Role of cyclin D1 in conferring malignant phenotypes on immortalized esophageal epithelial cells

Abstract

Cyclin D1 is commonly overexpressed in many types of human squamous cell carcinomas, including esophageal squamous cell carcinoma. Studies in the past mainly focused on the role of cyclin D1 in cell growth and regulation of the G1/S transition of the cell cycle. Recently, malignant transformation induced by classical oncogenes was shown to be associated with upregulation of cyclin D1. So far, there are still no reports on the direct contribution of cyclin D1 to malignant transformation of immortalized epithelial cells. In this study, the role of cyclin D1 in the early stage of esophageal carcinogenesis was investigated by stable overexpression of cyclin D1 in an hTERT-immortalized esophageal epithelial cell line, NE2-hTERT. Our results showed that ectopic expression of cyclin D1 stimulated anchorage-independent growth in soft-agar, which is a major hallmark of transformed cells. This finding was further supported by the decrease in colony number in an esophageal cancer cell line (KYSE510) with cyclin D1 knockdown by siRNA. Cyclin D1 overexpressing esophageal epithelial cells also acquired a higher ability to migrate through cellular matrix, and showed reduced adhesion to different substrates. Western blotting and pull-down assays were used to further explore the mechanism involved. The results showed that the overexpression of cyclin D1 was associated with upregulation of pERK1/2 and reduced Rac1 activity. Our findings suggest that cyclin D1 overexpression promotes invasive potential and migration ability in immortalized esophageal epithelial cells, and that these malignant phenotypes may be mediated by pERK1/2 and cytoskeletal signaling. [This study is supported by the Research Grants Council of the Hong Kong SAR, China (Central allocation Project No. HKUST 2/06C)