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 1

An integrative functional genomic strategy for identifying ER+ breast cancer dependency on cryptic lncRNA-encoded ORFs.

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An integrative functional genomic strategy for identifying ER+ breast ca...
Workflow diagram depicting the integrative strategy for (A) predicting cryptic lncRNA-encoded ORFs from ribo-seq data and (B) identifying ER+ BC dependency on these predicted cryptic ORFs by using the CRISPR/Cas9 screen. (C) Scatter plot showing the statistical significance [–log10(P value)] and the magnitude of change [log2(fold change)] between day 21 and day 0, for the representative negatively selected sgRNAs of the corresponding ORFs. The blue dots correspond to the cryptic lncRNA-encoded ORFs with at least 1 significantly and negatively selected targeting sgRNA in the CRISPR/Cas9 screen and the red dots correspond to the ORFs meeting the criterion of the blue dots, whose host lncRNA expression was significantly upregulated in luminal A (LumA) BC in comparison with the corresponding normal breast tissues, based on TCGA RNA-seq data. Puro, puromycion.