CRISPR/Cas9 screen uncovers functional translation of cryptic lncRNA-encoded open reading frames in human cancer
Caishang Zheng, … , Xi Chen, Yiwen Chen
Caishang Zheng, … , Xi Chen, Yiwen Chen
Published March 1, 2023
Citation Information: J Clin Invest. 2023;133(5):e159940. https://doi.org/10.1172/JCI159940.
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Research Article Genetics Oncology

CRISPR/Cas9 screen uncovers functional translation of cryptic lncRNA-encoded open reading frames in human cancer

Abstract

Emerging evidence suggests that cryptic translation within long noncoding RNAs (lncRNAs) may produce novel proteins with important developmental/physiological functions. However, the role of this cryptic translation in complex diseases (e.g., cancer) remains elusive. Here, we applied an integrative strategy combining ribosome profiling and CRISPR/Cas9 screening with large-scale analysis of molecular/clinical data for breast cancer (BC) and identified estrogen receptor α–positive (ER+) BC dependency on the cryptic ORFs encoded by lncRNA genes that were upregulated in luminal tumors. We confirmed the in vivo tumor-promoting function of an unannotated protein, GATA3-interacting cryptic protein (GT3-INCP) encoded by LINC00992, the expression of which was associated with poor prognosis in luminal tumors. GTE-INCP was upregulated by estrogen/ER and regulated estrogen-dependent cell growth. Mechanistically, GT3-INCP interacted with GATA3, a master transcription factor key to mammary gland development/BC cell proliferation, and coregulated a gene expression program that involved many BC susceptibility/risk genes and impacted estrogen response/cell proliferation. GT3-INCP/GATA3 bound to common cis regulatory elements and upregulated the expression of the tumor-promoting and estrogen-regulated BC susceptibility/risk genes MYB and PDZK1. Our study indicates that cryptic lncRNA-encoded proteins can be an important integrated component of the master transcriptional regulatory network driving aberrant transcription in cancer, and suggests that the “hidden” lncRNA-encoded proteome might be a new space for therapeutic target discovery.

Authors

Caishang Zheng, Yanjun Wei, Peng Zhang, Longyong Xu, Zhenzhen Zhang, Kangyu Lin, Jiakai Hou, Xiangdong Lv, Yao Ding, Yulun Chiu, Antrix Jain, Nelufa Islam, Anna Malovannaya, Yun Wu, Feng Ding, Han Xu, Ming Sun, Xi Chen, Yiwen Chen

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Figure 4

GT3-INCP is upregulated in ER+ tumors and exerts a tumor-promoting function.

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GT3-INCP is upregulated in ER+ tumors and exerts a tumor-promoting funct...
(A) qRT-PCR analysis of LINC00992 RNA expression (n = 3) and (B) Western blot analysis of GT3-INCP expression in the indicated breast epithelial cells and BC cell lines. For qRT-PCR analysis, GAPDH served as an internal control and all expression was relative to that in MCF10A cells. For Western blot analysis, β-actin served as an internal control. (C) Western blot analysis of GT3-INCP expression in ER+ luminal tumors (T) and the matched normal (N) breast tissue (n = 12). (D) The GT3-INCP protein level relative to that of β-actin was quantified by densitometry and plotted. (E) MCF7 and (F) ZR75-1 cells stably transduced with GT3-INCP that has a wild-type (ATG) or mutant (AGG) start codon or the empty vector (EV) control were transfected with the negative control siRNA (siNC) or LINC00992-targeting siRNAs. Cell growth was monitored for 4 days via CCK-8 assay. (G) Representative pictures of clonogenic growth and a bar graph quantifying the colonies formed by the MCF7 cells that were transduced with wild-type or mutant (AGG start codon) GT3-INCP or the EV control and were transfected with siNC or siRNAs targeting LINC00992. (H) Volume of the orthotopic tumors formed by the ZR75-1 cells that were stably transduced with 3 different combinations (n = 6 per combination): EV and shNC, EV and shLINC00992, or GT3-INCP and shLINC00992, was monitored as indicated in the Methods. Data are shown as mean ± SD; n = 3 (E–G) or n = 6 (H). **P < 0.01 by 2-tailed, paired Student’s t test (D) or 1-way ANOVA with Tukey’s multiple-comparison test (E–H). NS, not significant (P > 0.05). Data in B and C are representative of 3 independent experiments.