Estrogen Attenuates the Growth of Human Papillomavirus-Positive Epithelial Cells

Abstract

Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that are significant risk factors in the development of cancer, and HPV accounts for approximately 5% of all worldwide cancers. Recent studies using data from The Cancer Genome Atlas (TCGA) have demonstrated that elevated levels of estrogen receptor alpha (ERα) are associated with improved survival in oropharyngeal cancers, and these elevated receptor levels were linked with human papillomavirus-positive cancers (HPV+cancers). There has been a dramatic increase in HPV-related head and neck squamous cell carcinomas (HPV+HNSCCs) over the last 2 decades, and therapeutic options for this ongoing health crisis are a priority; currently, there are no antiviral therapeutics available for combatting HPV+cancers. During our TGCA studies on head and neck cancer, we had also discovered the overexpression of ERα in HPV+cancers. Here, we demonstrate that 17β-estradiol (estrogen) attenuates the growth/cell viability of HPV+cancers in vitro, but not HPV-negative cancer cells. In addition, N/Tert-1 cells (foreskin keratinocytes immortalized with human telomerase reverse transcriptase [hTERT]) containing human papillomavirus 16 (HPV16) have elevated levels of ERα and growth sensitivity after estrogen treatment compared with parental N/Tert-1 cells. Finally, we demonstrate that there are potentially two mechanisms contributing to the attenuation of HPV+ cell growth following estrogen treatment. First, estrogen represses the viral transcriptional long control region (LCR) downregulating early gene expression, including E6/E7. Second, expression of E6 and E7 by themselves sensitizes cells to estrogen. Overall, our results support the recent proposal that estrogen could be exploited therapeutically for the treatment of HPV-positive oral cancers. IMPORTANCE Human papillomaviruses cause around 5% of all human cancers, yet there are no specific antiviral therapeutic approaches available for combatting these cancers. These cancers are currently treated with standard chemoradiation therapy (CRT). Specific antiviral reagents are desperately required, particularly for HPV+HNSCC whose incidence is increasing and for which there are no diagnostic tools available for combatting this disease. Using data from The Cancer Genome Atlas (TCGA), we and others determined that the estrogen receptor alpha (ERα) is overexpressed in HPV+HNSCC and that elevated levels are associated with an improved disease outcome. This has led to the proposal that estrogen treatment could be a novel therapeutic approach for combatting HPV+cancers. Here, we demonstrate that estrogen attenuates the growth of HPV+epithelial cells using multiple mechanisms, supporting the idea that estrogen has potential as a therapeutic agent for the treatment of HPV+HNSCC.

The Relationship between Estrogen-Related Signaling and Human Papillomavirus Positive Cancers

“…There is significant evidence suggesting that estrogen may serve as a treatment modality in HPV-related lesions and cancers. The HPV18+ cervical cancer cell line, HeLa, is particularly sensitive to estrogen treatment [138][139][140]. Moreover, our laboratory confirmed HeLa estrogen sensitivity and further compared a number of HPV+ and HPV− epithelial cell lines for estrogen sensitivity; it was found that HPV expression confers specific sensitivity to estrogen, both through the expression of the viral LCR, and through expression of the viral oncogenes E6 and E7 [139].…”

“…The HPV18+ cervical cancer cell line, HeLa, is particularly sensitive to estrogen treatment [138][139][140]. Moreover, our laboratory confirmed HeLa estrogen sensitivity and further compared a number of HPV+ and HPV− epithelial cell lines for estrogen sensitivity; it was found that HPV expression confers specific sensitivity to estrogen, both through the expression of the viral LCR, and through expression of the viral oncogenes E6 and E7 [139]. This sensitivity to estrogen occurred in foreskin, cervical, tonsil, and HNSCC cell lines [139].…”

“…While there is limited evidence in animal models, patient studies provide evidence that the expression of the ER correlates with improved clinical outcomes, and that estrogen treatment could potentially enhance these outcomes. Recently, a number of studies have analyzed the expression of ERα in the TCGA [139,146,147]. These studies have all shown that higher ERα expression correlates with increased patient survival for HPV+ oropharyngeal cancer patients.…”

“…Cervical cancer cell culture studies indicate that estrogen can increase colony formation in some lines, and upregulated expression of a truncated version of ERα, ER-α36, increased the invasion, migration, and proliferation of some cell lines [99,122]. Whereas, other studies have shown, HeLa cells are particularly sensitive to estrogen, and estrogen specifically sensitizes numerous HPV+ cells when compared to HPV− cells, regardless of tissue of origin [138][139][140]. The overwhelming majority of animal studies in this area, have been conducted with the K-14-E7 transgene; these studies have made it abundantly clear that the expression of ERα is necessary for the formation of tumors in these models [117,[126][127][128][129][130][131][132].…”

Human papillomavirus 16 E2 repression of TWIST1 transcription is a potential mediator of HPV16 cancer outcomes

“…Transcription Assay. 5 x 10 5 N/Tert-1 cells were plated out on 100-mm 2 plates and transfected 24 hours later with either 1μg HPV16 E2 plasmid and 1μg pTWIST1-Luc, or 1μg pTWIST1-Luc alone using Lipofectamine 3000 (ThermoFisher Scientific) according to the manufacturer's instructions as previously described (73). pTWIST1-luc contains the human promoter and has been described previously (74,75).…”