Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Messa L

PROVIDER: S-EPMC5902497 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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

Messa Lorenzo L Celegato Marta M Bertagnin Chiara C Mercorelli Beatrice B Nannetti Giulio G Palù Giorgio G Loregian Arianna A

Scientific reports 20180416 1

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-ass ...[more]

PMID: 29662081

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Project description:Cervical cancer is a significant public health problem, especially in low- and middle-income countries, where women have little access to cervical cancer screening; consequently 80% of cervical cancer related mortality occurs in these regions. The development of screening methods that need less infrastructure thus represents an urgent medical need. The study aims to compare the detection rates of high-risk human papillomavirus 16 and 18 E6 oncoprotein in urine, vaginal self-collected, and cervical scrapes of women using the OncoE6™ Cervical Test and compare the HPV16 and/or HPV18 E6 detection rates with the HPV DNA testing. Paired urine, vaginal self-collected and cervical specimens were collected from 124 women who participated in cervical cancer screening or treatment in this proof-of-concept study and underwent to HPV16/18-E6 testing and high-risk HPV DNA testing prior to treatment of cervical neoplasia or cancer. Concordance between urinary, vaginal and cervical HPV16/18-E6 and HPV-DNA testing was evaluated for patients classified as negative group (<CIN2) and histological positive group (CIN2, CIN3 and invasive carcinoma). Overall, HPV16/18-E6 oncoprotein was detected in 30.6% of cervical samples, 20.3% of self-collected vaginal samples and 21% of urine samples. Regarding the clinical sensitivity, the HPV16/18-E6 oncoprotein was not detected in CIN2 cases, and was detected at low rates in CIN3 cases. The clinical sensitivity of the HPV16/18-E6 oncoprotein for detecting invasive cervical cancer was 70% for cervical scrapes, 55% for self-collected vaginal samples and 52% for urine samples. This study reports the urinary detection of E6 oncoprotein in vivo for the first time and our results suggest that this detection is only for invasive/microinvasive lesions. Then, further protocol development and standardization to achieve a clinical sensitivity for CIN2/3 detection close to what can be achieved for invasive lesions using the physician collected cervical is needed.

Project description:Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1?) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1? production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1? processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1? regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1? was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1? that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1? is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1? levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1? regulation which ultimately inhibits the secretion of IL-1? in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1? towards cervical cancer could be discerned. Hence, attenuation of IL-1? by the HPV16 E6 oncoprotein in immortalized cells is apparently a crucial step in viral immune evasion and initiation of malignancy.

Dataset Information

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

Publications

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

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