Abstract
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.
Highlights
Medullary thyroid cancer (MTC) is a neuroendocrine tumor originating from parafollicular C cells, and it is highly resistant to chemo- and radiotherapy (Vitale et al, 2001)
It is being increasingly recognized that aberrant DNA methylation seems to be involved in the development of drug resistance through transcriptional suppression of genes implicated in drug metabolism, apoptosis, cell cycle control, and other biological processes (Hervouet et al, 2013)
Taking into consideration that DNA methylation is a reversible event unlike genetic mutations, the use of demethylating agents has been recently proposed as a new therapeutic strategy in cancer
Summary
Medullary thyroid cancer (MTC) is a neuroendocrine tumor originating from parafollicular C cells, and it is highly resistant to chemo- and radiotherapy (Vitale et al, 2001). The hyperactivation of the PI3K/Akt/ mTOR cascade has a relevant role in the pathogenesis and progression of MTC. Most of the Abbreviations AZA, 5-Aza-20-deoxycytidine; BDNF, brain-derived neurotrophic factor; CI, combination index; DEGs, differentially expressed genes; DRI, dose reduction index; FDR, false discovery rate; MTC, medullary thyroid cancer; NGF, nerve growth factor; NT-3, neurotrophins-3; NT-4/5, neurotrophins-4/5; NT-6, neurotrophins-6; PARP, poly(ADP-ribose) polymerase; PF, potentiation factor; PI, propidium iodide; SAM, significance analysis of microarray; SEM, structural equation modeling; SPIA, signaling pathway impact analysis.
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