CN04-03

Tobacco carcinogen-treatment of immortalized human bronchial epithelial (HBE) cells uncovered novel cancer chemoprevention targets. Treatment of these cells with classical and non-classical retinoid receptor agonists highlighted induced cyclin D1 proteasomal degradation as a molecular pharmacologic target for cancer chemoprevention. We previously reported cyclin D1 was often aberrantly expressed in human pre-malignant and malignant lung tissues. This helped provide a rationale to target cyclin D1 in clinical trials. To understand engaged mechanisms, the role of cyclin D1 in chemoprevention was studied in relevant pre-clinical models. Chemoprevention of tobacco-carcinogen exposed HBE cells by retinoic acid receptor (RAR) and retinoid X receptor (RXR) agonists, among other agents, was linked to cyclin D1 proteasomal degradation. This was proposed as a chemopreventive mechanism since induced cyclin D1 proteolysis conferred cell cycle arrest at G1 and in turn permitted repair of genomic DNA damage by carcinogens. Threonine 286 mutation stabilized cyclin D1 protein, implicating a phosphorylation event in this regulation. A phospho-specific anti-cyclin D1 antibody that can recognize phosphorylation changes at threonine 286 was used to confirm in immunoblot analysis that phosphorylation occurred. Glycogen synthase kinase (GSK) inhibitors revealed this kinase regulated post-translational regulation of cyclin D1, but not other D-type cyclins. To elucidate which of the 18 lysines present in cyclin D1 mediated ubiquitin-dependent degradation, these residues were engineered with individual or multiple mutations. Domains responsible for triggering cyclin D1 degradation were identified and these results will be presented. These stabilizing mutations shared an ability to preferentially localize cyclin D1 to the nucleus likely sequestering this protein from cytosolic degradation enzymes. An important role for cyclin D1 in chemoprevention was independently shown using small interfering RNAs (siRNAs) that targeted this species for repression. To uncover other involved degradation programs, gene profiling experiments were performed using carcinogen-transformed and chemoprevented HBE cells. Those studies independently identified the E1-like ubiquitin-activating enzyme (UBE1L) and its physical partner, ISG15, as engaged in regulating cyclin D1 stability. Since treatment with epidermal growth factor (EGF) augmented HBE cell growth and cyclin D1 expression, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) was used to block these effects. Together, these in vitro experiments provided a strong rationale to conduct proof of principle trials using agents that targeted cyclin D1. Proof of principle trials monitor changes in pharmacodynamic targets in pre-treatment versus post-treatment tumor biopsies. Changes are then related to pharmacokinetic measurements of a drug in plasma and tumor. Results of proof of principle trials will be presented that use a rexinoid (bexarotene), an EGFR-TKI (erlotinib), or a bexarotene and erlotinib combination regimen to target effectively cyclin D1 expression in lung cancer cases. A phase I clinical trial using bexarotene and erlotinib to target lung and aerodigestive tract cancers showed encouraging anti-tumor activity against lung cancer. This set the stage for an ongoing phase II combination therapy trial with bexarotene and erlotinib for relapsed lung cancer, as will be discussed. This led to translating this work back to the laboratory using useful animal models for lung cancer chemoprevention studies. One that will be presented is a human surfactant C promoter-driven cyclin E mouse transgenic model that targets expression to the lung and spontaneously activated cyclin D1 expression. Notably, these mice develop pre-malignant and malignant (adenocarcinoma) lung lesions with features highly reminiscent of those found in clinical lung carcinogenesis. Stable cell lines have been derived from these murine lung cancers. This makes possible the study of anti-neoplastic effects of chemopreventive agents ex vivo. These cells are able to form lung tumors following tail vein injections into syngeneic mice. This opens up the prospect for rapid assessment of chemopreventive agent effects in vivo. Taken together, these and other findings that will be presented strongly implicate cyclin D1 as a novel molecular pharmacological target for cancer chemoprevention.

First AACR Centennial Conference on Translational Cancer Medicine-- Nov 4-8, 2007; Singapore