Nanog Mediates Tobacco Smoke-induced Enhancement Of Renal Cancer Stem Cell Properties

Background: Renal cell carcinoma(RCC)is one of the most commonly urinary cancers,and accounts for around 3.5% of all human caners.Due to its insensitivity to chemotherapy and radiotherapy,leading to patients with metastatic disease a poor clinical outcome.Cancer stem cells(CSCs)are associated with cancer initiation,growth,metastasis,relapse and therapy resistance.Elimination of CSCs may cure cancer.Established risk factors of RCC include tobacco smoking,hypertension,and obesity.Tobacco smoke contains many carcinogens,and can promote development of cancer via different mechanisms.Transcript factor Nanog plays an important role in maintaining and regulating characteristics of CSCs.The expression level of Nanog is negatively related with prognosis of patients with renal cancer.However,few researches have focused on the potential role of Nanog in renal CSCs.Purpose: In this study,the regulatory role of Nanog in tobacco smoke induced-enhancement of renal cancer stem cell properties was investigated.Methods:(1)Immunohistochemistry(IHC)staining and Western blotting assay were used to detect the protein level of Nanog in RCC patients with or without smoking history.(2)Serum-free medium(SFM)was used to enrich renal CSCs.Sphere formation ability and CSCs markers(Nanog,CD133,CD44,Oct4,ALDH1A1)expression were used to evaluate CSCs properties.(3)Renal cancer cells(ACHN and 786-O)were transfected with plasmids to overexpress Nanog,and cultured in SFM to examine CSCs properties.(4)ACHN and 786-O cells were continuously exposed to different concentrations of cigarette smoke extract(CSE)for 8 weeks and then cultured in SFM.Flow cytometric analysis was used to detected the percentage of CD133+ population and cell cycle distribution.PCR and Western blotting assay were used to examine the expression levels of relevant genes.(5)CSE-exposed ACHN and 786-O cells were transfected with Nanog-si RNA,and cultured in SFM to examine CSCs properties.Results: The level of Nanog was elevated in RCC patients with smoking history.Cell spheres cultured in SFM showed the characteristics of CSCs.Overexpression of Nanog enhanced sphere forming ability and increased the levels of stem cell markers,including CD133,CD44,Oct4 and ALDH1A1.CSE increased the size and number of renal cell spheres,CD133+ cell population,and stimulated stem cell markers and proliferation-related proteins expression.Meanwhile,CSE accelerated G1 phase to S phase transition in cell cycle.Downregulation of Nanog counteracted the promotive effects of CSE on renal CSCs.Conclusion: These data suggested that Nanog mediated tobacco smoke induced-renal CSCs properties.Our findings may provide new insights into the molecular mechanism of tobacco smoke-related renal tumorigenesis and search for new targets of renal cancer.