Activation of microRNA-494-targeting Bmi1 and ADAM10 by silibinin ablates cancer stemness and predicts favourable prognostic value in head and neck squamous cell carcinomas

Abstract

Tumor initiating cells (TICs) possessing cancer stemness were shown to be enriched after therapy, resulting in the relapse and metastasis of head and neck squamous cell carcinomas (HNC). An effective therapeutic approach suppressing the HNC-TICs would be a potential method to improve the treatments for HNC. We observed that the treatment of silibinin (SB) dose dependently down-regulated the ALDH1 activity, CD133 positivity, stemness signatures expression, self-renewal property, and chemoresistance in ALDH1+CD44+ HNC-TICs. Using miRNA-microarray and mechanistic studies, SB increased the expression of microRNA-494 (miR-494) and both Bmi1 and ADAM10 were identified as the novel targets of miR-494. Moreover, overexpression of miR-494 results in a reduction in cancer stemness. However, knockdown of miR-494 in CD44−ALDH1−non-HNC-TICs enhanced cancer stemness and oncogenicity, while co-knockdown of Bmi1 and ADAM10 effectively reversed these phenomena. Mice model showed that SB treatment by oral gavage to xenograft tumors reduced tumor growth and prolonged the survival time of tumor-bearing mice by activation of miR-494-inhibiting Bmi1/ADAM10 expression. Survival analysis indicated that a miR494highBmi1lowADAM10low phenotype predicted a favourable clinical outcome. We conclude that the inhibition of tumor aggressiveness in HNC-TICs by SB was mediated by up-regulation miR-494, suggesting that SB would be a valuable anti-cancer drug for treatment of HNC.

Sulforaphane targets cancer stemness and tumor initiating properties in oral squamous cell carcinomas via miR-200c induction

“…Red (> 650 nm) fluorescence emission from 10,000 cells illuminated with blue (488 nm) excitation light was measured with a FACSCalibur (Becton Dickinson, Mountain View, CA, USA) using CellQuest software. 21 …”

Aberrant SSEA-4 upregulation mediates myofibroblast activity to promote pre-cancerous oral submucous fibrosis

“…Mounting evidences have demonstrated that the dys-regulation of stemness marker expression contributes to the pathogenesis of diseases, especially its roles in tumorigenesis2021. Some aberrant stemness marker expressions such as Bmi1, CD133, or Sox2 have been found to be involved in the progression of fibrosis.…”

“…qRT–PCRs of mRNAs are reverse-transcribed using the Superscript III first-strand synthesis system for RT–PCR (Invitrogen Life Technologies, Carlsbad, CA, USA). qRT-PCR reactions on resulting cDNAs were performed on an ABI StepOne™ Real-Time PCR Systems (Applied Biosystems)20. The primer sequences listed below:…”

“…Red (>650 nm) fluorescence emission from 10,000 cells illuminated with blue (488 nm) excitation light is measured with a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) using CellQuest software (Becton Dickinson, San Jose, CA, USA)20.…”

“…Western blot analysis was performed by previously described protocols20. The primary antibodies will be those against α-SMA (1A4), vimentin (9E7E7), and rabbit ployclonal anti-human COL1A1 antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA)20.…”