Purpose: In head and neck squamous cell carcinoma (HNSCC) cells, Rap1 shuttles between the nucleus and cytoplasm. Prior findings suggested that Rap1 may modulate the β-catenin–independent Wnt pathway in some settings, but the role of Rap1 in β-catenin–dependent Wnt signaling remains undefined.

Experimental Design and Results: We observed that β-catenin bound to active Rap1 in vitro and Rap1 activated β-catenin/T-cell factor (TCF)–dependent transcription. Immunofluorescence studies showed that ectopic expression of Rap1 increased nuclear translocation of β-catenin. Overexpression of active Rap1 facilitated an increase in β-catenin–mediated transcription that was abrogated by dominant-negative TCF4. Conversely, small interfering RNA–mediated inhibition of endogenous Rap1 expression inhibited β-catenin/TCF–mediated transcription as well as invasion of HNSCC. Furthermore, inhibition of Rap1 expression downregulated the expression of matrix metalloproteinase 7, a transcriptional target of β-catenin/TCF. In HNSCC cells stably transfected with β-catenin or treated with lithium chloride or Wnt3A to stabilize endogenous β-catenin, inhibition of Rap1 expression led to decreases in the free pool of β-catenin. Immunohistochemical studies of tissue from HNSCC patients revealed that increased β-catenin intensity correlated with higher tumor stage. Furthermore, the prognostic effect of active Rap1 on tumor N stage was found to depend on cytosolic β-catenin expression (P < 0.013). When β-catenin is high, higher Rap1GTP intensity is associated with more advanced N stage.

Conclusions: The findings suggest that Rap1 enhances β-catenin stability and nuclear localization. In addition to indicating that Rap1 has a significant role in regulating β-catenin and β-catenin–dependent progression to more advanced N-stage lesions, these data highlight Rap1 as a potential therapeutic target in HNSCC. Clin Cancer Res; 16(1); 65–76