title: SMURF1 silencing diminishes a CD44-high cancer stem cell-like population in head and neck squamous cell carcinoma.
abstract: BACKGROUND
  Bone morphogenetic protein (BMP) signaling is thought to play key roles in regulating the survival and maintenance of cancer stem cells (CSCs), which contribute to disease recurrences and treatment failures in many malignances, including head and neck squamous cell carcinoma (HNSCC). Intracellular BMP signaling is regulated by SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) during cellular development. However, little is known about the role or regulation of BMP signaling in HNSCC CSCs.
  
  
  METHODS
  Two CSC-like populations, CD44(high)/BMI1(high) and CD44(high)/ALDH(high), were enriched from HNSCC cell lines and evaluated for the expression of SMURF1 by qRT-PCR, flow cytometry, and immunoblotting. The activation status of BMP signaling in these populations was determined by using immunoblotting to detect phosphorylated SMAD1/5/8 (pSMAD1/5/8) levels. Knockdown of SMURF1 transcripts by RNA interference was used to assess the role of SMURF1 in BMP signaling and CSC maintenance. Loss of CSC-like phenotypes following SMURF1 knockdown was determined by changes in CD44(high) levels, cellular differentiation, and reduction in colony formation.
  
  
  RESULTS
  Populations of enriched CSC-like cells displayed decreased levels of pSMAD1/5/8 and BMP signaling target gene ID1 while SMURF1, CD44, and BMI1 were highly expressed when compared to non-CSC populations. Stable knockdown of SMURF1 expression in CSC-like cells increased pSMAD1/5/8 protein levels, indicating the reactivation of BMP signaling pathways. Decreased expression of SMURF1 also promoted adipogenic differentiation and reduced colony formation in a three-dimensional culture assay, indicating loss of tumorigenic capacity. The role of SMURF1 and inhibition of BMP signaling in maintaining a CSC-like population was confirmed by the loss of a CD44(high) expressing subpopulation in SMURF1 knockdown cells.
  
  
  CONCLUSIONS
  Our findings suggest that inhibition of BMP signaling potentiates the long-term survival of HNSCC CSCs, and that this inhibition is mediated by SMURF1. Targeting SMURF1 and restoring BMP signaling may offer a new therapeutic approach to promote differentiation and reduction of CSC populations leading to reduced drug resistance and disease recurrence.
authors.forename: Ali
authors.initials: A
authors.lastname: Khammanivong
authors.forename: Raj
authors.initials: R
authors.lastname: Gopalakrishnan
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota, 1352 Boyd Avenue, Saint Paul, MN 55108, USA. edickers@umn.edu.
authors.forename: Erin B
authors.initials: EB
authors.lastname: Dickerson
mesh_terms: D019485:Bone Morphogenetic Proteins / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D020868:Gene Silencing / Q000502:physiology; D006258:Head and Neck Neoplasms / Q000235:genetics*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics*; D009364:Neoplasm Recurrence, Local / Q000235:genetics; D014411:Neoplastic Stem Cells / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics; D051785:Smad Proteins / Q000235:genetics; D000077195:Squamous Cell Carcinoma of Head and Neck; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*
chemical_list: D019485:Bone Morphogenetic Proteins; D018960:Hyaluronan Receptors; D051785:Smad Proteins; C498342:SMURF1 protein, human; D044767:Ubiquitin-Protein Ligases