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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.42 No.2 pp.23-32
DOI : https://doi.org/10.17779/KAOMP.2018.42.2.001

MAPRE1 Amplification Correlates with Immortalization in Human Papilloma Virus 16 E6/E7-Transfected Oral Keratinocytes

Jue Young Kim, Young-Jin Park, Jin Kim*
Oral Cancer Research Institute, Department of Oral Pathology, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 03722, Korea
†

Both authors equally contributed to this work as the co-first authors.


Correspondence: Jin Kim, Oral Cancer Research Institute, Department of Oral Pathology, Yonsei University College of Dentistry, Yonsei-Ro 50, Seodaemun-gu, Seoul 03722, Korea. Tel: +82-2-2228-3031, Fax: +82-2-392-2959 E-mail: jink@yuhs.ac
February 26, 2018 March 9, 2018 April 6, 2018

Abstract


Immortalization is an essential process of the transformation of cells to a neoplastic growth. High risk human papillomavirus (hrHPV) infection has been the major cause of head and neck squamous cell carcinoma (HNSCC). The aim of this study was to search for a novel pathway causing immortalization in HPV16 E6/E7 transfected immortalized oral keratinocytes (IHOK). hrHPV integration sites were identified through DNA sequencing. HPV16 E6/E7 genes were integrated into 1q32.2, 12q21.2, 15q15.2, and 19q13.43 in IHOKs. Array-CGH was conducted to examine the deranged sites of the genes of IHOK. Of the 587 amplification genes, 70 genes were resided on chromosome 20. We selected PLAGL2 and MAPRE1 as the most amplified genes. PLAGL2 and MAPRE1 mRNA showed higher expression in IHOK than in normal keratinocytes. Knockdown of MAPRE1 significantly reduced telomerase activity. The analysis using a public database substantiated our data, showing the amplification of chromosome 20 and MAPRE1. In conclusion, our results suggest that MAPRE1 could play a crucial role in activating telomerase activity in hrHPV-infected cells. This finding may provide basic data to develop a novel target therapy for hrHPV-related HNSCC.


High risk Human papillomavirus (hrHPV) , Immortalization , Chromosome 20q , MAPRE1 , hTERT

MAPRE1 유전자 증폭이 사람 유두종 바이러스16 E6/E7을 도입한 구강각화세포의 불멸화에 미치는 영향

김 주영, 박 영진, 김 진*
연세대학교 치과대학 구강병리학 교실, 구강종양연구소, BK21 플러스 통합구강생명과학 사업단

초록

    Ⅰ. INTRODUCTION

    It is well known that mucosal human papillomavirus (HPV) infections cause a wide spectrum of human proliferative diseases from benign papillomas to invasive carcinomas in cervical, vulvar, vaginal, anal, penile, and head and neck areas.1) High risk-HPV (hrHPV) infections are related to at least 90% of human cervical cancer.2) Recently, hrHPV infections have emerged as the main cause of head and neck squamous cell carcinomas (HNSCC).3) Even 20~25% of oral SCC has been shown to relate to hrHPV infections in the United States.4) Further, hrHPV-related SCC demonstrates better prognosis than hrHPV-not related types.5) Therefore, future differentiated therapeutic interventions are required for patients with HPV-positive HNSCC on the basis of underlying carcinogenic mechanism.

    The classical carcinogenic mechanism of hrHPV DNA by two viral oncogenes E6 and E7 has been well established. hrHPV E6 protein constructs a complex with an E3 ubiquitin ligase and E6-associated protein (E6AP), and then ubiquitinates the p53 tumor suppressor protein, resulting in deregulation of both the G1/S and G2/M cell cycle checkpoints.6) Consequently, DNA-damaged cells are accumulated, leading to genomic instability.7) hrHPV E7 protein binds to the cullin 2 ubiquitin ligase complex and the ubiquitinates retinoblastoma (pRb) tumor suppressor protein. In turn, the ubiquitination induces the degradation of pRb, resulting in an uncontrolled G1/S phase of the cell cycle.8) Finally, free E2F family induces S-phase genes, leading to cell proliferation.

    As another immortalization mechanism, HPV16 E6 protein binds to the GC-rich sequences of the telomerase promoter with MYC proteins and SP1, leading to telomerase activation.9) However, telomerase activation by oncoprotein E6 and p53 degradation are not sufficient to cause immortalization or oncogenic transformation.10)

    As evidence of genetic instability by the operation of an abnormal cell cycle, hrHPV-positive cells frequently acquire a number of genetic imbalances and structural chromosome aberrations such as translocations, deletions, and amplification at early passages.7) Structural genomic changes of the host genome are frequently observed at the HPV integration site11) Genomic instability caused by HPV allows cells to accumulate additional genetic alterations, eventually leads to immortalization and malignant transformation.12) In cervical cancers, several additional genetic alterations have been reported. For example, the activation of the PI3-kinase /PKB/AKT pathway through increased PIK3CA expression is involved in transformation in hrHPV-immortalized cells and may therefore play a critical role in hrHPV-mediated carcinogenesis.13) In E6- transgenic mice, E6 activates transcription of the E2F-responsive genes, MCM7 and cyclin E, in the absence of E7.14) Despite these proven genetic alterations, only 0.3% of HPV-infected epithelial cells advance to cervical malignancy15), which means that only selected clones progress to invasive cancer through numerous genetic damage after HPV infection. Taken together, the aim of this study is to search a novel pathway of immortalization by genomic instability analysis in hrHPV- immortalized oral keratinocytes. This approach will contribute to the development of a differentiated therapeutic strategy in hrHPV-positive HNSCC.

    II. MATERIAL AND METHODS

    1. Cell culture

    Generation of normal human oral keratinocyte (NHOK) (16) and HPV16 E6/E7-transfected immortalized human oral keratinocytes (IHOK) (17) has previously been described. IHOKs were cultured with two types of culture media. When cultured in the keratinocyte growth medium (KGM; Lonza, Walkersville, MD, USA) supplemented with 2 ml of bovine pituitary extract (13 mg/ml), each 0.5 ml of hydrocortisone (0.5 mg/ml), human epidermal growth factor (0.5 μg/ml), insulin (5 mg/ml), epinephrine (0.5 mg/ml), transferrin (10 mg/ml), triiodothyronine (6.5 μ g/ml), GA-1000, and 0.05 mM CaCl2, the cells were labeled as IHOK-KGM. When cultured in EF medium consisting of Dulbecco’s Modified Eagles Medium (DMEM; Gibco BRL, Grand Island, NY, USA) and Ham’s Nutrient Mixture-F12 (Gibco BRL, Grand Island, NY, USA) at a ratio of 3:1 supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.01 μg/ml cholera toxin, 0.04 μg/ml hydrocortisone, 0.5 μg/ml insulin, 0.5 μg/ml apo-transferrin, and 0.2 μg/ml triodothyronine (Sigma, St. Louis, MO, USA), the cells were labeled as IHOK-EF. SiHa and CaSki were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. All cells were cultured at 37℃ in an incubator that contained 5% CO2.

    2. Polymerase Chain Reaction (PCR), Reverse Transcription-PCR (RT-PCR), and real-time PCR

    Total cellular DNA or RNA was extracted from IHOK-KGM and IHOK-EF using a QIAamp DNA mini kit or an RNeasy plus mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The cDNA was synthesized from 1 μg of the total RNA by using a First Strand cDNA Synthesis kit (Roche applied science, Mannheim, Germany) according to the manufacturer’s instructions. The cDNA product was amplified by PCR using AccuPower Hotstart PCR PreMix (Bioneer, Daejeon, South Korea). The primers were synthesized by Macrogen (Seoul, South Korea), and are listed in Table 1. The reaction mixture was subjected to 30 amplification cycles of 40 s at 94 °C, 40 s at 58 °C, and 40 s at 72 °C. The PCR products were visualized in 1.5% agarose gel using Stay Safe Nucleic Acid Gel Stain (Real Biotech Corporation, Taipei, Taiwan). Real-time PCR was performed using SYBR Green I Master (Roche applied science, Mannheim, Germany) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Light Cycler 480 Software (Roche Applied Science, Basel, Switzerland) was used for data analysis.

    3. Analysis of HPV16 E6/E7 integration sites

    DNA was purified using an Intron MEGA quick-spin kit (Intron, Seongnam, South Korea). The DNA sequencing of the amplified viral-host junctions was analyzed by Macrogen (Seoul, South Korea). The integration loci were determined by database alignments using the National Centre for Biotechnology Information (NCBI) blast tool and the University of California, Santa Cruz (UCSC) genome browser, release hg19. Detection of integrated papilloma virus sequences was performed using PCR to determine the HPV integration loci at the DNA level. The PCR products were visualized using ethidium bromide in 0.7% agarose gel. The oligonucleotide primers were synthesized by Macrogen (Seoul, South Korea), and are listed in Table 2.

    4. Array-CGH analysis

    Array-CGH was performed using a MacArray Kary 4000 basic access control (BAC)-chip (Macrogen, Seoul, South Korea) including 4000 duplicate BAC clones covering the whole human genome with a resolution of about 1 Mb. NA15510 (lymphoblastoid cell lines; Macrogen, Seoul, South Korea) was used as reference DNA. The threshold for determining chromosome gain or loss was defined as log2 ratio >0.25 or <−0.25.

    5. siRNA transfection

    Pleomorphic Adenoma Gene-Like 2 (PLAGL2) (siRNA No.1117727), Microtubule Associated Protein RP/EB family member 1 (MAPRE1) (siRNA No.1092257), and control siRNA were purchased from Bioneer Corporation (Daejeon, South Korea) (Table 3). The siRNA-mediated inhibition of gene expression was carried out using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Transfection efficiencies were analyzed at 48 h post-transfection.

    6. Telomeric repeat amplification protocol (TRAP) assay

    Telomerase activity was measured using the TeloTAGGG Telomerase PCR ELISA kit (Roche applied science, Mannheim, Germany). Extracts were obtained from 2 ×105 cells using a lysis buffer and centrifuged at 4 ℃ for 20 min at 13000 rpm. Each protein was divided into two aliquots before performing the assay: one aliquot as the negative control was prepared at 85 °C for 10 min and the other aliquot was used to evaluate the telomerase mediated addition of telomeric sequence. The products obtained were amplified by PCR. The PCR product was hybridized to a digoxigenin -(DIG)-labelled, telomeric repeat-specific detection probe. The resulting product was fixed using a biotin labelled primer on a streptavidin-coated microplate. The immobilized PCR product was detected by antibody against digoxigenin conjugated to peroxidase. Finally, the probe was visualized by peroxidase-metabolizing 3,3',5,5'-Tetramethylbenzidine (TMB) to form a colored reaction product. The absorbance of the samples was measured at 450 nm using an ELISA microtiter reader.

    7. Public database analysis

    Array CGH data described here are available from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm. nih.gov/gds/) through series accession numbers GSE72063 (18). This public database was obtained by transduction of ten hrHPV types in human foreskin keratinocytes.

    8. Statistical analysis

    Statistical analysis was performed as applicable using the Mann-Whitney U test with SPSS software version 23.0 (IBM Corporation, NY, USA). to determine the statistical significance of the differences between measurements. P-values < 0.05 were considered to be significant.

    III. RESULTS

    1. Integration of HPV16 E6/E7 in IHOKs

    Two cell lines of HPV16 E6/E7-transfected immortalized human oral keratinocytes, IHOK-KGM and IHOK-EF were used in this study (Fig. 1A). The detection of HPV16 E6 and E7 viral oncogene was confirmed by PCR (Fig. 1B). SiHa and CaSki cells were used as positive controls. We examined HPV16 E6/E7 integration in IHOKs; HPV16 E6/E7 genes were inserted in 4 sites, including 1q32.2, 12q21.2, 15q15.2, and 19q13.43. Three sites are located in intergenic regions, except for 15q15.2 located in TTBK2 intron 9 (Fig. 1C, D).

    2. Increased genomic instability in IHOKs

    To search for a genetic aberration in IHOKs, we used array CGH to screen genomic change in IHOK-KGM compared with NA15510 (reference cell). The status of chromosome for IHOK-KGM is shown in Figure 2A. Of the 4042 BAC clones, 587 copy number gains (14.5%) and 272 copy number losses (6.7%) were identified. Gains were found in chromosome 20 and chromosome 5, whereas chromosomal losses were found in chromosome X and 18. All 8 clones showing more than 1.0 of log2 ratio were resided in the region of 20q (Fig. 2B) (Table 4). Gains were also observed in two integration sites. The log2 ratio mean was 0.44 and 0.45 for 1q32.2 and 19q13.43, respectively (Table 5).

    3. MAPRE1 mRNA related to hTERT expression and activity in IHOKs

    To search candidate genes in chromosome 20 related to telomerase activity, the Wnt/β-Catenin signaling activated genes, PLAGL2 and MAPRE1, were selected as the candidate genes causing telomerase activation (19, 20).

    We conducted RT-PCR to validate the RNA expression of the final candidate genes, PLAGL2 and MAPRE1, in IHOKKGM and IHOK-EF (Fig. 3A). The mRNA expression of PLAGL2 and MAPRE1 increased in two IHOK cell lines compared with NHOK. We introduced siRNA to evaluate whether PLAGL2 and MAPRE1 affect hTERT expression in IHOK-EF. Knockdown efficiency of siRNA of PLAGL2 and MAPRE1 showed 74.6% and 75.2%, respectively (P < 0.05, Fig. 3B). Knockdown of PLAGL2 or MAPRE1 mRNA led to the reduction of hTERT expression (Fig. 3C). To determine whether PLAGL2 or MAPRE1 regulates telomerase activity, we conducted TRAP assay. The results showed that the telomerase activity was significantly reduced following transfection with siMAPRE1 (Fig. 3D). These results suggest that telomerase activity could be regulated by MAPRE1.

    4. Chromosome 20 instability in public data base

    With the array CGH dataset obtained from the public database (18), we searched for chromosomal instability. Among the datasets, we selected the HPV16-immortalized cell line and analyzed chromosomal instability. All copy number variations of the HPV16-immortalized cell line are shown in Figure 4A. The threshold for determining chromosomal gain or loss was defined as log2 ratio >0.25 or <−0.25. Copy number gains were detected only in chromosome 20. Of the 2208 probes, 2012 copy number gains (91.12%) and 4 copy number losses (0.0018%) were identified (Fig. 4B). The HPV16-immortalized cell line showed gains of copy numbers in PLAGL2 and MAPRE1. The log2 ratio mean was 0.40 and 0.67 for PLAGL2 and MAPRE1, respectively (Fig. 4C).

    IV. DISCUSSION

    The complete transformation process initiated by the expression of the E6 and E7 oncogenes requires the induction of genomic instability and selection for oncogenic mutations (21). Previously, we showed that the upregulation of hTERT expression and telomerase activity was essential for carcinomatous transformation in IHOK cells.22) However, telomerase activation by oncoprotein E6 and p53 degradation are not sufficient to cause immortalization or carcinomatous transformation.10) The goals of this study were to search a novel pathway of the immortalization process of IHOK transfected by HPV16 E6/E7

    Amplification of the chromosome 20q region has been reported in a wide variety of cancers, such as pancreatic cancers23), gastric cancers24), colorectal cancers25), and breast cancers26). Accumulating data revealed the chromosome 20q instability in oral squamous cell carcinoma (OSCC). The alterations of chromosome 20q11.21-q13.33 occurred in 52% of OSCC.27) More than half of OSCC cell lines showed loss of 3p, gain of 3q, 8q, and 20q.28) Correspondingly, the chromosome 20q was the most frequent site of genetic instability in our study. Taken together, we can extrapolate that the genes encoded on 20q play important roles in contributing to carcinogenesis in many human cancer as well as OSCC.26,29)

    Copy number change was observed in two integration sites, 1q32.2 and 19q13.43, of which the log2 ratio mean was 0.44 and 0.45, respectively. Statistically, the integration sites of HPV DNA is associated with genetic instability, even no selectivity of chromosomal sites occurs in HPV infection.11) In spite that no known oncogenes or tumor suppressor genes are resided in these regions, we could not exclude the possibility that amplification of these integration sites may play a role in immortalization of IHOKs.

    Our study focused on the amplification of chromosome 20q related to immortalization of IHOK, because the chromosome 20q was the most amplified region. The positive relationship between chromosome 20q11 and HPV infection has been reported, evidenced by the data that E2F1, cell cycle regulator, 19% was activated via amplification of chromosome 20q11 in HPV-positive HNSCC compared with only 2% in HPV-negative group.30) Reporting the amplification of chromosome 20q associated with immortalization of airway and anogenital epithelial cells, the investigators considered the possibility that the abrogation of pRb by HPV E7 pathway may lead to a growth block and consequently lead to be compensated by the amplification of the genes on chromosome 20.31)

    To determine whether the genes resided in chromosome 20q are specifically involved in the immortalization process, we selected PLAGL2 and MAPRE1 as the Wnt/β-Catenin signaling activated gene. PLAGL2 activates the canonical Wnt/β-Catenin pathway in neural stem cells/progenitor cells.19) PLAGL2 is an oncoprotein involved in various malignancies including lipoblastomas, hepatoblastomas, and acute myeloid leukemia. PLAGL2 interacts with P53 induced RING-H2 protein, regulating p53 stability, uncovering its novel function as an oncoprotein.32) MAPRE1 promotes cell growth and induces tumor formation by activating the Wnt/ β-Catenin pathway.20) MAPRE1 protein was first identified by its binding to the adenomatous polyposis coli (APC) protein. Because this protein localizes to microtubules, it is believed that it is involved in the regulation of microtubule structures and chromosome stability.33) Considering that MAPRE1 negatively regulates telomeric repeat-binding factor 1, MAPRE1 may confer telomere elongation.34) In our study, the knockdown of MAPRE1 led to a reduction of telomerase activity, suggesting that MAPRE1 is associated with immortalization of IHOK. MAPRE1 has also been known as a potential biomarker in detecting adenoma and early colorectal cancer.35) Recently, MAPRE1 overexpression has been reported to serve as a prognostic marker in OSCC.36) Taken together, MAPRE1 can be a target molecule to develop a differentiation strategy in HPV-positive HNSCC.

    In conclusion, our results suggest that chromosomal instability caused by the replication of integrated HPV lead to the amplification of chromosome 20q, in which amplified MAPRE1 induces hTERT expression and telomerase activity eventuating in immortalization of oral keratinocytes. This finding might provide basic data to develop a novel target therapy for hrHPV-related HNSCC.

    ACKNOWLEDGMENTS

    This work was supported by the Priority Research Centers Program through the National Research foundation (NRF) of Korea, funded by the Ministry of Education, Science and Technology (2011-0031396).

    Figure

    KAOMP-42-23_F1.gif

    HPV16 E6/E7 integrated in IHOKs. (A) Morphology of IHOK-KGM and IHOK-EF. Two cells exhibited polygonal shape. (B) HPV16 E6 and E7 DNA insert in IHOK-EF and IHOK-KGM. SiHa and CaSki were used as a positive control for HPV16 E6 and E7. (C) HPV16 E6/E7 integration sites as determined by PCR. GAPDH was used as loading control. (D) Schematic view of HPV16 E6/E7 integration sites at 1q32.2, 12q21.2, 15q15.2, and 19q13.43 in IHOKs. Red bars indicate the integration sites of HPV16 E6/E7. Each black arrow represents the location of an HPV and chromosome breakpoint.

    KAOMP-42-23_F2.gif

    Analysis of array CGH data identified genomic instability in IHOK-KGM. (A) Frequencies of copy number alteration were plotted as genome locations in IHOK. Alteration are shown using light gray (gain) and dark gray (loss) coloration. The highest gains are observed at chromosomes 20 and 5. The lowest losses are observed at chromosomes 18 and X. (B) Chromosome 20 array profile. Amplification was observed in all clones in 20q. Each point represents a single probe.

    KAOMP-42-23_F3.gif

    hTERT expression and telomerase activity were regulated by MAPRE1. (A) Expression level of PLAGL2 and MAPRE1 was analyzed by RT-PCR. We confirmed the overexpression of PLAGL2 and MAPRE1 in IHOK cell lines. (B) Knockdown efficiency of siRNA targeting PLAGL2 or MAPRE1 was confirmed by real-time PCR in IHOK-EF cells. The results are shown as mean ± SD (n = 3) and were analyzed using the Mann-Whitney U test (*P < 0.05). (C) Knockdown of PLAGL2 or MAPRE1 in IHOK-EF led to reduced expression of hTERT by real-time PCR. The results are shown as mean ± SD (n = 3) and were analyzed using the Mann-Whitney U test (*P < 0.05). (D) Knockdown of MAPRE1 in IHOK-EF led to reduced telomerase activity. The results are shown as mean ± SD (n = 3) and were analyzed using the Mann-Whitney U test (*P < 0.05).

    KAOMP-42-23_F4.gif

    The public data analysis showed chromosome 20 instability in HPV16 transfected cell lines. (A) Whole genome-wide frequencies of copy number alteration in HPV16-immortalized cell line. (B) Scatter plot of copy number alteration represented as log2 ratio in Chromosome 20. Alteration is shown using light gray (gain) and dark gray (loss) coloration. (C) Gains were also observed in PLAGL2 and MAPRE1. The HPV16-immortalized cell line had high-level gains of MAPRE1 than PLAGL2.

    Table

    Sequences of primers used for PCR, RT-PCR and real-time PCR

    Sequences of primers used for HPV16 E6/E7 integration sites

    Chr, chromosome; HPV16 (1) (2), pBABE 5 LTR vector of human papilloma virus 16

    Sequences of siRNAs used in this study

    Top 8 candidate genes list from amplificated clones in gain group (Log2 ratio > 1.0)

    Genetic alteration in integration sites

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