News Release

Title
TUG1-mediated R-loop resolution at microsatellite loci as a prerequisite forcancer cell proliferation
Key Points• Oncogene-induced DNA replication stress and consequent pathogenic R-loop
formation is known to impede S phase progression.
• Nonetheless, cancer cells continuously proliferate under such high-stressed
conditions through incompletely understood mechanisms.
• We found taurine upregulated gene 1 (TUG1) long noncoding RNA (lncRNA),
which is highly expressed in many types of cancers, as an important regulator of
intrinsic R-loop in cancer cells.
• Under RS conditions, TUG1 was rapidly upregulated via activation of theATR-CHK1 signaling pathway, interacted with RPA and DHX9, and engaged inresolving R-loops at certain loci, particularly at the CA repeat microsatellite loci.
• Depletion of TUG1 led to an overabundant R-loops and enhanced replicationstress, leading to substantial inhibition of tumor growth.
• Our data reveal a crucial role of TUG1 as an indispensable molecule forresolving the problem of R-loop accumulation in cancer cells and a strong
rationale for targeting TUG1 as a potent therapeutic approach for cancertreatment.
SummaryCancer cells exhibit elevated levels of "replication stress," wherein DNA
replication is frequently interrupted. A primary cause of replication stress is theformation of R-loops, caused by the conflicts between RNA transcription andDNA replication on the DNA strand, leading to DNA damage. However, themechanisms by which cancer cells resolve R-loops and continue DNA replication
under such high replication stress have remained unclear. In this study, wefocused on lncRNAs, which are highly expressed in cancer cells and known to be
involved in cancer development and malignancy. We discovered that TUG1(Taurine Upregulated Gene 1), a lncRNA of which expression is rapidly induced
under the replication stress, binds to the proteins RPA and DHX9 and resolves
R-loops containing microsatellite sequences. Suppression of TUG1 led to theaccumulation of R-loops and severe DNA damage, inducing apoptotic cell death.
We made a brain tumor mouse model using human glioblastoma cells expressing
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a high level of TUG1 and treated with an oligonucleotide therapeutics called
TUG1-DDS,

whichspecificallysuppresses

TUG1,incombination

withtemozolomide, a standard glioblastoma treatment. The results demonstrated
significant inhibition of tumor growth and a remarkable improvement in survivaltime. Overall, this study reveals a role of TUG1 as molecule important forresolving R-loop accumulation in cancer cells and suggests targeting TUG1 as apotent therapeutic approach for the treatment of glioblastoma.
Research BackgroundIn cancer cells, the high level of replication stress leads to DNA damage
and mutation, contributing to cancer development and rapid cancer growth.
However, the mechanism by which cancer cells reduce replication stress andcontinue growing despite such high replication stress is not fully understood.
Recently, lncRNAs, which are not translated into proteins, have been linked tocancer development, malignancy, and drug resistance, but their functions
remain unclear. This study aimed to identify lncRNA that reduces replicationstress in cancer cells and enhances cell proliferation.
Research Results
When we exposed cancer cells tochemicals that induce replication stress(hydroxyureaandcamptothecin),theexpression of lncRNA TUG1 significantlyincreased within 2 hours (Figure 1). This
upregulation of TUG1 was revealed to be adownstream

eventoftheATR/Chk1

pathway, which is activated in response toreplication stress. Then, we investigated
where

themolecules

newlygoinexpressed
thenucleus

TUG1
whenreplication stress occurs. We found thatTUG1 localizes at a DNA structure called
R-loop (Figure 2). R-loops consist ofRNA/DNA hybrid, leaving the other strandto form a loop-like structure (Figure 2).
The single-stranded DNA in the R-loop isprone

tobreaking

andcausingDNAdamage. TUG1 appears to play a role inresolving

theR-loop

topreventthis2

damage. To understand how TUG1 resolves the R-loop, we identified proteinsinteracting with TUG1. These proteins include RPA, which binds to R-loops andstalled replication forks, and DHX9, an RNA helicase that is known to resolve
R-loop structures (Figure 2). We found that the TUG1/RPA/DHX9 complex isparticularly effective at resolving R-loops in microsatellite regions, especiallythose containing CA repeats. These microsatellite regions are known to besusceptible to DNA damage and mutations.

In cells where we reduced the expression of TUG1 (TUG1 knockdown),
we observed severe DNA damage and the induction of cell death throughapoptosis (Figure 3a). Since TUG1 is highly expressed in glioblastoma, anaggressive brain tumor, we tested the effects of TUG1 knockdown in mousemodels of brain tumors. Using an antisense oligonucleotide therapeutics called
TUG1-DDS, which specifically targets cancer cells by drug delivery system, wefound that it significantly suppressed tumor growth and improved survival,
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especially when administered in combination with the standard treatment,temozolomide (TMZ) (Figure 3b).
In conclusion, our study revealed that TUG1 is essential for protecting
the genome DNA of cancer cells from damage and assists in cell proliferation byresolving R-loops. The TUG1-DDS is a potentially effective therapeutic agent
for treating glioblastoma, refractory brain cancer.
Publication
Journal: Nature Communications
Title: TUG1-mediated R-loop resolution at microsatellite loci as a prerequisite
for cancer cell proliferation
Author: Miho M. Suzuki1,†, Kenta Iijima1,2,†, Koichi Ogami3, Keiko Shinjo1,Yoshiteru Murofushi1, Jingqi Xie1, Xuebing Wang1, Yotaro Kitano4, Akira Mamiya1,Yuji Kibe1,4, Tatsunori Nishimura1, Fumiharu Ohka4, Ryuta Saito4, Shinya Sato5,Junya Kobayashi6, Ryoji Yao7, Kanjiro Miyata8, Kazunori Kataoka9,10, Hiroshi I.
Suzuki3,11,12, and Yutaka Kondo1,11,12*
Affiliation:
1

Division of Cancer Biology, Nagoya University Graduate School of Medicine, 65Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan
2
Laboratory Animal Facilities and Services, Preeminent Medical Photonics
Education and Research Center, Hamamatsu University School of Medicine,
1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka, 431-3192, Japan
3
Division of Molecular Oncology, Nagoya University Graduate School of Medicine,
65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan
4
Department of Neurosurgery, Nagoya University Graduate School of Medicine,
65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan
5
Molecular Pathology and Genetics Division, Kanagawa Cancer Center ResearchInstitute, 2-3-2 Nakao, Asahi-ku, Yokohama, Kanagawa, 241-8515, Japan.
6
School of Health Sciences at Narita, International University of Health andWelfare, 4-3, Kozunomori, Narita, Chiba, 286-8686, Japan
7
Department of Cell Biology, Japanese Foundation for Cancer Research, 3-8-31
Ariake, Koto-ku, Tokyo, 135-8550, Japan
8

Department of Materials Engineering, Graduate School of Engineering, The

University of Tokyo, 7-3-1 Hongo, Tokyo, Bunkyo-ku, Tokyo, 113-8656, Japan.
9

Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion,

3-25-14 Tono-machi, Kawasaki-ku, Kanagawa, 210-0821, Japan.
10

Institute for Future Initiative, Bunkyo-ku, The University of Tokyo, 7-3-1 Hongo,Bunkyo-ku, Tokyo, 113-0033, Japan.
11

Institute for Glyco-core Research (iGCORE), Tokai National Higher Education
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and Research SystemFuro-cho, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan.
12

Center for One Medicine Innovative Translational Research (COMIT), Nagoya

University, Nagoya, Japan†

co-first authors*correspoinding author
DOI: 10.1038/s41467-023-40243-8

Japanese ver.
https://www.med.nagoya-u.ac.jp/medical_J/research/pdf/Nat_230822.pdf

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