Thyrotropin (TSH) regulates thyroid cell proliferation and function through cAMP-mediated signaling pathways that activate protein kinase A (PKA) and Epac/Rap1. The respective roles of PKA versus Epac/Rap1 in TSH signaling remain unclear. We set out to determine whether PKA and/or Rap1 mediate extracellular signal-regulated kinase (ERK) activation by TSH. Neither blocking Rap1 activity nor silencing the expression of Rap1 impaired TSH or forskolin-induced ERK activation in Wistar rat thyroid cells. Direct activation of Epac1 failed to stimulate ERK activity in starved cells, suggesting that Epac-induced Rap1 activity is not coupled to ERK activation in rat thyroid cells. By contrast, PKA activity was required for cAMP-stimulated ERK phosphorylation and was sufficient to increase ERK phosphorylation in starved cells. Expression of dominant-negative Ras inhibited ERK activation by TSH, forskolin, and N 6 -monobutyryl (6MB)-cAMP, a selective activator of PKA. Silencing the expression of B-Raf also inhibited ERK activation by TSH, forskolin, and 6MB-cAMP, but not that stimulated by insulin or serum. Depletion of B-Raf impaired TSH-induced DNA synthesis, indicating a functional role for B-Raf in TSH-regulated proliferation. Collectively, these results position PKA, Ras, and B-Raf as upstream regulators of ERK activation and identify B-Raf as a selective target of cAMP-elevating agents in thyroid cells. These data provide the first evidence for a functional role for B-Raf in TSH signaling.

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Introduction: Rap1 and Rap2, both small GTPases of the Ras family, serve as crucial regulators in cell polarity, adhesion, migration, and lumen formation via crosstalk with cytoskeletal proteins. Rap1 GTPase activating protein (Rap1GAP), which negatively regulates Rap1 and Rap2 activity, has found to be downregulated in multiple cancers, including oropharyngeal squamous cell carcinoma, melanoma, papillary and follicular thyroid carcinoma, and colorectal carcinoma but has not yet been studied in breast cancer. In order to determine if Rap1GAP downregulation plays a role in breast cancer formation, we decided to expand our prior work to study its expression in breast cancer tissue. Methods: Twenty-one formalin-fixed paraffin-embedded human tissue blocks were selected on the basis that they contained normal breast tissue and either adjacent breast ductal carcinoma in situ (DCIS) or invasive ductal carcinoma. Using immunohistochemistry, we assessed the expression of Rap1GAP. We also assayed two human breast cancer cell lines with different levels of invasiveness, MCF7 (non-invasive) and MDA-MB-231 (invasive), to determine different levels of Rap1GAP expression and subsequent changes in Rap1 and Rap2 expression and activity. Results: Rap1GAP expression in both DCIS and invasive ductal carcinoma were found to be decreased compared to adjacent normal breast tissue. However, Rap1GAP expression was not statistically different between DCIS and invasive carcinoma lesions. Rap1GAP was found to be expressed highly in MCF7 but not in MDA-MB-231 cells. Moreover, only MDA-MB-231 cells exhibited high basal Rap2 activity. Activated Rap1 (RAP1-GTP) was not detected in either line. Conclusion: Our findings suggest that loss of Rap1GAP occurs as an early event in breast cancer tumorigenesis with a significant decrease in the transition from normal to DCIS, with a possible further decrement in the DCIS to invasive transition. Interestingly, while Rap1 and Rap2 were expressed in non-invasive and invasive cell lines, loss of Rap1GAP in the invasive cell line was associated with increase in active Rap2, but not active Rap1. These findings suggest that as in other cancers, loss of Rap1GAP may contribute to the invasive phenotype and thus further investigation is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3840. doi:10.1158/1538-7445.AM2011-3840