Genes Involved in Cell Adhesion, Cell Motility and Mitogenic Signaling Are Altered Due to HPV 16 E5 Protein Expression

Oncogene (2008) 27, 2532–2541 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Genes involved in cell adhesion, cell motility and mitogenic signaling are altered due to HPV 16 E5 protein expression N Kivi1, D Greco2, P Auvinen2 and E Auvinen1 1Department of Virology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland and 2Institute of Biotechnology, University of Helsinki, Helsinki, Finland We investigated the effects of the human papillomavirus leads to indefinite proliferation and immortalization of type 16 E5 oncogene on cellular gene expression in human keratinocytes (Munger and Howley, 2002) and induces epithelial cells using cDNA microarray. In a genome-wide premalignant neoplasms together with v-Ha-Ras in vivo microarray assay, the expression of 179 genes was found (Schreiber et al., 2004). Both E6 and E7 oncoproteins to be significantly altered due to E5 expression. The can also independently immortalize human cells, but expression of lamin A/C was downregulated at protein with reduced efficiency (Band et al., 1990; Halbert et al., level. The expression of protein kinase C-d and phosphoi- 1991). The role of the E5 protein in cellular transforma- nositide-3-kinase proteins was found to be upregulated. tion is much less understood. HPV 16 E5 is an 83-amino We also observed increased motility of E5-expressing acid membrane-associated protein found in the Golgi cells. We conclude that the E5 protein affects several apparatus, endoplasmic reticulum and nuclear mem- cellular pathways involved in cell adhesion, cell motility brane (Conrad et al., 1993). It is able to transform and mitogenic signaling. These alterations may together murine fibroblasts and keratinocytes (Valle and Banks, lead to inhibition of apoptosis and facilitate the establish- 1995; Chen et al., 1996). E5 also enhances the ment of persistent infection in the epithelium. immortalization potential of E6 and E7, and stimulates Oncogene (2008) 27, 2532–2541; doi:10.1038/sj.onc.1210916; the proliferation of human and mouse primary cells in published online 5 November 2007 cooperation with E7 (Bouvard et al., 1994; Sto¨ ppler et al., 1996). The E5 protein is known to inhibit gap- Keywords: HPV; papillomavirus; E5; microarray; cell junctional intercellular communication (Oelze et al., adhesion; cell motility 1995), which may lead to disturbance of normal epithelial maintenance and differentiation. It has also been shown that E5 alters the growth and differentiation of stratified epithelia and induces epithelial tumors at a high frequency in mice (Genther Williams et al., 2005). Introduction Moreover, important functions in late stages of viral replication have been suggested (Fehrmann et al., 2003; Cervical cancer is one of the most common causes of Genther et al., 2003). cancer-related deaths in women worldwide (Pisani et al., Alterations in cellular signaling pathways commonly 2002). A prerequisite for cervical cancer is infection by a contribute to cancer. A number of viruses have developed high-risk human papillomavirus (HPV; Walboomers mechanisms to modulate cellular signaling pathways for et al., 1999), out of which HPV 16 is the predominant reprogramming host cells and supporting their life cycles, type in all continents (Mun˜ oz et al., 2004). Cancer- or for controlling host defense responses (DiMaio et al., associated HPV types encode three oncogenes, E5, E6 1998; Burgert et al., 2002; Duerst and Morrison, 2003). and E7, and the E6 and E7 proteins have a significant We examined genome-wide gene expression by cDNA role in malignant transformation (Hawley-Nelson et al., microarrays in a stable epithelial cell line to investigate 1989; Munger et al., 1989; Leechanachai et al., 1992; holistic effects of the E5 oncogene. The expression of Pim et al., 1992; Straight et al., 1993). E6 and E7 several genes of the phosphatidylinositol phosphate stimulate cell proliferation by interfering with the kinase pathway appeared altered in microarray and were function of regulatory proteins in cells, including the validated by quantitative PCR. Protein kinase C-d tumor suppressors p53 and pRB (Dyson et al., 1989; (PKC-d) and phosphoinositide-3-kinase p55 regulatory Werness et al., 1990). Coexpression of both E6 and E7 subunit (PI3KR3) were found to be upregulated, and lamin A/C was downregulated at both mRNA and protein level. E5 protein seems to affect several cellular Correspondence: N Kivi, Department of Virology, Haartman pathways involved in cell adhesion, cell motility and Institute, University of Helsinki, PO 21 (Haartmaninkatu 3), Helsinki mitogenic signaling. We were also able to verify altered 00014, Finland. E-mail: niina.kivi@helsinki.fi cell motility of E5-expressing cells by live-cell imaging. Received 1 March 2007; revised 31 August 2007; accepted 10 October This is the first report on a large-scale transcriptome 2007; published online 5 November 2007 analysis of HPV E5 effects in epithelial cells. HPV 16 E5 alters cellular gene expression N Kivi et al 2533 Results Validation of gene expression profiling of selected genes by RT–PCR Gene expression profiling using cDNA microarrays On the basis of the microarray results, altogether 27 genes We performed a cDNA microarray experiment to screen were selected according to their biological relevance for for the expression of 16 000 transcripts in human qRT-PCR validation (Table 1). The 24-h time point was epithelial HaCaT cells expressing the E5 protein of used for validation. The microarray result was considered HPV 16 (HaCaT-E5). E5 expression was induced with valid, if the P-value of the qRT-PCR triplicates was o0.05. dexamethasone and confirmed by northern blotting and For 15 out of 27 genes (56%), the microarray result could quantitative real-time RT-PCR (qRT-PCR). No anti- be confirmed. Similar validation rates of cDNA microarray body to the HPV 16 E5 protein is available. E5 results have been reported by other authors (Rajeevan transcription was observed already after short induc- et al., 2001; Chuaqui et al., 2002). Upregulation of PI3K tion, and high expression level of E5 mRNA was and inositol polyphosphate-4-phosphatase was confirmed. reached at 24 h (Table 2). For this reason, 24 h induction Also upregulation of fibronectin (FN1), 67-kDa laminin was selected for the microarray experiment. Moreover, receptor and PKC-d transcripts was observed with both we have previously shown that prolonged expression of methods. Of matrix metalloproteinases (MMPs), down- the E5 protein for several days, albeit at a high level in regulation of MMP-7 was validated. Twelve genes gave transient transfections, is lethal to cells (Auvinen et al., discordant fold change as compared to microarray, 2004). Cellular gene expression in E5 cells was compared including MMP-16 and lamin A/C. to that in control cells transfected with the empty vector To obtain more profound information about the (HaCaT-pMSG), cultured and treated in a similar dynamics of E5 effect on host cell pathways, we selected manner. a set of further genes from the same Gene Ontology We initially assumed that even a minor change in gene families, including some downstream target genes. expression may be of importance, and thus no cutoff Another 34 genes were chosen, and the expression of was set as to the fold change of gene expression. Since these altogether 61 genes was studied by qRT-PCR after the availability of reliable statistical methods, P-values E5 induction for 0, 2, 4, 24, 48 and 72 h to follow the have been launched for validation. P-value o0.01 was effect on cellular gene expression along with time. We employed to infer statistical significance of the observa- found that already at earlier time points, E5 expression tions. Using this P-value cutoff, we identified 117 affects transcription of a majority of cellular genes upregulated and 62 downregulated genes in E5-expres- studied, including MMP-7, -12, -16 and PKC-d (Table 2). sing cells from the microarray assay (Supplementary The transcript levels of several genes oscillated along Table 1). These genes were functionally grouped with time. This may partly represent true alterations in according to the Gene Ontology Biological Process gene expression, although further analysis is needed to (BP) database (Ashburner et al., 2000; Figure 1). The better understand this finding. statistically significant over-represented gene categories (Fisher’s exact test P-value o0.05) were metabolism, Changes of the selected genes and their downstream protein modification and various responses to external effectors at protein level stimulus, for example stress, biotic stimulus and immune Of the genes whose altered expression was confirmed by responses. qRT-PCR, 15 were also investigated by western blotting histone deacetylation response to biotic phosphorylation response to pest\, stimulus pathogen or parasite defense response macromolecule metabolism immune response intracellular transport amino acid biosynthesis response to stimulus intracellular protein transport response to external biotic response to stress antigen processing antigen presentation protein modification phosphorus metabolism protein metabolism phosphate metabolism Figure 1 Gene Ontology chart. Clustering analysis was performed on 114 genes that were altered due to HPV 16 E5 expression in the microarray analysis. Distribution of major functional categories for biological process terms is shown in the pie chart. Oncogene HPV 16 E5 alters cellular gene expression N Kivi et al 2534 Table 1 Validation of microarray results by qRT-PCR at 0, 2, 4, 24, 48 and 72 h time points (Figure 2a). The Gene name PCR FC MA FC selection of these genes was largely determined by the (P-value) (P-value) availability of antibodies. E5 cells were compared to pMSG control cells induced in a similar manner. The PCR-validated, concordant expression of each protein was normalized against the Major histocompatibility 1.5 (0.0025) 1.8 (0.0053) complex, class II, DQ expression of b-actin, which was used as a loading beta 1 control in each blot.

Load more