Overexpression of Kpnβ1 and Kpnα2 Importin Proteins in Cancer Derives from Deregulated E2F Activity

The Karyopherin superfamily comprises nuclear transport proteins, involved in the shuttling of certain cargo proteins into and out of the nucleus. Karyopherin beta 1 (Kpn beta 1) and Karyopherin alpha 2 (Kpn alpha 2) are importin proteins, which work in concert to transport their cargo into the nucleus. We previously identified increased expression of Kpn beta 1 and Kpn alpha 2 in cervical tumours compared to normal epithelium and in transformed cells compared to their normal counterparts. This study therefore aimed to identify the transcription regulatory mechanisms associated with high Kpn beta 1 and Kpn alpha 2 levels in cancer cells. Kpn beta 1 (-2013 to + 100) and Kpn alpha 2 (-1900 to + 69) promoter fragments were separately cloned into the reporter vector, pGL3-basic, and luciferase assays revealed both as significantly more active in cancer and transformed cells compared to normal. A series of deletion constructs identified the -637 to -271 Kpn beta 1 and 2180 to 224 Kpn alpha 2 promoter regions as responsible for the differential promoter activity, and a number of highly conserved E2F binding sites were identified within these regions. Mutation analysis confirmed the requirement of E2F sites for promoter activity, and ChIP analysis confirmed E2F2/Dp1 binding to the Kpn beta 1 and Kpn alpha 2 promoters in vivo. Dp1 inhibition resulted in decreased levels of the respective proteins, confirming the role of E2F in the overexpression of Kpn beta 1 and Kpn alpha 2 proteins in cancer. E2F activity is known to be deregulated in cervical cancer cells due to the inhibition of its repressor, Rb, by HPV E7. The inhibition of E7 using siRNA resulted in decreased Kpn beta 1 and Kpn alpha 2 promoter activities, as did the overexpression of Rb. In conclusion, this study is a first to show that elevated Kpn beta 1 and Kpn alpha 2 expression in cancer cells correlates with altered transcriptional regulation associated with deregulated E2F/Rb activities