Strategies to identify oncogene-dependent long non-coding RNAs (lncRNAs) in liver cancer

The Hippo signaling pathway plays a crucial role in human hepatocarcinogenesis. Its key signaling protein, yes-associated protein (YAP) and its paralog WW domain containing transcription regulator 1 (WWTR1; synonym: TAZ) are highly expressed in hepatocellular carcinoma (HCC) and display high therapeutic potential. Therefore, noninvasive strategies to identify patients suitable for Hippo pathway-directed therapy are needed. Liquid biopsy testing has shown great promise for personalized medicine. Especially, the detection of long non-coding RNAs (lncRNAs) has proven itself to be a valuable method to diagnose and classify cancer. In this project, we aim to identify YAP/TAZ-regulated lncRNA networks, which could serve as biomarkers for YAP/TAZ activation in HCCs.

In order to identify YAP/TAZ-regulated lncRNAs, two complementary strategies were applied: a sequencing-based approach of genetically manipulated HCC cells (HLF cells) and a bioinformatic-based approach using ChIP-seq data. While the former aimed to identify significantly downregulated lncRNA candidates upon YAP/TAZ silencing, the latter was applied to detect lncRNAs harboring transcription factor binding sites of the TEA domain family members (TEAD), the main interaction partners of YAP and TAZ. Total RNA-sequencing after siRNA-mediated YAP/TAZ inhibition led to a significant reduction of 2708 mRNAs including known YAP/TAZ target genes such as CTGF and 119 lncRNAs (p-value < 0.05). For the bioinformatic approach, we utilized ChIP-Seq data of TEAD1, 3 and 4 from the ENCODE database, extracted the peaks, annotated them to the nearest gene and filtered for lncRNAs (n1 = 949, n3 = 1992 and n4 = 324, respectively). Comparing the NGS data with the results from the bioinformatic approach revealed 49 significantly downregulated lncRNAs (fold change < 0.75), of which 19 had predicted binding sites of at least two TEAD family members. Selected candidate lncRNAs such as shieldin complex subunit 2 pseudogene 2 (SHLD2P3) were confirmed in independent experiments.

In summary, we developed a novel and stringent strategy combining RNA-sequencing and bioinformatics to identify YAP/TAZ regulated mRNAs and lncRNAs in liver cancer. This approach might be a valuable tool to detect biomarkers that characterize HCC patients with aberrant YAP/TAZ activity.