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The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

Korzeniewski, Nina, Treat, Benjamin, Duensing, Stefan·2011·Molecular Cancer
DOI 10.1186/1476-4598-10-61PMC PMC3120798
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Abstract

Background: Infection with high-risk human papillomaviruses (HPVs) such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs) of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication). The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood. Findings: Here, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4) protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR) analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA. Conclusions: These results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted.

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Figure 1 HPV-16 E7 induces centriole overduplication through transcriptional deregulation and aberrant recruitment of PLK4 to maternal centrioles. (A) Quantification of centriole overduplication (>4 centrioles per cell) in U-2 OS/centrin-GFP cells transfected with either control (siControl) or PLK4 (siPLK4) siRNA duplexes for 24 h followed by ectopic expression of HPV-16 E7 for another 48 h. Mean and standard error of at least three independent experiments with at least 100 cells counted per experiment are shown. Asterisk indicates statistically significant differences (p ≤ 0.002). (B) Immunofluorescence microscopic analysis of human keratinocytes transduced with either empty vector (LXSN) or HPV-16 E7 for endogenous PLK4 following transiently transfected with centrin-GFP (48 h). Arrows indicate PLK4 dots at maternal centrioles. Note the presence of two dots in a HPV-16 E7-expressing cell (bottom panels). (C) Quantification of the percentage of cells with aberrant (two or more) PLK4 dots at maternal centrioles in human keratinocytes transduced with either a control plasmid, LXSN, or HPV-16 E7. Asterisk indicates statistically significant differences (p ≤ 0.05). (D) Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) analysis for PLK4 mRNA was performed on total RNA isolated from human keratinocytes expressing either a control plasmid, LXSN, or HPV-16 E7. βactin was used as a control housekeeping gene. Experiments were performed in triplicate and analyzed as described in the Materials and Methods section (Additional File 1). Asterisks indicate statistically significant differences (p ≤ 0.01). (E) Fluorescence microscopic analysis of U-2 OS/centrin-GFP cells transfected with either an empty vector control plasmid (pCMV-Neo) or E2F-1 for 48 h. Nuclei stained with DAPI. Scale bar indicates 10 μm. (F) Quantification of centriole multiplication (>4 centrioles, >1 daughter at one or more maternal centrioles) in U-2 OS/centrin-GFP cells ectopically expressing either a control plasmid or E2F-1 for 48 h. Mean and standard error of two independent experiments with a triplicate of at least 100 cells counted per experiment are shown. Asterisk indicates statistically significant differences (p ≤ 0.002). (G) Quantification of PLK4 promoter activity following transient co-transfection (48 h) of U 2-OS/centrin-GFP cells with the PLK4 promoter construct (pGL3-PLK4) and a transfection control (pRL-CMV) with either an empty vector control (Control) or E2F-1. The bar graph shows fold activation of the PLK4 promoter when compared with the empty luciferase construct, pGL-3, from three independent transfections, and expressed as mean and standard error. Asterisks indicate statistically significant differences (p ≤ 0.001). (H) Table comparing quantification of centriole overduplication (>4 centrioles), PLK4 promoter activation, and PLK4 mRNA upregulation in U-2 OS/centrin-GFP cells ectopically expressing the indicated construct for 48 h and analyzed as described in the Materials and Methods section (Additional File 1).
Figure 1 HPV-16 E7 induces centriole overduplication through transcriptional deregulation and aberrant recruitment of PLK4 to maternal centrioles. (A) Quantification of centriole overduplication (>4 centrioles per cell) in U-2 OS/centrin-GFP cells transfected with either control (siControl) or PLK4 (siPLK4) siRNA duplexes for 24 h followed by ectopic expression of HPV-16 E7 for another 48 h. Mean and standard error of at least three independent experiments with at least 100 cells counted per experiment are shown. Asterisk indicates statistically significant differences (p ≤ 0.002). (B) Immunofluorescence microscopic analysis of human keratinocytes transduced with either empty vector (LXSN) or HPV-16 E7 for endogenous PLK4 following transiently transfected with centrin-GFP (48 h). Arrows indicate PLK4 dots at maternal centrioles. Note the presence of two dots in a HPV-16 E7-expressing cell (bottom panels). (C) Quantification of the percentage of cells with aberrant (two or more) PLK4 dots at maternal centrioles in human keratinocytes transduced with either a control plasmid, LXSN, or HPV-16 E7. Asterisk indicates statistically significant differences (p ≤ 0.05). (D) Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) analysis for PLK4 mRNA was performed on total RNA isolated from human keratinocytes expressing either a control plasmid, LXSN, or HPV-16 E7. βactin was used as a control housekeeping gene. Experiments were performed in triplicate and analyzed as described in the Materials and Methods section (Additional File 1). Asterisks indicate statistically significant differences (p ≤ 0.01). (E) Fluorescence microscopic analysis of U-2 OS/centrin-GFP cells transfected with either an empty vector control plasmid (pCMV-Neo) or E2F-1 for 48 h. Nuclei stained with DAPI. Scale bar indicates 10 μm. (F) Quantification of centriole multiplication (>4 centrioles, >1 daughter at one or more maternal centrioles) in U-2 OS/centrin-GFP cells ectopically expressing either a control plasmid or E2F-1 for 48 h. Mean and standard error of two independent experiments with a triplicate of at least 100 cells counted per experiment are shown. Asterisk indicates statistically significant differences (p ≤ 0.002). (G) Quantification of PLK4 promoter activity following transient co-transfection (48 h) of U 2-OS/centrin-GFP cells with the PLK4 promoter construct (pGL3-PLK4) and a transfection control (pRL-CMV) with either an empty vector control (Control) or E2F-1. The bar graph shows fold activation of the PLK4 promoter when compared with the empty luciferase construct, pGL-3, from three independent transfections, and expressed as mean and standard error. Asterisks indicate statistically significant differences (p ≤ 0.001). (H) Table comparing quantification of centriole overduplication (>4 centrioles), PLK4 promoter activation, and PLK4 mRNA upregulation in U-2 OS/centrin-GFP cells ectopically expressing the indicated construct for 48 h and analyzed as described in the Materials and Methods section (Additional File 1).CC BY · PubMed Central

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Research works printed but not linked: 29

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Metadata

Authors
Korzeniewski, N., Treat, B., & Duensing, S.
Venue
Molecular Cancer
Publisher
Springer Science and Business Media LLC
Published
2011-05-24
Volume
10
Issue
1
ISSN
1476-4598
DOI
10.1186/1476-4598-10-61
PMC
PMC3120798
Cited by
50
References
0
Language
en

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