MicroRNA-485-5p targets keratin 17 to regulate oral cancer stemness and chemoresistance via the integrin/FAK/Src/ERK/ß-catenin pathway.

BACKGROUND: The development of drug resistance in oral squamous cell carcinoma (OSCC) that frequently leads to recurrence and metastasis after initial treatment remains an unresolved challenge. Presence of cancer stem cells (CSCs) has been increasingly reported to be a critical contributing factor in drug resistance, tumor recurrence and metastasis. Thus, unveiling of mechanisms regulating CSCs and potential targets for developing their inhibitors will be instrumental for improving OSCC therapy. METHODS: siRNA, shRNA and miRNA that specifically target keratin 17 (KRT17) were used for modulation of gene expression and functional analyses. Sphere-formation and invasion/migration assays were utilized to assess cancer cell stemness and epithelial mesenchymal transition (EMT) properties, respectively. Duolink proximity ligation assay (PLA) was used to examine molecular proximity between KRT17 and plectin, which is a large protein that binds cytoskeleton components. Cell proliferation assay was employed to evaluate growth rates and viability of oral cancer cells treated with cisplatin, carboplatin or dasatinib. Xenograft mouse tumor model was used to evaluate the effect of KRT17- knockdown in OSCC cells on tumor growth and drug sensitization. RESULTS: Significantly elevated expression of KRT17 in highly invasive OSCC cell lines and advanced tumor specimens were observed and high KRT17 expression was correlated with poor overall survival. KRT17 gene silencing in OSCC cells attenuated their stemness properties including markedly reduced sphere forming ability and expression of stemness and EMT markers. We identified a novel signaling cascade orchestrated by KRT17 where its association with plectin resulted in activation of integrin ß4/α6, increased phosphorylation of FAK, Src and ERK, as well as stabilization and nuclear translocation of ß-catenin. The activation of this signaling cascade was correlated with enhanced OSCC cancer stemness and elevated expression of CD44 and epidermal growth factor receptor (EGFR). We identified and demonstrated KRT17 to be a direct target of miRNA-485-5p. Ectopic expression of miRNA-485-5p inhibited OSCC sphere formation and caused sensitization of cancer cells towards cisplatin and carboplatin, which could be significantly rescued by KRT17 overexpression. Dasatinib treatment that inhibited KRT17-mediated Src activation also resulted in OSCC drug sensitization. In OSCC xenograft mouse model, KRT17 knockdown significantly inhibited tumor growth, and combinatorial treatment with cisplatin elicited a greater tumor inhibitory effect. Consistently, markedly reduced levels of integrin ß4, active ß-catenin, CD44 and EGFR were observed in the tumors induced by KRT17 knockdown OSCC cells. CONCLUSIONS: A novel miRNA-485-5p/KRT17/integrin/FAK/Src/ERK/ß-catenin signaling pathway is unveiled to modulate OSCC cancer stemness and drug resistance to the common first-line chemotherapeutics. This provides a potential new therapeutic strategy to inhibit OSCC stem cells and counter chemoresistance.

[An experimental study on the role of keratin in angiogenesis in vitro].

OBJECTIVE: To investigate the effect of keratin 17 (K-17) on the migration, proliferation and tube formation of human umbilical vein endothelial cell (HUVEC), and to realize the role of K-17 in angiogenesis. METHODS: After HUVEC were cultured in DMEM medium supplemented with 10%FBS overnight, K-17-siRNA-mixture (experimental group) and control-siRNA-mixture (negative control group) were added into HUVEC, respectively, by Lipofectamine 2000 transfection assay, and the final concentration of the siRNA was 50 nmol/L. Lipofectamine 2000 alone was used as the control. After the cells were cultured for 36 hours, the cell proliferation ability was detected by cell counting. After 30-hour culture, the cell's abilities of migration and differentiation to tube were detected by 24-well Millicell units and the collagen gel assay, respectively. In addition, non-siRNA-treated HUVEC were cultured for 24 hours in DMEM medium supplemented with 10%FBS (group A), 2%FBS (group B) and 2%FBS+10 ng/mL bFGF (group C), respectively, and then the expression of K-17 in HUVEC was detected by RT-PCR and Western blot. RESULTS: After the treatment with K-17-siRNA for 36 hours, HUVEC exhibited no significant difference in the proliferation, compared with both control and negative control groups (P > 0.05). After transfected with K-17-siRNA for 30 hours, the number of HUVEC in the experimental group which migrated from the upper chamber to the lower chamber of Millicell wells within 24 hours (3719.0 +/- 319.0) was smaller than both control (7 437.5 +/- 212.0) and negative control (7 356.3 +/- 795.7) groups, with significant difference (P < 0.01). However, there was no significant difference between the control group and the negative control group (P > 0.05). After HUVEC were transfected with K-17-siRNA for 30 hours, the number of tubes in the experimental group, the negative control group and the control group in 24 hours was (1.1 +/- 0.5), (3.6 +/- 0.5) and (3.2 +/- 0.6) per field, respectively. The experimental group was significantly different from both control and negative control groups (P < 0.01), and there was no significant difference between the negative control group and the control group (P > 0.05). The expression of K-17 protein in HUVEC in groups A, B and C was 0.25 +/- 0.02, 0.08 +/- 0.01 and 0.72 +/- 0.03, respectively. There was significant difference among these three groups (P < 0.01). CONCLUSION: K-17 has no impact on cell proliferation, but may augment endothelial cell migration, which may facilitate angiogenesis.