Cigarette smoke stimulates the stemness of renal cancer stem cells via Sonic Hedgehog pathway

Abstract

Cancer stem cells (CSCs) are essentially responsible for tumor initiation, growth, progression, metastasis and recurrence, and cigarette smoke (CS) is closely involved in the occurrence and development of kidney cancer. However, the effect of CS on renal CSCs has not been elucidated yet. In the present study, tumorsphere formation assay was used to enrich renal CSCs from 786-O and ACHN cells. We illustrated that CS effectively promoted renal CSCs stemness by enhancing tumorsphere formation, increasing the expression of renal CSCs markers (CD133, CD44, ALDHA1, Oct4, and Nanog) and elevating CD133⁺ cell population. Moreover, our results showed that CS triggered the activation of Sonic Hedgehog (SHH) pathway, while inhibition of SHH pathway dampened the promotive effects of CS on renal CSCs. Finally, higher levels of renal CSCs markers and SHH pathway-related proteins were observed in kidney cancer tissues from smokers than non-smoking cancer tissues. Taken together, these results demonstrated the important role of SHH pathway in regulating CS-induced renal CSCs stemness augment. Findings from this study could provide new insight into the molecular mechanisms of CS-elicited stemness of renal CSCs.

Fig. 1. Tumorsphere formation assay of renal CSCs in SFM condition.

786-O and ACHN cells were grown in serum-supplied medium (SSM) and serum-free medium (SFM) for 5 days, respectively. a Representative images of tumorspheres. Bar, 100 μm. b The protein expression of renal CSCs markers (CD133, CD44, ALDHA1, Oct4, and Nanog) was measured by Western blotting. c The mRNA expression level of renal CSCs was measured by qRT-PCR. d The number of CD133⁺ cells in 786-O and ACHN cells was detected by flow cytometry. Data are expressed as mean ± standard deviation (SD). *p < 0.05, **p < 0.01 compared with SSM group

Fig. 2. CSE promotes the characteristics of renal CSCs.

Different concentrations of CSE were added to 786-O and ACHN tumorspheres for 5 days. a Cell viability was examined by CCK-8 assay. b The representative images of 786-O and ACHN sphere-forming cells were obtained. Bar, 100 μm. c The number of tumorspheres was counted and normalized to the control group. d Percentage of CD133⁺ cells after CSE treatment for 5 days. e Western blotting and f qRT-PCR were used to analyze protein and mRNA levels of renal CSCs markers. g Expression of cell proliferation associated protein was determined by western blotting. h Immunofluorescent staining of CD44 in 786-O and ACHN spheroids. Bar, 100 μm. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 compared with control

Fig. 3. CSE activates SHH pathway in renal CSCs.

Western blotting revealed the upregulation of SHH pathway after CSE treatment for 5 days

Fig. 4. CSE promotes renal CSCs stemness via upregulating SHH pathway.

a–c 786-O and ACHN tumorspheres were treated with 0.1% CSE with/without 10 mM Vismodegib for 5 days. a Western blotting analysis of SHH pathway proteins. b Images of tumorsphere formation. Bar, 100 μm. c The number of tumorspheres was calculated and normalized to control group. d Western blotting analysis of renal CSCs markers

Fig. 5. Alteration of renal CSCs correlates with the smoking status of renal cell carcinoma patients.

Clinical kidney cancer specimens were collected from smoker and non-smoker renal cell carcinoma patients. a Western blotting of renal CSCs markers. b Quantitative analysis of Western blotting of renal CSCs markers. c Immunohistochemistry (IHC) assay of renal CSCs markers, 200× magnification. d Percentage of IHC positive cells in tissue samples. e Western blotting of SHH pathway proteins. f Quantitative analysis of Western blotting of SHH pathway proteins. non-CS: non-smoker cancer patients; CS: smoker cancer patients. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 compared with non-smoke cancer patients