KR20200030294A - Composition for preventing or treating lapatinib resistant cancer - Google Patents

Abstract

The present invention relates to: a composition for enhancing the sensitivity of lapatinib resistant cancer cells to lapatinib, which includes an agent for inhibiting the expression or activity of PAK2 (p21-activated kinase 2) protein or genes encoding the protein; an anticancer adjuvant composition; a pharmaceutical composition for preventing or treating lapatinib resistant cancer, which includes lapatinib and the agent; and a method for preventing or treating lapatinib resistant cancer, which includes a step of administering the composition to an individual other than human. The composition of the present invention enhances sensitivity to lapatinib of cancer cells having resistance to lapatinib which is a target treatment agent for a human epidermal growth factor receptor type2 (HER-2) by inhibiting the expression or activity of PAK2 protein or genes encoding the protein and thus, can be used to prevent or treat lapatinib resistant cancer.

Description

Composition for preventing or treating lapatinib resistant cancer}

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

In Korea, breast cancer occurred in about 38.9 people per 100,000 people in 2008, but as of 2012, the number of breast cancer surged to about 52.1 people per 100,000 people. The increasing trend has attracted attention as it occupies. In particular, the annual incidence of breast cancer has increased by more than 4.5 times over the 15 years from 1996 to 2011, which is the number one in East Asia. Compared to the West, the majority of postmenopausal patients, in Korea, the incidence of relatively young patients in their 40s is higher, and the average age is 51 years, about 15 years younger than those in the West, so breast cancer may not be a major social and economic burden. none. However, breast cancer is clinically and pathologically complex and shows various aspects, and the mechanism of its occurrence is still unclear, and the presumed risk factors include genetic factors, female hormones such as estrogen, age and childbirth, etc. This has been pointed out.

Breast cancer is a mass of cancer cells formed in the breast, and specifically, refers to a malignant tumor that can spread out of the breast and become a life threat unlike a benign tumor that stays in the breast. Depending on the site of occurrence, it can be divided into cancers that occur in parenchymal tissues such as the ducts and lobules, and cancers that occur in other interstitial tissues. Is divided into Recently, with the development of various targeted therapeutics that have an effect on the treatment of breast cancer, breast cancer has gradually been classified according to the presence or absence of the target. The target of breast cancer treatment, which is well known to date, is the overexpression of hormone receptors and HER2, and the treatment of these targets has improved the prognosis of breast cancer (J Korean Surg Soc 2010; 79: 14-19).

On the other hand, the overexpression of epithelial cell growth factor receptor (HER2) is known to have a poor prognosis compared to normal breast cancer without overexpression. These HER2-positive breast cancers account for about 25% of all breast cancer patients, and over-expression of HER2 promotes cell survival, proliferation, angiogenesis, invasion, and metastasis, showing aggressive biological properties. Do. However, in the case of HER2-positive breast cancer, it is known that a large number of patients relapse despite surgery after treatment with additional anti-cancer drugs, radiation therapy, and targeted therapeutic agent, trastuzumab. In addition, after relapse, the effect of re-treatment of trastuzumab and lapatinib treatment to overcome trastuzumab resistance was also confirmed in several months or only in some patients (Breast Cancer Res Treat 2008), or resistance to lapatinib It is difficult to treat such as indicating.

Under this background, the present inventors studied the resistance mechanism of target therapeutics for HER2-positive breast cancer and exerted their efforts to discover new therapeutic and diagnostic targets. As a result, they compared resistance expression by comparing the expression of proteins in the lapatinib-resistant HER2 positive breast cancer cell line The new target protein PAK2 involved in was first discovered. Thus, the sensitivity of lapatinib-resistant cancer cells to lapatinib is increased upon treatment of a PAK2 protein or an agent that inhibits the expression or activity of the gene encoding the protein, and when combined with lapatinib, it exhibits a significant anticancer effect against lapatinib-resistant cancer. Confirmed to complete the present invention.

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

Another object of the present invention is to provide an anti-cancer adjuvant composition comprising the agent.

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

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 a human.

Specifically, it is 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 the various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not considered to be limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object is Lapatinib (Lapatinib); And it provides a pharmaceutical composition for the prevention or treatment of lapatinib-resistant cancer, including a PAK2 (p21-activated kinase 2) protein or an agent that inhibits the expression or activity of the 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 targets through phosphorylated protein analysis of lapatinib sensitive and lapatinib resistant cell lines. It is based on the first excavation. Accordingly, the inventors of the present invention surprisingly improve the sensitivity of lapatinib-resistant cancer cells to lapatinib when treating the PAK2 protein or an agent that inhibits the expression or activity of the gene encoding the same, and when the agent is used in combination with lapatinib, It was first confirmed that the effect of prevention or treatment is remarkably excellent, and its significance is very large.

The term “Lapatinib” of the present invention is called Lapatinib ditosylate, GW572016 and the like, and is a targeted anti-cancer treatment and signal transduction inhibitor, and is classified as a tyrosine kinase inhibitor, HER2 and EGFR inhibitor. Lapatinib is mainly used for the treatment of HER-2 positive advanced or metastasized breast cancer, and the trade 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 families. PAK2-mediated signaling is known to be involved in the development and progression of cancer, including apoptosis, lumen formation in the endothelium, 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 a PAK2 protein or a gene-derived transcript encoding a PAK2 protein. Specifically, the agent is a transcription factor that binds to the gene and inhibits its expression at the transcription level; RNA, such as miRNA, siRNA, shRNA, that binds to the resulting transcript and degrades the transcript; Antibodies, aptamers, antagonists, or compounds capable of binding 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 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, which 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 suppress the expression and activity of the gene encoding the PAK2 protein, the siRNA is SEQ ID NO: 2 (5'-GGAACUGAUCAUUAACGAGAUUCTG-3 ') or SEQ ID NO: 3 (3'-UUCCUUGACUAGUAAUUGCUCUAAGAC- 5 ').

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 conventional methods known in the art. The form of the antibody is not particularly limited, as long as it has a polyclonal antibody, a monoclonal antibody, and antigen binding, a part thereof is included in the antibody of the present invention, and all immunoglobulin antibodies as well as humanized antibodies are specialized. Antibodies. In addition, the antibody includes a complete form with two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody. The functional fragment of the antibody means at least an antigen-binding fragment, and may be Fab, F (ab '), F (ab') 2, and Fv. For the purposes of the present invention, the antibody can be included without limitation, as long as it can inhibit its activity by binding to PAK2 protein.

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

The term "antagonist (antagonist)" of the present invention refers to a substance capable of directly or indirectly reducing the biological activity of a receptor, and when used with a ligand of a receptor, includes a molecule capable of reducing the action of the ligand However, it is not limited thereto. For the purpose of the present invention, the antagonist can be included without limitation, as long as it can inhibit its activity by binding to the PAK2 protein.

In the present invention, the agent may be a compound that inhibits the expression or activity of the PAK2 protein, specifically, the compound may be FRAX1036, FRAX486 or FRAX597, and more specifically FRAX597, but inhibits the expression or activity of the PAK2 protein It can be included without limitation as long as. FRAX1036, FRAX486 and FRAX597 can be represented by the following formulas 1, 2 and 3, respectively.

[Formula 1]

[Formula 2]

[Formula 3]

The term “resistant cancer” of the present invention is a breast cancer treatment targeting HER-2, more specifically, exhibits low sensitivity to lapatinib, thereby improving, alleviating, alleviating and alleviating the symptoms of cancer by administration of lapatinib. It refers to a cancer that is not shown, and may be used interchangeably with “lapatinib resistant cancer” in the present specification. The resistant cancer may be resistant before lapatinib administration, or may not initially exhibit resistance, but may no longer exhibit susceptibility to the same level of lapatinib administration due to mutations in the gene in cancer cells due to long-term lapatinib administration, It is not limited to this. 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 despite the lapatinib treatment of 500 nM, the cell activity was hardly reduced.

 The term "breast cancer" of the present invention refers to a mass composed of cancer cells formed in the breast, and is divided into various classifications according to the occurrence site and the degree of infiltration. Specifically, "HER-2 positive breast cancer" refers to the overexpression of HER-2 receptors on the surface of cancer cells, accounting for about 25% of all breast cancer patients. HER-2 positive breast cancer is known to have an aggressive and poor prognosis due to the promotion of cell survival, proliferation, angiogenesis, invasion and metastasis due to overexpression of HER2, and treatment with targeted therapies targeting only HER-2 receptors is recommended. However, there are problems of resistance and recurrence.

The pharmaceutical composition for preventing or treating lapatinib-resistant cancer of the present invention may be to enhance the sensitivity of lapatinib-resistant cancer cells to lapatinib. Specifically, the "enhancement of sensitivity" in cancer cells with low sensitivity to lapatinib means all phenomena such as cell activity of the cancer cells is reduced or apoptosis occurs according to the expression and activity inhibition of PAK2 protein. .

In one embodiment of the present invention, in order to confirm whether the sensitivity to lapatinib increases due to PAK2 knockdown in lapatinib-resistant cancer cells (SKBR3-LR), knock down PAK2 of SKBR3-LR cells and measure cell activity according to lapatinib treatment As a result, it was confirmed that the cell activity according to lapatinib treatment was significantly reduced in SKBR3-LR cells that knocked down PAK2 compared to cells that did not knock down PAK2 (control) (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 composition is formulated in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, and sterile injectable solution, respectively, according to a conventional method. You can. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used in the manufacture of pharmaceutical compositions. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, and the like. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, may be included. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. In addition, as non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The content of the carrier, excipient or diluent included in the pharmaceutical composition of the present invention is not particularly limited, 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 ratio of rational benefit / risk applicable to medical treatment or prevention, and the effective dose level is the severity of the disease, the activity of the drug, the age of the patient , Weight, health, gender, patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and rate of excretion, the duration of treatment, the elements including the drug used in combination with or concurrently with the composition of the present invention and other medicine It can be determined by factors well known in the art. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and 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 age, weight, sex, history of the patient, 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 one day, and the frequency of administration of the pharmaceutical composition of the present invention is not particularly limited, It can be administered once to three times a day or several times in divided doses.

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

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

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

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

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

The term “individual” of the present invention is an individual who may or may have developed the lapatinib-resistant cancer, and may specifically mean all animals except humans.

The term “administration” of the present invention means any act of introducing the composition of the present invention to 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 suppresses or delays the development or progression of lapatinib-resistant cancer by ingesting or administering the composition of the present invention.

The term “treatment” of the present invention means any act of ingesting or administering the composition of the present invention to improve or eliminate lapatinib resistant cancer and related symptoms.

The composition of the present invention enhances the sensitivity of cancer cells resistant to lapatinib, a targeted therapeutic agent for human epidermal growth factor receptor type2 (HER-2), to lapatinib by inhibiting the expression or activity of the PAK2 protein or the gene encoding the protein. And thus can be used for the purpose of preventing or treating lapatinib-resistant cancer.

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

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 lines

A SKBR3-LR cell line was constructed that obtained resistance to lapatinib by exposing lapatinib-sensitive cells SKBR3 to low-dose drugs (600 nM lapatinib) for about 6 months or more. As a result of measuring the sensitivity to lapatinib while increasing the concentration of lapatinib to 0 to 500 nM for the cells, as shown in FIG. 4 below, SKBR3 cells rapidly decreased in cell activity as the treatment concentration of lapatinib increased, It showed the lowest cell activity of about 20% from around 100 nM. On the other hand, in the case of SKBR3-LR cells, despite the 500 nM lapatinib treatment, the cell activity was hardly reduced, and thus, it was confirmed that SKBR3-LR cells acquired resistance to lapatinib treatment.

Experimental Example 2: Signaling system analysis of HER2 positive breast cancer cell line and Lapatinib resistant cell line through phosphorylated protein profiling

By comparing and analyzing the signaling system of the HER-2 positive breast cancer cell line and its lapatinib-resistant cell line through phosphorylated protein analysis, a mechanism related to lapatinib resistance of breast cancer cells is identified, from which target materials for diagnosis and treatment of lapatinib-resistant cancer I wanted to dig. To this end, the phosphorylated peptide (phosphopeptide) is separated from the cell lysate 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, and mass spectrometry. Based on the qualitative and quantitative analysis, the signaling system related to lapatinib resistance was confirmed.

Specifically, after the protein obtained from each cell cultured in SILAC medium is decomposed into peptides using trypsin, phosphorylation is performed 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. Then, based on the data analyzed by the mass spectrometer, the intracellular signaling system was analyzed using analysis programs such as MaxQuant and DAVID to identify the lapatinib resistance mechanism, and PAK2 was finally discovered as a candidate protein for treatment.

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

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

Experimental Example 4: Western blot for PAK2 knockdown confirmation

RIPA cell lysis buffer containing 1 mM Na ortho-vanadate, 2.5 mM sodium pyrophosphate, and 1 mM β-glycerophosphate was used to obtain cell lysates. SDS-PAGE gel electrophoresis was performed on 20 μg of cell lysate, and the proteins separated according to size were 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 for 1 hour with a primary antibody recognizing the phosphorylated site on serine 141 of PAK2 or PAK2, followed by treatment with HRP-coupled secondary antibody for 1 hour. Β-actin (abcam # Ab6728, Cambridge, UK) was used as the loading control. After the membrane was developed using ECL substrates (Bio-Rad), it was confirmed whether the PAK2 protein was knocked down 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

In order to check whether the sensitivity to lapatinib increases due to PAK2 knockdown in lapatinib resistant cells (SKBR3-LR), knock down PAK2 of SKBR3-LR cells by siRNA treatment of Experimental Example 3, and cell activity according to lapatinib treatment by concentration. It was measured.

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

Example 2: Lapatinib and PAK2 inhibitor combination treatment in lapatinib resistant cells (SKBR3-LR) 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 was treated in each well and cell activity was measured after 48 hours.

As a result, as can be seen from FIG. 6 below, when 1 μM of lapatinib was treated with SKBR3-LR cells, the cell activity was almost the same as that of the drug-free control group, and even when treated with 1 μM of PAK2 inhibitor (FRAX597). Although there was no significant change in activity, it was confirmed that when 1 μM lapatinib and 1 μM FRAX597 were used in combination, the cell activity of lapatinib resistant cells was significantly reduced. These results indicate that PAK2 is inhibited when lapatinib and PAK2 inhibitors are combined, and sensitivity of lapatinib-resistant cancer cells to lapatinib is enhanced, and that cancer cell killing effects by lapatinib are also significantly increased.

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

<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 cancer comprising an agent that inhibits the expression or activity of the gene encoding the protein.
The composition of claim 1, wherein the PAK2 protein consists of the amino acid sequence of SEQ ID NO: 1.
The composition of claim 1, wherein the agent is an antibody, aptamer, antagonist or compound that inhibits the expression or activity of PAK2 protein.
The composition of claim 3, wherein the compound is a compound represented by Formula 1 below.
[Formula 1]

The composition of claim 1, wherein the agent is a miRNA, siRNA, or shRNA that inhibits expression or activity of a gene encoding a PAK2 protein.
The composition of claim 5, wherein the siRNA is composed of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
The composition of claim 1, wherein the lapatinib resistant cancer is breast cancer.
The composition of claim 7, wherein the breast cancer is HER-2 positive breast cancer.
The composition of claim 1, wherein the composition enhances the sensitivity of lapatinib resistant cancer cells to lapatinib.
The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.
A composition for enhancing sensitivity of lapatinib-resistant cancer cells to lapatinib, comprising a PAK2 (p21-activated kinase 2) protein or an agent that inhibits the expression or activity of a gene encoding the protein.
An anticancer adjuvant composition for lapatinib resistant cancer, comprising a pK2 (p21-activated kinase 2) protein or an agent that inhibits the expression or activity of a gene encoding the protein.
A method for preventing or treating lapatinib-resistant cancer, comprising administering the composition of any one of claims 1 to 12 to an individual other than a human.
The method of claim 13, wherein the step of administering is characterized in that the lapatinib and PAK2 proteins or agents that inhibit the expression or activity of the gene encoding the protein are administered simultaneously, sequentially, or in reverse order.

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