KR102143974B1 - Composition for preventing or treating lapatinib resistant cancer - Google Patents

Abstract

The present invention is a composition for enhancing the sensitivity of lapatinib-resistant cancer cells to lapatinib, comprising an agent that inhibits the expression or activity of a PAK2 (p21-activated kinase 2) protein or a gene encoding the protein; Anticancer adjuvant composition; Lapatinib and a pharmaceutical composition for the prevention or treatment of lapatinib-resistant cancer comprising the formulation; And it relates to a method for preventing or treating lapatinib-resistant cancer comprising administering the composition to an individual other than a human.
The composition of the present invention enhances the sensitivity of cancer cells resistant to lapatinib, a human epidermal growth factor receptor type 2 (HER-2) target therapeutic agent, to lapatinib by inhibiting the expression or activity of the PAK2 protein or the gene encoding the protein. Therefore, it can be used for the purpose of preventing or treating lapatinib-resistant cancer.

Description

Composition for preventing or treating lapatinib resistant cancer {Composition for preventing or treating lapatinib resistant cancer}

The present invention is a composition for enhancing the sensitivity of lapatinib-resistant cancer cells to lapatinib, comprising an agent that inhibits the expression or activity of a PAK2 (p21-activated kinase 2) protein or a gene encoding the protein; Anticancer adjuvant composition; Lapatinib and a pharmaceutical composition for the prevention or treatment of lapatinib-resistant cancer comprising the formulation; And it relates to a method for preventing or treating lapatinib-resistant cancer comprising administering the composition to an individual other than a human.

In Korea, breast cancer occurred at about 38.9 per 100,000 people in 2008, but as of 2012, the incidence of breast cancer increased sharply to about 52.1 per 100,000 people, and is currently the second largest incidence of female cancer. The increasing trend is attracting attention enough to occupy. In particular, for 15 years from 1996 to 2011, the annual incidence of breast cancer increased by about 4.5 times, which is the number one incidence rate among East Asian countries. Compared to the West, where the majority of post-menopausal patients, in Korea, the incidence of patients in their 40s is higher, and the average age is 51, about 15 years younger than Western patients, so breast cancer is not a big social and economic burden. Can't. However, breast cancer is clinically and pathologically very complex and exhibits a variety of patterns, and the mechanism of its occurrence is still unknown, and representative risk factors that are assumed are genetic factors, female hormones such as estrogen, age and birth, etc. This has been pointed out.

Breast cancer is a mass made of cancer cells in the breast. Specifically, it refers to a malignant tumor that can spread outside the breast and become a life threat unlike a benign tumor that remains in the breast. Depending on the site of occurrence, it can be divided into cancer that occurs in parenchymal tissues such as ducts and lobules, and cancers that occur in other interstitial tissues.Cancers that occur in ducts and lobules are re-invasive breast cancer and non-invasive breast cancer depending on the extent to which cancer cells spread to surrounding tissue It is divided into. Recently, with the development of various targeted therapeutics that are effective in the treatment of breast cancer, breast cancer is gradually classified according to the expression of the therapeutic target. The targets of breast cancer treatment well known to date are the overexpression of hormone receptors and HER2, and treatment for these targets has improved the prognosis of breast cancer (J Korean Surg Soc 2010; 79:14-19).

On the other hand, overexpression of epithelial growth factor receptor (HER2) is known to have a poorer prognosis compared to general breast cancer without overexpression. These HER2-positive breast cancers account for about 25% of all breast cancer patients, and overexpression of HER2 promotes cell survival, proliferation, angiogenesis, invasion and metastasis, thereby exhibiting aggressive biological characteristics, requiring special management and treatment. Do. However, in the case of HER2-positive breast cancer, it is known that a number of recurrences occur despite additional anticancer treatment, radiation therapy, and trastuzumab treatment after surgery. In addition, re-treatment of trastzumab after recurrence and the effect of lapatinib treatment to overcome trastuzumab resistance is also only a few months, or the effect is confirmed only in some patients (Breast Cancer Res Treat 2008), or resistance to lapatinib. He is struggling with such treatment.

Under this background, the inventors of the present invention studied the resistance mechanism of the target therapeutic agent for HER2-positive breast cancer to discover new therapeutic and diagnostic targets. As a result, the present inventors compared the expression of the protein in the lapatinib-resistant HER2-positive breast cancer cell line to study the resistance mechanism The new target protein PAK2 involved in was discovered for the first time. Accordingly, the sensitivity of lapatinib-resistant cancer cells to lapatinib is increased when the PAK2 protein or an agent that inhibits the expression or activity of the gene encoding the protein is treated, and the combined treatment with lapatinib shows a remarkable anticancer effect against lapatinib-resistant cancer. By confirming the present invention was completed.

One object of the present invention is to provide a composition for enhancing the sensitivity of lapatinib-resistant cancer cells to lapatinib, comprising an agent that inhibits the expression or activity of a PAK2 (p21-activated kinase 2) protein or a gene encoding the protein. will be.

Another object of the present invention is to provide an anticancer adjuvant composition comprising the above formulation.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating lapatinib-resistant cancer comprising lapatinib and the above preparation.

Another object of the present invention relates to a method for preventing or treating lapatinib-resistant cancer comprising administering the composition to an individual other than humans.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the specific description described below.

One aspect of the present invention for achieving the above object is lapatinib (Lapatinib); And PAK2 (p21-activated kinase 2) provides a pharmaceutical composition for preventing or treating lapatinib-resistant cancer comprising an agent that inhibits the expression or activity of a protein or a gene encoding the protein.

The present invention is based on the results of research on the mechanism of resistance to breast cancer therapeutics targeting HER-2, such as lapatinib in the treatment of HER-2 positive breast cancer, and targets PAK2 protein through phosphorylated protein analysis of lapatinib sensitive cell lines and lapatinib resistant cell lines. Based on the first excavation. Accordingly, the inventors of the present invention surprisingly improve the sensitivity of lapatinib-resistant cancer cells to lapatinib when the agent that inhibits the expression or activity of the PAK2 protein or the gene encoding it is treated, and when the agent is treated in combination with lapatinib, breast cancer It was confirmed for the first time that the preventive or therapeutic effect was remarkably excellent, and its significance is very high.

The term "Lapatinib" of the present invention is called Lapatinib ditosylate, GW572016, etc., and is classified as a target anticancer therapeutic agent and a signal transduction inhibitor, a tyrosine kinase inhibitor, HER2 and EGFR inhibitor. Lapatinib is mainly used for the treatment of HER-2 positive advanced or metastatic breast cancer, and its brand name is known as Tykerb.

The term "PAK2 (p21-activated kinase 2)" of the present invention is a protein (enzyme) encoded by the PAK2 gene and belongs to one of the serine/threonine kinase group I PAK family. PAK2-mediated signaling is known to be involved in the onset and progression of cancers including apoptosis, endothelial lumen formation, breast cancer, liver cancer and gastric cancer. The PAK2 protein may be composed of the amino acid sequence of SEQ ID NO: 1.

The term “an agent that inhibits expression or activity” of the present invention includes all substances capable of inhibiting the production and expression of PAK2 protein or a gene-derived transcript encoding PAK2 protein. Specifically, the agent is a transcription factor that binds to a gene and inhibits its expression at a transcription level; RNA such as miRNA, siRNA, and shRNA that binds to the transcript generated by transcription and degrades the transcript; Antibodies, aptamers, antagonists, or compounds capable of binding to the expressed protein may be included, but are not limited thereto.

More specifically, the miRNA, siRNA, shRNA is about 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% of the phase with respect to a portion of the gene encoding the PAK2 protein. It may be composed of a sequence having homology, and the sequence portion having homology may be 21 nucleotides or more, 22 nucleotides or more, 23 nucleotides or more, 24 nucleotides or more, or 25 nucleotides or more, but is not particularly limited thereto.

More specifically, the term "siRNA (short interfering RNA)" of the present invention refers to a double-stranded RNA composed of 20 to 25 base pairs that inhibits the expression of a specific gene by lowering the activity of a specific mRNA after the transcription process. . For the purposes of the present invention, as long as it can inhibit the expression and activity of the gene encoding the PAK2 protein, the siRNA is not particularly limited, but the siRNA is SEQ ID NO: 2 (5'-GGAACUGAUCAUUAACGAGAUUCTG-3') or SEQ ID NO: 3 (3'-UUCCUUGACUAGUAAUUGCUCUAAGAC- 5′) may be composed of the nucleotide sequence.

The term "antibody" of the present invention refers to a substance capable of specifically binding to an antigenic site of a protein or peptide molecule, which can be prepared by a conventional method known in the art. The form of the antibody is not particularly limited, and if it has polyclonal antibody, monoclonal antibody, and antigen binding properties, some of them are also included in the antibody of the present invention, and not only all immunoglobulin antibodies, but also special antibodies such as humanized antibodies. It may include an antibody. In addition, the antibody includes not only a complete form having two full-length light chains and two full-length heavy chains, but also a functional fragment of the antibody. The functional fragment of the antibody refers to a fragment that has at least antigen binding, and may be Fab, F(ab'), F(ab')2, Fv, and the like. For the purposes of the present invention, the antibody may be included without limitation, as long as it binds to the PAK2 protein and can inhibit its activity.

The term “aptamer” of the present invention refers to a nucleic acid molecule having binding activity to a target substance, and may be RNA, DNA, and modified nucleic acid or a mixture thereof, and may be a linear or cyclic It may be in the form, but is not limited thereto. For the purposes of the present invention, the aptamer may be included without limitation, as long as it binds to the PAK2 protein and inhibits its activity.

The term “antagonist” of the present invention refers to a substance capable of directly or indirectly reducing the biological activity of a receptor, and includes molecules capable of reducing the action of the ligand when used together with a ligand of the receptor. However, it is not limited thereto. For the purposes of the present invention, the antagonist may be included without limitation, as long as it binds to the PAK2 protein and inhibits its activity.

In the present invention, the agent may be a compound that inhibits the expression or activity of PAK2 protein, specifically, the compound may be FRAX1036, FRAX486, or FRAX597, and more specifically, FRAX597, but inhibits the expression or activity of PAK2 protein. It is not limited thereto and may be included as long as possible. FRAX1036, FRAX486 and FRAX597 can be represented by the following Chemical Formulas 1, 2 and 3, respectively.

[Formula 1]

[Formula 2]

[Chemical Formula 3]

The term "resistant cancer" of the present invention exhibits a low sensitivity to breast cancer treatment targeting HER-2, more specifically lapatinib, so that the symptoms of cancer are improved, alleviated, alleviated and other treatment symptoms by administration of lapatinib. It means a cancer that does not appear, and may be used interchangeably with “lapatinib-resistant cancer” in the present specification. The resistant cancer may have resistance from before administration of lapatinib, or may not show resistance at first, but may no longer show susceptibility to administration of lapatinib at the same level due to mutations in the genes in cancer cells due to long-term administration of lapatinib, It is not limited thereto. In the present invention, the resistant cancer may be breast cancer, and more specifically, may be HER-2 positive breast cancer, but is not limited thereto.

In one embodiment of the present invention, as a result of measuring the sensitivity to lapatinib while increasing the concentration of lapatinib, the cell activity of lapatinib-sensitive cells (SKBR3) rapidly decreased as the treatment concentration of lapatinib increased, but lapatinib-resistant cells (SKBR3 In the case of -LR), it was confirmed that the cell activity was hardly decreased despite treatment with 500 nM of lapatinib.

The term "breast cancer" of the present invention refers to a mass made of cancer cells in the breast, and is classified into various classifications according to the site of occurrence and the degree of infiltration. Specifically, "HER-2 positive breast cancer" appears as the HER-2 receptor is overexpressed on the surface of cancer cells, accounting for about 25% of all breast cancer patients. HER-2-positive breast cancer is known to be aggressive and poor prognosis as HER2 overexpression promotes cell survival, proliferation, angiogenesis, invasion and metastasis, and treatment with targeted therapeutics that target only HER-2 receptors is recommended. However, there are problems of tolerance and recurrence.

The pharmaceutical composition for preventing or treating lapatinib-resistant cancer of the present invention may be one to enhance the sensitivity of lapatinib-resistant cancer cells to lapatinib. Specifically, the “enhancement of sensitivity” refers to all phenomena such as a decrease in cell activity or apoptosis in cancer cells having a low sensitivity to lapatinib, depending on the expression and inhibition of the PAK2 protein. .

In one embodiment of the present invention, in order to determine whether the sensitivity to lapatinib increases due to PAK2 knockdown in lapatinib-resistant cancer cells (SKBR3-LR), PAK2 of SKBR3-LR cells is knocked down and cell activity according to lapatinib treatment is measured. As a result, it was confirmed that the cell activity according to the lapatinib treatment was significantly reduced in SKBR3-LR cells in which PAK2 was knocked down compared to cells in which PAK2 was not knocked down (control group) (FIG. 5).

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, and the carrier may include a non-naturally occurring carrier. Specifically, the pharmaceutical compositions are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and other oral formulations, external preparations, suppositories, and sterile injectable solutions according to conventional methods. I can. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants commonly used in the manufacture of pharmaceutical compositions. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or lactose. lactose), gelatin, etc. can be mixed. Also, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, and the like.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. In addition, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used as the non-aqueous solvent and suspension. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The content of the carrier, excipient, or diluent included in the pharmaceutical composition of the present invention is not particularly limited thereto, but may be included in an amount of 0.0001 to 50% by weight, more preferably 0.01 to 10% by weight, based on the total weight of the final composition. However, it is not limited thereto.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention, and the effective dose level is the severity of the disease, the activity of the drug, and the age of the patient. , Weight, health, sex, sensitivity of the patient to the drug, the time of administration of the composition of the present invention used, the route of administration and the rate of excretion of the composition of the present invention, the duration of treatment, factors including drugs used in combination or concurrent with the composition of the present invention used, and other medicines It can be determined according to factors well known in the field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple.

The dosage of the pharmaceutical composition of the present invention can be determined by a person skilled in the art in consideration of the purpose of use, the degree of poisoning of the disease, the patient's age, weight, sex, history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered at 10 to 100 mg/kg, more preferably 10 to 30 mg/kg for a day, and the frequency of administration of the pharmaceutical composition of the present invention is not particularly limited thereto, It may be administered once to three times a day, or may be administered several times in divided doses.

Another aspect of the present invention for achieving the above object is a PAK2 (p21-activated kinase 2) protein or an agent that inhibits the expression or activity of a gene encoding the protein, for lapatinib-resistant cancer cells It provides a composition for enhancing sensitivity. The PAK2, an agent that inhibits expression or activity, lapatinib, resistant cancer, and sensitivity enhancement are as described above.

Another aspect of the present invention for achieving the above object is an anticancer adjuvant for lapatinib-resistant cancer, comprising an agent that inhibits the expression or activity of a PAK2 (p21-activated kinase 2) protein or a gene encoding the protein. The composition is provided. The PAK2, an agent that inhibits expression or activity, lapatinib, resistant cancer, and sensitivity enhancement are as described above.

The term "anticancer adjuvant" of the present invention is meant to include all substances capable of improving, improving or increasing the anticancer effect of an anticancer agent. For example, the substance itself does not exhibit anticancer activity, but when used together with an anticancer agent, an agent capable of improving, enhancing or enhancing the anticancer effect of the anticancer agent may be used as an anticancer adjuvant. Specifically, for the purposes of the present invention, the anticancer adjuvant may be to enhance the sensitivity of cancer cells to lapatinib when treated in combination with lapatinib for cancer cells resistant to lapatinib, and the cellular activity of lapatinib-resistant cancer cells according to the enhancement of sensitivity It may be to cause reduction and cell death, but is not limited thereto.

More specifically, the anticancer adjuvant includes an agent that inhibits the expression or activity of a PAK2 protein or a gene encoding the protein, and the agent may be FRAX1036, FRAX486 or FRAX597, and more specifically FRAX597. In addition, the agent may be an siRNA composed of the nucleotide sequence of SEQ ID NO: 2 or 3, but is not limited thereto.

In one embodiment of the present invention, there was little change in the cellular activity of lapatinib-resistant cells (SKBR3-LR) when treated with 1 μM of lapatinib or 1 μM of a PAK2 inhibitor (FRAX597), respectively, but 1 μM lapatinib and 1 μM FRAX597 were used. It was confirmed that the cell activity of SKBR3-LR significantly decreased when the combination treatment was performed. These results indicate that PAK2 is inhibited when lapatinib and a PAK2 inhibitor are used in combination, and the sensitivity of lapatinib-resistant cancer cells to lapatinib is enhanced, and the cancer cell killing effect by lapatinib is also significantly increased.

Another aspect of the present invention for achieving the above object provides a method for preventing or treating lapatinib-resistant cancer, comprising administering the composition of the present invention to an individual other than humans.

The term "subject" of the present invention may refer to an individual who is likely to develop or has developed the lapatinib-resistant cancer, and specifically, may refer to all animals except humans.

The term “administration” of the present invention refers to any act of introducing the composition of the present invention into an individual through an appropriate method, and the route is administered without limitation through various routes such as oral or parenteral as long as it can reach the target tissue. Can be.

The term "prevention" of the present invention means any action that inhibits or delays the occurrence or progression of lapatinib-resistant cancer by ingesting or administering the composition of the present invention.

The term "treatment" of the present invention refers to any action that improves or disappears lapatinib-resistant cancer and related symptoms by ingesting or administering the composition of the present invention.

The composition of the present invention enhances the sensitivity of cancer cells resistant to lapatinib, a human epidermal growth factor receptor type 2 (HER-2) target treatment, to lapatinib by inhibiting the expression or activity of the PAK2 protein or the gene encoding the protein. Therefore, it can be used for the purpose of preventing or treating lapatinib-resistant cancer.

1 shows the mechanism of resistance of HER-2 targeted anticancer therapeutics.
FIG. 2 is a flow chart illustrating an analysis of an intracellular signaling system through mass spectrometry-based phosphorylated protein analysis using a stable isotope labeling with amino acids in cell culture (SILAC) technique.
3 shows the results of confirming the change in the HER-2 signaling system in lapatinib-resistant cells (SKBR3-LR).
Figure 4 shows the measurement of the cellular activity of lapatinib-sensitive cells (SKBR3) and lapatinib-resistant cells (SKBR3-LR) according to the lapatinib treatment concentration.
Figure 5 shows the measurement of cell activity according to whether PAK2 knockdown in lapatinib-resistant cells (SKBR3-LR).
6 is a lapatinib, a PAK2 inhibitor (FRAX597); And it shows by measuring the cellular activity of lapatinib-resistant cells (SKBR3-LR) when treated in combination with lapatinib and FRAX597.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Experimental Example 1: Construction of lapatinib-resistant cancer cell line

Lapatinib-sensitive cells SKBR3 was exposed to a low concentration drug (600 nM lapatinib) for more than about 6 months to construct a SKBR3-LR cell line that acquired resistance to lapatinib. As a result of measuring the sensitivity to lapatinib while increasing the concentration of lapatinib to the cells from 0 to 500 nM, as can be seen from FIG. 4 below, the cell activity of SKBR3 cells rapidly decreased as the treatment concentration of lapatinib increased, It showed the lowest cell activity of about 20% from around 100nM. On the other hand, in the case of SKBR3-LR cells, despite the treatment of 500 nM lapatinib, the cell activity was almost not decreased. It was confirmed that SKBR3-LR cells acquired resistance to lapatinib treatment.

Experimental Example 2: Analysis of the signaling system of HER2-positive breast cancer cell lines and Lapatinib-resistant cell lines through phosphorylated proteomic profiling

By comparing and analyzing the signaling system of HER-2 positive breast cancer cell line and its lapatinib-resistant cell line through phosphorylated proteomic analysis, the mechanism related to lapatinib resistance of breast cancer cells was identified, and target substances for diagnosis and treatment of lapatinib-resistant cancer were identified from this. I wanted to excavate. To this end, phosphorylated peptides were isolated from cell lysates obtained by culturing HER2-positive cell line SKBR3 and lapatinib-resistant cell line SKBR3-LR in SILAC (stable isotope labeling with amino acids in cell culture) medium. Based on the qualitative and quantitative analysis, the signaling system related to lapatinib resistance was confirmed.

Specifically, proteins obtained from each cell cultured in SILAC medium were digested into peptides using trypsin, and then phosphorylated by High pH fractionation/TiO ₂ enrichment method to reduce sample complexity and detect more accurate and more phosphorylated peptides. The peptide was selectively concentrated and analyzed by mass spectrometry. Subsequently, based on the data analyzed by mass spectrometry, the intracellular signaling system was analyzed using analysis programs such as MaxQuant and DAVID to identify the mechanism of lapatinib resistance, and finally, PAK2 as a therapeutic candidate protein was discovered.

Experimental Example 3: PAK2 knockdown and cell activity measurement using siRNA

RNAiMAX (Thermo Fisher scientific) forward transfection method was used to suppress the expression of PAK2 at the gene level, which was identified as a target for diagnosis and treatment of lapatinib-resistant cancer. Specifically, cancer cells were cultured on a 60mm plate the day before the transfection (Day1), and 1.28 x 10 ⁶ cells/well corresponding to 40% of the 60mm plate were inoculated at the time of transfection (Day2). The siRNAs used were each having a nucleotide sequence of 5'-GGAACUGAUCAUUAACGAGAUUCTG-3' (SEQ ID NO: 2) and 3'-UUCCUUGACUAGUAAUUGCUCUAAGAC-5' (SEQ ID NO: 3), and 4 to 6 hours after transfection was completed. Replaced. On the next day of transfection (Day3), 5 x 10 ³ cells were inoculated into a 96 well plate, and 1% DMSO (control) and 1 μM of Lapatinib were respectively treated on the next day (Day4), and Celltiter-Glo (Promega) was treated on Day5. Cell activity was measured in each medium.

Experimental Example 4: Western blot (westernblot) to confirm PAK2 knockdown

To obtain the cell lysate, a RIPA cell lysis buffer containing 1 mM Na ortho-vanadate, 2.5 mM sodium pyrophosphate, and 1 mM β-glycerophosphate was used. SDS-PAGE gel electrophoresis was performed on 20 μg of the cell lysate, and the protein separated according to the size was transferred to a nitrocellulose membrane (Whattman), and then in TBST buffer containing 5% (w/v) BSA at room temperature. Blocked for 1 hour. The membrane was treated with a primary antibody that recognizes a site phosphorylated on serine 141 of PAK2 or PAK2 for 1 hour, and then treated with a secondary antibody bound with HRP for 1 hour. As a loading control, β-actin (abcam #Ab6728, Cambridge, UK) was used. After the membrane was developed using ECL substrates (Bio-Rad), the knockdown of PAK2 protein was confirmed using a Davinchi-Chemi™ imaging system (DAVINCH-K, Seoul, Korea).

Example 1: Confirmation of increased sensitivity to lapatinib in lapatinib-resistant cells (SKBR3-LR) according to PAK2 knockdown

To check whether the sensitivity to lapatinib increased due to PAK2 knockdown in lapatinib-resistant cells (SKBR3-LR), the siRNA treatment of Experimental Example 3 knocked down PAK2 of SKBR3-LR cells, and the cell activity according to the lapatinib treatment by concentration was determined. Was measured.

As a result, as can be seen from FIG. 5 below, it was confirmed that the cell activity according to lapatinib treatment was significantly reduced in SKBR3-LR cells that knocked down PAK2 compared to the cells (control) that did not knock down PAK2. It showed a tendency to decrease depending on the concentration. These results indicate that the knockdown of PAK2 has an effect of enhancing the sensitivity of SKBR3-LR cells to lapatinib.

Example 2: In lapatinib-resistant cells (SKBR3-LR) according to the combined treatment of lapatinib and PAK2 inhibitor Cell activity measurement

As in the procedure of Experimental Example 2, 5 x 10 ³ lapatinib-resistant cells (SKBR3-LR) were added to each well of a 96 well plate and cultured for 16 hours, followed by 1 μM lapatinib, 1 μM PAK2 inhibitor (FRAX597), and 1 μM lapatinib. And 1 μM FRAX597 were treated in each well, and cell activity was measured 48 hours later.

As a result, as can be seen from Figure 6 below, when lapatinib 1 μM was treated with SKBR3-LR cells, the cell activity was almost similar to that of the control untreated with the drug, and even when 1 μM of the PAK2 inhibitor (FRAX597) was treated, the cells Although there was no significant change in the activity, it was confirmed that the cellular activity of the lapatinib-resistant cells was significantly reduced when 1 μM lapatinib and 1 μM FRAX597 were co-treated. These results indicate that PAK2 is inhibited when lapatinib and PAK2 inhibitor are used in combination, and the sensitivity of lapatinib-resistant cancer cells to lapatinib is enhanced, and the effect of lapatinib-induced cancer cell death is also significantly increased.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY Korea University Research and Business Foundation <120> Composition for preventing or treating lapatinib resistant cancer <130> KPA180513-KR <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 524 <212> PRT <213> Homo sapiens <400> 1 Met Ser Asp Asn Gly Glu Leu Glu Asp Lys Pro Pro Ala Pro Pro Val 1 5 10 15 Arg Met Ser Ser Thr Ile Phe Ser Thr Gly Gly Lys Asp Pro Leu Ser 20 25 30 Ala Asn His Ser Leu Lys Pro Leu Pro Ser Val Pro Glu Glu Lys Lys 35 40 45 Pro Arg His Lys Ile Ile Ser Ile Phe Ser Gly Thr Glu Lys Gly Ser 50 55 60 Lys Lys Lys Glu Lys Glu Arg Pro Glu Ile Ser Pro Pro Ser Asp Phe 65 70 75 80 Glu His Thr Ile His Val Gly Phe Asp Ala Val Thr Gly Glu Phe Thr 85 90 95 Gly Met Pro Glu Gln Trp Ala Arg Leu Leu Gln Thr Ser Asn Ile Thr 100 105 110 Lys Leu Glu Gln Lys Lys Asn Pro Gln Ala Val Leu Asp Val Leu Lys 115 120 125 Phe Tyr Asp Ser Asn Thr Val Lys Gln Lys Tyr Leu Ser Phe Thr Pro 130 135 140 Pro Glu Lys Asp Gly Phe Pro Ser Gly Thr Pro Ala Leu Asn Ala Lys 145 150 155 160 Gly Thr Glu Ala Pro Ala Val Val Thr Glu Glu Glu Asp Asp Asp Glu 165 170 175 Glu Thr Ala Pro Pro Val Ile Ala Pro Arg Pro Asp His Thr Lys Ser 180 185 190 Ile Tyr Thr Arg Ser Val Ile Asp Pro Val Pro Ala Pro Val Gly Asp 195 200 205 Ser His Val Asp Gly Ala Ala Lys Ser Leu Asp Lys Gln Lys Lys Lys 210 215 220 Thr Lys Met Thr Asp Glu Glu Ile Met Glu Lys Leu Arg Thr Ile Val 225 230 235 240 Ser Ile Gly Asp Pro Lys Lys Lys Tyr Thr Arg Tyr Glu Lys Ile Gly 245 250 255 Gln Gly Ala Ser Gly Thr Val Phe Thr Ala Thr Asp Val Ala Leu Gly 260 265 270 Gln Glu Val Ala Ile Lys Gln Ile Asn Leu Gln Lys Gln Pro Lys Lys 275 280 285 Glu Leu Ile Ile Asn Glu Ile Leu Val Met Lys Glu Leu Lys Asn Pro 290 295 300 Asn Ile Val Asn Phe Leu Asp Ser Tyr Leu Val Gly Asp Glu Leu Phe 305 310 315 320 Val Val Met Glu Tyr Leu Ala Gly Gly Ser Leu Thr Asp Val Val Thr 325 330 335 Glu Thr Cys Met Asp Glu Ala Gln Ile Ala Ala Val Cys Arg Glu Cys 340 345 350 Leu Gln Ala Leu Glu Phe Leu His Ala Asn Gln Val Ile His Arg Asp 355 360 365 Ile Lys Ser Asp Asn Val Leu Leu Gly Met Glu Gly Ser Val Lys Leu 370 375 380 Thr Asp Phe Gly Phe Cys Ala Gln Ile Thr Pro Glu Gln Ser Lys Arg 385 390 395 400 Ser Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Val Thr 405 410 415 Arg Lys Ala Tyr Gly Pro Lys Val Asp Ile Trp Ser Leu Gly Ile Met 420 425 430 Ala Ile Glu Met Val Glu Gly Glu Pro Pro Tyr Leu Asn Glu Asn Pro 435 440 445 Leu Arg Ala Leu Tyr Leu Ile Ala Thr Asn Gly Thr Pro Glu Leu Gln 450 455 460 Asn Pro Glu Lys Leu Ser Pro Ile Phe Arg Asp Phe Leu Asn Arg Cys 465 470 475 480 Leu Glu Met Asp Val Glu Lys Arg Gly Ser Ala Lys Glu Leu Leu Gln 485 490 495 His Pro Phe Leu Lys Leu Ala Lys Pro Leu Ser Ser Leu Thr Pro Leu 500 505 510 Ile Met Ala Ala Lys Glu Ala Met Lys Ser Asn Arg 515 520 <210> 2 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> PAK2_siRNA <400> 2 ggaacugauc auuaacgaga uuctg 25 <210> 3 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> PAK2_siRNA <400> 3 uuccuugacu aguaauugcu cuaagac 27

Claims (14)

Lapatinib; And PAK2 (p21-activated kinase 2) protein or a pharmaceutical composition for the prevention or treatment of lapatinib-resistant breast cancer comprising an agent that inhibits the expression or activity of the gene encoding the protein,
The agent is one or more selected from the group consisting of FRAX597, FRAX1036, FRAX486, siRNA consisting of the nucleotide sequence of SEQ ID NO: 2, and siRNA consisting of the nucleotide sequence of SEQ ID NO: 3,
FRAX597 is represented by Formula 1 below;
[Formula 1]

FRAX1036 is represented by Formula 2 below; And
[Formula 2]

FRAX486 is the following formula (3)
[Chemical Formula 3]

Which is a compound represented by, the composition.
The composition of claim 1, wherein the PAK2 protein consists of the amino acid sequence of SEQ ID NO: 1.
delete delete delete delete delete The composition of claim 1, wherein the breast cancer is a HER-2 positive breast cancer.
The composition of claim 1, wherein the composition enhances the sensitivity of lapatinib-resistant breast cancer cells to lapatinib.
The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
delete As an anticancer adjuvant composition for lapatinib-resistant breast cancer, comprising an agent that inhibits the expression or activity of a PAK2 (p21-activated kinase 2) protein or a gene encoding the protein,
The agent is one or more selected from the group consisting of FRAX597, FRAX1036, FRAX486, siRNA consisting of the nucleotide sequence of SEQ ID NO: 2, and siRNA consisting of the nucleotide sequence of SEQ ID NO: 3,
FRAX597 is represented by Formula 1 below;
[Formula 1]

FRAX1036 is represented by Formula 2 below; And
[Formula 2]

FRAX486 is the following formula (3)
[Chemical Formula 3]

Which is a compound represented by, the composition.
A method of preventing or treating lapatinib-resistant breast cancer, comprising administering the composition of any one of claims 1, 2, 8 to 10, and 12 to an individual other than a human.
The method of claim 13, wherein the administering step is characterized in that the lapatinib and PAK2 protein or an agent that inhibits the expression or activity of the gene encoding the protein are administered simultaneously, sequentially, or in reverse order.

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