Synergistic activity of everolimus and 5‐aza‐2′‐deoxycytidine in medullary thyroid carcinoma cell lines

Medullary thyroid cancer (MTC) is a tumor highly resistant to chemo‐ and radiotherapy. Drug resistance can be induced by epigenetic changes such as aberrant DNA methylation. To overcome drug resistance, we explored a promising approach based on the use of 5‐aza‐2′‐deoxycytidine (AZA), a demethylating agent, in combination with the mTOR inhibitor everolimus in MTC cells (MZ‐CRC‐1 and TT). This combined treatment showed a strong synergistic antiproliferative activity through the induction of apoptosis. The effect of everolimus and/or AZA on genome‐wide expression profiling was evaluated by Illumina BeadChip in MZ‐CRC‐1 cells. An innovative bioinformatic pipeline identified four potential molecular pathways implicated in the synergy between AZA and everolimus: PI3K‐Akt signaling, the neurotrophin pathway, ECM/receptor interaction, and focal adhesion. Among these, the neurotrophin signaling pathway was most directly involved in apoptosis, through the overexpression of NGFR and Bax genes. The increased expression of genes involved in the NGFR‐MAPK10‐TP53‐Bax/Bcl2 pathway during incubation with AZA plus everolimus was validated by western blotting in MZ‐CRC‐1 cells. Interestingly, addition of a neutralizing anti‐NGFR antibody inhibited the synergistic cytotoxic activity between AZA and everolimus. These results open a new therapeutic scenario for MTC and potentially other neuroendocrine tumors, where therapy with mTOR inhibitors is currently approved.