Epidermal Growth Factor Receptor Tyrosine Kinase Inhibition Represses Cyclin D1 in Aerodigestive Tract Cancers

Abstract

Purpose: 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 alltrans-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.Experimental Design: The BEAS-2B immortalized HBE cell line was exposed to varying concentrations of erlotinib, and effects on proliferation, cell cycle distribution, G 1 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 G 1 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 mol/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.

Tumor penetration of gefitinib (Iressa), an epidermal growth factor receptor tyrosine kinase inhibitor

“…In preclinical studies, erlotinib, given orally at 92 mg/kg, did not produce marked tumor penetration, achieving only a tumor/plasma ratio of 0.4 in mouse HN5 xenografts at 6 hours after dosing (25), compared with a gefitinib tumor/plasma ratio of 5 to 14 across a range of mouse tumor xenografts. A similar tumor/plasma profile has also been observed in a recent clinical study, where the mean concentration of erlotinib in lung or larynx tumors (2.9 Amol/L) of cancer patients treated daily with erlotinib (150 mg) for 9 days was about 55% of the mean plasma concentration (26). Although based on data from only four subjects, this limited distribution profile was consistent with the much lower volume of distribution (136 L) observed with erlotinib in man (27).…”

Loss of homotypic cell adhesion by epithelial-mesenchymal transition or mutation limits sensitivity to epidermal growth factor receptor inhibition

“…Trough, peak, and average steady-state plasma concentrations in treated patients reach 9.7, 15, and 11 Amol/L, respectively. Therefore, responses at the 10 Amol/L concentration lie within the window of clinical relevance (25,36,37). Moreover, growth inhibition by erlotinib for sensitive cell lines is dose dependent (24).…”

Cotargeting Cyclin D1 Starts a New Chapter in Lung Cancer Prevention and Therapy

“…The prevalence of the GG genotype in cyclin D1 overexpression seems to be about 30%. Dual targeting of cyclin D1 with bexarotene and erlotinib potentially will overcome the retinoic acid/rexinoid limitation in patients with this common polymorphism because erlotinib can transcriptionally repress cyclin D1 expression (17). Cyclin D1 is a very relevant prevention target, with frequent overexpression, in premalignant lung carcinogenesis, as shown, for example, by preclinical and clinical results of the Dmitrovsky group (10,18).…”