title: Defective autophagy leads to the suppression of stem-like features of CD271+osteosarcoma cells.
abstract: BACKGROUND
  As an important stress-response mechanism, autophagy plays crucial role in the tumor formation and drug resistance of cancer cells including osteosarcoma (OS). OS cancer stem cells (CSCs) also are considered a key factor of tumorigenesis, drug resistance and tumor recurrence. However, the relationship between autophagy and OS CSCs still remains unclear.
  
  
  METHODS
  CD271+ OS CSCs and CD271- OS cells were isolated by magnetic activated cell sorting. The autophagy level was evaluated by the mRNA expression of autophagy genes, the protein level of LC3II and p62, and the mean number of GFP-LC3 dot per cell. Lentivirus-delivered specific shRNA was utilized to inhibit the corresponding gene expression. The cell viability was examined with CCK8 assay. The cell proliferation level was detected with BrdU staining assay. Cell death was determined by Annexin V/PI double staining of fluorescence activated cell sorting, lactate dehydrogenase release and caspase-3 activity. Tumorigenicity ability was evaluated by colony and sphere formation assay, the protein expression of stemness markers and tumor formation in nude mice.
  
  
  RESULTS
  Our data indicated that CD271+ OS CSCs had a similar basic autophagy level with CD271- OS cells. Autophagy deficiency had no observable effects on the levels of cell proliferation and death both in CD271+ and CD271- OS cells under normal condition. However, CD271+ OS cells showed a higher autophagy activity than CD271- OS cells under hypoxia and low nutrient (LH) condition. Moreover, autophagy-deficient CD271+ OS cells lost the advantage of tolerance to LH condition compared to CD271- OS cells. Meanwhile, autophagy deficiency enhanced the sensitivity to chemotherapeutics in the CD271+ cells to the comparable level in the CD271- cells. More importantly, deficient-autophagy decreased the protein expression of stemness markers and caused the disappearance of the superiority in tumorigenicity in vitro and vivo in CD271+ OS cells.
  
  
  CONCLUSIONS
  The results above demonstrated that autophagy contributes to the stem-like features of CD271+ OS CSCs. Inhibition of autophagy is a promising strategy in the CSCs-targeting OS therapy.
authors.affiliation: Department of Spinal Disease, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437, People's Republic of China.
authors.forename: Dong
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Department of Spinal Disease, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437, People's Republic of China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Department of Spinal Disease, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437, People's Republic of China.
authors.forename: Hao
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Yin
authors.affiliation: Department of Spinal Disease, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437, People's Republic of China.
authors.forename: Jiajun
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Spinal Disease, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437, People's Republic of China.
authors.forename: Jun
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Spinal Disease, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437, People's Republic of China.
authors.forename: Tianxiang
authors.initials: T
authors.lastname: He
authors.affiliation: Department of Spinal Disease, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437, People's Republic of China. yangcl_yy@yeah.net.
authors.forename: Chunlei
authors.initials: C
authors.lastname: Yang
authors.affiliation: Department of Orthopedics, Huadong Hospital Affiliated to Fudan University, No. 221 West Yan An Road, Shanghai, 200040, People's Republic of China. liang333cn@aliyun.com.
authors.forename: Chengwei
authors.initials: C
authors.lastname: Liang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001343:Autophagy / Q000235:genetics; D063646:Carcinogenesis / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics*; D009364:Neoplasm Recurrence, Local / Q000235:genetics / Q000473:pathology; D014411:Neoplastic Stem Cells / Q000473:pathology; D009419:Nerve Tissue Proteins / Q000235:genetics*; D012516:Osteosarcoma / Q000235:genetics* / Q000473:pathology; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000235:genetics*; D017475:Receptors, Nerve Growth Factor / Q000235:genetics*; D023041:Xenograft Model Antitumor Assays
chemical_list: C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; C484065:NGFR protein, human; D009419:Nerve Tissue Proteins; D016271:Proto-Oncogene Proteins c-myc; D017475:Receptors, Nerve Growth Factor
keywords: Autophagy; CD271; Cancer stem cells; Osteosarcoma