Abstract 1250: Crosstalk between Notch and MAPK signaling regulates proliferation in papillary thyroid cancer

Abstract

Abstract Background: RET/PTC, RAS and BRAF are present in about two thirds of papillary thyroid carcinomas (PTC), activating constitutively the mitogen activated protein kinase (MAPK) signaling pathway. There is practically no overlap between RET/PTC, RAS and BRAF mutations in PTC, the lack of concordance for these mutations provides compelling genetic evidence for the requirement of this signaling system for transformation to PTC. However, the effect of these genetic alterations in other signaling pathways remains unclear, which could, at least in part, cooperates in transformation. Recently, Notch signaling has been implicated in tumorigenesis in other cancer types. In this study, we test the hypothesis that RET/PTC and BRAFT1799A oncogenes crosstalks and regulates Notch pathway. Methods: TPC-1 and BCPAP (human PTC cells harboring RET/PTC1 rearrangement and BRAFT1799A mutation, respectively) were treated with gamma secretase inhibitor (GSI) and proliferation was analyzed by MTT assay and growth curve; cell cycle and apoptosis were analyzed by flow cytometry. PCCL3 cells harboring Doxycycline-inducible RET/PTC3 and BRAFT1799A (PC-BRAF) were used to analyze protein and gene expression by Western blotting and real-time PCR, respectively. Notch1 protein expression was analyzed in transgenic mice (Tg-BRAF) which harbor targeted expression of BRAFT1799A restricted to thyroid gland. Results: PTC cells treated with GSI decreased the percentage of viable cell in a dose-dependent manner and reduced the growth rate. Cell cycle analysis in PTC cells showed enhancement of DNA fragmentation and increased apoptotic cells. Furthermore, the GSI treatment in PTC cells modulates cell cycle related genes, increasing CDKN1A and CDKN1B gene expression and reducing CCND1, MCM6, CKS2, and MADL2 expression. Conditional activation of RET/PTC3 and BRAFT1799A in PTC3-5 and PC-BRAF cells, respectively, showed a sustained enhancement in pERK protein expression, increased Notch1 protein and gene expression and Hes1 gene expression, the target gene of Notch signaling. Furthermore, transgenic mice Tg-BRAF showed a stronger stain for Notch1 in follicular and stromal cells. Treating PTC cells with MEK inhibitors, U0126 or PD96059, reduces NOTCH1 and HES1 gene expression. The combination of GSI with MEK inhibitor enhances the growth suppression promote by MAPK signaling inhibition in PTC cell proliferation. CONCLUSION: Taken together, targeting Notch signaling resulted in anti-proliferative effect, suggesting an important role of Notch signaling in tumorigenesis of MAPK-induced PTC and our results indentify Notch signaling as a potential therapeutic target in thyroid cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1250. doi:1538-7445.AM2012-1250