Sp1 regulates osteopontin expression in SW480 human colon adenocarcinoma cells

Surgery. 2007 Aug;142(2):163-9. doi: 10.1016/j.surg.2007.02.015.

Abstract

Background: Osteopontin (OPN) mediates cancer metastasis. Mechanisms regulating OPN expression in human colorectal cancer are unknown. Using SW480 colon adenocarcinoma cells, we hypothesized that transcription determines OPN expression.

Methods: SW480 constitutively express OPN. Transient transfection and deletion analysis of human OPN promoter (full-length 2.1 kb)-luciferase constructs identified cis-regulatory regions. Gelshift and chromatin immunoprecipitation (ChIP) assays identified the trans-regulatory nuclear protein. Using in vitro adhesion, migration, and invasion studies, siRNA was used to determine the functional effect of decreased nuclear protein expression.

Results: A cis-regulatory promoter region, nt-80 to nt-108, upregulated OPN transcription. Gelshift assays demonstrated specific binding of nuclear proteins. Competition with unlabeled mutant oligonucleotides indicated that the region, nt-94 to nt-104 (TGGGCTGGGC), was essential for protein binding in gelshift assays. Confirmatory ChIP assays showed the corresponding nuclear protein to be Sp1. Sp1 expression was ablated with siRNA (si-Sp1), resulting in decreased OPN-dependent adhesion, migration, and invasion by 50%, 70%, and 65%, respectively. Exogenous addition of OPN to si-Sp1 cells restored adhesion, migration, and invasion indices.

Conclusions: In SW480 human colon cancer cells, we conclude that Sp1 mediated expression of the tumor metastasis protein, OPN, regulates in vitro functional correlates of tumor metastasis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenocarcinoma / genetics
  • Adenocarcinoma / physiopathology*
  • Adenocarcinoma / secondary
  • Cell Line, Tumor
  • Colonic Neoplasms / genetics
  • Colonic Neoplasms / pathology
  • Colonic Neoplasms / physiopathology*
  • Electrophoretic Mobility Shift Assay
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • Osteopontin / genetics*
  • Osteopontin / metabolism
  • Promoter Regions, Genetic / physiology
  • RNA, Small Interfering
  • Sp1 Transcription Factor / genetics
  • Sp1 Transcription Factor / metabolism*

Substances

  • RNA, Small Interfering
  • Sp1 Transcription Factor
  • Osteopontin