Dataset Information

Live cell, image-based high-throughput screen to quantitate p53 stabilization and viability in human papillomavirus positive cancer cells.

ABSTRACT: Approximately 5% of cancers are caused by high-risk human papillomaviruses. Although very effective preventive vaccines will reduce this cancer burden significantly over the next several decades, they have no therapeutic effect for those already infected and remaining at risk for malignant progression of hrHPV lesions. HPV-associated cancers are dependent upon the expression of the viral E6 and E7 oncogenes. The oncogenic function of hrHPV E6 relies partially on its ability to induce p53 degradation. Since p53 is generally wildtype in hrHPV-associated cancers, p53 stabilization arrests proliferation, induces apoptosis and/or results in senescence. Here we describe a live cell, image-based high-throughput screen to identify compounds that stabilize p53 and/or affect viability in HPV-positive cancer HeLa cells. We validate the robustness and potential of this screening assay by assessing the activities of approximately 6,500 known bioactive compounds, illustrating its capability to function as a platform to identify novel therapeutics for hrHPV.

SUBMITTER: Martinez-Noel G

PROVIDER: S-EPMC8206038 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Live cell, image-based high-throughput screen to quantitate p53 stabilization and viability in human papillomavirus positive cancer cells.

Martínez-Noël Gustavo G Vieira Valdimara Corrêa VC Szajner Patricia P Lilienthal Erin M EM Kramer Rebecca E RE Boyland Kathleen A KA Smith Jennifer A JA Howley Peter M PM

Virology 20210522

Approximately 5% of cancers are caused by high-risk human papillomaviruses. Although very effective preventive vaccines will reduce this cancer burden significantly over the next several decades, they have no therapeutic effect for those already infected and remaining at risk for malignant progression of hrHPV lesions. HPV-associated cancers are dependent upon the expression of the viral E6 and E7 oncogenes. The oncogenic function of hrHPV E6 relies partially on its ability to induce p53 degrada ...[more]

PMID: 34051479

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Project description:Etiologically, 5% of all cancers worldwide are caused by the high-risk human papillomaviruses (hrHPVs). These viruses encode two oncoproteins (E6 and E7) whose expression is required for cancer initiation and maintenance. Among their cellular targets are the p53 and the retinoblastoma tumor suppressor proteins. Inhibition of the hrHPV E6-mediated ubiquitylation of p53 through the E6AP ubiquitin ligase results in the stabilization of p53, leading to cellular apoptosis. We utilized a live cell high-throughput screen to determine whether exogenous microRNA (miRNA) transfection had the ability to stabilize p53 in hrHPV-positive cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of p53 stability. Among the miRNAs whose transfection resulted in the greatest p53 stabilization was 375-3p, which has previously been reported to stabilize p53 in HeLa cells, providing validation of the screen. The top 32 miRNAs, in addition to 375-3p, were further assessed using a second cell-based p53 stability reporter system, as well as in nonreporter HeLa cells to examine their effects on endogenous p53 protein levels, resulting in the identification of 23 miRNAs whose transfection increased p53 levels in HeLa cells. While a few miRNAs that stabilized p53 led to decreases in E6AP protein levels, all targeted HPV oncoprotein expression. We further examined subsets of these miRNAs for their abilities to induce apoptosis and determined whether it was p53-mediated. The introduction of specific miRNAs revealed surprisingly heterogeneous responses in different cell lines. Nonetheless, some of the miRNAs described here have potential as therapeutics for treating HPV-positive cancers. IMPORTANCE Human papillomaviruses cause approximately 5% of all cancers worldwide and encode genes that contribute to both the initiation and maintenance of these cancers. The viral oncoprotein E6 is expressed in all HPV-positive cancers and functions by targeting the degradation of p53 through the engagement of the cellular ubiquitin ligase E6AP. Inhibiting the degradation of p53 leads to apoptosis in HPV-positive cancer cells. Using a high-throughput live cell assay, we identified several miRNAs whose transfection stabilize p53 in HPV-positive cells. These miRNAs have the potential to be used in the treatment of HPV-positive cancers.

Dataset Information

Live cell, image-based high-throughput screen to quantitate p53 stabilization and viability in human papillomavirus positive cancer cells.

Publications

Live cell, image-based high-throughput screen to quantitate p53 stabilization and viability in human papillomavirus positive cancer cells.

Similar Datasets

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