WO2021172315A1 - Ｌａｍｃ２－ｎｒ６ａ１スプライシングバリアント及びその翻訳産物 - Google Patents

Ｌａｍｃ２－ｎｒ６ａ１スプライシングバリアント及びその翻訳産物 Download PDF

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WO2021172315A1
WO2021172315A1 PCT/JP2021/006747 JP2021006747W WO2021172315A1 WO 2021172315 A1 WO2021172315 A1 WO 2021172315A1 JP 2021006747 W JP2021006747 W JP 2021006747W WO 2021172315 A1 WO2021172315 A1 WO 2021172315A1
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cancer
disease
peptide
nucleic acid
splicing variant
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直彦 越川
元治 清木
大輔 星野
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University of Tokyo NUC
Kanagawa Prefectural Hospital Organization
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University of Tokyo NUC
Kanagawa Prefectural Hospital Organization
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Definitions

  • the present invention broadly provides novel LAMC2-NR6A1 splicing variants and translations thereof, as well as various uses using them.
  • Intractable cancer diseases often lead to death of patients, and one of the main factors is the difficulty in diagnosing and detecting micrometastases.
  • Lack of effective diagnostic methods for cancer infiltration and metastasis not only makes it difficult to cure cancer diseases, but also greatly affects the quality of life (QOL) of patients.
  • QOL quality of life
  • 20-30% of patients with stage II colorectal cancer do not detect metastases at diagnosis, and distant metastases are found after surgical treatment is selected.
  • the cancer patient will be treated for distant metastatic cancer again after the operation, which increases the physical and financial burden.
  • the establishment of an effective infiltration / metastatic evaluation method at the time of diagnosis of cancer disease is required not only for the establishment of a treatment method for intractable cancer in the future but also for the current clinical requirements. It is a thing.
  • Laminin is a group of heterotrimeric proteins found in the basement membrane and forms part of the basement membrane. These proteins are classified on the basis of three non-identical polypeptides that combine with each other to form a laminin structure. These three polypeptides are identified as alpha ( ⁇ ), beta ( ⁇ ) and gamma ( ⁇ ) chains, each of which has several molecular species (eg, ⁇ 1- ⁇ 5, ⁇ 1- ⁇ 3 and ⁇ 1- ⁇ 2). ).
  • Laminin 332 also referred to as laminin 5 or LN5
  • LN5 is known to be abundant in the basement membrane, which is present in the basal lamina and is located between epithelial cells and the connective tissue that lines the epithelial cells.
  • laminin 332 is unique among known laminins in that it is the only laminin having a structure containing a gamma-2 ( ⁇ 2) chain that forms laminin 332 when combined with the ⁇ 3 and ⁇ 3 chains.
  • laminin 332 is produced by epithelial cells and can promote cell adhesion, proliferation, differentiation and / or migration.
  • laminin 332 is secreted from epithelial cells, it is susceptible to protease degradation (eg, by membrane type 1 matrix metalloproteinase-1 (MT1-MMP)).
  • MT1-MMP membrane type 1 matrix metalloproteinase-1
  • laminin 332 is processed towards the N-terminus of the gamma-2 chain sequence to produce fragments with epidermal growth factor (EGF) -like activity, including promotion of cell migration and infiltration (Koshikawa). Et al., J. Cell Biol. (2000) 148: 615-624).
  • EGF epidermal growth factor
  • WO 2014/027701 is a method of providing a diagnosis, prognosis or risk classification for subjects with or at risk of having cancer, Laminin Gamma-2 in Samples Derived from Subjects.
  • a step of comparing the monomer concentration with the reference laminin gamma-2 monomer concentration value, where the laminin gamma-2 monomer concentration in the sample is higher than the reference laminin gamma-2 monomer concentration value causes the subject to have cancer or Methods for identifying an increased risk of developing cancer have been disclosed.
  • Japanese Patent Application Laid-Open No. 2011-209281 discloses a test method and a test kit for urological cancer, which comprises measuring a laminin ⁇ 2 single chain in urine collected from a subject.
  • the present invention is a splicing variant produced via alternative splicing after a part of the sense strand of the laminin ⁇ 2 (LAMC2) gene and a part of the antisense strand of the NR6A1 gene are bound, and a translation product of these splicing variants.
  • LAMC2 laminin ⁇ 2
  • NR6A1 NR6A1
  • exon 12 of the laminin ⁇ 2 (LAMC2) gene and intron 1 of the antisense strand of the NR6A1 gene, which is a kind of nuclear receptor, are bound to each other, and then a new gene is generated by alternative splicing.
  • the novel gene and its translation product are expressed in various cancer cells, and the present invention has been completed.
  • the LAMC2 gene and the NR6A1 gene linked at the genomic level are referred to as "LAMC2-NR6A1 gene" (SEQ ID NO: 1) (also referred to as “LAMC2 fusion gene”), and the splicing variant thereof is referred to as "LAMC2-”. It is called "NR6A1 splicing variant".
  • the translation product of the LAMC2-NR6A1 splicing variant is also referred to as a LAMC2 fusion protein or an Ln- ⁇ 2 fusion protein.
  • Such a splicing variant can also be expressed as a splicing variant derived from exon 12 of the laminin ⁇ 2 (LAMC2) gene and intron 1 of the antisense strand of the NR6A1 gene. Up to cytosine at position 1857 of SEQ ID NO: 1 is derived from the LAMC2 gene, and after adenine at position 1858, it is derived from the antisense strand intron 1 of the NR6A1 gene.
  • LAMC2 laminin ⁇ 2
  • LAMC2-NR6A1 splicing variants the one having a short base sequence of 2544 bases (SEQ ID NO: 2) is called the LAMC2-NR6A1 splicing variant of "SHORT FORM" (hereinafter, also referred to as “Ln- ⁇ 2F") and has 2651 bases (hereinafter, also referred to as “Ln- ⁇ 2F”).
  • the long sequence number 6) is called the LAMC2-NR6A1 splicing variant of "LONG FORM”.
  • CMFCNSRMDGNLA novel peptide having the amino acid sequence of SEQ ID NO: 4 contained in SHORT FORM is referred to as "LAMC2-NR6A1 peptide”.
  • LAMC2-NR6A1 splicing variants there are many other LAMC2-NR6A1 splicing variants, and SHORT FORM and LONG FORM are only some examples. Since the splicing client is considered to be involved in the activation of the RAS / MAPK / ERK pathway as well as PI3K and Akt downstream of the EGF receptor signaling pathway, it is considered to be involved in the activation of the EGF receptor in the state of a fusion gene. It is expected to bind and activate its downstream.
  • the ligand domain of the EGF receptor is excised with a protease and exhibits ligand activity, but since the fusion gene product exhibits ligand activity in the expressed form as it is, RSK downstream of ERK etc. It is also considered to contribute to activity control.
  • the splicing variant has the sequence of SEQ ID NO: 2, for example, the sequence shown at positions 1919 to 2544 of SEQ ID NO: 2, particularly the base sequence encoding the amino acid sequence of SEQ ID NO: 4, and the translation product thereof Those with EGF receptor ligand activity, namely PI3K and Akt downstream of the EGF receptor signaling pathway, and / or ERK downstream of the RAS / MAPK / ERK signaling pathway, and optionally further downstream RSK. Anything that activates expression and / or phosphorylation may be used.
  • [1] A splicing variant derived from exon 12 of the laminin ⁇ 2 (LAMC2) gene and intron 1 of the antisense strand of the NR6A1 gene.
  • the following peptides (a), (b) or (c): (A) Peptide having the amino acid sequence of SEQ ID NO: 4;
  • (B) A peptide containing an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence of SEQ ID NO: 4 and having an activity of enhancing the activity of the EGF receptor and its downstream signal pathway.
  • [6] A nucleic acid encoding the peptide according to [5] or a nucleic acid complementary thereto.
  • [7] A composition containing a compound or a salt thereof that inhibits the expression of the peptide according to [5] or the nucleic acid according to [6].
  • [8] The composition according to [7], wherein the compound is an antibody or an antigen-binding fragment thereof, or a nucleic acid.
  • the nucleic acid is siRNA that cleaves mRNA.
  • [15] A model animal transformed with the splicing variant according to any one of [1] to [3] or the nucleic acid according to [6].
  • [16] An antibody or antigen binding thereof that binds to the translation product of the splicing variant according to any one of [1] to [3] or the peptide according to [5] and does not bind to wild-type laminin ⁇ 2 (LAMC2). piece.
  • a pair of oligonucleotide primers comprising a sense primer and an antisense primer for detecting or amplifying a nucleic acid encoding the peptide according to [5].
  • [18] A nucleic acid having an activity of binding to an mRNA encoding the peptide according to [5] and inhibiting translation of the mRNA into a protein.
  • [20] A vector containing the nucleic acid according to [18] or [19].
  • [21] Recombinant cells containing the vector according to [20].
  • [22] A method for treating or preventing a disease related to the activity of an EGF receptor and its downstream signal pathway in a subject, and a compound that inhibits the expression of the peptide according to [5] or the nucleic acid according to [6]. Or a method comprising administering a salt thereof to a subject.
  • the biomarker according to [24] for diagnosing a disease associated with the activity of the EGF receptor and its downstream signaling pathway.
  • the disease is cancer, and the biomarker is a tumor marker for diagnosing the susceptibility to cancer, the presence or absence of cancer, or the presence or absence of cancer progression, [25] or The biomarker according to [26].
  • [28] A method for detecting a biomarker of a disease related to the activity of the EGF receptor and its downstream signal pathway, wherein in a sample derived from a subject, the following (a) to (e): (A) The splicing variant according to any one of [1] to [3], preferably the splicing variant having the base sequence of SEQ ID NO: 2 or 6; (B) A protein encoded by the splicing variant according to any one of [1] to [3], preferably the splicing variant having the base sequence of SEQ ID NO: 2 or 6; (C) Peptide having the amino acid sequence of SEQ ID NO: 4; (D) Nucleic acid encoding the peptide of (c) or a nucleic acid complementary thereto; (E) Antibodies to the protein of (b) or the peptide of (c); A method that includes one of the following.
  • [32] A method for diagnosing a disease related to the activity of the EGF receptor and its downstream signal pathway in the subject, wherein in the subject, the following (a) to (e): (A) The splicing variant according to any one of [1] to [3], preferably the splicing variant having the base sequence of SEQ ID NO: 2 or 6; (B) A protein encoded by the splicing variant according to any one of [1] to [3], preferably the splicing variant having the base sequence of SEQ ID NO: 2 or 6; (C) Peptide having the amino acid sequence of SEQ ID NO: 4; (D) Nucleic acid encoding the peptide of (c) or a nucleic acid complementary thereto; (E) Antibodies to the protein of (b) or the peptide of (c); A method comprising the step of detecting a biomarker of a disease associated with the activity of an EGF receptor and its downstream signaling pathway, including any of the above.
  • [36] A method for screening a drug for treating or preventing a disease associated with the activity of an EGF receptor and its downstream signaling pathway, the peptide according to [5] or the nucleic acid according to [6].
  • a method comprising the step of selecting a substance that inhibits the expression of the drug as the drug.
  • the disease is cancer, obesity, autoimmune disorder, inflammation, heart disease, neurodegenerative disease or diabetes.
  • the LAMC2-NR6A1 splicing variant of the present invention and its translation products, particularly the LAMC2-NR6A1 peptide are novel ones specifically detected in cancer cells, they are expected to be used for various purposes. For example, by constructing a detection system for a LAMC2-NR6A1 splicing variant or a translation product thereof, cancer can also be detected.
  • a detection system that theoretically does not have a non-specific reaction can be obtained by using a system that can detect this amino acid sequence specifically. ..
  • the LAMC2-NR6A1 splicing variant since the LAMC2-NR6A1 splicing variant is considered to control the motility of cancer cells, it can also be used as a marker for predicting the malignancy of cancer. Furthermore, substances that inhibit the expression of the LAMC2-NR6A1 splicing variant are expected to have the effect of suppressing cancer infiltration and metastasis.
  • FIG. 1 shows the results of confirming chromosomal-level gene binding in Skov3 cells by the FISH method.
  • the red signal eg, the signal shown in FIG. 1a
  • the green signal eg, the signal shown in FIG. 1b
  • the yellow signal indicates that LAMC2 and NR6A1 are bound.
  • FIG. 2 is a diagram schematically showing how the sense strand of the LAMC2 gene and the antisense strand of the NR6A1 gene bind at the genomic level.
  • FIG. 3 is a diagram illustrating how alternative splicing occurs at the mRNA level after binding of the LAMC2 gene and the NR6A1 gene to generate splicing variants of SHORT FORM and LONG FORM.
  • FIG. 4 shows the results of PCR examination of the expression distribution of LAMC2-NR6A1 splicing variants of LONG FORM and SHORT FORM in normal cells. From this result, it can be seen that the LAMC2-NR6A1 splicing variants of LONG FORM and SHORT FORM are not expressed in normal cells.
  • FIG. 5 shows the results of detecting the expression of LAMC2-NR6A1 splicing variants of LONG FORM and SHORT FORM in clinical specimens derived from cancer patients.
  • the LAMC2-NR6A1 splicing variant of SHORT FORM was 19 out of 20 ovarian cancer samples and 19 breast cancer samples. It was detected in 13 of the samples and 11 of 16 in colorectal cancer. On the other hand, the LONG FORM splicing variant was detected in 12 out of 20 samples of ovarian cancer.
  • FIG. 6 shows the functional analysis results of Ln- ⁇ 2F in the ovarian cancer cell line Ovcar8 introduced with Ln- ⁇ 2F (LAMC2-NR6A1 splicing variant of SHORT FORM) and the ovarian cancer cell Skov3 expressing Ln- ⁇ 2F.
  • FIG. 7 shows anti-ERK antibody, anti-phosphorylated ERK antibody, anti-Akt antibody, anti-phosphorylated Akt antibody, anti-EGFR antibody and anti-phosphorylated EGFR antibody to examine the effect of Ln- ⁇ 2F on intracellular signals. The results of Western blotting using the antibody are shown.
  • Ln- ⁇ 2F means a fusion gene product
  • Ln- ⁇ 2m means a laminin ⁇ 2 single chain.
  • FIG. 8 shows the results of microarray analysis of Skov3-scr and Skov3-kd1. Comparing the gene expression patterns of the cells in which Ln- ⁇ 2F expression was suppressed and the control cells by microarray analysis, the expression of the gene cluster controlled by Akt was decreased.
  • FIG. 9 compares the changes in cell proliferation when LAMC2 gene expression was suppressed while Scov3 cells were cultured under nutrient starvation (0.5% FBS) and normal conditions (10% FBS). Shown (scr: control; Kd1, kd2: suppression of Ln- ⁇ 2F expression). 1000 cells were sown and the number of cells after 1, 2 and 3 days was counted.
  • FIG. 10 shows a comparison of changes in cell proliferation when LAMC2 gene expression was forcibly expressed while culturing OVCAR8 cells under nutrient starvation (0.5% FBS) and normal conditions (10% FBS).
  • FIG. 11 shows the results of evaluating the motility of cancer cells in the Boyden chamber (scr: control; KD2, KD1: Ln- ⁇ 2F expression suppression). The vertical axis represents the number of cells that have moved.
  • FIG. 12A shows the results of evaluating the effect of Ln- ⁇ 2F on tumorigenicity in a mouse inoculated with ovarian cancer in the peritoneum (Scr: control; Kd1, kd2: suppression of Ln- ⁇ 2F expression; Kd2- mock: Ln- ⁇ 2F expression suppression + control; Ln- ⁇ 2F: Kd2 + Fusion: Ln- ⁇ 2F expression suppression + Ln- ⁇ 2F).
  • FIG. 12B shows the results of measuring the luminescence intensity of the tumorigenicity in FIG. 12A and graphing it over time.
  • FIG. 13A shows the results of evaluating the tumorigenicity in mice when Ln- ⁇ 2F was expressed in OVCAR8 cells not expressing Ln- ⁇ 2F and transplanted intraperitoneally into mice (Mock: control; Ln-).
  • ⁇ 2F Ln- ⁇ 2F).
  • Cells expressing luciferase were imaged 60 days after transplantation into the abdominal cavity of scid / beige mice.
  • FIG. 13B shows the result of quantifying the emission intensity of FIG. 13A.
  • FIG. 14 is a schematic diagram of translation products of LAMC2 and LAMC2-NR6A1 splicing variants.
  • FIG. 15 illustrates that the Ln- ⁇ 2 fusion protein contributes to the promotion of carcinogenesis by activating the EGF receptor as it is without being processed by MT1-MMP.
  • FIG. 16 shows the sequence determination results of SHORT FORM and LONG FORM (Ln- ⁇ 2F).
  • FIG. 17 shows the results of immunostaining of tissues recovered 4 weeks after intraperitoneal injection of OVCAR8 cells (Mock) and Ln- ⁇ 2F overexpressing cells into the abdominal cavity of nude mice. Luciferase (green); Phosphorylated AKT (red in FIG. 17A); Phosphorylated ERK (red in FIG. 17B); Nucleus (blue) Scale bar: 50 ⁇ m
  • the present embodiment will be described, but the scope of the present invention is not construed as being limited to the following embodiments.
  • the following peptides or salts thereof are provided.
  • B) The amino acid sequence of SEQ ID NO: 4 contains an amino acid sequence in which one or several, for example, 1 to 5 amino acids have been deleted, substituted and / or added, and the activity of the EGF receptor and its downstream signaling pathway.
  • Peptides with enhanced activity (C) A peptide containing an amino acid sequence having 80% or more, preferably 90% or more identity with the amino acid sequence of SEQ ID NO: 4 and having an activity of enhancing the activity of the EGF receptor and its downstream signal pathway.
  • the laminin ⁇ 2 single chain (also referred to as “Ln- ⁇ 2m” in the present specification) refers to a ⁇ 2 chain which is a component of laminin 332 expressed as a monomer.
  • Laminin 5 is one of the main constituents of the basement membrane, and is a heterotrimer in which three polypeptide chains of ⁇ 3 chain, ⁇ 3 chain, and ⁇ 2 chain are associated at a coiled coil structure part. Of the three polypeptide chains, the ⁇ 2 chain has been reported to be expressed as a monomer in malignant cancer cells.
  • the ⁇ 2 chain expressed as a trimer in order to distinguish the ⁇ 2 chain expressed as a trimer from the “laminin 332 ⁇ 2 chain”, the ⁇ 2 chain expressed as a monomer is referred to as “laminin ⁇ 2 single chain” or “laminin ⁇ 2 chain”. Called.
  • amino acid sequence in which one or several amino acids have been deleted, substituted and / or added loses the desired function as compared to the amino acid sequence identified by SEQ ID NO: Means a mutant amino acid sequence in which no number of amino acids has been deleted, substituted and / or added.
  • Amino acids may refer to natural and synthetic amino acids, and amino acid analogs and amino acid mimetics that function similarly to natural amino acids.
  • the amino acid may be either an L-amino acid or a D-amino acid.
  • Natural amino acids are amino acids encoded by the genetic code and amino acids modified after translation in the intracellular code.
  • the substitution is preferably a conservative amino acid substitution. This is because if it is a conservative amino acid substitution, it is highly likely that it has a structure or property substantially equivalent to that of the LAMC2-NR6A1 peptide.
  • the laminin ⁇ 2 (Ln- ⁇ 2) chain translated from LAMC2 is produced as a single chain or as laminin 332 associated with the laminin ⁇ 3 and ⁇ 3 chains.
  • MT1-MMP excises the laminin EGF-like domain (domain III) in the short arm of these Ln- ⁇ 2 chains to release the fragment containing domain III having the ligand activity of the epidermal growth factor receptor (EGFR). (Fig. 14 top), it is thought that it contributes to the motility of cancer cells and the enhancement of survival signals through activation of ErbB receptors (Fig. 15).
  • processing by MT1-MMP is essential.
  • the Ln- ⁇ 2 fusion protein which is a translation product of LAMC2-NR6A1 does not require the processing of MT1-MMP and contributes to the promotion of carcinogenesis by activating the EGF receptor as it is (Fig. 14, bottom).
  • the Ln- ⁇ 2 fusion protein which is a translation product of LAMC2-NR6A1 does not require processing by protease and can efficiently activate the ErbB receptor.
  • splicing variants with the LAMC2-NR6A1 peptide directly activate the epidermal growth factor receptor (EGFR) without processing MT1-MMP and signal downstream thereof. It has an activity that enhances the activity of the pathway, and it is considered that these enhancements are involved in cancer.
  • EGFR epidermal growth factor receptor
  • the PI3K-Akt pathway begins with the phosphorylation activity of PI3K and inhibits cell survival and induction of apoptosis through phosphorylation of Akt. That is, Akt expression and / or phosphorylation is associated with cancer.
  • the PI3K-Akt pathway has been confirmed to be constitutively hyperactive in many tumors. Akt is also known to be associated with diseases other than cancer, such as obesity, autoimmune disorders, inflammation, or diabetes.
  • the MAPK / ERK pathway begins with phosphorylation activation of RAS and induces cell proliferation through phosphorylation of Raf, MEK, and ERK. That is, expression and / or phosphorylation of the Ras-ERK pathway is associated with cancer. In fact, the Ras-ERK pathway plays an important role in promoting pathway cell proliferation in many tumors and is therefore considered to be involved in carcinogenesis. In addition, it has been confirmed that the function of ERK is constitutively enhanced in cancer cells. ERK is also known to be associated with diseases other than cancer, such as heart disease and neurodegenerative diseases associated with cardiomyocyte hypertrophy. Since the LAMC2-NR6A1 peptide is expressed in various cancer cells, it is expected to be applied to various cancer-related applications regardless of its mechanism of action.
  • the LAMC2-NR6A1 peptide can be obtained by artificial conventional methods in the technical field such as chemical synthesis and recombinant DNA technology.
  • the LAMC2-NR6A1 peptide is prepared by binding an amino acid to a solid-phase carrier insoluble in a reaction solvent according to the solid-phase method, sequentially performing a condensation reaction with this amino acid, and extending the peptide chain. Can be done.
  • nucleic Acid in the second aspect, a nucleic acid encoding the amino acid sequence of the LAMC2-NR6A1 peptide or a nucleic acid complementary thereto is provided.
  • the method for obtaining the nucleic acid is not particularly limited.
  • an appropriate probe or library is prepared based on the information on the base sequence of the nucleic acid corresponding to the amino acid sequence disclosed in the present specification, and a cDNA library is prepared using them. It can be obtained by screening rallies and genomic DNA libraries. For example, it can be produced by selecting a desired clone from a genomic DNA library using an appropriate probe or the like specific to the desired gene. Separation of total RNA from cell lines, separation and purification of mRNA, acquisition of genomic DNA and cloning thereof, etc. can all be carried out according to a conventional method.
  • DNA or the like chemically synthesized based on the information on the base sequence of the desired nucleic acid can be generally used.
  • a sense primer and an antisense primer set based on the nucleotide sequence information of the nucleic acid can be used as a screening probe.
  • sense primers and antisense primers designed to sandwich the region encoding the LAMC2-NR6A1 peptide are preferably used.
  • Examples of such a pair of oligonucleotide primers include the primers set forth in SEQ ID NO: 12 and SEQ ID NO: 13.
  • a DNA amplification method by PCR can be preferably used. Isolation and purification of the amplified DNA fragment can be performed according to a conventional method. For example, gel electrophoresis and the like can be mentioned.
  • the base sequence of the nucleic acid obtained according to the above method can be determined according to a conventional method such as the Dideoxy method or the Maxam-Gilbert method.
  • Nucleic acid is also a nucleic acid that hybridizes with a nucleic acid consisting of a base sequence specified by a corresponding SEQ ID NO: or a base sequence complementary thereto under highly stringent conditions and encodes a peptide having the same activity as the nucleic acid. Include.
  • the "high stringent condition” means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • the high stringent condition is described in a condition in which nucleic acids having high identity hybridize with each other and nucleic acids with lower identity do not hybridize with each other, for example, Molecular cloning a Laboratory manual 2nd edition (Sambrook et al., 1989). Conditions can be mentioned. Specifically, it hybridizes at 60 ° C., 1 ⁇ SSC, 0.1% SDS, preferably 0.1 ⁇ SSC, 0.1% SDS, which is a washing condition in normal Southern hybridization, at a corresponding salt concentration. Conditions for soaking can be mentioned.
  • the splicing variant LAMC2-NR6A1 gene is a combination of the sense strand of LAMC2 and the antisense strand of NR6A1, its translation product contains the amino acid sequence encoded by the sense strand of LAMC2, but the sense strand of the NR6A1 gene. Does not contain the amino acid sequence encoded by.
  • alternative splicing ultimately produces mature mRNA (FIGS. 2 and 3).
  • the transcript of the LAMC2-NR6A1 gene may contain multiple alternative splicing variants encoding different isoforms, such as those with 2544 bases (SEQ ID NO: 2), those with 2651 bases (SEQ ID NO: 6), and the like. Exists.
  • the splicing variant has the sequence of SEQ ID NO: 2, for example, the sequence shown at positions 1919 to 2544 of SEQ ID NO: 2, particularly the base sequence encoding the amino acid sequence of SEQ ID NO: 4, and the translation product thereof is an EGF receptor.
  • SEQ ID NO: 2 for example, the sequence shown at positions 1919 to 2544 of SEQ ID NO: 2, particularly the base sequence encoding the amino acid sequence of SEQ ID NO: 4, and the translation product thereof is an EGF receptor.
  • Those having body ligand activity that is, those that activate PI3K and Akt downstream of the EGF signal transduction pathway are preferable.
  • splicing variant refers to mature mRNA as an alternative splicing product of a gene.
  • the protein translated from mRNA has a sequence derived from laminin ⁇ 2 (up to isoleucine at position 618 of SEQ ID NO: 3) at the N-terminal, and a peptide sequence read from NR6A1-derived mRNA is added to the C-terminal.
  • the amino acid sequence added to the C-terminal changes depending on the difference in the splicing site, and a typical example is the amino acid sequence of SEQ ID NO: 4 (CMFCNSRMDGNLA).
  • a vector containing a nucleic acid encoding a LAMC2-NR6A1 peptide or a nucleic acid complementary thereto, a recombinant cell containing the vector, and a model animal transformed with the nucleic acid are provided.
  • the nucleic acid encoding the peptide of SEQ ID NO: 4 and other desired nucleic acids may all be contained in one expression vector, or may be divided into two or more groups. , Each may be configured to be included in a separate expression vector.
  • the nucleic acid can be inserted between the promoter and the terminator.
  • the vector also contains selectable marker genes for selection of transformed cells (genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinosricin, genes that complement auxotrophic mutations, etc.). It can also be further contained.
  • the vector may be a plasmid or a viral vector as an expression vector.
  • the vectors include adenovirus, retrovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendaivirus, Epstein. It may be a virus vector such as bar virus.
  • Model animals can be used to study diseases associated with the activity of the EGF receptor and its downstream signaling pathways, such as the development, treatment or prevention of cancer.
  • Model animals include non-human primates (eg monkeys such as cynomolgus monkeys, rhesus monkeys, chimpanzees) and other mammals such as cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs and cats. , Dogs, rats and mice), any vertebrate is intended.
  • the desired gene is introduced into the fertilized egg or early embryo, and the fertilized egg or early embryo into which the gene is introduced is introduced into the foster mother of the animal. It may be transplanted into the uterus and developed.
  • the homozygous transformation model animal uses pluripotent stem cells such as ES cells (embryonic stem cells) and iPS cells (induced pluripotent stem cells) into which the desired gene has been introduced into early embryos such as blastocysts. It can also be produced by inserting into a chimeric animal, transplanting the chimeric embryo obtained thereby into the uterus of a foster parent, and mating the chimeric animal obtained by developing the embryo.
  • compositions comprising a compound or a salt thereof that inhibits the expression of the peptide or nucleic acid.
  • a compound may be an antibody or an antigen-binding fragment thereof, or a nucleic acid.
  • antibody means an antibody molecule capable of immunospecifically binding to a desired antigen or the like. Antibodies include anti-serum, polyclonal antibody, monoclonal antibody, chimeric antibody, human antibody, humanized antibody, recombinant antibody, single-stranded Fvs (“scFv”), single-stranded antibody, single domain antibody, F (ab). ) Fragments, F (ab') fragments, disulfide-linked Fvs (“sdFv”), anti-idiotype antibodies and functionally active epitope-binding fragments of any of the above.
  • antigen-binding fragment refers to any fragment of an antibody that retains the ability to immunospecifically bind to a target peptide or the like.
  • Antigen-binding fragments include single-chain antibodies, Fab fragments, F (ab') 2 fragments, disulfide bond Fvs, light chain variable regions (VL) that specifically bind to target peptides, and heavy chain variable regions (VH). Fragments containing complementarity determining regions (CDRs) and the like are included.
  • the antibody-binding fragment can be obtained by a method known in the art.
  • the LAMC2-NR6A1 peptide or nucleic acid has an activity of enhancing the activity of the EGF receptor and its downstream signal pathway
  • the compound that inhibits the activity is associated with the activity of the EGF receptor and its downstream signal pathway. It can be suitably used for the treatment or prevention of diseases such as cancer, obesity, autoimmune disorder, inflammation, heart disease, particularly heart disease associated with myocardial cell hypertrophy, neurodegenerative disease or diabetes.
  • the compound having such activity may be, for example, an antibody or an antigen-binding fragment thereof, or an antisense drug such as siRNA or ribozyme.
  • any antisense nucleic acid can be used as long as it binds to the mRNA encoding the target peptide and has an activity of inhibiting the translation of the mRNA into a protein.
  • the antisense nucleic acid siRNA that cleaves mRNA or a nucleic acid that is transcribed into siRNA or a precursor thereof in the cell can also be preferably used.
  • SiRNA is usually a double-stranded RNA of about 19 to 30 bases, for example, about 21 to 25 bases, and in general, one of them has a base sequence complementary to a part of the target mRNA, and the other is complementary to this. However, it does not have to be completely complementary to the target mRNA.
  • the expression inhibition method using siRNA is a sequence-specific gene expression suppression mechanism induced by a double-stranded nucleic acid.
  • SiRNA also has high target specificity and is highly safe because it is a method that utilizes a gene expression suppression mechanism that originally exists in the living body.
  • siRNA is a double-stranded RNA with 21 base pairs, and the 3'portion of each RNA strand is a 2-base overhang.
  • SiRNA is produced by cutting out hairpin RNA (SHRNA) or longer double-stranded RNA by a Dicer.
  • SHRNA hairpin RNA
  • the shRNA or long double-stranded RNA before being cleaved by the Dicer can be suitably used in the present invention as a precursor of siRNA.
  • the siRNA can be designed according to a known method based on the base sequence of the target mRNA. Further, the siRNA may be a double-stranded RNA or a DNA-RNA chimeric double-stranded nucleic acid as long as it has an RNAi effect on the target mRNA, and is subjected to an artificial nucleic acid or various modifications. It may be nucleic acid.
  • compositions are broad and include diseases associated with the activity of EGF receptors and their downstream signaling pathways, such as cancer, obesity, autoimmune disorders, inflammation, heart disease, especially heart disease associated with myocardial cell hypertrophy, neurodegenerative diseases or It can be suitably used for the treatment or prevention of diabetes.
  • the composition is used for such pharmaceutical uses, particularly for the treatment or prevention of cancer, for example, suppression of cancer cell growth, suppression of cell canceration, suppression of cancer cell malignancy, or cancer invasion.
  • Can can be used to suppress metastasis or recurrence.
  • cancer is used in its broadest sense.
  • Cancers include, for example, brain cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer (excluding colon cancer), anal cancer, rectal cancer, liver cancer, hepatocellular carcinoma, lung cancer, etc.
  • compositions suitable for oral administration include, for example, granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions, liquids and the like.
  • Compositions suitable for parenteral administration include, for example, injections for intravenous administration, intramuscular administration, or subcutaneous administration, drip agents, suppositories, transdermal absorbents, transmucosal absorbents, nasal drops, and ear drops. Agents, eye drops, inhalants and the like can be mentioned. It is also intended that a preparation prepared as a pharmaceutical composition in the form of a dry powder such as a lyophilized product is dissolved at the time of use and used as an injection or an infusion.
  • the composition may include solid or liquid pharmaceutical additives.
  • the additive for preparation may be either an organic substance or an inorganic substance.
  • an excipient is added to a substance selected from the group consisting of the above compounds or salts thereof, which are active ingredients, and hydrates thereof and solvates thereof.
  • a binder, a disintegrant, a lubricant, a colorant, a flavoring agent, etc. are added, and then a preparation in the form of a tablet, a coated tablet, a granule, a powder, a capsule, etc. is prepared by a conventional method. be able to.
  • excipients include lactose, sucrose, sucrose, glucose, cornstarch, starch, talc, sorbitol, crystalline cellulose, dextrin, kaolin, calcium carbonate, silicon dioxide and the like.
  • binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragant, gelatin, shelac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, dextrin, pectin and the like.
  • the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hardened spot oil.
  • any one that is approved to be added to a pharmaceutical product can be used.
  • the flavoring agent cocoa powder, mint brain, aromatic acid, mentha oil, borneol, cinnamon powder and the like can be used. Tablets and granules can be sugar-coated, gelatin-coated, or otherwise coated as appropriate. In addition, preservatives, antioxidants and the like may be added as needed.
  • liquid preparations for oral administration such as emulsions, syrups, suspensions, or liquids
  • inert diluents such as water or vegetable oils
  • auxiliary agents such as wetting agents, suspension aids, sweeteners, fragrances, colorants, preservatives and the like can be added to the liquid preparation.
  • After preparing the liquid preparation it may be filled in a capsule such as gelatin.
  • Examples of the solvent or suspension used in the production of pharmaceutical compositions for parenteral administration, such as injections or suppositories, include water, propylene glycol, polyethylene glycol, benzyl alcohol, ethyl oleate, lecithin and the like. Can be mentioned.
  • Examples of the base used in the production of suppositories include cocoa butter, emulsified cocoa butter, lauric acid and the like.
  • the method for preparing the pharmaceutical product is not particularly limited, and any method widely used in the art can be used.
  • a carrier for example, a diluent such as water, ethyl alcohol, propylene glycol; a pH regulator or buffer such as sodium citrate, sodium acetate, or sodium phosphate.
  • Stabilizers such as ethylenediamine tetraacetic acid, thioglycolic acid, and thiolactic acid can be used. Sufficient amounts of salt, glucose, mannitol, glycerin, etc. may be added to the composition to prepare an isotonic solution, and solubilizers, soothing agents, local anesthetics, etc. may be added. You can also do it.
  • a pharmaceutical composition in the form of an ointment for example a paste, cream, or gel
  • bases for example, white petrolatum, polyethylene, paraffin, glycerin, cellulose derivative, polyethylene glycol, silicon, bentonite and the like
  • preservative for example, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like can be used.
  • the ointment, cream, gel, paste or the like can be applied to the surface of a normal support by a conventional method.
  • a woven fabric or non-woven fabric made of cotton or synthetic fibers a film such as soft vinyl chloride, polyethylene, or polyurethane, or a foam sheet can be preferably used.
  • the amount of the active ingredient in the composition is not particularly limited as long as it can be used for a desired purpose, and can be appropriately increased or decreased according to the age, weight, gender, purpose of administration, symptoms, etc. of the patient.
  • the above active ingredients are diseases associated with the activity of EGF receptors and their downstream signaling pathways, such as cancer, obesity, autoimmune disorders, inflammation, heart disease, especially heart disease associated with myocardial cell hypertrophy, neurodegenerative diseases.
  • diseases associated with the activity of EGF receptors and their downstream signaling pathways such as cancer, obesity, autoimmune disorders, inflammation, heart disease, especially heart disease associated with myocardial cell hypertrophy, neurodegenerative diseases.
  • it is administered to the subject in a manner for treating or preventing diabetes.
  • Biomarkers in another embodiment, further provided are diagnostic markers for assessing biomarkers, particularly diseases associated with the activity of the EGF receptor and its downstream signaling pathways, such as cancer.
  • the marker detects the expression of the target nucleic acid or its translation product, preferably the peptide of SEQ ID NO: 4, or measures its expression level in the sample derived from the subject, or the target nucleic acid or its translation product in the sample. Whether or not the subject suffers from a disease associated with the activity of the EGF receptor and its downstream signaling pathway, such as cancer, by detecting or measuring the presence of a specific antibody against the peptide of SEQ ID NO: 4, preferably. Used to determine.
  • the term "subject” refers to humans and non-human primates (eg, monkeys such as cynomolgus monkeys, rhesus monkeys, chimpanzees) and other mammals such as cows, pigs, camels, llamas and horses. , Goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice). Depending on the embodiment, the subject may be a human or a non-human animal.
  • non-human primates eg, monkeys such as cynomolgus monkeys, rhesus monkeys, chimpanzees
  • other mammals such as cows, pigs, camels, llamas and horses. , Goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice.
  • the subject may be a human or a non-human animal.
  • the subject will be presented with diseases associated with the activity of the EGF receptor and its downstream signaling pathways, such as cancer, obesity, autoimmune disorders, inflammation, heart disease, especially heart disease associated with myocardial cell hypertrophy, neurodegeneration. It may be a patient at risk of developing the disease or diabetes or a patient who has already developed such a disease.
  • diseases associated with the activity of the EGF receptor and its downstream signaling pathways such as cancer, obesity, autoimmune disorders, inflammation, heart disease, especially heart disease associated with myocardial cell hypertrophy, neurodegeneration. It may be a patient at risk of developing the disease or diabetes or a patient who has already developed such a disease.
  • a biomarker is detected in a sample derived from a subject, it is determined that the patient is susceptible to a disease related to the activity of the EGF receptor and its downstream signaling pathway, has the disease, or has an advanced disease. can do.
  • the biomarker may be a tumor marker used to diagnose the susceptibility to cancer, the presence or absence of cancer, or the presence or absence of cancer progression.
  • sample broadly refers to biological material that is believed to contain a target. Any cell, tissue or body fluid can be utilized to obtain the sample.
  • tissue sections such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood (such as whole blood), plasma, serum, sputum, stool, Tears, mucus, saliva, bronchial alveolar lavage (BAL) fluid, hair, skin, red blood cells, platelets, interstitial fluid, ocular crystal fluid, cerebrospinal fluid, sweat, nasal juice, synovial fluid, underbelly, sheep water, semen, etc.
  • BAL bronchial alveolar lavage
  • Cells and tissues may include lymph, ascites, gynecological fluids, urine, ascitic fluid, cerebrospinal fluid and the like.
  • the target peptide or the like may be isolated or purified from the sample.
  • the detection of the expression of the target peptide or the measurement of the expression level thereof can be carried out by, for example, an immunoassay, an agglutination method, a turbidimetric method, a Western blotting method, a surface plasmon resonance (SPR) method or the like. Immunoassays are particularly simple and preferred.
  • Immunoassays are classified into enzyme immunoassays (EIA or ELISA), radioimmunoassays (RIA), fluorescence immunoassays (FIA), fluorescence polarization immunoassays (FPIA), chemiluminescent immunoassays (CLIA), etc. according to the antibody labeling method. Can be used.
  • the ELISA method peroxidase, an enzyme such as alkaline phosphatase, the RIA method, 125 I, 131 I, 35 S, 3 radioactive substances such as H, in the FPIA method, fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine Fluorescent substances such as isothiocyanate and near-infrared fluorescent materials, and in the ELISA method, antibodies labeled with luminescent substances such as luciferase, luciferin, and equolin are used. In addition, antibodies labeled with nanoparticles such as colloidal gold and quantum dots can be detected.
  • the antibody in the immunoassay, can be labeled with biotin, and avidin or streptavidin labeled with an enzyme or the like can be bound and detected.
  • the ELISA method using an enzyme label is preferable because the antigen can be measured easily and quickly.
  • the enzyme substrate used in the ELISA method may be 3,3'-diaminobenzidine (DAB), 3,3'5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), or the like.
  • DAB 3,3'-diaminobenzidine
  • TMB 3,3'5,5'-tetramethylbenzidine
  • OPD o-phenylenediamine
  • NPP p-nitrophenyl phosphate
  • the solid phase carrier is not particularly limited as long as it is a carrier on which an antibody can be immobilized, and examples thereof include microtiter plates made of glass, metal, resin and the like, substrates, beads, nitrocellulose membranes, nylon membranes, PVDF membranes and the like.
  • the target substance can be immobilized on these solid phase carriers according to a known method.
  • the agglutination method is also preferable as a method for easily detecting a trace amount of protein in the above immunoassay.
  • the agglutination method include a latex agglutination method in which latex particles are bound to an antibody.
  • Both monoclonal antibody and polyclonal antibody can be produced according to a known method.
  • Monoclonal antibodies can be obtained, for example, by isolating antibody-producing cells from target-immunized non-human mammals, fusing them with myeloma cells, etc. to prepare hybridomas, and purifying the antibodies produced by these hybridomas. Can be done.
  • polyclonal antibodies can be obtained from the serum of animals immunized with the target.
  • the marker is a gene such as LAMC2-NR6A1 gene or LAMC2-NR6A1 splicing variant
  • FISH Fluorescence in situ hybridization
  • RT-PCR Real-Time PCR
  • other known gene mutation testing method can be used.
  • a person skilled in the art can select an appropriate means according to the gene of interest, and for example, the state in which the LAMC2 gene and the NR6A1 gene are bound can be confirmed by the FISH method.
  • the probe used to detect the LAMC2-NR6A1 gene can be prepared from a bacterial artificial chromosome (BAC) clone containing these genes, for example RP11-158D24 and RP11-582A18 can be used.
  • BAC bacterial artificial chromosome
  • the probe signals are detected distantly in the normal case and overlapped in the case of gene binding.
  • the probe that can be used for detecting LAMC2-NR6A1 splicing and the like may have a sequence that specifically recognizes the gene of interest, such as the LAMC2-NR6A1 splicing variant.
  • specifically recognizing means binding to a particular LAMC2-NR6A1 splicing variant, but not to wild-type LAMC2.
  • a method for detecting a biomarker of a disease associated with the activity of the EGF receptor and its downstream signaling pathway is provided.
  • the detection method is as follows (a) to (e): in the sample derived from the subject.
  • LAMC2-NR6A1 splicing variant preferably a splicing variant having the nucleotide sequence of SEQ ID NO: 2 or 6
  • B A protein encoded by a LAMC2-NR6A1 splicing variant, preferably a splicing variant having the nucleotide sequence of SEQ ID NO: 2 or 6
  • C Peptide having the amino acid sequence of SEQ ID NO: 4
  • D Nucleic acid encoding the peptide of (c) or a nucleic acid complementary thereto
  • E Antibodies to the protein of (b) or the peptide of (c); Including any of.
  • the detection target is not limited to the above (a) to (e), and may be any that can suggest the existence of the LAMC2-NR6A1 splicing variant or its translation product.
  • the detection of the presence of the fusion gene is known to those skilled in the art and can be easily identified by using a next-generation sequencer or the like.
  • the read sequence of genomic DNA obtained from a sample derived from a subject is mapped to the standard sequence of the LAMC2 gene (or NR6A1 gene), and the unmapped portion is the NR6A1 gene (NR6A1 gene). If the mapped sequence is derived from the LAMC2 gene if it is the NR6A1 gene), the subject is determined to have the LAMC2-NR6A1 splicing variant.
  • Detection methods further include diseases associated with the activity of EGF receptors and their downstream signaling pathways, such as cancer, obesity, when biomarkers are detected or present in high concentrations compared to healthy individuals. Determines to be susceptible to disease, autoimmune disorder, inflammation, heart disease, especially heart disease associated with myocardial cell hypertrophy, neurodegenerative disease or diabetes, suffering from the disease, or progressing. Further steps may be included. For example, mapping the read sequence of genomic DNA from a sample from a subject suspected of having a disease associated with the activity of the EGF receptor and its downstream signaling pathway to the standard sequence of the LAMC2 gene (or NR6A1 gene).
  • the subject is determined to have a disease associated with the activity of the EGF receptor and its downstream signaling pathways. Will be done.
  • the biomarker may be a tumor marker used for diagnosing the susceptibility to cancer, the presence or absence of cancer, or the presence or absence of cancer progression.
  • the process of determining that a disease is predisposed to the activity of the EGF receptor and its downstream signaling pathways, is suffering from the disease, or is progressing is assisted by a clinical laboratory technician or medical device. Can be done.
  • Detection methods include diseases associated with the activity of EGF receptors and their downstream signaling pathways, such as cancer, obesity, autoimmune disorders, inflammation, heart disease, especially heart disease associated with myocardial cell hypertrophy, neurodegenerative disease or diabetes.
  • the detection method is optional: the step of determining that the subject has cancer, the step of determining the severity of the cancer, the risk of developing the subject's cancer (ie, the onset of the disease).
  • the process of determining the possibility) the process of determining the effectiveness of the cancer treatment regimen, the process of identifying the subject as a candidate for cancer treatment, and the process of assessing the risk of disease progression in subjects with cancer. It may be included.
  • diseases associated with the activity of EGF receptors and their downstream signaling pathways such as cancer, obesity, autoimmune disorders, inflammation, heart disease, especially heart disease associated with myocardial cell hypertrophy, neurodegeneration.
  • a method for screening a drug for treating or preventing a disease or diabetes is provided.
  • the screening method may optionally include a step of selecting as a drug a substance that inhibits the expression of the LAMC2-NR6A1 gene, the LAMC2-NR6A1 splicing variant or a translation product thereof, particularly the peptide of SEQ ID NO: 4.
  • a method is provided that comprises administering to the subject a compound or a salt thereof that inhibits the expression of the peptide of SEQ ID NO: 4.
  • Diseases associated with the activity of the EGF receptor and its downstream signaling pathways are not particularly limited, and examples thereof include cancer, obesity, autoimmune disorders, inflammation, heart disease, neurodegenerative diseases or diabetes.
  • the probe was prepared using a commercially available BAC clone on the centromere side of LAMC2 and the telomere side of NR6A1 (LAMC2: BAC clone RP11-158D24; NR6A1: BAC clone RP11-582A18, respectively).
  • the probe on the centromere side of LAMC2 was labeled with digoxigenin (red), and the probe on the telomere side of NR6A1 was labeled with biotin (green). These probes were applied to Skov3 cells, sections and probes were simultaneously metamorphosed for 5 minutes on a 70 ° C. hot plate and hybridized overnight at 37 ° C. Hybridized sections were strainency washes at 37 ° C. with 50% formamide / 2 ⁇ SSC and 1 ⁇ SSC. It was counter-stained with (4', 6-diamidino-2-phenylindole (DAPI) and mounted with an anti-fading agent.
  • DAPI 4-', 6-diamidino-2-phenylindole
  • the image was captured and the FISH data was analyzed by the LEICA CW-4000 site genetic workstation. 40x and 20x objective lenses were used for imaging. The results are shown in FIG.
  • a novel splicing variant in which the LAMC2 and NR6A1 genes were bound was identified from the ovarian cancer cultured cell line (Skov3) by the RACE-PCR method.
  • the full-length base sequence of SHORT FORM, which is one of the splicing variants of the present invention, and the amino acid sequence encoded by the full-length base sequence are represented as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • the sequence determination results of SHORT and LONG are shown in FIG.
  • SEQ ID NO: 3 CMFCNSRMDGNLA * (Note: The underlined part represents a novel peptide due to the binding of LAMC2 and NR6A1, and * represents a stop codon (taa).)
  • the amino acid sequence encoding the LAMC2-NR6A1 peptide was designated as SEQ ID NO: 4, and the nucleotide sequence encoding the same (tgcatgtttgtgcaacaggccgggatgggaacttagca) was designated as SEQ ID NO: 5.
  • SEQ ID NO: 6 tgctcccataacagcaggacctcaacgtccaagaagaataccacaccttacttgagcccatttacaagtcacctcctgaaaatccaagatgcctgtcagaagcagctactgagggaagtgaagatgtttttattttgttcattgtcattgtgtgaagactgactaaagtcttactgatcaaggagttttgttgaacatggtcagagagctttcaaagtcatttcagaaagttttcaaagtcatttcagaaagttttcaaagtcatttcagaaagttttcaacagatggtttgatggaagtagccag
  • SEQ ID NO: 7 I * (Note: The underlined part represents a novel peptide due to the binding of LAMC2 and NR6A1, and * represents a stop codon (taa).)
  • LAMC2-NR6A1 splicing variant of SHORT FORM was expressed in normal cells.
  • the human normal tissue cDNA used in this RT-PCR method was purchased from Filgen.
  • positive control cDNA prepared from Skov3 cells was used.
  • the LAMC2 sequence-derived primer was used, and for the detection of the LAMC2-NR6A1 splicing variant of SHORT FORM, only the LAMC2-NR6A1 splicing variant of SHORT FORM was detected using the LAMC2 sequence-derived primer and the NR6A1 sequence-derived primer.
  • GAPDH was used as an intrinsic control. The primers used are listed below.
  • GAPDH-sense aaggctgagaacgggaagcttgtcatcaat (SEQ ID NO: 8) GAPDH-anti sense ttcccgtctagctcagggatgaccttgccc (SEQ ID NO: 9)
  • LAMC2-WT sense gctacttcggggacccattg (SEQ ID NO: 10) LAMC2-WT anti-sense caagctggacagctgaatgc (SEQ ID NO: 11)
  • LAMC2-NR6A1 sense accagtgcaaagcaggctac (SEQ ID NO: 12) LAMC2-NR6A1 anti-sense tcagggttgctcttgaga (SEQ ID NO: 13)
  • the results are shown in FIG.
  • the LAMC2 wild type was expressed in all tissues.
  • the LAMC2-NR6A1 splicing variant of SHORT FORM was expressed only in Skov3 cells. From this, it was clarified that the LAMC2-NR6A1 splicing variant of SHORT FORM is cancer cell-specific.
  • RT-PCT was used to investigate whether the LAMC2-NR6A1 splicing variant of SHORT FORM actually exists in cancer patients.
  • CDNA was prepared from a patient with a predetermined cancer using the tissue at the time of surgery, and the LAMC2-NR6A1 splicing variant of SHORT FORM was detected by the PCR method.
  • the LAMC2-NR6A1 splicing variant of SHORT FORM was detected in 19 out of 20 samples for ovarian cancer, 13 out of 19 samples for breast cancer, and 11 out of 16 samples for colorectal cancer (Fig. 5).
  • LAMC2-NR6A1 splicing variant function of SHORT FORM Analysis of LAMC2-NR6A1 splicing variant function of SHORT FORM.
  • LAMC2-NR6A1 splicing variant of SHORT FORM by gene transfer of the LAMC2-NR6A1 splicing variant of SHORT FORM into the ovarian cancer cell line OVCAR8 that does not express the LAMC2-NR6A1 splicing variant of SHORT FORM using lentivirus.
  • a cell line that stably expresses the variant was established.
  • the LAMC2 protein was detected at about 150 kDa. It can be estimated from the amino acid sequence that the LAMC2-NR6A1 splicing variant of SHORT FORM is detected at about 80 kDa. In Ovcar8 cells that did not express the LAMC2-NR6A1 splicing variant of SHORT FORM, the (mock) endogenous LAMC2 protein was detected at about 150 kDa in the control vector-introduced cell line.
  • the band was increased by about 150 kDa, and in the cell line into which the LAMC2-NR6A1 splicing variant of SHORT FORM was introduced, the band was detected in about 80 kDa (LAMC2-NR6A1).
  • the LAMC2-NR6A1 splicing variant product of shRNA FORM was detected in (scr) about 80 kDa in the control cells.
  • Skov3 is expressed at the mRNA level in both the LAMC2 wild type and the LAMC2-NR6A1 splicing variant of SHORT FORM, but only the translation product of the LAMC2-NR6A1 splicing variant of SHORT FORM was detected at the protein level.
  • LAMC2-NR6A1 splicing variant of SHORT FORM affects intracellular signals
  • SKOV-3 cells were subjected to serum starvation under 0.5% serum culture, where the laminin ⁇ 2 single chain (laminin ⁇ 2 single chain ( Ln- ⁇ 2m), LAMC2-NR6A1 splicing variant of SHORT FORM (LAMC2 fusion protein (Ln- ⁇ 2F)) was added to 1.5 ⁇ g / mL, and then anti-ERK antibody, anti-phosphorylated ERK antibody, anti-Akt antibody, The expression and phosphorylation of EGFR, AKT, and ERK in cell lysates were examined using anti-phosphorylated Akt antibody, anti-EGFR antibody and anti-phosphorylated EGFR antibody (manufactured by cell signing).
  • Ln- ⁇ 2F induced phosphorylation of EGFR as well as positive control EGF. It was also revealed that Ln- ⁇ 2F induces phosphorylation of EGFR at a concentration lower than that of Ln- ⁇ 2m. Similarly, Ln- ⁇ 2F confirmed that AKT and ERK of EGFR downstream signals also induced phosphorylation. Therefore, Ln- ⁇ 2F contributes to activation of EGFR and its downstream signals in the same manner as Ln- ⁇ 2m. Western blots were performed using anti-ERK antibody, anti-phosphorylated ERK antibody, anti-Akt antibody, anti-phosphorylated Akt antibody, anti-EGFR antibody and anti-phosphorylated EGFR antibody (manufactured by cell signing).
  • SKOV-3 was cultured under normal serum containing 10% FCS, and cells in which the expression of Ln- ⁇ 2F expressed in SKOV-3 was knocked down (shKD1, shKD2), and mock and Ln- in shKD2 cells.
  • the expression and phosphorylation of EGFR and AKT were examined using cells that had returned ⁇ 2F.
  • FIG. 7B EGFR activity by endogenously produced Ln- ⁇ 2F was observed under serum content, but Ln- ⁇ 2F expression and EGFR phosphorylation disappeared in shKD1 and shKD2 (bottom panel) cells (top panel).
  • Akt and ERK are central to signal transduction networks that affect a wide range, and Akt activation acts as a master switch for these cellular signaling pathways, via a wide range of downstream target and interacting molecules. It is known to cause various intracellular reactions. It has been clarified that the activation of Akt and ERK is also enhanced in many cancer types.
  • Gene expression of Skov3-scr and Skov3-kd1 to investigate whether the activation of Akt and ERK controlled by the LAMC2 fusion protein (Ln- ⁇ 2F) we identified is sufficient to control downstream gene expression. The pattern was examined by the microarray method. The results are shown in FIG. Although various gene expression patterns were different, the expression of genes whose expression was known to be regulated by Akt was particularly decreased.
  • Ln- ⁇ 2F was expressed in OVCAR8 cells that did not express Ln- ⁇ 2F, and this was transplanted into the abdominal cavity of a mouse.
  • the tumorigenicity in the abdominal cavity of the mouse was evaluated, the tumor size and the tumor engraftment ability were remarkably enhanced in the Ln- ⁇ 2F expressing cells. The results are shown in FIG.
  • the effect of the present invention will be described assuming a multi-stage carcinogenesis model.
  • DNA is damaged by carcinogens, UV, etc. in the process of conversion of normal cells into cancer cells as the first stage (initiation).
  • the second step is to maintain and increase the growth of cancer cells converted by (promotional) initiation.
  • (progression) movement, infiltration, and metastatic potential increase and become malignant.
  • Ln- ⁇ 2F affects cell proliferation, cancer cell motility, and tumor engraftment, suggesting that Ln- ⁇ 2F is involved in promotion and progression. Will be done. That is, the function inhibitor (expression inhibition) of Ln- ⁇ 2F is expected to have an anticancer effect not only in the primary lesion (promotion stage) but also in the metastatic lesion (progression).
  • Ln- ⁇ 2F promotes AKT and ERK activation in vivo
  • OVCAR8 cells Mock
  • Ln- ⁇ 2F overexpressing cells were injected intraperitoneally in nude mice. After 4 weeks, the immunostaining result of the recovered tissue is shown in FIG. It was shown that the activation of AKT and ERK in cancer tissues formed in the abdominal cavity by Ln- ⁇ 2F overexpressing cells was enhanced as compared with MOCK. From the above, Ln- ⁇ 2F may contribute to tumorigenicity through proliferation and survival of cancer cells in vivo.
  • Ln- ⁇ 2F is involved in the malignant transformation of cancer and is thought to control the motility of cancer cells in particular, so it can also be used as a marker for predicting the malignancy of cancer. Furthermore, substances that inhibit the expression of Ln- ⁇ 2FF are expected to have the effect of suppressing cancer infiltration and metastasis.

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WO2024226899A1 (en) * 2023-04-28 2024-10-31 Abbott Laboratories Diagnosis of late-stage hepatocellular carcinoma

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JP2013520439A (ja) * 2010-02-19 2013-06-06 アセア バイオサイエンシズ インコーポレイテッド 抗癌剤としての複素環式化合物および使用
JP2015527562A (ja) * 2012-08-13 2015-09-17 アボットジャパン株式会社 がんの予後判定および診断方法
WO2016148215A1 (ja) * 2015-03-17 2016-09-22 国立大学法人東京大学 Ｍｉｎｔ３阻害剤
WO2018136416A1 (en) * 2017-01-17 2018-07-26 Illumina, Inc. Oncogenic splice variant determination

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JP2015527562A (ja) * 2012-08-13 2015-09-17 アボットジャパン株式会社 がんの予後判定および診断方法
WO2016148215A1 (ja) * 2015-03-17 2016-09-22 国立大学法人東京大学 Ｍｉｎｔ３阻害剤
WO2018136416A1 (en) * 2017-01-17 2018-07-26 Illumina, Inc. Oncogenic splice variant determination

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