title: Cholecystokinin down-regulation by RNA interference impairs Ewing tumor growth.
abstract: OBJECTIVE
  Tumors of the Ewing family are characterized by chromosomal translocations that yield chimeric transcription factors, such as EWS/FLI1, which regulate the expression of specific genes that contribute to the malignant phenotype. In the present study, we show that cholecystokinin (CCK) is a new target of the EWS/FLI1 oncoprotein and assess its functional role in Ewing tumor pathogenesis.
  
  
  METHODS
  Relevant EWS/FLI1 targets were identified using a combination of cell systems with inducible EWS/FLI1 expression, Ewing tumors and cell lines, microarrays, and RNA interference with doxycycline-inducible small hairpin RNA (shRNA) vectors. A doxycycline-inducible CCK-shRNA vector was stably transfected in A673 and SK-PN-DW Ewing cell lines to assess the role of CCK in cell proliferation and tumor growth.
  
  
  RESULTS
  Microarray analysis revealed that CCK was up-regulated by EWS/FLI1 in HeLa cells. CCK was overexpressed in Ewing tumors as compared with other pediatric malignancies such as rhabdomyosarcoma and neuroblastoma, with levels close to those detected in normal tissues expressing the highest levels of CCK. Furthermore, EWS/FLI1 knockdown in A673 and SK-PN-DW Ewing cells using two different doxycycline-inducible EWS/FLI1-specific shRNA vectors down-regulated CCK mRNA expression and diminished the levels of secreted CCK, showing that CCK is a EWS/FLI1 specific target gene in Ewing cells. A doxycycline-inducible CCK-specific shRNA vector successfully down-regulated CCK expression, reduced the levels of secreted CCK in Ewing cell lines, and inhibited cell growth and proliferation in vitro and in vivo. Finally, we show that Ewing cell lines and tumors express CCK receptors and that the growth inhibition produced by CCK silencing can be rescued by culturing the cells with medium containing CCK.
  
  
  CONCLUSIONS
  Our data support the hypothesis that CCK acts as an autocrine growth factor stimulating the proliferation of Ewing cells and suggest that therapies targeting CCK could be promising in the treatment of Ewing tumors.
authors: Laboratorio de Patología Molecular de Tumores Sólidos Infantiles, Departamento de Biología Molecular y Celular del Cáncer, Instituto de Investigaciones Biomédicas A. Sols (CSIC-UAM), Spain.
authors: Jaime
authors: J
authors: Carrillo
authors: Eva
authors: E
authors: García-Aragoncillo
authors: Daniel
authors: D
authors: Azorín
authors: Noelia
authors: N
authors: Agra
authors: Ana
authors: A
authors: Sastre
authors: Imelda
authors: I
authors: González-Mediero
authors: Purificación
authors: P
authors: García-Miguel
authors: Angel
authors: A
authors: Pestaña
authors: Soledad
authors: S
authors: Gallego
authors: Dolores
authors: D
authors: Segura
authors: Javier
authors: J
authors: Alonso
mesh_terms: D001859:Bone Neoplasms / Q000235:genetics / Q000473:pathology*; D002455:Cell Division; D045744:Cell Line, Tumor; D002766:Cholecystokinin / Q000235:genetics*; D003001:Cloning, Molecular; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006133:Growth Substances; D006367:HeLa Cells; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012512:Sarcoma, Ewing / Q000235:genetics / Q000473:pathology*
chemical_list: D006133:Growth Substances; D012333:RNA, Messenger; D002766:Cholecystokinin