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.

Keywords: head and neck squamous cell carcinomas; microRNA-494; silibinin; tumor initiating cells.

Figure 1. SB treatment suppresses the stem-like properties of ALDH⁺CD44⁺ HNC-TICs

A. SG and ALDH⁺CD44⁺ HNC-TICs were treated with various concentrations of SB up to 200 μM for 24 hours. Cell survival was assessed by MTT assay and was presented as percent survival relative to untreated cells. B. ALDH1⁺CD44⁺ HNC-TICs treated with or without SB were subjected to a self-renewal secondary sphere-forming assay. The number of secondary spheres was calculated and was presented. The CD133 positivity C. and ALDH1 activity D. of HNC-TICs treated with or without SB was assessed by flow cytometry. E. The indicated stemness markers expression levels (Oct4, Nanog, and Nestin) in the SB-treated ALDH⁺CD44⁺ HNC-TICs were analyzed by quantitative real-time PCR (upper panel) and western blotting (lower panel). The experiments were repeated three times and representative results were shown. Results are means ± SD. *, p < 0.05 vs. Control.

Figure 2. Oncogenicity and EMT traits of HNC-TICs are abolished by SB treatment

Different concentration SB-treated HNC-TICs were subjected to soft agar colony formation assay A., migration assay B., matrix invasion assay C., and vasculogenic mimicry assay D.. E. immunoblotting analysis of EMT-related markers (Snail, ZEB1, Vimentin, and E-cadherin) in control and SB-treated HNC-TICs was determined. The experiments were repeated three times and representative results were shown. Results are means ± SD. *, p < 0.05 vs. Control.

Figure 3. SB sensitized HNC-TICs to conventional chemo-treatment

A. Parental HNC cells and HNC-TICs with control or SB treatment were subjected to treatment with different concentrations of doxorubicin or cisplatin or 5-FU for cell viability assessment. *, p < 0.05 HNC-TICs vs. Parental; #, p < 0.05 SB vs. HNC-TICs. B. The expression levels of ABCG2 in the cells indicated were determined by flow cytometry analysis. C. Annexin V-positive apoptosis cells were assessed in HNC-TICs synergetically treated with SB combined with cisplatin chemotherapy. Colony-forming ability D., migration E., and invasion ability F. was assessed in HNC-TICs synergetically treated with SB combined with cisplatin chemotherapy. *, p < 0.05 SB vs. HNC-TICs; #, p < 0.05 SB+Cisplatin vs. SB alone.

Figure 4. SB activates miR-494 and miR-494 direct targets Bmi1 and ADAM10

A. The indicated miRNAs expression levels in the SB-treated HNC-TICs were analyzed by miRNAs microarray analysis. B. qPCR analysis was applied to analyzed the relative miR-494 expression level in SB dose-dependently treated HNC-TICs. C. The wild-type (WT) and serial deleted forms of the 3′UTR reporter Bmi1 and ADAM10 plasmids were constructed as shown in the schematic presentation. D. Schematic presentation of the constructed Bmi1 and ADAM10 3′UTR reporter plasmids were used in this study. E. The wild-type and deleted forms of the Bmi1 and ADAM10 reporters were co-transfected with miR-494 or empty vector into HNC-TICs. The luciferase activity of each combination was assessed and was presented; F. Similar reporter assays were performed in HNC-TICs with wild-type (WT) and mutated (Mut) reporter plasmids. The results of the luciferase assays indicated that only WT reporter activity was inhibited by miR-494. G. Expression level of miR-494 in HNC-TICs transfected with pLV-miR-scrambled (pLV-miR-Scr.) and pLV-miR-494. (H) The protein expression levels of ADAM10 and Bmi1 in miR-494-transfected HNC-TICs were analyzed by western blot.

Figure 5. miR-494 modulates cancer stemness through targeting Bmi1 and ADAM10

A. miR-494 expression in ALDH⁺CD44⁺ and ALDH1⁻CD44⁻ cells was assessed by quantitative real-time PCR and presented as relative fold-changes. B. HNC-TICs with pLV-miR-Scr. and pLV-miR-494 were subjected to a sphere formation assay. The quantitative sphere number was presented in the chart at the right. Only spheres with a diameter more than 50 μm were counted. C. HNC-TICs transfected with pLV-miR-Scr. or pLV-miR-494 were then subjected to invasion assay. D. Spg-miR-494-ALDH1-CD44- non-HNC-TICs with or without co-knockdown of Bmi1 and ADAM10 expression were subjected to western blotting to assess the expression level of Bmi1 and ADAM10. ALDH1⁻CD44⁻ HNC cells transfected with indicated plasmids were subjected to a sphere formation assay E., colony formation assay F., invasion assay G., and the tumor measurements in xenografts for 6 weeks H.. *, p < 0.05 Spg. 494 or Spg. 494+sh-Luc. vs. Spg. Ctrl.; #, p < 0.05 Spg. 494+sh-Bmi1+sh-ADAM10 vs. Spg. 494+sh-Bmi1+sh-ADAM10.

Figure 6. Oral delivery SB treatment suppresses tumor growth and increases animal survival

After subcutaneous implantation of HNC-TICs, BALB/c nude mice (N = 5 for each group) were oral-feeding treated with saline or SB and then analyzed for the bioluminescence signal A., average tumor weight B., growth of tumor C., and average mice body weight D.. The emitted by the implanted HNC-TICs was monitored for 20 days and was photographed. Mice were sacrificed, and tumor sections as indicated treatments were assessed for relative miR-494 expression E. and stained using specific antibodies against Bmi1 and ADA10 by immunohistochemistry F.. G. The survival rate of the mice treated with saline or SB was monitored for up to 12 weeks and is presented in the graph (each group; n = 12).

Figure 7. Clinical significance of miR494^lowBmi1^highADAM10^high signature expression in specimens from HNC patients

A. Adjacent adjacent noncancerous matched tissues (NCMT; n = 45), and paired tissue samples from tumor (T; n = 45) as well as lymph node metastatic (LN; n = 45) lesions in HNC patients were subjected to analysis for the expression levels of miR-494. B. Samples from patients with different stages (stage I to IV) of HNC were collected and subjected to qPCR for miR-494 level. C. IHC staining to assess the protein expression of Bmi1 and ADAM10 in high-grade and low grade HNC patient specimens. D. An overall survival correlation analysis was performed for HNC patient samples expressing different levels of the indicated molecules. The miR494^lowBmi1^highADAM10^high signature correlated with the worst survival rate. E. A schematic representation of the SB-activated miR-494-targeting Bmi1 and ADAM10 signaling proposed in the current study.