S. K. Odorico1, X. Yu1, A. Dammalapati1, A. Harrison1, A. Hundal1, J. A. Bibb2, H. Chen1 1University Of Wisconsin,Department Of Surgery,Madison, WI, USA 2University Of Texas Southwestern Medical Center,Department Of Psychiatry,Dallas, TX, USA
Introduction: Medullary thyroid cancer (MTC) is a neuroendocrine carcinoma that arises from C cells. Recently, it has been demonstrated that Cdk5 and its cofactor p25 promote C cell proliferation, leading to the development of MTC, while repressing p25 overexpression causes C cell growth arrest. In our previous studies, we have shown that activation of Notch intracellular domain inhibits MTC cell proliferation and alters the neuroendocrine phenotype. However, little is known about the possible interaction between Cdk5/p25 and pan Notch signaling. Therefore, the purpose of this study was to investigate the role of Notch isoforms including Notch1 and 2 in the p25–mediated inhibition of proliferation in MTC cells.
Methods: A murine thyroid cancer cell line MTCp25, which retains TetOp promoter-controlled p25-GFP expression, was used in our study. The exogenous p25-GFP is stably overexpressed under basal conditions and repressed by treatment with doxycycline. We treated MTCp25 cells with 3 different concentrations of doxycycline (1, 2 and 5 μg/mL) for 2, 4 and 6 days. Notch1, Notch 2, p25, GFP and beta actin expression were then evaluated by Western blot. We also quantified the mRNA levels of Notch isoforms during different time points using quantitative real-time polymerase chain reaction (PCR).
Results: Both p25 and GFP expression were consistently reduced during different time points in MTCp25 cells culturing in the presence of doxycycline. No significant changes were detected by Western blot for the intracellular domain of Notch 1 or Notch2 after 2-day treatment of doxycycline. Interestingly, protein expression of Notch1 and Notch2 intracellular domains profoundly increased in a dose dependent manner after repressing p25 for 4 and 6 days. Furthermore, we found that the mRNA levels of Notch 1 and Notch 2 did not change significantly with p25 suppression, suggesting that p25 may be involved in the regulation of protein cleavage or degradation of Notch1 and Notch2.
Conclusion: Cdk5/p25 signaling up-regulates the protein level of Notch1 and Notch2 intracellular domains. This intriguing finding could suggest the function of p25 involved in the post-translational processing of Notch1 and Notch2 receptor. These results call for further investigation into the mechanism on the crosstalk between Notch and Cdk5 signaling pathways, which may play an important role in medullary thyroid cancer development and growth.