KR101041308B1 - A composition for anticancer comprising appl1 inhibitor and a method for screening regulators of interaction between appl1 and egfr - Google Patents

Abstract

The present invention relates to an inhibitor of APPL1, and more particularly, to a method for screening a modulator for regulating the binding of APPL1 and EGFR, and a composition for preventing and treating cancer comprising a substance that inhibits the expression or activity of APPL1. .

APPL1, APPL, EGFR EGF, Cancer

Description

A COMPOSITION FOR ANTICANCER COMPRISING APPL1 INHIBITOR AND A METHOD FOR SCREENING REGULATORS OF INTERACTION BETWEEN APPL1 AND EGFR}

The present invention relates to an inhibitor of APPL1, and more particularly, to a method for screening a modulator for regulating the binding of APPL1 and EGFR, and a composition for preventing and treating cancer comprising a substance that inhibits the expression or activity of APPL1. .

Cancer is one of the most deaths in the world, with about 7.6 million people dying from cancer in 2007, and it is predicted that the age of cancer will gradually decrease, and the incidence of cancer will increase as life expectancy increases. According to the American Cancer Society (ACS) data, more than 12 million new cases of cancer were diagnosed worldwide in 2007 alone, with 7.6 million deaths, killing about 20,000 people every day.

EGFR is a member of the tyrosine kinase growth factor receptor protein family, and has recently been described in extracellular domains (Ogiso H et al. Cell 2002, 110: 775-787; Garrett TP et al. Cell 2002, 110: 763-773; Ferguson KM et. al. Cell 2003; 11: 507) and structural determination studies of intracellular kinase domains (Stamos J et al. J. Biol. Chem. 2002, 277: 46265-46272). This study provided important information on the action of receptors and their ligands. Specifically, EGFR is a cell surface related molecule that is activated through the binding of highly specific ligands such as EGF and transforming growth factor alpha (TGF-α). After ligand binding, the receptor dimerizes to phosphorylate the intracellular tyrosine kinase region. This induces downstream signaling and activates cascade reactions to grow and proliferate cells. EGFR is usually expressed in liver and skin and is often useful as a prognostic marker, as increased activity is often found in solid cancers. Overexpression of EGFR is accompanied by an increase in TGF-α production which favors self-secreting loop growth in tumors, and the fact that EGFR gene amplification and rearrangement observed in some tumors is often associated with the development of mutant forms of EGFR. Libermann TA, et al Nature 1985, 313: 144-147; Wong AJ, Proc Natl Acad Sci USA 1992, 89: 2965-2969; Frederick L, et al. Cancer Res 2000, 60: 1383-1387.

APPL1 refers to an adapter protein comprising a PH domain, a PTB domain and a Leucine zipper motif, also known as DIP13α. These APPL1 proteins are known to interact with RAB5A, DCC, AKT2, PIK3FCA, adiponectin receptors, and NuRD / MeCP1 complexes, and have recently been reported to interact with HDAC, but still interact with EGFR. Is unknown.

Accordingly, the present inventors have made diligent efforts to find a molecule that APPL1 interacts with, and found that the protein interacts with EGFR, and that inhibiting APPL1 effectively inhibits cancer caused by overexpression of EGFR. It was.

An object of the present invention to provide a composition for the prevention and treatment of cancer comprising a substance that inhibits the expression or activity of APPL1.

Another object of the present invention is to determine the binding amount of APPL1 and EGFR in the cells of the individual suspected of cancer; (b) administering the candidate; (c) identifying the binding amount of APPL1 and EGFR; And (d) comparing the expression of steps (a) and (c).

In one embodiment, the present invention relates to a composition for the prevention and treatment of cancer comprising a substance that inhibits the expression or activity of APPL1.

The term “APPL1” is an abbreviation of Adaptor protein, Phosphotyrosine interaction, PH domain and Leucine zipper containing 1. It is also called APPL or DIP13alpha. The protein is RAB5A, DCC, AKT2, PIK3CA, adiponectin receptors. , And are known to interact with the NuRD / MeCP1 complex protein, are found on the endosomal membrane and are secreted by EGF and translocated into the nucleus, but are still known to interact with EGFR. We first found out that APPL1 binds to EGFR, and thus we found that EGFR can effectively cure cancer if it inhibits APPL1 in cancers that are overexpressed, preferably APPL1 interacts with EGFR. More preferably, APPL1 binds the site with EGFR because its specific site interacts with EGFR. By effectively blocking cancer, the sites of APPL1 were identified by experiments as the BAR domain of APPL1, and specifically, in the preferred embodiment of the present invention, APPL1 expressing only the BAR domain (amino acid 1-273 site) was It was confirmed that the binding force with EGFR was significantly increased (Example 1) In addition, when siRNA was used to reduce the expression of APPL1, the expression of EGFR was also reduced, and Akt and the involved in the downstream signaling pathway of EGFR It was also confirmed that phosphorylation of Erk was also significantly reduced (Example 3).

In the present invention, the term "epidermal growth factor receptor (EGFR)" is a cell-surface receptor for EGF (Epidermal growth factor), also referred to as ERBB, ERBB1, HER1 or mENA. EGFR is a member of the ErbB receptor family and mutations that affect EGFR expression or activity are known to cause cancer, but no interactions between EGFR and APPL1 have been reported to date.

As used herein, the term "cancer" includes all cancers induced by overexpression of EGFR and can be broadly classified into solid and hematological cancers, of which "solid cancer" refers to breast or prostate. Abnormal growth of cells in the solid organs of the back, as opposed to leukemia, which affects the liquid blood, specifically the bladder, breast, intestine, kidney, liver, brain, Carcinomas such as esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid gland, prostate and skin (including squamous cell carcinoma); tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; astrocytoma, neuroblastoma, glioma and Tumors of the central and peripheral nervous system, including schwannomas, and other tumors, including melanoma, normal adenomas, teratomas, osteosarcomas, pigmentosa, keratinocytosis, thyroid follicular cancer and Kaposi's sarcoma, preferably Lung cancer, ovarian cancer, lung cancer, head and neck cancer, cervical cancer, neuroblastoma, osteosarcoma, cervical cancer, prostate cancer or rectal cancer, more preferably gastric cancer, liver cancer, skin cancer, lung cancer, colon cancer, uterine cancer, ovarian cancer, cervical cancer, prostate Cancer, or rectal cancer, including, but not necessarily limited to, "hematologic cancer" as described above, also referred to as myeloid leukemia, means a disease in which the leukocyte-producing tissues of bone marrow proliferate indefinitely. If the cancer develops, not only solid cancer but also such blood cancer may be included.

As a specific aspect, the pharmaceutical composition for preventing and treating cancer of the present invention may include a substance that inhibits the activity of APPL1. The activity inhibitory substance (inhibitor) of APPL1 according to the present invention is not limited as long as it is a substance capable of inhibiting the activity of APPL1. Preferably, the activity inhibitory substance is an antibody that specifically recognizes APPL1 expressed in cancer cells. Such antibodies may include polyclonal antibodies as well as monoclonal antibodies and chimeric antibodies, humanized antibodies and human antibodies thereto, as well as novel antibodies, as well as antibodies already known in the art. Preferably, the antibody can prevent or treat cancer due to overexpression of EGFR by inhibiting binding to EGFR.

Such antibodies include functional fragments of antibody molecules as well as complete forms having the full length of two heavy and two light chains, as long as they have the property of binding specifically to APPL1. The functional fragment of the molecule of an antibody means the fragment which has at least an antigen binding function, and includes Fab, F (ab '), F (ab') ₂ , Fv, etc. In the preferred embodiments and experimental examples of the present invention in more detail confirming the binding site of the EGFR of APPL1 of the present invention, as a result, it was confirmed that the BAR domain and EGFR in APPL1 can interact by binding. Thus, the inhibitory activity against APPL1 of the present invention may be an antibody against APPL1, more preferably an antibody against the BAR domain of APPL1. In addition, in the preferred experimental examples and examples of the present invention, the substance that interacts with the PH domain of APPL1 is phosphoinositide produced by PI3K, these substances affect the binding of APPL1 and EGFR As a result of the study, it was revealed that the site of APPL1 is essential for binding of APPL1 and EGFR. Thus, the active substance of APPL1 of the present invention may be an antibody against APPL1, more preferably an antibody acting on the PH domain of APPL1. Confirmed.

In another specific aspect, the pharmaceutical composition for preventing and treating cancer of the present invention may include an oligonucleotide that inhibits the expression of APPL1. Preferably said oligonucleotide is an antisense nucleic acid sequence complementary to siRNA, shRNA or mRNA of APPL1 protein for mRNA of APPL1.

As used herein, the term "siRNA" refers to a short double-chain RNA capable of inducing RNA interference (RNAi) through the cleavage of a particular mRNA. It consists of a sense RNA strand having a sequence homologous to mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. siRNAs can be provided as an efficient gene knockdown method or as a method of gene therapy because they can inhibit the expression of a target gene.

siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases. The siRNA terminal structure can be either blunt or cohesive, as long as the expression of the target gene can be inhibited by the RNAi effect. The cohesive end structure can be either a 3 'end projecting structure or a 5' end projecting structure. The number of protruding bases is not limited. In addition, siRNA is a low-molecular RNA (for example, natural RNA molecules such as tRNA, rRNA, viral RNA or artificial RNA molecules) in the protruding portion of one end to the extent that can maintain the expression inhibitory effect of the target gene It may include. The siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a step loop structure in which the terminal portion of the double-stranded RNA is connected by linker RNA.

The siRNA used in the present invention is itself a complete form with polynucleotide pairing, that is, a form introduced into a cell through two transformation processes in which the siRNA is directly synthesized in vitro, or one form to have such a form after administration in vivo. Single-chain oligonucleotide fragments and their reverse complements may be derived from single-chain polynucleotides separated by spacers, for example siRNA expression vectors or PCR-derived siRNAs prepared to be expressed in cells The expression cassette may be in a form introduced into a cell through a transformation or infection process. Determination of how to prepare an siRNA and introduce it into a cell or animal may depend on the purpose and cellular biological function of the target gene product. According to one embodiment of the present invention, the siRNA may have a sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

As used herein, the term "shRNA" is intended to overcome the disadvantages of the high cost of biosynthesis of siRNA, the short-term maintenance of RNA interference effect due to low cell transfection efficiency, and the adenovirus, lenti from the promoter of RNA polymerase III. Viral and plasmid expression vector systems can be used to introduce them into cells for expression, and these shRNAs are converted into siRNAs with the correct structure by siRNA processing enzymes (Dicer or Rnase III) present in the cells to silence the target genes. It is well known to induce.

As used herein, the term "antisense nucleic acid" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to the complementary sequence within the mRNA to translate the mRNA into a protein. There is a characteristic that inhibits. An antisense sequence of the invention refers to a DNA or RNA sequence that is complementary to APPL1 mRNA and capable of binding to APPL1 mRNA, and that is responsible for translation, translocation into the cytoplasm, maturation or any other overall biological function of APPL1 mRNA. May inhibit essential activity. The antisense nucleic acid has a length of 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases.

Antisense RNA can be synthesized in vitro and administered in vivo by conventional methods, or the antisense RNA can be synthesized in vivo. One example of synthesizing antisense RNA in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin is in the opposite direction of the recognition site (MCS). Such antisense RNA is desirable to ensure that there is a translation stop codon in the sequence so that it is not translated into the peptide sequence.

The composition of the present invention may comprise an additional substance that inhibits the proliferation of cancer cells of a patient, in addition to comprising at least one of siRNA or APPL1 antisense nucleic acid of APPL1, or promoting the influx of siRNA or antisense nucleic acid molecules. Agents, for example liposomes (US Pat. Nos. 4,897,355, 4,394,448, 4,23,871, 4,231,877, 4,224,179, 4,753,788, 4,673,567, 4,247,411, 4,814,270) or It can also be combined with one of the lipophilic carriers of many sterols, including cholesterol, cholate and deoxycholic acid. Antisense nucleic acids can also be conjugated to peptides that are taken up by cells. Examples of useful peptides include peptide hormones, antigens or antibodies and peptide toxins.

The compositions of the present invention can be used alone, but radiation therapy or chemotherapy (cell growth arrest or cytotoxic substances, antibiotic-like substances, alkylating agents, anti-metabolic substances, hormonal agents, immunizing agents, interferon type) to increase the treatment efficiency Substances, cyclooxygenase inhibitors, metallomatrix protease inhibitors, telomerase inhibitors, tyrosine kinase inhibitors, anti-growth factor receptor substances, anti-HER substances, anti-EGFR substances, anti-angiogenic substances, farnesyl transferase inhibitors , ras-raf signal conduction pathway inhibitors, cell cycle inhibitors, other cdk inhibitors, tubulin conjugates, topoisomerase I inhibitors, topoisomerase II inhibitors, etc.).

The composition of the present invention may be administered with a pharmaceutically acceptable carrier, and when used orally, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, and the like may be used. In the case of injections, buffers, preservatives, analgesic agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used. For topical administration, bases, excipients, lubricants, preservatives and the like can be used. Formulations of the compositions of the present invention can be prepared in a variety of mixtures with the pharmaceutically acceptable carriers described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, they may be prepared in unit dosage ampoules or in multiple dosage forms.

As used herein, the term administration refers to introducing a composition of the invention to a patient in any suitable manner, and the route of administration of the composition of the invention may be administered via any general route as long as it can reach the desired tissue. have. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration, rectal administration, intranasal administration, intraperitoneal administration, intradural administration, but are not limited thereto. Do not.

The range of effective dosages of the compositions of the present invention may range from sex, body surface area, type and severity of disease, age, sensitivity to drugs, route of administration and rate of release, time of administration, duration of treatment, target cells, expression levels, etc. It may vary according to various well-known factors, and can be easily determined by those skilled in the art.

In another aspect, the present invention provides a method for treating cancer, comprising: (a) identifying the amount of binding of APPL1 and EGFR in cells of a subject suspected of having cancer; (b) administering a candidate; (c) identifying the binding amount of APPL1 and EGFR; And (d) comparing the expression of steps (a) and (c).

Based on the fact that APPL1 binds to EGFR and regulates EGFR protein levels, we have found that APPL1 can be used as a therapeutic for these cancers. Therefore, when screening including an agent capable of measuring the binding level of APPL1 and EGFR, it can be effectively used as a method for developing a therapeutic agent for cancer induced by overexpression of EGFR.

The term "candidate" of the present invention refers to an agent that inhibits or promotes the binding of APPL1 and EGFR, and measures the binding level of APPL1 and EGFR before and after administration of the candidate, and after treatment than before treatment of the candidate. If the binding level of the two molecules is increased, the agent promoting cancer, and in the opposite case (when the binding level decreases before and after treatment of the candidate material), the candidate may be determined as an agent capable of preventing or treating cancer. have. The binding level is a concept including the binding amount, and means that not only the binding amount of the two molecules is increased, but also all quantitative or qualitative actions related to the binding of the two molecules are increased, such as an increase in binding activity.

The “agent capable of measuring the binding level of APPL1 and EGFR” is not limited as long as it is an agent capable of quantitatively or qualitatively determining the binding, and preferably, may be an antibody against each protein of APPL1 and EGFR. In a preferred embodiment of the present invention, using a polyclonal antibody against APPL1 and an antibody against EGFR, it was confirmed that the two proteins bind to each other in the region through the reaction of both antibodies.

In addition, preferably the measurement of the binding level of APPL1 and EGFR can be confirmed by additionally measuring the phosphorylation level of Akt or Erk protein involved in the cellular signal transduction pathway by overexpression of EGFR. The phosphorylation degree of Akt and Erk can be used without limitation in the methods commonly used in the art, it is possible to measure either Akt or Erk, or both can be measured simultaneously. The degree of phosphorylation may be used in addition to measuring the binding level of APPL1 and EGFR, or may be used as a means of verifying the binding level of APPL1 and EGFR.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for carrying out the present invention by way of example and the scope of the present invention is not limited to these Examples and Experimental Examples.

The use of the APPL1 inhibitor of the present invention not only reduces the expression of EGFR but also regulates the stabilization of EGFR, thereby effectively treating cancer caused by overexpression of EGFR. In addition, when using the screening method of the present invention, it is possible to conveniently search for a substance that inhibits or promotes the binding of APPL1 and EGFR, which are essential for cell growth, proliferation and differentiation by cell signal transduction, and thus an effective cancer therapeutic agent. Can be screened.

Experimental Example 1. Plasmid Structure and RNA Interference

Human APPL1 gene was amplified by RT-PCR using mRNA purified from human embryonic kidney fibroblast cells. Full length of APPL1 (# 1), BAR-PH (# 2, aa 1-499), BAR (# 3, aa 1-273), PH-PTB (# 4, aa 265-709), and PTB (# 5 , aa 486-709) domains were inserted into pCMV6-HA or pCMV6-SRT mammalian expression vectors (Lee, JR, Chang, YY, and Hahn, MJ (2001) Biotechniques 31, 541-545). Human EGFR gene was provided by Dr P.G., Suh (Postech Pohang, Korea). Duplex small interfereing RNAs (APPL1) (5'-GGGAGGCAGGCGTACAAAT (SEQ ID NO: 1) and 5'-AUUUGUACGCCUGCCUCCC (SEQ ID NO: 2)) for APPL1 were synthesized from Dharmacon (Lafayette, CO, USA). Negative control scrambled siRNA was purchased from Ambion (Austin, TX, USA).

Experimental Example 2. Cell Culture and Transfection

HEK293 cells and HeLa cells were grown in DMEM filled with 10% fetal bovine serum. For immunoprecipitation, HEK293 cells were transfected with the expression plasmid using the calcium phosphate precipitation method. For siRNA and expression plasmid transfection in HeLa cells, a microporation method was performed according to the manufacturer's protocol.

Experimental Example 3. Antibodies and Chemicals

APPL1 rabbit polyclonal antibodies were prepared against the C-terminal PTB domain of human APPL1. Serum was purified using Sulfolink beads (Pierce, Rockford IL, USA). Other antibodies used in this experiment were: anti-HA (Roche, Basel, Switzerland), anti-β-actin (Sigma-Aldrich, St Louis, MO, USA), mouse anti-EGFR (Zymed, San Francisco, CA, USA), rabbit anti-EGFR (Santa Cruz, Santa Cruz, CA, USA), anti-Akt, anti-phosphate-Akt, anti-Erk1 / 2, anti-phosphate-Erk1 / 2 (Cell Signaling Technology, Laboratories , Franklin Lakes, NJ, US), anti-c-Cbl (BD Transduction Laboratories, Franklin Lakes, NJ, USA) and FITC-fused goat anti-rabbit IgG (ICN, Irvine, CA, USA). Rhodamine-fused EGF was purchased from Molecular Probes (Eugene, OR, USA). EGF and Hoechst stains were purchased from Sigma-Aldrich (St Louis, Mo, USA).

Experimental Example 4. Immunoprecipitation and Immunoblotting

Immunoblot and immunoprecipitation assays were performed according to previously known methods (Lee, SJ, Lee, JR, Hahn, HS, Kim, YH, Ahn, JH, Bae, CD, Yang, JM, and Hahn, MJ ( Exp Mol Med 39, 450-457). Cells were dissolved in lysis buffer (50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM PMSF, 1 mM sodium sorbovadate) orthovanadate), 2 mM leupeptin) for 30 min. For ubiquitination assay, 10 mM NEM was added to the lysis buffer. After centrifugation, the protein concentration of the supernatant was confirmed by BSA kit (Pierce, Rockford, IL, USA). Cell lysates were incubated with the indicated antibodies. After 1 hour of incubation at 4 ° C., A-Sepharose beads were added and absorbed for several hours. Immune complexes were pelleted, washed twice with lysis buffer and then washed twice with lysis buffer containing 100 mM NaCl. Proteins were separated and transferred by SDS-PAGE. After blocking, membranes were probed with appropriate antibodies and detected by an ECL system (GE Healthcare, Buckinghamshire, UK). Densitometric quantification was performed using the LAS-3000 and analyzed using Gauge image analysis software (Fuji Photo Film).

Experimental Example 5. Immunofluorescence

HeLa cells were fixed with 4% paraformaldehyde in PBS. After blocking, immobilized cells were incubated with anti-APPL1 antibodies. FITC-fusion anti-rabbit antibodies were used for primary antibody detection. Nuclei were stained with Hoechst reagent. Confocal microscopy analysis was performed using LSM510 META (Zeiss) fmf.

Experimental Example 6 EGFR Internalization Analysis

HeLa cells treated with mixed siRNA or siRNA for APPL1 were incubated with ¹²⁵ I-EGF 1.7 nM in DHB (DMEM with 25 mM HEPES, 0.2% fatty acid free BSA) at 4 ° C. for 1 hour. Unbound ligand was washed with cold PBS (ice-cold PBS). Cells were transferred at 37 ° C. and incubated for several passages. At the end of each time point, cells were washed with acid strip buffer (0.2M acetic acid, 0.5M NaCl, pH 2.8) and measured with a gamma counter for surface bound fragments. The remaining cells were lysed with lysis buffer (1M NaOH) and internalized fragments were measured.

Example 1 Combination of APPL1 and EGFR

In DNA replication through nucleosome remodeling and interaction with histone deacetylase complex NuRD / MeCP1, for EGF stimulation, EGF is responsible for APPL1-containing endosomes (APPL1) that induce translocation of APPL1 in the nucleus. It was previously known that it rapidly internalized into -contatining endosomes (Miaczynska, M., Christoforidis, S., Giner, A., Shevchenko, A., Uttenweiler-Joseph, S., Habermann, B., Wilm, M). , Parton, RG, and Zerial, M. (2004) Cell 116, 445-456). Recently, it has been reported that APPL1 binds to TrkA in endosomes and regulates neuritogenesis induced by NGF (Varsano, T., Dong, MQ, Niesman, I., Gacula, H., Lou, X., Ma, T , Testa, JR, Yates Iii, JR, and Farquhar, MG (2006) Molecular and Cellular Biology 26, 8942-8952, Lin, DC, Quevedo, C., Brewer, NE, Bell, A., Testa, JR, Grimes, ML, Miller, FD, and Kaplan, DR (2006) Mol Cell Biol 26, 8928-8941). This interaction in the endosomal compartments has been shown to require NGF-mediated activity of MEK, ERK and Akt without any significant translocation of APPL1 in the nucleus. We therefore assumed that APPL1 binds to EGFR and modulates EGFR-initiated signaling events in the endosomal compartment. To test this hypothesis, we cotransfected HEK293 cells with expression vectors for SRT-tagged human APPL1 and HA-tagged EGFR. After 48 hours, cells were obtained and cell lysates were co-immunoprecipitated with SRT antibodies followed by immunoblot analysis with antibodies against HA and SRT. As shown in FIG. 1A, APPL1 precipitated with EGFR. In order to identify the domain of APPL1 mediating interaction with EGFR, various deletion constructs of APPL1 according to the domain structure were prepared (FIG. 1B). As shown in FIG. 1C, 1-499 constructs and 1-273 constructs comprising the N-terminal BAR domain of APPL1 were co-immunoprecipitated with EGFR. However, the 1-273 construct containing the BAR domain of APPL1 bound EGFR much more effectively than full length APPL1. Interestingly, the 1-499 construct mutations, including both the BAR and PH domains of APPL1, showed reduced EGFR binding compared to the 1-273 construct, where the PH domain of APPL1 plays an important role in EGFR binding of the BAR domain. Imply that it can (FIG. 1C, lanes 2 and 3 in the bottom panel). On the other hand, in 265-709 and 486-709 mutations, EGFR binding was greatly reduced, which means that the BAR domain containing the 1-273 amino acid region of APPL1 contains the EGFR binding site. To analyze whether internal endogenous APPL1 interacts with EGFR, we prepared polyclonal antibodies to APPL1 and characterized their specificity. HEK293 cells were transfected with a vector for SRT control or SRT-tagged APPL1 and immunoblot analysis was performed with antibodies to SRT or APPL1. SRT-tagged APPL1 protein was recognized by anti-SRT as well as anti-APPL1, resulting in a single band of ˜80 kD molecular mass. Internal APPL1 was also recognizable as APPL1 antibody in the transfected cells of the control group (FIG. 1D). In addition, this signal was reduced by specific siRNA for APPL1 in both immunoblot and immunocytochemistry analysis (FIGS. 1E and 1F). From these results, we concluded that these antibodies were specific for APPL1 and used them for further studies. To verify whether the binding between internal APPL1 and EGFR is EGF stimulatory dependent, HeLa cells were treated with 100ng / ml EGF for a period of time. Cell lysates were immunoprecipitated with APPL1 antibody followed by immunoblot analysis with EGFR antibody. After 5 minutes of EGF stimulation, the binding of EGFR to internal APPL1 increased significantly, indicating that the binding of these proteins was EGF stimulatory dependent (FIG. 1G). The PH domain of APPL1 is known to interact with phosphoinositide produced by PI3K (Chial, HJ, Wu, R., Ustach, CV, McPhail, LC, Mobley, WC, and Chen, YQ ( 2008) Traffic 9, 623-624. To investigate this possibility, cells were treated with wortmannin, a PI3K inhibitor, 30 minutes before EGF stimulation. Five minutes after EGF treatment, cell lysates were immunoprecipitated with APPL1 antibody, followed by immunoblot analysis using EGFR antibody. PI3K inhibition by wortmannin markedly reduced binding between internal APPL1 and EGFR in HeLa cells, supporting that the PH domain of APPL1 plays a regulatory role in binding to EGFR (FIG. 1H).

Example 2 Temporary Colocalization of APPL1 and Rh-EGF

To investigate the intracellular localization of this interaction, HeLa cells were incubated for 12 hours in serum-free and incubated with 100ng / ml rhodamine-fusion EGF (Rh-EGF) for 30 minutes at 4 ° C. The cells were then transferred to 37 ° C., further incubated for some time, followed by immunostaining with APPL1 antibody. In cells incubated at 4 ° C. for 30 minutes, Rh-EGF was weakly detected along the plasma membrane, while APPL1 was detected throughout the cytoplasm. Interestingly, when cells were moved at 37 ° C. for 5 min, Rh-EGF and some co-localized APPL1 were attracted close to the plasma membrane. After that, most of the APPL1 was relocalized in cytoplasmic vesicles, which made it difficult to observe co-localization with Rh-EGF. However, no significant translocation of APPL1 was observed in the nucleus. These data along with immunoprecipitation analyzes indicate that APPL1 is associated with EGFR dependent on EGF stimulation.

Example 3 APPL1 Regulates EGFR Signaling by Controlling EGFR Stabilization

We investigated the role of APPL1 in EGFR-mediated signaling and trafficking. The first HeLa cells were transfected with specific siRNA for APPL1, and cell lysates were subjected to immunoblot analysis with antibodies against β-actin as APPL1, EGFR and loading controls. Interestingly, APPL1 knockdown HeLa cells showed a significant reduction in EGFR protein as compared to cells transfected with control siRNA. These cells were then placed in serum-free 12 hours and stimulated with 100ng / ml EGF for some time. Most of the EGFR in the control group showed slow migration to EGF stimulation due to the phosphorylation of its intracellular region. In these cells, EGFR protein was markedly reduced 30 minutes after EGF stimulation. In contrast, APPL1 knockdown cells showed a pronounced EGFR reduction 5 minutes after EGF stimulation (FIG. 3B).

Next we analyzed the EGFR levels for EGF in APPL1 overexpressing cells. EGFR protein levels were significantly higher in APPL1 overexpression in HeLa cells than in control cells (FIG. 3C, upper panel). However, APPL1 overexpressing cells showed an increase in EGFR protein at early time points. Therefore, we measured the EGFR signal by densitometry and set the initial 0 (time) time point to 100% for each cell type. Relative EGFR signal intensity was measured compared to the control (FIG. 3C, bottom panel). After 30 minutes of EGF treatment, about 44% of EGFR was maintained in control cells transformed with the vector, whereas 72% of EGFR was detected in APPL1 overexpressed cells (FIG. 3C), indicating that APPL1 is induced by EGF stimulation. This means that the rate of degradation of EGFR can be controlled.

To demonstrate the functional importance of EGFR stabilization by APPL1, we analyzed the activation state of downstream signal molecules. APPL1 overexpressing HeLa cells were treated with EGF for a period of time, and activation status of Akt and Erk was determined using phosphorus-specific antibodies against Akt and ERK1 / 2 and antibodies against total Akt and Erk. Analyzed. Five minutes after EGF stimulation, both kinases were strongly phosphorylated without altering the expression of kinase in control and APPL1 overexpressing cells. However, APPL1 overexpression increased 3 times of Akt phosphorylation and 1.55 times of Erk phosphorylation in response to EGF treatment for 5 minutes (FIG. 4A). In contrast, APPL1 knockdown showed a decrease in phosphate-Akt (65%) and phosphate-ERK1 / 2 (32%) after 5 minutes of EGF treatment. These data indicate that APPL1 is required for the effective activation of Akt and Erk1 / 2 induced by EGFR in response to EGF.

Example 4 APPL1 Regulating EGFR Transport

Internalization of EGFR is the first step in its transport and degradation, so a reduction in EGFR internalization by APPL1 can affect EGFR degradation. To investigate whether internalization of EGFR is affected by depletion of APPL1, HeLa cells were transfected with control mixed siRNA or APPL1 siRNA, incubated for 1 hour with 1.7 nM ¹²⁵ I-EGF at 4 ° C. Ligands not washed away. Cells were then incubated at 37 ° C. for a period of time, washed, and the surface-bound radioligand measured. Internalized fragments were also obtained by lysing the cells. Internalized ¹²⁵ I-EGF peaked near 5 minutes in both control and APPL1 knockdown cells, but depletion of APPL1 reduced internalization of ¹²⁵ I-EGF by 40% at this point (FIG. 5A). We confirmed the rate of EGF internalization by the ratio between cell lysate and radiation from surface-bound ¹²⁵ I-EGF, and found no significant difference found in the control and APPL1 knockdown cells. These results indicate that the reduced internalization of EGFR in APPL1-knockdown cells is due to a decrease in the initial capacity of EGFR for ¹²⁵ I-EGF and is not due to a change in the rate of EGF internalization.

To further investigate the role of APPL1 in regulating EGFR stability, we analyzed the shift of Rh-EGF. HeLa cells were transfected with mixed siRNA or APPL1 siRNA and after 48 hours these cells were treated with Rh-EGF for a period of time, followed by internalization of Rh-EGF and APPL1. In the control group, Rh-EGF migrated into the perinuclear late endosomal compartments at 30 minutes of incubation time, whereas EGF was concentrated in the perinuclear compartment at 15 minutes of incubation time in APPL1-knockdown cells. (FIG. 6). These results indicate that APPL1 is required for EGF signal transmission by regulating EGFR transport and stability. Given that internalization of EGFR and APPL1 occurs in the endosomal compartment around the cytoplasmic membrane, it suggests that APPL1 can regulate receptor transport in this localization.

Example 5 c-Cbl Binding to EGFR is Regulated by APPL1

Prolonged presence of EGFR after EGF stimulation caused by impaired EGFR degradation is known to cause cancer progression. It is also known that E3 ligase c-Cbl plays a key role in EGFR degradation. c-Cbl has been reported to interact with activated EGFR and has been reported to downregulate these receptors via multiple mono-ubiquitations by subsequent targeting for lysosomal degradation (Swaminathan , G., and Tsygankov, AY (2006) J Cell Physiol 209, 21-43, Kirisits, A., Pils, D., and Krainer, M. (2007) The International Journal of Biochemistry & Cell Biology 39, 2173- 2182). Therefore, we hypothesized that APPL1 will affect the interaction between c-Cbl and EGFR, thus affecting EGFR degradation. To demonstrate this hypothesis, we expressed an increased amount of APPL1 in HeLa cells and analyzed the interaction of internal EGFR and c-Cbl. Transfected cells were treated with EGF for 5 minutes, and cell lysates were immunoprecipitated with EGFR antibodies followed by immunoblotting with antibodies against c-Cbl and ubiquitin. EGFR protein levels were very closely associated with an increase in APPL1. Overexpression of APPL1 reduced ubiquitination of EGFR (FIG. 7B) as well as binding of c-Cbl and EGFR in these cells (FIG. 7A). These data indicate that APPL1 inhibits the binding of EGFR and c-Cbl, leading to increased stability of EGFR. This can occur through the negative regulation of EGFR targeting for lysosomal compartments in its degradation.

1 is a diagram showing the binding of EGFR and APPL1.

Figure 2 shows colocalization of EGFR and APPL1 after EGF stimulation.

Figure 3 shows the effect of APPL1 on the protein level of EGFR.

4 is a diagram confirming that APPL1 can adjust the EGF signal.

5 shows that APPL1 does not affect the internalization rate of EGFR.

Figure 6 shows that APPL1 induces rapid migration of EGF vesicles to the perinuclear endosome.

Figure 7 shows that the binding of EGFR and c-Cbl is inhibited by APPL1.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> A COMPOSITION FOR ANTICANCER COMPRISING APPL1 INHIBITOR AND A          METHOD FOR SCREENING REGULATORS OF INTERACTION BETWEEN APPL1 AND          EGFR <130> PA090135 / KR <140> 10-2009-0051125 <141> 2009-06-09 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 1 gggaggcagg cgtacaaat 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 2 auuuguacgc cugccuccc 19  

Claims (10)

A composition for preventing and treating cancer comprising a substance that inhibits the expression or activity of APPL1. The composition of claim 1, wherein the substance that inhibits the expression of APPL1 is siRNA, shRNA or antisense nucleic acid. The composition of claim 1, wherein the siRNA has a sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. The composition of claim 1, wherein the substance that inhibits the activity of APPL1 is an antibody. The composition of claim 4, wherein the antibody is an antibody against the BAR domain of APPL1 or an antibody against the PH domain. The composition of claim 1, wherein the cancer is gastric cancer, skin cancer, lung cancer, liver cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, or colon cancer. (a) identifying the binding amount of APPL1 and EGFR in the cells of the individual suspected of having cancer; (b) treating the candidate; (c) identifying the binding amount of APPL1 and EGFR; And (d) comparing the expression of steps (a) and (c). The method of claim 7, wherein the binding amount of APPL1 and EGFR is confirmed using an antibody to APPL1 and an antibody to EGFR. The method of claim 8, wherein the antibody is a monoclonal antibody. 8. The method of claim 7, further comprising the step of (a) and (c) further confirming the amount of change in Akt or Erk protein phosphorylation.

Images