ΔNp63α promotes cigarette smoke-induced renal cancer stem cell activity via the Sonic Hedgehog pathway

Abstract

Cigarette smoke (CS) has been generally recognized as a chief carcinogenic factor in renal cell carcinoma (RCC). The stimulative effect of CS on renal cancer stem cells (RCSCs) has been described previously. The Sonic Hedgehog (SHH) pathway plays an essential role in self-renewal, cell growth, drug resistance, metastasis, and recurrence of cancer stem cells (CSCs). Renal cancer-related gene ΔNp63α is highly expressed in renal epithelial tissues and contributes to the RCSCs characteristics of tumors. The aim of this study was to elucidate the role of ΔNp63α and the SHH pathway on the activity of RCSCs induced by CS through a series of in vivo and in vitro studies. It was shown that in renal cancer tissues, ΔNp63α and RCSCs markers in smokers are expressed higher than that in non-smokers. RCSCs were effectively enriched by tumor sphere formation assay. Besides, CS increased the expression of RCSCs markers and the capability of sphere-forming in vitro and in vivo. Moreover, the SHH pathway was activated, and the specialized inhibitor alleviated the promotion of CS on RCSCs. ΔNp63α activated the SHH pathway and promoted CS-induced enhancement of RCSCs activity. These findings indicate that ΔNp63α positively regulates the activity of CS-induced RCSCs via the SHH pathway.

Figure 1-. CS promotes the activity expression of ΔNp63α and CSCs markers in patients with kidney cancer. Western blot (A) and immunohistochemistry (B) detected the expression levels of ΔNp63α and RCSCs markers in the two groups (H&E, x200). *p<0.05, **p<0.01.

Figure 2 -. Enrichment of RCSCs by SFM. Renal cancer cells 786-O and ACHN were cultured with SFM and SFM for 5 days, respectively. (A) Microscopy was performed to observe the morphological changes of renal cancer cells (H&E, x100). The protein and mRNA expression levels of RCSCs markers were detected by western blot (B) and qRT-PCR (C). (D) The quantity of CD44-labeled positive cells in renal cancer cells was detected by flow cytometry. (E) Tumor size and weight were measured in SSM and SFM groups. (F) The expression of CD44 and ΔNp63α in SSM and SFM tissues was analyzed by immunohistochemistry (H&E, x200). *p<0.05, **p<0.01.

Figure 3 -. CS promotes the activity of RCSCs. RCSCs were administrated with various concentrations of CSE. (A) The effects of different concentrations of CSE on 786-O and ACHN tumor spheres were detected by CCK-8 assay. (B) The changes in the morphology and number of RCSCs treated with different concentrations of CSE were observed under a microscope (H&E, x100). The effects of CSE on the protein and mRNA expression levels of RCSCs markers were detected by western blot (C) and qRT-PCR (D). (E) Flow cytometry was utilized to determine the number of CD44-labeled positive cells in RCSCs treated with CSE. (F) The size and weight of tumors in the armpit of nude mice in the CSE-treated group were measured. (G) The immunohistochemical technique was used to detect the expression of CD44 and ΔNp63α in tumor tissues of the CSE-treated group (H&E, x200). *p<0.05, **p<0.01.

Figure 4 -. ΔNp63α promotes CSE-induced activity in RCSCs. 786-O and ACHN tumor spheres were transfected with overexpression plasmids and targeted small interfering RNA. (A) Western blot analysis of ΔNp63α expression in CSE-treated tumorspheres. (B) Tumor spheres transfected with overexpression plasmid and siRNA were observed by microscopy (H&E, x200). (C) western blot analysis of the expression of ΔNp63α, CD44, Oct4, and SOX2 in tumorspheres transfected with overexpression plasmids. (D) Western blot analysis of the expression of ΔNp63α, CD44, Oct4, and SOX2 in the tumorspheres after siRNA transfection. *p<0.05, **p<0.01.

Figure 5-. SHH signaling pathway is promoted in the CSE-induced acquisition of activity in RCSCs. (A) Western blot analysis of the expression levels of SHH signaling pathway-related proteins Shh, Smo, Gli1, and Gli2 in CSE-treated RCSCs. (B) Changes in the size and number of tumor spheres after treatment with SHH signaling pathway inhibitor Vismodegib were observed under a microscope (H&E, x100). (C) The expression levels of CD44, Oct4, and SOX2 in tumor spheres treated with Vismodegib were detected by Western blot. * p <0.05, ** p <0.01.

Figure 6 -. ΔNp63α positively regulates CS-induced activity in RCSCs through the SHH pathway. (A, B) Western blot was used to determine the expression levels of SHH signaling pathway-related proteins Shh, Smo, Gli1, and Gli2 in tumor spheres with or without ΔNp63α overexpression. (C) Co-immunoprecipitation assay examined the interaction between ΔNp63α and the SHH signaling pathway. * p <0.05, ** p <0.01.

Figure 7 -. Schematic diagram.