A novel function of HPV16-E6/E7 in epithelial–mesenchymal transition

https://doi.org/10.1016/j.bbrc.2013.04.060Get rights and content

Highlights

  • HPV16 is among the important etiologies in HNSCCs patients.

  • HPV16-E6 and E7 oncogenes induce the EMT-like process in MDCK cells.

  • Correlation between HPV16 positivity and ZEB1 expression in HNSCC cell lines.

  • HPV16 may contribute to tumor cell invasion via EMT-like processes.

Abstract

Human papillomavirus (HPV) 16 is among the most important etiological factors in many human cancers, including head and neck squamous cell carcinomas (HNSCCs) not associated with alcohol or tobacco use. HPV16-E6 and E7 oncoproteins target intracellular signaling networks, altering key molecular and cellular events during tumor progression. The present study investigates the role of HPV16-E6 and E7 oncogenes on the epithelial–mesenchymal transition (EMT), a cellular process thought to be critical for tumor cell invasion and metastasis. Using the epithelial MDCK cell line as an in vitro model, we show that the stable expression of HPV16-E6 or E7 induces morphological conversion from cobblestone-shaped epithelium to spindle-shaped mesenchyme-like phenotype. Consistent with these morphological changes, both E6 and E7 induce expression of the EMT-activating transcriptional factors Slug, Twist, ZEB1 and ZEB2, especially ZEBs, accompanied with switch from epithelial to mesenchymal markers. Importantly, E6 and E7 expression results in induction of the migratory and invasive potential, a functional hallmark of EMT. When we examined the association between HPV16 and the EMT signature in HNSCC cell lines derived from head and neck cancer patients, we found a correlation between HPV16 positivity and the expression of EMT transcription factor ZEB1. Taken together, our findings suggest HPV16 induces EMT-like processes via induction of the EMT transcription factors which may contribute to tumor progression and metastasis.

Introduction

Human papillomavirus (HPV) is a small circular double-stranded DNA virus with a tropism for squamous epithelium and is known to be the primary cause of cervical cancer [1], [2]. In addition, 90% of anal cancers, 40% of vaginal, vulvar, and penile cancers as well as more than 60% of oropharyngeal cancer are related to HPV infection [3]. HPV 16 and 18, among approximately a dozen oncogenic HPV types, are most frequently associated with human cancers such as cervical cancer. HPV 16 in particular is found in approximately 90% of HPV-associated noncervical cancers including head and neck (HN) cancers [3], [4]. Squamous cell carcinoma (SCC) is by far the most common histologic type in HN cancers. Despite advances in the management of HNSCC, mortality and morbidity associated with the disease continue to be high. In fact, world-wide incidence and mortality rates from HNSCC are higher than those for cervical cancer [5]. In the United States, despite the overall declining trend in the incidence of head and neck cancer reflecting the decrease in tobacco consumption, incidence of cancer from the oropharyngeal sites, especially the tonsil and the base of the tongue, are rising, most notably in ages 40–55 [6]. Recent studies identified HPV as a causative agent for oropharyngeal SCC particularly in individuals with no history of heavy consumption of alcohol or tobacco [7], [8], [9]. However, the oncogenic consequences of oropharyngeal HPV infection and its implications for the best clinical management of patients with HPV-associated HNSCC are just beginning to be understood [10].

It has been well demonstrated that the HPV early gene products E6 and E7 play crucial roles in HPV-mediated carcinogenesis by targeting tumor suppressor p53 and retinoblastoma protein (pRb), respectively. E6 binds to the ubiquitin/protein ligase E6AP and p53, resulting in ubiquitination of p53 and its subsequent proteolytic degradation. The E7 protein, on the other hand, promotes cell cycle progression by destabilizing the pRb-E2F complex [11], [12].

HNSCC patients with distant metastasis have extremely poor prognosis with mortality rate of approximately 90% [13]. Although E6 and E7 regulation of cell survival and growth are fairly well studied, their effects on tumor cell invasion and metastasis are not well understood. While normal epithelial cells are stationary, carcinomas often lose their epithelial characteristics and acquire more migratory mesenchymal properties through epithelial–mesenchymal transition (EMT). EMT-regulating genes are often conserved in development of different organs and throughout evolution. Importantly, studies show that carcinoma cells can re-activate the developmental EMT-like process during tumor cell invasion and metastasis [14]. Gene expression profiling studies suggested activation of the NF-B pathway, EMT, and deregulation of cell adhesion as prominent genetic alterations for the development and progression of HNSCC [15]. A recent study reported that primary HNSCC tumors with EMT signatures have a twofold increase in the average tumor satellite distance compared to primary HNSCC tumors without an EMT signature [16]. Interestingly, HPV positive HNSCC tends to be poorly differentiated and often diagnosed with regional spread [17]. Although HPV is an important etiological factor in oropharyngeal HNSCC, it is unknown whether HPV oncogenes regulate its invasive phenotype.

In the present study, we determined the effects of HPV16-E6 and E7 on the EMT-like process utilizing the Madin-Darby canine kidney (MDCK) cell line, a well-known in vitro model for study of EMT. In addition, we examined the expression levels of EMT-related markers in HNSCC cell lines derived from patients with known HPV status. We report herein that both E6 and E7 induces the expression of EMT transcription factors including SLUG, TWIST, ZEB1 and ZEB2, resulting in the down-regulation of epithelial marker E-cadherin and concomitant up-regulation of the mesenchymal markers N-cadherin, fibronectin, and vimentin. Functionally, both E6 and E7 are capable of inducing fibroblast-like morphological changes of MDCK cells and more importantly promoting cell migration and invasion. In patient-derived HNSCC cell lines, expression levels of EMT genes, in particular ZEB1, were significantly higher in HPV16 positive HNSCC cell lines, validating our findings using MDCK cells.

Section snippets

Reagents and antibodies

Mitomycin C, anti-vimentin mAb, and peroxidase conjugated antibodies against mouse or rabbit IgG were purchased from Sigma–Aldrich (St. Louis, MO). Anti-E-cadherin mAb was purchased from BD Transduction Laboratories (San Jose, CA). Anti-GAPDH mAb was purchased from Santa-Cruz Biotechnology (Santa Cruz, CA).

Cell culture

Canine kidney epithelial MDCK cells were purchased from the American Type Tissue Collection (ATCC) and cultured DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml

HPV16-E6. and E7 induce an EMT-like process

To determine the role of HPV16-E6 and E7 oncogenes on the EMT-like process, MDCK cells that stably express E6 (MDCK-E6), E7 (MDCK-E7), both E6 and E7 (MDCK-E6/E7) or control vector (MDCK-neo) were established as described in Section 2. Expression of E6 and/or E7 oncogenes was confirmed by reverse transcription-PCR analysis (Fig. 1B). As shown in Fig. 1A, MDCK-neo cells retained the morphological characteristics of clustered cobblestone-shaped epithelial cells, whereas MDCK-E6, MDCK-E7, and

Discussion

In the present study, we demonstrated a novel function of HPV16-E6/E7 oncoproteins in inducing the expression of EMT activating transcriptional factors leading to activation of the EMT-like process in MDCK cells. The ZEB family (ZEB1, ZEB2), the Snail superfamily of zinc finger transcription factors (Slug, Snail), and the Twist family of bHLH factors are the typical developmental EMT master transcription factors that bind to the promoters of epithelial and mesenchymal specific genes for the

Acknowledgment

This work was supported by WSU institutional funding to Dr. Ikuko Kato and Dr. Hyeong-Reh Choi Kim.

References (27)

  • S. Marur et al.

    HPV-associated head and neck cancer: a virus-related cancer epidemic

    Lancet Oncol.

    (2010)
  • T.L. Yang et al.

    Significance of tumor satellite variables in reflecting the epithelial–mesenchymal transition of tongue cancer

    Oral Oncol.

    (2011)
  • J.P. Thiery et al.

    Epithelial–mesenchymal transitions in development and disease

    Cell

    (2009)
  • J.M. Walboomers et al.

    Human papillomavirus is a necessary cause of invasive cervical cancer worldwide

    J. Pathol.

    (1999)
  • H. zur Hausen

    Papillomaviruses and cancer: from basic studies to clinical application

    Nat. Rev. Cancer

    (2002)
  • M.L. Gillison et al.

    HPV prophylactic vaccines and the potential prevention of noncervical cancers in both men and women

    Cancer

    (2008)
  • J.S. Cooper et al.

    I. Radiation Therapy Oncology Group, Postoperative concurrent radiotherapy and chemotherapy for high-risk squamous-cell carcinoma of the head and neck

    N. Engl. J. Med.

    (2004)
  • A.K. Chaturvedi et al.

    Incidence trends for human papillomavirus-related and -unrelated oral squamous cell carcinomas in the United States

    J. Clin. Oncol.

    (2008)
  • M.L. Gillison et al.

    Evidence for a causal association between human papillomavirus and a subset of head and neck cancers

    J. Natl. Cancer Inst.

    (2000)
  • G. D’Souza et al.

    Case-control study of human papillomavirus and oropharyngeal cancer

    N. Engl. J. Med.

    (2007)
  • T. Andl et al.

    Etiological involvement of oncogenic human papillomavirus in tonsillar squamous cell carcinomas lacking retinoblastoma cell cycle control

    Cancer Res.

    (1998)
  • C. Fakhry et al.

    Clinical implications of human papillomavirus in head and neck cancers

    J. Clin. Oncol.

    (2006)
  • R. Ghittoni et al.

    The biological properties of E6 and E7 oncoproteins from human papillomaviruses

    Virus Genes

    (2010)
  • Cited by (52)

    • Mechanical profile of human keratinocytes expressing HPV-18 oncogenes

      2023, Biochemical and Biophysical Research Communications
    • Dynamic changes of the EMT spectrum between circulating tumor cells and the tumor microenvironment in human papillomavirus-positive head and neck squamous cell carcinoma

      2023, Oral Oncology
      Citation Excerpt :

      Two viral oncoproteins, HPV-E6 and E7, play pivotal roles in HPV-mediated carcinogenesis and subsequent tumor progression. HPV-E6 and E7 act as inactivators of p53 and pRb tumor suppressor genes, as well as contribute to the acquisition of malignant traits, including epithelial-mesenchymal transition (EMT), cancer stemness, immune evasion, and therapeutic resistance [7,11–13]. It is well-known that EMT is a key process that transforms tumor cells from the epithelial to mesenchymal phenotype and facilitates all stages of tumor progression, including initiation, growth, invasion, dissemination, and metastasis.

    • Mechanistic role of HPV-associated early proteins in cervical cancer: Molecular pathways and targeted therapeutic strategies

      2022, Critical Reviews in Oncology/Hematology
      Citation Excerpt :

      HPV16-E6/E7 prompts EMT-induced by FGF-2 and FGF-4 to increase invasive cancer capacity (Cheng et al., 2012). Simultaneous expression of E6/E7 and ErbB-2 reduces the E-cadherin function enhancing the expression of transcription factors including SLUG, SNAIL, and TWIST which activate the EMT pathway and consequently leads to the overexpression of cyclin D1 and c-Myc genes and expression of SFRP1/2 (Al Moustafa et al., 2004; Jung et al., 2013). Protein 1 (AP-1) is a transcription factor activator that plays a critical role in the progression of HPV-associated CC.

    • Survival-related epithelial-mesenchymal transition proteins in oropharyngeal squamous cell carcinoma: A systematic review and meta-analysis

      2021, Archives of Oral Biology
      Citation Excerpt :

      In this systematic review, although the loss of E-cadherin resulted in poor survival of the patients, its expression was not significantly associated with the HPV status of the tumors. The loss of E-cadherin expression favors the process of invasion since it confers high plasticity to the cell, promoting migration, invasion, dedifferentiation and a poor prognosis (Jung et al., 2013; Wakisaka et al., 2015; Yang et al., 2020). This loss of expression is correlated with distant metastasis in HNSCC (De Morais et al., 2020).

    • Lipid metabolism and oxidative stress in HPV-related cancers

      2021, Free Radical Biology and Medicine
      Citation Excerpt :

      Moreover, Jung et al. showed that E6 and E7 induce the expression of Slug, Twist, but especially ZEBs (ZEB1 and ZEB2), which are TFs markers associated with EMT activation. This group also showed that E6 and E7 expression induces migration and cell invasion [99]. Xu et al. [42], showed that HPV-positive tumors and cell lines induce lipogenesis via SIRT3, which was associated with cell invasion and migration, creating a favorable environment for metastasis [100].

    View all citing articles on Scopus
    View full text