Connexin43 (Cx43) is often deregulated in breast cancer tissue compared with normal adjacent tissue. Stable reexpression of Cx43 in cancer slows growth and renders the cells more sensitive to cytotoxic chemotherapeutics. Pseudogenes are often considered nonfunctional copies of DNA. The Cx43 pseudogene (ΨCx43) possesses all the features of an expressed gene and is exclusively transcribed in breast cancer cell lines and not in normal cells. ΨCx43 can be translated in vivo, and its protein exhibits growth-suppressive behavior similar to Cx43. We showed that ΨCx43 binds to the polyribosomes in breast cancer cells and that exogenous expression of ΨCx43 induces translational inhibition of Cx43. Furthermore, ΨCx43 is translated and binds more efficiently to the translational machinery than does Cx43 in an in vitro system. Following knockdown of ΨCx43 in breast cancer cells, we observed an increase in Cx43 RNA and protein. This results in increased cellular sensitivity to cytotoxic chemotherapy. Our results show that ΨCx43 acts as a posttranscriptional regulator of Cx43 in breast cancer cells, and that this represents an example of the regulation of genes by pseudogenes with potential therapeutic implications in cancer. [Mol Cancer Ther 2009;8(4):786-93]

Connexin43 (Cx43) expression is lost in cancer cells and many studies have reported that Cx43 is a tumor suppressor gene. Paradoxically, in a cellular NIH3T3 model, we have previously shown that Ha-Ras-mediated oncogenic transformation results in increased Cx43 expression. Although the examination of transcriptional regulation revealed essential regulatory elements, it could not solve this paradox. Here we studied post-transcriptional regulation of Cx43 expression in cancer using the same model in search of novel gene regulatory elements. Upon Ras transformation, both Cx43 mRNA stability and translation efficiency were increased. We investigated the role of Cx43 mRNA 3′ and 5′Untranslated regions (UTRs) and found an opposing effect; a 5′UTR-driven positive regulation is observed in Ras-transformed cells (NIH-3T3Ras), while the 3′UTR is active only in normal NIH-3T3Neo cells and completely silenced in NIH-3T3Ras cells. Most importantly, we identified a previously unknown regulatory element within the 3′UTR, named S1516, which accounts for this 3′UTR-mediated regulation. We also examined the effect of other oncogenes and found that Ras- and Src-transformed cells show a different Cx43 UTRs post-transcriptional regulation than ErbB2-transformed cells, suggesting distinct regulatory pathways. Next, we detected different patterns of S1516 RNA-protein complexes in NIH-3T3Neo compared to NIH-3T3Ras cells. A proteomic approach identified most of the S1516-binding proteins as factors involved in post-transcriptional regulation. Building on our new findings, we propose a model to explain the discrepancy between the Cx43 expression in Ras-transformed NIH3T3 cells and the data in clinical specimens.