099 Novel roles for plakophilin 3 in cell cycle regulation in head and neck squamous cell carcinoma cells

Abstract

Analysis via automated microscopy of human head and neck squamous cell carcinoma (HNSCC) tissue microarrays revealed loss of the armadillo protein plakophilin 3 (Pkp3) in tongue, nasal, and larynx SCC. HNSCC progression is driven in part by aberrant DNA replication. Upon silencing Pkp3 in DOK (derived from a dysplastic oral lesion) and SCC9 (HNSCC) cells, proliferation increased and a multinucleated phenotype was observed. We explored whether Pkp3 loss altered the cell cycle and hypothesized that Pkp3 is involved in regulating DNA replication. Knocking down Pkp3 in SCC9 cells changed expression of several cell cycle regulators; G1 checkpoint proteins p21 and p27 were downregulated, the DNA replication protein PCNA was upregulated, as was cyclin B, which drives G2 to M phase progression. When SCC9 cells were synchronized at the G1/S transition and released, 50% more cells with suppressed Pkp3 incorporated 5-ethynyl-2’-deoxyuridine into DNA within 2 hours compared to control cells, indicating an increase in DNA replication after Pkp3 loss. We found using co-immunoprecipitation from SCC9 cells that both Pkp2 and Pkp3 were in complex with the replication licensing protein mini-chromosome maintenance protein 7 (MCM7), consistent with a recent report in breast cells that found Pkp2 and Pkp3 peptides in MCM7 immunoprecipitates subjected to mass spectrometry. Furthermore, knocking down Pkp3 increased expression of geminin, a regulator of DNA replication licensing. We propose that Pkp3 presence in MCM complexes and/or regulation of geminin expression helps regulate proper DNA replication licensing and that Pkp3 loss during HNSCC progression allows DNA over-replication. In conclusion, we have uncovered new roles for Pkp3 in cell cycle regulation in HNSCC cells. Understanding how Pkp3 regulates DNA replication may lead to novel therapeutics for HNSCC.