Inhibitors of Raf kinase activity block growth of thyroid cancer cells with RET/PTC or BRAF mutations in vitro and in vivo.
OBJECTIVE
Papillary thyroid carcinomas are associated with nonoverlapping activating mutations of RET, NTRK, RAS and BRAF, which altogether are present in approximately 70% of cases. We postulated that compounds that inhibit a distal effector in the mitogen-activated protein kinase (MAPK) pathway would inhibit growth and tumorigenicity of human thyroid cancer cell lines with mutations of RET or BRAF.


RESULTS
We first examined the effects of AAL-881 and LBT-613, two inhibitors of RAF kinase activity, on RAF-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK activation in thyroid PCCL3 cells after conditional induction of expression of H-RAS(G12V) or BRAF(V600E). Both compounds blocked RAS and RAF-dependent MEK and ERK phosphorylation. They also potently blocked MEK phosphorylation in human thyroid cancer cell lines with either RET/PTC1 (TPC1) or BRAF(V600E) (NPA, ARO, and FRO) mutations. Inhibition of ERK phosphorylation was transient in TPC1 and ARO cells, with recovery of ERK phosphorylation associated with concomitant down-regulation of the MAPK phosphatases MKP-3 and DUSP5. Both compounds inhibited growth of all cell lines, with LBT-613 being approximately 10-fold more potent than AAL-881. TPC1 cells were more sensitive to growth inhibition (IC50 0.1-0.25 and approximately 0.05 micromol/L for AAL-881 and LBT-613, respectively) than BRAF + lines (IC50 2.5-5 and 0.1-0.5 micromol/L, respectively). Growth inhibition was associated with G1 arrest, and induction of cell death. Growth of ARO and NPA tumor xenografts was inhibited by LBT-613 or AAL-881. MEK and ERK phosphorylation was inhibited by both compounds in ARO but not in NPA cell xenografts.


CONCLUSIONS
Compounds that inhibit kinase activity are effective growth inhibitors for poorly differentiated thyroid cancer cell lines with either RET or RAF mutations, and hold promise for treatment of most forms of papillary thyroid carcinoma.
Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0547, USA.
Bin
B
Ouyang
Jeffrey A
JA
Knauf
Eric P
EP
Smith
Lei
L
Zhang
Tim
T
Ramsey
Naeem
N
Yusuff
David
D
Batt
James A
JA
Fagin
D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D054642:Dual Specificity Phosphatase 6; D054637:Dual-Specificity Phosphatases; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology*; D051379:Mice; D008819:Mice, Nude; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013964:Thyroid Neoplasms / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D023041:Xenograft Model Antitumor Assays; D048490:raf Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
C514876:AAL 881; D007546:Isoquinolines; C514877:LBT 613; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D048490:raf Kinases; C442320:DUSP5 protein, human; C517035:DUSP6 protein, human; D054642:Dual Specificity Phosphatase 6; D054637:Dual-Specificity Phosphatases; C517036:Dusp6 protein, mouse; D017027:Protein Tyrosine Phosphatases