uORF-encoded Peptides as Novel Regulators of Protein Kinase Activity: New Opportunities for Cancer Therapy

Introduction. Approximately 40% of human mRNAs contain upstream open reading frames (uORFs) in their 5’ untranslated regions. Many of these uORF sequences, thought to impact translation or degradation of the primary ORF, were recently shown to be translated, but the function of encoded peptides remained unknown. We have previously published the presence of uORFs in PKCs. PKCs are involved in cell proliferation, differentiation, and apoptosis, among other processes impaired in cancer. Here we show for the first time that PKCeta, a signaling and anti-apoptotic stress kinase of PKC family, encodes for a uORF peptide with novel kinase inhibitory functions and therapeutic potential in cancer.

Materials and Methods. Bio-informatics analysis was used to identify uORFs in PKCs of different species and their conservation. The translational probability was determined by ribosome profiling data analysis and by generating uORF-luciferase fusion plasmids with point mutations in their respective initiation and stop codons. The ability of the peptides to inhibit PKCs kinase activity was investigated using in vitro kinase assays. Effects on cell viability and synergy with chemotherapy were studied as well as effects on cell migration and response to DNA damage.

Results and Discussion. We previously identified two uORFs upstream PKCeta that regulate its translation under normal growth conditions and upon stress. Here, we demonstrate that one of these uORFs possesses the typical pseudosubstrate motif present in all PKCs, auto-inhibiting their kinase activity. We show that this uORF-encoded peptide (uPEP2) inhibits the kinase activity of PKCeta and of other members of the novel PKC sub-family, implying for network regulation in this family of kinases. Functionally, the peptide inhibits proliferation and migration of different aggressive tumors including breast cancers and leukemia cells. uPEP2 synergizes with a chemotherapeutic agent by interfering with the response to DNA damage by interfering with gH2AX phosphorylation that marks DNA double strand breaks for repair. We show that uPEP2 interferes with DNA repair processes upstream of H2AX phosphorylation, resulting in enhanced cell death.

Conclusion. Our studies introduce uORFs as new players in protein networks regulation, adding another layer of complexity to eukaryotic protein control mechanisms. Furthermore, these novel peptides may provide potential therapeutic agents for cancer treatment.