Abstract 4403: Next-generation RNA sequencing identifies UHRF1 as a key regulatory protein in triple-negative breast cancer survival.
Authors: Solzak Jeffrey P. , Atale Rutuja , Radovich Milan 
Published: August 2014

Abstract
Introduction: Triple-negative breast cancer (TNBC) is a particularly aggressive form of breast cancer that presently lacks specific treatment. Using next-generation RNA sequencing of 94 TNBC tumors and 20 normal breast tissues, we have identified UHRF1, an E3 ubiquitin ligase, as significantly overexpressed. UHRF1 is involved in DNA methylation, is upregulated via mutated p53, and plays a role in the repression of RB1 & BRCA1. Epigallocatechin gallate (EGCG), a polyphenol found in green tea, has been previously investigated in its role of downregulating UHRF1.
Methods: cDNA libraries from 20 normal breast tissues from the Susan G. Komen Tissue Bank at the IU Simon Cancer Center and 10 TNBC tumors were sequenced on an Applied Biosystems SOLiD3 sequencer. Mapping of reads to the human genome (hg19) was performed using the LifeScope software. RNA-seq data from an additional 84 TNBCs was downloaded from The Cancer Genome Atlas (TCGA) data portal. Differential gene expression was analyzed using Partek Genomics Suite; and network, pathway, and transcription factor analysis was performed using Ingenuity Systems IPA 9.0. To investigate the effects of EGCG a known downregulator of UHRF1 in vitro, nine TNBC cell lines were utilized representing the known TNBC molecular subtypes. TNBC cell lines were chosen based on differing subtypes which include different genetic and morphological characteristics. Cells were seeded in 96 well plates at 10,000 cells per well and dosed with increasing concentrations of EGCG from 0-400μM, in increments of 50μM. CellTiter-Glo was used to assess the viability of all cells lines after 72 hours. The IC50 was ascertained using percent changes from control luminosity of untreated cells.
Results: Transcription factor analysis of RNA-seq data identified RB1 activity as significantly inhibited in TNBC. A search for upstream inhibitors identified UHRF1 as highly overexpressed in TNBC (Fold-change =7.96, p-value=8.34x10−6). Further network analysis revealed that upregulation of UHRF1 is mediated by mutated p53 (which is mutated &gt;80% of TNBCs) and is an inhibitor of BRCA1 which is known to be suppressed in TNBC. Using a publically available gene expression database, high expression of UHRF1 is correlated with poor survival in breast cancer (p&lt;0.001). As EGCG is a known downregulator of UHRF1, we tested EGCG on nine TNBC cell lines and assessed for cell viability. EGCG resulted in significant cell death with a median LD50 of 109μM. Furthermore, in two of the cell lines, HCC70 and HCC1143, 100% cell death was observed at 250μM.
Conclusions: UHRF1 is a key member of the p53/RB1/BRCA1 network and is important in TNBC survival. EGCG, a natural compound that downregulates UHRF1, significantly decreases the viability of TNBC cell lines in-vitro. Investigation into more potent small molecule inhibitors of UHRF1 is currently underway.
Citation Format: Jeffrey P. Solzak, Rutuja Atale, Milan Radovich. Next-generation RNA sequencing identifies UHRF1 as a key regulatory protein in triple-negative breast cancer survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4403. doi:10.1158/1538-7445.AM2013-4403