KR102146047B1 - Tumor suppressor gene PIP4K2A and uses thereof - Google Patents

Abstract

Regarding the tumor suppressor gene PIP4K2A and its use, the tumor suppressor gene PIP4K2A according to an aspect is derived through an in vivo RNAi screening technique, and can be a useful new target for cancer treatment, so it is useful for cancer diagnosis, treatment, and therapeutic screening methods There is an effect that can be used in a way.

Description

Tumor suppressor gene PIP4K2A and uses thereof {Tumor suppressor gene PIP4K2A and uses thereof}

To the tumor suppressor gene PIP4K2A and its use.

Glioma (glioma) is a tumor that accounts for 60% of primary brain tumors, and it is a malignant tumor that does not have any special treatment other than radiation therapy. Glioblastoma accounts for 12-15% of all brain tumors, and is the most common single tumor in the brain that accounts for 50-60% of glioma. In addition, brain pressure rises rapidly, resulting in headache (severe in the morning), nausea (nausea), vomiting, convulsions, decreased nerve function due to brain swelling, decreased limb movement or sensation, facial paralysis, speech disorder, cognitive decline, left -Shows various symptoms such as right classification disorder. In the case of glioblastoma (GBM), compared with other cancers, the resistance to radiation and chemotherapy is very high, and once diagnosed, the expected survival period is only 1 year. In addition, in the case of such a brain tumor, it is difficult to deliver drugs for treatment due to the cerebral vascular barrier. In particular, due to a relatively lack of understanding of cranial nerve biology, the development of therapeutic agents for it has been slow. Moreover, glioblastoma exhibits an aggressive variant when compared to other brain tumors, which can lead to lethal outcomes within weeks if not treated as soon as possible. In order to treat a patient with glioblastoma, it is necessary to consider the patient's genetic, physiological and environmental characteristics, as well as to match the progression level of glioblastoma, chemotherapy, radiation therapy, surgical therapy, and immune cell therapy. Various treatment methods such as, etc. should be applied. However, these treatments have reached their limits due to the occurrence of resistant mutants and recurrences due to tumor stem cells, and thus the need to develop new treatments is required.

One aspect is to provide the tumor suppressor gene PIP4K2A.

Another aspect is to provide a composition for preventing, improving or treating cancer comprising the PIP4K2A gene or protein or an active fragment thereof.

Another aspect is to provide a method for diagnosing cancer using the PIP4K2A.

Another aspect is to provide a method for screening cancer therapeutic candidates using the PIP4K2A.

One aspect provides a composition for preventing, improving, or treating cancer comprising an agent for increasing the expression or activity of the PIP4K2A gene or protein as an active ingredient.

The agent for increasing the expression or activity of the PIP4K2A protein may include the PIP4K2A protein or an active fragment thereof.

As used herein, "treat" refers to an individual suffering from or at risk of developing a disease, improving the condition of the individual (eg, one or more symptoms), delaying the progression of the disease, symptoms It refers to any form of treatment or prevention that provides an effect, including delaying occurrence or slowing the progression of symptoms. Thus, the term “treatment” also includes prophylactic treatment of an individual that prevents the occurrence of symptoms.

As used herein, "treatment" and "prevention" are not intended to mean cure or complete elimination of symptoms. They refer to any form of treatment that provides an effect to a patient suffering from the disease, including improving the patient's condition (eg, one or more symptoms), delaying the progression of the disease, and the like.

As used herein, a "treatment-effective amount" is in a patient suffering from cancer such as glioblastoma, including improvement of the patient's condition (eg, one or more symptoms), delaying the progression of the disease, etc. It means an amount sufficient to produce the desired effect.

Cancer of the present specification is glioblastoma, cerebrospinal cancer, head and neck cancer, lung cancer, breast cancer, thymoma, esophageal cancer, brittle cancer, colon cancer, liver cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, Cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, or skin cancer. In addition, in one embodiment, the PIP4K2 may play a specific role in PTEN-deleted cancer cells. Therefore, the cancer may be a PTEN-deleted cancer.

In addition, the glioblastoma may be any one subtype selected from the group consisting of a classic glioblastoma, a proneural glioblastoma, a neural glioblastoma, and a mesenchymal glioblastoma. .

The PIP4K2A ((Phosphatidylinositol-5-phosphate 4-kinase type-2 alpha) protein or fragment thereof refers to a natural or recombinant PIP4K2A protein or a protein having substantially equivalent physiological activity to the protein. A protein having substantially equivalent physiological activity. Includes native/recombinant PIP4K2A protein or fragments thereof, functional equivalents and functional derivatives thereof.

The "functional equivalent" refers to an amino acid sequence variant in which some or all of the natural protein amino acids are substituted, or a part of the amino acid is deleted or added, and has substantially the same physiological activity as the natural PIP4K2A protein or a fragment thereof. The "functional derivative" refers to a protein or fragment thereof that has been modified to increase or decrease the physicochemical properties of the PIP4K2A protein or fragment thereof, and has substantially the same physiological activity as the native PIP4K2A protein.

The PIP4K2A protein or fragment thereof of the present invention may be derived from a mammal, and the mammal includes a human. For example, the PIP4K2A protein may include the amino acid sequence of SEQ ID NO: 1. Further, the active fragment of PIP4K2A may include any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5.

In another embodiment, the PIP4K2A gene expression increasing agent may be a vector containing a polynucleotide encoding a PIP4K2A protein or an active fragment thereof, or a cell containing the vector.

The term “polynucleotide” refers to a polymer of deoxyribonucleotides or ribonucleotides present in single-stranded or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and unless specifically stated otherwise, includes analogs of natural polynucleotides.

The polynucleotide includes not only a nucleotide sequence encoding the amino acid sequence of the PIP4K2A protein, but also a sequence complementary to the sequence. The complementary sequence includes not only a perfectly complementary sequence, but also a substantially complementary sequence, which under stringent conditions known in the art, for example, a nucleotide encoding the amino acid sequence of the fusion protein. It refers to a sequence capable of hybridizing with the nucleotide sequence of the sequence.

The term “vector” refers to a means for expressing a gene of interest in a host cell. For example, viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, lentiviral vectors, retroviral vectors and adeno-associated viral vectors. Vectors that can be used as the recombinant vector are, for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19 Plasmids often used in the art, such as λgt4λB, λ-Charon, λΔz1, and phages such as M13, or viruses such as SV40 may be fabricated.

In the recombinant vector, the polynucleotide sequence encoding the fusion protein is operably linked to a promoter. The term “operatively linked” refers to a functional linkage between a nucleotide expression control sequence, such as a promoter sequence, and another nucleotide sequence, whereby the control sequence facilitates transcription and/or translation of the other nucleotide sequence. Will be adjusted.

The recombinant vector may be an expression vector capable of stably expressing the PIP4K2A protein in a host cell. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms. The recombinant vector can be constructed through various methods known in the art.

The recombinant vector can be constructed using prokaryotic or eukaryotic cells as a host. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc. ), a ribosome binding site for initiation of translation and a transcription/translation termination sequence are generally included. In the case of eukaryotic cells as a host, the origin of replication operating in eukaryotic cells included in the vector includes the f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adeno origin of replication, AAV origin of replication, BBV origin of replication, etc. It is not limited. In addition, a promoter derived from the genome of a mammalian cell (e.g., metallotionine promoter) or a promoter derived from mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus promoter and the tk promoter of HSV) can be used and generally have a polyadenylation sequence as the transcription termination sequence.

In another embodiment, the cell penetrating peptide may be linked to the PIP4K2A protein or an active fragment thereof. The cell penetrating peptide may be a cationic cell penetrating peptide. The cationic cell penetrating peptide contains at least 70% or more, 80% or more, for example, 70-90%, or 70-90% of the total amino acid sequence, of any one or more amino acid residues selected from the group consisting of arginine, lysine and histidine. It may be included as. In addition, the cell penetrating peptide may have an amino acid in the L-type or D-type in consideration of stability in the body. Specifically, the cell penetrating peptide may be composed of 10 to 40 amino acid residues in length including any one or more amino acid residues selected from the group consisting of arginine, lysine, and histidine. Specifically, the cell penetrating peptide is TAT (SEQ ID NO: 6: YGRKKRRQRRR), penetratin (Penetratin; SEQ ID NO: 7: RQIKIWFQNRRMKWKK), polyarginine (SEQ ID NO: 8: RRRRRRR), polylysine (SEQ ID NO: Number 9:KKKKKKKKKK), LMWP (low molecular weight protamine; SEQ ID NO: 10: VSRRRRRRGGRRRR), protamine (protamine) fragment, and antenna pedia (Antennapedia, ANTP) may be any one selected from the group consisting of. Peptides or peptide analogs other than the aforementioned peptides may also be used as long as they can penetrate the cell membrane.

In another embodiment, the composition may be administered in combination with an additional anticancer agent. The anticancer agent may be, for example, a target anticancer agent. Targeted anticancer agent may refer to an anticancer agent that selectively kills only cancer cells by blocking signals involved in the growth and development of cancer by targeting specific proteins or specific gene changes that appear only in specific cancer cells.

In one embodiment, the PIP4K2A protein or an active fragment thereof may bind to p85 of PI3K. Through this, the PIP4K2A protein or its active fragment may promote the degradation of p85 and p110.

The composition of the present invention may be prepared by including at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers can be used by mixing saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and if necessary, antioxidants , Buffers, bacteriostatic agents, and other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to form injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets, and can act specifically on target organs. Thus, a target organ-specific antibody or other ligand may be used in combination with the carrier. Furthermore, it can be preferably formulated according to each disease or component by an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA. have.

The composition of the present invention may be prepared for oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal administration, or the like. For example, it can be used in an injectable form. Thus, it can be mixed with any pharmaceutically acceptable vehicle for injectable compositions, especially for direct injection into the area to be treated. The composition of the present invention may comprise a lyophilized composition which enables the composition of an injectable solution, in particular upon addition of sterile isotonic solutions or sterile water or appropriate physiological saline. Direct injection into the patient's tumor is advantageous as it focuses treatment efficiency on the infected tissue. The dosage used can be adjusted by various parameters, in particular the protein, gene, vector, mode of administration used, the disease in question or alternatively the duration of treatment required. In addition, the range varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage is about 0.0001 to 10 mg/kg, preferably 0.001 to 1 mg/kg, and may be administered once to several times a day.

Another aspect provides a method for diagnosing cancer using PIP4K2A.

The term "diagnosis" as used in the present invention is used to measure the presence or absence of the PIP4K2A protein of the present invention and the polynucleotide encoding the same in a biological sample or tissue sample to determine the presence or characteristics of a disease related to the expression or activity of the gene or protein. Means to check.

In detail, it provides a composition for diagnosis of cancer comprising an agent measuring the activity or expression of the PIP4K2A protein or gene.

In addition, measuring the activity or expression of the PIP4K2A protein or gene in a sample derived from the subject as an experimental group; Comparing the activity or expression of the PIP4K2A protein or gene with the activity or expression of the PIP4K2A protein or gene of a normal control; And when the activity or expression of the PIP4K2A protein or gene is decreased compared to the control, it provides a protein or gene detection method for providing information on cancer diagnosis comprising determining that the risk of developing cancer is high.

The activity of the PIP4K2A protein may be measured through an antibody.

The expression of the PIP4K2A gene may include an agent measuring the mRNA expression level of the gene.

In the present invention, "measurement of mRNA expression level" refers to a process of confirming the presence and expression level of mRNA of a cancer marker gene in a biological sample to diagnose cancer, and can be determined by measuring the amount of mRNA. Analysis methods for this include RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), and Northern blotting. blotting), DNA chips, etc., but are not limited thereto.

In the present invention, "measurement of protein expression or activity level" refers to a process of checking the presence and expression level of a protein expressed in a cancer marker gene in a biological sample to diagnose cancer, and specifically binds to the protein of the gene. Check the amount of protein using the antibody. Analysis methods for this include western blot, ELISA (enzyme linked immunosorbent asay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion, and rocket immunoelectrophoresis. , Tissue immunostaining, immunoprecipitation assay, Complement Fixation Assay, FACS, protein chip, etc., but are not limited thereto.

Another aspect is the step of processing the candidate material to the PIP4K2A protein or gene; Measuring the activity or expression of the PIP4K2A protein or gene; And it provides a method for screening a cancer treatment candidate material comprising the step of selecting a candidate material that has increased the activity or expression of the PIP4K2A protein or gene compared to a control group that has not been treated with the candidate material.

Measurement of the activity or expression level of the protein or gene is as described above.

The candidate material may be, but is not limited to, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, or animal tissue extracts, and may be not only novel substances but also widely known ones. The treatment of the candidate material may be treatment on cells expressing the PIP4K2A protein or gene. The cell may be a cell transformed with a recombinant expression vector containing the gene, and such a transformation method is well known in the art.

The expression level of the gene is not limited thereto, but preferably, in the case of using an antibody as a method for determining the amount of protein according to the expression, a second antibody linked to a label that is specific and detectable to the target protein may be added, The detection also has a binding affinity for the second antibody, after the addition of the second antibody, and a third antibody linked to a detectable label may be added. As the label detectable in the second or third antibody, an appropriate chromogenic substrate and an enzyme that exhibits color development upon culture may be used. The detectable moiety may include a composition detectable by spectroscopic, enzymatic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means, for example, fluorescent markers and dyes, magnetic labels. , Linked enzymes, mass spectrometric tags, spin labels, electron transfer donors and acceptors, but are not limited thereto.

When the expression level of the protein or gene in the cells treated with the candidate substance is increased compared to the control group not treated with the candidate substance, the candidate substance may be selected as a cancer treatment.

In the case of using the screening method of the present invention, a prophylactic agent and/or therapeutic agent useful for patients in need of prophylaxis and/or treatment of cancer disease through an experiment that selects a therapeutic agent involved in the mechanism in a short time and demonstrates this Can provide.

The tumor suppressor gene PIP4K2A according to an aspect is derived through an in vivo RNAi screening technique, and can be a useful new target for cancer treatment, and thus has an effect that can be usefully used in cancer diagnosis, treatment, and treatment screening methods.

1 is a diagram showing an in vivo RNAi screening technique using cells derived from glioblastoma patients to screen for tumor suppressor genes; A: A diagram for selecting the highest amplified gene candidate among cancer suppressor gene candidates; B: A diagram showing the presence of the PIP4K2A gene among shRNA candidates injected and amplified into GBM1 tumors; C: A diagram showing gene amplification of PIP4K2A shRNA among three brain tumor patient-derived cells and LN428 cells; D: A diagram showing that among the shRNA candidates amplified in three brain tumor patient-derived cells, the PIP4K2A gene shRNA was commonly amplified in all cells.
2 is a diagram showing the expression of PIP4K2A in patients with glioblastoma and in normal brain tissue using A: Oncomine dataset; B and C: Phillips data set (mRNA) and TCGA dataset CNA is a diagram showing the survival rate in patients with low PIP4K2A expression or copy number; D: A diagram showing the expression of PIP4K2A in glioblastoma patient-derived cells and normal neural progenitor cells (NPC) using RNAseq data.
3 is a view confirming the expression of PIP4K2A using Immunohistochemistry (IHC) in tissue microarrays of glioblastoma; A: This is a diagram showing the protein expression level of PIP4K2A in the tumor and non-tumor tissues of each patient through TissueFAX; B: A diagram of quantification of the expression level of the PIP4K2A protein measured in Figure A; C: This is a graph showing the expression level of PIP4K2A protein in the tumor and non-tumor tissues of 202 patients; D: It is a figure measuring the expression level of PIP4K2A protein in tumor and non-tumor tissues in 68 patient tissues.
4 is a diagram confirming that the cell growth and stemness are significantly decreased by overexpressing PIP4K2A in cells derived from glioblastoma patients; A: As a result of comparing cell growth after overexpressing PIP4K2A in cells derived from glioblastoma patients, this is a diagram showing that cell growth is inhibited in cells overexpressing PIP4K2A; B: As a result of comparing stem cell properties after overexpressing PIP4K2A in cells derived from glioblastoma patients, it is a diagram showing that stem cell features are suppressed in cells overexpressing the PIP4K2A gene; C: It is a result of numerically comparing the stem cell properties measured in Fig. B; D: Figure C is a graph showing the number of stem cell properties measured in Figure C.
5 is a view confirming that cancer growth is significantly reduced when PIP4K2A is overexpressed using a glioblastoma animal model (in vivo competition assay). A: Cells that overexpress PIP4K2A in glioblastoma patient-derived cells are made red, and cells that are not green are made to be injected 1:1 into an animal model; B: A diagram showing the results of the PIP4K2A protein overexpression experiment using Western blot; C: As a result of comparing the color of tumor cells obtained after the animal model experiment, it is a diagram confirming that the distribution of cells (green color) in which the PIP4K2A gene is not overexpressed is greater; D: It is a figure measured by quantifying the result shown in Figure C; E: As a result of comparing the color of tissues from animal models, it is a diagram showing a higher distribution of cells (green) that do not overexpress PIP4K2A; F: This is a diagram confirming the distribution of cells in an animal model tissue for the results shown in Figure E.
6 is a result of confirming that phosphorylation of AKT and S6K decreases when PIP4K2A is overexpressed in cells derived from glioblastoma patients; A: A diagram showing the results of an immunofluorescence assay; B: A diagram showing the results of Western blotting; C: It is a graph quantitatively showing the Western blotting results.
Fig. 7 is a diagram confirming that the expression of the regulatory (p85) and catalytic (p110) subunits of PI3K is decreased when PIP4K2A is overexpressed in cells derived from glioblastoma patients; A: A diagram showing the results of Western blotting; B: It is a graph quantitatively showing the Western blotting results; C: It is a figure showing the result of an immunofluorescence assay.
Fig. 8 is a result of confirming that the reduction of p85 expression in cells derived from glioblastoma patients using MG-132, a proteasome inhibitor, is not simply reduced by expression but by protein degradation; B to E are the results of confirming through the relative number of cells (B) and Western blotting that p85 degradation due to overexpression of PIP4K2A using p85 shRNA, Cbl shRNA, or MG-132 is cbl dependent.
Figure 9 shows that PIP4K2A inhibits cancer growth in animal models using glioblastoma patient-derived animal models and PIK3CA activating mutations (E545K), relative number of cells (A) and number of survival days (B), and tissue examination ( This is the drawing confirmed by C).
Fig. 10 is a diagram showing the expression of the regulatory (p85) and catalytic (p110) subunits of PI3K according to the expression of PIP4K2A using Immunohistochemistry (IHC) in glioblastoma patient tissues (Tissue microarrays) (A); B: This is a diagram of the expression of the regulatory (p85) and catalytic (p110) subunits of PI3K according to the expression of PIP4K2A in the tissues of 68 glioblastoma patients.
FIG. 11 is a result of verifying that the phenomenon that PIP4K2A promotes the degradation of the regulatory (p85) and catalytic (p110) subunits of PI3K is found only in patients with glioblastoma in which the PTEN gene has been deleted, and the PTEN gene is wild- In the type of glioblastoma, even when PIP4K2A is expressed, AKT and S6K phosphorylation and p85 and p110 proteins are not changed.
Figure 12 is a patient with high PIP4K2A expression without PTEN in the TCGA dataset (copy number), where PIP4K2A binds to p85 and PTEN interferes with this binding (A and B) using a co-immunoprecipitation method. Is a diagram showing that the survival rate is higher than that of the patient group with low PIP4K2A expression without PTEN (C).
FIG. 13 is a diagram (B) showing that mimic TAT-peptide (A) of PIP4K2A binds to the regulatory (p85) subunit of PI3K and promotes degradation through Western blotting.
14 is a diagram illustrating inhibition of cell growth and phosphorylation of AKT using the PIP4K2A mimic TAT-peptide of FIG. It is a diagram verified through immunofluorescence that the expression levels of pAKT and pS6K are decreased; B: A diagram showing the results of analysis of the decrease in pAKT protein by Western blotting; C: A diagram showing that the peptide inhibits cell growth and stem cell characteristics; D: It is a figure which quantified that a peptide inhibits cell growth.
FIG. 15 is a result of confirming that genes related to EGFR are higher in patients with low PIP4K2A expression through Gene Set Enrichment Analysis (GSEA) analysis of A: Glioblastoma patients with low and high PIP4K2A gene expression; B, C: A diagram confirming the results of correlation analysis between the PIP4K2A gene and the EGFR gene in glioblastoma patients.
Fig. 16 is a diagram showing the lowest expression in the Classical patient group as a result of comparing the expression of the PIP4K2A gene in four subtypes of A: glioblastoma patients; B: A diagram showing the results of correlation analysis between PIP4K2A expression and Classical subtype scores in glioblastoma patients; C: In glioblastoma patients, when the overall gene expression levels of the patients with high and low PIP4K2A gene expression levels were analyzed, it was confirmed that the Classical subtype score was high in the patients with low PIP4K2A expression; D: A diagram showing the results of correlation analysis in which the expression of PIP4K2A and the expression is opposite to the Classical score in glioblastoma patients.
Fig. 17 is a diagram showing that the expression of the PIP4K2B gene in four subtypes of glioblastoma patients is not correlated with the subtype; A: A diagram showing the RNA expression of the gene of PIP4K2B in four subtypes of glioblastoma patients; B: A diagram showing the DNA expression of the PIP4K2B gene in four subtypes of glioblastoma patients.

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

Example 1. Selection of novel tumor suppressor genes through in vivo RNAi screening technique

To screen for new tumor suppressor genes, in vivo RNAi screening techniques were performed. Specifically, through the genome analysis of glioblastoma patients in the public database, 23 cancer suppressor gene candidates were selected and RNAi pools were prepared. The prepared pool was injected into cells derived from various types of brain tumor patients, and the injected cells were injected in an in vivo mouse model to compare shRNA amplification properties in the tumor model. Thereafter, the new tumor suppressor gene PIP4K2A was selected through the highest amplification shRNA, and the results are shown in FIG. 1.

As shown in Fig. 1, as novel tumor suppressor candidates, PIP4K2A and MXI1 were selected, and an experiment for PIP4K2A was performed in subsequent experiments.

Example 2. Analysis of expression of PIP4K2A in cells derived from glioblastoma patients

To analyze the expression of PIP4K2A in cells derived from glioblastoma patients, Oncomine dataset, Philips data set, TCGA dataset CNA, and RNA seq data were used.

Specifically, the expression of PIP4K2A was measured by comparing the expression level of the gene of PIP4K2A from the public database, and the results are shown in FIG. 2.

In addition, the expression of PIP4K2A was additionally measured through the IHC method. Specifically, the expression of PIP4K2A was measured by measuring the expression level of the antibody through the antibody targeting PIP4K2A in the patient tissue, and the results are shown in FIG. 3.

In addition, in order to confirm the relationship between PIP4K2A overexpression and cell growth, a vector containing the PIP4K2A gene sequence was prepared, and then the vector was directly transformed into cells, and the expression levels of the PIP4K2A gene in each cell were compared. For cell growth, after the vector containing the PIP4K2A gene sequence was introduced, the cells were cultured for 5 to 7 days, and then the growth of the cells was confirmed using a substance called ATPLite, and the results are shown in FIG. 4.

As shown in Fig. 2, the survival rate was very low in the patient group with low PIP4K2A expression or the number of copies, and the expression of PIP4K2A was expressed in glioblastoma patient-derived cells and normal neural progenitor cells (NPC). Appeared low.

In addition, as shown in FIG. 3, the expression of PIP4K2A was significantly lower in cancer tissues than in normal tissues, and as a result of overexpressing PIP4K2A as shown in FIG. 4, cell growth and stemness were significantly increased. It was confirmed that it decreased.

As a result of the above, it can be seen that PIP4K2A plays a role as a tumor suppressor gene.

Example 3. Analysis of mechanism of action through overexpression of PIP4K2A in cells derived from glioblastoma patients

The phenomenon that occurs when PIP4K2A is overexpressed in cells derived from glioblastoma patients was confirmed by Western blotting and IHC methods.

Western blotting and IHC methods measured the amount of PIP4K2A gene expression using an antibody targeting the PIP4K2A gene after accumulating the protein of the cell, and the results are shown in FIGS. 6 to 8, 10, and 11 and 12. .

As shown in FIG. 6, it was confirmed that phosphorylation of AKT and S6K decreased when PIP4K2A was overexpressed. In addition, as shown in FIG. 7, when overexpressing PIP4K2A, it was confirmed that the expression of the regulatory (p85) and catalytic (p110) subunits of PI3K decreased. As shown in Fig. 8, it was confirmed that the decrease in the expression of p85 was not caused by simple expression reduction, but by protein degradation. In addition, it was found that the degradation of p85 due to overexpression of PIP4K2A was cbl-dependent.

In addition, as shown in FIG. 10, it was found that the expression of p85 and p110 proteins was reduced in patient tissues with high PIP4K2A expression.

In addition, as shown in Fig. 11, PIP4K2A promotes the degradation of the regulatory (p85) and catalytic (p110) subunits of PI3K, but even expressing PIP4K2A derived from some glioblastoma patients, phosphorylation of AKT and S6K and proteins of p85 and p110 It was confirmed that there was no change. Based on these results, as shown in FIG. 12, PIP4K2A and p85 bind, and PTEN interferes with their binding, so that patients with high PIP4K2A expression without PTEN have a higher survival rate than patients with low PIP4K2A expression without PTEN. Could be confirmed.

Example 4. Confirmation of cancer growth inhibitory ability through overexpression of PIP4K2A in animal models

The ability to inhibit cancer growth through overexpression of PIP4K2A in an animal model was confirmed by the following method.

First, the animal model was produced by injecting cells directly into the brain of a mouse by intracranial injection. In order to overexpress PIP4K2A, a vector containing the PIP4K2A sequence was introduced into the cells as described above, and then the cells were directly injected into the mouse brain. Subsequently, the cancer cell growth inhibitory ability was confirmed by measuring the survival rate period of the mouse, and the results are shown in FIGS. 5 and 9.

As shown in FIGS. 5 and 9, it was confirmed that cancer growth significantly decreased and the survival rate period of the mouse was increased when PIP4K2A was overexpressed using an animal model derived from glioblastoma (in vivo competition assay).

Example 5. Confirmation of cancer growth inhibitory ability using active fragment and cell penetrating peptide

SEQ ID NOs: 2 to 5 were used as the active fragment of PIP4K2A, and TAT was used as the cell penetrating peptide.

Thereafter, these cells were treated with glioblastoma patient-derived cells, and the expression levels were measured using antibodies targeting PIP4K2A or p85 subunit as before to determine whether they bind to the p85 subunit and inhibit cell growth. Is shown in FIGS. 13 and 14.

13 and 14, it was confirmed that the mimic TAT-peptide of PIP4K2A binds to the regulatory (p85) subunit of PI3K, promotes degradation, and inhibits cell growth and phosphorylation of AKT.

Example 6. Analysis of PIP4K2A gene expression in glioblastoma patients

It was confirmed how the gene expression of PIP4K2A actually appeared in patients with glioblastoma.

Specifically, GSEA analysis was performed through an algorithm used in GSEA by measuring the expression level of PIP4K2A RNA, and the results are shown in FIG. 15.

In addition, the expression analysis of PIP4K2A in the four subtypes of glioblastoma patients was performed by determining the subtypes of glioblastoma patients previously described in TCGA, and determining the expression level of PIP4K2A expressed in each group of patients with the quantified expression level of the gene already described in TCGA. Compared and performed, the results are shown in FIGS. 16 and 17.

As shown in Fig. 15, it was confirmed that genes related to EGFR appear higher in the patient group with low PIP4K2A expression.

In addition, as shown in Figs. 16 and 17, when analyzing the total gene expression level of the patient group with high and low PIP4K2A gene expression in glioblastoma patients, it was confirmed that the Classical subtype score was high in the patient group with low PIP4K2A expression, It was confirmed that the expression of the PIP4K2B gene in the four subtypes of glioblastoma patients was not correlated with the subtype.

<110> Samsung Life Public Welfare Foundation <120> Tumor suppressor gene PIP4K2A and uses thereof <130> PN119066 <160> 10 <170> KopatentIn 2.0 <210> 1 <211> 406 <212> PRT <213> Homo sapiens <400> 1 Met Ala Thr Pro Gly Asn Leu Gly Ser Ser Val Leu Ala Ser Lys Thr 1 5 10 15 Lys Thr Lys Lys Lys His Phe Val Ala Gln Lys Val Lys Leu Phe Arg 20 25 30 Ala Ser Asp Pro Leu Leu Ser Val Leu Met Trp Gly Val Asn His Ser 35 40 45 Ile Asn Glu Leu Ser His Val Gln Ile Pro Val Met Leu Met Pro Asp 50 55 60 Asp Phe Lys Ala Tyr Ser Lys Ile Lys Val Asp Asn His Leu Phe Asn 65 70 75 80 Lys Glu Asn Met Pro Ser His Phe Lys Phe Lys Glu Tyr Cys Pro Met 85 90 95 Val Phe Arg Asn Leu Arg Glu Arg Phe Gly Ile Asp Asp Gln Asp Phe 100 105 110 Gln Asn Ser Leu Thr Arg Ser Ala Pro Leu Pro Asn Asp Ser Gln Ala 115 120 125 Arg Ser Gly Ala Arg Phe His Thr Ser Tyr Asp Lys Arg Tyr Ile Ile 130 135 140 Lys Thr Ile Thr Ser Glu Asp Val Ala Glu Met His Asn Ile Leu Lys 145 150 155 160 Lys Tyr His Gln Tyr Ile Val Glu Cys His Gly Ile Thr Leu Leu Pro 165 170 175 Gln Phe Leu Gly Met Tyr Arg Leu Asn Val Asp Gly Val Glu Ile Tyr 180 185 190 Val Ile Val Thr Arg Asn Val Phe Ser His Arg Leu Ser Val Tyr Arg 195 200 205 Lys Tyr Asp Leu Lys Gly Ser Thr Val Ala Arg Glu Ala Ser Asp Lys 210 215 220 Glu Lys Ala Lys Glu Leu Pro Thr Leu Lys Asp Asn Asp Phe Ile Asn 225 230 235 240 Glu Gly Gln Lys Ile Tyr Ile Asp Asp Asn Asn Lys Lys Val Phe Leu 245 250 255 Glu Lys Leu Lys Lys Asp Val Glu Phe Leu Ala Gln Leu Lys Leu Met 260 265 270 Asp Tyr Ser Leu Leu Val Gly Ile His Asp Val Glu Arg Ala Glu Gln 275 280 285 Glu Glu Val Glu Cys Glu Glu Asn Asp Gly Glu Glu Glu Gly Glu Ser 290 295 300 Asp Gly Thr His Pro Val Gly Thr Pro Pro Asp Ser Pro Gly Asn Thr 305 310 315 320 Leu Asn Ser Ser Pro Pro Leu Ala Pro Gly Glu Phe Asp Pro Asn Ile 325 330 335 Asp Val Tyr Gly Ile Lys Cys His Glu Asn Ser Pro Arg Lys Glu Val 340 345 350 Tyr Phe Met Ala Ile Ile Asp Ile Leu Thr His Tyr Asp Ala Lys Lys 355 360 365 Lys Ala Ala His Ala Ala Lys Thr Val Lys His Gly Ala Gly Ala Glu 370 375 380 Ile Ser Thr Val Asn Pro Glu Gln Tyr Ser Lys Arg Phe Leu Asp Phe 385 390 395 400 Ile Gly His Ile Leu Thr 405 <210> 2 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Fragments of PIP4K2A <400> 2 Gly Ser Ser Val Leu Ala Ser Lys Thr Lys Thr Lys Lys Lys His Phe 1 5 10 15 Val Ala Gln Lys Val 20 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Fragments of PIP4K2A <400> 3 Thr His Pro Val Gly Thr Pro Pro Asp Ser Pro Gly Asn Thr Leu Asn 1 5 10 15 Ser Ser Pro Pro Leu 20 <210> 4 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Fragments of PIP4K2A <400> 4 Val Glu Arg Ala Glu Gln Glu Glu Val Glu Cys Pro Leu Ala Pro Gly 1 5 10 15 Glu Phe <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Fragments of PIP4K2A <400> 5 Lys Val Asp Asn His Leu Phe Asn Lys Glu Asn 1 5 10 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Cell penetrating peptide: TAT <400> 6 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Cell penetrating peptide: Penetratin <400> 7 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Cell penetrating peptide: polyarginine <400> 8 Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Cell penetrating peptide: polylysine <400> 9 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Cell penetrating peptide: LMWP (low molecular weight protamine) <400> 10 Val Ser Arg Arg Arg Arg Arg Arg Gly Gly Arg Arg Arg Arg 1 5 10

Claims (13)

PIP4K2A (Phosphatidylinositol-5-phosphate 4-kinase type-2 alpha) protein or a pharmaceutical for the prevention and treatment of glioblastoma containing an active fragment containing any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5 as an active ingredient Ever composition. The pharmaceutical composition according to claim 1, wherein the PIP4K2A protein comprises the amino acid sequence of SEQ ID NO: 1. delete The pharmaceutical composition according to claim 1, wherein the cell penetrating peptide is linked to the PIP4K2A protein or active fragment. The method of claim 4, wherein the cell penetrating peptide is TAT (SEQ ID NO: 6: YGRKKRRQRRR), penetratin (Penetratin; SEQ ID NO: 7: RQIKIWFQNRRMKWKK), polyarginine (SEQ ID NO: 8: RRRRRRR), polylysine (SEQ ID NO: 9:KKKKKKKKKK), LMWP (low molecular weight protamine; SEQ ID NO: 10: VSRRRRRRGGRRRR), protamine (protamine) fragment and antenna pediatric (Antennapedia, ANTP) any one selected from the group consisting of a pharmaceutical composition. The pharmaceutical composition of claim 1, wherein the glioblastoma is PTEN (Phosphatase and tensin homolog) deleted. The method according to claim 1, wherein the glioblastoma is any one subtype selected from the group consisting of a classical glioblastoma, a proneural glioblastoma, a neural glioblastoma, and a mesenchymal glioblastoma. The pharmaceutical composition that is. The pharmaceutical composition according to claim 1, which is administered in combination with an additional anticancer agent. The pharmaceutical composition of claim 1, wherein the PIP4K2A protein or active fragment binds to p85 of PI3K to promote degradation of p85 and p110. A vector comprising a polynucleotide encoding an active fragment comprising an amino acid sequence selected from the group consisting of PIP4K2A (Phosphatidylinositol-5-phosphate 4-kinase type-2 alpha) protein or SEQ ID NO: 2 to 5, or the vector Cancer prevention and treatment pharmaceutical composition containing the cells containing as an active ingredient. The pharmaceutical composition of claim 10, wherein the vector is a linear DNA, a plasmid DNA or a recombinant viral vector. delete delete

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