Prostate apoptosis response protein 4 sensitizes human colon cancer cells to chemotherapeutic 5-FU through mediation of an NF kappaB and microRNA network.
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.
Wang
Bi-Dar
BD
Department of Pharmacology and Physiology, The George Washington University Medical Center, Washington, DC 20037, USA.
Kline
Christina Leah B
CL
Pastor
Danielle M
DM
Olson
Thomas L
TL
Frank
Bryan
B
Luu
Truong
T
Sharma
Arun K
AK
Robertson
Gavin
G
Weirauch
Matthew T
MT
Patierno
Steven R
SR
Stuart
Joshua M
JM
Irby
Rosalyn B
RB
Lee
Norman H
NH
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
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