Rybakova et al. Cell Death and Disease (2021) 12:178 https://doi.org/10.1038/s41419-021-03453-w Cell Death & Disease ARTICLE Open Access Identification of a long non-coding RNA regulator of liver carcinoma cell survival Yulia Rybakova 1,2, John T. Gonzalez 1, Roman Bogorad 1, Vikash P. Chauhan1,YizeL.Dong3,4, Charles A. Whittaker1, Timofei Zatsepin 2, Victor Koteliansky2 and Daniel G. Anderson 1,5,6,7 Abstract Genomic studies have significantly improved our understanding of hepatocellular carcinoma (HCC) biology and have led to the discovery of multiple protein-coding genes driving hepatocarcinogenesis. In addition, these studies have identified thousands of new non-coding transcripts deregulated in HCC. We hypothesize that some of these transcripts may be involved in disease progression. Long non-coding RNAs are a large class of non-coding transcripts which participate in the regulation of virtually all cellular functions. However, a majority of lncRNAs remain dramatically understudied. Here, we applied a pooled shRNA-based screen to identify lncRNAs essential for HCC cell survival. We validated our screening results using RNAi, CRISPRi, and antisense oligonucleotides. We found a lncRNA, termed ASTILCS, that is critical for HCC cell growth and is overexpressed in tumors from HCC patients. We demonstrated that HCC cell death upon ASTILCS knockdown is associated with apoptosis induction and downregulation of a neighboring gene, protein tyrosine kinase 2 (PTK2), a crucial protein for HCC cell survival. Taken together, our study describes a new, non-coding RNA regulator of HCC. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction dysregulated in HCC samples compared to healthy tis- Liver cancer is one of the leading causes of cancer sues4. Further Gene Ontology analysis of these genes mortality worldwide, accounting for more than 700,000 identified several gene networks associated with HCC deaths per year1. Hepatocellular carcinoma (HCC) is the progression. Among them were upregulation of cell pro- most frequent subtype of liver cancer. Despite recent liferation, downregulation of apoptosis, loss of hepatocyte progress in HCC treatment it remains one of the deadliest differentiation, immunosuppression, and activation of types of cancer1,2. Notably, the incidence of HCC has been proteins acting at an epigenetic level4. Comprehensive increasing in recent decades, making HCC one of the genomic profiling of patient HCC samples and their fastest-growing causes of death worldwide3. This poor comparison with healthy tissues have helped uncover prognosis underlines the need for new effective therapies. molecular changes promoting the above phenotypic fea- – Better understanding of the molecular mechanisms reg- tures of HCC5 9. Among them, mutations leading to ulating HCC progression may yield new potential drug activation of the WNT signaling pathway were most targets. common5,6, implicating the WNT pathway as a major A meta-analysis of human HCC datasets revealed 935 driver of hepatocarcinogenesis7. Moreover, activation of genes for which RNA expression was significantly the WNT pathway is associated with an immunosup- pressive microenvironment, another hallmark of HCC progression8,9, which emphasizes the role of WNT path- Correspondence: Daniel G. Anderson ([email protected]) way activity in HCC progression. Other common muta- 1David H. Koch Institute for Integrative Cancer Research, Massachusetts tions affected the TERT promoter, TP53, genes regulating Institute of Technology, Cambridge, MA 02142, USA 2 cell cycle, PI3K-AKT-mTOR signaling and cell differ- Skolkovo Institute of Science and Technology, Moscow 121205, Russia 5,6 Full list of author information is available at the end of the article entiation . Notably, up to 50% of clinical HCC samples Edited by B. Rotblat © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Rybakova et al. Cell Death and Disease (2021) 12:178 Page 2 of 14 reported in different studies have a mutation in chromatin silencing is restricted to the cytoplasm, limiting targeting modifiers5,6, indicating the importance of epigenetic reg- of nuclear transcripts. However, recent studies suggest ulation in HCC development. RNAi presence and activity in the mammalian nucleus as – Besides shedding light on the roles of protein-coding well, although with less efficiency28 30. Clustered regularly genes, integrative genomic studies have revealed that the interspaced short palindromic repeat interference (CRIS- majority (>70%) of transcribed sequences in the human PRi) is another potent technique for lncRNA silen- genome participate in cell function regulation without cing21,23,25. Given that majority of lncRNA promoters is producing a protein10. Long non-coding RNAs (lncRNAs) poorly annotated and lncRNAs often overlap with are defined as non-coding transcripts longer than 200 protein-coding genes (or their promoters/enhancers), nucleotides and represent a large class of non-coding application of CRISPRi to regulate a lncRNA overlapping elements, comprising more than 50,000 annotated tran- with other transcripts might distort expression of those scripts to date11,12. Pertinently, hundreds of lncRNAs are transcripts, confounding data interpretation31. Thus, in recurrently deregulated in HCC, suggesting potential our screen we have chosen to perturb lncRNA at the roles in hepatocarcinogenesis. Co-expression network transcript level. We performed an shRNA-based pooled analysis determined that these lncRNAs were associated screen to identify lncRNAs essential for the survival of the with cell proliferation, metastasis, immune response, and human HCC cell line HUH7. Based on the lncRNA liver metabolism—hallmarks of HCC progression13,14. expression profile of these cells, we designed a lentiviral While the pathogenic roles of some of these lncRNAs (e.g. shRNA library targeting all identified lncRNAs. Using this HULC, H19, HOTAIR, HOTTIP, DANCR) have already library, we performed a loss-of-function genetic screen been described15, a plurality of lncRNA transcripts remain and found that lncRNA ENST00000501440.1 is critical for largely uncharacterized. Discovery of novel lncRNAs and HUH7 cell growth. We named this lncRNA ASTILCS their intracellular functions promises to expand our (AntiSense Transcript Important for Liver Carcinoma knowledge of HCC cellular physiology and may provide Survival). Importantly, in patient data, ASTILCS is sig- the basis for new therapeutic modalities. nificantly overexpressed in HCC compared to normal Currently, lncRNA functions can hardly be predicted tissues. Further, using gene expression manipulation based on their sequence. Instead, subcellular localization, techniques, we demonstrate that ASTILCS knockdown transcript abundance, and functional genomic screens can results in apoptosis induction and HCC cell death. Finally, help to efficiently narrow down possible lncRNA biolo- we show that ASTILCS knockdown correlates with – gical roles and molecular functions16 19. For instance, downregulation of a neighboring gene expressing Protein lncRNAs located mainly in the nucleus typically function Tyrosine Kinase 2 (PTK2), the silencing of which results as transcription regulators of local genes (in cis) or distant in HCC cell death. genes (in trans)18. Cytoplasmic lncRNAs are more likely to regulate protein production, formation of post- Materials and methods translational modifications, and sequestration of miR- Cell culture NAs or RNA-binding proteins19. Transcript abundance Human HCC HUH7 cell line was a gift from Prof. Jay can provide another hint about lncRNA function. For Horton (UT Southwestern Medical Center). HUH7 and example, low-abundance transcripts tend to function in HEK293ft cell lines were grown in Dulbecco’s modified cis because their low concentration makes diffusion a Eagle’s medium with L-glutamine (DMEM, Gibco™) sup- barrier to activity at long distances from the transcription plemented with 4.5 mg/ml glucose, 50 µg/ml gentamicin site. Abundant lncRNAs, on the other hand, can achieve sulfate (Sigma), 25 mM HEPES (Gibco™), and 10% heat- high concentrations at multiple target regions, including inactivated fetal bovine serum (FBS, Gibco™). All cells those outside of the nucleus and therefore often function were cultured at 37 °C, 5% CO2. When the cells reached a in trans16,17. Finally, pooled functional genetic screens are 70–80% monolayer, they were detached from the flask a powerful tool allowing for parallel perturbation of using 0.25% Trypsin–EDTA (Gibco™) solution and split multiple genes to select for those that are critical for a 1:10. Concentrations for selection agents were determined phenotype or