KR20200009503A - Biomarker for predicting therapeutic response of pancreatic cancer to PTEN inhibitor and use thereof - Google Patents

Abstract

The present invention relates to a biomarker for predicting the therapeutic response of a phosphatase and tensin homologue (PTEN) inhibitor to pancreatic cancer, and to a use thereof. More specifically, where pancreatic cell lines are divided into the cell line whose growth, migration, and infiltration are accelerated, and the cell line whose growth, migration, and infiltration are suppressed when the activity of PTEN protein is inhibited, the present invention has confirmed an increase in the expression of PLK1 protein in the cell line showing anticancer effects. Accordingly, the PLK1 protein may be beneficially used as a biomarker for predicting the therapeutic response of a PTEN inhibitor to pancreatic cancer, and furthermore, in pancreatic cancer with increased expression of PLK1 protein, the PTEN inhibitor may be beneficially used as a composition for treating pancreatic cancer.

Description

Biomarker for predicting therapeutic response of pancreatic cancer to PTEN inhibitor and use approximately}

The present invention relates to a biomarker for predicting pancreatic cancer therapeutic reactivity against a phosphatase and tensin homologue (PTEN) inhibitor and its use.

Pancreatic cancer is the 14th most common cancer in the world, with a significant increase in the frequency of cancer and the fourth largest cause of cancer deaths in the United States. Pancreatic cancer is not specific for early symptoms, and clinical symptoms such as weakness, loss of appetite, and weight loss occur after systemic metastasis. Therefore, regular diagnosis is required.

Pancreatic cancer has a poor prognosis, with only 1 to 4% of patients having a 5-year survival rate after surgery and a median survival of 5 months. Treatment is largely dependent on chemotherapy because 80-90% of patients are found in a state where curative resection is not possible to expect a cure at diagnosis. To date, anti-cancer drugs known to be effective in pancreatic cancer include 5-fluorouracil, gemcitabine, and tarceva, but the effects are extremely low, and the response rate is only about 15%. Therefore, the development of more effective early diagnosis and treatment is urgently needed to improve the prognosis of pancreatic cancer patients.

PTEN (phosphatase and tensin homologue) is a protein consisting of 403 amino acids, also called mutated in multiple advanced cancers 1 (MMAC1) or TGF-βregulated and epithelial cell enriched phosphatase 1 (TEP1). PTEN has the function of dephosphorylation of phosphatidylinositol-3,4,5-trisphosphate, which activates Phosphoinositide-dependent kinase (PDK) and protein kinase B (AKT). Loss of PTEN function increases the phosphatidylinositol-3,4,5-triphosphate, activating the PI3K ()-AKT pathway. In other words, PTEN acts on the amplification signaling cascade induced by PI3K to regulate tumor proliferation, cell survival, cell migration, cell invasion, etc. It is known to function (Besson A et al , Eur J Biochem , 263: 605, 1990).

However, it has recently been reported that compounds that inhibit PTEN can be applied to the treatment of liver cancer by inhibiting cell viability, proliferation and colony formation in liver cancer cell lines and inducing cell aging (Giuseppa Augello, et al. , Cell Cycle , 15, No. 4, 573-583, 2016). In other words, it was confirmed that inhibition of PTEN could rather inhibit the PI3K-AKT pathway and that PTEN inhibitor could rather inhibit the proliferation of cancer cells.

On the other hand, PLK1 (Polo-like kinase 1) protein is involved in the initiation of mitosis or spindle formation to regulate the cell cycle and overexpressed in various tumors, and has been studied as a cancer diagnostic marker and therapeutic target. . In this regard, it has been reported that PLK1 protein acts as an upstream signal of PTEN protein and phosphorylates PTEN protein and inhibits tumor suppressor function (Li, Z. et al ., Molecular and cellular biology , 34: 3642-3661, 2014 ).

Therefore, based on the hypothesis that PTEN may not maintain tumor suppressor function in a situation where the balance of homeostasis in the body is not maintained due to the occurrence of cancer, the present inventors confirmed the therapeutic responsiveness to PTEN inhibitor in normal cell lines and pancreatic cancer cell lines. Increasing expression of PLK1 in pancreatic cancer, which is highly responsive to PTEN inhibitors, suggests that PLK1 gene or protein can be used as a co-marker to predict drug reactivity in the treatment of pancreatic cancer with PTEN inhibitor. The present invention was completed by confirming.

It is an object of the present invention to provide a composition for predicting pancreatic cancer therapeutic responsiveness to a PTEN inhibitor comprising a PLK1 protein or a detection reagent specifically binding to a gene encoding the same and a use thereof.

In order to achieve the above object, the present invention provides a composition for predicting pancreatic cancer therapeutic response to a PTEN inhibitor comprising a detection reagent that specifically binds to a PLK1 protein or a gene encoding the same.

In addition, the present invention provides a kit for predicting pancreatic cancer treatment reactivity to a PTEN inhibitor comprising the composition.

In addition, the present invention comprises the steps of 1) measuring the expression level of PLK1 protein or gene encoding the same from a sample isolated from an individual with pancreatic cancer; And 2) comparing the expression level of the protein of the step 1) or the gene encoding the same with the expression level of a normal individual, thereby providing information for predicting pancreatic cancer treatment responsiveness to a PTEN inhibitor.

The present invention also provides a pharmaceutical composition for preventing or treating pancreatic cancer containing an inhibitor of expression of PTEN gene or an inhibitor of activity of PTEN protein as an active ingredient.

The present invention also provides a dietary supplement for the prevention or improvement of pancreatic cancer containing an inhibitor of PTEN gene expression or an inhibitor of PTEN protein activity as an active ingredient.

In addition, the present invention comprises the steps of 1) measuring the expression level of PLK1 protein or gene encoding the same from a blood sample; And 2) comparing the expression level of the protein of the step 1) or the gene encoding the same with the expression level of a normal individual, the method for providing pancreatic cancer diagnostic information.

In addition, the present invention provides a pancreatic cancer diagnostic kit for blood comprising a detection reagent that specifically binds to a PLK1 protein or a gene encoding the same.

In the present invention, when the pancreatic cancer cell line is divided into a cell line that promotes growth, metastasis and invasion when the PTEN protein activity is inhibited, and a cell line where growth, metastasis and invasion is inhibited, the expression of PLK1 protein is increased in the cell line showing anticancer effect It was confirmed that it is done. Therefore, the PLK1 protein is a biomarker for predicting pancreatic cancer treatment responsiveness to a PTEN inhibitor, and the PTEN inhibitor may be usefully used as a composition for treating pancreatic cancer in pancreatic cancer in which the expression of the PLK1 protein is increased.

1 is a diagram confirming the expression level of PTEN mRNA in various normal tissues or pancreatic cancer tissues of human males.
2 is a diagram confirming the expression level of PTEN mRNA in various normal tissues or pancreatic cancer tissues of a human female.
3 is a diagram confirming the expression level of PTEN protein in various normal or cancerous tissues of humans.
Figure 4 is a diagram confirming the expression level of PTEN protein in normal pancreatic cells (H6c7) or pancreatic cancer cells (AsPC-1, Capan-2, SNU-213, CFPAC-1, Panc-1, Miapaca-2).
5 is a diagram confirming the change in survival rate of normal pancreatic cells (H6c7) according to SF1670 treatment.
Figure 6 is a diagram confirming the degree of movement of normal pancreatic cells (H6c7) in accordance with SF1670 treatment.
7 is a diagram confirming the survival rate change of pancreatic cancer cells (AsPC-1, Capan-2, SNU-213, CFPAC-1, Panc-1, Miapaca-2) following SF1670 treatment.
8 is a diagram confirming the degree of migration of pancreatic cancer cells (AsPC-1, Capan-2, SNU-213, CFPAC-1, Panc-1, Miapaca-2) according to SF1670 treatment.
Figure 9 shows the change in survival rate of normal pancreatic cells (H6c7) or pancreatic cancer cells (AsPC-1, Capan-2, SNU-213, CFPAC-1, Panc-1, Miapaca-2) according to the inhibition of PTEN protein expression using siRNA This is the figure.
Figure 10 shows the degree of migration of normal pancreatic cells (H6c7) or pancreatic cancer cells (AsPC-1, Capan-2, SNU-213, CFPAC-1, Panc-1, Miapaca-2) according to the inhibition of PTEN protein expression using siRNA This is the figure.
11 is a diagram confirming the change in the expression level of proteins related to the signaling system in normal pancreatic cells (H6c7) or pancreatic cancer cells (AsPC-1, Panc-1) according to the inhibition of PTEN protein expression using siRNA.
12 is a diagram confirming the expression level of PLK1 protein in normal pancreatic cells (H6c7) or pancreatic cancer cells (AsPC-1, Capan-2, SNU-213, CFPAC-1, Panc-1, Miapaca-2).
Figure 13 is a diagram confirming the relative expression of PLK1 protein in normal pancreatic cells (H6c7) or pancreatic cancer cells (AsPC-1, Capan-2, SNU-213, CFPAC-1, Panc-1, Miapaca-2).
14 is a diagram showing the expression level of PLK1 protein in normal pancreatic cells (H6c7) or pancreatic cancer cells (AsPC-1, Panc-1) according to the inhibition of PTEN protein expression using siRNA.
15 is a diagram confirming the volume change of pancreatic cancer tumor (Panc-1) according to SF1670 treatment (DPI: days post inoculation).
16 is a diagram confirming the weight change of pancreatic cancer tumor (Panc-1) according to SF1670 treatment (DPI: days post inoculation).
17 is a view showing the volume change of pancreatic cancer tumor (AsPC-1) following SF1670 treatment (DPI: days post inoculation).
18 is a diagram confirming the weight change of pancreatic cancer tumor (AsPC-1) following SF1670 treatment (DPI: days post inoculation).
19 is a diagram confirming the change in survival rate of pancreatic cancer patients according to PTEN gene expression in the PAAD-US-TCGA database.
20 is a diagram confirming the change in survival rate of pancreatic cancer patients according to PTEN gene expression in the GSE78229 database.
21 is a diagram confirming the change in survival rate of pancreatic cancer patients according to PTEN gene expression in the GSE62452 database.
Figure 22 is a diagram confirming the expression level of PLK1 protein according to the PTEN gene expression in the GSE62452 database.
Figure 23 is a diagram confirming the expression level of PLK1 protein according to PTEN gene expression in the GSE78229 database.
24 is a diagram confirming the change in survival rate of pancreatic cancer patients according to PTEN gene expression in PACA-AU (TNM: T = 3) database.
25 is a diagram confirming the change in survival rate of pancreatic cancer patients according to PTEN gene expression in PACA-AU (Grade 2) database.
FIG. 26 shows changes in survival rate of pancreatic cancer patients according to PTEN gene expression in the GSE21501 database.
Figure 27 is a diagram confirming the expression level of PLK1 protein according to PTEN gene expression in PACA-AU (TNM: T = 3) database.
28 is a diagram confirming the expression level of PLK1 protein according to PTEN gene expression in PACA-AU (Grade 2) database.
29 is a diagram confirming the expression level of PLK1 protein according to PTEN gene expression in GSE21501 database.
30 is a diagram showing the content of soluble PLK1 protein in the blood of normal people (N1 to N12) or blood of pancreatic cancer patients (T1 to T11). Among the pancreatic cancer patients, T7 was stage 1, T8 was stage 2, T9 and T10 stage 3, and T11 stage 4 pancreatic cancer patients.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for predicting pancreatic cancer treatment responsiveness to a PTEN inhibitor, including a detection reagent that specifically binds to a Polo-like kinase 1 (PLK1) protein or a gene encoding the same.

The PLK1 protein is also called STPK13 (Serine / threonine-protein kinase 13) protein and is a serine / threonine kinase that acts on cell dividers. Human PLK1 protein is a 66kDa-sized protein composed of 603 amino acids, which is involved in the functional maturation of centrosomes and is an important protein for G2 / M cell cycle switching. It is mainly expressed in tissues. In addition, it has been reported that expression is increased in cancer tissues such as non-small cell lung cancer, head and neck cancer, laryngeal cancer, breast cancer, liver cancer, endometrial cancer, colon cancer, ovarian cancer, pancreatic cancer, prostate cancer. The PLK1 protein may be a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 1 and may be a fragment thereof. In addition, the gene encoding the PLK1 protein may be a polynucleotide consisting of the nucleotide sequence described in SEQ ID NO: 2.

The PTEN protein has been reported to function as a tumor suppressor protein by dephosphorylation of phosphatidylinositol-3,4,5-triphosphate that activates enzymes that mediate the proliferation of cells such as PDK and AKT. Recently, however, it has been reported that compounds that inhibit PTEN can be applied to the treatment of liver cancer by inhibiting cell viability, proliferation and colony formation in liver cancer cell lines and inducing senescence of cells, wherein the PTEN protein is set forth in SEQ ID NO: 3. It may be a polypeptide consisting of an amino acid sequence and a fragment thereof. The gene encoding the PTEN protein may be a polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO: 4.

The PTEN inhibitor may be an expression inhibitor of PTEN gene or an activity inhibitor of PTEN protein. The inhibitor of expression of the PTEN gene may include any substance known in the art as long as it inhibits the expression or translation of the mRNA encoding the PTEN protein or promotes the degradation of the mRNA, and may chemically synthesize or in vitro transcription. It can be prepared by a variety of methods such as synthesis using. The expression inhibitor is any one selected from the group consisting of antisense nucleotides (combined antisense nucleotide), siRNA (small interfering RNA), shRNA (short hairpin RNA) and ribozyme complementary to the polynucleotide constituting the PTEN gene It may include one or more.

In addition, the activity inhibitor of the PTEN protein is any one selected from the group consisting of compounds, peptides, peptide mimetics, matrix analogs, aptamers and antibodies that complementarily bind to the polypeptide constituting the PTEN protein. It may include one or more. In one example, the activity inhibitor of the PTEN protein may be a compound represented by the following formula (1), (2), (3) or (4):

[Formula 1]

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[Formula 2]

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,

[Formula 3]

,

,

[Formula 4]

.

.

The detection reagent in the composition according to the present invention may be any one or more selected from the group consisting of antibodies, antibody fragments, aptamers, primers, probes and anti-sense nucleotides. If the PLK1 to be detected is a protein, the detection reagent may be any one or more selected from the group consisting of antibodies, antibody fragments and aptamers. If the PLK1 is a gene, the detection reagent is selected from the group consisting of primers, probes and antisense nucleotides. It may be any one or more.

The antibody may be a monoclonal antibody, polyclonal antibody or recombinant antibody. Antibodies to a particular protein can be readily prepared using techniques well known in the art, provided that the sequence of the protein is known. The antibody fragment may comprise a functional fragment of an antibody molecule. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and may be, but is not limited to, Fab, F (ab '), F (ab') 2 and Fv.

The monoclonal antibodies can be prepared using hybridoma methods, or phage antibody library techniques. Generally, hybridoma cells secreting monoclonal antibodies can be made by fusing cancer cell lines with immune cells isolated from immunologically suitable host animals, such as mice injected with antigenic proteins. These two populations of cell fusion can be fused using polyethylene glycol or the like and antibody-producing cells can be propagated by standard culture methods. Subcloning can be performed using limiting dilution, and a homogeneous cell population can be obtained and then hybridoma cells capable of producing antibodies specific for the antigen can be prepared by culturing in large quantities in vitro or in vivo. Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The polyclonal antibody can be prepared by injecting a biomarker protein or fragment thereof that is an immunogen into an external host. The external host can be a mammal such as a mouse, rat, sheep, rabbit. When the immunogen is injected by intramuscular, intraperitoneal or subcutaneous injection methods, it can be administered with an adjuvant to increase antigenicity. Thereafter, blood may be collected periodically from an external host to obtain serum showing improved titer and specificity for the antigen, from which the antibody may be isolated and purified.

The aptamer refers to a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and has a characteristic of binding to a target molecule with high affinity and specificity. It can bind and block its function, so it can be regarded as a kind of chemical antibody that replaces a single antibody. In addition, the aptamer may be obtained by separating oligomers having high affinity and selectivity for a specific chemical or biological molecule by using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). .

The primer is a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with complementary templates and serving as a starting point for template strand copying. It consists of a pair of forward and reverse primers, usually 7-50, specifically 10-30 nucleic acids. Primers can initiate DNA synthesis in the presence of enzymes, reagents and four different nucleoside triphosphates for polymerization in appropriate buffers and temperatures. The primer is complementary to the gene encoding the PLK1 protein of the present invention, any primer designed to amplify it can be used.

The probe is a nucleic acid fragment corresponding to short to several hundreds of bases, which can form specific binding with DNA or RNA, and can be used to confirm the presence or absence of specific DNA or RNA. The probe may be prepared in the form of oligonucleotide probe, single-stranded DNA probe, double-stranded DNA probe, RNA probe, etc., and may be labeled with biotin, FITC, rhodamine, digoxigenin, or the like, or labeled with radioisotopes. have.

The antisense nucleotide means to bind (hybridize) the complementary sequencing of DNA, immature-mRNA or mature mRNA, as defined in Watson-click base pair, to disrupt the flow of genetic information as a protein in DNA. In addition, since the antisense nucleotides are long chains of monomer units, they can be easily synthesized for the target RNA sequence.

The detection reagent may further include a ligand that can specifically bind to the detection reagent. The ligand may be a conjugate labeled with a chromophore, a fluorescent substance, a radioisotope or a colloid, and a ligand treated with streptavidin or avidin.

The composition for predicting pancreatic cancer treatment reactivity to the PTEN inhibitor may include distilled water or a buffer solution that stably maintains their structure in addition to the detection reagents described above.

In addition, the present invention provides a kit for predicting pancreatic cancer treatment reactivity to a PTEN inhibitor comprising the composition.

The composition included in the kit for predicting pancreatic cancer treatment reactivity to the PTEN inhibitor of the present invention may have the characteristics as described above. For example, the composition may include a detection reagent that specifically binds to a PLK1 protein or a gene encoding the same.

The kit not only makes it possible to determine whether a treatment responsiveness is administered to a patient with pancreatic cancer, but also monitors the patient's response to the treatment and alters the treatment according to the outcome and the prognosis of the patient with pancreatic cancer. Make it predictable.

The kit for predicting pancreatic cancer treatment reactivity for the PTEN inhibitor may be prepared by conventional methods known to those skilled in the art, and may further include a buffer, a stabilizer, an inactive protein, and the like.

The kit can be used for ultra-fast screening through fluorescence, which is performed by detecting the fluorescence of a fluorescent substance attached as a detector to detect the amount of detection reagent, or by radiation, which is performed by detecting radiation of a radioisotope attached as a detector. Throughput screening (HTS) system, surface plasmon resonance (SPR) method for measuring the plasmon resonance change of the surface in real time without labeling of the detector, or surface plasmon resonance imaging (SPRI) method for imaging and confirming the SPR system can be used.

In addition, the present invention comprises the steps of 1) measuring the expression level of PLK1 protein or gene encoding the same from a sample isolated from an individual with pancreatic cancer; And 2) comparing the expression level of the protein of the step 1) or the gene encoding the same with the expression level of a normal individual, thereby providing information for predicting pancreatic cancer treatment responsiveness to a PTEN inhibitor.

The sample of step 1) may include a biological sample capable of identifying a disease-specific polypeptide that can be distinguished from a normal state such as urine, blood, serum, plasma or tissue. In one example, the sample may be blood.

In addition, the sample may be pretreated using a method such as anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, continuous extraction, centrifugation or gel electrophoresis.

The method may be characterized in that the pancreatic cancer treatment responsiveness to the PTEN inhibitor is good if the expression level of the PLK1 protein or gene encoding the same in the sample isolated from the individual with pancreatic cancer is higher than that of the normal individual.

The protein expression level of step 1) is enzyme-linked immunosorbent assay (ELISA), radioimmunoassays, sandwich enzyme immunoassay, Western blot, immunoprecipitation, immune tissue It can be measured by any one or more methods selected from the group consisting of chemical staining (immunohistochemistry) and fluoroimmunoassay.

Gene expression level of step 1) is reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (competitive RT-PCR), real-time reverse transcription polymerase chain reaction (real-time RT) -PCR), RNase protection assay, Northern blot and microarray can be measured by any one or more of the methods selected from the group consisting of.

In a specific embodiment of the present invention, the inventors of the present invention show that the PTEN protein is expressed in normal tissues and cancer tissues indiscriminately (FIGS. 1 to 4), and when the PTEN protein is inhibited in the pancreatic cancer cell line, a cell line and an anticancer effect appear. Cell lines are divided (Figs. 5 to 11), PLK1 protein is relatively strongly expressed in the cell line showing the anti-cancer effect (Fig. 12 to 13), and the expression level of PLK1 protein decreases when PTEN protein is inhibited in the cell line showing the anti-cancer effect (FIG. 14), the tumor size decreased when the PTEN was inhibited in the xenograft model transplanted with a cell line exhibiting an anticancer effect (FIGS. 15 to 18), and the higher the expression of the PTEN protein, the worse the prognosis. The expression level of the protein is high (Figs. 19 to 23), and the higher the expression of the PTEN protein, the lower the expression level of the PLK1 protein in the patient group. A negative (FIGS. 24-29) was confirmed. Therefore, the PLK1 protein may be usefully used as a biomarker for predicting pancreatic cancer treatment responsiveness to PTEN inhibitor.

The present invention also provides a pharmaceutical composition for preventing or treating pancreatic cancer containing an inhibitor of expression of PTEN gene or an inhibitor of activity of PTEN protein as an active ingredient.

The PTEN gene may include all of the polynucleotides consisting of any sequence known in the art, for example, may be a polynucleotide consisting of the nucleotide sequence described in SEQ ID NO: 4.

The inhibitor of expression of the PTEN gene may include any substance known in the art as long as it inhibits the mRNA of the gene encoding the PTEN protein, and may be prepared by various methods such as chemical synthesis or synthesis using in vitro transcription. Can be. The expression inhibitor is any one selected from the group consisting of antisense nucleotides (combined antisense nucleotide), siRNA (small interfering RNA), shRNA (short hairpin RNA) and ribozyme complementary to the polynucleotide constituting the PTEN gene It may include one or more.

The antisense nucleotide means to bind (hybridize) the complementary sequencing of DNA, immature-mRNA or mature mRNA, as defined in Watson-click base pair, to disrupt the flow of genetic information as a protein in DNA. In addition, since the antisense nucleotides are long chains of monomer units, they can be easily synthesized for the target RNA sequence.

The siRNA refers to a short double-stranded RNA capable of inducing interference of RNA through cleavage of specific mRNA. In addition, the siRNA is not limited to completely paired double-stranded RNA portion paired with RNA, mismatch (corresponding base is not complementary), bulge (there is no base corresponding to one chain), etc. It may also include a portion that is not paired by. The siRNA terminal structures can be either smooth or protruding ends as long as the expression of the target gene can be suppressed by RNA interference effects, and the adhesive end structures can be both 3 'and 5' terminal protruding structures.

The shRNA refers to a sequence of RNA that makes a robust hairpin turn, which can be used to silence gene expression through RNA interference. In addition, the shRNA can be delivered to the cell using a vector for cell introduction, such a vector can always be delivered to the daughter cells so that gene silencing can be inherited. The shRNA hairpin structure is broken down into siRNA by intracellular mechanisms, which are bound to RNA-induced silencing complexes, which can bind to and degrade mRNAs corresponding to siRNAs.

Ribozyme means an enzyme RNA molecule capable of catalyzing specific RNA cleavage. Specific hybridization of the target RNA complementary to the molecular sequence of the ribozyme may lead to insoluble nucleic acid cleavage, wherein the ribozyme is a known sequence that is responsible for cleavage of one or more sequences or functional equivalent sequences complementary to the target RNA. It may include. In addition, the ribozyme may be a hammer-head ribozyme or a check type ribozyme, which is an endoribonuclease RNA, and is formed by an altered oligonucleotide, resulting in safety, targeting, or the like. This can be improved. On the other hand, the ribozyme can be distributed in cells expressing the target gene in vivo, and strong constitutive polymerase such that the transfected cells produce a sufficient amount of ribozyme to destroy the endogenous target messenger and inhibit translation DNA constructs encoding ribozymes can be used under the control of III or polymerase II promoters. Because ribozymes have catalytic activity, unlike other antisense molecules, they may have to be maintained at low concentrations in cells.

In addition, the PTEN protein may include a polypeptide consisting of any sequence known in the art, for example, may be a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3.

The activity inhibitor of the PTEN protein is any one or more selected from the group consisting of compounds, peptides, peptide mimetics, matrix analogs, aptamers and antibodies that complementarily bind to the polypeptide constituting the PTEN protein It may include.

The compound may be a compound represented by Formula 1, 2, 3 or 4.

[Formula 1]

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[Formula 2]

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[Formula 3]

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[Formula 4]

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The peptide and peptide mimetics may be to inhibit the activity of the PTEN protein by inhibiting the binding of the PTEN protein to other proteins. The peptide mimetics may be peptides or non-peptides and may consist of amino acids bound by non-peptide bonds such as psi bonds. Non-hydrolyzable peptide mimetics can be prepared using β-turn dipeptide cores, keto-methylene pseudopeptides, azepins, benzodiazepines, β-aminoalcohols or substituted gammalactam rings as major residues.

The aptamer refers to a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) having a stable tertiary structure and having a characteristic that can bind to a target molecule with high affinity and specificity. It can bind to membrane proteins and block its function, so it can be regarded as a kind of chemical antibody that replaces a single antibody. In addition, the aptamer may be obtained by separating oligomers having high affinity and selectivity for a specific chemical or biological molecule by using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). .

The antibody may be a monoclonal antibody, polyclonal antibody or recombinant antibody. Antibodies to a particular protein can be readily prepared using techniques well known in the art, provided that the sequence of the protein is known. Specifically, they can be prepared using hybridoma methods, or phage antibody library techniques. Generally, hybridoma cells secreting monoclonal antibodies can be made by fusing cancer cell lines with immune cells isolated from immunologically suitable host animals, such as mice injected with antigenic proteins. These two populations of cell fusion can be fused using polyethylene glycol or the like and antibody-producing cells can be propagated by standard culture methods. Subcloning can be performed using limiting dilution, and a homogeneous cell population can be obtained and then hybridoma cells capable of producing antibodies specific for the antigen can be prepared by culturing in large quantities in vitro or in vivo. Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The polyclonal antibody can be prepared by injecting a biomarker protein or fragment thereof that is an immunogen into an external host. The external host can be a mammal such as a mouse, rat, sheep, rabbit. When the immunogen is injected by intramuscular, intraperitoneal or subcutaneous injection methods, it can be administered with an adjuvant to increase antigenicity. Thereafter, blood may be collected periodically from an external host to obtain serum showing improved titer and specificity for the antigen, from which the antibody may be isolated and purified.

The pharmaceutical composition may be to inhibit the growth, metastasis or invasion of pancreatic cancer cells.

In a specific embodiment of the present invention, the inventors of the present invention show that the PTEN protein is expressed in normal tissues and cancer tissues indiscriminately (FIGS. 1 to 4), and when the PTEN protein is inhibited in the pancreatic cancer cell line, a cell line and an anticancer effect appear. Cell lines are divided (Figs. 5 to 11), PLK1 protein is relatively strongly expressed in the cell line showing the anti-cancer effect (Fig. 12 to 13), and the expression level of PLK1 protein decreases when PTEN protein is inhibited in the cell line showing the anti-cancer effect (FIG. 14), the tumor size decreased when the PTEN was inhibited in the xenograft model transplanted with a cell line exhibiting an anticancer effect (FIGS. 15 to 18), and the higher the expression of the PTEN protein, the worse the prognosis. The expression level of the protein is high (Figs. 19 to 23), and the higher the expression of the PTEN protein, the lower the expression level of the PLK1 protein in the patient group. A negative (FIGS. 24-29) was confirmed. Accordingly, the expression inhibitor of the PTEN gene or the activity inhibitor of the PTEN protein may be usefully used for the prevention or treatment of pancreatic cancer in which the expression of the PLK1 protein is increased.

The pharmaceutical composition may include 10 to 95% by weight of the PTEN gene expression inhibitor or PTEN protein activity inhibitor according to the present invention as an active ingredient with respect to the total weight of the pharmaceutical composition. In addition, the pharmaceutical composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the above-mentioned active ingredients.

Compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. Pharmaceutically acceptable carriers are not particularly limited so long as they are suitable for delivery of the composition in vivo, see, eg, Merck Index, 13th ed., Merck & Co. Inc. The compound, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol or one or more of these components may be mixed. At this time, other conventional additives, such as antioxidant, buffer, bacteriostatic agent, can be added as needed.

When formulating the composition, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants.

The composition of the present invention may be formulated as an oral or parenteral preparation. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, troches, and the like, which may comprise at least one excipient such as starch, calcium carbonate, sucrose, It may be prepared by mixing lactose and gelatin. In addition, lubricants such as magnesium styrate and talc may also be added. Meanwhile, liquid preparations include suspensions, solvents, emulsions, or syrups, and may include excipients such as wetting agents, sweeteners, fragrances, and preservatives.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions and injections. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

The composition of the present invention may be administered orally or parenterally according to a desired method, and parenteral administration may be performed by external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection. Can be selected.

The composition according to the invention is administered in a pharmaceutically effective amount. This may vary depending on the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, the drug being used concurrently, and the like. The composition of the present invention may be administered alone or in combination with other therapeutic agents. In combination administration, administration may be sequential or simultaneous. However, for the desired effect, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.001 to 10,000 mg / kg, specifically 0.1 to 5 g / kg. The administration may be once daily, or divided into several times.

The present invention also provides a dietary supplement for the prevention or improvement of pancreatic cancer containing an inhibitor of PTEN gene expression or an inhibitor of PTEN protein activity as an active ingredient.

The PTEN gene and the inhibitor of expression of the PTEN gene may have the characteristics as described above. In one example, the PTEN gene may be a polynucleotide consisting of the nucleotide sequence described in SEQ ID NO: 4. In addition, the expression inhibitor may include any one or more selected from the group consisting of antisense nucleotides, siRNA, shRNA and ribozyme complementary to the polynucleotide constituting the PTEN gene.

The PTEN protein and the inhibitor of activity of the PTEN protein may have the characteristics as described above. In one example, the PTEN protein may be a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3. In addition, the activity inhibitor may include any one or more selected from the group consisting of compounds, peptides, peptide mimetics, matrix analogs, aptamers and antibodies that complementarily bind to the polypeptides constituting the PTEN protein, wherein The compound may be a compound represented by Formula 1, 2, 3 or 4.

[Formula 1]

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,

[Formula 2]

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,

[Formula 3]

,

,

[Formula 4]

.

.

In a specific embodiment of the present invention, the inventors of the present invention show that the PTEN protein is expressed in normal tissues and cancer tissues indiscriminately (FIGS. 1 to 4), and when the PTEN protein is inhibited in the pancreatic cancer cell line, a cell line and an anticancer effect appear. Cell lines are divided (Figs. 5 to 11), PLK1 protein is relatively strongly expressed in the cell line showing the anti-cancer effect (Fig. 12 to 13), and the expression level of PLK1 protein decreases when PTEN protein is inhibited in the cell line showing the anti-cancer effect (FIG. 14), the tumor size decreased when the PTEN was inhibited in the xenograft model transplanted with a cell line exhibiting an anticancer effect (FIGS. 15 to 18), and the higher the expression of the PTEN protein, the worse the prognosis. The expression level of the protein is high (Figs. 19 to 23), and the higher the expression of the PTEN protein, the lower the expression level of the PLK1 protein in the patient group. A negative (FIGS. 24-29) was confirmed. Therefore, the expression inhibitor of the PTEN gene or the activity inhibitor of the PTEN protein can be usefully used for the prevention or improvement of pancreatic cancer in which the expression of the PLK1 protein is increased.

The inhibitor of expression of PTEN gene or the inhibitor of activity of PTEN protein of the present invention may be added to a food as it is or used with other food or food ingredients. At this time, the amount of the active ingredient added may be determined according to the purpose. In general, the content in the dietary supplement may be from 0.01 to 90 parts by weight of the total food weight.

In addition, the form and type of the health functional food is not particularly limited. The health functional food to which the substance can be added may be tablets, capsules, powders, granules, liquids and pills.

The health functional food of the present invention may contain various flavors or natural carbohydrates and the like as additional ingredients, as in the general health functional food. The above-mentioned natural carbohydrates are sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame can be used.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Alcohol and the like. These components can be used independently or in combination. The proportion of such additives may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention comprises the steps of 1) measuring the expression level of PLK1 protein or gene encoding the same from a blood sample; And 2) comparing the expression level of the protein of the step 1) or the gene encoding the same with the expression level of a normal individual, the method for providing pancreatic cancer diagnostic information.

The method may be characterized by diagnosing pancreatic cancer when the expression level of the PLK1 protein or gene encoding the same in the blood sample is higher than that of the normal individual.

The method for measuring the expression level of the PLK1 protein or the gene encoding the same in step 1) may have the characteristics as described above.

In addition, the present invention provides a pancreatic cancer diagnostic kit for blood comprising a detection reagent that specifically binds to a PLK1 protein or a gene encoding the same.

The detection reagent or diagnostic kit may have the features as described above.

In a specific embodiment of the present invention, the inventors of the present invention have higher content of soluble PLK1 protein in the blood of pancreatic cancer patients, and higher levels of soluble PLK1 protein in pancreatic cancer patients. It was confirmed (Fig. 30). Therefore, the PLK1 protein may be usefully used as a biomarker for diagnosing pancreatic cancer.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Experimental Example 1.Comparison of Expression of PTEN Protein

It was confirmed by the following method whether the PTEN protein having a tumor suppressor function shows differential expression in normal tissue and cancer tissue.

1-1. Expression of PTEN mRNA

Expression of PTEN mRNA in various normal tissues or pancreatic cancer tissues was confirmed by Gene Expression Omnibus (GEO) microarray database (www.oncopression.com).

As a result, as shown in Figs. 1 and 2, PTEN mRNA was sporadically expressed without specificity in various normal tissues or pancreatic cancer tissues.

1-2. Expression of PTEN Protein

First, the expression level of PTEN protein in various normal or cancerous tissues was confirmed by Western blot. Specifically, 20 μg of protein for normal tissues of the human brain, colon, heart, kidney, liver, lung, pancreas or spleen and cancer tissues of the brain, colon, kidney, liver, pancreas, skin, spleen, stomach or lung. Was electrophoresed on SDS-PAGE and transferred to transfer membrane (ProSci, USA, normal tissue; # 1522, cancer tissue: # 1542), 5% skim milk overnight at 4 ° C. Blocking with TBST. The primary antibody was treated with anti-PTEN antibody (Cell signaling, # 9188) and reacted at 4 ° C. for 12 hours, followed by treatment with anti-rabbit IgG HRP antibody (santacruz) with secondary antibody for 1 hour at room temperature. Development was carried out.

In addition, the expression level of PTEN protein in normal pancreatic cell lines or pancreatic cancer cell lines was confirmed by Western blot. Specifically, H6c7 (Kerafast, USA) cells, which are normal pancreatic cell lines, were transferred to KBM (lonza, USA) medium kits, pancreatic cancer cell lines AsPC-1 (Korea Cell Line Bank), Capan-2 (Korea Cell Line Bank), or SNU-213 (Korea Cell Line Bank) cells were placed in RPMI-1640 (gibco, USA) medium containing 10% of FBS (invitrogen, USA), CFPAC-1 (American Type Culture Collection, USA), Miapaca-2 (Korea Cell Line Bank) , Or Panc-1 (Korea Cell Line Bank) cells were dispensed in DMEM (gibco, USA) medium containing 10% of FBS, and cultured in an incubator at 37 ° C., 5% CO ₂ . Cultured cells were lysed with M-PER lysis buffer (Thermo, USA), and protein concentration was measured using bicinchoninic acid (BCA) protein assay kit (Pierce). The same amount of protein was electrophoresed on SDS-PAGE and then transferred to nitrocellulose membrane at 35V for 15 hours and blocked with 5% skim milk at 4 ° C. overnight. The primary antibody was treated with anti-PTEN antibody (Cell signaling, 1: 1000) and reacted overnight in 1% skim milk, followed by the anti-rabbit IgG HRP antibody (Cell Signaling, 1: 10000) with secondary antibody. The reaction was performed for a period of time, and washed with TBST to confirm PTEN protein expression using Enhanced Chemiluminescence (ACL).

As a result, as shown in Figure 3, except for lung tissue, PTEN protein was expressed higher in cancer tissue than normal tissue. In addition, as shown in FIG. 4, both PTEN protein was expressed in normal pancreatic cells and pancreatic cancer cells.

From the above results, it was found that PTEN mRNA and protein are expressed in a similar pattern without distinguishing between normal tissue and cancer tissue.

Experimental Example 2 Comparison of Cell Growth and Metastasis Changes by PTEN Protein Inhibition

The growth and metastasis of normal pancreatic cells and pancreatic cancer cells by PTEN protein inhibition was confirmed by cell viability assay and cell migration assay.

2-1. Changes in Cell Growth and Metastasis by SF1670

The growth and metastasis of normal pancreatic cells and pancreatic cancer cells by SF1670, a PTEN protein inhibitor, was confirmed by the following method.

First, SF1670 (N- (9, 9,7), a PTEN protein inhibitor, was applied to H6c7 cells, a normal pancreatic cell line, and AsPC-1, Capan-2, SNU-213, CFPAC-1, Panc-1, or Miapaca-2 cells, a pancreatic cancer cell line. 10-Dihydro-9,10-dioxo-2-phenanthrenyl) -2,2 dimethylpropanamide, Sigma-Aldrich, USA) at a concentration of 0, 0.1, 0.3, 0.5, or 1 μM and confirmed cell viability and metastasis changes It was. Specifically, after treating the SF1670-treated cells with water soluble tetrazolium-1 (Nalgene, USA) reagent for 10 minutes at room temperature, the microplate reader (Bio-Rad, USA) ), The absorbance was measured at a wavelength of 450 nm to confirm the survival rate.

In addition, each cell treated with SF1670 was subjected to an upper chamber of a 24-well trnas-well apparatus (Corning, USA) containing serum-free Roswell Park Memorial Institute (RPMI) medium. 6 hours later, the cells transferred to the lower part of the filter were stained. The stained cells were measured by absorbance at a wavelength of 560 nm with an ELISA reader (Bio-Rad, USA) to confirm the degree of migration.

As a result, as shown in Figures 5 and 6, the survival rate of H6c7 cells increased concentration-dependently of SF1670 to a concentration of 0.3 μM, and the degree of migration of H6c7 cells increased concentration-dependently of SF1670 to a concentration of 0.5 μM. In addition, as shown in FIGS. 7 and 8, the survival and migration of AsPC-1, Capan-2 and SNU-213 cells increased depending on the concentration of SF1670 to a concentration of 0.5 μM, but CFPAC-1, Panc-1 and Survival and migration of Miapaca-2 cells decreased.

2-2. Changes in Cell Growth and Migration by siRNA (small Interfering RNA)

Similarly, when siRNA was used to inhibit PTEN protein expression, growth and migration of normal pancreatic cells and pancreatic cancer cells were changed by the following method.

Specifically, 1 × 10 ⁵ H6c7, AsPC-1, Capan-2, SNU-213, CFPAC-1, Panc-1 or Miapaca-2 cells were dispensed in a 6-well plate and the PTEN gene the next day. Specific binding oligonucleotides (Soligonucleotide, Santa Cruz Biotechnology, sc-29459), or control scrambled control (S Santa Cruz Biotechnology, sc-37007) using lipofectamine (lipofectamine, invitrogen) Each cell line was transfected according to the protocol. The transfected cells were cultured in a medium not containing FBS for 72 hours, and then cell viability was measured by the same method as Experimental Example 2-1. Also, the transfected cells were cultured with 10% FBS. After 48 hours of incubation, and replaced with a medium without FBS, overnight culture for 15 hours to make starvation (starvation) state, the degree of cell migration was measured in the same manner as in Experimental Example 2-1.

As a result, as shown in Figures 9 and 10, even when the expression of PTEN protein using siRNA, the survival rate and migration of H6c7, AsPC-1, Capan-2 and SNU-213 cells, similar to the case of treatment with SF1670 Increased, but survival and migration of CFPAC-1, Panc-1 and Miapaca-2 cells decreased.

From the above results, it can be seen that the pancreatic cancer cell line can be classified into two groups which show different effects on the growth and metastasis of cells when the PTEN protein is inhibited.

Experimental Example 3 Comparison of Intracellular Signal Transduction System Changes by PTEN Protein Inhibition

Changes in the expression patterns of intracellular signaling molecules of normal pancreatic and pancreatic cancer cells by PTEN protein inhibition were investigated by Western blot.

Specifically, siRNA for PTEN protein was transfected into H6c7 cells, which are normal pancreatic cell lines, and AsPC-1 or Panc-1 cells, which are pancreatic cancer cell lines, and subjected to Western blot to perform HGFR (hepatocyte growth). expression of a factor receptor, AKT (protein kinase B), phospho-AKT (Ser473), focal adhesion kinase (FAK), phospho-FAK (Y397), proto-oncogene tyrosine-protein kinase (Src), or phospho-Src protein The amount was confirmed. Anti-HGFR antibody (# 3127), anti-AKT antibody (# 9272), anti-phospho-AKT antibody (# 9171), anti-FAK antibody (# 3285), anti-phospho-FAK antibody (# 3283), anti-Src antibody (# 2108), and anti-phospho-Src antibody (# 2108) were purchased from Cell signaling.

As a result, as shown in FIG. 11, the expression of HGFR protein and phosphorylation of AKT, FAK and Src proteins were increased in H6c7 and AsPC-1 cells when siRNA was used to inhibit PTEN protein expression, but in Panc-1 cells. Decreased.

The results suggest that the effect of PTEN protein inhibition by pancreatic cancer cell line type is related to the activation of intracellular molecules involved in cell proliferation and migration.

Experimental Example 4. Comparison of Expression of PLK1 Protein

We investigated whether PLK1 protein is expressed differently in normal and pancreatic cancer cell lines.

4-1. Expression of PLK1 Protein

The expression level of PLK1 protein in normal cell line and pancreatic cancer cell line was confirmed by Western blot.

Specifically, Western blots were obtained by a conventional known method for H6c7 cells, which are normal pancreatic cell lines, and AsPC-1, Capna-2, CFPAC-1, Miapaca-2, Panc-1, or SNU-213 cells, which are pancreatic cancer cell lines. It was performed to confirm the expression level of PLK1 protein and to calculate the relative expression level compared with the expression level of GAPDH protein. Antibodies used in the experiment were purchased from Cell Signaling Technology (USA).

As a result, as shown in Figs. 12 and 13, PLK1 protein was strongly expressed in CFPAC-1, Miapaca-2, Panc-1 cells compared to H6c7, AsPC-1, Capna-2 and SNU-213 cells.

4-2. Confirmation of PLK1 Protein Expression by PTEN Protein Inhibition

Western blot confirmed that the expression level of PLK1 protein in normal cell line and pancreatic cancer cell line was changed by PTEN protein inhibition.

Specifically, siRNA for PTEN protein was transfected into H6c7 cells, which are normal pancreatic cell lines, and AsPC-1 or Panc-1 cells, which are pancreatic cancer cell lines, in the same manner as in Experiment 2-2. Was performed to confirm the expression level of PLK1 protein.

As a result, as shown in FIG. 14, when the expression of PTEN protein was suppressed by siRNA, the expression level of PLK1 protein was not changed in H6c7 and AsPC-1 cells, but decreased in Panc-1 cells.

The expression level of PLK1 protein is high in the cell line where antitumor effect is observed when PTEN protein is inhibited, and the expression is decreased by PTEN inhibition. The results suggest that PTEN protein acts as an upstream signal of PLK1 protein expression. .

Experimental Example 5. Confirmation of anticancer effect by PTEN protein inhibition

It was confirmed by xenograft model whether the antitumor effect by PTEN protein inhibition.

Specifically, 1 × 10 ⁷ Panc-1 or AsPC-1 cells were injected subcutaneously in the right flank of 6-8 week old BALB / c nude mice (Orient, Korea), and when the tumor size was about 200 mm ³ The group was injected with PBS in the control group and 10 mg / kg or 30 mg / kg SF1670 in the control group. Tumor volume and weight were measured at 28, 32, 35, 38 or 40 days after infusion.

As a result, as shown in Figs. 15 to 18, SF1670 showed an anticancer effect in Panc-1 cells, but not in AsPC-1 cells.

The results suggest that the effects on the viability and metastatic activity, which are different for pancreatic cancer cell lines when PTEN protein is inhibited, are similarly reproduced in experiments through xenograft models.

Experimental Example 6. Analysis of the prognosis or expression level of PLK1 gene in patients with pancreatic cancer

The correlation between the expression of the PTEN gene and the prognosis of pancreatic cancer patients or the expression of the PLK1 gene was analyzed using a database.

Specifically, PAAD-US-TCGA, GSE78229, GSE62452, PACA-AU (TNM: T = 3), PACA-AU (Grade 2), and GSE21501 data including prognostic information are downloaded from GEO and each probe is converted into an EntrezID. In addition, probes corresponding to the same EntrezID were applied as average values. Quantile-Quantile normalization was applied to all samples to eliminate the effects of each batch in which the experiment was performed. After dividing the data into two groups based on the average value of PTEN gene expression, a Log-rank test was performed with GraphPad Prism ver. 5, GraphPad, USA.

As a result, as shown in Figs. 19 to 23, the higher the PTEN gene was expressed, the more poor the prognosis of pancreatic cancer patients was, and the expression of the PLK1 gene was also increased in the high PTEN gene group. On the other hand, as shown in Figure 24 to 29, the higher the expression of the PTEN gene, the better the prognosis of the pancreatic cancer patients, the higher the expression of PLK1 gene in the group of high PTEN gene expression.

The higher the expression of PTEN gene, the poorer the prognosis of pancreatic cancer patients, suggests that PTEN inhibition may have a positive therapeutic effect. In this group, the expression of PLK1 is increased. It is suggested that it is a co-marker that allows for predicting therapeutic reactivity by an inhibitor.

Experimental Example 7. Detection of PLK1 Protein

Western blots were confirmed for normal human blood or for blood of pancreatic cancer patients to see if the PLK1 protein could be detected in the blood.

Specifically, blood was collected from 10 normal patients and 11 pancreatic cancer patients, diluted 1: 5 with PBS buffer, respectively, and the diluted solution and Laemmli sample buffer (Biorad, USA) were mixed at a ratio of 1: 2. The mixture was electrophoresed on SDS-PAGE and then transferred for 18 hours at 35 V, 4 ° C., which was blocked with TBST containing 5% skim milk at 4 ° C. overnight. The primary antibody was treated with anti-PTEN antibody (Cell signaling, # 9188) and reacted at 4 ° C. overnight, and then treated with anti-rabbit IgG HRP antibody with secondary antibody for 1 hour at room temperature.

As a result, as shown in FIG. 30, the content of soluble PLK1 protein was higher in the blood of pancreatic cancer patients than the blood of normal persons, and the content of the soluble PLK1 protein was higher in patients with advanced stages of pancreatic cancer.

These results suggest that by measuring the PLK1 protein content in the blood, antitumor effects by PTEN protein inhibition can be predicted and pancreatic cancer can be diagnosed.

<110> CHO, Sung Eun <120> Biomarker for predicting therapeutic response of pancreatic          cancer to PTEN inhibitor and use <130> 2018P-05-060 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 603 <212> PRT <213> Homo sapiens <400> 1 Met Ser Ala Ala Val Thr Ala Gly Lys Leu Ala Arg Ala Pro Ala Asp   1 5 10 15 Pro Gly Lys Ala Gly Val Pro Gly Val Ala Ala Pro Gly Ala Pro Ala              20 25 30 Ala Ala Pro Pro Ala Lys Glu Ile Pro Glu Val Leu Val Asp Pro Arg          35 40 45 Ser Arg Arg Arg Tyr Val Arg Gly Arg Phe Leu Gly Lys Gly Gly Phe      50 55 60 Ala Lys Cys Phe Glu Ile Ser Asp Ala Asp Thr Lys Glu Val Phe Ala  65 70 75 80 Gly Lys Ile Val Pro Lys Ser Leu Leu Leu Lys Pro His Gln Arg Glu                  85 90 95 Lys Met Ser Met Glu Ile Ser Ile His Arg Ser Leu Ala His Gln His             100 105 110 Val Val Gly Phe His Gly Phe Phe Glu Asp Asn Asp Phe Val Phe Val         115 120 125 Val Leu Glu Leu Cys Arg Arg Arg Ser Leu Leu Glu Leu His Lys Arg     130 135 140 Arg Lys Ala Leu Thr Glu Pro Glu Ala Arg Tyr Tyr Leu Arg Gln Ile 145 150 155 160 Val Leu Gly Cys Gln Tyr Leu His Arg Asn Arg Val Ile His Arg Asp                 165 170 175 Leu Lys Leu Gly Asn Leu Phe Leu Asn Glu Asp Leu Glu Val Lys Ile             180 185 190 Gly Asp Phe Gly Leu Ala Thr Lys Val Glu Tyr Asp Gly Glu Arg Lys         195 200 205 Lys Thr Leu Cys Gly Thr Pro Asn Tyr Ile Ala Pro Glu Val Leu Ser     210 215 220 Lys Lys Gly His Ser Phe Glu Val Asp Val Trp Ser Ile Gly Cys Ile 225 230 235 240 Met Tyr Thr Leu Leu Val Gly Lys Pro Pro Phe Glu Thr Ser Cys Leu                 245 250 255 Lys Glu Thr Tyr Leu Arg Ile Lys Lys Asn Glu Tyr Ser Ile Pro Lys             260 265 270 His Ile Asn Pro Val Ala Ala Ser Leu Ile Gln Lys Met Leu Gln Thr         275 280 285 Asp Pro Thr Ala Arg Pro Thr Ile Asn Glu Leu Leu Asn Asp Glu Phe     290 295 300 Phe Thr Ser Gly Tyr Ile Pro Ala Arg Leu Pro Ile Thr Cys Leu Thr 305 310 315 320 Ile Pro Pro Arg Phe Ser Ile Ala Pro Ser Ser Leu Asp Pro Ser Asn                 325 330 335 Arg Lys Pro Leu Thr Val Leu Asn Lys Gly Leu Glu Asn Pro Leu Pro             340 345 350 Glu Arg Pro Arg Glu Lys Glu Glu Pro Val Val Arg Glu Thr Gly Glu         355 360 365 Val Val Asp Cys His Leu Ser Asp Met Leu Gln Gln Leu His Ser Val     370 375 380 Asn Ala Ser Lys Pro Ser Glu Arg Gly Leu Val Arg Gln Glu Glu Ala 385 390 395 400 Glu Asp Pro Ala Cys Ile Pro Ile Phe Trp Val Ser Lys Trp Val Asp                 405 410 415 Tyr Ser Asp Lys Tyr Gly Leu Gly Tyr Gln Leu Cys Asp Asn Ser Val             420 425 430 Gly Val Leu Phe Asn Asp Ser Thr Arg Leu Ile Leu Tyr Asn Asp Gly         435 440 445 Asp Ser Leu Gln Tyr Ile Glu Arg Asp Gly Thr Glu Ser Tyr Leu Thr     450 455 460 Val Ser Ser His Pro Asn Ser Leu Met Lys Lys Ile Thr Leu Leu Lys 465 470 475 480 Tyr Phe Arg Asn Tyr Met Ser Glu His Leu Leu Lys Ala Gly Ala Asn                 485 490 495 Ile Thr Pro Arg Glu Gly Asp Glu Leu Ala Arg Leu Pro Tyr Leu Arg             500 505 510 Thr Trp Phe Arg Thr Arg Ser Ala Ile Lele His Leu Ser Asn Gly         515 520 525 Ser Val Gln Ile Asn Phe Phe Gln Asp His Thr Lys Leu Ile Leu Cys     530 535 540 Pro Leu Met Ala Ala Val Thr Tyr Ile Asp Glu Lys Arg Asp Phe Arg 545 550 555 560 Thr Tyr Arg Leu Ser Leu Leu Glu Glu Tyr Gly Cys Cys Lys Glu Leu                 565 570 575 Ala Ser Arg Leu Arg Tyr Ala Arg Thr Met Val Asp Lys Leu Leu Ser             580 585 590 Ser Arg Ser Ala Ser Asn Arg Leu Lys Ala Ser         595 600 <210> 2 <211> 1812 <212> DNA <213> Homo sapiens <400> 2 atgagtgctg cagtgactgc agggaagctg gcacgggcac cggccgaccc tgggaaagcc 60 ggggtccccg gagttgcagc tcccggagct ccggcggcgg ctccaccggc gaaagagatc 120 ccggaggtcc tagtggaccc acgcagccgg cggcgctatg tgcggggccg ctttttgggc 180 aagggcggct ttgccaagtg cttcgagatc tcggacgcgg acaccaagga ggtgttcgcg 240 ggcaagattg tgcctaagtc tctgctgctc aagccgcacc agagggagaa gatgtccatg 300 gaaatatcca ttcaccgcag cctcgcccac cagcacgtcg taggattcca cggctttttc 360 gaggacaacg acttcgtgtt cgtggtgttg gagctctgcc gccggaggtc tctcctggag 420 ctgcacaaga ggaggaaagc cctgactgag cctgaggccc gatactacct acggcaaatt 480 gtgcttggct gccagtacct gcaccgaaac cgagttattc atcgagacct caagctgggc 540 aaccttttcc tgaatgaaga tctggaggtg aaaatagggg attttggact ggcaaccaaa 600 gtcgaatatg acggggagag gaagaagacc ctgtgtggga ctcctaatta catagctccc 660 gaggtgctga gcaagaaagg gcacagtttc gaggtggatg tgtggtccat tgggtgtatc 720 atgtatacct tgttagtggg caaaccacct tttgagactt cttgcctaaa agagacctac 780 ctccggatca agaagaatga atacagtatt cccaagcaca tcaaccccgt ggccgcctcc 840 ctcatccaga agatgcttca gacagatccc actgcccgcc caaccattaa cgagctgctt 900 aatgacgagt tctttacttc tggctatatc cctgcccgtc tccccatcac ctgcctgacc 960 attccaccaa ggttttcgat tgctcccagc agcctggacc ccagcaaccg gaagcccctc 1020 acagtcctca ataaaggctt ggagaacccc ctgcctgagc gtccccggga aaaagaagaa 1080 ccagtggttc gagagacagg tgaggtggtc gactgccacc tcagtgacat gctgcagcag 1140 ctgcacagtg tcaatgcctc caagccctcg gagcgtgggc tggtcaggca agaggaggct 1200 gaggatcctg cctgcatccc catcttctgg gtcagcaagt gggtggacta ttcggacaag 1260 tacggccttg ggtatcagct ctgtgataac agcgtggggg tgctcttcaa tgactcaaca 1320 cgcctcatcc tctacaatga tggtgacagc ctgcagtaca tagagcgtga cggcactgag 1380 tcctacctca ccgtgagttc ccatcccaac tccttgatga agaagatcac cctccttaaa 1440 tatttccgca attacatgag cgagcacttg ctgaaggcag gtgccaacat cacgccgcgc 1500 gaaggtgatg agctcgcccg gctgccctac ctacggacct ggttccgcac ccgcagcgcc 1560 atcatcctgc acctcagcaa cggcagcgtg cagatcaact tcttccagga tcacaccaag 1620 ctcatcttgt gcccactgat ggcagccgtg acctacatcg acgagaagcg ggacttccgc 1680 acataccgcc tgagtctcct ggaggagtac ggctgctgca aggagctggc cagccggctc 1740 cgctacgccc gcactatggt ggacaagctg ctgagctcac gctcggccag caaccgtctc 1800 aaggcctcct aa 1812 <210> 3 <211> 403 <212> PRT <213> Homo sapiens <400> 3 Met Thr Ala Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr   1 5 10 15 Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile              20 25 30 Ile Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn          35 40 45 Asn Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His      50 55 60 Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys  65 70 75 80 Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro                  85 90 95 Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu             100 105 110 Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys         115 120 125 Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys     130 135 140 Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr 145 150 155 160 Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr                 165 170 175 Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala             180 185 190 Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly         195 200 205 Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile     210 215 220 Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr 225 230 235 240 Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu                 245 250 255 Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His             260 265 270 Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu         275 280 285 Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys     290 295 300 Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu 305 310 315 320 Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr                 325 330 335 Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu             340 345 350 Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr Pro Asp         355 360 365 Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp     370 375 380 Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln Ile 385 390 395 400 Thr Lys Val             <210> 4 <211> 1212 <212> DNA <213> Homo sapiens <400> 4 atgacagcca tcatcaaaga gatcgttagc agaaacaaaa ggagatatca agaggatgga 60 ttcgacttag acttgaccta tatttatcca aacattattg ctatgggatt tcctgcagaa 120 agacttgaag gcgtatacag gaacaatatt gatgatgtag taaggttttt ggattcaaag 180 cataaaaacc attacaagat atacaatctt tgtgctgaaa gacattatga caccgccaaa 240 tttaattgca gagttgcaca atatcctttt gaagaccata acccaccaca gctagaactt 300 atcaaaccct tttgtgaaga tcttgaccaa tggctaagtg aagatgacaa tcatgttgca 360 gcaattcact gtaaagctgg aaagggacga actggtgtaa tgatatgtgc atatttatta 420 catcggggca aatttttaaa ggcacaagag gccctagatt tctatgggga agtaaggacc 480 agagacaaaa agggagtaac tattcccagt cagaggcgct atgtgtatta ttatagctac 540 ctgttaaaga atcatctgga ttatagacca gtggcactgt tgtttcacaa gatgatgttt 600 gaaactattc caatgttcag tggcggaact tgcaatcctc agtttgtggt ctgccagcta 660 aaggtgaaga tatattcctc caattcagga cccacacgac gggaagacaa gttcatgtac 720 tttgagttcc ctcagccgtt acctgtgtgt ggtgatatca aagtagagtt cttccacaaa 780 cagaacaaga tgctaaaaaa ggacaaaatg tttcactttt gggtaaatac attcttcata 840 ccaggaccag aggaaacctc agaaaaagta gaaaatggaa gtctatgtga tcaagaaatc 900 gatagcattt gcagtataga gcgtgcagat aatgacaagg aatatctagt acttacttta 960 acaaaaaatg atcttgacaa agcaaataaa gacaaagcca accgatactt ttctccaaat 1020 tttaaggtga agctgtactt cacaaaaaca gtagaggagc cgtcaaatcc agaggctagc 1080 agttcaactt ctgtaacacc agatgttagt gacaatgaac ctgatcatta tagatattct 1140 gacaccactg actctgatcc agagaatgaa ccttttgatg aagatcagca tacacaaatt 1200 acaaaagtct ga 1212

Claims (16)

A composition for predicting pancreatic cancer treatment responsiveness to a PTEN (phosphatase and tensin homologue) inhibitor comprising a detection reagent specifically binding to a PLK1 (Polo-like kinase 1) protein or a gene encoding the same.
The PTEN of claim 1, wherein the detection reagent is any one or more selected from the group consisting of an antibody, an antibody fragment, an aptamer, a primer, a probe, and an anti-sense nucleotide. A composition for predicting pancreatic cancer treatment reactivity to an inhibitor.
A kit for predicting pancreatic cancer treatment reactivity to a PTEN inhibitor comprising the composition of claim 1.
1) measuring the expression level of PLK1 protein or gene encoding the same from a sample isolated from a subject with pancreatic cancer; And
2) comparing the expression level of the protein of the step 1) or the gene encoding the same with the expression level of a normal individual, the method for providing information of predicting pancreatic cancer treatment responsiveness to the PTEN inhibitor.
The method of claim 4, wherein the sample of step 1) is urine, blood, serum, plasma or tissue.
The method according to claim 4, wherein when the expression level of PLK1 protein or gene encoding the same in a sample isolated from an individual with pancreatic cancer is higher than that of a normal individual, the pancreatic cancer treatment response to the PTEN inhibitor is predicted to be good. Way.
The method of claim 4, wherein the protein expression level of step 1) is enzyme-linked immunosorbent assay (ELISA), radioimmunoassays, sandwich enzyme immunoassay, Western blot, immunoprecipitation ( A method for providing information of predicting pancreatic cancer treatment responsiveness to a PTEN inhibitor measured by any one or more methods selected from the group consisting of immunoprecipitation, immunohistochemistry and fluoroimmunoassay.
The method of claim 4, wherein the gene expression level of step 1) is reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (competitive RT-PCR), real time reverse transcription polymerase chain reaction. treatment of pancreatic cancer for PTEN inhibitors measured by one or more methods selected from the group consisting of real-time RT-PCR, RNase protection assay, Northern blot and microarray Method for providing information of reactivity prediction.
A pharmaceutical composition for preventing or treating pancreatic cancer containing an inhibitor of expression of PTEN gene or an inhibitor of activity of PTEN protein as an active ingredient.
The group of claim 9, wherein the inhibitor of expression of the PTEN gene is a group consisting of antisense nucleotides, small interfering RNAs, siRNAs, short hairpin RNAs, and ribozymes for the polynucleotides constituting the PTEN gene. Pharmaceutical composition for the prevention or treatment of pancreatic cancer comprising any one or more selected from.
The method of claim 9, wherein the inhibitor of PTEN protein activity is selected from the group consisting of compounds, peptides, peptide mimetics, matrix analogs, aptamers, and antibodies that complementarily bind to the polypeptides that make up the PTEN protein. Pharmaceutical composition for the prevention or treatment of pancreatic cancer comprising any one or more selected.
The pharmaceutical composition for preventing or treating pancreatic cancer according to claim 11, wherein the compound is a compound represented by Formula 1, 2, 3, or 4.
[Formula 1]

,
[Formula 2]

,
[Formula 3]

,
[Formula 4]

.
The pharmaceutical composition for preventing or treating pancreatic cancer according to claim 9, wherein the pharmaceutical composition inhibits the growth, metastasis, or invasion of pancreatic cancer cells.
A dietary supplement for the prevention or improvement of pancreatic cancer containing an inhibitor of PTEN gene expression or an inhibitor of PTEN protein activity as an active ingredient.
1) measuring the expression level of PLK1 protein or gene encoding the same from a blood sample; And
2) comparing the expression level of the protein of the step 1) or the gene encoding the same with the expression level of a normal subject, pancreatic cancer diagnostic information.
A pancreatic cancer diagnostic kit for blood containing a detection reagent that specifically binds to a PLK1 protein or a gene encoding the same.

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