KR20240000032A - Composition for preventing, improving or treating breast cancer comprising LRRK2 protein - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating breast cancer containing LRRK2 protein as an active ingredient. When using a composition containing LRRK2 protein as an active ingredient according to an embodiment of the present invention, the LRRK2 protein binds to Fbw7 protein, which plays an important role in decomposing Notch1 protein, thereby increasing the binding between Notch1 protein and Fbw7 protein, thereby increasing Notch1 By controlling protein degradation, Notch1-dependent breast cancer can be effectively prevented or treated.

Description

Composition for preventing, improving or treating breast cancer comprising LRRK2 protein {Composition for preventing, improving or treating breast cancer comprising LRRK2 protein}

The present invention relates to a composition for preventing, improving or treating breast cancer containing the LRRK2 protein.

Breast cancer is one of the cancer diseases that occurs most frequently in women worldwide. According to the World Health Organization, as of 2012, nearly 1.6 million new cases of breast cancer were reported, of which approximately 550,000 deaths were reported. Breast cancer treatment is carried out differently depending on the type of breast cancer. For example, selective estrogen receptor modulators (SERMs), which are drugs that inhibit estrogen receptor activation, are mainly used for estrogen receptor-positive breast cancer, and hormonal treatments such as tamoxifen are representative examples. However, hormone therapy such as tamoxifen has problems such as resistance and risk of developing endometrial cancer, and triple-negative breast cancer treatment has problems such as recurrence and poor prognosis, so the development of new breast cancer treatment continues to be required.

Meanwhile, the LRRK2 (leucin-rich repeat kinase-2) protein is a protein belonging to the leucin-rich repeat kinase family, and is implicated in the transmission of mild cognitive impairment related to Alzheimer's disease, L-dopa-induced dyskinesia, and neurons. It has been reported to be associated with CNS disorders related to progenitor differentiation. In addition, it has been reported that the G2019S variant of the LRRK2 protein is associated with an increase in the incidence of non-skin cancers such as kidney cancer, breast cancer, lung cancer, and prostate cancer, as well as acute myeloid leukemia.

The present inventors completed the present invention by confirming that LRRK2 protein stabilizes Fbw7 protein, which suppresses Notch1-dependent breast cancer tumor formation, and confirmed that LRRK2 protein can be usefully used in the treatment of breast cancer.

Republic of Korea Patent No. 10-2061142

One object of the present invention is to provide a pharmaceutical composition for preventing or treating breast cancer containing LRRK2 (leucine-rich repeat kinase 2) protein as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of breast cancer containing as an active ingredient a vector containing a polynucleotide encoding the LRRK2 protein, an LRRK2 overexpressing cell line transformed with the vector, or a culture medium thereof. .

Another object of the present invention is to provide a pharmaceutical composition for inhibiting metastasis of breast cancer containing LRRK2 protein as an active ingredient.

Another object of the present invention is a) treating breast cancer cells expressing LRRK2 protein with a test substance; b) measuring the expression level of LRRK2 protein in the cells of step a); and c) selecting a test substance in which the expression level of the LRRK2 protein in step b) is increased compared to a control group not treated with the test substance.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating breast cancer containing LRRK2 (leucine-rich repeat kinase 2) protein as an active ingredient.

As used herein, the term "LRRK2 (leucin-rich repeat kinase-2) protein" is a protein belonging to the leucin-rich repeat kinase family, and is composed of 2,527 amino acid sequences with high similarity between species. there is. The LRRK2 protein has the characteristic of having both guanosine triphosphatase (GTPase) and serine-threonine kinase activities in one protein. Expressed LRRK2 protein is observed in various organs and tissues, including the brain, and at the cellular level, it exists in the cytoplasm or cell membrane and outer mitochondrial membrane. In addition, the LRRK2 protein has five functionally important domains and regulates cell function through self-activation regulation through autophosphorylation, protein interaction, and enzyme action.

As used herein, the term “active ingredient” refers to an ingredient that can exhibit the desired activity alone or in combination with a carrier that is not active on its own.

As used herein, the term “breast cancer” refers to cancer that occurs in the milk ducts and lobules of the breast.

As used herein, the term “prevention” refers to all actions that suppress or delay the onset of breast cancer by administering the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which symptoms of breast cancer are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

According to one embodiment of the present invention, the LRRK2 protein promotes the degradation of Notch1 protein.

Regarding the Notch1 protein, the Notch signaling pathway is a genetically conserved signaling mechanism that regulates embryonic development and cell proliferation or death, and is important for maintaining adult homeostasis. It plays a role. Unlike other signal transduction pathways, the Notch signaling system does not transmit signals through secondary messengers or other proteins, but transmits signals through decomposition of receptors through proteolytic cleavage and movement of the degraded receptors. . The Notch signaling system is known to play a central role in regulating cellular homeostasis in conjunction with various signaling pathways in various tissues and organs. Mammals have Notch (Notch-1, -2, -3, -4), which acts as a receptor in the Notch signaling system, and its ligands, Jagged (Jagged-1, -2) and Delta (Delta-1, -3, It has -4). The regulation of the expression level of target genes due to Notch is controlled through the degradation of Notch ICD. In the generally known method of degradation of Notch ICD, CDK8 phosphorylates the PEST domain of Notch ICD, and ubiquitination of Fbw7 promotes degradation. There is a way. The LRRK2 protein binds to Fbw7 and regulates the activity of Fbw7, thereby promoting the degradation of Notch1 protein. Referring to Experimental Results 4 of the Example, LRRK2 inhibited the interaction between Fbw7 and Pin1, thereby reducing polyubiquitination of basal Fbw7. Therefore, a pharmaceutical composition containing LRRK2 protein as an active ingredient can be usefully used for the prevention or treatment of breast cancer.

Another aspect of the present invention is to provide a pharmaceutical composition for the prevention or treatment of breast cancer, comprising as an active ingredient a vector containing a polynucleotide encoding the LRRK2 protein, an LRRK2 overexpressing cell line transformed with the vector, or a culture medium thereof. .

According to one embodiment of the present invention, the vector may be any one selected from the group consisting of linear DNA, plasmid DNA, and recombinant viral vector.

According to one embodiment of the present invention, the cells may be any one selected from the group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells, and established tumor cells.

According to one embodiment of the present invention, the recombinant virus may be any one selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and lentivirus.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. In the above, 'pharmacologically acceptable' refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., and those for parenteral administration, such as water, suitable oils, saline solutions, aqueous glucose and glycols, etc. Carriers, etc., and may additionally contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.

The pharmaceutical composition according to the present invention can be formulated in a suitable form according to methods known in the art along with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various forms for parenteral or oral administration according to known methods. Representative formulations for parenteral administration include an isotonic aqueous solution or suspension, preferably an injectable formulation. Injectable formulations can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be dissolved in saline solution or buffer solution and formulated for injection. Additionally, dosage forms for oral administration include, but are not limited to, powders, granules, tablets, pills, and capsules.

The pharmaceutical composition formulated in the above manner can be administered in an effective amount through various routes including orally, transdermally, subcutaneously, intravenously, or intramuscularly. In the above, ‘effective amount’ refers to the amount that exhibits a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention can be appropriately selected depending on the administration route, administration target, age, gender, weight, individual differences, and disease state.

Another aspect of the present invention provides a pharmaceutical composition for inhibiting metastasis of breast cancer comprising LRRK2 protein as an active ingredient.

Here, explanations that are common to the above-mentioned content are omitted to avoid excessive complexity.

Referring to Experimental Results 5 of Example 5 and FIGS. 5A and 5B, LRRK2 protein mediates Fbw7 to inhibit the migration of cancer cells. Therefore, a composition containing LRRK2 protein as an active ingredient can be used to inhibit metastasis of breast cancer.

According to one embodiment of the present invention, the LRRK2 protein promotes the degradation of Notch1 protein.

Here, explanations that are common to the above-mentioned content are omitted to avoid excessive complexity.

Another aspect of the present invention includes the steps of a) treating breast cancer cells expressing LRRK2 protein with a test substance; b) measuring the expression level of LRRK2 protein in the cells of step a); and c) selecting a test substance in which the expression level of the LRRK2 protein in step b) is increased compared to a control group not treated with the test substance.

The breast cancer cells in step a) consist of Hs578T, SUM159PT, 4T1, MCF7, BT474, ZR75B, MDA-MB-435, MDA-MB-468, MDA-MB-231, SK-BR-3, 67NR and 4T07 cells. It may be any one selected from the group, preferably MDA-MB-231 cells.

According to one embodiment of the present invention, the expression level of the protein in step b) is determined by Western blot, immunoprecipitation, dual luciferase reporter assay, and enzyme-linked immunosorbent assay (ELISA). and immunohistochemistry.

The measurement may be performed using an antibody. “Antibody” refers to a proteinaceous molecule that can specifically bind to the antigenic site of a protein or peptide molecule. Such an antibody can be used to express a gene according to a conventional method. A protein encoded by the marker gene is obtained by cloning into a vector, and the obtained protein can be produced by a conventional method. The form of the antibody is not particularly limited, and as long as it is a polyclonal antibody, a monoclonal antibody, or has antigen binding properties, a part of it is included in the antibody of the present invention, and not only all immunoglobulin antibodies can be included, but also humanized antibodies, etc. It may also contain special antibodies. Additionally, the antibodies include intact forms with two full-length light chains and two full-length heavy chains as well as functional fragments of the antibody molecule. A functional fragment of an antibody molecule refers to a fragment that possesses at least an antigen-binding function and may include Fab, F(ab'), F(ab') 2, and Fv.

When using a composition containing LRRK2 protein as an active ingredient according to an embodiment of the present invention, the LRRK2 protein binds to Fbw7 protein, which plays an important role in decomposing Notch1 protein, thereby increasing the binding between Notch1 protein and Fbw7 protein, thereby increasing Notch1 By controlling protein degradation, Notch1-dependent breast cancer can be effectively prevented or treated.

Figures 1A to 1C show the results of integrated analysis of mRNA expression of clinical data published by the TCGA database to identify the relationship between LRRK2 expression and Notch signaling pathway target genes.
Figures 2A-2C show the results of luciferase reporter assay of the transcriptional activity of Notch1-IC in transfected HEK293T cells. 2A is the result of transfection with 4xCSL-Luc, 2B is the result of transfection with Hes1-Luc, and 2C is the result of transfection with Hes5-Luc.
Figure 2D shows the results of luciferase reporter assay of the transcriptional activity of Notch1-IC in the presence or absence of shLRRK2 in HEK293T cells.
Figure 2E shows the results of luciferase reporter analysis of the transcriptional activity of Notch1-IC after HEK293T cells were treated with IN-1 for 12 hours.
Figure 2F shows the results of a luciferase reporter assay of the transcriptional activity of Notch1-IC in HEK293T cells transfected with Nothch1-IC, LRRK2 WT, or pathogenic variants of LRRK2.
Figures 2G and 2H show the results of confirming the binding of Notch-IC and LRRK2.
Figure 2G shows the results of co-immunoprecipitation analysis with anti-Myc along with anti-Flag in transiently transfected HEK293 cells.
Figure 2H shows the results of analyzing the simultaneous endogenous co-immunoprecipitation of Notch1 and LRRK2 in HEK293T cells.
Figure 3A shows immunoblot results of Notch1-IC, Hes1, Hes5, and LRRK2 in HEK239T cells transfected with different doses of LRRK2.
Figure 3B shows the results of measuring the level of Notch1-IC by immunoblot after treatment with 100 mmol/L cycloheximide in MDA-MB 231 cells transfected with shCon and LRRK2.
Figure 3C shows the results of measuring the levels of Notch1-IC and LRRK2 by immunoblot after transiently transfected HEK293T cells were treated with 20 μM ALLN, 5 μM MG132, and 5 μM Epoxomicin for 6 hours.
Figure 3D shows the results of luciferase reporter analysis of Notch1-IC transcriptional activity in transiently transfected HEK293T cells.
Figure 4A shows the results of analysis of co-immunoprecipitation of anti-Flag and anti-HA from transiently transfected HEK293 cells.
Figure 4B is an immunoblot result of Fbw7, Pin1, and LRRK2 in transiently transfected HEK293T cells.
Figures 4C and 4D show the results of transiently transfecting HEK293T cells, treating them with MG132 for 6 hours, and analyzing the ubiquitination of Fbw7 in vivo.
Figure 4E shows the results of co-immunoprecipitation analysis of anti-Xpress and anti-HA from transiently transfected HEK293 cells.
Figure 4F shows the results of transiently transfecting HEK293T cells, treating them with MG132 for 6 hours, and analyzing Pin1 ubiquitination in vivo.
Figure 5A is a photomicrograph of a wound repair assay in MDA-MB-231 cells expressing LRRK2 or shFbw7.
Figure 5B is a photomicrograph of Matrigel invasion assay of MDA-MB-231 cells expressing LRRK2 or shFbw7.
Figure 5C is the result of colony formation assay of MDA-MB-231 cells expressing LRRK2 or shFbw7.
Figure 5D shows the results of measuring the size of the tumor after injecting MDA-MB-231-SQ cells expressing LRRK2 or shFbw7 into mice.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.

Experimental method

1. Cell culture

HEK293 (human embryonic kidney 293) cells were grown in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin at 95% O ₂ . Cultured in a humidified incubator under 5% CO ₂ conditions. MDA-MB-231 cells were humidified under 95% CO ₂ conditions in RPMI (Rosewell Park Menorial Institute) medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cultured in an incubator. Lipofectamine 2000 (Invitrogen) was used for plasmid DNA transfection into cells.

2. Transfection of shRNA

pSUPER-derived expression vectors inserted with short hairpin RNA (hereinafter referred to as shRNA) targeting the Notch1 gene were constructed according to the method described in Mo, Yoon et al., PNAS (110), 2013. .

The shRNA sequence targeting Fbw7 is 5'-CCTTCTCTGGAGAGAGAAA-3' (SEQ ID NO: 1). The shRNA sequence targeting LRRK2 is 5′-CAATGTCAGGTGTTATAATAT-3′ (SEQ ID NO: 2). The shRNA sequence targeting Pin1 is 5'-CCTTCTCTGGAGAGAGAAA-3' (SEQ ID NO: 3). Sequence specificity was confirmed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) search, and a scrambled shRNA sequence (5'-AACAGTCGCGTTTGCGACTGG-3', sequence that did not match any known mammalian sequence) was identified. Number 4) was used to verify in GenBank. Control shRNA, Fbw7 shRNA, Pin1 shRNA, or LRRK2 shRNA were each transfected into cells using Lipofectamine 2000 according to the manufacturer's protocol.

3. Immunoblot analysis

에서 20분 동안 원심분리하여 수용성 분획을 분리하였다. 상기 수용성 분획에 램리 버퍼(Laemmli buffer)를 첨가하여 끓인 후, 황산 도데실 나트륨-폴리아크릴아미드 전기영동(sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 이하, SDS-PAGE로 기재함)을 수행하였다. 전기영동이 끝난 후 단백질을 PVDF 막(polyvinylidene difluoride membrane)으로 semi-dry transfer cell system을 이용하여 이동시켰다. PVDF 막을 0.1% 트윈-20 및 5% 탈지유(non-fat milk)를 포함하는 PBS(pH 7.4)로 블록킹하였다. 이후 PVDF 막을 1차 항체와 반응시키고, 항 마우스 HRP(horseradish peroxidase)가 연결된 2차 항체와 추가로 반응시켰다. 48 hours after transfection, cells were recovered and incubated in RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 Cells were lysed with mM dithiothreitol (DTT) and 2 μg/ml each of leupeptin and aprotinin. Cell lysates were weighed at 12,000xg, 4

The water-soluble fraction was separated by centrifugation for 20 minutes. Laemmli buffer was added to the water-soluble fraction and boiled, and then sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE) was performed. After electrophoresis was completed, the protein was transferred to a PVDF membrane (polyvinylidene difluoride membrane) using a semi-dry transfer cell system. The PVDF membrane was blocked with PBS (pH 7.4) containing 0.1% Tween-20 and 5% non-fat milk. Afterwards, the PVDF membrane was reacted with a primary antibody and further reacted with a secondary antibody linked to anti-mouse horseradish peroxidase (HRP).

The primary antibodies used were anti-Myc (9E10), anti-HA (12CA5), anti-Flag M2 (Sigma-Aldrich), anti-Hes1 (R&D system), anti-LRRK2 (Novus Biologicals), and anti-Hes5 (EMD). Millipore), anti-Notch1, anti-RBP-Jk, anti-Fbw7, and anti-β-actin (Santa Cruz Biotechnology) antibodies.

4. Luciferase reporter analysis

HEK293 cells were transfected with luciferase reporter plasmids (4xCSL-Luc, Hes1-Luc, Hes5-Luc, and β-galactosidase). After 48 hours, the cells were collected, lysed with chemiluminescence lysis buffer (1 MK ₂ HPO ₄ 18.3%, 1 M KH ₂ PO ₄ 1.7%, 1 mM PMSF, and 1 mM DTT), and measured with a luminometer. ; Berthold). The activity of the luciferase reporter gene was corrected by β-galactosidase activity.

5. Co-immunoprecipitation analysis

48 hours after transfection, cells were washed with cold PBS. Cells were lysed with RIPA buffer, and the cell lysate was centrifuged at 12,000xg at 4°C for 20 minutes. The supernatant was separated and reacted with the primary antibody, and then further reacted with protein A-agarose beads. After washing the beads three times with cold PBS, Laemrebuffer was added and boiled. The pellet was heated at 95°C for 5 minutes and SDS-PAGE was performed. Afterwards, immunoblot was performed in the same manner as in 3 above.

6. In vitro binding assay

Recombinant GST-Notch1-IC and its mutant proteins were expressed in E. coli BL21 strain using the pGEX system and isolated using glutathione (GSH)-agarose beads (Sigma) according to the manufacturer's instructions. and purified. Lysates of HEK293 cells transfected with different expression vectors and the same amount of GST or GST-Notch1-IC were reacted at 4°C for 2 hours. After the reaction was over, the beads were washed three times with cold PBS, then Laemmli buffer was added and boiled. Precipitates were separated by SDS-PAGE, and the pulled-down proteins were identified by immunoblotting in the same manner as in 3 above.

7. Immunofluorescence staining

Cells were fixed with 4% paraformaldehyde diluted in PBS, and cell permeability was restored with 0.1% Triton X-100 diluted in PBS. Afterwards, the cells were blocked with 1% bovine serum albumin (BSA). The primary antibody was diluted 1:100 and reacted. After reacting with the primary antibody, the cells were washed three times with PBS. Alexa Fluor® 488 or Alexa Fluor® 532 (Invitrogen) coupled with anti-mouse secondary antibody was diluted 1:100 for reaction. Nuclei were localized with DNA stain TO-PRO®-3 Iodide. The localization of stained cells was evaluated through confocal microscopy (Leica TCS SPE). Each image was an image of a single Z section of the same cell level. The final image was produced by analysis using a confocal microscope with LAS AF software (Leica).

8. Cell viability analysis

MCF-7 cells, which are breast cancer cells, were grown for 12 hours and treated with 1 μM DAPT (N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester). . After treatment with DAPT, the cells were trypsinized and resuspended in complete medium. A trypan blue solution was mixed with each sample. The number of stained (non-viable) cells and cells excluded from staining (viable) were counted with a hemocytometer. Controls were treated with the same final DMSO concentration in the same medium as the samples. The ratio of surviving cells, that is, the ratio of the number of blue-stained cells to the total number of cells, was determined by counting 100 cells per field three times.

9. Wound recovery analysis

Cells were grown to confluence in a 12-well plate, and the monolayer was wounded with a P200 micropipette tip. After measurement, DMEM supplemented with 10% FBS was immediately replaced. Wound healing images were taken at 6-hour intervals using a microscope system. The movement speed of cells was calculated by measuring the distance of the sketched area at different time points using the Image J program.

10. Colony formation and soft agar analysis

Colony formation analysis was performed by seeding 500 cells on a cell growth matrix consisting of lower agar and upper agarose in a 6-well culture plate. The lower layer (1.5 ml) contained DMEM medium, 10% FBS, and 0.5% agar, and the upper layer (1.5 ml) contained DMEM medium, 10% FBS, 0.35% agarose, and cell suspension (1 x 10 ³ ). Cells were supplied every 3 days, and the number of colonies was measured after 2 to 3 weeks.

11. Cell migration and invasion assay

Transwell migration analysis was performed using a transwell chamber (8 μm polycarbonate membrane, Corning). Matrigel invasion assay was performed with BD BioCoat Growth Factor. _After ^{dispensing 2.5} It was removed. Afterwards, the cells were fixed with 3.7% formaldehyde or 100% methanol, and then stained with DAPI to measure the number of cells on the bottom surface. The number of migrated cells was determined as the number of stained cells within multiple randomly selected microscope fields.

12. Xenograft mouse metastasis model

Female C57BL/6 mice (Orient Bio, Korea) were maintained in the animal laboratory of the National Cancer Center. shCon (control), the MDA-MB-231-SQ breast cancer cell line stably expressing specific shRNAs for LRRK2 and ^Fbw7 (1 It was injected subcutaneously in the lower flank. After 4 weeks, the mice were euthanized via anesthetic overdose. Tumor size was measured with a digital caliper, and tumor volume was calculated.

13. Statistical analysis

Graphs of the experimental results were created using SigmaPlot software (SYSTAT). Statistical comparison of means was performed using the unpaired Student's 2-tailed t-test for the two data sets. In all statistical tests, if the p value was less than 0.05, it was judged to be statistically significant. For all experimental results, the standard deviation (SD) was calculated, and variations in the experiment and related data were displayed as error bars. Data are expressed as mean ± mean deviation of values from three independent experiments.

Experiment result

1. Relationship between LRRK2 expression and breast cancer Notch signaling pathway

To identify the relationship between LRRK2 expression and Notch signaling pathway target genes, an integrated analysis of mRNA expression of clinical data published by The Cancer Genome Atlas (TCGA) database was performed. As a result, as shown in Figure 1A, a significant negative correlation was found between LRRK2 and Hes1 and Hes5, suggesting that LRRK2 expression is related to the activity of the Notch signaling pathway.

To determine the mRNA level of LRRK2 in tumor cells, we analyzed data published by the TCGA database. As a result, as shown in Figure 1B, LRRK2 was found to be downregulated in breast cancer cell lines.

To evaluate the potential impact of LRRK2 on patient survival, we analyzed LRRK2 variants in clinical data related to survival of cancer patients published by the TCGA database. Kaplan-Meier survival analysis showed that LRRK2 variants significantly reduced overall survival in breast cancer. As shown in Figure 1C, LRRK2 variants were associated with poor patient survival. Additionally, the mRNA level of LRRK2 was also correlated with cancer progression.

2. Regulation of Notch1 signaling pathway by LRRK2

We investigated whether the level of LRRK2 affects the transcriptional activity of Notch-IC.

As a result, as shown in Figures 2A to 2C, ectopic expression of LRRK2 decreased the transcriptional activity of Notch-IC in a dose-dependent manner. As shown in Figure 2D, Notch1-induced expression of the luciferase reporter gene was higher in LRRK2 knockdown cells than in control cells. Additionally, as shown in Figure 2E, the transcriptional activity of the Notch signaling pathway in HEK293 cells was increased by IN-1 and LRRK2 inhibitors in a dose-dependent manner. Overexpression of wild-type LRRK2 suppressed the transcriptional activity of Notch1-IC as well as the transcriptional activity of in vivo mutants of LRRK2. However, as shown in Figure 2F, LRRK2 1906M, a kinase dominant recessive mutant in somatic variants of PD (Parkinson's disease), did not inhibit the transcriptional activity of Notch1-IC.

Afterwards, the interaction ability of LRRK2 with Notch1-IC was analyzed. Next, Myc-Notch1-IC was expressed alone or simultaneously in the presence of Flag-GFP-LRRK2, and co-immunoprecipitation assays were performed. As a result, as shown in Figure 2G, it was confirmed that LRRK2 effectively co-precipitates with Notch1-IC. Additionally, Notch1-IC was confirmed to co-precipitate with endogenous LRRK2 in HEK293 cells. As shown in Figure 2H, Notch1-IC showed strong interaction with LRRK2.

These results clearly show a direct interaction between Notch1-IC and LRRK2, and it was confirmed that this interaction inhibits the Notch1 signaling pathway.

3. Promotion of Fbw7-mediated degradation of Notch1-IC by LRRK2

We assessed the physiological relevance of the effect of LRRK2 on Notch1-IC protein levels. As shown in Figure 3A, the levels of endogenous Notch1-IC, Hes1, and Hes5 proteins decreased in a dose-dependent manner as LRRK2 increased. Additionally, as shown in Figure 3B, the steady-state level and half-life of endogenous Notch1-IC were lower in LRRK2-overexpressing cells than in control cells.

To determine whether the decrease in protein levels was mediated by protein degradation, the results were confirmed in the presence or absence of proteasome inhibitors ALLN, MG132, or Epoxomicin. As shown in Figure 3C, proteasome inhibitor treatment significantly prevented LRRK2-mediated Notch1-IC protein degradation and stabilized protein levels. These results indicate that LRRK2 reduced the stability of Notch1-IC through a proteasome-dependent pathway.

To analyze the role of FBXW7 in LRRK2-mediated Notch1-IC degradation, LRRK2 and Notch1-IC were co-expressed in the presence or absence of LRRK2 siRNA. As shown in Figure 3D, knockdown of Fbw7 restored the transcriptional activity of Notch1-IC, which was reduced by LRRK2. The physical association between Fbw7 and Notch1-IC was significantly higher in the LRRK2-overexpressing group than in the control group. Additionally, the endogenous interaction between Notch1-IC and Fbw7 was significantly lower in LRRK2 knockdown cells than in MDA-MB 231 cells. These results indicate that Fbw7 plays an important role in the degradation of Notch1-IC mediated by LRRK2.

4. Analysis of the effect of LRRK2 on Fbw7 protein stability through inhibition of interaction between Pin1 and Fbw7

The present inventors investigated whether LRRK2 affects the regulatory action of Fbw7. As shown in Figure 4A, LRRK2 interacts with Fbw7. We analyzed whether LRRK2 kinase activity is essential to affect the regulatory action of Fbw7. A kinase active variant (LRRK2 G2019S) or a kinase dominant recessive variant (LRRK2 K1906M) was expressed in somatic variants of PD. As shown in Figure 4B, LRRK2 G2019S, unlike the kinase dominant recessive variant (LRRK2 K1906M), increased endogenous Fbw7 and decreased endogenous Pin1.

Additionally, the effect of LRRK2 on the ubiquitination of Fbw7 was examined. Flag-Fbw7 and HA-Ubiquitin were simultaneously expressed in the presence or absence of LRRK2 G2019S and LRRK2 K1906M in the presence of MG-132, and the ubiquitination status of Fbw7 was analyzed. As shown in Figure 4C, it was confirmed that polyubiquitination of Fbw7 was reduced in the presence of LRRK2 G2019S.

Additionally, in the presence of MG-132, we compared wild-type Fbw7 and Fbw7 T205A to examine the effect on basal levels of LRRK2-mediated polyubiquitination of Fbw7 by Pin1. As shown in Figure 4D, the level of LRRK2-mediated Fbw7 ubiquitination was significantly low when wild-type Fbw7 was expressed, but not when Fbw7 T205A was expressed. Additionally, LRRK2 suppressed the basal level of Notch1 transcriptional activity when wild-type Fbw7 was expressed, but not in Fbw7 T205A. Next, we investigated the effect of LRRK2 on the physical association between Fbw7 and Pin1. As a result, LRRK2 significantly inhibited the interaction between Fbw7 and Pin1. Additionally, we investigated the effect of Pin1 on LRRK2-mediated regulation of Fbw7. As shown in Figure 4E, LRRK2 was physically associated with Pin1. The effect of LRRK2 on Pin1 ubiquitination was investigated. Xpress-Pin1 and HA-Ubiquitin were simultaneously expressed in the presence of MG-132, and the ubiquitination status of Pin1 was analyzed in the presence or absence of LRRK2 G2019S and LRRK2 K1906M. As shown in Figure 4F, polyubiquitination of Pin1 increased in the presence of LRRK2 G2019S. Additionally, LRRK2 inhibited the basal level of Notch1 transcriptional activity more in the case of Pin1 knockdown than in the control group. In conclusion, these data suggest that LRRK2 inhibits the interaction between Fbw7 and Pin1, thereby reducing polyubiquitination of basal Fbw7.

5. Analysis of LRRK2 mediating Fbw7-dependent tumorigenesis inhibition

As shown in Figure 5A, cell migration experiments in MDA-MB-231 cells showed that overexpression of LRRK2 decreased cell migration of Fbw7 knockout cells, although the protein level of LRRK2 significantly affected cancer cell migration in the presence of Fbw7. was significantly increased, which means that LRRK2 mediates Fbw7 to inhibit the migration of cancer cells. Moreover, knockdown of LRRK2 and Fbw7 co-expressed in MDA-MB-231 cells inhibited LRRK2-induced cell invasion. As shown in Figure 5B, Fbw7 is essential for suppressing cancer invasion driven by LRRK2. Finally, as shown in Figure 5C, overexpressing LRRK2 in MDA-MB-231 cells significantly reduced colony forming activity, but the inhibition of colony forming activity induced by LRRK2 was reduced in Fbw7 knockdown MDA-MB-231 cells. It didn't work. Additionally, tumor growth was significantly suppressed in mice injected with LRRK2 overexpressing cells compared to mice injected with control cells. However, as shown in Figure 5D, no effect on tumor growth was observed in mice injected with cells co-expressing LRRK2 and shFbw7. These results indicate that LRRK2 inhibits tumor formation both in vitro and in vivo, and that LRRK2 can effectively prevent breast cancer tumor formation by inhibiting Notch1-IC through Fbw7. Taken together, these results suggest a novel role for LRRK2 in cancer cell migration/invasion through regulation of Fbw7 levels.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Composition for preventing, improving or treating breast cancer comprising LRRK2 protein <130> BPN221008 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Fbw7 shRNA <400> 1 ccttctctgg agagagaaa 19 <210> 2 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> LRRK2 shRNA <400> 2 caatgtcagg tgttataata t 21 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Pin1 shRNA <400> 3 ccttctctgg agagagaaa 19 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> scramble shRNA <400> 4 aacagtcgcg tttgcgactg g 21

Claims (10)

Containing LRRK2 (leucine-rich repeat kinase 2) protein as an active ingredient,
Pharmaceutical composition for preventing or treating breast cancer.
In claim 1,
The LRRK2 protein promotes the degradation of Notch1 protein,
Pharmaceutical composition for preventing or treating breast cancer.
Containing as an active ingredient a vector containing a polynucleotide encoding the LRRK2 protein, an LRRK2 overexpressing cell line transformed with the vector, or a culture medium thereof,
Pharmaceutical composition for preventing or treating breast cancer.
In claim 3,
The vector is any one selected from the group consisting of linear DNA, plasmid DNA, and recombinant viral vector,
Pharmaceutical composition for preventing or treating breast cancer.
In claim 3,
The cells are any one selected from the group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells, and established tumor cells,
Pharmaceutical composition for preventing or treating breast cancer.
In claim 4,
The recombinant virus is any one selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus and lentivirus,
Pharmaceutical composition for preventing or treating breast cancer.
A pharmaceutical composition for inhibiting metastasis of breast cancer comprising LRRK2 protein as an active ingredient.
In claim 7,
The LRRK2 protein promotes the degradation of Notch1 protein,
Pharmaceutical composition for inhibiting metastasis of breast cancer.
a) treating breast cancer cells expressing LRRK2 protein with a test substance;
b) measuring the expression level of LRRK2 protein in the cells of step a); and
c) comprising the step of selecting a test substance in which the expression level of the LRRK2 protein in step b) is increased compared to the control group that did not treat the test substance.
Screening method for breast cancer therapeutics or metastasis inhibitors.
In claim 9,
The expression level of the protein in step b) was determined by Western blot, immunoprecipitation, dual luciferase reporter assay, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. Measured by any one method selected from the group consisting of,
Screening method for breast cancer therapeutics or metastasis inhibitors.