WO2023157776A1 - NOUVEL AGENT THÉRAPEUTIQUE CONTRE LE CANCER DU FOIE CONTENANT UN INHIBITEUR D'INTERACTION INHIBANT L'INTERACTION ENTRE PKCδ ET E-SYt1 - Google Patents

NOUVEL AGENT THÉRAPEUTIQUE CONTRE LE CANCER DU FOIE CONTENANT UN INHIBITEUR D'INTERACTION INHIBANT L'INTERACTION ENTRE PKCδ ET E-SYt1 Download PDF

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WO2023157776A1
WO2023157776A1 PCT/JP2023/004659 JP2023004659W WO2023157776A1 WO 2023157776 A1 WO2023157776 A1 WO 2023157776A1 JP 2023004659 W JP2023004659 W JP 2023004659W WO 2023157776 A1 WO2023157776 A1 WO 2023157776A1
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pkcδ
syt1
liver cancer
cells
interaction
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PCT/JP2023/004659
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Japanese (ja)
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幸司 山田
沙耶 本橋
清嗣 吉田
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学校法人慈恵大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

  • the present invention relates to a novel liver cancer therapeutic drug containing an interaction inhibitor between protein kinase C delta (PKC ⁇ ) and Extended-synaptotagmin 1 (E-Syt1).
  • PLC ⁇ protein kinase C delta
  • E-Syt1 Extended-synaptotagmin 1
  • protein secretion is a fundamental mechanism required for intercellular communication in developmental processes, maintenance of homeostasis, and development and progression of cancer. Transmembrane mechanisms are important for the movement of secreted proteins out of the cell.
  • General secretory proteins have a signal peptide at the N-terminus, bind to signal recognition particles, move into the endoplasmic reticulum (ER) through the pore of the translocon, and then exit the cell after being transported to the Golgi apparatus. secreted (ER-Golgi secretory pathway).
  • Non-Patent Document 1 a pathway via the plasma membrane (PM) directly (Non-Patent Document 1) and a pathway via vesicular transport (Non-Patent Document 1) Patent Document 2), and it has been reported to be involved in various types of organelles such as autophagosomes, lysosomes, and ER Golgi intermediate compartment (ERGIC) (Non-Patent Document 3).
  • the mechanisms of secretion of these cytoplasmic proteins have been previously investigated in the context of inflammatory and neurodegenerative diseases, but it is unclear whether this mechanism also applies to other diseases such as cancer. It wasn't.
  • the ER is the largest intracellular organelle that synthesizes proteins with a signal peptide at the N-terminus. It is widely known that ER membranes are distributed throughout the cell, often forming contact sites near the membranes of different organelles, controlling organelle dynamics and triggering the biogenesis of organelles such as autophagosomes. ing. Autophagosome formation is thought to originate from the ER membrane at ER-mitochondria or ER-PM contact sites. Interestingly, autophagosomes have been reported to be involved in the secretion of IL-1 ⁇ , a secreted protein lacking an N-terminal signal peptide, in inflammatory cells.
  • Non-Patent Document 3 SEC22B
  • E-Syt1 is known to be a tethering factor at the ER-PM contact site together with STIM1 (Non-Patent Document 4. ).
  • STIM1 Non-Patent Document 4.
  • An object of the present invention is to provide a novel therapeutic agent for liver cancer whose mechanism of action is inhibition of extracellular secretion of PKC ⁇ .
  • the present inventors have found that PKC ⁇ is secreted from hepatoma cells through a pathway via vesicle transport by interacting E-Syt1 on the ER with PKC ⁇ in hepatoma cells, and that PKC ⁇ and E-Syt1 interact.
  • the present inventors have found that by inhibiting the action of PKC ⁇ , the secretion of PKC ⁇ from liver cancer cells can be inhibited, and as a result, the proliferation of liver cancer cells is suppressed.
  • the present invention was completed based on the above findings.
  • the present invention is as follows.
  • the therapeutic drug for liver cancer according to [1], wherein the interaction inhibitor is an anti-PKC ⁇ antibody or an antigen-binding fragment thereof.
  • the anti-PKC ⁇ antibody or antigen-binding fragment thereof has 90% or more identity with the amino acid sequence of amino acid numbers 601 to 676 in SEQ ID NO: 1 or the amino acid sequence at positions corresponding thereto, or those amino acid sequences
  • the therapeutic drug for liver cancer according to [1] or [2] which recognizes an epitope sequence contained in the sequence.
  • liver cancer drug any one of [1] to [3], wherein the antigen-binding fragment is Fab, Fab', F(ab') 2 , scFab, scFv, diabodies, triabodies, minibodies, or nanobodies; liver cancer drug.
  • the interaction inhibitor is a nucleic acid that targets E-Syt1.
  • the drug for treating liver cancer according to [7], wherein the nucleic acid is siRNA or a gene construct used in the CRISPR/Cas9 system.
  • the genetic construct used in the siRNA or CRISPR / Cas9 system is [8], wherein the genetic construct comprises at least one siRNA among siRNA having a nucleotide sequence represented by any one of SEQ ID NOS: 5 to 8, or a gRNA having a nucleotide sequence represented by SEQ ID NO: 4; The drug for treating liver cancer as described.
  • a PKC ⁇ secretion inhibitor including an intracellular interaction inhibitor between PKC ⁇ and E-Syt1.
  • a growth inhibitor for liver cancer cells comprising an inhibitor of intracellular interaction between PKC ⁇ and E-Syt1.
  • a therapeutic drug for liver cancer comprising an E-Syt1 expression inhibitor.
  • the drug for treating liver cancer of [12], wherein the E-Syt1 expression inhibitor is a nucleic acid that targets E-Syt1.
  • the nucleic acid is siRNA or a gene construct used in the CRISPR/Cas9 system.
  • the genetic construct used in the siRNA or CRISPR/Cas9 system is [14], wherein the genetic construct comprises at least one siRNA among siRNA having a nucleotide sequence represented by any one of SEQ ID NOS: 5 to 8, or a gRNA having a nucleotide sequence represented by SEQ ID NO: 4;
  • the drug for treating liver cancer as described.
  • a method of treating liver cancer comprising administering an inhibitor of intracellular interaction between PKC ⁇ and E-Syt1 to a subject in need thereof.
  • a method of suppressing secretion of PKC ⁇ which comprises administering an inhibitor of intracellular interaction between PKC ⁇ and E-Syt1 to a subject in need thereof.
  • a method for suppressing proliferation of liver cancer cells which comprises administering an inhibitor of intracellular interaction between PKC ⁇ and E-Syt1 to a subject in need thereof.
  • a method of treating liver cancer comprising administering an E-Syt1 expression inhibitor to a subject in need thereof.
  • the present invention provides a novel therapeutic agent for liver cancer containing an inhibitor of the interaction between PKC ⁇ and E-Syt1.
  • FIG. 1 shows that the BioID screen identified E-Syt1 as a membrane protein that interacts with cytoplasmic PKC ⁇ .
  • FIG. 1a shows the results of two-dimensional image transformation analysis (2DICAL) of biotinylated proteins purified from membrane fractions of two types of HepG2 cells (clone E5 or clone A8) stably expressing PKC ⁇ -BioID2. 360 counts of 2DICAL were calculated comparing untreated cells not expressing PKC ⁇ -BioID2 with doxycycline treated cells. Specificity was defined when the number of spectra for both E5 and A8 clones was greater than or equal to 3-fold.
  • FIG. 1 shows that the BioID screen identified E-Syt1 as a membrane protein that interacts with cytoplasmic PKC ⁇ .
  • FIG. 1a shows the results of two-dimensional image transformation analysis (2DICAL) of biotinylated proteins purified from membrane fractions of two types of HepG2 cells (clone E5 or clone A8)
  • FIG. 1c is a confocal micrograph showing the co-localization of PKC ⁇ and E-Syt1 in HepG2 cells. Pictures are representative of three independent experiments. Scale bar: 10 ⁇ m, inset: Enlarged photograph within a white frame.
  • FIG. 1d shows that doxycycline-inducible PKC ⁇ -GFP stably expressing HepG2 cells were incubated with 0.5 ⁇ g/mL doxycycline for 24 hours, then fixed and stained with Sec61 ⁇ as an ER marker, and the stained cells were examined by super-resolution microscopy (3D). It is a photograph imaged by SIM). A planar image (xy) and a three-dimensional reconstructed image (inset: an enlarged photograph in a white frame; yz) are shown. Arrowheads suggest co-localization of PKC ⁇ -GFP and ER. Pictures are representative of three independent experiments. FIG.
  • 1e is a photograph of detection of the interaction between PKC ⁇ and E-Syt1 in HepG2 cells and AGS cells using confocal microscopy.
  • Each cell was fixed and a combination of mouse anti-PKC ⁇ antibody and rabbit anti-E-Syt1 antibody (PKC ⁇ E-Syt1), a combination of mouse IgG and rabbit IgG (control ⁇ control), or mouse anti-PKC ⁇ antibody and rabbit anti-PKC ⁇ Duolink In Situ proximity ligation assay (PLA) was performed by reacting with a combination of antibodies (PKC ⁇ PKC ⁇ ).
  • PKA in situ proximity ligation assay
  • FIG. 1f is a photograph showing the detection of the interaction between PKC ⁇ and Sec61 ⁇ (ER marker) in HepG2 cells (control), E-Syt1 knockout HepG2 cells (E-Syt1 KO) and AGS cells using a confocal microscope. Each cell was fixed, reacted with a combination of mouse anti-PKC ⁇ antibody and rabbit anti-Sec61 ⁇ antibody (PKC ⁇ Sec61 ⁇ ), and Duolink In Situ proximity ligation assay (PLA) was performed. In the graph on the right, the data are mean ⁇ s.e.m. d.
  • FIG. 2 shows that E-Syt1 is required for PKC ⁇ secretion.
  • FIG. 2c shows a schematic representation of human wild-type PKC ⁇ (WT), partial deletion mutants of PKC ⁇ ( ⁇ 451-676 and ⁇ 601-676). These PKC ⁇ constructs were fused with BioID2 and HA-epitope tags.
  • FIG. 2d shows human wild-type PKC ⁇ (WT) or partially deleted mutants of PKC ⁇ ( ⁇ 451-676 and ⁇ 601-676) fused with an empty vector (empty) or with BioID2 and HA-epitope tags.
  • FIG. 2e is a confocal micrograph of PKC ⁇ -BioID2-HA vector containing WT or a partially deleted mutant of PKC ⁇ ( ⁇ 601-676) transfected into doxycycline-inducible HepG2 cells. Pictures are representative of two independent experiments. Scale bar: 10 ⁇ m. FIG.
  • FIG. 3 shows that autophagy-related proteins are utilized for secretion of PKC ⁇ .
  • Figure 3a shows the results of HiBiT extracellular assay for PKC ⁇ secretion in doxycycline-induced HepG2 cells.
  • FIG. 3b is a flow cytometric analysis of doxycycline-treated HepG2 cells incubated in 10% FBS-containing medium, 0.1% FBS-containing medium, or EBSS medium for 6 hours. The figure is representative of three independent experiments.
  • FIG. 3d is a photograph showing the results of immunoblot analysis of lysate and medium of doxycycline-induced HepG2 cells treated with scrambled (Scr), ATG5 or LC3B siRNA (2 ⁇ M) for 48 hours. Pictures are representative of three independent experiments. GAPDH and Ponceau-S staining were used as loading controls for lysate and medium, respectively.
  • Figure 3e is a confocal micrograph showing the co-localization of intracellular PKC ⁇ and LC3B in HepG2 cells cultured in medium containing 10% FBS. Pictures are representative of three independent experiments. Scale bar: 10 ⁇ m, inset: Enlarged photograph within a white frame. Arrows suggest co-localization of PKC ⁇ and LC3B.
  • FIG. 3f is a confocal micrograph of HepG2 cells expressing GFP and mCherry-LC3B cultured in EBSS medium for 3 hours. Pictures are representative of three independent experiments. Scale bar: 10 ⁇ m, inset: Enlarged photograph within a white frame.
  • FIG. 3h is a confocal micrograph for detecting the interaction of PKC ⁇ and LC3B (an autophagosome marker) in HepG2 cells, AGS cells.
  • FIG. 4b is a confocal micrograph to show the close proximity of PKC ⁇ and SEC22B in HepG2 (control), E-Syt1 KO HepG2 (E-Syt1 KO) or AGS cells.
  • Each cell was fixed, reacted with a combination of mouse anti-PKC ⁇ antibody and rabbit anti-SEC22B antibody (PKC ⁇ SEC22B), and Duolink In Situ PLA was performed.
  • FIG. 4c is an electron micrograph of HepG2 cells. The left panel shows the presence of PKC ⁇ (arrows) within SEC22B + vesicles (arrowheads) around PMs, and the right panel shows the moment PKC ⁇ is secreted upon fusion of vesicles and PMs. Pictures are representative of two independent experiments. Scale bar: 100 nm. FIG. 4d.
  • FIG. 4e is a photograph of cancer tissue from a patient with hepatocellular carcinoma (HCC).
  • Duolink In Situ PLA shows the co-localization of cancer cell-specific PKC ⁇ and SEC22B (middle panel and right panel. Cancer (T) and non-cancer (NT) are hematoxylin and eosin staining of each cancer section. (left panel) Photographs are two representatives of independent experiments in sections of 5 liver cancer patients Scale bar: 50 ⁇ m.
  • FIG. 4f is a schematic depicting the role of E-Syt1 with respect to ER-PM contact sites, showing secretion of cytoplasmic proteins through vesicles. PKC ⁇ uptake into SEC22B + vesicles is dependent on the expression of E-Syt1 and autophagy-related proteins.
  • FIG. 5 shows HCC cytostatic effects targeting the interaction of PKC ⁇ and E-Syt1.
  • FIG. 5a is a photograph showing intracellular transport of anti-PKC ⁇ antibody (C-20) in HepG2 cells using Ab (antibody)-DeliverIN transfection reagent (DeliverIN is a drug delivery system). Scale bar: 10 ⁇ m.
  • FIG. 5b is a confocal micrograph of HepG2 cells treated with DeliverIN and control IgG or anti-PKC ⁇ antibody (C-20).
  • Figures 5d-g show the results of proliferation assays by counting the number of cells of cell lines treated with DeliverIN and control IgG or anti-PKC ⁇ antibody (C-20) for 48 hours.
  • FIG. 5g shows the results of the proliferation assay using AGS cells, which are cells that do not secrete PKC ⁇ .
  • FIG. 5h shows photographs of 3D multicellular spheroids of HepG2 cells treated with control IgG or anti-PKC ⁇ antibody (C-20) in the presence/absence of DeliverIN. Scale bar: 20 ⁇ m.
  • Figure 5i Number of 3D multicellular spheroids of HepG2 cells were counted based on size >25 ⁇ m. Three independent experiments were performed. Data are mean ⁇ s.d. d. indicated by *: p ⁇ 0.002 (ANOVA). n. s. No significant difference.
  • FIG. 6 shows that E-Syt1 is involved in cell proliferation of liver cancer cell lines.
  • Figure 6a shows tumorigenicity.
  • FIG. 6d shows tumorigenicity. Photomicrographs of 3D multicellular spheroids after 5 days of culture of HuH7 cells treated with scrambled (Scr) siRNA or E-Syt1 siRNA. Scale bar: 100 ⁇ m.
  • FIG. 7 shows that E-Syt1 is required for PKC ⁇ secretion in hepatoma cell lines.
  • FIG. 7d are confocal micrographs for detection of interaction of SEC22B and STX3 (SNARE in PM) in control HepG2 cells or E-Syt1 KO HepG2 cells (left panel).
  • FIG. 8a is a confocal micrograph to confirm the localization of PKC ⁇ in the ER of HepG2 cells. Sec61 ⁇ was used as an ER marker and DAPI as a nuclear marker. Scale bar: 10 ⁇ m. inset: 2 ⁇ m.
  • FIG. 8b is a schematic diagram of a split-GFP assay, showing that GFP is reconstituted by co-localization of PKC ⁇ and E-Syt1.
  • Fig. 3 is a micrograph showing the results of split-GFP assay using GFP 1-10 -V5-E-Syt1 construct and PKC ⁇ -GFP 11 -HA construct. Scale bar: 10 ⁇ m. inset: 2 ⁇ m.
  • FIG. 9 shows that E-Syt1 is involved in activation of PKC ⁇ -IGF1R signaling.
  • Figure 9a shows phospho-IGF1R (Y1135/1136), phospho-ERK1/2, total IGF1R, total ERK1/2,
  • Fig. 12 shows the results of immunoblot analysis of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (loading control) (left panel).
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • FIG. 9b shows phospho-IGF1R (Y1135/1136), phospho-ERK1/2, total IGF1R in medium of scrambled (Scr) siRNA-treated HuH7 cells or E-Syt1 siRNA-treated HuH7 cells under low nutrition for 16 h. , total ERK1/2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (loading control) (left panel). Three independent experiments were performed. Relative signal densities were quantified (middle and right panels).
  • FIG. 10 shows that E-Syt1 is involved in tumorigenesis of liver cancer.
  • FIG. 10c shows the results of a comparison of overall survival between HCC patients expressing high and low levels of E-Syt1 mRNA. Patients who were negative for hepatitis virus infection were subdivided by clinical grade as grade I (left panel) or grade I+II (right panel). Hazard ratio (HR) and log-rank P value are shown in each panel.
  • HR Hazard ratio
  • One embodiment of the present invention is a therapeutic agent for liver cancer comprising an intracellular interaction inhibitor between PKC ⁇ and E-Syt1.
  • PKC Protein kinase C
  • DAG diacylglycerol
  • CA 2+ calcium ions
  • novel PKC isozymes that require only DAG for their activation ( ⁇ , ⁇ , ⁇ , ⁇ ), etc.
  • a new PKC isozyme, PKC ⁇ is an intracellular signaling kinase of approximately 78 kilodaltons and is known to be expressed in a variety of cells.
  • PKC ⁇ is preferably human PKC ⁇ , and examples thereof include those having the amino acid sequence represented by SEQ ID NO:1.
  • E-Syt1 is a protein involved in intracellular transport of lipids and the like. It is also localized on the endoplasmic reticulum (ER) membrane and is known as a tethering factor that participates in binding between the ER and the cell membrane.
  • E-Syt1 is preferably human E-Syt1, and examples thereof include those having the amino acid sequence represented by SEQ ID NO:2.
  • the method for measuring the interaction between PKC ⁇ and E-Syt1 is not particularly limited as long as the interaction can be measured;
  • the inhibition of the interaction between PKC ⁇ and E-Syt1 is, for example, the interaction between PKC ⁇ and E-Syt1 when the inhibitor is added is 50% or more compared to when the inhibitor is not added. It may be disappearance, it may be disappearance by 70% or more, or it may be disappearance by 90% or more.
  • the PKC ⁇ and E-Syt1 interaction inhibitor is not particularly limited as long as it is introduced into cells and inhibits the interaction between PKC ⁇ and E-Syt1 in cells, and may be antibodies or antigen-binding fragments, It may be a peptide, compound, nucleic acid, or the like. These various interaction inhibitors may be used alone or in combination.
  • the interaction inhibitor is an antibody or antigen-binding fragment thereof, for example, it may be an antibody or antigen-binding fragment thereof targeting PKC ⁇ , an antibody or antigen-binding fragment thereof targeting E-Syt1, PKC ⁇ -targeting antibodies or antigen-binding fragments thereof are preferred.
  • the epitope sequence recognized by the anti-PKC ⁇ antibody or antigen-binding fragment thereof is not particularly limited as long as it targets PKC ⁇ in cells and inhibits the interaction between PKC ⁇ and E-Syt1.
  • the epitope recognized by the anti-PKC ⁇ antibody or antigen-binding fragment thereof preferably recognizes the C-terminal amino acid sequence of PKC ⁇ . For example, having 90% or more, preferably 95% or more, more preferably 98% or more identity with the amino acid sequence of amino acid numbers 601 to 676 in SEQ ID NO: 1 or the amino acid sequence at the position corresponding thereto, or those amino acid sequences It may recognize an epitope sequence contained in an amino acid sequence, or may recognize an epitope sequence consisting of those amino acid sequences.
  • the position corresponding to the amino acid sequence of amino acid numbers 601 to 676 in SEQ ID NO: 1 means the position corresponding to 601 to 676 of SEQ ID NO: 1 in PKC ⁇ that does not have SEQ ID NO: 1.
  • the anti-PKC ⁇ antibody may be either a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody from the viewpoint of stability of therapeutic effects.
  • the anti-PKC ⁇ antibody is preferably a chimeric antibody, a humanized antibody, or a fully human antibody from the viewpoint of reducing antigenicity.
  • the animal from which the antibody is derived may be humans, mice, rats, rabbits, monkeys, etc., camelids such as dromedary, Bactrian camel, llama, alpaca, guanaco, vicuna, cartilage such as shark and chimaera. It may be fish or the like.
  • an antibody produced by a method known to those skilled in the art can also be used.
  • a method for producing an antibody is to immunize an animal such as a mouse with PKC ⁇ as an antigen, collect cells that produce an antibody against the PKC ⁇ antigen protein, and collect the collected cells to treat allogeneic or xenogeneic myeloma. It can be obtained from the culture supernatant of hybridoma cells by selecting hybridoma cells that fuse with the cells and produce anti-PKC ⁇ monoclonal antibodies.
  • chimeric antibodies or humanized antibodies can be obtained by further modifying the above hybridoma cells.
  • the portion encoding the Fc region in this gene is converted to a human Fc region by a gene recombination technique that is a method known to those skilled in the art.
  • a target antibody can be obtained by manipulation such as gene replacement.
  • a fully human anti-PKC ⁇ antibody is obtained by immunizing a genetically modified mouse or the like capable of producing a human antibody with PKC ⁇ as an antigen, collecting anti-PKC ⁇ antibody-producing cells obtained from the genetically modified mouse, It can be obtained from the culture supernatant of hybridoma cells by fusing with myeloma cells and selecting hybridoma cells that produce anti-PKC ⁇ antibodies.
  • Fully human anti-PKC ⁇ antibodies can also be produced using phage display methods, methods known to those skilled in the art.
  • the interaction inhibitor is introduced into cells and inhibits the interaction between PKC ⁇ and E-Syt1 in cells, it should have a small molecular weight to facilitate introduction into cells. preferable.
  • the interaction inhibitor when it is an antigen-binding fragment, it can include, for example, Fab, Fab', F(ab') 2 , scFab, scFv, diabodies, triabodies, minibodies, nanobodies, and the like.
  • Any antigen-binding fragment can be produced by utilizing genetic modification techniques known to those of skill in the art.
  • antigen binding fragments may be obtained from transgenic animals carrying genes capable of producing these antigen binding fragments.
  • the antigen-binding fragment may be a VHH (variable domain of heavy chain of heavy chain antibody) derived from camelids or a VNAR (variable domain of new antigen receptor) derived from cartilaginous fish.
  • the interaction inhibitor when it is a peptide, it may be, for example, a peptide at the interaction site of PKC ⁇ or E-Syt1.
  • the length of the peptide is not particularly limited as long as it can bind to these interaction sites and inhibit the interaction between PKC ⁇ and E-Syt1, but the upper limit may be, for example, 300 amino acids or less, or 200 amino acids or less. well, it may be 100 amino acids or less, it may be 50 amino acids or less, it may be 30 amino acids or less, or it may be 20 amino acids or less, and the lower limit may be, for example, 10 amino acids or more, or it may be 20 amino acids or more, or it may be 30 amino acids or less. It may be amino acids or more, or may be 50 amino acids or more.
  • the upper and lower limits of these lengths may be arbitrarily combined, for example, 10 amino acids or more and 100 amino acids or less.
  • a peptide having a part of the amino acid sequence of amino acid numbers 601 to 676 in SEQ ID NO:1 can be used.
  • the interaction inhibitor when it is a compound, it is preferably, for example, a middle-molecular-weight compound or a low-molecular-weight compound.
  • a commercially available compound may be used as the compound, but a compound prepared by a method known to those skilled in the art may also be used.
  • the interaction inhibitor when it is a nucleic acid, it may be, for example, a nucleic acid targeting PKC ⁇ or E-Syt1, preferably a nucleic acid targeting E-Syt1.
  • Any nucleic acid can be used as long as it can inhibit the expression of a target gene, and examples thereof include siRNA, miRNA, antisense nucleic acids, and nucleic acid constructs for use in genome editing by the CRISPR/Cas9 system.
  • siRNA In order to efficiently knock down the target gene, it is preferable to use siRNA, and in order to efficiently knock out the target gene, it is preferable to use a nucleic acid construct for use in genome editing by the CRISPR / Cas9 system.
  • a commercially available nucleic acid may be used as the nucleic acid, but a nucleic acid prepared by a method known to those skilled in the art may also be used.
  • the siRNA targeting E-Syt1 is not particularly limited as long as it can inhibit the expression of the target gene. It may target E-Syt1 represented by Session Number: NM_015292.3. Specifically, at least one siRNA among siRNAs having a nucleotide sequence represented by any one of SEQ ID NOs: 5 to 8 can be used. One type of siRNA may be used, or multiple siRNAs may be used. A target gene can be knocked down more efficiently when multiple siRNAs are used.
  • the genetic construct used in the CRISPR / Cas9 system is not particularly limited as long as it can inhibit the expression of the target gene by the CRISPR / Cas9 system known to those skilled in the art, for example, gRNA having a nucleotide sequence represented by SEQ ID NO: 4 ( Gene constructs containing guide (RNA) can be used.
  • Screening for interaction inhibitors can be performed, for example, by binding assays.
  • the cells into which the interaction inhibitor is introduced are preferably liver cancer cells.
  • liver cancer to be treated examples include hepatocellular carcinoma, cholangiocellular carcinoma, mixed liver cancer, metastatic liver cancer, hepatoblastoma, fibrolamellar HCC, and the like. It is not limited. In addition, liver cancer is not particularly limited in specific disease sites, disease stages, etc. in the liver, and includes any disease site, disease stage, and the like.
  • the liver cancer to be treated is a cancer type that secretes PKC ⁇ extracellularly through interaction of PKC ⁇ with E-Syt1 on the ER in the cell.
  • This extracellular secretion of PKC ⁇ preferably involves an autophagy-inducing factor, and examples of autophagy-inducing factors include ATG5, ATG7, ATG16L1, LC3B, and p62.
  • extracellular secretion of PKC ⁇ is preferably carried out by a pathway involving vesicles in which SEC22B is expressed. By interacting with STX4 etc., PKC ⁇ incorporated into vesicles is extracellularly secreted.
  • the means for introducing the interaction inhibitor into cells is not particularly limited as long as the interaction inhibitor can be introduced into the target cells.
  • the interaction inhibitor is an antibody or antigen-binding fragment thereof, it can be introduced into cells, for example, using an introduction agent.
  • Delivery agents include lipid-based ones, such as liposomes.
  • lipid carriers such as liposomes having surface-bound cell surface antigens specifically expressed in liver tissue may also be used.
  • the interaction inhibitor is a nucleic acid, it can be introduced into cells using lipid-based nanoparticle-based transfection reagents such as Lipofectamine, or it can be introduced into cells by electroporation. can.
  • the therapeutic drug for liver cancer of this embodiment may contain other active ingredients and pharmacologically acceptable carriers.
  • the content of the interaction inhibitor in the therapeutic agent for liver cancer is not particularly limited as long as it inhibits the interaction between PKC ⁇ and E-Syt1 in cells and can treat liver cancer, for example, 1 ng / mL to 10 ⁇ g / mL.
  • the therapeutic drug for liver cancer of the present invention may be formulated in any dosage form.
  • solutions, suspensions, injections and the like can be mentioned.
  • the mode of administration is not particularly limited, but local administration to the affected area or its surroundings by injection or the like, or intravenous/arterial injection, etc. can be mentioned.
  • active ingredients include, for example, immunostimulants such as cytokines, chemotherapeutic agents, and the like. These other active ingredients can be used appropriately in appropriate amounts.
  • Examples of pharmacologically acceptable carriers include solvents, distilled water, physiological saline, diluents, surfactants, stabilizers, solubilizers, suspending agents, emulsifiers, buffers, preservatives and the like. is mentioned. Furthermore, additives such as preservatives, antioxidants, coloring agents, adsorbents, wetting agents and the like can be used as necessary. These carriers can be used in appropriate amounts.
  • the administration subject is a mammal with liver cancer, preferably a human with liver cancer.
  • the liver cancer patient is preferably a liver cancer patient whose blood PKC ⁇ level is significantly increased compared to healthy subjects. Therefore, the amount of PKC ⁇ is measured before administration of the drug for treating liver cancer of the present invention, and if the amount of PKC ⁇ in the blood is significantly increased compared to healthy subjects, administration may be determined. good.
  • the dosage of the therapeutic drug for liver cancer should be an amount that allows the interaction inhibitor, which is an active ingredient, to inhibit the interaction between PKC ⁇ and E-Syt1 in cells.
  • the dosage can be appropriately adjusted according to the age, sex, body weight, symptoms, therapeutic effect, treatment site area, administration method, etc. of the subject to be administered.
  • it is preferably about 0.01 mg to 5000 mg, more preferably about 0.1 mg to 500 mg per day.
  • the total daily dose may be given in single or divided doses.
  • the results of treatment with a therapeutic drug for liver cancer were compared with those before treatment with a therapeutic drug for liver cancer, or compared with a control without treatment with a therapeutic drug for liver cancer.
  • the analysis method is not particularly limited, and can be performed by a method known to those skilled in the art.
  • Cell biological analysis it is possible to predict the therapeutic effect of a therapeutic agent in vivo by comparing the sample after treatment with the therapeutic agent and the sample before treatment with the therapeutic agent.
  • Cell biological analysis is not particularly limited, and examples thereof include cell proliferation assay, cell cluster (spheroid) formation assay, Western blotting, etc., and can be performed by methods known to those skilled in the art. .
  • liver cancer comprising administering an inhibitor of intracellular interaction between PKC ⁇ and E-Syt1 to a subject in need thereof;
  • PKC ⁇ secretion inhibitor Another embodiment of the present invention is a PKC ⁇ secretion inhibitor, including an intracellular interaction inhibitor between PKC ⁇ and E-Syt1.
  • PKC ⁇ secretion inhibitors descriptions of inhibitors of interaction between PKC ⁇ and E-Syt1, descriptions of cells into which interaction inhibitors are introduced, descriptions of liver cancer, descriptions of means for introducing into cells, PKC ⁇ and E- Descriptions regarding the content of the inhibitor of interaction with Syt1, descriptions regarding dosage form, mode of administration, other ingredients, subjects of administration, dosage, etc. are described in the above-mentioned therapeutic drug for liver cancer containing the inhibitor of interaction between PKC ⁇ and E-Syt1. The description corresponding to each in can be used.
  • PKC ⁇ secretion inhibitory effect was analyzed, for example, as a result of treatment with a PKC ⁇ secretion inhibitor, compared with before treatment with a PKC ⁇ secretion inhibitor or without treatment with a PKC ⁇ secretion inhibitor.
  • the analysis method is not particularly limited, and can be performed by a method known to those skilled in the art.
  • a method of suppressing secretion of PKC ⁇ comprising administering an inhibitor of intracellular interaction between PKC ⁇ and E-Syt1 to a subject in need thereof; an intracellular PKC ⁇ -E-Syt1 interaction inhibitor for use in inhibiting the secretion of PKC ⁇ ; Use of an intracellular interaction inhibitor between PKC ⁇ and E-Syt1 in the production of a PKC ⁇ secretion inhibitor.
  • Another embodiment of the present invention is an agent for suppressing the growth of liver cancer cells, comprising an inhibitor of intracellular interaction between PKC ⁇ and E-Syt1.
  • the analysis method is not particularly limited, and can be performed by a method known to those skilled in the art.
  • examples of this embodiment include the following: A method for suppressing the proliferation of hepatoma cells, comprising administering an inhibitor of intracellular interaction between PKC ⁇ and E-Syt1 to a subject in need thereof; An inhibitor of intracellular interaction between PKC ⁇ and E-Syt1 for use in suppressing proliferation of hepatoma cells; Use of an intracellular interaction inhibitor between PKC ⁇ and E-Syt1 in the production of a growth inhibitor for liver cancer cells.
  • Another embodiment of the present invention is a therapeutic drug for liver cancer, comprising an E-Syt1 expression inhibitor.
  • the E-Syt1 expression inhibitor is not particularly limited as long as it inhibits the expression of E-Syt1, but is preferably a nucleic acid that targets E-Syt1.
  • nucleic acid can be used as long as it can inhibit the expression of a target gene.
  • examples include siRNA, miRNA, antisense nucleic acids, and nucleic acid constructs for use in genome editing by the CRISPR/Cas9 system.
  • siRNA In order to efficiently knock down the target gene, it is preferable to use siRNA, and in order to efficiently knock out the target gene, it is preferable to use a nucleic acid construct for use in genome editing by the CRISPR / Cas9 system.
  • a commercially available nucleic acid may be used as the nucleic acid, but a nucleic acid prepared by a method known to those skilled in the art may also be used.
  • the siRNA targeting E-Syt1 is not particularly limited as long as it can inhibit the expression of the target gene. It may target E-Syt1 represented by the number: NM_015292. Specifically, at least one siRNA among siRNAs having a nucleotide sequence represented by any one of SEQ ID NOs: 5 to 8 can be used. One type of siRNA may be used, or multiple siRNAs may be used. A target gene can be knocked down more efficiently when multiple siRNAs are used.
  • the genetic construct used in the CRISPR / Cas9 system is not particularly limited as long as it can inhibit the expression of the target gene by the CRISPR / Cas9 system known to those skilled in the art, for example, gRNA having a nucleotide sequence represented by SEQ ID NO: 4 ( Gene constructs containing guide (RNA) can be used.
  • E-Syt1 The expression level of E-Syt1 can be measured by a method known to those skilled in the art. Inhibition of E-Syt1 expression is preferably complete inhibition of expression in target cells, but may be partial inhibition of expression.
  • the means for introducing the E-Syt1 expression inhibitor into cells is not particularly limited as long as the interaction inhibitor can be introduced into the target cells.
  • it can be introduced into cells using lipid-based nanoparticle-based transfection reagents such as Lipofectamine, or it can be introduced into cells by electroporation.
  • the content of the E-Syt1 expression inhibitor in the therapeutic drug for liver cancer is not particularly limited as long as it can inhibit the expression of E-Syt1 in the cells of interest and treat liver cancer. mL.
  • the dosage of the therapeutic drug for liver cancer may be any amount as long as the E-Syt1 expression inhibitor, which is an active ingredient, can inhibit the expression of E-Syt1 in the cells of interest.
  • the dosage can be appropriately adjusted according to the age, sex, body weight, symptoms, therapeutic effect, treatment site area, administration method, etc. of the subject to be administered. For the target, it is preferably about 0.01 mg to 5000 mg, more preferably about 0.1 mg to 500 mg per day.
  • the total daily dose may be given in single or divided doses.
  • liver cancer descriptions regarding liver cancer, dosage form, mode of administration, other ingredients, subjects of administration, etc. correspond to the aforementioned drug for treating liver cancer including the PKC ⁇ -E-Syt1 interaction inhibitor. The description to do can be cited.
  • liver cancer comprising administering an E-Syt1 expression inhibitor to a subject in need thereof; E-Syt1 expression inhibitor for use in the treatment of liver cancer; Use of an E-Syt1 expression inhibitor in the manufacture of a drug for treating liver cancer.
  • pcDNA3.1-mCherry-hLC3B (plasmid no.40827), pEGFP-C1-EGFP-ESYT1 (plasmid no.66830), pHRm-NLS-sCas9-GFP 11 ⁇ 7-NLS (plasmid no.70224), and pHRm- GFP1-10-VP64-NLS (plasmid no. 70228) was purchased from Addgene.
  • BioID2-HA was cloned from MCS-BioID2-HA plasmid 1 (plasmid no.
  • PKC ⁇ (WT) and PKC ⁇ ( ⁇ 601-676) were amplified and cloned into pTRE using the DNA Assembly kit (NEB).
  • NEB DNA Assembly kit
  • a HiBiT sequence (5′-GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC-3′ (SEQ ID NO: 3)) was amplified under license from Promega and transformed into pTRE-PKC ⁇ -13 ⁇ GS vector and pTRE-importin ⁇ 1-13 ⁇ . Cloned into the GS vector.
  • the regions of PKC ⁇ -13 ⁇ GS-GFP 11 -V5 and GFP 1-10 -HA-E-Syt1 were amplified and cloned into pTRE using DNA Assembly Kit (NEB, MA, USA ).
  • HepG2 and human gastric cancer cell line AGS were obtained in 2017 from the Japan Research BioResource Collection (Osaka, Japan).
  • HepG2 Tet-On® Advanced cells were purchased from Takara (Shiga, Japan) in 2018.
  • HepG2 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma).
  • FBS fetal bovine serum
  • AGS cells were maintained in RPMI1640 (Sigma) supplemented with 10% FBS.
  • HepG2 Tet-On® Advanced cells were maintained in ⁇ -MEM (Nacalai, Kyoto, Japan) supplemented with 0.1 mM NEAA, 500 g/mL G418, and 10% Tet system approved FBS (Takara). For undernutrition (starvation) conditions, each cell was washed and cultured in Earle's Balanced Salt Solution (EBSS; Sigma) for the indicated time. Cell lines were routinely monitored for mycoplasma (4A Biotech Co.). Cell lines were confirmed by short tandem repeat profiling every 6 months. The cells used in the experiment were those that had been passaged 10 times or less after thawing in Examples 1-5, and those that had been passaged 10 times after thawing in Examples 6-9.
  • E-Syt1 gRNA 5′-GATGCGCAGGAACGGCGCCATGG-3′ (SEQ ID NO: 4) was designed with CRISPRdirect (https://crispr.dbcls.jp). The gRNA was cloned in a lentiviral lentiCRISPR v2 vector (Addgene plasmid no. 98290). The construct was confirmed by DNA sequencing. E-Syt1 knockout cell lines were generated by methods known to those skilled in the art.
  • Biotinylated proteins were isolated by methods known to those skilled in the art using BioID2. Specifically, it is as follows. TRE-induced HepG2 cells containing BioID2 constructs were cultured in two 100 cm 2 dishes. Biotin (50 ⁇ M) and 1 ⁇ g/mL doxycycline were reacted for 24 hours before cell lysis.
  • Cells were washed with PBS, harvested with a cell scraper, and added with 1 mL of lysis buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1 mM PMSF, 1 mM DTT, 10 ⁇ g/mL aprotinin, 1 ⁇ g/mL leupeptin, 1 ⁇ g/mL pepstatin A, and 1% NP-40). After centrifugation at 20,000 g for 15 minutes, the supernatant was used as cell lysate for the following operations. Protein concentrations of cell lysates were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo).
  • BCA bicinchoninic acid
  • Reagents are Phorbol 12-myristate 13-acetate (PMA; Abcam, Cambridge, UK), Doxycycline (Sigma or Merck), LY2109761 (MedChemExpress, NJ, USA), Chloroquine (CQ; FUJIFILM, Tokyo, Japan), Ba filomycin A1 (Baf A1; InvivoGen, San Diego, CA, USA) and BaptaAM (Dojindo, Kumamoto, Japan) were used.
  • PMA Phorbol 12-myristate 13-acetate
  • Doxycycline Sigma or Merck
  • LY2109761 MedChemExpress, NJ, USA
  • Chloroquine CQ
  • Ba filomycin A1 Baf A1; InvivoGen, San Diego, CA, USA
  • BaptaAM Dojindo, Kumamoto, Japan
  • ⁇ Cell-based HiBiT assay> Extracellularly localized HiBiT fusion proteins were assessed using the Nano-Glo HiBiT Extracellular Detection System (Promega) according to the manufacturer's instructions. Tet-On HepG2 cells were plated in 96-well plates and treated with doxycycline for the indicated times. Nano-Glo HiBiT Extracellular Detection reagents were added to all wells and incubated for 5 minutes prior to reading on an Infinite 200PRO plate reader (Tecan, Zurich, Switzerland).
  • Membrane fractions were extracted using the Qproteome cell compartment kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions with minor modifications.
  • the membrane fraction contains endosomes, membrane compartment organelles such as mitochondria and endoplasmic reticulum (ER), and cell surface proteins.
  • Cell lysates were obtained by harvesting cells and resuspending in lysis buffer with or without phosphatase inhibitors (10 mM NaF and 1 mM Na 3 VO 4 ). The supernatant was isolated by centrifugation and used as cell lysate.
  • Equal amounts of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes.
  • the transferred membranes were respectively ESYT1 (ATLAS HPA016858; 1:1,000), HA (Roche 3F10; 1:500), LC3B (MBL 4E12; 1:1,000), PKC ⁇ (Abcam ab181076; 1:2,000), ATG5 (Abcam ab108327; 1:1,000), GAPDH (Sigma MAB374; 1:2,000) and Actin (Thermo MS1295; 1:2,000).
  • the transferred membrane was PKC ⁇ (Abcam ab181076; 1:2,000), importin ⁇ 1 (BD Bioscience, NJ, USA, 610486; 1:1,000), and GAPDH (Merck MAB374), respectively. ; 1:2,000). Signals were measured by enhanced chemiluminescence (ECL, Thermo Fisher Scientific).
  • Flow cytometry Cells were dissociated with Accutase (Thermo Fisher Scientific) and pelleted by centrifugation at 500 xg for 5 minutes at 4°C. Cell suspensions were incubated with 7-aminoactinomycin D (7-AAD, diluted 1:2,000; Thermo Fisher Scientific) in phosphate-buffered saline (PBS) containing 0.1% FBS at 4°C. Incubated for 30 minutes. Flow cytometry analysis was performed using MACS Quant (Miltenyi Biotec, North Rhine-Westphalia, Germany). At least three independent experiments were performed.
  • mice anti-PKC ⁇ monoclonal antibody (BD Bioscience 610398; 1:500), rabbit anti-E-Syt1 monoclonal antibody (ATLAS HPA016858; 1 :200), rat anti-HA monoclonal antibody (Roche 3F10; 1:500), rabbit anti-SEC61B monoclonal antibody (CST 14648; 1:200), rabbit anti-Calnexin monoclonal antibody (CST 2679; 1:200), rabbit anti-PDI monoclonal antibody antibody (CST3501; 1:200), and rabbit anti-LC3B monoclonal antibody (MBL 4E12; 1:100).
  • mouse anti-PKC ⁇ monoclonal antibody (BD Bioscience 610398; 1:500)
  • rabbit anti-E-Syt1 monoclonal antibody ATLAS HPA016858; 1 :200
  • rat anti-HA monoclonal antibody (Roche 3F10; 1:500)
  • rabbit anti-SEC61B monoclonal antibody (CST
  • Example 7 the following primary antibodies were added and incubated at 37° C. for 1 hour: mouse anti-HA monoclonal antibody (mouse Bioscience; 1:500), mouse anti-importin ⁇ 1 monoclonal antibody (BD HPA016858; 1:200). , rat anti-HA monoclonal antibody (Roche 3F10; 1:500), rabbit anti-Sec61 ⁇ monoclonal antibody (CST #14648; 1:200), and rabbit anti-calnexin monoclonal antibody (CST #2679; 1:200).
  • mouse anti-HA monoclonal antibody mouse Bioscience; 1:500
  • mouse anti-importin ⁇ 1 monoclonal antibody BD HPA016858; 1:200
  • rat anti-HA monoclonal antibody Roche 3F10; 1:500
  • rabbit anti-Sec61 ⁇ monoclonal antibody CST #14648; 1:200
  • rabbit anti-calnexin monoclonal antibody CST #2679; 1:200
  • Alexa Fluor 488-conjugated, Alexa Fluor 594-conjugated secondary antibodies, or Alexa Fluor 647-conjugated secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI) for nuclear detection were added. was incubated for 30 minutes at room temperature. Cells were observed by a confocal laser fluorescence microscope (LSM880, Zeiss, Germany) or fluorescence microscope (BZ-X800; Keyence, Osaka, Japan).
  • PKC ⁇ BD Bioscience 610398; 1:500
  • LC3B MBL 4E12; 1:1,000
  • importin ⁇ 1 BD Bioscience 610485; 1:200
  • SEC22B Santa Cruz sc101
  • PKC ⁇ santacruz sc937; 1:200
  • E-Syt1 ATLAS HPA016858; 1:200
  • Sec61B CST 14648; 1:200
  • SEC22B Abcam ab181076; 1:200
  • STX3 Abcam ab133750; 1:500
  • Example 5 ⁇ Proliferation assay>
  • cells were plated in 96-well plates with Ab-DeliverIN (OZ Biosciences, San Diego, Calif., USA) in medium alone, with isotype control IgG, or with anti-PKC ⁇ antibody (santacruz sc937; 100 ng/mL, C-20). ) with or without 100 ⁇ L (5 ⁇ 10 3 cells/well). Cultured at 37° C. for 24 hours or 48 hours. Also in Example 6, cells were cultured in 96-well plates at 100 ⁇ L (5 ⁇ 10 3 cells/well).
  • Example 5 ⁇ Spheroid formation assay>
  • cells were incubated with Ab-Deliver IN (OZ Biosciences) were cultured with or without. Eight days after treatment, spheroidal cell growth was observed and measured with a BZ-9000 fluorescence microscope. Spheroids with a major axis of 40 ⁇ m or more were randomly observed in multiple fields and counted. Also in Example 6, cells were cultured in ultra-low attachment 96-well plates (Corning) to form spheres. Five days after treatment, spheroidal cell growth was observed and measured with a BZ-9000 fluorescence microscope. Spheroids larger than 80 or 100 ⁇ m in diameter were randomly observed in multiple fields and counted.
  • the Split-GFP assay is an assay based on the GFP fragments GFP ⁇ -chain 1-10 (GFP 1-10 ) and GFP ⁇ -chain 11 (GFP 11 ), and when the GFP fragments are sufficiently close, GFP is completely
  • the assay is based on the rearrangement of the ⁇ -barrel structure, resulting in fluorescence emission. Single constructs were transfected into doxycycline-induced HepG2 cells to confirm signal expression, localization and loss in GFP channels. Figures are rendered in pseudocolor using a color-blind palette.
  • Example 1 BioID screening identified E-Syt1 as a membrane protein that interacts with cytosolic PKC ⁇ > Using liver cancer cultured cells that constantly secrete cytoplasmic proteins under normal culture conditions (Non-Patent Document 7), interaction of intracellular membrane proteins and secretory cytoplasmic proteins by the proximal-dependent biotinylation enzyme (BioID) method was performed. explored the effects. Based on the high specificity of PKC ⁇ in liver cancer, PKC ⁇ was used as a model for secreted cytoplasmic proteins (Non-Patent Document 5).
  • BioID2 proximal-dependent biotinylation enzyme 2
  • E-Syt1 Extended-synaptotagmin 1
  • CLCC1 chloride Channel CLIC like 1
  • STBD1 star binding domain 1
  • STIM1 stromal interaction molecule 1
  • E-Syt1 which has the highest value, is known to be a tethering factor of the ER-PM contact site together with STIM1.
  • E-Syt1 allows the cytoplasmic protein to be readily accessible to the ER, and the results of the interactome analysis were analyzed by immunoassay of streptavidin-purified biotinylated proteins using a PKC ⁇ -BioID2-expressing HepG2 cell line. It was verified by experiments such as blotting, co-localization analysis of conventional immunofluorescence observation, and proximity ligation assay (PLA) (FIGS. 1b to 1d). In order to confirm the interaction between ER and PKC ⁇ , colocalization analysis using the ER marker Sec61 ⁇ was further performed. Super-resolution imaging by structured illumination microscopy (SIM) revealed that some PKC ⁇ were adjacent to the ER (Fig. 1e).
  • SIM structured illumination microscopy
  • PLA analysis also yielded similar observations suggesting that PKC ⁇ interacts with the ER membrane (Fig. 1f). Also importantly, localization of PKC ⁇ in the ER was shown to be markedly reduced upon loss of E-Syt1 (Fig. 1f), suggesting that PKC ⁇ localization in the ER is dependent on E-Syt1. It was suggested. Moreover, when the gastric cancer cell line AGS, which is incapable of secreting PKC ⁇ , was used, almost no colocalization of PKC ⁇ with E-Syt1 or Sec61 ⁇ was observed (Fig. 1e). These results suggested that E-Syt1-mediated localization of PKC ⁇ in the ER contributes to PKC ⁇ secretion.
  • E-Syt1 is required for the secretion of PKC ⁇ >
  • Fig. 2a the importance of E-Syt1 in PKC ⁇ secretion due to E-Syt1 deficiency was examined.
  • Immunoblot analysis showed clearly reduced levels of PKC ⁇ in cultures of E-Syt1 knockout cells (Fig. 2a), suggesting that E-Syt1 is required for PKC ⁇ secretion.
  • AFP ⁇ -fetoprotein
  • Fig. 2a was suggested to be different from the previously known ER-Golgi body secretory pathway.
  • Example 4 PKC ⁇ is secreted via SEC22B + vesicles Given that a series of autophagic pathways are associated with membrane trafficking, we previously verified that PKC ⁇ secretion is involved in vesicle trafficking. As a result, intracellular membrane-bound PKC ⁇ was detected by a biochemical protection assay using membrane fractions treated with protease K, which digests membrane-bound surface proteins. Evidence is also accumulating implicating autophagosomes in immune cell cytoplasmic protein secretion. Furthermore, these secretory autophagosomes are known to retain SEC22B as an R-SNARE on the membrane surface instead of STX17, which is required to promote autophagosome-lysosome fusion and cargo degradation.
  • Non-Patent Document 11 SEC22B is generally known to be a member of the ER-PM contact site. Therefore, it was verified whether SEC22B is involved in the secretion of PKC ⁇ by cancer cells. The results showed that PKC ⁇ secretion was markedly reduced by knocking down SEC22B (Fig. 4a), suggesting that SEC22B is essential for PKC ⁇ secretion. The proximity interaction between PKC ⁇ and SEC22B could be observed around the PM of HepG2 cells, but not in other cells that do not secrete PKC ⁇ (e.g., E-Syt1 knockout HepG2 cells and AGS cells) (Fig.
  • Example 5 HCC cell proliferation inhibitory effect targeting interaction between PKC ⁇ and E-Syt1>
  • an anti-PKC ⁇ antibody was transported into cells using DeliverIN transfection reagent as a drug delivery system.
  • the anti-PKC ⁇ antibody (C-20) that recognizes the C-terminus of PKC ⁇ , which inhibits the PKC ⁇ -ER interaction was intracellularly delivered, the interaction between PKC ⁇ and E-Syt1 was inhibited, and PKC ⁇ secretion was marked. (Fig. 5a-c). Intracellular accumulation of anti-PKC ⁇ antibody (C-20) was not observed in HepG2 cells treated with Ab alone.
  • E-Syt1 affects the proliferation of hepatoma cells>
  • a HepG2 cell line in which E-Syt1 expression is knocked out was used.
  • Three-dimensional tumorsphere formation assays showed that knocking out (KO) E-Syt1 significantly decreased the number of spheres compared to control cells (FIGS. 6a-b).
  • RNA interference (RNAi) studies confirmed that knockdown of E-Syt1 significantly reduced proliferation of adherent cells in HepG2 cells (Fig. 6c).
  • HuH7 another liver cancer cell line, the anti-tumor growth effect of E-Syt1 knockdown was confirmed (FIGS. 6d to 6f).
  • E-Syt1 contributes to PKC ⁇ secretion>
  • PKC ⁇ is secreted in liver cancer through a pathway different from that previously known, that is, PKC ⁇ is secreted atypically. shown to be important.
  • PKC ⁇ secretion was monitored in hepatoma cell lines. Immunoblot analysis showed that loss of E-Syt1 markedly decreased the amount of PKC ⁇ in cultures of HepG2 and HuH7 cells under low-nutrient conditions (FIGS. 7a-b).
  • E-Syt1 is previously known to be a major component of the ER-PM contact site and function in a Ca 2+ -dependent manner. Since the secretion of some proteins such as neurofactors is affected by intracellular Ca 2+ , we previously investigated whether the secretion of PKC ⁇ is Ca 2+ -dependent using the Ca 2+ chelator BaptaAM. The results showed that PKC ⁇ secretion was not affected by BaptaAM treatment, ie PKC ⁇ secretion was independent of intracellular Ca 2+ concentration.
  • Atypically secreted proteins including PKC ⁇
  • PKC ⁇ are previously known to be taken up by the SEC22B organelle by autophagic mechanisms before secretion and fused with SNAREs such as STX3 in PMs, and indeed SEC22B and STX3 were knocked down by RNAi and analyzed, it was shown that the secretion of PKC ⁇ was remarkably suppressed (Fig. 7c).
  • proximity ligation assay showed that knocking out E-Syt1 clearly reduced the interaction of SEC22B and STX3 (Fig. 7d). This suggested that E-Syt1 is involved in the upstream part in the atypical secretion of PKC ⁇ . That is, it was suggested that PKC ⁇ performs atypical secretion through interaction with E-Syt1.
  • E-Syt1 Since E-Syt1 is also localized in the intracellular cytoplasmic ER membrane, it was confirmed that PKC ⁇ , a secreted cytoplasmic protein, co-localizes with E-Syt1 in the ER (Fig. 8a).
  • a split-GFP assay was constructed using doxycycline-induced HepG2 cells. GFP 1-10 -V5-E-Syt1 and PKC ⁇ -GFP 11 -HA constructs were generated (FIG. 8b). Then, by co-introducing these constructs into cells, signals of GFP reconstituted throughout the cytoplasm were confirmed (Fig. 8c). Furthermore, confocal microscopic analysis confirmed the co-localization of reconstituted GFP and Sec61 ⁇ (an ER marker) (Fig. 8d). These results suggested that PKC ⁇ localizes to E-Syt1 in the ER.
  • E-Syt1 is involved in the activation of PKC ⁇ -IGF1R signaling> E-Syt1-deficient liver cancer cells were used to examine the involvement of E-Syt1 in liver cancer cell growth. We previously confirmed that loss of E-Syt1 is independent of apoptosis in HepG2 cells. Also, previously, extracellular PKC ⁇ has been shown to activate IGF1R signaling involving glypican 3 (GPC3). Therefore, we examined the involvement of E-Syt1 in extracellular PKC ⁇ signals.
  • GPC3 glypican 3
  • E-Syt1 is involved in tumorigenesis of liver cancer>
  • E-Syt1 KO HepG2 cells or control HepG2 cells were inoculated subcutaneously into NOD/SCID mice.
  • mice engrafted with E-Syt1 KO HepG2 cells had less tumorigenesis than mice engrafted with control cells (FIGS. 10a-b), indicating that E-Syt1 It has been suggested that PKC ⁇ secretion by cytogenes contributes to tumorigenesis.

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Abstract

Dans la présente invention, il a été découvert qu'en raison de l'interaction de E-Syt1 sur ER avec PKCδ dans des cellules cancéreuses hépatiques, PKCδ est sécrétée à partir des cellules cancéreuses hépatiques par des voies via un transport vésiculaire, et que la sécrétion de PKCδ à partir des cellules cancéreuses hépatiques peut être inhibée par inhibition de l'interaction entre PKCδ et E-Syt1, et il a également été découvert qu'en conséquence, la prolifération des cellules cancéreuses hépatiques est supprimée.
PCT/JP2023/004659 2022-02-21 2023-02-10 NOUVEL AGENT THÉRAPEUTIQUE CONTRE LE CANCER DU FOIE CONTENANT UN INHIBITEUR D'INTERACTION INHIBANT L'INTERACTION ENTRE PKCδ ET E-SYt1 WO2023157776A1 (fr)

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