CN109718374B - Use of IRF3 inhibitor for preparing medicine for treating or preventing YAP over-activated cancer - Google Patents

Abstract

The invention relates to the use of an IRF3 inhibitor for the manufacture of a medicament for the treatment or prevention of YAP overactivated cancer. The present invention finds that IRF3 can activate YAP, which provides a target and more accurate therapeutic approach for treatment of YAP-initiated tumors.

Description

Use of IRF3 inhibitor for preparing medicine for treating or preventing YAP over-activated cancer

Technical Field

The invention relates to the technical field of biology, in particular to application of an IRF3 inhibitor in preparing a medicine for treating or preventing YAP (Yap-activated cancer).

Background

The Hippo-YAP signaling pathway has important roles in regulation of tissue and organ size and maintenance of homeostasis, and dysfunction of this signaling pathway has been shown to be associated with a variety of cancers. YAP has been currently used as a target for tumor therapy because of the detection of excessive activation of YAP in clinical specimens of a subset of tumor patients. YAP is phosphorylated by the upstream kinase LAT1/2 and then retained in the cytoplasm. After entering the nucleus, the transcription factor TEAD4 is combined to form a transcription activation complex to regulate the transcription of the target gene. However, the mechanism of entry and activation is not clear.

The classical function of IRF3 (interferon regulatory factor 3) is to modulate the antiviral immune response in response to stimulation by viral nucleic acids in the cytoplasm. Phosphorylation of IRF3 upstream of TBK1 in the TLR signaling pathway is essential for IRF3 dimerization and activation, phosphorylated IRF3 is activated, inducing production of type I interferon.

Amlexanox (Amlexanox), CAS number 68302-57-8, molecular formula: c₁₆H₁₄N₂O₄Is a benzopyran-pyridine carboxylic acid derivative. Amlexanox has antiinflammatory and antiallergic effects, and can inhibit delayed anaphylaxis caused by chemical mediator, and antagonize inflammatory substances such as leukotriene. Biological functions are now known to include TBK1 inhibitors. Currently, the national drug catalogue of the national food and drug administration (CFDA) has nine records in common, including: 2 amlexanox oral patches suitable for treating oral ulcer of adults with normal immune system and teenagers older than 12 years old; 3 amlexanox pastes, suitable for treating aphthous ulcer with normal immune system; 4 amlexanox are used as raw material medicaments. In the prior art, the role of amlexanox in the treatment of gastric cancer has not been reported.

Disclosure of Invention

The invention provides the use of an IRF3 inhibitor for the manufacture of a medicament for the treatment or prevention of YAP mediated diseases.

In certain embodiments, the YAP-mediated disease is cancer, preferably YAP-overactivated cancer.

In certain embodiments, the cancer is a solid tumor or a hematologic tumor.

In certain embodiments, the cancer is selected from: gastric cancer, non-small cell lung cancer, colon cancer, cervical cancer, leukemia, breast cancer and lymphoma.

In certain embodiments, the IRF3 inhibitor is a nucleic acid molecule, a protein molecule, or a compound; wherein, the nucleic acid molecule is selected from gene knockout vectors, siRNA and expression vectors of interfering RNA, and the protein molecule is a specific antibody of IRF 3.

In certain embodiments, the IRF3 inhibitor is a TBK1 inhibitor.

In certain embodiments, the TBK1 inhibitor is a nucleic acid molecule, a protein molecule, or a compound; wherein, the nucleic acid molecule is selected from a gene knockout carrier, an expression carrier of siRNA and interfering RNA, the protein molecule is a specific antibody of IRF3, and the compound is selected from amlexanox and BX 795.

In certain embodiments, the IRF3 inhibitor is selected from amlexanox and BX795, and the YAP-mediated disease is YAP-overactivated gastric cancer.

The invention also provides the use of a detection reagent for IRF3 and optionally a detection reagent for YAP in the preparation of a diagnostic kit for diagnosing the survival of a patient having a YAP-mediated disease.

In certain embodiments, the detection reagent is a detection reagent for mRNA expression levels and/or a detection reagent for proteins.

In certain embodiments, the YAP-mediated disease is a cancer, preferably a YAP-overactivated cancer, more preferably a solid or hematologic tumor, more preferably the cancer is selected from: gastric cancer, non-small cell lung cancer, colon cancer, cervical cancer, leukemia, breast cancer and lymphoma.

Drawings

FIG. 1: YAP in gastric cancer is positively correlated with the level of IRF3 expression. (A) Scatter plots indicate a positive correlation of YAP and IRF3 transcript levels in different gastric cancer cell lines. The correlation of YAP mRNA levels with IRF3 mRNA levels was measured by spearman rank correlation. (B) Immunoblot experiments of YAP with IRF3 in gastric cancer cell lines. (C) Co-immunoprecipitation experiments in gastric cancer cell lines examined the interaction of YAP/IRF3 with TEAD 4. (D) Boxplot-characterized mRNA levels of YAP and IRF3 in MNNG/h. (E) The database contains a hotspot map of the expression levels of YAP, IRF3 and TAZ in gastric cancer. (F) Scattergrams of the mRNA levels of YAP and IRF3 in the gastric cancer database GSE 13911. (G) YAP and IRF3 mRNA levels in gastric cancer. (H) Phosphorylation level of IRF3 in gastric cancer samples. (I) YAP and IRF 3. (J) In the histochemical staining, the levels of YAP and IRF3 staining were characterized as negative (-), weak (+), general (+ +), strong (+++) in normal colon samples as well as in colon cancer samples. (K) Survival analysis the Kaplan-Meier method characterizes the correlation of patient survival with the mRNA expression levels of YAP/IRF3 in tissue samples.

FIG. 2: targeting IRF3 inhibited gastric tumor growth. (A) The chemical structural formula of amlexanox. (B) Proliferation levels of cells following treatment of HGC27, BGC-823 and MKN45 cell lines with various doses of amlexanox. (C) cloning of cells after treatment with amlexanox. (D) Effect of amlexanox treatment on proliferation of transplanted gastric cancer cell lines. (E) Amlexanox treatment was on tumor size after transplantation of gastric cancer cell line. (F) mRNA levels of target genes downstream of YAP. (G) MNNG/h.pylori induced gastric cancer mouse model number of tumors after treatment with different doses of amlexanox. (H) Ki-67 staining of adenomas. (I) Cell viability following amlexanox treatment at different doses for different gastric cancer cell lines.

FIG. 3: targeting IRF3 inhibited gastric tumor growth dependence and regulation of YAP levels. (A) After amlexanox treatment of the cells, QPCR detected mRNA levels of IFNB as well as CTGF. (B) Cells were clonally formed after amlexanox treatment. (C) Proliferation of cells treated with different doses of BX795, HGC27, BGC-823 and MKN 45. (D) The level of YAP transcription and half-inhibitory concentration of amlexanox in different cancer cell lines (IC 50). (E) After overexpression of YAP in MKN cells, cells were treated with amlexanox, and cell viability was examined. (F) After knocking out YAP in HGC27 cells, treating the cells with amlexanox, and detecting the cell viability. (G) ChIP-QPCR detected antibody levels of IRF3 in gastric cancer cell lines.

Detailed Description

It is understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the embodiments) can be combined with each other to constitute a preferred technical solution.

YAP (Yes-associated protein), is a multifunctional intracellular connexin and transcription co-activator. The YAP protein is a switch protein which plays a central role in a Hippo signal path, and the YAP combines with a transcription factor TEAD to promote the expression of downstream target genes in the normal growth and development process of cells, thereby promoting the growth of the cells and inhibiting the apoptosis of the cells.

IRF3 (interferon regulatory factor 3) is one of the interferon regulatory factor family members and is closely related to the expression of interferon genes upon viral infection. IRF3 is constitutively expressed in a variety of cells, primarily in the cytoplasm.

The invention discovers that IRF3, a key molecule in natural immunity, activates YAP under both physiological and pathological conditions. IRF3 increased YAP entry and activation. In one aspect, IRF3 binds YAP directly, rendering LATS unable to bind and phosphorylate YAP, allowing YAP to be transferred from the cytoplasm into the nucleus. On the other hand, IRF3 interacts with both YAP and YEAD4 to stabilize the transcription complex and enhance YAP regulation of the gene of interest. Meanwhile, the overexpression of IRF3 in cancer cells further aggravates YAP-mediated cell proliferation, and the knockout of IRF3 can down-regulate YAP activation and thus inhibit tumor growth. IRF3 can activate YAP, which provides a target and more precise treatment for YAP-initiated tumor treatment.

Thus, the present invention targets YAP for the treatment and prevention of YAP-mediated diseases via inhibition of IRF3 activation. YAP-mediated diseases are herein especially cancers, including solid and hematological tumors, such as gastric cancer, non-small cell lung cancer, colon cancer, cervical cancer, leukemia, breast cancer and lymphoma. Preferably, said YAP-mediated diseases are especially those in which YAP is over-activated (YAP overexpression). In certain embodiments, YAP over-expressed diseases also exhibit IRF3 over-expression.

Herein, "overexpression", "overactivation" and "high expression" are used interchangeably to mean that the expression (mRNA level or protein level) of YAP or IRF3 is higher than the normal expression level of normal tissue, e.g. at least 1-fold, 2-fold or more than 3-fold higher. In certain embodiments, overexpression or high expression as described herein refers to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, even 1-fold, 2-fold, or more than 3-fold higher expression of YAP or IRF3 in cells of the same class of diseased tissue (e.g., other gastric cancer cells resulting from a different cause) but likely resulting from a different cause as compared to expression of YAP or IRF3 in cells of the same class of diseased tissue (e.g., gastric cancer cells).

The expression level of YAP or IRF3 can be characterized by detecting the number of transcribed copies of YAP or IRF 3. The number of transcribed copies can be detected using methods conventional in the art. For example, detection by reverse transcription can be performed with reference to the experimental method (1) of example 1 of the present application. In general, "overexpression", "overactivation" and "overexpression" can be considered if the number of copies of YAP or IRF3 transcribed in a sample is 1-fold or more higher than the normal value. The normal value may be the expression level of YAP or IRF3 in the corresponding tissue of the normal population, or the expression level of YAP or IRF3 in the peripheral tissue of the diseased tissue (e.g., paracancerous tissue).

In certain embodiments, YAP overexpression may be considered when the number of copies of YAP transcript measured as described herein is greater than or equal to 600; preferably, YAP overexpression is considered when the YAP transcript copy number is equal to or greater than 700, equal to or greater than 800, equal to or greater than 900, equal to or greater than 1000, equal to or greater than 1100, or equal to or greater than 1200. In certain embodiments, IRF3 is considered to be overexpressed when the number of transcribed copies of IRF3 tested to be greater than or equal to 500 using the methods described herein; preferably, IRF3 is considered to be overexpressed when the YAP transcript copy number is 600. gtoreq.700. gtoreq.800 or 900. gtoreq..

Thus, the YAP-mediated disease is preferably a YAP-overexpressing cancer, more preferably a cancer in which both YAP and IRF3 are overexpressed, such as a YAP-overexpressing and optionally IRF 3-overexpressing gastric cancer.

Based on the findings of the present invention, inhibitors of IRF3 can be used to inhibit the activation of IRF3, thereby reducing the activation of YAP to treat or prevent various diseases mediated by YAP as described herein. IRF3 inhibitors can be a variety of inhibitors known in the art, including inhibitors that reduce IRF3 expression from the gene level and inhibitors that inhibit IRF3 activity from the protein level. Herein, IRF3 activity generally refers to its activity to bind YAP. The expression of IRF3 can be reduced by administration of a suitable nucleic acid molecule. For example, a suitable nucleic acid molecule may be a suitable gene knockout vector to knock out IRF3 gene sequences from the genome such that the cell of interest does not express IRF 3; or introducing a mutation in the IRF3 gene sequence such that the cell of interest expresses inactive or weakly active IRF 3. Suitable nucleic acid molecules may also be siRNA capable of inhibiting the expression of IRF3 and RNA interference vectors for IRF3 gene. Suitable knock-out vectors, siRNA and RNA interference vectors can be constructed using methods conventional in the art. In certain embodiments, the inhibitor of IRF3 may also be a protein-based inhibitor, such as an antibody specific for IRF 3.

Alternatively, an inhibitor that inhibits the protein activity of IRF3 may be administered. Such inhibitors may be small molecule inhibitors of IRF3 known in the art. In certain embodiments, since phosphorylation of IRF3 by TBK1(TANK binding kinase 1) is necessary for IRF3 dimerization and activation, inhibition of TBK1 also inhibits IRF3 activity. Thus, in the present invention, IRF3 inhibitors also include small molecule inhibitors (compounds) of TBK1 activity (especially its activity to phosphorylate IRF 3) as well as inhibitors that inhibit or reduce TBK1 expression. The present invention may be practiced using TBK1 inhibitors known in the art, such inhibitors including, but not limited to, for example, the compounds disclosed in CN103748086A, CN103119025A, CN103596938A, CN103930416A, and the like. In certain embodiments, the TBK1 inhibitors of the invention are BX795 and amlexanox. Inhibitors that inhibit or reduce TBK1 expression can be nucleic acid or protein molecules, such as knock-out vectors, siRNA, RNA interference vectors, and antibodies specific for TBK 1.

In certain embodiments, the invention is particularly directed to the use of amlexanox for the treatment or prevention of YAP-mediated diseases, such as cancers described herein, preferably cancers that overexpress YAP, more preferably cancers that overexpress both YAP and IRF3, such as gastric cancer that overexpresses YAP and optionally IRF 3.

Accordingly, the present application provides for the use of an IRF3 inhibitor in the manufacture of a medicament for the treatment or prevention of YAP-mediated diseases as described herein. The present application also provides IRF3 inhibitors for use in treating or preventing YAP-mediated diseases as described herein.

In certain aspects, the invention also provides a method of treating or preventing a YAP-mediated disease described herein, comprising administering to a subject in need thereof a therapeutically effective amount of an IRF3 inhibitor or a pharmaceutical composition thereof.

Herein, "patient" and "subject" are used interchangeably and refer to the mammal, particularly a human, to be treated. As used herein, a "therapeutically effective amount" or "effective amount" refers to the amount of an inhibitor or pharmaceutical composition that is effective to effect treatment in the prevention or treatment of a disease. The "therapeutically effective amount" or "effective amount" may vary depending on the polypeptide, the mode of administration, the disease and its severity, health, age, body weight, family history, genetic makeup, stage of pathological development, type of pre-and concurrent therapy, etc., as well as other individual characteristics of the subject to be treated.

Although the requirements vary from person to person, the optimal dosage of each part of the pharmaceutical composition can be determined by the skilled person. In general, where the inhibitor is a small molecule compound, the amount administered orally to the mammal per day may be in the range of about 0.0025 to 200 mg/kg of body weight. But preferably about 1 to 150 mg/kg per kg of oral dosage. A unit oral dosage may include from about 0.01 to 50mg, preferably from about 0.1 to 100 mg of the inhibitor. The unit dose may be administered one or more times per day in one or more tablets, each tablet containing from about 0.1 to 50mg, some from about 0.25 to 10 mg, of the inhibitor.

The inhibitors or pharmaceutical compositions thereof may be administered using administration means well known in the art. Administration may be topical, pulmonary, epidermal, transdermal, oral or parenteral. Parenteral administration includes intravenous, subcutaneous, intraperitoneal or intramuscular injection or infusion, or intracranial, e.g., intrathecal or intraventricular administration. The pharmaceutical compositions may be prepared in suitable dosage forms for various modes of administration, including but not limited to tablets, capsules, gelcaps, powders or granules, solutions, suspensions, emulsions or mixed media.

The pharmaceutical composition may contain a suitable pharmaceutically acceptable carrier. "pharmaceutically acceptable carrier" refers to an inactive ingredient, such as a solid, semi-solid, or liquid filler, diluent, coating material, formulation accessory, excipient, or carrier, which, in combination with a therapeutic agent, constitutes a "pharmaceutical composition" for administration to a subject. The pharmaceutically acceptable carrier is non-toxic to the subject at the dosages and concentrations employed, and is compatible with the other ingredients in the formulation. Pharmaceutically acceptable carriers are appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, it is desirable that the carrier be non-irritating to the skin and not cause injection site reactions.

Pharmaceutically acceptable carriers can include, for example, buffers (e.g., phosphates, citrates and other organic acids); antioxidants (such as ascorbic acid and methionine); preservatives (such as octadecyl benzyl dimethyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol); low molecular weight (e.g., less than about 10 residues) polypeptides; proteins (such as serum albumin, gelatin or immunoglobulins); hydrophilic polymers (such as polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); a monosaccharide; a disaccharide; and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; and/or a non-ionic surfactant, such as polyethylene glycol (PEG).

Exemplary pharmaceutical carriers may also include binders such as pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, and the like; fillers such as lactose or other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates or dibasic calcium phosphate, and the like; lubricants, such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate and the like; disintegrants, such as starch, sodium starch glycolate, and the like; and wetting agents such as sodium lauryl sulfate and the like.

In certain aspects, the invention also provides the use of a detection reagent for IRF3 and optionally a detection reagent for YAP in the preparation of a diagnostic kit for diagnosing survival of a cancer patient. The detection reagent may be a detection reagent for mRNA expression level or a detection reagent for protein. The reagents for detecting the expression level of mRNA may include primer and probe sequences referred to the coding sequence of IRF3 or YAP. The detection reagent of the protein can be specific antibody of IRF3 protein or YAP protein. Preferably, the cancer is a YAP-mediated cancer as described herein, more preferably gastric cancer.

The present invention will be illustrated below by way of specific examples. It is to be understood that these examples are illustrative only and not limiting of the invention. The methods and materials mentioned in the examples are, unless otherwise indicated, conventional in the art.

Basic experimental methods:

1. immunoblotting

Protein samples were prepared according to the experimental requirements, denatured at 100 ℃ for 10min, centrifuged at 13200rpm for 2 min, and equal amounts of supernatant were added to the loading wells of SDS-PAGE gels. The voltage of the protein sample in the concentrated gel was 80V, and the voltage in the separation gel was 120V. After electrophoresis, taking down the gel, and installing a film transfer device according to the following sequence: (negative electrode), filter paper, gel, nitrocellulose membrane, filter paper, and (positive electrode). And (3) putting the film rotating device into a refrigerator with the temperature of 4 ℃, and rotating for 1h at constant pressure of 100V. After the membrane transfer was complete, the nitrocellulose membrane was removed, the membrane was soaked in 5% skim milk prepared with TBST buffer solution and incubated on a shaker at room temperature for 1 h. The membrane was washed with TBST buffer solution 5min X3 times.

Primary antibody diluted with 5% BSA solution at the specified ratio was added and incubated overnight on a shaker at 4 ℃ in a freezer. The membrane was washed with TBST buffer solution 10min X3 times. Adding secondary antibody diluted with 5% milk at a given ratio, and incubating on a shaker at room temperature for 40-60 min. The membrane was washed with TBST buffer solution 10min X3 times. The chromogenic substrate was overlaid on a nitrocellulose membrane and developed for 2 minutes at room temperature. The images were taken with a LAS4000 luminescence/bioluminescence image analyzer.

Preparation of required reagents: 5% BSA solution: 5g of BSA powder were weighed into 100ml of 1 XPBS solution, and 0.02% sodium azide was added and stored at 4 ℃. 10 XWestern blot membrane transfer buffer: 30.3g Tris base and 144g glycine were weighed, 300ml methanol was added, and 1L volume was made up with deionized water.

2. Extraction of RNA

The transfected cells were discarded from the medium and washed once with pre-cooled PBS. Each well of the six-well plate was treated with 500. mu.l Trizol Reagent at room temperature for 5 minutes. The cell lysate was transferred to a 1.5ml Eppendorf tube. 100 μ l of chloroform was added to each sample, and the mixture was stirred at low speed by vortex and allowed to stand for 5 minutes. Centrifuge at 12000g for 15 min at 4 ℃ and transfer 240. mu.l of the upper aqueous phase to a fresh Eppendorf tube. To each sample was added 240. mu.l of isopropyl alcohol, mixed by inversion, and left at room temperature for 10 minutes. After centrifugation at 12000g for 15 minutes at 4 ℃ the supernatant was discarded, leaving a white RNA precipitate. 500. mu.l of 75% ethanol was added to each sample and mixed by inversion. Centrifuge at 12000g for 5min at 4 ℃ and discard the supernatant. And (5) drying at room temperature. Adding proper amount of DEPC treated deionized water, and fully dissolving. The concentration of the extracted RNA was measured by using NanoDrop ND1000, and the ratio of the absorbance at 260nm to the absorbance at 280nm was maintained between 1.9 and 2.0. The extracted RNA is used for reverse transcription or directly frozen to-80 ℃ for storage.

Preparation of required reagents: phosphate Buffered Saline (PBS): 800ml of distilled water dissolved 0.2g of KCl, 8g of NaCl, 0.24g of KH₂PO₄And 1.44g Na₂HPO₄. The pH of the solution was adjusted to 7.4 with HCl and the volume was 1L. Autoclaving or filter sterilization.

3. Immunoprecipitation

The transfected cells were discarded from the medium and washed once with pre-cooled PBS. Adding appropriate amount of cell lysis buffer RIPA (containing protease inhibitor), performing ice lysis for 30min, centrifuging cell lysate at 4 deg.C and maximum rotation speed for 30min, and collecting supernatant. The required amount of protein A/G agarose beads was taken and washed 3 times with the appropriate amount of lysis buffer. A small amount of lysate was taken for Western blot analysis, and 20. mu.l of pretreated protein A/G agarose beads and 1. mu.g of the corresponding antibody were added to the remaining lysate, which was incubated overnight at 4 ℃ with slow rotation on a stirrer in a freezer. After the immunoprecipitation was completed, the agarose beads were centrifuged to the bottom of the EP tube by centrifugation at 7000rpm for 1min at 4 ℃. The supernatant was carefully aspirated, and the agarose beads were washed 2 times with 300. mu.l lysis buffer and 2 times with 300. mu.l PBS. Finally, 20. mu.l of 2 XSDS loading buffer was added and the mixture was boiled at 100 ℃ for 10 minutes. Proteins bound to the antibodies were analyzed by SDS-PAGE and Western blot.

Preparation of required reagents: RIPA buffer: 150mM NaCl,100mM Tris pH8.0, 1% TritonX-100,5mM EDTA,10mM NaF.

4. Cell culture

HEK293FT, HeLa, MCF-7, SW480 and HCT116 cells were cultured in DMEM (Invitrogen, Carlsbad, Calif.) medium supplemented with 10% serum, 100ug/mL penicillin, 100 ug/mL. The cells were cultured at 37 ℃ with a carbon dioxide concentration of 5%.

A549, Jurkat, Raji, HGC-27, MGC-803, KATOIII, SNU-1, ZGC-1, BGC-823, SGC-7901, MKN-1, GES, ZGC-2, NCI-N87, MKN-45, AGS, SNU-216, ZGC-3, ZGC-4 cells were cultured in RPMI1640(Invitrogen, Carlsbad, CA) culture medium to which 10% serum, 100ug/mL penicillin, 100ug/mL was added. The cells were cultured at 37 ℃ with a carbon dioxide concentration of 5%.

5. Real-time quantitative fluorescent PCR

The relative CT value was measured by real-time quantitative fluorescent PCR using a two-step real-time PCR (real-PCR) system manufactured by Applied Biosystems. Quantitative fluorescent PCR premix (Toyobo Co., Ltd., 2X) was used

Green real PCR reagent) configured reaction system to detect and quantify the expression level of the target gene, and GAPDH as an internal reference.

6. Immunohistochemistry

Tissue samples were fixed with Zinc (BD Biosciences) and paraffin-embedded according to the BD Pharmingen IHC Zinc Fixative manual (manual No.: 550523). Tissue sections (thickness 5 μm) were fixed by heating, and the sections were deparaffinized in xylene for 5 minutes, and then deparaffinized with fresh xylene for a total of 3 times. Absolute ethanol for 5 minutes, twice. 90% ethanol 5min, twice, 70% ethanol 5min, once. Distilled water for 5 minutes twice. Depending on the antigen and antibody, the sections are optionally placed in an antigen retrieval solution of 10mM sodium citrate, pH6.0, or 1mM EDTA, pH8.0, or 10mM Tris, pH10.0, heated at 95 ℃ for 12 minutes and slowly cooled to room temperature over approximately 30 minutes. 5% skim milk was added and blocked for 60 minutes.

All steps from the closing are carried out, and the moisture retention of the sample is necessarily required to avoid the drying of the sample, otherwise, a higher background is easily generated. Primary antibody was diluted in appropriate proportions, incubated overnight at 4 ℃ with slow shaking, primary antibody recovered, PBST added and washed for 5 minutes. After the washing solution was completely absorbed, the washing solution was added thereto and washed for 5 minutes. The total number of washes was 3. Horseradish peroxidase (HRP) or Biotin (Biotin) or Alkaline Phosphatase (AP) labeled secondary antibodies were diluted in appropriate proportions. Incubate for one hour at room temperature or 4 ℃ on a side-shaking table with slow shaking. And (5) recovering the secondary antibody. PBST wash was added and washed on a side-shaking table with slow shaking for 5 minutes. After the washing solution was completely absorbed, the washing solution was added thereto and washed for 5 minutes. The total number of washes was 3. Selecting DAB for subsequent detection. And performing HE dyeing after DAB dyeing. Finally, dehydrating, and sealing the transparent neutral resin.

7. Cell proliferation assay

Cell proliferation detection Using ATP cell viability detection kit (Promega Co., Ltd.)

Luminescent Cell visual Assay). Cells were prepared with medium in 100. mu.l/well in a 96-well plate with opaque walls, while control wells containing medium alone and no cells were prepared to obtain background luminescence. The test compound is added to the test well and incubated under appropriate conditions.

Equilibrate the plate and its contents to room temperature, taking approximately 30 minutes. Adding to each well a volume of cell culture medium

Reagent 100. mu.l. The contents were mixed on an orbital shaker for 2 minutes to induce cell lysis, and the plates were incubated at room temperature for 10 minutes to stabilize the fluorescence signal value and the luminescence signal was recorded.

8. Soft agarose cell clone formation assay

Cells transfected with the corresponding plasmid reached a cell density of 10⁴Thereafter, the cells were inoculated on soft agarose in a 6-well plate, and clones having a diameter of more than 0.05 mm were counted after 14 days.

9. Tumor transplantation experiment

Six-week male nude mice (BALB/cA-nu/nu) were purchased from Shanghai laboratory animal center and cultured in a sterile environment. Bare chipMice were divided into four groups and each was injected subcutaneously with a gastric cancer cell line. The amount of injected HGC-27 was 1X 10⁶(ii) a BGC-823 is 1X 10⁶(ii) a MKN-45 of 2X 10⁶Cells were injected in the flank of mice. Following tumor formation, mice were given amlexanox coated in corn oil daily by intragastric drench. Mice were injected randomly with 5 mg/kg/day or 50 mg/kg/day amlexanox. In addition, mice were treated with intravenous 5-fluorouracil at 50 mg/kg/day as a positive control. After four weeks, the mice were sacrificed and their tumor sizes were measured. The experiment adopts an experimental method of a double-blind experiment. Animal culture and animal experiments were in compliance with the rules and animal welfare policies of the institute of biochemistry and cell biology, national institute of sciences, shanghai, china.

10. MNNG/helicobacter pylori induction establishment of mouse gastric cancer model

The mice were housed in a culture chamber dedicated to an animal model of infectious diseases, and received a light irradiation for 12 hours and a dark day and night rhythm for 12 hours. Scraping helicobacter pylori SS1 from the cultured and solid culture medium, and injecting into mouse with single intragastric injection amount of 1 × 10⁷CFU/ml. Mice in the control group were injected with an equal amount of physiological saline and kept in isolation.

11. Cloning of the Gene of interest

Designing a primer aiming at a required target gene, cloning the target gene from a plasmid containing the target gene or cDNA of Hela cells by a PCR method, wherein two sides of a target gene fragment are provided with two different enzyme cutting sites. Detecting whether the size of the PCR product is consistent with the size of the required target gene by an agarose gel electrophoresis method, and recovering the fragment by using an agarose gel DNA recovery kit. Adding enzymes corresponding to the enzyme cutting sites on both sides of the PCR product and a proper buffer solution into the gel recovery product for double enzyme cutting, and incubating overnight at 37 ℃. Simultaneously, the vector to which the fragment is to be ligated is double digested with the same enzyme. And recovering the digested fragments by using a common DNA product purification kit, recovering the digested vector by using an agarose gel DNA recovery kit, connecting the fragments with the vector by using T4 ligase, and converting the connecting product into DH5 alpha. And (4) obtaining the clone of the target gene by resistance screening, verification and sequencing.

12. Transfection

Invitrogen (San Diego, Calif.) transfection reagent was used

Transient transfection was performed on cells and the effect of overexpression on cells was examined after 24 hours and the effect of gene knock-out on cells was examined after 48 hours.

13. Chromatin immunoprecipitation technique (ChIP)

The cells were suspended in 5-fold volume of cell lysate (10mM Hepes-KOH, pH 7.8,10mM KCl,0.1mM EDTA, 0.1% NP-40) and incubated on ice for 5 minutes. The lysate was centrifuged at 700g for 3 minutes and resuspended using 3 volumes of cell lysate. The pelleted nuclei were again centrifuged at 700g for 3 minutes and resuspended in 9.5ml PBS. 0.5ml of 20mM DSP was added and mixed at 25 ℃ for 30 minutes to immobilize the nuclei. After centrifugation at 190g for 3 minutes, nuclei were obtained, and fixed with 1% formaldehyde at 25 ℃ for 10 minutes. 0.5ml of 2.5M glycine was added thereto, and the mixture was mixed for 5 minutes to terminate the reaction. The pellet was centrifuged at 700g for 3 minutes and resuspended in 0.3ml of protease inhibitor-added cell nuclear lysate (10mM Tris-HCl, pH 7.5, 200mM NaCl,10mM EDTA, 1% SDS). And (3) carrying out ultrasonic treatment on the lysate to obtain a 300-1,000 bp DNA fragment. After removing cell debris by centrifugation, 1.8ml of ChIP dilution buffer was added, followed by mixing with 10. mu.l of protein A-Sepharose (50% concentration) filler at 4 ℃ for 30 minutes. 2-5 μ g of YAP antibody and IRF3 antibody are incubated with the YAP antibody and IRF3 antibody for obtaining cross-linked chromatin for immunoprecipitation, and Q-PCR detection is performed by using ChIP primers of related genes.

14. Data analysis

Data were analyzed using the SAS data software analysis package (9.1.3) and the mean ± standard deviation of the data was counted. One-way variational analysis (ANOVA) and Student's t-test were used to analyze continuous variables. The confidence interval was P < 0.05.

Example 1: IRF3 is YAP-related gastric cancer diagnostic target

1. Purpose of the experiment: determination of the Association of IRF3 with YAP expression levels in gastric carcinoma

2. The experimental method comprises the following steps:

(1) detection of mRNA expression levels of YAP and IRF 3: cell extraction of total RNA As described in basic Experimental method 2, AMV reverse transcriptase synthesizes a first strand, and PCR amplification is performed with corresponding primers under the action of DNA taq enzyme by using the first strand as a template; the reverse transcription system is shown in Table 1, the reverse transcription reaction procedure is shown in Table 2, the cell sample amplification PCR primer is shown in Table 3, the Realtime-PCR reaction system is shown in Table 4, and the Realtime-PCR amplification procedure is shown in Table 5.

Table 1: reverse transcription reaction system

Table 2: reverse transcription procedure

37℃

15min

50℃

5min

95℃

5min

4℃

20min

Table 3: primer sequences

Table 4: Realtime-PCR reaction system

Table 5: PCR reaction System Programming

(2) Detection of protein levels of YAP and IRF3 and phosphorylation level of IRF 3: as described in basic experimental method 1.

(3) Co-immunoprecipitation assay to detect YAP and binding levels of IRF3 to TEAD 4: as described in basic experimental method 3.

(4) Expression levels of IRF3 with YAP immunohistochemical staining: as described in basic experimental method 6.

3. Experimental results and analysis:

since the expression level of YAP in cancer cells is sensitive to amlexanox, we investigated whether YAP and IRF3 have an association in tumors.

We first examined the expression profiles of YAP and IRF3 in different cell lines. In the gastric cancer cell lines, YAP was associated with the mRNA expression level of IRF3, and the same results were obtained in other cancer cells, such as A549 (non-small cell lung cancer), HCT116 (colon cancer), SW480 (colon cancer), Hela (cervical cancer), Jurkat (leukemia), MCF-7 (breast cancer) and Raji (lymphoma) (FIG. 1, A). Furthermore, the protein level of IRF3 was also correlated with YAP (fig. 1, B).

Since activation of YAP requires not only its nuclear entry but also binding to the transcription factor TEAD4, IRF3 may serve to stabilize the YAP-TEAD4 transcription complex. We examined the level of YAP and IRF3 binding to TEAD4 in gastric cancer cells. The co-IP experiments showed that the amount of YAP obtained by endogenous IP of TEAD 4-specific antibodies correlates with the amount of IRF3 obtained by co-immunoprecipitation in the gastric cancer cell lines HGC-27, MGC-803, MKN-1, BGC-823 and MKN-45cells, indicating that IRF3 is an activator of YAP (FIG. 1, C).

Subsequently, we examined the expression profile of YAP and IRF3 in a helicobacter pylori-infected mouse gastric cancer model, and we found that the expression level of YAP was significantly increased (fig. 1, D), and the expression level of IRF3 was also significantly increased, and positively correlated with the change in YAP level (r ═ 0.612, p <0.001) (fig. 1, D). Through analysis of clinical sample data in the Gene Expression Omnibus (GEO) database (http:// www.ncbi.nlm.nih. gov/gds), we found that mRNA levels of YAP and IRF3 were significantly upregulated in gastric cancer patients (FIG. 1, E). In addition, IRF3 mRNA levels were positively correlated with YAP (p 0.002) in samples from gastric cancer patients (fig. 1, F).

To further validate the association of IRF3 with YAP, we examined clinical samples of 90 gastric cancer patients and univariate analysis showed up-regulation of mRNA levels of YAP in 50% of samples and IRF3 in 39% of samples from gastric cancer patients (fig. 1, G). Also, the up-regulation of IRF3 was correlated with YAP (table 6). In addition, we tested the phosphorylation level of IRF3 in two clinical samples, the protein levels of IRF3 and YAP in gastric cancer samples were increased compared to normal samples, and the phosphorylation of IRF3 was also increased in gastric cancer samples, and YAP was over-activated (fig. 1, H).

TABLE 6

To investigate the clinical relevance of IRF3 expression levels, we performed immunohistochemical staining of tissue chip samples from 88 patients with long-term follow-up gastric cancer and examined IRF3 and YAP expression levels. By analytically comparing representative images of gastric tissue staining of cancer and normal tissues of the same patient, we found that protein levels of IRF3 as well as YAP were significantly upregulated in both cytoplasm and nucleus (fig. 1, I). At the same time, we collected and processed patient information and extracted clinical background (age, sex) and tumor stage information (tumor size, lymph node metastasis rate, distant metastasis, tumor stage) from it. Our results show that staining of IRF3 was significantly correlated with tumor size (p <0.05), but not with age, sex, lymph node metastasis, tumor metastasis and staging (fig. 1, J, table 7). Similar results were also found in the detection of YAP (fig. 1, J). Kaplan-Meier survival analysis showed that the expression levels of IRF3 and YAP negatively correlated with 5-year survival in gastric cancer patients (FIG. 1, K). The above data indicate that IRF3 is a diagnostic indicator for the overall survival rate of gastric cancer patients (relative risk: 0.589, 95% confidence interval [ CI ]: 0.351-0.960, p ═ 0.037).

TABLE 7

Example 2: the IRF3 is used as a therapeutic target for inhibiting gastric cancer cell proliferation

1. Purpose of the experiment: the effect of IRF3 as a therapeutic target on gastric cancer cell proliferation was investigated.

2. The experimental method comprises the following steps:

(1) detection of cell proliferation: as described in basic experimental method 7.

(2) Detection of transcription level of YAP downstream target gene, i.e. IFNB, detection of expression level of CTGF: as shown in example 1, the amplification primers are shown in Table 8.

Table 8: primer sequences

(3) ChIP experiment: : as described in basic experimental method 13. ChIP primers are shown in Table 9.

Table 9: primer sequences

3. Experimental results and analysis:

since phosphorylation and activation of IRF3 can promote the entry and activation of YAP, we speculate that the IRF3 upstream kinase TBK1 can regulate the activation of IRF3 and further regulate the activity of YAP, and inhibit the proliferation of gastric cancer cells caused by YAP. To test this hypothesis, we used the inhibitor amlexanox of TBK 1. Amlexanox is a small molecular compound with immunoregulatory function, and is clinically used for treating recurrent oral ulcer. Our results show that the proliferation of the gastric cancer cell lines HGC-27 and BGC-823 cells can be significantly inhibited by amlexanox, but there is no inhibition of the MKN-45cell line (FIG. 2, B). Meanwhile, amlexanox not only inhibited IRF3 signaling pathway, we found that amlexanox also blocked YAP activation by detecting the transcription level of YAP downstream target genes (fig. 3, a). Likewise, amlexanox reduced the clonogenic activity of HGC-27 and BGC-23 cells (FIG. 2, C). In addition, HGC-27 and BGC-823 cells treated with small molecule BX795 (also a TBK1 inhibitor) significantly inhibited cell viability, but had no significant effect on MKN-45 (FIG. 3, C):

further transplantation experiments showed that amlexanox can inhibit cell proliferation of YAP-overactivated gastric cancer cell lines, namely HGC-27 and BGC-823, with dose-dependent effects, as with 5-FU, a traditional gastric cancer treatment (FIG. 2, D, E). The transcriptional levels of the YAP target gene CTGF and CYR61 were down-regulated in amlexanox-treated tumor tissues (fig. 2, F). In agreement with the results of the transplantation experiments, amlexanox reduced the number of tumors and had a dose-dependent effect (fig. 2, G), which was also confirmed using Ki67 staining in a helicobacter pylori-induced mouse gastric cancer model (fig. 2, H).

To further validate the inhibitory effect of amlexanox on gastric cancer cell proliferation at different YAP expression levels, we performed 12 gastric cancer cell lines and 4 gastric cancer primary cell lines (ZGC-1)ZGC-2, ZGC-3, ZGC-4) (FIG. 2, I) examined the effect of amlexanox on cell viability. As shown in FIG. 2(I), amlexanox can significantly inhibit HGC-27 (IC)₅₀＝3.90μM),MGC-803(IC₅₀＝3.2μM), KATOIII(IC₅₀＝8.7μM),SNU-1(IC₅₀7.6 μ M) and ZGC-1 (IC)₅₀4.3 μ M), for BGC-823 (IC)₅₀＝33.0μM),SGC-7901(IC₅₀＝31.5μM),MKN-1 (IC₅₀＝12.4μM),GES(IC₅₀17.3 μ M) and ZGC-2 (IC)₅₀21.7 μ M) has certain inhibiting effect on cell viability; but in NCI-N87, MKN-45, AGS, SNU-216, ZGC-3 and ZGC-4 cells (IC)₅₀>100 μ M) cell line only slightly decreased cell viability. These results indicate that amlexanox is selective for inhibiting tumor cell growth. While the expression level of YAP is directly related to the sensitivity of tumor cells to amlexanox treatment (FIG. 2, I; 3, D). That is, YAP is highly expressed in HGC-27, MGC-803 and ZGC-1 cell lines, and sensitivity to amlexanox treatment is higher than in cell lines with low YAP expression such as MKN-45 and ZGC-4. To further validate the correlation of amlexanox sensitivity with YAP expression levels, we overexpressed YAP in the MKN-45cell line, whose expression resulted in increased sensitivity of the MKN-45cell line to amlexanox (fig. 3, E). Similarly, transfection of shRNA in HGC-27 cell line to knock out YAP reduced sensitivity of HGC-27 cell line to amlexanox (FIG. 3, F). Furthermore, ChIP experiments showed that the activation of YAP by IRF3 was dependent on the expression level of YAP by detecting the level of IRF3 binding to the CTGF promoter in HGC-27, BGC-823 and MKN-45cells (FIG. 3, G).

Sequence listing

<110> Shanghai Life science research institute of Chinese academy of sciences

<120> use of IRF3 inhibitor for preparing a medicament for treating or preventing YAP overactivated cancer

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Claims (1)

1. Use of BX795 in the manufacture of a medicament for inhibiting the proliferation and/or viability of a gastric cancer cell, said gastric cancer cell being an HGC-27, BGC-823 or MKN-45 cell.

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