KR102505355B1 - 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 combination of THRAP3 and DDX5, and more particularly, THRAP3 and methylated DDX5 bind in an R-loop (DNA:RNA hybrid) of a cell, and through the binding, nucleic acid exohydrolysis XRN2, an enzyme (exonuclease), is mobilized to degrade R-loop RNA, thereby suppressing R-loop accumulation and thereby inhibiting cell DNA damage. It is intended to provide a method for screening a candidate substance capable of inducing as a cancer treatment.

Description

Method for screening therapeutic agents of cancer using the combination of THRAP3 and DDX5 {Method for screening therapeutic agents of cancer by interaction THRAP3 and DDX5}

The present invention relates to a method for screening cancer therapeutics using the combination of Thyroid hormone receptor-associated protein 3 (THRAP3) and DEAD box RNA helicase DDX5 (DDX5).

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

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

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

Republic of Korea Patent Publication No. 10-2017-0088462 (2017.08.02. Publication)

The present invention relates to a cancer treatment screening method for selecting a candidate drug that inhibits the binding of THRAP3 and DDX5 to induce DNA damage in cancer cells.

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

Checking the binding levels of THRAP3 and DDX5 in cancer cells in contact with the test substance; and

It provides a cancer therapeutic screening method comprising the step of selecting a test substance with a reduced binding level of the THRAP3 and DDX5 compared to a control sample.

In addition, the present invention provides a method for inducing DNA damage in cancer cells, comprising reducing the binding levels of THRAP3 and DDX5 in 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 reduced binding of THRAP3 and DDX5 reduced the expression of the RNA degrading enzyme XRN2, thereby reducing R-loop accumulation and DNA damage. As confirmed the effect of inhibiting cancer cell proliferation by induction of THRAP3, when the binding level of THRAP3 and DDX5 is inhibited, R-loop accumulation in cancer cells is induced and DNA is damaged, thereby increasing cancer cell death. , To provide a screening method for providing a candidate substance capable of inducing cancer cell death by regulating the binding of the THRAP3 and DDX5 as a cancer treatment.

Figure 1 is a result of confirming the co-localization of THRAP3 and R-loop and R-loop accumulation according to THRAP3 deficiency, Figure 1a is a result confirming that THRAP3 is present in the same position as R-loop in the nucleus, 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 the absence of THRAP3 due to siRNA transfection.
Figure 2 is a result of confirming DNA replication inhibition and DSB induction according to THRAP3 deficiency, Figure 1a is a cofactor of DNA polymerase, PCNA protein known to accumulate in the process of R-loop generation due to transcription-replication conflict THRAP3 deficiency It was confirmed that the formation of R-loop was increased through the increase in the nucleus in the situation of 2d and 2e show that γH2AX and 53BP1, which are representative indicators, are fluorescently labeled in the nucleus to confirm DNA double-strand damage that can be induced by R-loop, and increase with THRAP3 deficiency Results , from the above results, it was confirmed that the overexpression of RNaseH1, which releases the R-loop, restores to normal.
Figure 3 is the result of confirming the binding of THRAP3 with DDX5 and XRN2. Figure 3a classifies proteins related to the resolution of R-loop among 250 proteins that bind to THRAP3 found through mass spectrometry, and DDX5, the most significant protein , RNA helicase is confirmed, and FIGS. 3b, 3c, 3d, 3e and 3f show that THRAP3 binds to DDX5 as well as DDX5 through immunoprecipitation using the THRAP3 antibody to form R-loop RNA. As a result of confirming that it is also bound to XRN2, which is responsible for the direct R-loop removal function by cutting it, methylation of DDX5 plays an important role when DDX5 and XRN2 form a complex to resolve the R-loop. Therefore, the degree of binding to THRAP3 was confirmed using a methylation inhibitor (g) and DDX5 in which the methylated arginie residue was substituted with lysine (h, i). As a result, methylation of DDX5 also mediates binding to THRAP3, and when methylation is hindered, the binding This is the result of confirming this decrease.
Figure 4 is a result of confirming that DDX5 interacts with the N-terminus of THRAP3. When the THRAP3 protein was expressed in cells and the association with DDX5 was confirmed using immunoprecipitation, the binding to the N-terminal region of THRAP3 was confirmed. This is the result of checking
5 is a result of confirming XRN2 as a sub-target of THRAP3 for R-loop degradation. In order to find a direct sub-factor that causes an increase in R-loop formation due to a deficiency of THRAP3, XRN2, whose binding was confirmed in FIG. This is the result of confirming that access to the R-loop is reduced when the phenomenon is confirmed in a state where THRAP3 is deficient.
Figure 6 is a result of confirming the effect of THRAP3 to degrade R-loop in breast cancer cells. Figure 6a shows that siRNA transfection of THRAP3 was performed to confirm the effect of R-loop degradation through THRAP3 in MCF7 cells, a breast cancer cell line. It is a result confirming that DNA replication is reduced in knockdown cells, and FIG. 6b confirms that R-loop is increased in THRAP3-deficient cells, thereby inducing DNA double-strand damage by increasing γH2AX and 53BP1 fluorescence staining In addition, it was confirmed that cell proliferation was reduced in MCF7 cells selected in a THAP3-deficient state, and cell proliferation was restored by overexpression of RNaseH1, which relieves the R-loop. This is the result of confirming that the control of the loop has an effect on regulating the proliferation of cancer cells.
Figure 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 complex with methylated DDX5 and induces the recruitment of XRN2 to cut RNA, thereby inducing R-loop indicates that it plays a role in relieving

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

In the present invention, THRAP3 and methylated DDX5 are combined in the cell's R-loop (DNA:RNA hybrid), and through this binding, XRN2, an exonuclease, is mobilized to degrade the RNA of the R-loop. As it was confirmed that DNA damage in cells is inhibited by suppressing the accumulation of -loop, a method for screening candidates that can induce apoptosis by regulating the combination of THRAP3 and DDX5 in cancer cells as a cancer treatment method is provided. .

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

Checking the binding levels of THRAP3 and DDX5 in cancer cells in contact with the test substance; and selecting a test substance having a reduced binding level of THRAP3 and DDX5 compared to a control sample.

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

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

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

The reduced binding level of THRAP3 and DDX5 may 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, which includes reducing the binding levels of THRAP3 and DDX5 in cancer cells in vitro.

The THRAP3 and DDX5 binding levels may be confirmed by immunoprecipitation and immunoblotting, but are not limited thereto.

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

The term "test substance" used while referring to the screening method of the present invention is used to examine whether or not it affects the expression level of a gene, affects the expression or activity of a protein, or affects the binding between proteins. It means an unknown candidate substance used in screening. The samples include, but are not limited to, chemical substances, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural product extracts.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but 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 skilled 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 American Type Culture Collection (ATCC) and cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum (FBS). maintained in a CO ₂ incubator.

To confirm the proliferative ability of MDA-MB-231, 10 ⁵ cells were aliquoted and counted every day. THRAP3 knockdown MDA-MB-231 cells were prepared through lentiviral shRNA transfection. For lentivice 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 and infected with MDA-MB-231, and after infection, the cells were selected with 2 μg/mL of puromycin (VWR Chemicals).

2. Plasmid and siRNA transfection

RNaseH1-V5, Flag/Myc-DDX5, HA-THRAP3 plasmids and 20 nM of the following siRNA were transfected with Lipofectamine 2000 and RNAiMAX transfection reagent (Invitrogen), respectively, according to the transfection procedure described by the manufacturer.

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) shRNAs

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 Assays

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 and 4°C for 15 minutes. The concentration of the removed cell protein extract was confirmed using the Pierce BCA Protein Assay Kit.

Equal amounts of protein were loaded on SDS-PAGE and transferred to a nitrocellulose membrane (GE healthcare). 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 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 to precipitate.

4. Immunofluorescence analysis

U2OS cells were seeded on chamber slides. After 1 day of division, the cells were washed twice, extracted with 0.5% Triton X-100 on ice for 5 minutes, and then 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, the step was changed to extraction with 0.5% Triton X-100 for 2 minutes and fixation 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 fluorescence microscopy images of cells washed with PBS were obtained.

5. Proximity Ligation Assay

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

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

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 sample was incubated with a 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, they were placed on slides with DAPI and subjected to confocal microscopy analysis using a 63× oil immersion objective lens, and the number of nuclear PLA foci in the obtained images was quantified.

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

Cells were incubated on ice in lysis buffer containing 85 mM KCl, 5 mM PIPES (pH8.0) and 0.5% NP-40 for 10 minutes. The lysed cells were collected and centrifuged at 4° C. and 3000 g for 5 minutes.

Immediately prior to use, the nuclei pellet was prepared in RSB buffer (10 mM Tris-HCl (pH7.5), 200 mM NaCl, 2.5 mM RSB buffer) supplemented with 0.2% deoxycholate salts, 0.1% SDS and 0.5% Triton X-100 and protease inhibitor cocktail. MgCl ₂ ).

After sonicating the nuclei 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 assay

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

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

For nuclei visualization, DAPI staining was performed for 15 minutes. EdU incorporation was confirmed by fluorescence microscopy and EdU positive cells and fluorescence intensity were analyzed.

8. Purification and Mass Spectrometry

HEK293 cells were ectopically 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).

Eluted proteins were separated by SDS-PAGE and molecular features were analyzed by performing 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 related to DNA damage response through involvement in 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, colocalization was confirmed in the nuclear region, as shown in Fig. 1A, and ligation signal was reduced due to THRAP3 deficiency using siRNA.

In addition, as a result of 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 confirmed 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 and regulates the R-loop.

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

In the previous experiment, it was confirmed that R-loop was accumulated by THRAP3 reduction, and PCNA increased at the replication fork in the TRC (Transcription replication collision) region, and TRC-induced R-loop suppressed PCNA accumulation. When THRAP3 was deficient, PCNA levels in the R-loop strand were checked. Referring to the PLA results of Figure 2A, it was confirmed that PCNA significantly increased in the R-loop.

In addition, as it has been reported that the unseparated R-loop induces DNA damage by promoting DNA strand scission, EdU hybridization to U2OS cells was confirmed under control and THRAP3 knockdown conditions to evaluate DNA replication.

As a result, as shown in FIGS. 2B and 2C, the reduced 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 to DDX5 and XRN2

To understand the mechanism by which THRAP3 regulates R-loop level and genome stability, mass spectrometry was performed to identify proteins interacting with THRAP3.

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

As a result, DDX5 was co-immunoprecipitated with THRAP3 in the lysed cells incubated with the THRAP3 antibody and the 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 binds to THRAP3 endogenously.

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

On the other hand, when cells are stimulated with camptothecin (CPT), a topoisomerase known to increase R-loop formation, 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 bound very strongly to THRAP3 30 minutes after CPT treatment, and XRN2 followed about 3 hours after treatment.

Next, it was confirmed whether THRAP3 affects the association between DDX5 and XRN2. THRAP3 siRNA and control scramble 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, to confirm whether the binding of THRAP3 and DDX5 is changed by methylation change of DDX5, 20 μM of EPZ015666 (PRMT5 inhibitor, Sigma Aldrich, Cat# 1421) was applied to U2OS cells for 72 hours to inhibit methylation induced by PRMT5. 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 changed to lysine residues.

As a result, it was confirmed that the interaction between the RK mutant DDX5 and THRAP3 was reduced under 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 identify which domains of THRAP3 are essential for DDX5 interaction, Cells were co-transfected with Flag/Myc tagged DDX5 and 5 types of HA-tagged THRAP3 domain truncation mutants, as shown in Figure 4A, and immunoprecipitated with Flag antibody. was performed.

As a result, as shown in FIG. 4B, ΔN and ΔNC THRAP3 failed to 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> Confirmation of the role of XRN2 in R-loop resolution

In previous experiments, interactions between THRAP3 and R-loop degradation proteins DDX5 and XRN2 were confirmed, but the basic mechanism for 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 did not change DDX5. In addition, a significantly low PLA signal between S9.6 and XRN2 was confirmed in THRAP3 knockout cells as shown in Fig. 5C.

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

<Example 6> Confirmation of the effect of THRAP3 on 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 Figures 6C and 6D, THRAP3 knockdown in MCF7 cells induced R-loop accumulation in the nucleus, and as shown in Figures 6E and 6F, THRAP3-mediated R-loop accumulation induced γH2AX and 53BP1 localization in the nucleus, , R-loops degradation through RNaseH1 overexpression mitigated DNA double-strand breaks by THRAP3 knockdown.

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

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, 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 a test substance to cancer cells isolated from humans;
Checking the binding levels of THRAP3 and DDX5 in cancer cells in contact with the test substance; and
A cancer therapeutic screening method comprising the step of selecting a test substance having a reduced binding level of the THRAP3 and DDX5 compared to a control sample. The method according to claim 1, wherein the cancer drug screening method 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 the R-loop (DNA:RNA hybrid) and recruits an exonuclease to degrade the R-loop. The method of claim 1, wherein arginine of DDX5 is methylated by PRMT5, and the methylated DDX5 binds to the N-terminus of THRAP3. The method according to claim 1, wherein the reduced 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 of claim 1, wherein the cancer is 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. A method of inducing DNA damage in cancer cells comprising reducing the binding levels of THRAP3 and DDX5 in cancer cells in vitro.

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