The HIV protease inhibitor nelfinavir down-regulates RET signaling and induces apoptosis in medullary thyroid cancer cells.
BACKGROUND
Mutations of RET tyrosine kinase are associated with the development of medullary thyroid cancer (MTC). The heat shock protein (HSP) 90 chaperone is required for folding and stability of RET mutants. HSP90 is a molecular target for the HIV protease inhibitor nelfinavir (NFV).


OBJECTIVE
We hypothesized that treatment with NFV may lead to the inhibition of RET signaling and induction of apoptosis in MTC cells.


METHODS
Two human MTC cell lines, TT and MZ-CRC-1, which harbor endogenous C634W or M918T RET mutations, respectively, were exposed to clinically achievable concentrations of NFV. JC-1 staining and caspase-3 cleavage assays were performed to measure mitochondrial membrane potential and apoptosis. Activation of RET signaling was examined by Western blot. Autophagy was monitored by the detection of the light-chain 3BII. Expression of HSP90 and LC3B were examined in 36 human MTCs.


RESULTS
At a therapeutic serum concentration (10 μM), NFV inhibited the viability of TT and MZ-CRC-1 cells by 55% and 10%, respectively. In a dose-dependent manner, NFV inhibited cyclin D1 and caused caspase-3 cleavage. NFV decreased the level of RET protein and blocked the activation of RET downstream targets (phosphorylated ERK, phosphorylated AKT, and p70S6K/pS6). NFV induced metabolic stress, activated AMP-activated protein kinase and increased autophagic flux. Pharmacological inhibition of autophagy (chloroquine) augmented NFV-inducible cytotoxicity, suggesting that autophagy was protective in NFV-treated cells. NFV led to mitochondrial membrane depolarization and induced both oxidative stress and DNA damage. An antioxidant (n-acetylcysteine) attenuated DNA damage and prevented NFV-inducible apoptosis. HSP90 overexpression was found in 17 of 36 human MTCs and correlated with metastases and RET mutations. LC3B was detected in 20 of 36 human MTCs.


CONCLUSIONS
NFV has a wide spectrum of activity against MTC cells, and its cytotoxicity can be augmented by inhibiting autophagy. Expression of NFV molecular targets in metastatic MTC suggests that NFV has a potential to become a thyroid cancer therapeutic agent.
Department of Pediatrics (K.J., A.R., J.C., A.P., V.V.), Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814; Department of Medicine (Y.K., J.K.-G., K.B.), Division of Endocrinology, Washington Hospital Center, and Department of Surgery (L.B.), Medstar Georgetown University Hospital, Washington, DC 20010.
Yevgeniya
Y
Kushchayeva
Kirk
K
Jensen
Antony
A
Recupero
John
J
Costello
Aneeta
A
Patel
Joanna
J
Klubo-Gwiezdzinska
Lisa
L
Boyle
Kenneth
K
Burman
Vasyl
V
Vasko
D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects; D018276:Carcinoma, Medullary / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects*; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D005260:Female; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D019888:Nelfinavir / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D013964:Thyroid Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
D017320:HIV Protease Inhibitors; D018841:HSP90 Heat-Shock Proteins; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D019888:Nelfinavir