Rap1 stabilizes beta-catenin and enhances beta-catenin-dependent transcription and invasion in squamous cell carcinoma of the head and neck.

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 beta-catenin-independent Wnt pathway in some settings, but the role of Rap1 in beta-catenin-dependent Wnt signaling remains undefined. EXPERIMENTAL DESIGN AND RESULTS: We observed that beta-catenin bound to active Rap1 in vitro and Rap1 activated beta-catenin/T-cell factor (TCF)-dependent transcription. Immunofluorescence studies showed that ectopic expression of Rap1 increased nuclear translocation of beta-catenin. Overexpression of active Rap1 facilitated an increase in beta-catenin-mediated transcription that was abrogated by dominant-negative TCF4. Conversely, small interfering RNA-mediated inhibition of endogenous Rap1 expression inhibited beta-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 beta-catenin/TCF. In HNSCC cells stably transfected with beta-catenin or treated with lithium chloride or Wnt3A to stabilize endogenous beta-catenin, inhibition of Rap1 expression led to decreases in the free pool of beta-catenin. Immunohistochemical studies of tissue from HNSCC patients revealed that increased beta-catenin intensity correlated with higher tumor stage. Furthermore, the prognostic effect of active Rap1 on tumor N stage was found to depend on cytosolic beta-catenin expression (P < 0.013). When beta-catenin is high, higher Rap1GTP intensity is associated with more advanced N stage. CONCLUSIONS: The findings suggest that Rap1 enhances beta-catenin stability and nuclear localization. In addition to indicating that Rap1 has a significant role in regulating beta-catenin and beta-catenin-dependent progression to more advanced N-stage lesions, these data highlight Rap1 as a potential therapeutic target in HNSCC.

Rap1GAP promotes invasion via induction of matrix metalloproteinase 9 secretion, which is associated with poor survival in low N-stage squamous cell carcinoma.

The objective of the current study was to investigate the effects of Rap1GAP on invasion and progression of head and neck squamous cell carcinoma (SCC) and the role of matrix metalloproteinase (MMP) 9 and MMP2 in this process. Rap1GAP functions by switching off Rap1, the Ras-like protein that has been associated with carcinogenesis. Previous findings suggest that Rap1GAP acts as a tumor suppressor protein in SCC by delaying the G(1)-S transition of the cell cycle. However, cells transfected with Rap1GAP exhibit a more invasive phenotype than corresponding vector-transfected control cells. MMP2 and MMP9 are enzymes that mediate SCC invasion via degradation of the extracellular matrix. Using SCC cells transfected with empty vector or Rap1GAP, cell invasion and MMP secretion were determined by Matrigel assays and gelatin zymography, respectively. Rap1GAP up-regulated transcription and secretion of MMP2 and MMP9, as assayed by quantitative reverse transcription-PCR and zymography. Furthermore, chemical and RNA interference blockade of MMP2/MMP9 inhibited invasion by Rap1GAP-transfected cells. Immunohistochemical staining of a human oropharyngeal SCC tissue microarray showed that Rap1GAP and MMP9 expression and staining intensity are correlated (P < 0.0001) and that, in early N-stage lesions of SCC, high MMP9 is prognostic of poor disease-specific survival (P < 0.05). Furthermore, Rap1GAP staining is correlated with MMP2 (P < 0.03). MMP2 in combination with N stage has a prognostic effect on time to indication of surgery at primary site. MMP2 intensity is also positively correlated with T stage (P < 0.015). In conclusion, Rap1GAP inhibits tumor growth but induces MMP2- and MMP9-mediated SCC invasion and tumor progression, suggesting a role for this protein as a biomarker for early N-stage, aggressive SCCs.

Rap1GAP inhibits tumor growth in oropharyngeal squamous cell carcinoma.

Rap1, a growth regulatory protein that is strongly expressed in human squamous cell carcinoma (SCC), is inactivated by rap1GAP. Recent evidence in normal rat cells suggests that rap1GAP regulates proliferation. The objective of the current study was to investigate whether rap1GAP functions as a tumor suppressor in SCC. Using a pull-down assay, active GTP-bound rap1 was up-regulated in SCC compared to normal or immortalized keratinocytes. Because both rap1A and rap1B isoforms of rap1 are expressed in SCC, the rap1GAP inactivation of both rap1 isoforms was verified using cells transfected with EGFP-rap1A or EGFP-rap1B or co-transfected with FLAG-tagged rap1GAP. The results demonstrate that expression of rap1GAP in oropharyngeal SCC down-regulated active rap1, ERK activation, and proliferation. Incubation of stably transfected SCC cells with nocodazole, an inhibitor of mitosis, caused a slower accumulation of rap1GAP-transfected cells in the G2 phase, in comparison to the vector control, indicating that rap1GAP-transfected cells have slower progression through the cell cycle. This was supported by down-regulation of cyclin D1, cdk4, and cdk6 in rap1GAP-transfected SCC cells. Furthermore, SCC cells transfected with rap1GAP produced significantly smaller tumors in nude mice as compared to controls (P < 0.01). These novel findings suggest that rap1GAP acts as a tumor suppressor protein in SCC.

Rap1A and rap1B ras-family proteins are prominently expressed in the nucleus of squamous carcinomas: nuclear translocation of GTP-bound active form.

We recently showed that rap1 regulates growth and proliferation in normal keratinocytes, which provoked us to investigate its expression and regulation in malignant cells. Rap1 is variably expressed in whole cell lysates of squamous cell carcinoma (SCC) cell lines. Immunoblot analysis of nuclear and cytosolic fractions and immunohistochemistry revealed that in addition to cytoplasmic expression, SCC cells also exhibit prominent punctate rap1 expression in the nucleus. This unexpected nuclear distribution was confirmed by the evaluation of human oral cancer specimens by immunohistochemistry, which showed both nuclear and cytoplasmic localization. Cytoplasmic rap1 expression was observed mostly in large differentiated cells, whereas nuclear localization was found in morphologically less differentiated cells. Quantitative reverse transcriptase polymerase chain reaction and Northern blot analysis showed that both rap1A and rap1B are expressed in SCC cell lines although rap1B signals are more prominent. Transfection with enhanced GFP-tagged constitutively active and inactive forms of rap1B demonstrated that the active GTP-bound form translocates to the nucleus whereas inactive rap1B(GDP) is retained in the cytoplasm, much of which is in a perinuclear distribution. Furthermore, growth factors induce nuclear translocation of rap1 in oral cancer cells. This novel discovery that active, GTP-bound rap1 translocates to the nucleus makes it only the second of over 100 small GTP-binding proteins to be identified in the nucleus, and the striking prominence of rap1 expression in the nucleus of SCC cells suggests that activated rap1 plays a role in the malignant process.