KR20220143261A - Method for screening therapeutic agents of cancer by interaction THRAP3 and DDX5 - Google Patents

Abstract

The present invention relates to a method for screening cancer therapeutics using the binding of THRAP3 and DDX5. More particularly, the present invention is to provide a method for screening, as cancer therapeutics, candidate substances which can induce apoptosis by reducing the binding of THRAP3 and DDX5 in cancer cells, as it is confirmed that the accumulation of R-loops is suppressed and DNA damage of cells is inhibited as THRAP3 and methylated DDX5 bind in a cellular R-loop (DNA:RNA hybrid), and XRN2, an exonuclease, is brought to degrade the RNA of the R-loop through the binding.

Description

Method for screening therapeutic agents of cancer by interaction THRAP3 and DDX5

The present invention relates to a cancer therapeutic screening method using THRAP3 (Thyroid hormone receptor-associated protein 3) and DDX5 (DEAD box RNA helicase DDX5) binding.

All living things store their genetic information in the form of DNA. Preserving the genome is essential for the survival and smooth functioning of all living things. However, cells are continuously exposed to risks due to stress from the external environment such as reactive oxygen species (ROS) or ultraviolet rays generated during normal metabolism. Various internal and external risk factors cause mutations in DNA nucleotide sequences, resulting in genome instability. Fortunately, there is a DNA repair system in the cell that can correct the mutations that occur on a daily basis, but sometimes the damage is so severe that the intracellular system alone cannot repair it. A change in the basic structure that is not restored during DNA self-replication due to physical and chemical causes is defined as DNA damage.

A DNA-RNA hybrid (DNA:RNA hybrid, R-loop) is a nucleic acid molecule composed of a DNA chain and an RNA chain whose nucleotide sequence is complementary thereto, and has a double helix structure similar to type A DNA and double-stranded RNA. RNA-DNA hybrids and their formation reactions are used in gene analysis technologies such as cDNA synthesis by reverse transcriptase, Northern aspiration, and S1 mapping. to form

However, if the R-loop (DNA:RNA hybrid) formed during the DNA transcription-replication process is not resolved, DNA damage occurs, and this DNA damage causes cancer disease. Therefore, the R-loop formed after the DNA transcription-replication process Degradation could be a major target for the treatment of cancer diseases.

Republic of Korea Patent Publication No. 10-2017-0088462 (published on Aug. 2, 2017)

The present invention relates to a cancer therapeutic screening method for selecting a candidate drug that induces DNA damage in cancer cells by inhibiting THRAP3 and DDX5 binding.

The present invention comprises the steps of contacting a test substance to cancer cells;

checking the binding level of THRAP3 and DDX5 in cancer cells contacted with the test substance; and

Provided is a method for screening a cancer therapeutic agent, comprising selecting a test substance having a reduced binding level of THRAP3 and DDX5 compared to a control sample.

In addition, the present invention provides a method of inducing DNA damage in cancer cells, comprising reducing the THRAP3 and DDX5 binding levels of the cancer cells in vitro.

According to the present invention, the binding of THRAP3 and DDX5 was reduced in cancer cells in which the expression of THRAP3 was suppressed, and the expression of XRN2, an RNA degrading enzyme, was reduced due to the decrease in the binding of THRAP3 and DDX5. Accordingly, R-loop accumulation and DNA damage were reduced. As this induction confirms the effect of inhibiting cancer cell proliferation, when the binding level of THRAP3 and DDX5 is inhibited, R-loop accumulation in cancer cells is induced, DNA is damaged, and thus cancer cell death can be increased. , to provide a screening method for providing a candidate substance capable of inducing cancer cell death by regulating the THRAP3 and DDX5 binding as a cancer therapeutic agent.

1 is a result of confirming the co-localization of THRAP3 and R-loop and R-loop accumulation according to THRAP3 deficiency. Figure 1b is the result of confirming that THRAP3 binds to R-loop through immunoprecipitation using R-loop antibody and that R-loop formation is increased in a THRAP3 deficient situation due to siRNA transfection.
Figure 2 is the result of confirming the inhibition of DNA replication and DSB induction according to THRAP3 deficiency. It was confirmed that the increase in the formation of R-loop through the increase in the nucleus under the current situation, and FIGS. 2b and 2c show that when looking at newly replicated DNA by hybridizing EdU, the number decreased in THRAP3 deficient cells compared to the control group. 2d and 2e show that γH2AX and 53BP1, which are representative indicators, are fluorescently labeled in the nucleus to confirm the DNA double-stranded damage that can be induced by R-loop, and it is confirmed that the increase due to THRAP3 deficiency As a result, it was confirmed that normal recovery was achieved through overexpression of RNaseH1 that releases R-loop from the above results.
Figure 3 is the result of confirming the binding of THRAP3 to DDX5 and XRN2, Figure 3a is the most significant protein DDX5 by classifying the protein related to the resolution of R-loop among the 250 THRAP3-binding proteins found through mass spectrometry. , are the results of confirming the RNA helicase, and FIGS. 3b, 3c, 3d, 3e and 3f show that THRAP3 binds to DDX5 as well as DDX5 and RNA constituting the R-loop through immunoprecipitation using the THRAP3 antibody. As a result of confirming that it is also bound to XRN2, which is responsible for the direct R-loop removal function after cutting, DDX5 methylation plays an important role when DDX5 and XRN2 form a bond to resolve R-loop. Therefore, as a result of confirming the degree of binding to THRAP3 using DDX5 in which the methylation inhibitor (g) and the methylation arginie residue were substituted with lysine (h, i), DDX5 methylation also mediates binding to THRAP3, and binding when methylation is disturbed It is the result of confirming that this decrease.
4 is a result confirming that DDX5 interacts with the N-terminal of THRAP3. When the THRAP3 protein is expressed in cells and the binding to DDX5 is confirmed using the immunoprecipitation method, the binding to the N-terminal site of THRAP3 is shown. This is the confirmed result.
5 is a result of confirming XRN2 as a sub-target of THRAP3 for R-loop degradation, XRN2, which was confirmed binding in FIG. It is the result of confirming that the access to the R-loop is decreased when the phenomenon is confirmed in the state of THRAP3 deficiency.
6 is a result of confirming the effect of THRAP3 to degrade R-loop in breast cancer cells. It is a result of confirming that DNA replication is decreased in knockdown cells, and FIG. 6b is a result of confirming that R-loop is increased in THRAP3-deficient cells, thereby inducing DNA double-stranded damage. Increase in γH2AX, 53BP1 fluorescence staining In addition, by confirming that cell proliferation was reduced in MCF7 cells selected in a THAP3-deficient state, and cell proliferation was restored by overexpression of RNaseH1 that resolves R-loop, R- caused by a decrease in THRAP3 This is the result of confirming that the control of the loop affects the regulation of the proliferation of cancer cells.
7 is a schematic diagram showing the action of THRAP3 in R-loop degradation. THRAP3 binds to the RNA site of R-loop to form a conjugate with methylated DDX5, and induces the recruitment of RNA-cleaving XRN2 to R-loop. indicates that it plays a role in resolving

Hereinafter, the present invention will be described in more detail.

In the present invention, THRAP3 and methylated DDX5 are bound in R-loop (DNA:RNA hybrid) of cells, and XRN2, an exonuclease, is mobilized through the binding to degrade R-loop RNA. By inhibiting the accumulation of -loop and confirming that it inhibits cell DNA damage, it is intended to provide a method for screening candidate substances capable of inducing apoptosis as a cancer treatment agent by regulating the binding of THRAP3 and DDX5 in cancer cells. .

The present invention comprises the steps of contacting a test substance to cancer cells;

checking the binding level of THRAP3 and DDX5 in cancer cells contacted with the test substance; and selecting a test substance having a reduced binding level of THRAP3 and DDX5 as compared to a control sample.

The cancer therapeutic agent screening method may further include further confirming a decrease in THRAP3 and XRN2 binding levels in cancer cells contacted with a test substance.

The THRAP3 may bind to DDX5 in R-loop (DNA: RNA hybrid) and mobilize exonuclease to degrade R-loop.

The DDX5 may be one in which arginine is methylated by PRMT5, and the methylated DDX5 binds to the N-terminus of THRAP3.

The decrease in the binding level of THRAP3 and DDX5 may be to inhibit R-loop (DNA:RNA hybrid) degradation in cancer cells, thereby inducing DNA damage and killing cancer cells.

The cancer may be selected from the group consisting of osteosarcoma, breast cancer, colon cancer, lung cancer, liver cancer, kidney cancer, stomach cancer, brain cancer, prostate cancer and pancreatic cancer.

In addition, the present invention may provide a method for inducing DNA damage in cancer cells, comprising reducing the THRAP3 and DDX5 binding levels of the cancer cells in vitro.

The THRAP3 and DDX5 binding level may be confirmed by immunoprecipitation (Immunoprecipitation) and immunoblotting (Immunoblotting), but is not limited thereto.

"THRAP3" of the present invention is NCBI accession no. 9967, "DDX5" is the NCBI accession no. 1655, "XRN2" is the NCBI accession no. 22803, but is not limited thereto.

The term "test substance" used while referring to the screening method of the present invention affects the expression level of a gene, affects the expression or activity of a protein, or affects the binding between proteins. It refers to an unknown candidate substance used in screening. The sample includes, but is not limited to, chemicals, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural product extracts.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Cell Culture

U2OS cells and MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum (FBS). It was maintained in a CO ₂ incubator.

To confirm the proliferative ability of MDA-MB-231, 10 ⁵ cells were aliquoted and counted daily. THRAP3 knockdown MDA-MB-231 cells were prepared through lentiviral shRNA transfection. For lentivis production, HEK-293T cells (ATCC) were transfected with 4 μg of lentiviral pLKO.1 shRNA, packaging and envelope vectors.

The transfected cell culture medium was collected, infected with MDA-MB-231, and cells were selected with 2 μg/mL of puromycin (VWR Chemicals) after infection.

2. Plasmid and siRNA transfection

Transfections of RNaseH1-V5, Flag/Myc-DDX5, HA-THRAP3 plasmids and the following siRNA at 20 nM were performed with Lipofectamine 2000 and RNAiMAX transfection reagent (Invitrogen), respectively, according to the transfection procedure of the manufacturer's instructions.

1) siRNA

Negative control siRNA: 5'-UUC UCC GAA CGU GUC ACG UTT-3'

human THRAP3 siRNA: 5'-GGU AUA AGC UCC GAG AUG A-3'

2) shRNA

human THRAP3 MISSION shRNA,

shTHRAP3 #1 sequence: CCG GAG TAT ATC AGA ATC GGG ATT TCT CGA GAA ATC CCG ATT CTG ATA TAC TTT TTT G

shTHRAP3 #2 sequence: CCG GGC CTT GAT ATT GAA CGT CGT ACT CGA GTA CGA CGT TCA ATA TCA AGG CTT TTT G

shTHRAP3 #3 sequence: CCG GAG ATC TCG TTC TCG TTC ATT TCT CGA GAA ATG AAC GAG AAC GAG ATC TTT TTT G

shTHRAP3 #4 sequence: CCG GTG GAG CTA AGA TGA CTA ATT TCT CGA GAA ATT AGT CAT CTT AGC TCC ATT TTT G

3. Cell Lysis, Western Blotting and Immunoprecipitation Analysis

Whole cell lysates were prepared by treating cells washed with cold PBS with RIPA lysis buffer containing 1% deoxycholate salt, protease inhibitor (Roche) and phosphatase inhibitor cocktail (Sigma Aldrich).

The collected cell suspension was vortexed vigorously and centrifuged at 15000 rpm, 4° C. for 15 minutes. The concentration of the removed cell protein extract was confirmed with the Pierce BCA Protein Assay Kit.

Equal amounts of protein were loaded onto SDS-PAGE and transferred to a nitrocellulose membrane (GE healthcare). The membranes were blocked with 5% skim milk and each primary antibody was incubated overnight at 4°C.

The membrane was washed with TBST buffer for 30 minutes and incubated with HRP-conjugated secondary antibody diluted at 1:10000 for 1 hour at room temperature. Protein expression was confirmed with chemiluminescent HRP substrate solutions (Advansta).

For immunoprecipitation assays, cellular protein extracts were spin-incubated with specific antibodies overnight at 4°C. Thereafter, the protein-antibody sample was mixed with protein A/G agarose beads at 4° C. for 1 hour for sedimentation.

4. Immunofluorescence Assay

U2OS cells were seeded on chamber slides. After 1 day of dispensing, the cells were washed twice, extracted with 0.5% Triton X-100 on ice for 5 minutes, and fixed with 3% paraformaldehyde for 15 minutes at room temperature.

After washing twice with PBS, 0.5% Triton X-100 was added to permeabilize the cells.

In particular, in the case of chromatin-binding protein, extraction was performed with 0.5% Triton X-100 for 2 minutes and fixed with 100% methanol at -20°C for 30 minutes.

Thereafter, the cells were treated with blocking buffer for 1 hour and incubated overnight at 4° C. with the primary antibody diluted 1:1000.

After antibody washing, Alexa Fluor-conjugated secondary antibody was treated and incubated for 1 hour, and a fluorescence microscope image of cells washed with PBS was obtained.

5. Proximity Ligation Assay

Proximity junction analysis was performed according to standard protocols using a Duolink® PLA fluorescence kit (Sigma Aldrich).

Briefly, after extraction, fixation, and permeabilization of U2OS cells seeded on slides, cells were blocked with PBS-added 2% BSA at room temperature for 1 hour.

Diluted primary antibody was added to the cells and incubated overnight at 4°C.

Antibodies were washed three times with PBS and incubated with premixed PLUS and MINUS PLA probes at 37° C. for 1 hour. Thereafter, the samples were incubated with a fluorescence ligation solution and an amplification solution for 30 minutes and 100 minutes, respectively, in a constant temperature and humidity chamber at 37°C.

After final washing with the washing buffer provided in the kit, the number of nuclear PLA foci was quantified in the images obtained by placing on slides with DAPI and performing confocal microscopy analysis using a 63× oil immersion objective.

6. DRIP (S9.6 DNA:RNA immunoprecipitation) analysis

Cells were incubated for 10 min in lysis buffer containing 85 mM KCl, 5 mM PIPES (pH 8.0) and 0.5% NP-40 on ice. Lysed cells were collected and centrifuged at 4° C., 3000 g for 5 minutes.

Prior to use, nuclear pellets were prepared in RSB buffer (10 mM Tris-HCl (pH7.5), 200 mM NaCl, 2.5 mM) with 0.2% deoxycholate salt, 0.1% SDS and 0.5% Triton X-100 and protease inhibitor cocktail added MgCl ₂ ) was resuspended.

After sonicating the nucleus for 10 minutes using a diagenode bioruptor, the extract was diluted in RSB buffer containing 0.5% Triton X-100 and immunoprecipitated with S9.6 antibody.

7. EdU Hybridized DNA Replication Analysis

DNA replication was confirmed through EdU (5-ethynyl-2’-deoxyurinidine) hybridization using Click-iT™ EdU Cell Proliferation Kit (Invitrogen) and Alexa Fluor™ 488 dye.

THRAP3 knockdown, RNaseH1 overexpressed U2OS cells were labeled with EdU working solution for 15 minutes. After incubation, the medium was removed immediately, and cells were fixed with PBS containing 4% formaldehyde for 15 minutes at room temperature, and then permeabilized with PBS containing 0.5% Triton X-100 for 20 minutes.

The permeation buffer was removed from the cells and the cells were washed twice. A reaction buffer containing a Click-iT™ reaction cocktail containing CuSO ₄ and Alexa Fluror azide was added to the cells and incubated at room temperature for 30 minutes while blocking light.

For nuclear visualization, DAPI staining was performed for 15 min. EdU hybridization was confirmed by fluorescence microscopy, and EdU-positive cells and fluorescence intensity were analyzed.

8. Purification and Mass Spectrometry

HEK293 cells were ectopic transfected with FLAG/Myc tagged THRAP3 and lysates were prepared and mixed with immobilized anti-FLAG M2 agarose (Sigma Aldrich).

After incubation, FLAG tagged THRAP3 and interacting proteins were washed with binding buffer and eluted with 3× FLAG peptide (Sigma Aldrich).

The eluted proteins were separated by SDS-PAGE, and molecular characteristics were analyzed by reverse-phase LC-MS/MS using a high-resolution hybrid mass spectrometer (LTQ-Orbitrap, Thermo Fisher Scientific).

<Example 1> Confirmation of association between THRAP3 and DNA:RNA hybrid (R-loop)

Since THRAP3 is an RNA-binding protein and is involved in DNA damage response through RNA metabolism, it was hypothesized that THRAP3 is related to R-loop-structured RNA.

To confirm this, Proximity Ligation Assay (PLA) was performed using the S9.6 antibody capable of detecting R-loop and THRAP3 antibody.

As a result, co-localization was confirmed in the nuclear region as shown in FIG. 1A, and a decrease in the ligation signal according to THRAP3 deficiency using siRNA was confirmed.

In addition, as a result of performing DNA-RNA immunoprecipitation analysis using S9.6 and THRAP3 antibodies in U2OS cells, the association between R-loops and THRAP3 was confirmed as shown in FIG. 1B.

Next, it was checked whether THRAP3 could regulate the R-loop level.

As a result, when THRAP3 expression was suppressed by siRNA in the immunofluorescence image as shown in FIG. 1C, it was confirmed that the amount of R-loops was significantly increased.

From the above results, it was confirmed that THRAP3 interacts with R-loop and regulates R-loop.

<Example 2> Confirmation of replication inhibition and DSB (double-strand break) induction by THRAP3 deficiency

In the previous experiment, it was confirmed that the R-loop was accumulated due to the decrease in THRAP3, and the proliferation cell nuclear antigen (PCNA) increased at the replication fork in the transcription replication collision (TRC) region, and the TRC-induced R-loop decreased the accumulation of PCNA. As reported to induce induction, the level of PCNA in the R-loop strand was confirmed when THRAP3 was deficient. Referring to the PLA results of FIG. 2A, it was confirmed that PCNA was significantly increased in R-loop.

In addition, as it is reported that unseparated R-loop promotes DNA strand breakage and induces DNA damage, EdU hybridization to U2OS cells was confirmed under control and THRAP3 knockdown conditions for DNA replication evaluation.

As a result, as shown in FIGS. 2B and 2C , the decreased level of THRAP3 inhibited DNA replication, but overexpression of RNaseH1, a ribonuclease that induces R-loop degradation, restored the replication ability.

Meanwhile, as shown in FIGS. 2D and 2E , THRAP3 knockdown increased γH2AX and 53BP1 foci per nucleus, which are representative markers of double-strand break (DSB). In addition, RNaseH1 overexpression reduced DNA double-strand breaks induced by THRAP3 deficiency.

In addition, as shown in FIG. 2F , it was confirmed that reduced DNA replication and severe DNA damage in cells transfected with THRAP3 siRNA delayed cell proliferation.

<Example 3> Confirmation of binding of THRAP3 with DDX5 and XRN2

To understand the mechanism by which THRAP3 regulates R-loop levels and genomic stability, mass spectrometry was performed to identify proteins that interact with THRAP3.

As shown in FIG. 3A, after separating the protein showing a two-fold or more significant expression change along with THRAP3 expression, it was classified into 7 RNA helicases and 5 DNA damage-related proteins, and DDX5 was identified as a major THRAP3 interacting protein. In addition, we confirmed the endogenous interaction between THRAP3 and DDX5 in U2OS cells.

As a result, DDX5 co-immunoprecipitated with THRAP3 in lysed cells incubated with THRAP3 antibody and control IgG antibody as shown in FIG. 3B. In addition, it was confirmed that XRN2, which is involved in R-loop degradation by exonuclease function, also endogenously binds with THRAP3.

On the other hand, when RNaseH1 was overexpressed as shown in FIG. 3C, it was confirmed that the protein binding to DDX5 and XRN2 was weakened, and XRN2 recruitment by THRAP3 was also significantly reduced as shown in FIG. 3D.

On the other hand, when cells were stimulated with camptothecin (CPT), known as a topoisomerase, which could increase R-loop formation, it was confirmed that THRAP3 dynamically binds to DDX5 and XRN2 in a time-dependent manner after CPT treatment. there was. Referring to FIG. 3E , it was confirmed that DDX5 was strongly bound to THRAP3 30 minutes after CPT treatment, and XRN2 followed approximately 3 hours after treatment.

Next, it was checked whether THRAP3 affects the association between DDX5 and XRN2. THRAP3 siRNA and control scrambled siRNA transfected U2OS cells were lysed and immunoprecipitated with DDX5.

As a result, as shown in FIG. 3F, it was confirmed that XRN2 interacting with DDX5 was reduced in the THRAP3-deficient condition.

DDX5 recruits nucleases from arginine-guanine motifs methylated by PRMT5 to resolve R-loops and inhibit R-loop accumulation.

Accordingly, in order to check whether the binding of THRAP3 and DDX5 is changed by the change in DDX5 methylation, 20 μM of EPZ015666 (PRMT5 inhibitor, Sigma Aldrich, Cat# 1421) was administered to U2OS cells to suppress PRMT5 induced methylation for 72 hours. treated during

As a result, it was confirmed that inhibition of DDX5 arginine methylation also inhibited protein interaction with THRAP3 as shown in FIG. 3G.

To further confirm the effect of EPZ015666, wild-type Flag/Myc-DDX5 and RK mutants were generated in which five arginine sequences known to be methylated by PRMT5 were replaced with lysine residues.

As a result, it was confirmed that the interaction between the RK mutant DDX5 and THRAP3 was reduced in the CPT condition as shown in FIG. 3G.

From the above results, it was confirmed that THRAP3 physically interacts with RNA helicase DDX5 and exonuclease XRN2 required for R-loop degradation.

<Example 4> Confirmation of interaction with DDX5 at the N-terminus of THRAP3

Next, to confirm which domain of THRAP3 is essential for DDX5 interaction, as shown in FIG. 4A, Flag/Myc tagged DDX5 and 5 types of HA-tagged THRAP3 domain truncated mutants were co-transfected into cells and immunoprecipitated with Flag antibody. was performed.

As a result, as shown in FIG. 4B, ΔN and ΔNC THRAP3 did not interact with DDX5.

From the above results, it was confirmed that the N-terminal domain contributes to the binding of THRAP3 and DDX5 proteins.

<Example 5> Confirm the role of XRN2 in R-loop resolution

In the previous experiment, the interaction between THRAP3 and the R-loop degradation proteins DDX5 and XRN2 was confirmed, but the basic mechanism for the R-loop degradation mediated by THRAP3 was not clearly identified. Accordingly, from the results of each interaction between THRAP3, DDX5 and XRN, it was hypothesized that THRAP3 could regulate the recruitment of R-loop resolution proteins, and to confirm this, DRIP analysis was performed under THRAP3 knockdown conditions.

As a result, as shown in FIG. 5B, THRAP3 deficiency in R-loop reduced XRN2 binding, but no change in DDX5. In addition, as shown in FIG. 5C , a significantly low PLA signal between S9.6 and XRN2 was confirmed in THRAP3 knockout cells.

From the above results, it was confirmed that R-loop accumulation induced by THRAP3 was partially caused by a decrease in XRN2 recruitment. Therefore, it was confirmed that XRN2 is a sub-target of THRAP3 in R-loop resolution.

<Example 6> Confirmation of the effect of THRAP3 in breast cancer cells

The role of THRAP3 on R-loop resolution in pathological conditions was confirmed.

Referring to FIG. 6A , as in the previously identified U2OS cells, THRAP3 deficiency reduced EdU hybridization cells in MCF7 breast adenocarcinoma cells, which means reduced DNA replication.

In addition, as shown in FIGS. 6C and 6D , THRAP3 knockdown in MCF7 cells induced nuclear R-loop accumulation, and THRAP3-mediated R-loop accumulation induced nuclear γH2AX and 53BP1 localization as shown in FIGS. 6E and 6F , and , R-loops degradation via RNaseH1 overexpression mitigated DNA double-strand breaks caused by THRAP3 knockdown.

In addition, it was confirmed that the cell proliferation of the MCF7 breast cancer cell line in which THRAP3 is knocked down is inhibited as shown in FIGS. 6G and 6H.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

contacting the test substance to the cancer cells;
checking the binding level of THRAP3 and DDX5 in cancer cells contacted with the test substance; and
A cancer treatment screening method comprising the step of selecting a test substance having a reduced binding level of THRAP3 and DDX5 compared to a control sample. The method according to claim 1, wherein the screening method for a cancer treatment agent further comprises the step of further confirming a decrease in the binding level of THRAP3 and XRN2 in cancer cells contacted with the test substance. The method according to claim 1, wherein the THRAP3 binds to DDX5 in an R-loop (DNA: RNA hybrid) and mobilizes an exonuclease to degrade the R-loop. The method according to claim 1, wherein in DDX5, arginine is methylated by PRMT5, and the methylated DDX5 binds to the N-terminus of THRAP3. The method according to claim 1, wherein the reduction of the binding level of THRAP3 and DDX5 inhibits R-loop (DNA:RNA hybrid) degradation in cancer cells, thereby inducing DNA damage and killing cancer cells. The method according to claim 1, wherein the cancer is selected from the group consisting of osteosarcoma, breast cancer, colorectal cancer, lung cancer, liver cancer, kidney cancer, stomach cancer, brain cancer, prostate cancer and pancreatic cancer. A method of inducing DNA damage in cancer cells, comprising reducing the level of binding of THRAP3 and DDX5 in the cancer cells in vitro.

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