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The Karyopherin Proteins, Crm1 and Karyopherin B1, Are Overexpressed in Cervical Cancer and Are Critical for Cancer Cell Survival and Proliferation Pauline J

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The Karyopherin Proteins, Crm1 and Karyopherin B1, Are Overexpressed in Cervical Cancer and Are Critical for Cancer Cell Survival and Proliferation Pauline J Int. J. Cancer: 124, 1829–1840 (2009) ' 2008 Wiley-Liss, Inc. The Karyopherin proteins, Crm1 and Karyopherin b1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation Pauline J. van der Watt1, Christopher P. Maske2, Denver T. Hendricks1, M. Iqbal Parker1,4, Lynette Denny3, Dhirendra Govender2, Michael J. Birrer5 and Virna D. Leaner1* 1Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, South Africa 2Division of Anatomical Pathology, Faculty of Health Sciences, University of Cape Town, South Africa 3Department of Obstetrics and Gynaecology, Faculty of Health Sciences, University of Cape Town, South Africa 4International Center of Genetic Engineering and Biotechnology (ICGEB), Cape Town, (ICGEB) Cape Town Component, South Africa 5Department of Cell and Cancer Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD The Karyopherin proteins are involved in nucleo-cytoplasmic lular genes.10,11 The progression of cervical cancer is thus a com- trafficking and are critical for protein and RNA subcellular local- plex biological process, accompanied by many genetic alterations. ization. Recent studies suggest they are important in nuclear enve- We used cDNA microarray analysis to profile normal and cancer lope component assembly, mitosis and replication. Since these are tissue of the cervix, to identify potential cancer biomarkers and all critical cellular functions, alterations in the expression of the Karyopherins may have an impact on the biology of cancer cells. genes functionally relevant to cervical tumorigenesis. Amongst In this study, we examined the expression of the Karyopherins, the genes that were found to be differentially expressed in the cer- Crm1, Karyopherin b1 (Kpnb1) and Karyopherin a2 (Kpna2), in vical cancer tissues were members of the Karyopherin superfamily cervical tissue and cell lines. The functional significance of these of nuclear transport proteins. Specifically, we identified increased proteins to cancer cells was investigated using individual siRNAs expression of Crm1 (Exportin1), Karyopherin b1 (Importin b1) to inhibit their expression. Microarrays, quantitative RT-PCR and Karyopherin a2 (Importin a1) in cervical cancer tissue com- and immunofluorescence revealed significantly higher expression pared to normal cervical epithelium. of Crm1, Kpnb1 and Kpna2 in cervical cancer compared to nor- mal tissue. Expression levels were similarly elevated in cervical The Karyopherin proteins are involved in the shuttling of cargo cancer cell lines compared to normal cells, and in transformed proteins, and certain RNAs, across the nuclear pore complex into epithelial and fibroblast cells. Inhibition of Crm1 and Kpnb1in and out of the cell nucleus. They have also been shown to have a cancer cells significantly reduced cell proliferation, while Kpna2 key role in transit through the cell cycle, mitosis and replication.12 inhibition had no effect. Noncancer cells were unaffected by the Crm1 is a Karyopherin protein involved in nuclear export. It rec- inhibition of Crm1 and Kpnb1. The reduction in proliferation of ognizes the leucine-rich nuclear export signal (NES) of cargo pro- cancer cells was associated with an increase in a subG1 population teins that require shuttling out of the nucleus, and carries them by cell cycle analysis and Caspase-3/7 assays revealed increased into the cytoplasm.13 Nuclear import of proteins is mediated by apoptosis. Crm1 and Kpnb1 siRNA-induced apoptosis was accom- panied by an increase in the levels of growth inhibitory proteins, the recognition of a nuclear localization signal (NLS) on the cargo protein by Karyopherin b1 (Kpnb1), which subsequently binds p53, p27, p21 and p18. Our results demonstrate that Crm1, 14 Kpnb1 and Kpna2 are overexpressed in cervical cancer and that the cargo and transports it into the cell nucleus. Kpnb1 can inhibiting the expression of Crm1 and Kpnb1, not Kpna2, induces import proteins directly, or, in the case of the classical nuclear cancer cell death, making Crm1 and Kpnb1 promising candidates import pathway, an adaptor molecule, of the Karyopherin a fam- as both biomarkers and potential anticancer therapeutic targets. ily, is required to bridge the interaction between Kpnb1 and its ' 2008 Wiley-Liss, Inc. cargo. The concerted action of Kpnb1 and Karyopherin a2 (Kpna2) is necessary for the nuclear import of proteins containing 14 Key words: cervical cancer; nuclear transport proteins; Crm1; a classical NLS. Nuclear transport also requires Ran, a small Karyopherin b1; Karyopherin a2 Ras-like GTPase involved in both import and export processes.15 In the case of cervical cancer cells, HPV proteins utilize the nu- clear transport proteins for transport into and out of the host cell Cervical cancer is the second most common cancer among nucleus, and the nuclear transport proteins hence play a role in women worldwide,1 with nearly 80% of cases occurring in devel- promoting viral pathogenesis. HPV capsid proteins, L1 and L2, oping countries.2 The primary risk factor in the development of enter the nucleus via interaction with Karyopherins b1 and a2, the disease is infection with the Human Papillomavirus (HPV),3,4 amongst other importin proteins, and it is suggested that the L2 and more than 90% of cervical cancers carry high-risk HPV protein facilitates nuclear import of the viral DNA.16–18 Once in DNA.4,5 The HPV E6 and E7 oncoproteins are responsible for the nucleus the viral DNA and capsid proteins can be assembled cancer development, and evidence has shown that they alone are into virion particles. HPV E6 is also known to enter the nucleus sufficient to immortalize human foreskin keratinocytes.6 Their via interaction with various Karyopherin proteins, whereafter it continued expression is essential for maintaining the transformed can interact with nuclear transcription factors.19 While several state.6 HPV E6 and E7 promote cellular transformation by binding studies have focused on understanding the import strategies of to and blocking the functions of the cell cycle regulatory proteins, p53 and pRb, respectively.7,8 Prophylactic vaccines against low- risk (HPV6, 11) and high-risk (HPV16 and 18) HPV types have recently been developed.9 They rely on the vaccination of women Grant sponsors: University of Cape Town, Carnegie Corporation of New before exposure to the virus; hence, their benefit to women already York, MRC South Africa, CANSA, NHLS Research Trust. infected with HPV is still unclear, as well as their benefit to *Correspondence to: Division of Medical Biochemistry, Faculty of women infected with HPV types other than HPV6, 11, 16 and 18. Health Sciences, University of Cape Town, Observatory, Cape Town Although the high-risk HPV proteins are the causative agents 7925, South Africa. Fax: 127-21-4066061. E-mail: [email protected] behind cervical cancer development, they initiate a cascade of on- Received 10 July 2008; Accepted after revision 17 October 2008 cogenic events within the cell, which ultimately drive its transfor- DOI 10.1002/ijc.24146 mation. For example, the cell develops an increased telomerase Publishedonline 11 November 2008 in Wiley InterScience (www.interscience. activity, which results in the deregulation of many important cel- wiley.com). Publication of the International Union Against Cancer 1830 VAN DER WATT ET AL. HPV proteins, less is known regarding their interaction with the Prof. A. K. Rustgi (University of Pennsylvania, Philadelphia, export receptor, Crm1. However, it has been reported that the USA). These cells were maintained in keratinocyte growth me- HPV E2 protein uses Crm1 for nuclear export,20 and that high-risk dium supplemented with 1 ng/ml EGF and 50 lg/ml pituitary HPV E6 proteins require Crm1 function for their role in the degra- extract. All cells were cultured in 95% air and 5% CO2 at 37°C. dation of p53.21 In this study, we focus on the analysis of the Karyopherin pro- RNA isolation, amplification and microarray analysis teins in cervical cancer patient specimens and cell lines. This RNA was isolated from the patient tissues using Trizol reagent study provides evidence for a role of these proteins in contributing (Invitrogen, Rockville, MD, USA), according to the manufac- to the cancer phenotype. Our data suggests that while Crm1, turer’s protocols. RNA was amplified using the Eberwine RNA Kpnb1 and Kpna2 are all overexpressed in cancer, Crm1 23,24 b amplification procedure and used to prepare cDNA probes as and Kpn 1 in particular have functional significance to cancer described previously.25 Human reference RNA (Stratagene, La biology. Jolla, Ca, USA) served as a control on all of the arrays. Amplified human reference RNA was labeled with Cy3-dUTP and amplified Material and methods RNA from tissue specimens was labeled with Cy5-dUTP. Labeled reference cDNA was combined with labeled patient cDNA in a 40 Tissue specimens and histology of cervical carcinomas ll reaction containing 2 lg human COT1 DNA (Gibco), 16 lg Twenty-six cervical specimens, including sixteen cervical can- polyA (Sigma, St. Louis, MO, USA) and 8 lg yeast tRNA in 13 cer biopsies and 10 normal cervical specimens, were collected hybridization solution (53 SSC, 25% formamide and 0.2% SDS). from patients at Groote Schuur Hospital (South Africa). Tissue After denaturation at 95°C for 1 min and snap cooling on ice, the specimens were obtained with patient consent and the Research probes were hybridized to cDNA microarray slides containing Ethics Committee of the University of Cape Town approved all 11,000 elements produced by the Microarray Core Facility, aspects of the project (REC REF153/2004). Normal specimens National Cancer Institute, using Incyte Genomics UniGEM were collected from women undergoing hysterectomies for rea- clones. Slides had been prehybridized (53 SSC, 0.1% SDS, 10 sons other than cervical cancer. Approximately 2 mm of the epi- mg/ml BSA) for 1 hr and washed in water followed by isopropa- thelial cell layer at the transformation zone of the normal speci- nol, before hybridization with the probe.
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