Long non-coding RNA DARS-AS1 promotes tumor progression by directly suppressing PACT-mediated cellular stress

Long non-coding RNA DARS-AS1 promotes tumor progression by directly suppressing PACT-mediated cellular stress
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Long non-coding RNAs (LncRNAs) are transcripts greater than 200 nucleotides without protein-coding potential. Since thousands of lncRNAs have been identified by advanced genome-wide sequencing projects[1,2], numerous efforts have been made to elucidate their biological functions. Accumulating numbers of studies demonstrate that lncRNAs are involved in a multitude of biological processes, including the regulation of X chromosome inactivation[3], imprinting[4], transcription[5], translation[6], and even cancer growth[7]. How to identify functional candidates from hundreds of thousands of lncRNAs is crucial for lncRNA studies.

In previous study, by taking advantage of pooled shRNA library, Prof. Wang's group (Tsinghua University & Chengdu University of Traditional Chinese Medicine) identified a lncRNA, LINC00973[8], which directly interacted with Lactate dehydrogenase A (LDHA), an essential enzyme in aerobic glycolysis. This interaction led to enhanced enzyme activity and aerobic glycolysis and thus resulted in the promotion of cancer cell proliferation in vitro and tumor growth in vivo.

Recently, Prof. Wang's group performed large-scale loss-of-function screening via a CRISPRi system and discovered that lncRNA DARS-AS1 is an important player in helping cancer cells overcome cellular stresses, and thus prevents apoptotic cell death and promotes cancer cell proliferation.

 

DARS-AS1 was identified by CRISPRi screening

To pinpoint promising lncRNA candidates involved in cancer progression, we performed loss-of-function screening for reduced proliferation in the colorectal cancer cell line SW620 using the CRISPRi system. A unique feature of SW480 and SW620 colon carcinoma cell lines is that they are derived from the primary and secondary tumors of a single patient. This enables a valuable comparison for examining the genetic changes in late colon cancer progression. Accordingly, we profiled the transcriptome of colorectal cancer cell lines (SW480 and SW620) using RNA-sequencing and also collected some potential functional lncRNAs from published literatures. From these results, we designed a pooled sgRNA library, containing 7355 sgRNA oligos targeting 971 cancer-related lncRNAs and 500 non-targeting sgRNA oligos for negative controls. Among all lncRNAs tested, DARS-AS1 was screened out with three of its associated sgRNA oligos significantly reduced after 18 days culture, indicating that the knockdown of this lncRNA results in reduced cancer proliferation.

 

DARS-AS1 promotes multiple cancer growth

Knocking down DARS-AS1 inhibits cell proliferation of multiple cancer cell lines and promotes cancer cell apoptosis in vitro. Results of xenograft mouse models show that knockdown of DARS-AS1 dramatically decreased tumor growth in mice. Accordingly, significant reductions of mean tumor volumes and weights in the DARS-AS1-knockdown group were observed. These results strongly suggest that DARS-AS1 could significantly promote tumor growth in vivo.

 

DARS-AS1 involves in PACT-mediated cellular stress responses

DARS-AS1 directly binds to PACT, an essential responder of cellular stress. The binding of DARS-AS1 to PACT blocks the interaction between PACT and PKR, further suppresses stress signaling transduction to their downstream effector, eIF2α, consequently inhibits stress-induced apoptosis and promotes cancer cell proliferation eventually. Heterogenous expression of DARS-AS1 in cells reduces PKR activation and eIF2α phosphorylation under serum starvation or treatment of thapsigargin, an ER stressor. These discoveries enrich our understanding of the regulation of the PACT/PKR axis and the role of lncRNAs in stress responses.

 

DARS-AS1 is a potential prognosis biomarker for multiple cancers

DARS-AS1 is broadly expressed and significantly upregulated over healthy cells in a variety of tumors, including colon adenocarcinoma (COAD), kidney renal clear cell carcinoma (KIRC), and kidney renal papillary cell carcinoma (KIRP). Meanwhile, a higher expression of DARS-AS1 is significantly correlated with poorer survival in uveal melanoma (UVM), kidney chromophobe (KICH), kidney renal papillary cell carcinoma (KIRP), mesothelioma (MESO), glioblastoma multiforme (GBM), and brain lower grade glioma (LGG) patients. The analysis of clinical data underscores the significance of DARS-AS1/PACT/PKR axis in cancer prognosis, indicating the potential value of DARS-AS1 in clinical therapy.

 

In summary, this study illustrates that DARS-AS1 is a significant regulator of PACT/PKR signaling axis, which promotes cancer cell proliferation and inhibits cell apoptosis during stress responses. Moreover, this study also provides a promising potential therapeutic target for anti-cancer research.

 

[1]   Derrien, T. et al. The GENCODE v7 catalog of human long noncoding RNAs: Analysis of their gene structure, evolution, and expression. Genome research 22, 1775-1789 (2012).

[2]   Djebali, S. et al. Landscape of transcription in human cells. Nature 489, 101-108 (2012).

[3]   Jegu, T., Aeby, E. & Lee, J. T. The X chromosome in space. Nature Reviews Genetics 18 (2017).

[4]   Thorvaldsen, J. L., Duran, K. L. & Bartolomei, M. S. Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2. Genes & development 12, 3693-3702 (1998).

[5]   Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390-+ (2011).

[6]   Yoon, J.-H. et al. LincRNA-p21 Suppresses Target mRNA Translation. Molecular cell 47, 648-655 (2012).

[7]   Prensner, J. R. et al. The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex. Nature genetics 45, 1392-+ (2013).

[8] Wang, H. & Lin, K. et al. Oncogenic lncRNA LINC00973 promotes Warburg effect by enhancing LDHA enzyme activity. Science Bulletin, 66(13), 1330-1341 (2021).

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