Nrf2 is the key transcription factor for cytoprotective gene programs. Nrf2 is normally maintained at very low concentrations by proteasomal degradation, through its interaction with the adapter protein Keap1 and the Cul3 E3 ligase. Increased Nrf2 concentration resulting from loss of function Keap1 mutations has been described in chemoresistant non-small cell lung cancer. Previous studies in breast cancer showed low levels of some Nrf2-regulated detoxification genes, but the mechanism has not been systematically examined. We found that half of the breast cancer cell lines examined have decreased concentration of Nrf2 compared with normal mammary epithelial cell lines, associated with variable but detectable levels in Keap1 levels, and consistently increased Cul3 mRNA and protein.

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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]