One approach that recently has been studied with exciting results will be to target the constitutively active Ret kinase and/or its key downstream Gemcitabine Cancer signaling pathways. Mutated Ret in MTC invokes several downstream signaling pathways, like the Ras/ Raf/Mek/Erk and phosphatidylinositol 3 kinase /Akt/mammalian target of rapamycin cascades leading to cancer development and probably progression rendering it a rational therapeutic target for this disease. Sorafenib can be a multikinase inhibitor that prevents activity of Ret kinase, other tyrosine kinases, and Raf serine threonine kinase members making it a compound of interest in MTC. We recently reported results of the phase 2 clinical test for patients with advanced level MTC in which a partial response rate of six months was seen and 500-square of patients demonstrated stable disease 15 months, with cyst shrinkage ranging from 8 to 27-yr. Nevertheless, like other tyrosine kinase inhibitors, all of the people in this study eventually developed progressive disease. Thus, we were interested in exploring combinatorial strategies in Digestion MTC cells using sorafenib like a base ingredient due emphasizing compounds with rational combinatorial signaling inhibiting features including compounds in clinical trial or already approved for clinical use in the United States. Included in these are the mTOR inhibitor everolimus and the Mek inhibitor AZD6244. Our results suggest that the anti-proliferative activity of sorafenib was synergistically enhanced when it was coupled with a Mek inhibitor however not everolimus. This result was predicted by dose related signaling inhibition studies using sorafenib alone GW0742 for both cell lines. Our data also show that AZD6244 and everolimus, when used together were not synergistic in either mobile line despite inhibition of Mek and TORC1 respectively. Curiously, everolimus was shown to cause both Akt and Ret phosphorylation and this effect was increased by co treatment with AZD6244, indicating a possible mechanism of resistance. Taken together, our results underscore the potential of a mixed therapeutic approach with sorafenib and Mek inhibitors for treating MTC as well as the requirement for correlative studies to higher determine rational combinatorial strategies. Materials and methods Cell lines and reagents The human medullary thyroid cancer cell lines, TT and MZ CRC 1, were kindly provided from Bary Robert, PhD and Nelkin Gagel, MD respectively. The TT cells have a heterozygous C634W Ret mutation and the MZ CRC 1 cells have a heterozygous M918T Ret mutation. Cells were managed in RPMI 1640 medium supplemented with heat inactivated 1 nonessential proteins and 2005-2010 fetal bovine serum at 37 C and humidified five hundred CO2. For MZ CRC 1 culture, we used collagen fiber to induce a thin layer on tissue culture surfaces to enhance cell attachment and growth.