Alex Shimura Yamashita
CROSS-TALK BETWEEN NOTCH AND MAPK SIGNALING DURING RET/PTC3 AND BRAFT1799A ONCOGENE ACTIVATION
BACKGROUND: Mutually exclusive mutations in members of MAPK signaling pathway (RET/PTC or BRAF) are present in about 70% of papillary thyroid carcinomas (PTC), activating constitutively the MAPK signaling pathway. However, the effect of this activation in other signaling pathways remains unclear. Recently, Notch signaling has been implicated in tumorigenesis in other cancer types. Therefore, the aim of this study was to evaluate the influence of RET/PTC and BRAFT1799A oncogene activation in Notch signaling pathway in normal thyroid rat cell line and in TPC-1 cell line.
METHODS: Cell lines. PCCl3 (rat normal thyroid cell line) harboring doxycycline-inducible RET/PTC3 (PTC3-5) and BRAFT1799A (PC-BRAF), TPC-1 (human PTC cell line harboring RET/PTC1 mutation) were treated with MEK inhibitor U0126 (10 mM). RNA and protein expression: total RNA extract to perform quantitative real-time PCR (NOTCH1, HES1, P21, P27, and CCND1) and total protein were extract to perform Western blotting (phospho-ERK1/2, ERK1/2, NOTCH1, and alpha-actin). Flow cytometry analysis: TPC-1 were treated with GSI (1 mM) and proliferation (growth curve and viable cells), cell viability, cell cycle (stained with propidium iodide) and apoptosis (Anexin-FITC) analysis were performed using Guava System.
RESULTS: Conditional activation of RET/PTC3 and BRAFT1799A by doxyclycline (Dox) showed a sustained enhancement in ERK phosphorylation (24h, 48h, and 72h) in PTC3-5 and PC-BRAF cell lines. The time course of Notch1 gene expression was raised soon after 24 hours RET/PTC3 activation, and still higher 48 hours and 72 hours after oncogene induction in PTC3-5 cell line (24h: +8.3 fold, p<0.001; 48h: +7.0 fold, p<0.001; 72h: +3.8 fold, p<0.01). During BRAFT1799A activation in PC-BRAF cell line the gene expression of Notch1 also increased after 24 hours, with the higher expression in 48 hours, and still higher in 72 hours (24h: 1.3 fold, p<0.05; 48h: 2.6 fold, p<0.05; and 72: 56%, p<0.05). In addition, RET/PTC3 and BRAFT1799A activation increased the HES1 gene expression (60%, p<0.05 and 81%, p<0.05, respectively), the target gene of Notch signaling. To further understand the effect of MAPK signaling in Notch pathway, TPC1 were treated with MEK inhibitor U0126 (1mM), which reduced NOTCH1 protein expression and HES1 gene expression (78%, p<0.05). Then we treated the TPC-1 cell line with GSI (1mM), the Notch signaling inhibitor, the treatment diminished cell proliferation, reducing the number of cells after 48 hours of treatment and the percentage of viable cells (-30%, p<0.05), and increasing apoptosis (60%, p<0.01). In cell cycle analysis we observed the enhancement of DNA fragmentation (1.1 fold, p<0.01). Furthermore, the GSI (1mM) treatment in TPC-1 cell line modulates the cell cycle related genes: increasing the gene expression of P21 (26%, p<0.05) and P27 (71%, p<0.05), and reducing CCND1 gene expression (27%, p<0.05).
CONCLUSION: The induction of PTC related oncogenes, acting through MAPK signaling, enhanced Notch pathway, wich are more potent under RET/PTC activation. Targeting Notch signaling showed a anti-proliferative effect, suggesting an important role in tumorigenesis of MAPK-induced PTC, with a therapeutic implication in thyroid cancer.