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Prostate apoptosis response protein 4 sensitizes human colon cancer cells to chemotherapeutic 5-FU through mediation of an NFκB and microRNA network

Bi-Dar Wang1, Christina Leah B Kline2, Danielle M Pastor23, Thomas L Olson2, Bryan Frank1, Truong Luu1, Arun K Sharma4, Gavin Robertson4, Matthew T Weirauch5, Steven R Patierno1, Joshua M Stuart5, Rosalyn B Irby2* and Norman H Lee1*

Author Affiliations

1 Department of Pharmacology and Physiology, The George Washington University Medical Center, Washington, DC 20037, USA

2 Penn State Hershey Cancer Institute, Hershey, PA 17033, USA

3 Department of Surgery, Penn State College of Medicine, Hershey, PA 17033, USA

4 Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA

5 Biomolecular Engineering Department, UC Santa Cruz, Santa Cruz, CA 95064, USA

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Molecular Cancer 2010, 9:98  doi:10.1186/1476-4598-9-98

Published: 30 April 2010

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 NFκB 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 ~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 NFκB, 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 NFκB 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.

Conclusion

Our findings suggest that the initiation of apoptotic sensitivity in colon cancer cells can be mediated by Par-4 binding to NFκB in the cytoplasm with consequential changes in the expression of microRNA pathway components.