KR101267580B1 - Transglutaminase2 as a marker for diagnosis of renal cell carcinoma and use thereof for diagnosis of renal cell carcinoma - Google Patents

Abstract

The present invention relates to TGase2 (Transglutaminase 2) and VHL (Von Hippel-Lindau) as a biomarker of renal cancer, and more particularly to a composition for diagnosing renal cancer comprising an agent measuring the expression level of one or more of TGase2 and VHL. It relates to a kit for diagnosing kidney cancer comprising the composition. In another aspect, the present invention comprises the steps of measuring the expression level of one or more of TGase2 and VHL from a sample of a patient suspected of renal cancer to provide information necessary for diagnosing kidney cancer; And comparing the expression level of one or more of TGase2 and VHL with the expression level of one or more of TGase2 and VHL of a normal control sample.

Description

Transglutaminase2 as a marker for diagnosis of renal cell carcinoma and use about for diagnosis of renal cell carcinoma}

The present invention relates to TGase2 (Transglutaminase 2) and VHL (Von Hippel-Lindau) as a biomarker of renal cancer, and more particularly to a composition for diagnosing renal cancer comprising an agent measuring the expression level of one or more of TGase2 and VHL. It relates to a kit for diagnosing kidney cancer comprising the composition. In another aspect, the present invention comprises the steps of measuring the expression level of one or more of TGase2 and VHL from a sample of a patient suspected of renal cancer to provide information necessary for diagnosing kidney cancer; And comparing the expression level of one or more of TGase2 and VHL with the expression level of one or more of TGase2 and VHL of a normal control sample.

Kidney cancer is a renal cell carcinoma that occurs mostly in the parenchyma of the kidneys (the part of the kidney where the cells that make urine are composed of the medulla and the cortex).

Kidney cancer has little symptoms when the size of the tumor is small, and it only occurs when the tumor grows to some extent and pushes out the organ. Therefore, the diagnosis is often delayed, so when the first diagnosis, about 30% of the patients are already metastasized. The most common symptom is hematuria, but this only occurs in 60% of patients. Rather, depending on the site of the transition, symptoms such as dyspnea, cough, headache, and so on, the diagnosis of kidney cancer due to such metastases in 30% of the total patients. Kidney cancers can cause high blood pressure, hypercalcemia, and liver dysfunction, especially due to certain hormones produced by cancer cells. In recent years, however, many patients have been diagnosed without any symptoms and accidentally found on imaging tests. These cases are mainly found early, so the treatment results are relatively good.

Kidney cancer accounts for about 3% of adult cancers in the United States, with approximately 32,000 new cases per year. It is estimated that about 12,000 people die from kidney cancer, and the frequency is increasing every year worldwide.

In Korea, the incidence of incidence is lower. According to the 2002 Central Cancer Registry, 1,578 patients were newly registered, accounting for 1.6% of all cancers. In particular, men are twice as likely as women, and in 2002, 1,104 patients occurred in men, representing 2.0% of men's cancers by organ, and recorded as the 10th most common cancer. It is.

Kidney cancer is common in people in their 40's and 60's, and according to the age-specific incidence (2002 Central Cancer Registration Data), 60's are the most common (479, 30.2%), 50's (412, 26.0%), 40's. (268 people, 16.9%).

Therefore, the development of a marker for early diagnosis of kidney cancer can be said to be an important task.

Against this background, the inventors found that TGase2 is overexpressed in kidney cancer and that overexpressed TGase2 downregulates VHL by mediating the polymer formation of VHL. As a result, the expression level of TGase2 and VHL was confirmed in renal cancer cells and tissues, and thus, the present invention was found to be useful for diagnosing renal cancer.

One object of the present invention is to provide a composition for diagnosing kidney cancer comprising an agent for measuring the expression level of one or more of TGase2 and VHL.

Another object of the present invention to provide a kit for diagnosing kidney cancer comprising the composition.

Another object of the present invention is to measure the expression level of one or more of TGase2 and VHL from a sample of a suspected kidney cancer to provide information necessary for diagnosing kidney cancer; And comparing the expression level of one or more of TGase2 and VHL with the expression level of one or more of TGase2 and VHL of a normal control sample.

As one embodiment for achieving the above object, the present invention relates to a composition for diagnosing kidney cancer comprising an agent for measuring the expression level of one or more of TGase2 and VHL.

As used herein, the term "TGase2" refers to transglutaminase 2 (EC 2.3.2.13, protein-glutamine γrglutamyltransferase; Transglutaminase 2; TGase2), and the Nε- (γ-L-glutamyl) -L-lysine between lysine and glutamine. iso refers to Ca ^{+ 2} dependent enzymes to promote peptide bond formation. Nε- (γ-L-glutamyl) -L-lysine crosslinks are known to form protective barrier function, cell death, extracellular matrix in stabilizing epithelial cells and to stabilize proteins present in and outside of cells. TGase2 is normally expressed at low levels in various tissues and is abnormally activated in various pathological conditions. In particular, TGase2 expression levels are known to increase in inflammatory diseases.

As used herein, the term "von Hippel-Lindau" (VHL) is a cancer suppressor protein that is part of the ubiquitin-ligase complex and downregulates HIF-1α through poly-ubiquitination and proteosomal degradation. In addition, VHL enhances apoptosis through the transcriptional activity of p53 and inhibits the cell cycle, thus functioning as a cancer suppressor. In renal cancer cells, VHL also downregulates NF-κB by inhibiting nf-κB targeted anti-apoptotic genes such as c-FLIP, subvavin, c-IAP-1, c-IAP-2. In the present invention, it was first identified that TGase2 downregulates VHL.

As used herein, the term "agent for measuring the expression level of TGase2" refers to an agent that directly or indirectly measures the expression of the TGase2 gene in a biological sample to diagnose kidney cancer. It may be a composition comprising a primer, a probe or an antisense oligonucleotide capable of binding to. In addition, the agent may be an antibody that specifically binds to TGase2 protein.

As used herein, the term "agent for measuring the expression level of VHL" is an agent for determining the expression level by directly or indirectly measuring the expression of VHL protein in a biological sample in order to diagnose kidney cancer. It is an antibody that binds specifically. The antibody can detect the expression level of the VHL protein or the expression level of the VHL protein polymer.

As used herein, the term "primer" is a single chain oligonucleotide, generally consisting of 15 to 30 bases. Primers are commonly synthesized but can also be used on naturally occurring polynucletide. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but only if it is sufficiently complementary to hybridize with the template. The position or primer binding site of the primer refers to the target DNA fragment to which the primer hybridizes. Preferably the primer used in the amplification step of the present invention is a primer for the TGase2 gene, can be easily designed by those skilled in the art by a known method.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which is short to several bases to hundreds of bases, which is capable of specific binding with mRNA. You can check the presence. Probes may be made in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, and the like. In the present invention, hybridization may be performed using a probe complementary to the marker polynucleotide of the present invention to diagnose kidney cancer through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

Antisense oligonucleotides, primers or probes of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosphoro Amidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).

As used herein, the term "antibody" refers to a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, an antibody means an antibody that specifically binds to a marker of the present invention, and the form of the antibody of the present invention is not particularly limited and has a polyclonal antibody, a monoclonal antibody or an antigen binding property. If part of it is included in the antibody of the present invention and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies. Antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. The functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, and Fv.

Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, and Rocket immunoelectrolysis Yeongdong, tissue immunostaining, immunoprecipitation assay (immunoprecipitation assay), complement fixation assay (complete fixation assay), FACS, protein chip (protein chip) and the like, but are not limited thereto.

In a specific embodiment of the present invention, as a result of confirming the expression of TGase2 and VHL in normal kidney tissue and kidney cancer tissue by immunohistochemical staining, the expression of TGase2 is increased in the kidney cancer tissue and vice versa. As shown in FIG. 5, Western blot was performed in normal kidney cells and various kidney cancer cell lines, and unlike normal kidney cells, TGase2 was increased and VHL was decreased in all kidney cancer cells (FIG. 12). It was also confirmed that TGase2 mediates cross-linking between VHL, reducing monomer of VHL protein and forming polymer of VHL protein (FIG. 2). Therefore, it can be seen that by measuring the expression level of TGase2 or the expression level of the VHL protein can be utilized in the diagnosis of renal cancer.

As another aspect, the present invention relates to a kit for diagnosing kidney cancer comprising an agent for measuring the expression level of at least one of TGase2 and VHL.

The kit of the present invention can confirm the expression level of the mRNA expression level or protein of the TGase2 gene, or diagnose the kidney cancer by confirming the expression level of the VHL protein. The kit for diagnosing kidney cancer of the present invention is a primer, a probe, or an antibody that selectively recognizes a marker for measuring the expression level of TGase2 specifically expressed in renal cancer diagnosis, as well as one or more other components suitable for analytical methods. Component compositions, solutions or devices may be included. In addition, the kit may further include an antibody, composition, solution or device for measuring the expression level of the VHL protein.

As a specific example, the kit for measuring the mRNA expression level of the TGase2 gene in the present invention may be a kit containing the essential elements for performing RT-PCR. RT-PCR kits include test tubes or other appropriate containers, reaction buffers, enzymes such as deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, DNase, RNase inhibitors, in addition to each primer pair specific for the gene. , DEPC-water, sterile water, and the like.

In addition, the kit of the present invention may be a kit for detecting a kidney cancer specific gene diagnosis including essential elements necessary for performing a microarray chip. The microarray chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached with a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. More specifically, the microarray chip of the present invention may be a kit for diagnosing kidney cancer comprising a DNA microarray chip for diagnosing kidney cancer, in which an oligonucleotide or a complementary strand molecule corresponding to 10 or more genes or fragments thereof selected from is integrated. have. The oligonucleotide or complementary strand molecule thereof may comprise 18 to 30 nucleic acids of the gene, preferably 20 to 25 nucleic acids. The microarray chip of the present invention can be easily produced by a manufacturing method commonly used in the art using the kidney cancer specific expression gene of the present invention. In order to fabricate a microarray chip, a micropipetting method or a pin using a piezoelectric method is used to immobilize the detected marker gene as a probe DNA molecule on a substrate of a DNA chip. It is preferable to use a method using a spotter of the form, but is not limited thereto. The substrate of the microarray chip is preferably coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, but is not limited thereto. . In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon film, and nitrocellulose membrane, but is not limited thereto.

In addition, the kit for measuring the protein expression level in the present invention may include a substrate, a suitable buffer, a secondary antibody labeled with a coloring enzyme or a fluorescent substance, and a coloring substrate for immunological detection of the antibody. The substrate may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like. The chromophore may be a peroxidase or an alkaline force. Alkaline phosphatase may be used, and the fluorescent material may be FITC, RITC, or the like, and the colorant substrate is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfur). Phonic acid)) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine) can be used.

In another aspect, the present invention provides a method of treating cancer in a subject, comprising: (a) measuring the expression level of one or more of TGase2 and VHL from a sample of a patient suspected of having kidney cancer; And (b) comparing the expression level of one or more of TGase2 and VHL to the expression level of one or more of TGase2 and VHL of a normal control sample.

Determination of the expression level of VHL in step (a) comprises measuring the expression level of monomer of VHL protein or polymer of VHL protein.

Preferably, the present invention can treat TGase2 and VHL antibodies in a sample and compare the degree of response to diagnose whether the individual with the target sample develops kidney cancer.

As used herein, the term "sample" includes samples such as whole blood, plasma, blood, saliva, urine sputum, lymphatic fluid, cerebrospinal fluid, and intercellular fluid with different expression levels of TGase2 and VHL in cancer tissues of an individual, and cell division. It will not restrict | limit if it is a sample containing the cell which can detect whether or not.

The information providing method for diagnosing cancer of the present invention can compare the reaction level of the antigen-antibody reaction after the anti-TGase2 and VHL antibodies to the control group and the experimental group as described above. The amount is associated with the protein antigen and the antibody or antigen-binding portion thereof of the present invention that recognizes the antigen, and the amount of the antigen-antibody complex. The response levels of these antigen-antibodies can be compared using, without limitation, measurement methods commonly used in the art. The response levels of these antigen-antibodies can be compared using, without limitation, measurement methods commonly used in the art. Examples of methods for measuring the level of antigen-antibody reaction include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion But are not limited to, immunoassays, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, complement fixation assays, FACS, agglutination, and protein chips.

As used herein, the term “antigen-antibody complex” means a combination of a marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formed can be measured quantitatively through the size of a signal of a detection label. Do.

The protein expression level can be measured by ELISA. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex, a labeled antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antigen with the antibody attached to the solid support Various ELISA methods include indirect sandwich ELISA using secondary antibodies. For example, by attaching an antibody to a solid support and reacting a sample, a labeled antibody that recognizes an antigen of the antigen-antibody complex is enzymatically developed or labeled for an antibody that recognizes an antigen of the antigen-antibody complex. It can be detected by the sandwich ELISA method which attaches the secondary antibody thus enzymatically and colors it. By confirming the degree of complexation of the marker protein with the antibody, it is possible to diagnose the presence of renal cancer.

In addition, Western blot using one or more antibodies to the marker can be used. The whole protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the generated antigen-antibody complex can be confirmed by using a labeled antibody to confirm the amount of the protein produced by the expression of the gene, thereby confirming the presence of kidney cancer. The detection method consists of comparing the expression levels of the TGase2 and VHL proteins in the kidney cancer patients with the expression levels of the protein in the normal control group. Protein levels can be expressed as absolute (eg μg / ml) or relative (eg relative intensity of signals) differences of the marker proteins described above. In addition, immunohistostaining with one or more antibodies to the marker can be performed. After collecting and immobilizing the tissues of a suspicious kidney cancer subject, paraffin embedding blocks are prepared by methods well known in the art. These are cut into slices of several μm thickness, adhered to glass slides, and reacted with one of the above antibodies by a known method. The unreacted antibody is then washed and labeled with one of the above-mentioned detection labels to read whether or not the antibody is labeled on a microscope.

In addition, a protein chip in which one or more antibodies against the marker are arranged at predetermined positions on a substrate and immobilized at high density can be used. In the method of analyzing a sample using a protein chip, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex, which is read to confirm the presence or expression level of the protein, Diagnose the development of kidney cancer.

The present invention is expected to find an effect that can be used as an indicator useful in the treatment and prognosis of kidney cancer by discovering and providing a diagnostic marker for kidney cancer. Furthermore, it can be used to study tumorigenesis of renal cancer, and it is expected to contribute to the early diagnosis of renal cancer.

1 is a diagram showing that HIF-1α expression is reduced by TGase2 knockdown in normal and cancer cell lines, and that the expression of VHL is inversely related to TGase2 expression. (a) HIF-1α expression was inhibited by TGase2 knockdown in cancer cells. TGase2 was inhibited by treatment with TGase2 specific siRNA, and levels of HIF-1α were analyzed in immunoblot. VEGF expression in cancer cells was inhibited by TGase2 knockdown using TGase2-specific siRNA. VEGF levels were analyzed by RT-PCR. GAPDH mRNA levels were measured as controls. (b) The expression of endogenous VHL was decreased in ovarian carcinoma and breast cancer cells overexpressing TGase2. Cytoplasmic fractions of HEK293, MCF7, NCI / ADR-RES, and MDA-MB-231 cells were analyzed by immunoblot using anti-TGase2 and anti-VHL antibodies. β-actin was analyzed as a loading control. (c) Endogenous VHL protein levels were down regulated by overexpression of TGase2 and recovered by TGase2 inhibitor treatment or expression of TGase2 mutants (C277S). HEK293 and MCF7 cells were transfected stepwise for 24 hours with an expression vector of TGase2 or TGase2 C277S mutant and with or without cystamine for 24 hours (CTM; 0.5 mM). Cytoplasmic fractions of cells were recovered and analyzed by immunoblot using anti-TGase2 and anti-VHL antibodies. (d) Expression of ectopic VHL and TGase2 in HEK-293 and MCF7 cells. Cells were concurrently infected with the expression plasmids presented for 24 hours, and with or without cystamine for 24 hours (CTM; 0.5 mM). TGase2 and VHL levels were analyzed via immunoblot using anti-TGase2 and anti-VHL antibodies. (e) Ectopically expressed VHL was down regulated by dose dependent overexpression of ectopic TGase2. The cells were simultaneously infected with the VHL expression vector and the amount of TGase2 expression vector increased for 48 hours. Cell extracts were prepared and analyzed by immunoblot using anti-TGase2 and VHL antibodies.
FIG. 2 shows that direct physical interaction of TGase2 and VHL results in TGase2-VHL polymer formation and proteasome-dependent degradation. (a) TGase2 induces the accumulation of degraded VHL monomers in the proteasome. Cells were transfected with the plasmids presented for 24 hours, and various inhibitors (calpeptin, calpein inhibitor, 50 μM; leupeptin, lysosomal inhibitor, 50 μM; MG 132, proteasome inhibitor, 5 μM) for 12 hours prior to recovery. Treated and untreated. TGase2 and VHL levels were analyzed by immunoblot using anti-TGase2 and anti-HA antibodies. (b) VHL interacts with TGase2, and this interaction is further increased in the presence of proteasome inhibitors. Cells were simultaneously infected with the indicated expression plasmids for 24 hours, followed by or without MG132 (5 mM) for 12 hours. Whole cell extracts were analyzed by immunoprecipitation using anti-HA antibodies and then immunoblot using anti-TGase2 and anti-VHL antibodies. Simultaneous immunoblot analysis of whole cell extracts with anti-TGase2 and anti-HA antibodies showed that TGase2 and VHL were stably expressed in a proteosome dependent method. (c) TGase2-VHL complexes undergo proteosome-dependent degradation. Cells were simultaneously infected with the indicated expression vectors for 24 hours and MG132 (5 mM) was treated with or without 12 hours. VHL was immunoprecipitated from whole cell extracts with anti-HA antibody (5 times more cell lysate than b) and immune complexes were analyzed by immunoblot with anti-TGase2 and anti-VHL antibodies.
3 is a diagram showing that expression of ectopic VHL with inhibition of TGase2 inhibits NF-κB activity and reduces IGF1R-β levels. (a) NF-κB activity was reduced by ectopic expression of VHL and inhibition of TGase2. Cells were transfected with the indicated expression vectors for 24 hours and either with or without cystamine (CTM; 0.5 mM) (right panel) or TGase2-specific siRNA (left panel). The culture medium was recovered and NF-κB transcriptional activity was measured using SEAP reporter assay. Results are expressed as base error (SEM) of the mean ± 3 sets of experimental means (p <0.05). (b) Nuclear extracts of the cells described in (a) were analyzed by EMSA using a ³² P-labeled NF-kB probe. (c) Cell death was analyzed by annexin V / propidium iodide staining (PI) and immunoblot of cleaved caspase 3 and PARP. Cells were transfected with VHL expressing plasmids and TGase2-specific-siRNA. Annexin V / PI staining was determined by flow cytometry. The percentage of cells recovered in the quadrant corresponding to early and late cell death was shown (top panel). Whole cell extracts were analyzed by immunoblot using anti-cleaved caspase-3, anti-PARP and anti-β-actin antibodies. (d) Inhibition of TGase2 restored VHL protein levels and downregulated endogenous IGF1R-β protein levels. Cells were transfected with the indicated plasmids for 24 hours, and then treated or untreated with cystamine (CTM; 0.5 mM) for 24 hours before recovery (left panel). Alternatively, cells were transfected 24 hours with TGase2-specific siRNA using reversible infection, followed by 24 hours transfection with the indicated expression plasmids (right panel). Whole cell extracts were analyzed by immunoblot with anti-TGase2, anti-IGF1R-β and anti-VHL antibodies.
Figure 4 shows that overexpression of TGase2 induces inhibition of VHL and activity of IFG1R-β and NF-κB in the body. (a) The schematic of the TGase2-pCAGGS rpsha construct used to generate TGase2 transgenic mouse species is shown. (b) The potential founder mice to which the TGase2 transplant gene was introduced by PCR were identified. (c) After perfusion, frozen tissues derived from wild-type and TGase2 transgenic mice were homogenized and then TGase2, VHL and IGF1R-β levels were immunized with anti-TGase2, anti-VHL and anti-IGF1R-β antibodies. Relative to blot. (d) Tissues were prepared as described in (a) and nuclear extracts were analyzed by EMSA using ³² P-labeled NF-κB probes. NF-κB DNA binding activity was quantified using a density analyzer. Results are shown as SEM of mean ± three set samples (p <0.05).
Figure 5 shows that TGase2 and VHL expression is in a reversible relationship in human RCC carcinoma samples. In 9 of 11 RCC cell carcinoma samples, TGase2 and VHL expression were inversely related. For samples with high expression of TGase2, VHL expression was low or absent (a and b). For samples with low expression of TGase2, the expression of VHL was relatively high (c and d). The expression of TGase2 was high in invasive progenitor cells (e, arrow), while the expression of VHL was relatively low or absent (f, arrow), providing additional evidence indicating the inverse of TGase2 and VHL expression. All image amplification rates are 100X.
6 shows a model presented for the role of VHL and TGase2 in NF-κB activity. TGase2 is an I-κBα (Kim, DS, Park, SS, Nam, BH, Kim, IH & Kim, SY Reversal of drug resistance in breast cancer cells by transglutaminase 2 inhibition and nuclear factor-kappaB inactivation. Cancer Res 66 , 10936-43 (2006)) and reduction of VHL induces or expands NF-κB activity. It is a well known major inhibitor of NF-κB, and VHL is a tumor inhibitor that modulates HIF-1α and IGF-1R. Reduction of I-κBα and VHL increases resistance to survival, growth, migration, drug resistance and apoptosis in cancer cells.
Figure 7 shows that expression of VHL in TGase2 overexpressing NCI / ADR-RES and MDA-MB-231 is due to inhibition of TGase2 expression of ectopic VHL restored by inhibition of cystamine or TGase2-specific siRNA. Cells were transfected with TGase-2 specific siRNA and the plasmids presented above (a. Right panel) or transfected with the plasmids presented above with or without cystamine (b. Left panel). Overexpression of VHL was confirmed by immunoblot analysis using anti-VHL antibodies.
8 shows that localization of ectopic VHL is regulated by TGase2 gene in NCI / ADR-RES and MDA-MB-231 cancer cells. Cells were transfected for 24 hours with reverse transformation and reverse transcription TGase2-specific siRNA using the plasmids disclosed above. The cells were recovered and reinoculated on two chamber slides for 16 hours. Cells were analyzed using immuno-chemical analysis using anti-HA primary antibodies followed by fluorescently bound anti-mouse immunoglobulin secondary antibodies for detection of VHL.
9 shows that TGase2 is induced by polymerization of VHL in a dose dependent manner in vitro. Purified recombinant VHL was polymerized by guinea pig liver TGase2 in vitro. VHL polymers were separated by SDS-PAGE, visualized by Coomassie blue staining (left panel) and analyzed by immunoblot with anti-VHL antibodies (right panel).
FIG. 10 shows that mass spectrometry of peptides is due to proteolytic hydrolysis assumed to be monomers, dimers and polymers of VHL. Purified recombinant VHL was polymerized by TGase2 in vitro. Multiple binding VHL complexes were separated by SDS-PAGE and visualized by Coomassie Blue staining. The quantified bands were collected, reduced and resolved by trypsin treatment. The mass of peptides was determined using MALDI-TOF MS.
FIG. 11 shows analysis of VHL monomers and polymers purified by MS to identify crosslinking sites in VHL. 4F shows amino acid sequences of VHL monomers and polymers, in which the red letters represent residues identified by MS and the blue letters (in VHL polymer) represent residues blocked after polymerization. This result indicates that glutamine 164 and 203 are included in the TGase2-induced VHL polymer.
FIG. 12 is a Western blot showing that TGase2 and VHL expression are inverse in human RCC carcinoma samples.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only examples of the present invention and the present invention is not limited thereto.

Example  1: Antibodies and Reagents

Anti-VHL antibodies from BD Pharmingen (USA, CA, San Diego), anti-TGase2 antibodies from NeoMarkers (USA, CA, Fremont, clone CUB 7402), anti-β-actin was Abcam (UK, Cambridge, Head) Quarter, anti-IGF1R-β and anti-HA-probe (F7) antibodies from Santa cruz Biotechnology (Santa Cruz, CA, USA), anti-PARP and anti-cutting caspase-3 (Asp175) antibodies from Cell It was purchased from Signaling Technologies (Beverly, MA, USA). Lipofectamine RNAiMAX, including Lipofectamine 2000, Lipofectamine RNAiMAX Transfection Reagent, and Stealth Negative Control, was purchased from Invitrogen, Calpeptin (Calbiochem), Leupeptin (USA, MO, St. Louis, Sigma-Aldrich). Co), MG132 (Calbiochem) and FITC-hybrid anti-mouse immunoglobulin (US, Pennsylvania, West Grove, Jackson Immunology Laboratory) were purchased commercially.

Example  2: siRNA ( small interfering RNA ) Treatment TGase2 Suppression of silencing )

SiRNA double strands targeting human TGase2 (5′-AAGAGCGAGAUGAUCUGGAACTT-3 ′: SEQ ID NO: 1) were introduced into cells using lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. Cells were harvested 48 hours post infection and cytoplasmic fractions or whole cell extracts were prepared for immunoblot analysis. As a negative control, cells were incubated with lipofectamine RNAiMAX alone and with a universal negative siRNA (Invitrogen).

Example  3: Immunoblotting ( Immunoblotting )

Standardized methods (Kim, DS, Park, SS, Nam, BH, Kim, IH & Kim, SY Cancer Res 66 , 10936-43 (2006)) to perform immunoblot analysis.

Example  4: Electrophoresis  Change analysis method Electrophoretic mobility shift assay , EMSA )

A double stranded oligonucleotide probe (5'-AGT TGA GGG GAC TTT CCC AGG C-3 ': SEQ ID NO: 2) containing an NF-kB DNA binding site was purchased from Santa Cruz Biotech. EMSA analysis was performed according to the manufacturer's protocol.

Example  5: Apoptosis Assay

Apoptosis analysis by Annexin V binding was performed using the Annexin V-FITC Apoptosis Detection Kit (CA, Mountain View, BD Biosciences) according to the manufacturer's instructions.

Example  6: TGase2 ⁺  Preparation of Transgenic Mice

The mouse transglutaminase 2 gene was inserted into the native EcoRI-BglII site of pCAGGS comprising human CMV enhancer, chicken β-actin promoter-intron and rabbit β-globin polyadenylation sequence. The 5.8 kb DNA fragment product was cut and prepared following the basic method for pronuclei DNA injection. TGase2 transgene was selected by PCR analysis of genomic DNA extracted from a 1 cm tail clip. PCR was performed using the following primers.

5'-CAC CCT TCG TGT TTG CCG AGG TCA-3 '(SEQ ID NO: 3)

5'-TCT CCG AGG TGT CGT TGC CGA TGT-3 '(SEQ ID NO: 4)

Transglutaminase 2 transgene and wild type allele were obtained with 334 basepairs (pb) and 3,056 bp, respectively. In this way, we identified four founder mice with gene copies of the TGase2 transgene.

Example  7: Human RCC  In the sample TGase2  And VHL Immunochemical staining

Eleven kidney cancer samples obtained by surgical operation at Ilsan Paik Hospital (Korea, Goyang) were selected from pathology records. Use of the recorded organization was approved by the Ethics Association and the Institutional Review Board (IRB number: 1B-2-1003-010). Immunohistochemical staining was performed using the Ultravision LP detection kit and DAB (Lab Vision Corporation Fremont, CA, USA). Paraffinized human RCC tissue was stained. Tissue sections were deparaffinized in xylene and antigen recovery was heat treated at 121 ° C. for 15 minutes in 10 mM Tris buffer at pH 9.0 with 1 mM EDTA. Sections were fixed in 95% ethanol, treated with 0.3% hydrogen peroxide and fixed by irradiating Ultra V for 15 minutes. Sections were reacted for 1 hour at room temperature with anti-TGase2 and anti-VHL primary antibodies in 0.05% TBS-T with 20% FBS. After washing TBS-T, the sections were sequentially reacted with a primary antibody amplifier and then reacted with horseradish peroxidase (HRP) polymers for 30 minutes each. After TBS-T washing, slides were counterstained using DAB and Mayer's hematoxylin (DakoCytomation).

Example  8. In normal kidney cells and kidney cancer cells Western Blat

The expression of VHL and TGase2 proteins in HEK293 cell line, which is a normal kidney cell, and 786-O, A498, ACHN, CAKI-1, SN12C, TK10, and U031 cell lines, which were normal kidney cells, was measured by Western blot. The normal kidney cell line and various kidney cancer cell lines were treated with anti-TGase2 and anti-VHL primary antibodies and reacted at room temperature, and then the secondary labeled antibodies were reacted at room temperature to determine whether TGase2 and VHL proteins were detected.

Example 9: immunochemistry

HA-VHL was visualized by immunochemistry and phase contrast microscopy. Cells were seeded in a 2-well slide chamber in the presence of TGase2 specific siRNA, or in the presence of universal negative siRNA the day before transfection as a control. Cells were transfected with a control empty vector (mock) or HA-VHL expression vector for 24 hours and then washed with PBS (Phosphate buffered saline). Immunchemistry was performed according to standard protocols.

Example  10: Human VHL &Lt; / RTI &gt;

Human VHL cDNA was subcloned into modified pET32 (Novagen) using BamHI / XhoI restriction enzymes, a 23-residue polyhistidine tag (MHHHHHHGSLVPRSENLYFQGS, 6xHis) at the amino terminus of VHL, and a 10-residue polyhistidine tag at the carboxy terminus (LEHHHHHHHH, 8 × His) was added. Recombinant VHL was purified by established protocol after overexpression in E. coli strain Rosetta 2 (DE3) (Novagen).

Example  11: Guinea Pig  liver TGase2 On by VHL Polymerization

Add guinea pig liver TGase2 (5 μl, U / mL) to 30 μl of reaction buffer (50 mM Tris-HCL, pH 7.5, 150 mM NaCl, 15 mM CaCl ₂ ) containing purified human VHL (5 μg). It reacted at 2 degreeC. Proteins were separated by SDS-PAGE and visualized by Coomassie Blue staining or analyzed by immunoblot.

Experiment result

Experimental Example  One: VHL  And TGase2 of Inverse relationship

In previous studies we have shown that increased TGase2 expression plays a key role in obtaining drug resistance of cancer cells. In particular, HIF-1α was increased in drug resistant cancer cells under normal oxygen conditions (FIG. 1A). Knockdown of the TGase2 gene reduced the expression level of endogenous HIF-1α protein in NCI / ADR-RES and MDA-MB-231 cells (FIG. 1A). The expression levels of VHL in NCI / ADR-RES and MDA-MB-231 breast cancer cells were lower than in HEK-293 and MCF7 cells (FIG. 1B), and endogenous VHL levels in cancer cell lines were inversely related to TGase2 (FIG. 1B). . Endogenous VHL was inhibited by ectopic TGase2 expression in HEK293 and MCF7 breast cancer cells, and this reduction was either by inhibition of TGase2 with cystamine or by expression of catalytically inactivated TGase2 variant (C277S). It was reversed by inhibition of TGAse2 (FIG. 1C). Levels of ectopic VHL were reduced by co-expression of TGase2, and this effect was lost by inhibition of TGase2 using expression of cystamine or catalytically inactivated TGase2 C277S (FIG. 1D). These results indicate that cross-linking catalytic activity of TGase2 is a major cause of VHL reduction.

Experimental Example  2: TGase  Suppression in cancer cells VHL Increases stability

To determine whether the TGase2 inhibitor increases the level of VHL by regulating VHL stability, the present invention confirmed the inhibitory effect of endogenous TGase2 activity on VHL protein levels. NCI / ADR-RES and MDA-MB-231 cells expressing high levels of TGase2 were transiently transfected with HA-VHL expression vectors with TGase2-specific siRNA or cystamine. Levels of ectopic HA-VHL increased in response to TGase2 inhibition by cystamine or gene knockdown, compared to control cells expressing only HA-VHL without treatment with TGase2-specific siRNA or cystamine (FIG. 7). It is consistent with the result of FIG. 2A. Immunohistochemical staining of HA-VHL-transfected NCI / ADR-RES and MDA-MB-231 cells with an anti-HA monoclonal antibody showed that VHL was localized in the nucleus. In comparison, cells co-transfected with TGase2-specific siRNA appeared in the cytoplasmic and nuclear regions of HA-VHL (FIG. 8). Thus, TGase2 appears to target VHL in the cytoplasm where TGase2 is most abundant.

Experimental Example  3: VHL  Polymer Proteasome  Dependent decomposition ( proteasome - dependent degradation )

In order to identify whether the VHL polymer produced via TGase2-catalyzed cross linking is metabolically degraded, we have identified three different proteolytic inhibitors (proteasome inhibitors, lysosomes) for their ability to inhibit VHL degradation. Inhibitors, calpine inhibitors). Reduction of VHL monomers by TGase2 was inhibited by proteasome inhibitor MG132 treatment, but not by calpain or lysosomal inhibitors (FIG. 2A). To determine whether TGase2 and VHL interact directly, cells were co-transfected with expression vectors of HA-VHL and TGase2, with or without MG132 and using anti-HA antibodies for immunoblot analysis. By immunoprecipitation (Fig. 2b). TGase2 was detected in the VHL precipitate, and the amount of precipitated VHL was increased by MG132 treatment (FIG. 2B). These results indicate that VHL interacts with TGase2 and that the VHL complex is degraded via proteasome-dependent mechanisms (FIG. 2A).

VHL polymer was easily detected and the expression level of the polymer was increased in the cells treated with MG132. In addition, polymerized TGase2 was also detected in anti-HA precipitation, indicating that TGase2 acts as a substrate for polymerization with the ability to catalyze polymerization (FIG. 2C). To confirm that VHL is a substrate of TGase2, purified recombinant human VHL was incubated in vitro with purified guinea pig liver TGase. TGase2 induced the polymerization of VHL in a dose dependent manner (FIG. 9). Recovery by high molecular weight gel fraction (arrow head in FIG. 9) and analysis by MS / MS confirmed that VHL was a major component of the polymerized protein complex (FIG. 10). To identify the crosslinking site of VHL, the analyzed gels were purified by MS into VHL monomers and polymers. 4F shows amino acid sequences of VHL monomers and polymers, red letters represent residues identified by MS, and blue letters (in VHL polymer) represent residues blocked after polymerization. These results indicate that glutamine 164 and 203 are associated with TGase2-induced VHL polymerization (FIG. 11).

Experimental Example  4: TGase  By inhibition VHL  Manifestation recovery NF results in inhibition of -κB activity

The activity of NF-κB has been reported to be downregulated by VHL. NF-κB activity was reduced by Tgase2 inhibition in VHL expressed cells. As shown in FIG. 3, NF-κB activity in HA-VHL expressing cells treated with TGase2-specific siRNA or Tgase2 inhibitor cystamine was lower than in HA-VHL-only untreated cells (two-fold and 2.5 times) (FIG. 3A). Simultaneously with the SEAP reporter assay, we performed an electrophoretic mobility shift assay (EMSA) to assess NF-κB DNA binding activity. Reduced NF-κB DNA binding activity expressed with ectopic VHL expression alone or with TGase2 inhibition (FIG. 3B). Apoptosis was induced more efficiently in HA-VHL expressing cells treated with TGase2-specific siRNA compared to cells treated with siRNA alone, measured by relative levels of annexin-V positive staining and cleaved PARP (FIG. 3c).

Previous reports have shown that VHL inhibits the expression of IGF-1R. TGase2 inhibition by cystamine or gene knockdown results in the stability of ectopic VHL, which is associated with reduced IGF-1R levels in drug resistant cancer cell lines (FIG. 3D). These results indicate that downregulation of VHL by TGase2 is associated with Indicates that IGF-1R protein expression is essential.

Experimental Example  5: TGase2 + In transgenic mice VHL Down-regulation of

In order to confirm the relationship with VHL and TGase2 in vivo, we prepared TGase2 + transgenic mice into which the mouse TGase2 + transplant gene was introduced (FIGS. 4A and 4B). The kidneys of TGase2 + transgenic mice showed no histological abnormalities and the appearance was normal. Analysis of VHL protein levels in kidneys of three different wild-type and TGase2 ⁺ transgenic mice via immunoblot showed lower VHL expression levels in TGase2 + transgenic mice than in wild type mice (FIG. 4C). To determine whether VHL reduction by TGase2 + overexpression affected IGF-1R protein levels, we analyzed kidney samples from wild-type and TGase2 + transgenic mice via immunoblot with anti-IGF-1R antibodies. IGF-1R protein levels were expressed higher in TGase2 + transgenic mice than wild type mice as expected (FIG. 4C). As a result of analysis of NF-κB DNA binding activity by EMSA, NF-κB activity was increased almost three-fold in TGase2 + transgenic mice compared to wild type mice (FIG. 4D). Therefore, VHL levels were decreased in the kidneys of TGase2 + transgenic mice compared to wild type mice, which is associated with high expression levels of IGF-1R. These results indicate that TGase2 + inhibits VHL, thereby inducing NF-κB activity and expression of IGF-1R in vivo.

Experimental Example  6: human kidney cells carcinoma  In the sample TGase2 + And VHL  Protein level Decorrelating  relation

In order to confirm the validity of the experimental results in the cancer cell line and the transformed TGase2 + mouse model, that is, to confirm the inverse relationship between TGase2 + and VHL expression, the present inventors have immunohistochemically analyzed a sample of renal clear cell carcinoma tissue. Expression levels of VHL and TGase2 were confirmed in (FIG. 5). In normal kidney tissues, TGase2 was present in glomerular cells and relatively low in tubular cells, whereas VHL was restricted in renal tubular cells. In RCC carcinoma tissues, expression levels of TGase2 generally increased and expression of VHL decreased. Nine out of eleven human RCC carcinomas showed an inverse relationship between TGase2 and VHL levels. As shown in FIGS. 5A-D, almost complete inversion was seen in cancer 6, and in cancer 3, it was shown in positive regions of TGase2 and VHL. In general, for samples with high expression of TGase2, the expression of VHL was low or absent (Figures 5a, b). In the samples with low expression of TGase2, the expression level of VHL was high (Fig. 5C, d). In invasive front cells, TGase2 expression was higher than the rest of the cancer (FIG. 5E) and the expression of VHL was low or absent (FIG. 5F).

In addition, the expression of VHL and TGase2 protein in HEK293 cell line, which is normal kidney cell, and 786-O, A498, ACHN, CAKI-1, SN12C, TK10, and U031 cell lines, which were normal kidney cells, was measured by Western blot. Unlike kidney cells, it was confirmed that TGase2 was increased and VHL was decreased in all kidney cancer cells (FIG. 12).

These results further demonstrate the inverse relationship between TGase2 and VHL expression.

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Claims (11)

A composition for diagnosing kidney cancer comprising an agent for measuring the expression level of TGase2 (Transglutaminase 2) and the amount of protein in VHL (Von Hippel-Lindau).
The composition of claim 1, wherein the agent for measuring the expression level of TGase2 comprises a primer, probe or antisense oligonucleotide that specifically binds to the TGase2 gene.
The composition of claim 1, wherein the agent for measuring the expression level of TGase2 comprises an antibody specific for TGase2 protein.
The composition of claim 1, wherein the agent for measuring the protein amount of VHL comprises an antibody specific for the VHL protein.
The composition of claim 1, wherein the protein amount of the VHL is the protein amount of a monomer of the VHL protein or a polymer of the VHL protein.

Kit for diagnosing kidney cancer comprising a composition according to any one of claims 1 to 5.
The kit of claim 6, wherein the kit is a protein chip kit.
In order to provide information necessary for diagnosing renal cancer, (a) measuring the expression level of TGase2 and the amount of VHL protein from isolated samples of patients suspected of renal cancer; And (b) comparing the expression level of TGase2 and the protein amount of VHL with the expression level of TGase2 and the protein amount of VHL in a normal control sample.
The method of claim 8, wherein the measuring of the protein amount of the VHL in step (a) is measuring the protein amount of the monomer of the VHL protein or the polymer of the VHL protein.

The method of claim 8, wherein measuring the expression level of TGase2 and the protein amount of VHL is performed using an antibody specific for the protein.
According to claim 8, The method for measuring the expression level of TGase2 and protein amount of VHL is Western blot, ELISA, radioimmunoassay, radioimmunoassay, Oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunity staining , Immunoprecipitation assay, complement fixation assay, FACS or protein chip method.

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