title: Prostate apoptosis response protein 4 sensitizes human colon cancer cells to chemotherapeutic 5-FU through mediation of an NF kappaB and microRNA network.
abstract: BACKGROUND
  Diminished expression or activity of prostate apoptosis response protein 4 (Par-4) has been demonstrated in a number of cancers, although reports on Par-4 expression during colon cancer progression are lacking. An understanding of the molecular events in conjunction with the genetic networks affected by Par-4 is warranted.
  
  
  RESULTS
  Colon cancer specimens derived from patients have significantly diminished expression of Par-4 mRNA relative to paired normal colon. Hence, the functional consequences of reintroducing Par-4 into HT29 colon cancer cells were assessed. Overexpression augmented the interaction of Par-4 with NF kappaB in the cytosol but not nucleus, and facilitated apoptosis in the presence of 5-fluorouracil (5-FU). Analogous findings were obtained when AKT1 pro-survival signaling was inhibited. Transcriptome profiling identified approximately 700 genes differentially regulated by Par-4 overexpression in HT29 cells. Nearly all Par-4-regulated genes were shown by promoter analysis to contain cis-binding sequences for NF kappaB, and meta-analysis of patient expression data revealed that one-third of these genes exist as a recurrent co-regulated network in colon cancer specimens. Sets of genes involved in programmed cell death, cell cycle regulation and interestingly the microRNA pathway were found overrepresented in the network. Noteworthy, Par-4 overexpression decreased NF kappaB occupancy at the promoter of one particular network gene DROSHA, encoding a microRNA processing enzyme. The resulting down-regulation of DROSHA was associated with expression changes in a cohort of microRNAs. Many of these microRNAs are predicted to target mRNAs encoding proteins with apoptosis-related functions. Western and functional analyses were employed to validate several predictions. For instance, miR-34a up-regulation corresponded with a down-regulation of BCL2 protein. Treating Par-4-overexpressing HT29 cells with a miR-34a antagomir functionally reversed both BCL2 down-regulation and apoptosis by 5-FU. Conversely, bypassing Par-4 overexpression by direct knockdown of DROSHA expression in native HT29 cells increased miR-34a expression and 5-FU sensitivity.
  
  
  CONCLUSIONS
  Our findings suggest that the initiation of apoptotic sensitivity in colon cancer cells can be mediated by Par-4 binding to NF kappaB in the cytoplasm with consequential changes in the expression of microRNA pathway components.
authors.affiliation: Department of Pharmacology and Physiology, The George Washington University Medical Center, Washington, DC 20037, USA.
authors.forename: Bi-Dar
authors.initials: BD
authors.lastname: Wang
authors.forename: Christina Leah B
authors.initials: CL
authors.lastname: Kline
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Pastor
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Olson
authors.forename: Bryan
authors.initials: B
authors.lastname: Frank
authors.forename: Truong
authors.initials: T
authors.lastname: Luu
authors.forename: Arun K
authors.initials: AK
authors.lastname: Sharma
authors.forename: Gavin
authors.initials: G
authors.lastname: Robertson
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Weirauch
authors.forename: Steven R
authors.initials: SR
authors.lastname: Patierno
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Stuart
authors.forename: Rosalyn B
authors.initials: RB
authors.lastname: Irby
authors.forename: Norman H
authors.initials: NH
authors.lastname: Lee
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D005472:Fluorouracil / Q000494:pharmacology; D015870:Gene Expression; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D019073:HT29 Cells; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D043244:Ribonuclease III / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D016328:NF-kappa B; C101296:prostate apoptosis response-4 protein; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt; C517918:DROSHA protein, human; D043244:Ribonuclease III; D005472:Fluorouracil