JP6436477B2 - Pharmaceutical composition for cancer treatment - Google Patents

Description

  The present invention relates to a pharmaceutical composition for treating cancer. More specifically, the present invention relates to a pharmaceutical composition for cancer treatment containing a substance that suppresses the expression of a specific gene or the activity of a specific protein, and a screening method for a substance for cancer treatment based on the fluctuation of the expression of a specific gene or the activity of a specific protein.

  The most common feature of cancer cells is rapid and uncontrolled growth compared to normal cells. Taking advantage of this feature, chemotherapy using drugs that are toxic to proliferating cells has been performed in the oncology region. The types of drugs (anticancer agents) that suppress the growth of cancer cells include nucleic acid synthesis inhibitors such as cyclophosphamide, antibiotics such as actinomycin D and bleomycin, and antimetabolites such as 5-fluorouracil and methotrexate. In addition, microtubule depolymerization inhibitors such as paclitaxel and molecular target drugs such as imatinib and gefitinib are known. However, these drugs do not necessarily have a sufficient therapeutic effect on cancer, and have a problem that drug resistance is caused by use. In addition, since it acts on normal cells, it may have side effects. Therefore, it has been desired to develop an anticancer agent having a new mechanism of action, that is, an anticancer agent having a growth-inhibiting action against cancer cells and having little toxicity against normal cells.

  An approach for treating cancer by finding a gene that is a target for cancer treatment and suppressing the growth or growth of cancer cells by suppressing the expression or function of the target gene has been studied extensively. In recent years, RNA oligonucleotide strands of about 21 bases complementary to a desired gene (mRNA) and double-stranded RNA (small interfering RNA: siRNA) consisting of the antisense RNA oligonucleotide strand have been developed, and can be arbitrarily used in mammalian cells. It has become possible to suppress the expression of these genes (Non-patent Document 1: Nature, 411, 494-498, 2001). Currently, siRNA technology is actively used in life science research and is expected to be applied to various diseases including cancer. However, there are no siRNAs for treating diseases that are sold as pharmaceuticals including oncology.

Nature, 411, 494-498, 2001

  An object of the present invention is to find a novel target gene for cancer treatment, that is, a gene capable of suppressing cancer cell growth by suppressing its expression or suppressing its function, thereby enabling cancer treatment. It is to provide a pharmaceutical composition for use.

  As a result of diligent research focusing on the fact that the process of epithelial lumen structure formation and cancer cell growth are similar, the present inventors have found that expression of Arl4c is induced during the formation of epithelial lumen structure. The present inventors have found that expression is increased in colorectal cancer and lung adenocarcinoma, and have completed the present invention.

That is, the present invention relates to the following [1] to [15].
[1] A pharmaceutical composition for treating cancer comprising a substance that suppresses the expression of the Arl4c gene or a substance that suppresses the activity of the Arl4c protein.
[2] The pharmaceutical composition according to claim 1, wherein at least one selected from the group consisting of a substance that suppresses the expression of the Arl4c gene and a substance that suppresses the activity of the Arl4c protein is a nucleic acid.
[3] The pharmaceutical composition according to [1] or [2], wherein the substance that suppresses gene expression is siRNA, antisense oligonucleotide or ribozyme, or an expression vector thereof.
[4] The pharmaceutical composition according to any one of [1] to [3], wherein the substance that suppresses gene expression is siRNA or an siRNA expression vector.
[5] The pharmaceutical composition according to [3] or [4], wherein one or both strands of the siRNA contain a 3 ′ terminal overhang.
[6] The pharmaceutical composition according to any one of [1] to [5], wherein the cancer is colon cancer or lung cancer.
[7] A method for treating cancer, comprising a step of administering a substance that suppresses the expression of an Arl4c gene or a substance that suppresses the activity of an Arl4c protein.
[8] The method according to [7], wherein the substance that suppresses gene expression is siRNA or an siRNA expression vector.
[9] A substance that suppresses the expression of the Arl4c gene or a substance that suppresses the activity of the Arl4c protein for the treatment of cancer.
[10] The substance according to [9], wherein the substance that suppresses gene expression is siRNA or an siRNA expression vector.
[11] Use of a substance that suppresses the expression of the Arl4c gene or a substance that suppresses the activity of the Arl4c protein for the treatment of cancer.
[12] The use according to [11], wherein the substance that suppresses gene expression is siRNA or an siRNA expression vector.
[13] Use of a substance that suppresses the expression of the Arl4c gene or a substance that suppresses the activity of the Arl4c protein in the preparation of a therapeutic agent for cancer.
[14] The use according to [13], wherein the substance that suppresses gene expression is siRNA or an siRNA expression vector.
[15] A method for screening a substance for treating cancer comprising the following steps (a) to (c):
(A) contacting a cell capable of measuring Arl4c gene expression or Arl4c protein activity with a test substance;
(B) measuring the expression level of the gene or the activity of the protein in the cell contacted with the test substance and comparing the measured value with the expression level of the gene or the activity of the protein in the control cell not contacted with the test substance ;
Then, (c) when the expression level of the gene or the protein activity in the cell given the test substance is lower than the expression level of the gene or the protein activity in the cell not given the test substance, A step of selecting a test substance as a cancer therapeutic substance.

  According to the present invention, by suppressing the expression of the Arl4c gene or suppressing the activity of the Arl4c protein, it is possible to suppress the growth of cancer cells, and the substance that inhibits the expression or function of the gene is a pharmaceutical composition for cancer treatment It can be used as an active ingredient of a product. The pharmaceutical composition for cancer treatment of the present invention provides an anticancer agent having a new mechanism of action that specifically suppresses the expression of a specific gene or its function, and is less toxic than conventional anticancer agents. It is a big feature. Furthermore, the present invention also provides a method for screening a cancer therapeutic substance using the gene or protein as a target.

FIG. 1 is a diagram showing the expression of Arl4c in human colon cancer and lung adenocarcinoma pathological specimens. (A) is an example of a diagram in which a non-tumor region and a tumor region of a colon cancer tissue are stained with an anti-Arl4c antibody and hematoxylin, and a black square is an enlarged image. The right figure shows a diagram classified by the ratio of Arl4c positive areas in the tumor area (n = 118) and non-tumor area (n = 81). (B) is an example of a diagram in which lymph nodes of colorectal cancer patients (43 patients) with lymph node metastasis were stained with anti-Arl4c antibody and hematoxylin. The results of measuring the number of positive and negative Arl4c in the primary region and metastasized lymph nodes are shown together. (C) is an example of a diagram in which a non-tumor region and a tumor region of lung adenocarcinoma tissue are stained with an anti-Arl4c antibody and hematoxylin. A black square is a figure which shows an enlarged image, and the broken line in a figure shows the boundary line of a non-tumor area | region and a tumor area | region. The right figure shows a diagram categorized by the ratio of Arl4c positive areas in the tumor area (n = 68) and non-tumor area (n = 61). (D) is an example of a diagram in which an Arl4c positive hyperplastic region of atypical adenomatous hyperplasia (AAH) is stained with an anti-Arl4c antibody and hematoxylin. A black square is a diagram showing an enlarged image, and a broken line in the figure indicates a boundary line between the non-AAH region and the AAH region. The right figure shows a diagram classified by the ratio of Arl4c positive areas in the AAH region (n = 9) and the non-AAH region (n = 9). The scale bar in each figure is (A) 250 μm, (B) 250 μm, (C) 200 μm, and (D) 250 μm. FIG. 2 is a diagram showing that expression of Arl4c is necessary for invasive activity of cancer cells. (A) is a figure which shows the result of having performed the migration assay at the time of transfecting Arl4c siRNA into the HCT116 cell which stably expresses GFP or Arl4c-GFP. (B) is a figure which shows the result of having performed the invasion assay at the time of transfecting HCT116 cell which stably expresses GFP or Arl4c-GFP with Arl4c siRNA. (C) is a figure which shows the result of having performed the migration assay at the time of transfecting A549 cell which expresses GFP or Arl4c-GFP with Arl4c siRNA. (D) is a figure which shows the result of having performed the invasion assay at the time of transfecting Arl4c siRNA to the A549 cell which stably expresses GFP or Arl4c-GFP. The scale bar in each figure is 200 μm. Moreover, the result of calculating the ratio with respect to a control cell about migration activity and invasion activity is also shown on the right of each figure. The results are shown as the mean ± SE of three experimental results, and “*” in the figure means P <0.01. FIG. 3 is a diagram showing that Arl4c is necessary for proliferation in three-dimensional culture. (A) and (C) show the results of measuring the number of cells when HCT116 cells or A549 cells stably expressing Arl4c shRNA were cultured in a two-dimensional petri dish in the presence of 5% serum for the number of days shown on the right. It is a figure which shows the result of having detected the lysate with the antibody on the left. (B) and (D) are images taken using a phase-contrast microscope when HCT116 cells or A549 cells stably expressing Arl4c shRNA were cultured in 3D Matrigel for 5 days, and stained images, with the average of the stained regions. It is a figure which shows the result (n = 15) which calculated the area value on the right. The results are shown as the mean ± SE of three experimental results, and “*” in the figure means P <0.01. FIG. 4 is a diagram showing Arl4c required for tumor formation in vivo. (A) And (B) is a figure which shows an example of the photograph of the nude mouse which transplanted the HCT116 cell or A549 cell (5x10 ⁶ cell) which stably expressed Arl4c shRNA subcutaneously for the designated number of days (n = 6). Tumor volume and weight are shown in the right figure, the broken line in the photograph shows the outline of the xenograft, and the arrow tip indicates the position of the tumor. (C) and (D) show a section prepared from HCT116 cell or A549 cell graft with HE staining, Arl4c antibody (including enlarged view), and cell proliferation marker Ki67. . The graph shows the number of Ki67 positive cells with respect to the total number of cells included in the imaging field (n = 5). The broken line in the figure shows the outline of the tumor. The scale bar in each figure is 1 cm for (A) and (B), the left figure for (C) and (D), the middle figure is 100 μm, and the right figure is 50 μm. FIG. 5 is a diagram showing an in vivo tumor growth inhibitory effect by suppressing the expression of Arl4c using siRNA. (A) is a view showing a tumor part in a nude mouse 10 days after injection of Arl4c siRNA into the graft 3 days after subcutaneous implantation of HCT116 cells (1 × 10 ⁶ cells) into a nude mouse. In the left figure, the broken line indicates the outline of the graft, and the tip of the arrow indicates the position of the tumor. The right figure shows tumor volume and weight. (B) is a diagram showing the expression level of Arl4c mRNA in the tumor site, a diagram of HE-stained sections prepared from the grafts, and a diagram of staining with Arl4c antibody. (C) is a diagram showing a section prepared from a graft with a Ki67 antibody and the number of Ki67 positive cells. The scale bars in each figure are (A) 1 cm, (B) and (C) 100 μm.

  The pharmaceutical composition of the present invention is characterized by containing, as an active ingredient, a substance that suppresses the expression of the Arl4c gene or a substance that suppresses the activity of the Arl4c protein. In addition, the term used in this specification is used by the meaning normally used in the said field except the case where it mentions especially.

  The present inventors have so far induced the expression of Arl4c protein by co-stimulation of Wnt and EGF signals, thereby changing the morphology of epithelial cells, and the growth regulators are transferred into the nucleus to promote cell proliferation. As a result, it has been clarified that the formation of epithelial lumen tissue is induced. On the other hand, it is known that Wnt and EGF signal abnormalities are involved in various cancers. Therefore, the present inventors examined the relationship between cancer and Arl4c, and as a result, EGF and Wnt3a signal promoted the binding between Arl4c gene and Ets, β-catenin, Tcf4, etc. It was found that expression of Arl4c is induced by improving acetylation. Therefore, it is speculated that the expression of Arl4c is improved in cancers in which Wnt and EGF signals are continuously activated. As a result of further investigation that Arl4c could be suppressed by suppressing its expression, It was found as a novel cancer target gene.

  In one aspect, the present invention provides a pharmaceutical composition comprising a substance that suppresses the expression of the Arl4c gene or a substance that suppresses the activity of the Arl4c protein. Such a pharmaceutical composition can be used, for example, for the treatment of cancer.

  Arl4c is a low molecular weight G-protein, and includes Arl4c isoform 1 and Arl4c isoform 2 having a shorter C-terminal than isoform 1, and any of Arl4c in the present invention can be used. Arl4c is presumed to be activated by binding to GTP, but the details of the function are unknown. So far, Arl4c is involved in promoting cholesterol efflux in Hela cells and in transporting transferrin from early endosomes to recycling endosomes by interacting with α-tubulin in human renal cell carcinoma. Are known. However, the relationship between Arl4c and cancer is not known.

  In the present specification, “Arl4c gene” means a gene encoding human Arl4c protein. The base sequence of the human Arl4c gene and the amino acid sequence of the human Arl4c protein are known. For example, for Arl4c isoform 1, GenBank Accession ID: NM_001282431.1 is the human Arl4c gene base sequence (SEQ ID NO: 1). GenBank Accession ID: NP_001269360.1 is the protein amino acid sequence (SEQ ID NO: 2), and for Arl4c isoform 2, GenBank Accession ID: NM_005737.3 is the human Arl4c gene base sequence (SEQ ID NO: 3). GenBank Accession ID: NP — 005728.2 is registered and published in GenBank as a sequence (SEQ ID NO: 4).

  In the present specification, the human Arl4c protein includes not only a protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4, but also a variant that can occur in a human individual. Proteins in which several amino acids have been deleted, substituted and / or added and which are caused by mutations based on polymorphisms or mutations are included. However, these mutant human Arl4c proteins have functions equivalent to those of the protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4.

  In the present specification, the “Arl4c gene” includes not only a gene consisting of the nucleotide sequence of SEQ ID NO: 1 or 3, but also a mutant that can occur in a human individual. For example, in the gene consisting of the nucleotide sequence of SEQ ID NO: 1 or 3 Examples include genes in which one or several bases have been deleted, substituted and / or added, which are caused by mutations based on polymorphisms or mutations. Furthermore, the “Arl4c gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more with respect to the nucleotide sequence of SEQ ID NO: 1 or 3. Variants consisting of nucleotide sequences having 99.5% or more, 99.7% or more, or 99.9% or more identity are included. The identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA. Furthermore, the “Arl4c gene” includes a variant consisting of a base sequence that hybridizes under stringent conditions to the gene consisting of the base sequence of SEQ ID NO: 1 or 3. Examples of stringent hybridization conditions include, for example, conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” as washing conditions after hybridization. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although it is not particularly limited, a more severe hybridization condition is about “0.5 × SSC, 0.1% SDS, 42 ° C.”, and a more severe hybridization condition is “0.1 × SSC, 0.1% SDS, 65 ° C.” Can do. Hybridization: Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocol in Coil in Amplification (Clinic): Current Protocols in Molecular Biology (1994) It can be carried out according to the method described in Edition (1995) (Oxford University Press). However, these mutant genes encode proteins having functions equivalent to those of the protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4.

  In the present specification, the “substance that suppresses gene expression” refers to a substance that suppresses the transcription of mRNA of the target gene, a substance that degrades the transcribed mRNA, or a substance that suppresses the translation of the protein from the mRNA. There is no particular limitation. Such substances include nucleic acids, and examples include siRNA, antisense oligonucleotides or ribozymes, or expression vectors thereof. Among them, antisense oligonucleotides and siRNA and expression vectors thereof are preferable, and antisense oligonucleotides and siRNA are more preferable. In addition to the above, “substances that suppress gene expression” include proteins, peptides, and other small molecules. In the present invention, the target gene is the Arl4c gene.

  In this specification, “siRNA” is an RNA molecule having a double-stranded RNA portion consisting of about 15 to 40 bases, and cleaves the mRNA of the target gene having a sequence complementary to the antisense strand of the siRNA. And has a function of suppressing the expression of the target gene. Specifically, the siRNA in the present invention comprises two sense RNA strands consisting of a sequence homologous to a continuous RNA sequence in the mRNA of the Arl4c gene and an antisense RNA strand consisting of a sequence complementary to the sense RNA sequence. RNA comprising a single-stranded RNA portion. The design and production of such siRNAs and mutant siRNAs described below are within the skill of the artisan.

  The length of the double-stranded RNA portion is preferably about 15 to 40 bases, more preferably 15 to 30 bases, still more preferably 15 to 25 bases, still more preferably 18 to 23 bases, and still more preferably 19 as bases. ~ 21 bases. The end structure of the sense strand or antisense strand of siRNA is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it may have a blunt end or a protruding end (overhang) It is preferable that the 3 ′ end protrudes. An siRNA having an overhang consisting of several bases, preferably 1 to 3 bases, more preferably 2 bases, at the 3 ′ end of the sense RNA strand and the antisense RNA strand suppresses the expression of the target gene. In many cases, the effect is large, which is preferable. The type of the overhanging base is not particularly limited, and may be either a base constituting RNA or a base constituting DNA.

  Furthermore, siRNA in which 1 to several nucleotides are deleted, substituted, inserted and / or added in one or both of the sense strand or antisense strand of the siRNA is also a pharmaceutical composition for treating cancer of the present invention. It can be used for things. Here, 1 to several bases are not particularly limited, but preferably 1 to 4 bases, more preferably 1 to 3 bases, and still more preferably 1 to 2 bases. Specific examples of such mutations include those in which the number of bases in the 3 ′ end overhang portion is 0 to 3, or the base sequence in the 3 ′ end overhang portion is changed to another base sequence, or Those in which the length of the sense RNA strand differs from that of the antisense RNA strand by 1 to 3 bases due to insertion, addition or deletion, or in which the base is substituted with another base in the sense strand and / or antisense strand, etc. For example, but not limited to. However, it is necessary that the sense strand and the antisense strand can hybridize in these mutant siRNAs, and that these mutant siRNAs have the same ability to suppress gene expression as siRNA having no mutation.

  Further, the siRNA may be a molecule having a closed structure at one end, for example, an siRNA having a hairpin structure (Short hairpin RNA; shRNA). A shRNA is a RNA comprising a sense strand RNA of a specific sequence of a target gene, an antisense strand RNA consisting of a sequence complementary to the sense strand RNA, and a linker sequence connecting both strands. The sense strand portion and the antisense strand portion Hybridize to form a double stranded RNA portion.

  It is desirable that siRNA does not exhibit a so-called off-target effect in clinical use. The off-target effect refers to the action of suppressing the expression of another gene that is partially homologous to the siRNA used in addition to the target gene. In order to avoid the off-target effect, it is possible to confirm in advance that there is no cross-reaction for the candidate siRNA using a DNA microarray or the like. In addition, by using a known database provided by NCBI (National Center for Biotechnology Information) etc., by confirming that there is no gene containing a portion that is highly homologous to the candidate siRNA sequence other than the target gene, It is possible to avoid the off-target effect.

  In order to prepare the siRNA of the present invention, a known method such as a method using chemical synthesis or a method using a gene recombination technique can be appropriately used. In the synthesis method, double-stranded RNA can be synthesized by a conventional method based on sequence information. In the method using gene recombination technology, an expression vector incorporating a sense strand sequence or an antisense strand sequence is constructed, and the sense strand RNA or antisense strand RNA generated by transcription after introducing the vector into a host cell. It can also be produced by acquiring each of the above. In addition, by expressing a sense strand RNA of a specific sequence of the target gene, an antisense strand RNA consisting of a sequence complementary to the sense strand RNA, and a linker sequence connecting both strands, and expressing a shRNA that forms a hairpin structure, Desired double-stranded RNA can also be prepared.

  As long as the siRNA has an activity of suppressing the expression of the target gene, a part of the nucleic acid constituting the siRNA may be DNA. The siRNA may be a nucleic acid in which all or part of the nucleic acid constituting the siRNA is modified as long as it has the activity of suppressing the expression of the target gene.

  The modified nucleic acid means a nucleic acid having a structure different from that of a natural nucleic acid, in which a nucleoside (base site, sugar site) and / or internucleoside binding site is modified. Examples of the “modified nucleoside” constituting the modified nucleic acid include, for example, abasic nucleoside; arabino nucleoside, 2′-deoxyuridine, α-deoxyribonucleoside, β-L-deoxyribonucleoside, and other sugars Examples include nucleosides having modifications; peptide nucleic acids (PNA), peptide nucleic acids to which phosphate groups are bound (PHONA), locked nucleic acids (LNA), morpholino nucleic acids and the like. Examples of the nucleoside having a sugar modification include substituted pentose monosaccharides such as 2′-O-methylribose, 2′-deoxy-2′-fluororibose, and 3′-O-methylribose; 1 ′, 2′-deoxyribose Arabinose; substituted arabinose sugars; nucleosides with hexose and alpha-anomeric sugar modifications are included. These nucleosides may be modified bases with modified base sites. Examples of such modified bases include pyrimidines such as 5-hydroxycytosine, 5-fluorouracil and 4-thiouracil; purines such as 6-methyladenine and 6-thioguanosine; and other heterocyclic bases.

  Examples of the “modified internucleoside linkage” constituting the modified nucleic acid include, for example, alkyl linker, glyceryl linker, amino linker, poly (ethylene glycol) linkage, methylphosphonate internucleoside linkage; methylphosphonothioate, phosphotriester , Phosphothiotriester, phosphorothioate, phosphorodithioate, triester prodrug, sulfone, sulfonamide, sulfamate, formacetal, N-methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidate, etc. Non-natural internucleoside linkages.

  An oligonucleotide complementary to the mRNA of the target gene is called an “antisense oligonucleotide”, and the function of the mRNA is suppressed by forming a double strand with the gene (mRNA) targeted by the antisense oligonucleotide. “Antisense oligonucleotides” are not limited to those that are completely complementary to the target gene (mRNA), but may contain some mismatches as long as they can be stably hybridized with mRNA.

  Antisense oligonucleotides may be modified. By applying an appropriate modification, the antisense oligonucleotide becomes difficult to be degraded in the living body, and the expression of the target gene can be inhibited more stably. Such modified oligonucleotides include S-oligo type (phosphorothioate type), C-5 thiazole type, D-oligo type (phosphodiester type), M-oligo type (methyl phosphonate type), peptide nucleic acid And modified antisense oligonucleotides such as phosphodiester bond type, C-5 propynyl pyrimidine type, 2-O-propyl ribose, 2′-methoxyethoxy ribose type. Furthermore, the antisense oligonucleotide may be one in which at least a part of the oxygen atom constituting the phosphate group is substituted or modified with a sulfur atom. Such an antisense oligonucleotide is particularly excellent in nuclease resistance and affinity for RNA. Examples of the antisense oligonucleotide in which at least a part of the oxygen atom constituting the phosphate group is substituted or modified with a sulfur atom include oligonucleotides such as S-oligo type.

  An antisense oligonucleotide (or a derivative thereof) can be synthesized by a conventional method, and can be easily synthesized by, for example, a commercially available DNA synthesizer (for example, Applied Biosystems). Examples of the synthesis method include a solid phase synthesis method using phosphoramidite and a solid phase synthesis method using hydrogen phosphonate.

  “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligoDNA having the base sequence of the enzyme active site also has a nucleic acid cleaving activity. In the book, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage activity. Specifically, the ribozyme can specifically cleave mRNA or an initial transcription product encoding a target gene within the coding region (including an intron portion in the case of the initial transcription product). The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends adjacent to the portion having the hammerhead structure (about 10 bases in total) are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. Furthermore, when ribozymes are used in the form of expression vectors containing the DNA that encodes them, they should be hybrid ribozymes in which tRNA-modified sequences are further linked in order to promote the transfer of transcripts to the cytoplasm. (Nucleic Acids Res., 29 (13): 2780-2788 (2001)).

  The substance that suppresses the expression of the target gene may be a nucleic acid molecule such as siRNA, antisense oligonucleotide or ribozyme, and an expression vector encoding the nucleic acid molecule. In the expression vector, the oligonucleotide or polynucleotide encoding the nucleic acid molecule must be operably linked to a promoter capable of exhibiting promoter activity in a mammalian cell to be administered. The promoter to be used is not particularly limited as long as it can function in the mammal to be administered, but for example, polIII promoter (eg, tRNA promoter, U6 promoter, H1 promoter), mammalian promoter (eg, CMV promoter, CAG promoter, SV40 promoter) and the like.

  The expression vector preferably contains a transcription termination signal, ie a terminator region, downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule. In addition, selectable marker genes for selection of transformed cells (such as genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, genes that complement auxotrophic mutations, etc.) You can also

  The basic backbone vector used as the expression vector is not particularly limited, and examples thereof include a plasmid vector and a viral vector. Suitable vectors for administration to mammals such as humans include viral vectors such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, and the like. .

Examples of the expression vector of the present invention include siRNA, antisense oligonucleotide or ribozyme expression vectors. Among them, siRNA expression vectors are preferable.

  These “substances that suppress gene expression” suppress the growth of a wide range of cancer cells including lung cancer, colon cancer, pancreatic cancer, and the like, and the degree of suppression is large. The effect of things is great.

  In the present specification, the “substance that suppresses the activity of the protein” is not particularly limited as long as it is a substance that suppresses the activity of the target protein. Examples of such substances include proteins, peptides, antibodies, and other low molecular compounds. Among these, peptides, antibodies, and low molecular compounds are preferable, and antibodies and low molecular compounds are more preferable. In the present invention, the target protein is Arl4c protein.

  In the present invention, a substance that suppresses the expression of the Arl4c gene or a substance that suppresses the activity of the Arl4c protein can be used as an active ingredient of a pharmaceutical composition. The pharmaceutical composition of the present invention can be used as a pharmaceutical composition for cancer treatment by administering the pharmaceutical composition in vivo.

  The type of cancer that is the subject of treatment with the pharmaceutical composition of the present invention is not particularly limited. Moreover, it may be solid cancer or invasive cancer. Specifically, for example, bladder cancer, breast cancer, colon cancer, colon cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid gland Mention may be made of cancer, prostate cancer, squamous cell carcinoma, skin cancer, bone cancer, lymphoma, leukemia and brain tumor. In particular, the application of the pharmaceutical composition of the present invention is expected for lung cancer and colon cancer.

  The pharmaceutical composition for cancer treatment of the present invention can be used not only for cancer treatment but also for prevention of recurrence and metastasis after cancer treatment.

  The pharmaceutical composition of the present invention can employ both oral and parenteral dosage forms. In the case of parenteral administration, it is also possible to administer directly to the tumor site.

  The pharmaceutical composition of the present invention can be formulated according to a conventional method, and may contain a pharmaceutically acceptable carrier or additive. Such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl. Sodium starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, pharmaceutical Examples of acceptable surfactants include additives.

  The additive is selected from the above alone or in appropriate combination depending on the dosage form of the pharmaceutical composition of the present invention. As for the dosage form, in the case of oral administration, it can be administered as a tablet, capsule, fine granule, powder, granule, liquid, syrup or the like, or in an appropriate dosage form. In the case of parenteral administration, pulmonary dosage forms (for example, those using a nephriser etc.), nasal dosage forms, transdermal dosage forms (for example, ointments, creams), injection dosage forms and the like can be mentioned. In the case of an injection dosage form, it can be administered systemically or locally by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection or the like.

  When the substance that suppresses the expression of the target gene is a nucleic acid molecule such as siRNA, antisense oligonucleotide, or ribozyme, or an expression vector that encodes the nucleic acid molecule, the expression inhibitor is introduced into a phospholipid endoplasmic reticulum such as a liposome. In addition, the endoplasmic reticulum can be used as the pharmaceutical composition of the present invention.

  The dosage of the pharmaceutical composition of the present invention varies depending on age, sex, symptom, administration route, administration frequency, and dosage form. The administration method is appropriately selected depending on the age and symptoms of the patient. The effective dose is 0.01 μg to 1000 mg, preferably 0.1 μg to 100 μg, per kg of body weight at a time. However, the therapeutic agent is not limited to these doses.

The invention in a further aspect,
(I) a method for treating cancer, comprising a step of administering a substance that suppresses the expression of the Arl4c gene or a substance that suppresses the activity of the Arl4c protein;
(Ii) a substance that suppresses the expression of the Arl4c gene or a substance that suppresses the activity of the Arl4c protein, and (iii) a substance that suppresses the expression of the Arl4c gene or Arl4c in the preparation of a therapeutic agent for cancer. Use of a substance that inhibits the activity of a protein is provided.
The “substance that suppresses gene expression” or “substance that suppresses protein activity” is as described above. Further, when siRNA is used as the “substance that suppresses gene expression”, preferred siRNA is as described above.

  In another embodiment, the present invention screens a therapeutic agent for cancer using, as an index, suppression of Arl4c gene expression or Arl4c protein activity, and a substance that suppresses expression or activity as a candidate compound. Provide a method.

  The test substance to be subjected to the screening method may be any known compound and novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, compound prepared using combinatorial chemistry technology Examples include libraries, random peptide libraries prepared by solid phase synthesis and phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.

In one embodiment, the screening method of the present invention includes the following steps (a) to (c):
(A) contacting a cell capable of measuring Arl4c gene expression or Arl4c protein activity with a test substance;
(B) measuring the expression level of the gene or the activity of the protein in the cell contacted with the test substance and comparing the measured value with the expression level of the gene or the activity of the protein in the control cell not contacted with the test substance ;
Then, (c) when the expression level of the gene or the protein activity in the cell given the test substance is lower than the expression level of the gene or the protein activity in the cell not given the test substance, A step of selecting a test substance as a cancer therapeutic substance.

  In step (a) of the above method, a cell capable of measuring the expression level of the Arl4c gene or the activity of the Arl4c protein and the test substance are placed under contact conditions. Contact of the test substance with the cells whose expression can be measured is performed in a culture medium.

  A cell that can measure the expression level of Arl4c gene or the activity of Arl4c protein is a cell that can directly or indirectly evaluate the expression level of Arl4c gene products, such as transcripts and translation products, and the activity of Arl4c protein. Say. A cell capable of directly evaluating the expression level of the gene product can be a cell capable of naturally expressing the gene, while a cell capable of indirectly evaluating the expression level of the gene product includes: Examples include cells that allow reporter assay for the gene transcription regulatory region. As the cells capable of measuring the expression of the gene, animal cells such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys or human mammalian cells can be used, and human-derived cells are particularly preferable.

  A cell enabling a reporter assay for a gene transcription regulatory region is a cell comprising a target gene transcription regulatory region and a reporter gene operably linked to the region. The target gene transcription regulatory region and the reporter gene can be inserted into an expression vector. The transcriptional regulatory region of the target gene is not particularly limited as long as it can control the expression of the target gene. For example, a region from the transcription start point to about 2 kbp upstream, or one or more bases in the base sequence of the region Examples include a region consisting of a base sequence deleted, substituted or added and having the ability to control the transcription of the target gene. The reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance. For example, a GFP (green fluorescent protein) gene, GUS (β-glucuronidase) gene, LUC (luciferase) gene, CAT (Chloramphenicol acetyltransferase) gene and the like.

  A cell into which a gene transcription regulatory region and a reporter gene operably linked to the region are introduced can be used for quantitative analysis of the expression level of the reporter gene as long as the transcriptional regulatory function of the target gene can be evaluated. As long as it is, it is not particularly limited.

  The culture medium in which the cell capable of measuring the expression level of the Arl4c gene or the activity of the Arl4c protein and the test substance is contacted is appropriately selected according to the type of the cell used, for example, about 5 to 20% Examples include minimal essential medium (MEM) containing fetal calf serum, Dulbecco's modified minimal essential medium (DMEM), RPMI 1640 medium, and 199 medium. The culture conditions are also appropriately determined according to the type of cells used, etc. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 144 hours.

  In step (b) of the above method, first, the expression level of the Arl4c gene or the activity of the Arl4c protein in the cell contacted with the test substance is measured. The expression level or activity can be measured by a method known per se in consideration of the type of cells used. For example, when a cell capable of naturally expressing any of the Arl4c gene is used as a cell capable of measuring the expression of the gene, the expression level is known per se for a gene product such as a transcription product or a translation product. It can be measured by this method. For example, the expression level of a transcription product can be measured by preparing total RNA from cells and performing RT-PCR, Northern blotting, or the like. The expression level of the translation product can be measured by preparing an extract from the cells and using an immunological technique. As an immunological technique, a radioisotope immunoassay (RIA method), an ELISA method (Methods in Enzymol. 70: 419-439 (1980)), a fluorescent antibody method, or the like can be used. On the other hand, when a cell capable of performing a reporter assay for the gene transcription regulatory region is used as a cell capable of measuring the expression of the Arl4c gene, the expression level can be measured based on the signal intensity of the reporter.

  Next, the gene expression level or protein activity in the cell contacted with the test substance is compared with the gene expression level or protein activity in the control cell not contacted with the test substance (step (c) of the above method). . The comparison of expression level or activity is preferably performed based on the presence or absence of a significant difference. For example, the expression level of the gene in the control cell not contacted with the test substance is the expression level measured at the same time, even if the expression level is measured in advance, compared to the measurement of the gene expression level in the cell contacted with the test substance. Although it may be present, it is preferably the expression level measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

  Hereinafter, the present invention will be described based on examples. However, the examples are provided for better understanding of the present invention, and the scope of the present invention is intended to be limited to these examples. It is not a thing.

Example 1 (Expression of Arl4c in colon cancer and lung cancer)
During the period from January 2007 to March 2013, tissue was collected from patients with colorectal cancer (118 patients) and lung adenocarcinoma patients (68 patients) aged 28 to 86 years (median 68 years) at Osaka University Hospital did. The tissue was fixed in 10% formalin and treated with paraffin, and then a section sample having a thickness of 4 μm was prepared.

  The obtained sample was immunostained according to Dako Real (registered trademark) EnVision (registered trademark) Detection System. Specifically, first, antigen activation was performed using a Pascal pressure chamber (manufactured by Dako), and Dako REAL Peroxidase-Blocking Solution was reacted for 5 minutes to block nonspecific binding sites. Subsequently, after making it react overnight at 4 degreeC using anti- Arl4c antibody (made by Abcam), it reacted for 60 minutes at room temperature using anti-rabbit IgG-horseradish peroxidase (HRP), and detected. The signal was confirmed using a substrate-chromogen solution. Counterstaining was also performed with 0.1% hematoxylin.

  When the Arl4c staining area is 5% or more, the expression of Arl4c is judged to be positive, and the percentage of the staining area occupying the tumor area is 5% or more, less than 20% is “Low”, 20% or more, Less than 50% was classified as "Middle", 50% or more and less than 95% as "High". The results are shown in FIG. 1A. Table 1 shows the correlation between the expression results of Arl4c and the staging classification of patients conducted according to the International Union of Cancer (UICC) TNM stage classification, and the P-value between stages with Fisher's exact probability. Also shown in FIG.

  As a result, expression of Arl4c was not observed in the non-tumor region of colorectal cancer patients, but expression of Arl4c was observed in the tumor region, and Arl4c expression was positive in 55 of 118 patients (47%) . Although the positive rate and expression rate did not correlate with the T classification (depth penetration) and N classification (degree of lymph node metastasis), which are cancer stages, Arl4c expression may be involved in the early stage of tumor formation It is suggested. In addition, in colon cancer patients (43 patients) with metastasis to lymph nodes, Arl4c expression was found to be higher in metastatic lymph nodes than in the primary lesion (see FIG. 1B). This suggests that Arl4c positive cancer cells grow in the metastasis destination. Of the 118 pathological specimens, one specimen contained only the T classification.

  Similarly, the expression of Arl4c was not observed in the alveolar epithelium of the non-tumor region of lung adenocarcinoma patients, but the expression of Arl4c was observed in the tumor region, and the area where Arl4c was expressed was larger than that of colon cancer patients I understand. In addition, the positive rate and expression rate did not correlate with the T stage (deep penetration) and N classification (the degree of lymph node metastasis), which are cancer stages, but the expression of Arl4c in 52 of 68 patients Was positive (76.5%). In addition, Arl4c expression was confirmed in 8 out of 9 patients with atypical adenomatous hyperplasia (AAH), which is a preinvasive lesion of adenocarcinoma (see FIG. 1D). AAH is milder than adenocarcinomas, but AAH cells proliferate along the alveolar epithelial framework, the cell shape increases in a cubic shape, and nucleoli protrude. Atypical. The expression of Arl4c was confirmed in the cytoplasm of AAH cells. Of the 68 pathological specimens, 3 specimens contained only the T classification.

Example 2 (Involvement of Arl4c in cancer cell migration and invasive activity)
We examined the expression of Arl4c mRNA in various cell lines derived from colorectal cancer and lung cancer, and HCT116 cells of colon cancer cell lines with gene mutations in β-catenin and Ras and lung adenocarcinoma cell lines with gene mutations in Ras Since A549 cells highly expressed Arl4c, the role of Arl4c in cancer cells was investigated.

  First, a plasmid for preparing cells that stably express Arl4c was prepared. Specifically, using Gateway technology (manufactured by Invitrogen), the base sequence of the human Arl4c isoform 1 gene (SEQ ID NO: 1) is incorporated into CS-RfA-EVBsd together with the H1 promoter to prepare a lentiviral vector. did. The vector was transfected into X293T cells together with the packaging vector, and the resulting lentiviral supernatant was used to introduce the plasmid into HCT116 cells and A549 cells to prepare cells that stably express Arl4c.

Next, siRNA was introduced into the cells expressing Arl4c. Specifically, the following target sequences were used.
Randomized control: 5'-CAGTCGCGTTTGCGACTGG-3 '(SEQ ID NO: 5)
human Arl4c: 5'-CCTCTAACATCTCGGCCTT-3 '(SEQ ID NO: 6)
The HCT116 cells and A549 cells obtained above were transfected with a mixture of siRNAs (20 nM each) against the above sequence using RNAiMAX (Invitrogen) to obtain cells (SiControl, SiArl4c). The obtained cells were transfected 36-48 hours later and evaluated as follows.

The migratory activity of the obtained cells was measured using a modified Boyden chamber (Costar). Specifically, the lower surface of the filter was treated with 10 μg / mL type I collagen for 2 hours, and HCT116 cells (5 × 10 ⁵ cells in 100 μL) or A549 cells (2.5 × 10 ⁴ cells in 100 μL) were treated with 0.1% bovine serum albumin (BSA). ) Contained-Suspended in serum-free DMEM medium was applied to the upper chamber. After culturing at 37 ° C. for 4 to 8 hours, the cells that migrated to the lower surface of the upper chamber were fixed with PBS containing 4 w / v% paraformaldehyde (PFA), and the number of cells was measured.

Invasion activity was measured using a modified Boyden chamber (BD Biosciences). That is, HCT116 cells (5 × 10 ⁵ cells in 100 μL) or A549 cells (2.5 × 10 ⁴ cells in 100 μL) suspended in 0.1% BSA-serum-free DMEM medium in the Matrigel-coated Boyden chamber After adding DMEM medium containing 10% FBS to the lower chamber, the cells were cultured at 37 ° C. for 24 hours. Cells infiltrating the lower surface from the upper chamber via Matrigel were fixed with PBS containing 4 w / v% PFA, and the number of cells was measured.

  As a result, it was found that HCT116 cells in which Arl4c was knocked down by siRNA were inhibited in both migration and invasive activity as compared to cells in which Arl4c expression was not suppressed by siRNA (see FIGS. 2A and 2B). In addition, it was found that A549 cells in which Arl4c was knocked down by siRNA inhibited both migration and invasive activity as compared to cells in which Arl4c expression was not suppressed by siRNA (see FIGS. 2C and 2D). In addition, suppression of migration and invasion activity by suppression of Arl4c expression by siRNA was rescued by stable expression of Arl4c (see FIGS. 2A, 2B, 2C, and 2D). This suggests that Arl4c is involved in migration and invasive activity in cancer cells.

Example 3 (Involvement of Arl4c in cancer cell growth)
First, a plasmid for preparing cells stably expressing Arl4c shRNA was prepared. Specifically, using Gateway technology (Invitrogen), the following sequences were incorporated into CS-RfA-EVBsd together with the H1 promoter to prepare a lentiviral vector.
Control shRNA targeting luciferase: 5'-GTGCGTTGCTAGTACCAAC-3 '(SEQ ID NO: 7)
human Arl4c # 1: 5'-GGCTGTGAAGCTGAGTAAT-3 '(SEQ ID NO: 8)
human Arl4c # 2: 5'-GATGATCCTGAAACGCAGGAA-3 '(SEQ ID NO: 9)
These vectors are transfected into X293T cells together with the packaging vector, and the resulting lentiviral supernatant is used to introduce the plasmid into HCT116 and A549 cells to stably express Arl4c shRNA (shControl, shArl4c # 1 , ShArl4c # 2) was prepared. The degree of proliferation of these cells in a two-dimensional petri dish and 3D matrigel was examined.

Specifically, in two-dimensional culture, cells were suspended at a concentration of 1.0 × 10 ⁵ / ml cells and seeded, and the number of cells was measured on the designated day. In the three-dimensional culture, 40 μL of Matrigel was mounted on a cover slip, and then incubated at 37 ° C. for 30 minutes to measure with 3D Matrigel solidified. After seeding HCT116 cells or A549 cells (4 × 10 ⁴ cells) in 1 mL of growth medium containing 2% Matrigel on the Matrigel, the medium was changed after 3 days, and then the cell shape (area) ) Was measured. In addition, cells were fixed on 3D Matrigel with PBS containing 4 w / v% PFA, permeabilized and blocked with PBS containing 0.5 w / v% Triton X-100 and 40 mg / mL BSA, and then confocal microscope ( LSM510, Carl-Zeiss, Jana).

  As a result, there was no difference from the control in the two-dimensional culture (see FIGS. 3A and 3C), but there was a difference in the growth in the cell mass area in the three-dimensional culture (see FIGS. 3B and 3D).

Example 4 (Involvement of Arl4c in tumor formation in vivo)
5-week-old male BALB / cAnNCrj-nu nude mice (Charles River Laboratories) were anesthetized with medetomidine (0.3 mg / kg body weight) and midazolam (4 mg / kg body weight), and then the HCT116 cells prepared in Example 2 (5 × 10 ⁶ cells) or A549 cells (5 × 10 ⁶ cells) suspended in 100 μL of PBS were subcutaneously administered to the back surface. After transplantation, the transplanted sample was taken out 14 days later (HCT116) or 38 days later (A549), the diameter and weight were measured, and histological examination was performed in the same manner as in Example 1.

  As a result, in both HCT116 cells and A549 cells, the tumor increased when shControl was transplanted, but the cell growth was suppressed when shArl4c # 1 cells were transplanted (Fig. 4A, 4B). Immunohistologically, it was also found that Ki67, which is a marker for cell proliferation, was decreased when shArl4c # 1 cells were transplanted (see FIG. 4D).

Example 5 (Inhibition of tumor growth by in vivo treatment with Arl4c siRNA)
5-week-old male BALB / cAnNCrj-nu nude mice (Charles River Laboratories) were anesthetized in the same manner as in Example 4, and then HCT116 cells (1 × 10 ⁶ cells) suspended in 100 μL of PBS Was administered subcutaneously. 3 days after transplantation, a solution prepared by adding 30% mol / L of control siRNA or Arl4c siRNA to 0.5% (v / v) atelocollagen (AteloGene (registered trademark), manufactured by Koken Co., Ltd.) on the tumor site (diameter 4 mm or less) 200 μL was injected by direct injection. As siRNA in this Example, the human Arl4c # 1 sequence used in Example 3 was synthesized in a laboratory using CUGA (registered trademark) 7 in vitro Transcription Kit (Nippon Gene). Ten days after siRNA administration, transplanted samples were extracted from nude mice, diameters and weights were measured, and histological examination was performed in the same manner as in Example 1 and Example 4.

  As a result, in the mice administered with Arl4c siRNA, the volume and weight of the tumor part decreased (see FIG. 5A), and a decrease in the expression level at the Arl4c mRNA level was also confirmed as compared with the control mice (see FIG. 5B). . Immunohistochemically, it was found that both the number of Arl4c positive cells and the number of Ki67 positive cells were decreased in mice administered with Arl4c siRNA (see FIGS. 5B and 5C).

  The present invention can be used in the field of pharmaceuticals, particularly in the field of development and production of anticancer agents.

Sequence number 1 of a sequence table is a base sequence of mRNA of human Arl4c isoform 1.
SEQ ID NO: 2 in the sequence listing is a polypeptide of human Arl4c isoform 1.
Sequence number 3 of a sequence table is a base sequence of mRNA of human Arl4c isoform 2.
SEQ ID NO: 4 in the sequence listing is a polypeptide of human Arl4c isoform 2.
SEQ ID NO: 5 in the sequence listing is a control sequence targeted in the Arl4c siRNA experiment.
Sequence number 6 of a sequence table is the arrangement | sequence targeted by the experiment of Arl4c siRNA.
Sequence number 7 of a sequence table is the arrangement | sequence introduced in Control vector.
Sequence number 8 of a sequence table is the arrangement | sequence introduced in human Arl4c # 1 vector.
Sequence number 9 of a sequence table is the arrangement | sequence introduced in human Arl4c # 2 vector.

Claims (4)

A pharmaceutical composition for treating cancer comprising a nucleic acid that suppresses the expression of the Arl4c gene. The pharmaceutical composition according to claim 1, wherein the cancer is colon cancer or lung cancer.   A method for screening a substance for cancer treatment, wherein a substance that suppresses expression or suppresses activity is used as a candidate compound with the expression of Arl4c gene or the activity of Arl4c protein as an index. The screening method according to claim 3, wherein the cancer is colon cancer or lung cancer.