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Stabilization of p53 by microRNAs in HPV-positive cervical cancer cells

By Gustavo Martínez-Noël, Patricia Szajner, Jennifer A. Smith, Kathleen Boyland, Rebecca E. Kramer, Peter M. Howley

Posted 21 Sep 2020
bioRxiv DOI: 10.1101/2020.09.21.305946

Etiologically 5% of 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 the cellular targets of these viral oncoproteins are the p53 and the retinoblastoma tumor suppressor proteins. Inhibition of 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 assess the ability of 885 microRNAs (miRNAs) to stabilize p53 in human papillomavirus (HPV)-positive cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of p53 stability. The 32 miRNAs whose expression resulted in the greatest p53 stabilization were further assessed in validation experiments using a second cell-based p53 stability reporter system as well as in HeLa cells to examine their effects on endogenous p53 protein levels. The positive miRNAs identified included 375-3p that has previously been reported as stabilizing p53 in HeLa cells, providing validation of the screen. Additional miRNAs that stabilized p53 led to decreases in E6AP protein levels, while others, including members of the 302/519 family of miRNAs, targeted HPV oncoprotein expression. We further examined a subset of these miRNAs for their abilities to induce apoptosis and determined whether the apoptosis was p53-mediated. The miRNAs described here have potential as therapeutics for treating HPV-positive cancers. Author summary Human papillomaviruses cause approximately 5% of cancers worldwide and encode genes that contribute to both the initiation and maintenance of these cancers. The viral gene E6 is expressed in all HPV-positive cancers and functions by targeting the degradation of the p53 protein through its engagement of the cellular ubiquitin ligase E6AP. Inhibiting the degradation of p53 results in apoptosis in HPV-positive cancer cells. We have developed a high-throughput live cell assay to identify molecules that stabilize p53 in HPV-positive cells and we present the results of a screen we have carried out examining miRNAs for their abilities to stabilize p53 and induce apoptosis in HPV-positive cervical cancer cells. These miRNAs have the potential to be used in the treatment of HPV-positive cancers.

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