Microarray Analyses Uncover UBE1L as a Candidate Target Gene for Lung Cancer Chemoprevention

Ian Pitha-Rowe,Tom Turi,Debra A Dimattia,Lynn Pinder,W Jeffrey Petty,Natalie Memoli,Konstantin H Dragnev,Qing Feng,Vincent Memoli,Petra H Koza-Taylor,Jean Beebe,Ethan Dmitrovsky,Sutisak Kitareewan

doi:10.1158/0008-5472.can-03-3938

Abstract

Retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and chemoprevention. We reported previously that all-trans-retinoic acid (RA) treatment prevented carcinogen-induced transformation of immortalized human bronchial epithelial (HBE) cells. To identify cancer chemopreventive mechanisms, immortalized (BEAS-2B), carcinogen-transformed (BEAS-2B(NNK)), and RA-chemoprevented (BEAS-2B(NNK/RA)) HBE cells were used to conduct microarray analyses independently. Species increased in chemoprevented as compared with immortalized HBE cells (group I) and those augmented in chemoprevented as compared with transformed HBE cells (group II) included known RA-target genes as well as previously unrecognized RA-target genes in HBE cells. Unexpectedly, both groups were also enriched for interferon-stimulated genes. One interferon-stimulated gene of particular interest was UBE1L, the ubiquitin-activating enzyme E1-like protein. UBE1L expression was also induced after prolonged RA-treatment of immortalized HBE cells. UBE1L mRNA was shown previously as repressed in certain lung cancer cell lines, directly implicating UBE1L in lung carcinogenesis. Notably, UBE1L immunoblot expression was reduced in a subset of malignant as compared with adjacent normal lung tissues that were examined. Immunohistochemical analyses were performed using a new assay developed to detect this species using rabbit polyclonal anti-UBE1L antibodies independently raised against the amino- or carboxyl-termini of UBE1L. Studies done on paraffin-embedded and fixed tissues revealed abundant UBE1L, but low levels of cyclin D1 expression in the normal human bronchial epithelium, indicating an inverse relationship existed between these species. To study this further, cotransfection into HBE cells of wild-type or mutant UBE1L species was accomplished. In a dose-dependent manner, wild-type but not mutant UBE1L species repressed cyclin D1 expression. This implicated UBE1L in a retinoid chemoprevention mechanism involving cyclin D1 repression described previously. Taken together, these findings directly implicate UBE1L as a candidate-pharmacologic target for lung cancer chemoprevention. These findings also provide a mechanistic basis for the tumor suppressive effects of UBE1L through cyclin D1 repression.

Highlights

IntroductionThese in vitro findings were confirmed and extended in our prior work that revealed cyclin D1, cyclin E, and epidermal growth factor receptor were each frequently overexpressed in bronchial preneoplasia and in non–small-cell lung cancers (2, 8 –11)
Lung cancer is the leading cause of cancer related deaths for men and women in the United States
We reported previously that acute N-nitrosamine-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment of the immortalized BEAS-2B human bronchial epithelial (HBE) cell line caused cellular transformation to occur with increased proliferation, anchorage-independent growth, and tumor formation in athymic mice

Summary

Introduction

These in vitro findings were confirmed and extended in our prior work that revealed cyclin D1, cyclin E, and epidermal growth factor receptor were each frequently overexpressed in bronchial preneoplasia and in non–small-cell lung cancers (2, 8 –11) These and other findings raised the prospect that these or other differentially overexpressed species in bronchial preneoplasia or malignant lung cancers were chemopreventive targets. Affymetrixbased microarray analyses of immortalized, carcinogen-transformed, and RA-chemoprevented HBE cells were used to uncover target genes having increased expression during chemoprevention. One of these augmented species was the ubiquitin-activating enzyme E1-like (UBE1L), a potential tumor suppressor in lung cancers [12]. Immunohistochemical analyses revealed that UBE1L was abundantly expressed in the histologically normal human bronchial epithelium where low levels of cyclin D1 expression were known to be evident

Methods

Results

Conclusion