Epidermal growth factor receptor tyrosine kinase inhibition represses cyclin D1 in aerodigestive tract cancers.
OBJECTIVE
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are active in cancer therapy. Mechanisms engaged during these clinical responses need to be determined. We reported previously that epidermal growth factor stimulation markedly increased cyclin D1 protein expression in human bronchial epithelial (HBE) cells, and this was opposed by chemoprevention with all-trans-retinoic acid. The current study sought to determine whether the EGFR TKI erlotinib repressed cyclin D1 protein expression in immortalized HBE cells, lung cancer cell lines, and clinical aerodigestive tract cancers.


METHODS
The BEAS-2B immortalized HBE cell line was exposed to varying concentrations of erlotinib, and effects on proliferation, cell cycle distribution, G1 cyclin expression, and cyclin D1 reporter activity were measured. Non-small-cell lung cancer cell lines were also evaluated for changes in proliferation and cyclin protein expression after erlotinib treatments. A proof of principle clinical trial was conducted. During this study, patients underwent a 9-day course of erlotinib treatment. Pretreatment and posttreatment tumor biopsies were obtained, and changes in candidate biomarkers were determined by immunostaining. Plasma pharmacokinetics and tumor tissue erlotinib concentrations were measured.


RESULTS
Erlotinib, at clinically achievable dosages, repressed BEAS-2B cell growth, triggered G1 arrest, and preferentially reduced cyclin D1 protein expression and transcriptional activation. Erlotinib also preferentially repressed proliferation and cyclin D1 protein expression in responsive, but not resistant, non-small-cell lung cancer cell lines. This occurred in the presence of wild-type EGFR sequence at exons 18, 19, and 21. Five patients were enrolled onto an erlotinib proof of principle clinical trial, and four cases were evaluable. Pharmacokinetic studies established therapeutic erlotinib plasma levels in all patients, but tissue levels exceeding 2 micromol/L were detected in only two cases. Notably, these cases had pathological evidence of response (necrosis) in posttreatment biopsies as compared with pretreatment biopsies. In these cases, marked repression of cyclin D1 and the proliferation marker Ki-67 was detected by immunohistochemical assays. Cases without pathological response to erlotinib did not exhibit changes in cyclin D1 or Ki-67 immunohistochemical expression and had much lower erlotinib tissue levels than did responding cases.


CONCLUSIONS
Taken together, these in vitro and in vivo findings provide direct evidence for repression of cyclin D1 protein as a surrogate marker of response in aerodigestive tract cancers to erlotinib treatment. These findings also provide a rationale for combining an EGFR TKI with an agent that would cooperatively repress cyclin D1 expression in clinical trials for aerodigestive tract cancer therapy or chemoprevention.
Petty
W Jeffrey
WJ
Department of Pharmacology, Norris Cotton Cancer Center, Hanover, New Hampshire 03755, USA.
Dragnev
Konstantin H
KH
Memoli
Vincent A
VA
Ma
Yan
Y
Desai
Neil B
NB
Biddle
Adrian
A
Davis
Thomas H
TH
Nugent
William C
WC
Memoli
Natalie
N
Hamilton
Marta
M
Iwata
Kenneth K
KK
Rigas
James R
JR
Dmitrovsky
Ethan
E
D014408:Biomarkers, Tumor / Q000378:metabolism; D001980:Bronchi / Q000473:pathology; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002986:Clinical Trials as Topic; D019938:Cyclin D1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D000069347:Erlotinib Hydrochloride; D005091:Exons; D016193:G1 Phase; D005770:Gastrointestinal Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D041981:Gastrointestinal Tract / Q000473:pathology*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D019394:Ki-67 Antigen / Q000096:biosynthesis; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D009336:Necrosis; D009369:Neoplasms / Q000378:metabolism; D011799:Quinazolines / Q000493:pharmacokinetics / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D013997:Time Factors; D015533:Transcriptional Activation
D014408:Biomarkers, Tumor; D019394:Ki-67 Antigen; D011799:Quinazolines; D019938:Cyclin D1; D004247:DNA; D000069347:Erlotinib Hydrochloride; D008156:Luciferases; D066246:ErbB Receptors