Identification of translational targets of tumor suppressor Pdcd4 through analysis of polysomal mRNA recruitment

A95 Programmed Cell Death 4 (Pdcd4) encodes a novel tumor suppressor that inhibits tumorigenesis in mouse models and invasion in human cancer cells. Pdcd4 exerts its effects by inhibiting protein translation initiation. Eukaryotic translational initiation depends on the formation of an active initiation complex (eIF4F), which includes the scaffold protein, eIF4G and the RNA helicase, eIF4A. The eIF4A helicase activity is essential for the efficient translation of specific mRNAs having structured 5’untranslated regions. Pdcd4 interacts with eIF4A, prevents association of eIF4A with eIF4G (eIF4F), and thereby inhibits eIF4A helicase activity and protein synthesis. Pdcd4 expression is often decreased in progressed carcinomas of human lung, breast, colon, and prostate. We hypothesize that Pdcd4 inhibits tumor promotion by regulating the translation of specific mRNA transcripts that are necessary for tumorigenesis. We anticipate that proteins encoded by Pdcd4-regulated mRNAs will be attractive molecular targets for cancer prevention and therapy. In order to identify mRNA targets of Pdcd4, we performed sucrose gradient fractionation of whole cell extracts from human breast cancer (T47D) cells attenuated for Pdcd4 function using siRNA and shRNA to Pdcd4. Decreased Pdcd4 activity is expected to result in increased translation of a subset of cellular mRNAs. We performed comparative microarray analysis of mRNA isolated from light fractions containing monosomes (i.e., mRNAs associated with 1-2 ribosomes) and heavy fractions containing polysomes (i.e., mRNAs associated with multiple ribosomes). We have identified several Pdcd4 target mRNAs that preferentially (>2-fold, p