title: Nicotine-mediated invasion and migration of non-small cell lung carcinoma cells by modulating STMN3 and GSPT1 genes in an ID1-dependent manner.
abstract: BACKGROUND
  Inhibitor of DNA binding/Differentiation 1 (ID1) is a helix loop helix transcription factor that lacks the basic DNA binding domain. Over-expression of ID1 has been correlated with a variety of human cancers; our earlier studies had shown that reported ID1 is induced by nicotine or EGF stimulation of non-small cell lung cancer (NSCLC) cells and its down regulation abrogates cell proliferation, invasion and migration. Here we made attempts to identify downstream targets of ID1 that mediate these effects.
  
  
  METHODS
  A microarray analysis was done on two different NSCLC cell lines (A549 and H1650) that were transfected with a siRNA to ID1 or a control, non-targeting siRNA. Cells were stimulated with nicotine and genes that were differentially expressed upon nicotine stimulation and ID1 depletion were analyzed to identify potential downstream targets of ID1. The prospective role of the identified genes was validated by RT-PCR. Additional functional assays were conducted to assess the role of these genes in nicotine induced proliferation, invasion and migration. Experiments were also conducted to elucidate the role of ID1, which does not bind to DNA directly, affects the expression of these genes at transcriptional level.
  
  
  RESULTS
  A microarray analysis showed multiple genes are affected by the depletion of ID1; we focused on two of them: Stathmin-like3 (STMN3), a microtubule destabilizing protein, and GSPT1, a protein involved in translation termination; these proteins were induced by both nicotine and EGF in an ID1 dependent fashion. Overexpression of ID1 in two different cell lines induced STMN3 and GSPT1 at the transcriptional level, while depletion of ID1 reduced their expression. STMN3 and GSPT1 were found to facilitate the proliferation, invasion and migration of NSCLC cells in response to nAChR activation. Attempts made to assess how ID1, which is a transcriptional repressor, induces these genes showed that ID1 down regulates the expression of two transcriptional co-repressors, NRSF and ZBP89, involved in the repression of these genes.
  
  
  CONCLUSIONS
  Collectively, our data suggests that nicotine and EGF induce genes such as STMN3 and GSPT1 to promote the proliferation, invasion and migration of NSCLC, thus enhancing their tumorigenic properties. These studies thus reveal a central role for ID1 and its downstream targets in facilitating lung cancer progression.
authors.forename: Sajitha
authors.initials: S
authors.lastname: Nair
authors.forename: Namrata
authors.initials: N
authors.lastname: Bora-Singhal
authors.forename: Deepak
authors.initials: D
authors.lastname: Perumal
authors.affiliation: Department of Tumor Biology, H, Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA. Srikumar.Chellappan@moffitt.org.
authors.forename: Srikumar
authors.initials: S
authors.lastname: Chellappan
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects* / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D051549:Glutathione S-Transferase pi / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000378:metabolism*; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology; D009361:Neoplasm Invasiveness; D009538:Nicotine / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D034741:RNA, Small Interfering / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D050777:Stathmin / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D014945:Wound Healing / Q000187:drug effects; D000099229:RE1-Silencing Transcription Factor
chemical_list: D004268:DNA-Binding Proteins; D004815:Epidermal Growth Factor; D051549:Glutathione S-Transferase pi; D051798:Inhibitor of Differentiation Protein 1; D009538:Nicotine; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D050777:Stathmin; D014157:Transcription Factors; D000099229:RE1-Silencing Transcription Factor; C112285:STMN3 protein, human; C110951:ZNF148 protein, human; C496556:GSTP1 protein, human