Abstract 4221: Plakophilin 3 (Pkp3) increases oral squamous carcinoma cell-cell adhesion while inhibiting cell motility and proliferation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Desmosomal junctions are major contributors to the stability of mechanically stressed tissues including oral epithelium. Plakophilins are a subfamily of Armadillo proteins with a critical role in desmosomal assembly. They have emerged as candidates for a number of regulatory roles both dependent and independent of their function as cell-cell adhesion molecules. These include potential regulation of transcription and translation, as well as control of various signaling cascades. However, there is little known about the biological consequences of their actions. We show here that plakophilin 3 (Pkp3) plays a prominent regulatory role in oral squamous carcinoma cells (OSCC). OSCCs present a great clinical challenge with poor prognosis and no improvement in survival rates for the last 20 years. Using 141 patient tissue samples we demonstrated, by immunoblotting and immunofluorescence, an inverse correlation between Pkp3 and tumor aggressiveness. In addition, >40% of tumor samples completely lost Pkp3 expression. In order to test its role in OSCC cell-cell adhesion, we ablated Pkp3 in the oral cancer cell line SCC9. Pkp3 loss disrupted desmosomal assembly by preventing proper cell border localization of the obligate desmosomal component desmoplakin (DP). Not surprisingly, Pkp3 dependent disruption of desmosomal assembly severely diminished cell-cell adhesion, while at the same time increasing cell motility in a wound healing assay. Interestingly, the activators of cAMP dependent signaling partially rescued the Pkp3 deficient phenotype, demonstrating a role for cAMP in Pkp3 dependent maintenance of oral keratinocyte cell-cell adhesion strength, an important impediment to OSCC growth, motility and invasion. Moving beyond cell-cell adhesion, our preliminary data indicated that Pkp3 down-regulation increases cell cycle progression and proliferation in OSCC cells. The levels of cell cycle inhibitors p21 and p27 were decreased in the absence of Pkp3 whereas the proliferation marker PCNA was increased. Correspondingly, over-expression of Pkp3 in the aggressive OSCC cell line SCC 22B led to an increase in p21 and p27. Both of these results point to Pkp3 acting as a regulator of cell cycle progression, thus we further tested the ability of Pkp3 to regulate cell cycle using flow cytometry. Decreased Pkp3 expression led to an increase in S and G2/M phases of the cell cycle and a concomitant decrease in G0/G1 phase in SCC9 cells indicating an increase in cell cycle progression. Furthermore, a marker of G2 progression, Cyclin B1 is increased in Pkp3 depleted SCC9 cells, indicating that the cells are not experiencing G2/M arrest. In summary, these results support the hypothesis that Pkp3 attenuates OSCC tumor progression towards more aggressive phenotype through both adhesion and cell cycle regulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4221. doi:1538-7445.AM2012-4221