A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells

High-risk human papillomaviruses (HR-HPVs) are the causative agents for the onset of several epithelial cancers in humans. The deregulated expression of the viral oncoproteins E6 and E7 is the driving force sustaining the progression of malignant transformation in pre-neoplastic lesions. Targeting the viral E6 oncoprotein through inhibitory compounds can counteract the survival of cancer cells due to the reactivation of p53-mediated pathways and represents an intriguing strategy to treat HPV-associated neoplasias. Here, we describe the development of a quantitative and easy-to-perform assay to monitor the E6-mediated degradation of p53 in living cells to be used for small-molecule testing. This assay allows to unbiasedly determine whether a compound can protect p53 from the E6-mediated degradation in cells, through a simple 3-step protocol. We validated the assay by testing two small molecules, SAHA and RITA, reported to impair the E6-mediated p53 degradation. Interestingly, we observed that only SAHA efficiently rescued p53, while RITA could not provide the same degree of protection. The possibility to specifically and quantitatively monitor the ability of a selected compound to rescue p53 in a cellular context through our LumiFluo assay could represent an important step towards the successful development of anti-HPV drugs

Cellular binding partners of the human papillomavirus E6 protein

The high-risk strains of human papillomavirus (HR-HPV) are known to be causative agents of cervical cancer and have recently also been implicated in cancers of the oropharynx. E6 is a potent oncogene of HR-HPVs, and its role in the progression to malignancy has been and continues to be explored. E6 is known to interact with and subsequently inactivate numerous cellular proteins pivotal in the mediation of apoptosis, transcription of tumor suppressor genes, maintenance of epithelial organization, and control of cell proliferation. Binding of E6 to these proteins cumulatively contributes to the oncogenic potential of HPV. This paper provides an overview of these cellular protein partners of HR-E6, the motifs known to mediate oncoprotein binding, and the agents that have the potential to interfere with E6 expression and activity and thus prevent the subsequent progression to oncogenesis

Therapy of human papillomavirus-related disease

This chapter reviews the current treatment of chronic and neoplastic HPV-associated conditions and the development of novel therapeutic approaches. Surgical excision of HPV-associated lower genital tract neoplasia is very successful but largely depends on secondary prevention programmes for identification of disease. Only high-risk HPV-driven chronic, preneoplastic lesions and some very early cancers cannot be successfully treated by surgical procedures alone. Chemoradiation therapy of cervical cancer contributes to the 66–79% cervical cancer survival at 5 years. Outlook for those patients with persistent or recurrent cervical cancer following treatment is very poor. Topical agents such as imiquimod (immune response modifier), cidofovir (inhibition of viral replication; induction apoptosis) or photodynamic therapy (direct damage of tumour and augmentation of anti-tumour immunity) have all shown some useful efficacy (~50–60%) in treatment of high grade vulvar intraepithelial neoplasia. Provider administered treatments of genital warts include cryotherapy, trichloracetic acid, or surgical removal which has the highest primary clearance rate. Patient applied therapies include podophyllotoxin and imiquimod. Recurrence after “successful” treatment is 30–40%. Further improvements could derive from a rational combination of current therapy with new drugs targeting molecular pathways mediated by HPV in cancer. Small molecule inhibitors targeting the DNA binding activities of HPV E1/E2 or the anti-apoptotic consequences of E6/E7 oncogenes are in preclinical development. Proteasome and histone deacetylase inhibitors, which can enhance apoptosis in HPV positive tumour cells, are being tested in early clinical trials. Chronic high-risk HPV infection/neoplasia is characterised by systemic and/or local immune suppressive regulatory or escape factors. Recently two E6/E7 vaccines have shown some clinical efficacy in high grade VIN patients and this correlated with strong and broad systemic HPV-specific T cell response and modulation of key local immune factors. Treatments that can shift the balance of immune effectors locally in combination with vaccination are now being tested. This article forms part of a special supplement entitled “Opportunities for comprehensive control of HPV infections and related diseases” Vaccine Volume 30, Supplement X, 2012