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. 2017 May;39(5):840-852.
doi: 10.1002/hed.24729. Epub 2017 Feb 25.

Integration of high-risk human papillomavirus into cellular cancer-related genes in head and neck cancer cell lines

Heather M Walline  1   2 , Christine M Goudsmit  2 , Jonathan B McHugh  3 , Alice L Tang  2   4 , John H Owen  2 , Bin T Teh  5 , Erin McKean  2 , Thomas W Glover  6 , Martin P Graham  2 , Mark E Prince  2 , Douglas B Chepeha  2 , Steven B Chinn  2 , Robert L Ferris  7 , Susanne M Gollin  8 , Thomas K Hoffmann  9   10 , Henning Bier  9   11 , Ruud Brakenhoff  12 , Carol R Bradford  2 , Thomas E Carey  2 ; University of Michigan Head and Neck Specialized Program of Research Excellence (SPORE) Program
Affiliations

Integration of high-risk human papillomavirus into cellular cancer-related genes in head and neck cancer cell lines

Heather M Walline et al. Head Neck. 2017 May.

Abstract

Background: Human papillomavirus (HPV)-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16- and HPV18-positive squamous cell carcinoma lines.

Methods: E6/E7 alternate transcripts were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites.

Results: All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 head and neck squamous cell carcinoma (HNSCC) lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration.

Conclusion: HPV integration into cancer-related genes occurred in 7 of 9 HPV-positive cell lines and of these 6 were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. © 2017 Wiley Periodicals, Inc. Head Neck 39: 840-852, 2017.

Keywords: cancer; head and neck squamous cell carcinoma (HNSCC); human papillomavirus (HPV); integration; oropharynx.

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Conflict of interest statement

The authors disclose no potential conflicts of interest.

Figures

Figure 1
Figure 1
HPV In Situ Hybridization in HNSCC cell lines. Panel A: UM-SCC-47 fluorescence in situ hybridization for HPV16, Panels B-H show hrHPV In Situ Hybridization indicated by dark blue signals. Panel B. UD-SCC-2 (43 HPV copies/cell), Panel C. UM-SCC-47 (5 HPV copies/cell), Panel D. UM-SCC-104 (1.6 HPV copies/cell), Panel E. UPCI:SCC90 (284 HPV copies/cell), Panel F. UPCI:SCC152 (150 HPV copies/cell), Panel G. UPCI:SCC154 (1.3 HPV copies/cell), Panel H. VU-SCC-147 (218 HPV copies/cell).
Figure 2
Figure 2
HPV Oncogene Transcript- Specific Quantitative RT-PCR and E6–E7 RT-PCR in HPV-Positive HNSCC Cell Lines. Bar graphs represent TaqMan quantitative PCR relative expression, and electrophoretic gel images represent E6-E7 RT-PCR. Panel A. Depiction of HPV16 and HPV18 splicing. Primers within the splice region or across splice junctions allow for exclusive amplification of full length E6 or alternate E6 transcripts. Panel B. UD-SCC-2, Panel C. UM-SCC-47, Panel D. UM-SCC-104, Panel E. UPCI:SCC90, Panel F. UPCI:SCC152, Panel G. UPCI:SCC154, Panel H. VU-SCC-147, Panel I. UM-GCC-1, Panel J. UM-SCC-105. Arrows indicate sizes of expected amplicon bands: HPV16 E6 FullLength_E7=499bp, HPV16 E6*I_E7= 454bp, and HPV16 E6*II_E7= 338bp, HPV18 E6_E7 FullLength=723bp, HPV18 E6*I_E7= 540bp, NO RT=no reverse transcriptase negative control.
Figure 3
Figure 3
Representative Cell Line DIPS PCR gels. A. HPV16-E1a/Adapter primers, B. HPV16-E1c/Adapter primers, C. HPV16-E5a/Adapter primers, D. HPV16-L2a/Adapter primers, E. HPV16-E2b/Adapter primers, F. HPV16-E6a/Adapter primers, G. HPV18-L2f/Adapter primers. M=100 bp marker ladder.
Figure 4
Figure 4
Schematic representation of integration events in HPV-positive HNSCC cell lines. Panel A. Linear organization of the HPV genome, Panel B. UD-SCC-2, Panel C. UM-SCC-47, Panel D. UM-SCC-104, Panel E. UPCI:SCC90, Panel F. UPCI:SCC152, Panel G. UPCI:SCC154, Panel H. VU-SCC-147, Panel I. UM-GCC-1, Panel J. UM-SCC-105.
Figure 5
Figure 5
Gel Electrophoresis of Cell Line Integration Transcript Analysis. Bold text= Viral/cellular fusion transcript, Italicized text=Transcript spans integration site. Panel A. UD-SCC-2, Panel B. UM-SCC-47, Panel C. UM-SCC-104, Panel D. UPCI:SCC090, Panel E. UPCI:SCC152, Panel F. UPCI:SCC154, Panel G. VU-SCC-147. M= 100bp ladder.
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