were also determined using cross‑linking assay, nuclear and cytoplasmic fractionation assay, reverse transcription‑quantitative PCR and chromatin immunoprecipitation assay. In the present study, it was observed that human HSC‑3 and HSC‑4 OSCC cells exhibited higher levels of phosphorylated (p)‑STAT3 compared with those in immortalized oral keratinocytes (iHOK cells). BBI608 inhibited cell proliferation in a concentration‑dependent manner and triggered caspase 3‑dependent apoptosis in HSC‑3 and HSC‑4 cells. Additionally, BBI608 reduced the nuclear translocation of p‑STAT3 in HSC‑3 and HSC‑4 cells compared with that in DMSO‑treated cells. Mechanistically, BBI608 modulated anti‑apoptotic STAT3 downstream genes: Survivin expression was regulated at the transcriptional level, while myeloid cell leukemia‑1 expression was modulated post‑translation via proteasomal degradation. Consistent with the results from 2D culture, BBI608 showed effective anticancer effects against OSCC spheroids in 3D culture. These results suggest that BBI608 effectively inhibits STAT3 activation in both 2D and 3D models, offering a promising therapeutic strategy and supporting its potential for repurposing in patients with OSCC who exhibit elevated STAT3 activity.