Guohui Wan

The ATM Kinase Induces MicroRNA Biogenesis in the DNA Damage Response

The DNA damage response involves a complex network of processes that detect and repair DNA damage. Here we show that miRNA biogenesis is globally induced upon DNA damage in an ATM-dependent manner. About one-fourth of miRNAs are significantly upregulated after DNA damage, while loss of ATM abolishes their induction. KH-type splicing regulatory protein (KSRP) is a key player that translates DNA damage signaling to miRNA biogenesis. The ATM kinase directly binds to and phosphorylates KSRP, leading to enhanced interaction between KSRP and pri-miRNAs and increased KSRP activity in miRNA processing. Mutations of the ATM phosphorylation sites of KSRP impaired its activity in regulating miRNAs. These findings reveal a mechanism by which DNA damage signaling is linked to miRNA biogenesis.

Oncogenic Wip1 Phosphatase Is Inhibited by miR-16 in the DNA Damage Signaling Pathway

Wild-type p53-induced phosphatase 1 (Wip1) was identified as an oncogene amplified and overexpressed in several human cancers. Recent evidence suggested that Wip1 is a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 dephosphorylates several key DNA damage-responsive proteins and reverses DNA damage-induced cell cycle checkpoints. Previous reports showed that Wip1 was transcriptionally induced by p53 at the early stage of the DNA damage response. To investigate the temporal and functional regulation of Wip1, we identified a microRNA, miR-16, that specifically targets the mRNA of Wip1 and thus negatively regulates the expression level of Wip1. miR-16 itself is induced immediately after DNA damage. Therefore, the increase in Wip1 protein level is significantly postponed compared with that of its mRNA level, preventing a premature inactivation of ATM/ATR signaling and allowing a functional completion of the early DNA damage response. To better understand miR-16 biological functions in the context of cancer cells, we examined its expression in mammary tumor stem cells and found it to be markedly downregulated in mammary tumor stem cells. Overexpression of miR-16 or inhibition of Wip1 suppresses the self-renewal and growth of mouse mammary tumor stem cells and sensitizes MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin. Together, our results suggest an important role of miR-16 in the regulation of Wip1 phosphatase in the DNA damage response and mammary tumorigenesis.

Long non-coding RNA ANRIL (CDKN2B-AS) is induced by the ATM-E2F1 signaling pathway

The maintenance of genome integrity is essential for the proper function and survival of all organisms. Human cells have evolved prompt and efficient DNA damage response to eliminate the detrimental effects of DNA lesions. The DNA damage response involves a complex network of processes that detect and repair DNA damage, in which long non-coding RNAs (lncRNAs), a new class of regulatory RNAs, may play important roles. Recent studies have identified a large number of lncRNAs in mammalian transcriptomes. However, little is known about the regulation and function of lncRNAs in the DNA damage response. In the present study, we demonstrate that one specific lncRNA, ANRIL, is transcriptionally up-regulated by the transcription factor E2F1 in an ATM-dependent manner following DNA damage, and elevated levels of ANRIL suppress the expression of INK4a, INK4b and ARF at the late-stage of DNA damage response, allowing the cell to return to normal at the completion of the DNA repair.

miRNA response to DNA damage.

Faithful transmission of genetic material in eukaryotic cells requires not only accurate DNA replication and chromosome distribution but also the ability to sense and repair spontaneous and induced DNA damage. To maintain genomic integrity, cells undergo a DNA damage response using a complex network of signaling pathways composed of coordinate sensors, transducers and effectors in cell cycle arrest, apoptosis and DNA repair. Emerging evidence has suggested that miRNAs play a crucial role in regulation of DNA damage response. In this review, we discuss the recent findings on how miRNAs interact with the canonical DNA damage response and how miRNA expression is regulated after DNA damage.

miR-203 induces oxaliplatin resistance in colorectal cancer cells by negatively regulating ATM kinase

Chemotherapy for patients with metastatic colorectal cancer (CRC) is the standard of care, but ultimately nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may help identify novel targets and drugs that will, in turn, improve therapy. Oxaliplatin is a common component combination therapeutic regimen for use in patients with metastatic CRC, but is also used as a component of adjuvant therapy for patients at risk for recurrent disease. In this study, unbiased microRNA array screening revealed that the miR‐203 microRNA is up‐regulated in three of three oxaliplatin‐resistant CRC cell lines, and therefore we investigated the role of miR‐203 in chemoresistance. Exogenous expression of miR‐203 in chemo‐naïve CRC cells induced oxaliplatin resistance. Knockdown of miR‐203 sensitized chemoresistant CRC cells to oxaliplatin. In silico analysis identified ataxia telangiectasia mutated (ATM), a primary mediator of the DNA damage response, as a potential target of miR‐203. ATM mRNA and protein levels were significantly down‐regulated in CRC cells with acquired resistance to oxaliplatin. Using TCGA database, we identified a significant reverse correlation of miR‐203 and ATM expression in CRC tissues. We validated ATM as a bona fide target of miR‐203 in CRC cells. Mutation of the putative miR‐203 binding site in the 3′ untranslated region (3′UTR) of the ATM mRNA abolished the inhibitory effect of miR‐203 on ATM. Furthermore, stable knockdown of ATM induced resistance to oxaliplatin in chemo‐naïve CRC cells. This is the first report of oxaliplatin resistance in CRC cells induced by miR‐203‐mediated suppression of ATM.

TP53 loss creates therapeutic vulnerability in colorectal cancer

TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody–drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

A prompt and efficient DNA damage response (DDR) eliminates the detrimental effects of DNA lesions in eukaryotic cells. Basic and preclinical studies suggest that the DDR is one of the primary anti‐cancer barriers during tumorigenesis. The DDR involves a complex network of processes that detect and repair DNA damage, in which long non‐coding RNAs (lncRNAs), a new class of regulatory RNAs, may play an important role. In the current study, we identified a novel lncRNA, lncRNA‐JADE, that is induced after DNA damage in an ataxia‐telangiectasia mutated (ATM)‐dependent manner. LncRNA‐JADE transcriptionally activates Jade1, a key component in the HBO1 (human acetylase binding to ORC1) histone acetylation complex. Consequently, lncRNA‐JADE induces histone H4 acetylation in the DDR. Markedly higher levels of lncRNA‐JADE were observed in human breast tumours in comparison with normal breast tissues. Knockdown of lncRNA‐JADE significantly inhibited breast tumour growth in vivo. On the basis of these results, we propose that lncRNA‐JADE is a key functional link that connects the DDR to histone H4 acetylation, and that dysregulation of lncRNA‐JADE may contribute to breast tumorigenesis.

DNA-damage-induced nuclear export of precursor microRNAs is regulated by the ATM-AKT pathway.

Expression of microRNAs (miRNAs) involves transcription of miRNA genes and maturation of the primary transcripts. Recent studies have shown that posttranscriptional processing of primary and precursor miRNAs is induced after DNA damage through regulatory RNA-binding proteins in the Drosha and Dicer complexes, such as DDX5 and KSRP. However, little is known about the regulation of nuclear export of pre-miRNAs in the DNA-damage response, a critical step in miRNA maturation. Here, we show that nuclear export of pre-miRNAs is accelerated after DNA damage in an ATM-dependent manner. The ATM-activated AKT kinase phosphorylates Nup153, a key component of the nucleopore, leading to enhanced interaction between Nup153 and Exportin-5 (XPO5) and increased nuclear export of pre-miRNAs. These findings define an important role of DNA-damage signaling in miRNA transport and maturation.

Noncoding RNAs in DNA Repair and Genome Integrity

SIGNIFICANCE The well-studied sequences in the human genome are those of protein-coding genes, which account for only 1%-2% of the total genome. However, with the advent of high-throughput transcriptome sequencing technology, we now know that about 90% of our genome is extensively transcribed and that the vast majority of them are transcribed into noncoding RNAs (ncRNAs). It is of great interest and importance to decipher the functions of these ncRNAs in humans. RECENT ADVANCES In the last decade, it has become apparent that ncRNAs play a crucial role in regulating gene expression in normal development, in stress responses to internal and environmental stimuli, and in human diseases. CRITICAL ISSUES In addition to those constitutively expressed structural RNA, such as ribosomal and transfer RNAs, regulatory ncRNAs can be classified as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), and long noncoding RNAs (lncRNAs). However, little is known about the biological features and functional roles of these ncRNAs in DNA repair and genome instability, although a number of miRNAs and lncRNAs are regulated in the DNA damage response. FUTURE DIRECTIONS A major goal of modern biology is to identify and characterize the full profile of ncRNAs with regard to normal physiological functions and roles in human disorders. Clinically relevant ncRNAs will also be evaluated and targeted in therapeutic applications.

Crosstalk between the DNA damage response pathway and microRNAs

MicroRNAs (miRNAs) are a family of small, non-coding RNAs that control gene expression at the post-transcriptional level by destabilizing and inhibiting translation of their target messenger RNAs. MiRNAs are involved in the regulation of a number of fundamental biological processes, and their dysregulation is thought to contribute to several disease processes. Emerging evidence suggests that miRNAs also play a critical role in protecting the heritable genome by contributing to the regulation of the DNA damage response. Consequently, much recent investigative effort has been directed towards an improved understanding of how miRNAs are regulated in response to DNA damage. In this review, we discuss the most recent findings regarding the regulation of miRNA expression and the functional roles of miRNAs in the DNA damage response.