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Overexpression of Kpnb1 and Kpna2 Importin Proteins in Cancer Derives from Deregulated E2F Activity

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Overexpression of Kpnb1 and Kpna2 Importin Proteins in Cancer Derives from Deregulated E2F Activity Overexpression of Kpnb1 and Kpna2 Importin Proteins in Cancer Derives from Deregulated E2F Activity Pauline J. van der Watt, Ellen Ngarande, Virna D. Leaner* Division of Medical Biochemistry, Faculty of Health Sciences, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa Abstract The Karyopherin superfamily comprises nuclear transport proteins, involved in the shuttling of certain cargo proteins into and out of the nucleus. Karyopherin b1 (Kpnb1) and Karyopherin a2 (Kpna2) are importin proteins, which work in concert to transport their cargo into the nucleus. We previously identified increased expression of Kpnb1 and Kpna2 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 Kpnb1 and Kpna2 levels in cancer cells. Kpnb1(22013 to +100) and Kpna2(21900 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 2637 to 2271 Kpnb1 and 2180 to 224 Kpna2 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 Kpnb1 and Kpna2 promoters in vivo. Dp1 inhibition resulted in decreased levels of the respective proteins, confirming the role of E2F in the overexpression of Kpnb1 and Kpna2 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 Kpnb1 and Kpna2 promoter activities, as did the overexpression of Rb. In conclusion, this study is a first to show that elevated Kpnb1 and Kpna2 expression in cancer cells correlates with altered transcriptional regulation associated with deregulated E2F/Rb activities Citation: van der Watt PJ, Ngarande E, Leaner VD (2011) Overexpression of Kpnb1 and Kpna2 Importin Proteins in Cancer Derives from Deregulated E2F Activity. PLoS ONE 6(11): e27723. doi:10.1371/journal.pone.0027723 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received October 13, 2011; Accepted October 23, 2011; Published November 18, 2011 Copyright: ß 2011 van der Watt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the University of Cape Town, the Carnegie Corporation of New York, CANSA, and the Medical Research Council (SA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: This work was supported by the University of Cape Town/Carnegie Corporation of New York Development grants, CANSA, and the Medical Research Council (SA). This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: [email protected] Introduction We have recently shown that the expression of Kpnb1 and the Kpna protein, Kpna2, is elevated in cervical cancer and Karyopherin proteins are soluble nuclear transport receptors transformed cells at both the mRNA and protein levels [4]. We that function in transporting cargo proteins and certain RNAs into found too that inhibition of Kpnb1 expression in cervical cancer and out of the cell nucleus, via the nuclear pore complex (NPC) cells leads to cell death via apoptosis, suggesting that cervical [1]. Karyopherins may act as importins of exportins, depending of cancer cells become functionally dependent on Kpnb1 overex- their direction of transport. Karyopherin beta 1 (Kpnb1, also pression, highlighting the importance of Kpnb1 upregulation in known as Importin b or p97) is an importin protein that plays a maintaining cancer biology. In our study Kpna2 inhibition had no key role in the nuclear import process. Nuclear import via Kpnb1 effect on cervical cancer cell viability [4]; it is possible that its can occur either by Kpnb1 acting as an autonomous nuclear inhibition resulted in functional compensation by other Kpna transport receptor, or through its association with an adaptor family members. Noetzel et al. [5] showed that breast cancer cells protein, like Karyopherin alpha (Kpna, also known as Importin a), underwent apoptotic cell death when Kpna2 was inhibited [5], in which case the import process is known as classical nuclear suggesting that in some cell lines it might be functionally import [2]. In the classical nuclear import pathway, the nuclear redundant, while in others it is functionally relevant. In addition, localisation sequence (NLS) present in the cargo is recognised and many recent studies have identified high Kpna2 in tumour tissue, bound by the adaptor protein, Kpna, which then forms a trimer suggesting that the upregulation of Kpna2 associates with cancer complex with Kpnb1. The cargo:Kpna:Kpnb1 complex is development. Such cancers include melanoma [6], breast cancer transported across the nuclear pore complex into the nucleus, [7;8], oesophageal cancer [9], ovarian cancer [10], non-small cell where upon it is dissociated by the binding of RanGTP. There are lung cancer [11], prostate cancer [12], bladder cancer [13] and six Kpna isoforms (Kpna1-6), which have been determined to liver cancer [14]. Interestingly, Wang et al. [11] showed that have preferential binding to specific substrates [3]. The function- Kpna2 is secreted into the serum, suggesting it has potential ing of all six isoforms thus broadens the substrate range carried clinical usefulness as a cancer biomarker. In addition, several across the NPC. studies have reported that high Kpna2 expression associates with PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27723 E2F Regulates Kpnb1 and Kpna2 Overexpression poor patient survival [6–10;12;13], suggesting a potential prog- level of the empty vector, with the decrease in activity being more nostic role for Kpna2. pronounced in SVWI38 and CaSki cells, compared to WI38 While the overexpression of Kpnb1 and Kpna2 in cancer cells (Fig. 2C, D). This suggests that important functional elements appear to be significant events, little is known regarding their necessary for high basal Kpna2 promoter activity in transformed transcriptional regulation and the factors that control their and cervical cancer cells reside in the 2180 to 224 region of the expression and lead to their upregulation in cancer cells. In this promoter. study, therefore, we cloned and analysed the Kpnb1 and Kpna2 promoters, and identified an important role for the transcription The Kpnb1 and Kpna2 promoters contain functional E2F factor, E2F, in the induction of Kpnb1 and Kpna2 expression in sites cancer cells. A bioinformatic analysis of the regions 2637 to 2271 of the Kpnb1 promoter and 2180 to 224 of the Kpna2 promoter (using Results MatInspector and Conreal software programmes) identified a number of putative binding sites for E2F. Deregulated E2F activity Increased Kpnb1 and Kpna2 protein expression in is known to play a key role in cancer development; hence the transformed and cancer cells correlates with increased functionality of these putative E2F sites was investigated. Five promoter activity putative E2F binding sites were identified in the Kpnb1(2637 to Based on our previous findings showing elevated Kpnb1 and 2271) promoter region, and using site-directed mutagenesis (and Kpna2 expression in cervical cancer cells and transformed cells mutating at least three bases within the consensus binding sites), compared to normal, we investigated Kpnb1 and Kpna2 protein mutant constructs were generated. Three E2F sites were found to expression in a representative normal, transformed and cervical be functional, as their mutation resulted in significantly decreased cancer cell line. These included the normal fibroblast cell line, Kpnb1 promoter activity (Fig. 3A). This included the putative sites WI38, the SV40-transformed fibroblast cell line, SVWI38, and the labelled as E2F(a), E2F(b) and E2F(c). Interestingly, mutation of cervical cancer cell line, CaSki. Western Blot analysis revealed these three sites simultaneously did not confer any further decrease elevated Kpnb1 and Kpna2 expression in the transformed and in promoter activity, compared to mutation of the E2F sites cancer cells, compared to the normal WI38 cells (Fig. 1A). To individually, suggesting that they may work in concert to regulate determine the transcriptional regulatory mechanisms that associ- Kpnb1 gene expression (Fig. 3A). Of note, too, was the finding ate with elevated Kpnb1 and Kpna2 expression in cancer and that mutation of the E2F sites resulted in promoter activity transformed cells, an approximate 2 Kb region upstream of the comparable to that of the 2271 to +100 construct, suggesting that transcription start site of the Kpnb1 and Kpna2 genes, together it is the E2F sites that are responsible for the increase in promoter with approximately 100 bp of their 59 untranslated regions activity in the region from 2271 to 2637. Mutation
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