Bristol, M. L. | bioRxiv Science

bioRxiv2020

Estrogen attenuates the growth of human papillomavirus positive epithelial cells

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 (ER) 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 two decades and therapeutic options for this ongoing health crisis are a priority; currently there are no anti-viral therapeutics available for combating HPV+cancers. During our own TGCA studies on head and neck cancer we had also discovered the overexpression of ER in HPV+cancers. Here we demonstrate that 17{beta}-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 hTERT) containing HPV16 have elevated levels of ER and growth sensitivity following estrogen treatment when compared with parental N/Tert-1. 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. ImportanceHuman papillomaviruses cause around 5% of all human cancers, yet there are no specific anti-viral therapeutic approaches available for combating these cancers. These cancers are currently treated with standard chemo-radiation therapy (CRT). Specific anti-viral reagents are desperately required, particularly for HPV+HNSCC whose incidence is increasing and for which there are no diagnostic tools available for combating this disease. Using data from The Cancer Genome Atlas (TCGA) ourselves and others determined that the estrogen receptor (ER) 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 combating 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.

cancer biology↗

bioRxiv2019

Human papillomavirus 16 E6 and E7 synergistically repress innate immune gene transcription

Human papillomaviruses are causative agents in 5% of all cancers, including the majority of anogenital and oropharyngeal cancers. Downregulation of innate immune genes (IIGs) by HPV to promote the viral life cycle is well documented; E6 and E7 are known repressors of these genes. More recently we demonstrated that E2 could also repress IIGs. These studies have been carried out in cells over-expressing the viral proteins and to further investigate the role of individual viral proteins in this repression we introduced stop codons into E6 and/or E7 in the entire HPV16 genome and generated N/Tert-1 cells stably maintaining the HPV16 genomes. We demonstrate that E6 or E7 individually are not sufficient to repress IIG expression in the context of the entire HPV16 genome, both are required for a synergistic repression. The DNA damage response (DDR) is activated by HPV16 irrespective of E6 and E7 expression, presumably due to viral replication; E1 is a known activator of the DDR. In addition, replication stress was apparent in the HPV16 positive cells lacking E6 and E7, manifested by attenuated cellular growth and activation of replication stress genes. These studies lead us to the following model. Viral replication per se can activate the DDR following infection, and this activation is a known inducer of IIG expression which could induce cellular senescence. To combat this, E6 and E7 synergistically combine to manipulate the DDR and actively repress innate immune gene expression promoting cellular growth; neither protein by itself is able to do this. ImportanceThe role of HPV16 in human cancers is well established; however, to date there are no anti-viral therapeutics that are available for combatting these cancers. To identify such targets, we must enhance understanding of the viral life cycle. Innate immune genes (IIGs) are repressed by HPV16, and we have reported that this repression persists through to cancer. Reversal of this repression would boost the immune response to HPV16 positive tumors, an area that is becoming more important given the advances in immunotherapy. This report demonstrates that E6 and E7 synergistically repress IIG expression in the context of the entire HPV16 genome. Removal of either protein activates the expression of IIGs by HPV16. Therefore, gaining a precise understanding of how the viral oncogenes repress IIG expression represents an opportunity to reverse this repression and boost the immune response to HPV16 infections for therapeutic gain.

molecular biology↗