WO2016060407A1 - Method for producing lung cancer inducible animal model lacking emp2 gene, and use thereof - Google Patents

Abstract

The present invention relates to a method for producing a lung cancer inducible animal model using an epithelial membrane protein 2 (EMP2) gene, and a use thereof. An effect of promoting metastasis of cancer in the lung, as well as a lung cancer inducing effect, was observed in the animal model produced by the method. Thus, it is expected that the animal model of the present invention can be diversely used in clinical research to screen drug candidates for treating lung cancer.

Description

Method for preparing lung cancer-induced animal model lacking EMP2 gene and use thereof

The present invention is a method for producing a lung cancer-induced animal model through the lack of EMP2 gene, More specifically, the present invention relates to a method for producing an animal model of lung cancer disease in which lung cancer is induced and metastasis to the lung, and a use thereof.

Lung cancer is the second most common cancer in both sexes, accounting for 15% of all cancers worldwide. According to a 2011 American cancer society report, more than 220,000 cases of lung cancer are diagnosed each year, of which about 70% have died, accounting for 27% of cancer mortality. .

These lung cancers are classified into primary lung cancer, which is a malignant tumor originating in the lung, and metastatic cancer of the lung that has metastasized from other organs to lung, and largely small cell lung cancer and non-small cell lung cancer depending on the tissue type. cancer). In the treatment of lung cancer, early non-metastatic lung cancer has very low sensitivity to chemotherapy and radiation, and therefore surgically in combination with ancillary chemotherapy involving cisplatin containing platinum. In the case of metastatic lung cancer beyond the initial stage, various chemotherapy and radiation treatments are used.

The symptoms of lung cancer include persistent coughing, chest pain, weight loss, nail damage, joint pain, and shortness of breath. Early detection and treatment are difficult and are likely to be detected after metastasis to the whole body, such as bone, liver, small intestine, and brain. Therefore, as an early diagnosis method for metastasis and prognosis of lung cancer, tumor size, lymph node metastasis, etc. are examined, and biopsy of lung tumor tissue or lymph node is performed by immunohistochemical analysis, chest X-ray, chest computed tomography. Although techniques such as photography and bronchoscopy are used, despite the high incidence and mortality, no effective drug has yet been developed to completely overcome lung cancer.

Therefore, there is a need for an effective diagnosis and treatment of lung cancer, and a suitable animal model is essential for the development of biological samples for the study of the mechanism and treatment of lung cancer. Therefore, studies have recently been actively conducted on animal models that cause primary lung cancer or metastatic cancer to the lungs (Korean Patent Publication No. 10-2010-0015667), but are still inadequate.

The present invention has been made to solve the above problems, the present inventors in the mice knocked out epithelial membrane protein 2 (EMP2) gene, lung cancer-inducing effect, crosses between the mouse and cancer disease mouse In the mouse prepared through, the increase of tumor cells and nodules in lung tissue was confirmed and based on this, the present invention was completed.

Accordingly, an object of the present invention is to provide a method for producing an animal model of lung cancer disease, except human, comprising knocking out the epithelial membrane protein 2 (EMP2) gene.

In addition, another object of the present invention is to prepare a animal model knocked out epithelial membrane protein 2 (EMP2) gene; And b) to provide a method for producing a lung cancer disease animal model other than human, comprising the step of crossing the animal model and cancer disease animal model.

In addition, another object of the present invention to provide a lung cancer disease animal model produced by the method.

In addition, another object of the present invention is to provide a method for screening a candidate drug for treating lung cancer using the animal model.

In addition, another object of the present invention is to provide an epithelial membrane protein 2 (Epithelial membrane protein 2; EMP2) or a gene composition for inhibiting cancer metastasis comprising the gene encoding the protein as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object of the present invention, the present invention provides a method for producing an animal model of lung cancer disease, except human, comprising knocking out the epithelial membrane protein 2 (EMP2) gene. .

In addition, the present invention comprises the steps of a) preparing an animal model in which epithelial membrane protein 2 (EMP2) gene knocked out; And b) crossing the animal model with a cancer disease animal model.

In one embodiment of the present invention, the cancer disease animal model may be a mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) mouse or K-Ras mutant mouse (KRAS ^G12D ).

In another embodiment of the present invention, the cancer may be any one selected from the group consisting of breast cancer, stomach cancer, colon cancer, and pancreatic cancer.

The present invention also provides a lung cancer disease animal model produced by the method.

In addition, the present invention comprises the steps of: a) treating a test substance to the animal model; b) measuring the growth of lung cancer cells in the animal model treated with the test substance; And c) selecting a substance that inhibits the growth of lung cancer cells as a lung cancer treatment material as compared to the non-treated group.

The present invention also provides an epithelial membrane protein 2 (Epithelial membrane protein 2; EMP2) or a gene comprising the gene encoding the protein as an active ingredient, cancer pharmaceutical composition for inhibiting metastasis.

The present invention also provides a method for inhibiting cancer metastasis, comprising administering the pharmaceutical composition to a subject.

The present invention also provides a novel use of epithelial membrane protein 2 (EMP2) or a gene encoding the protein for preparing a composition for inhibiting cancer metastasis.

The present invention comprises the steps of knocking out the epithelial membrane protein 2 (EMP2) gene or crossing the knockout animal model and a cancer disease animal model, the method of producing a lung cancer disease animal model, In the animal model produced by the method, as well as the effect of inducing lung cancer as well as promoting the cancer metastasis to the lung, it is expected to be useful in clinical studies such as screening candidate drugs for the treatment of lung cancer.

1 is a schematic diagram showing a process of knocking out the EMP2 gene.

Figure 2 shows the results of confirming the knockout of the EMP2 gene knockout for mouse embryonic stem cells into which the target vector is introduced.

Figure 3 shows the results confirmed by Western blot EMP2 expression of EMP2 knockout mice.

Figure 4 shows the histopathological results of EMP2 knockout mice (EMP2-/-) and control (EMP2 + / +) induced lung cancer by urethane treatment.

Figure 5 is a comparison of lung tumor cell distribution between EMP2 knockout mice (EMP2-/-) and control (WT) induced lung cancer by urethane treatment.

6 is a result of comparing lung tumor cell numbers between EMP2 knockout mice (EMP2 − / −) and control (WT) induced lung cancer by urethane treatment.

7 is a result of visual observation of cancer metastasis of the lungs of EMP2 knockout mice (EMP2 − / −) and control (WT) by Lewuis lung carcinoma cell injection.

8 is a comparison of lung tumor cell distribution by metastasis between EMP2 knockout mice (EMP2 − / −) and control (WT) by Lewuis lung carcinoma cell injection.

9 is a comparison of the number of lung nodules due to metastasis between EMP2 knockout mice (EMP2 − / −) and control (WT) by Lewuis lung carcinoma cell injection.

Figure 10a is a comparison of lung tumor cell distribution of lung cancer disease mice (K-Ras / EMP (-/-)) and control K-ras / EMP (+ / +) mice.

Figure 10b is the result of comparing the number of pulmonary nodules of lung cancer disease mice (K-Ras / EMP (-/-)) and control K-ras / EMP (+ / +) mice.

FIG. 11 shows the pathological histologically confirmed invasion of lung cells of tumor cells of lung cancer disease mice (K-Ras / EMP (-/-)) and control K-ras / EMP (+ / +) mice.

FIG. 12 shows the pathological histological results of breast cancer in lung cancer diseased mice (EMP2 (-/-) MMTV-pyMT) and cancer-controlled WT / MMTV-PyMT mice.

13 is a result of visually confirming the formation of nodule in lung tissue of lung cancer disease mouse (EMP2 (-/-) MMTV-pyMT) and cancer control WT / MMTV-PyMT mouse due to metastasis.

14 shows pathologically histologically confirmed invasion of lung cells of tumor cells of lung cancer disease mice (EMP2 (-/-) MMTV-pyMT) and control WT / MMTV-PyMT mice by cancer metastasis.

Figure 15 is a comparison of lung tumor cell distribution by metastasis of lung cancer disease mouse (EMP2 (-/-) MMTV-pyMT) due to cancer metastasis and control WT / MMTV-PyMT mouse.

Figure 16 is a result of comparing the number of pulmonary nodules due to metastasis of lung cancer disease mice (EMP2 (-/-) MMTV-pyMT) due to cancer metastasis and control WT / MMTV-PyMT mice.

The present inventors confirmed the excellent lung cancer inducing effect and cancer metastasis ability in the mouse knocked out EMP2 gene. In addition, in an animal model prepared by crossing the EMP2 knockout mouse and a cancer disease animal model (MMTV-PyMT mouse, K-Ras gene mutant mouse), the area of tumor cells infiltrated into lung tissue and cancer metastasis to the lung After confirming this, the present invention was completed.

Hereinafter, the present invention will be described in detail.

The present invention includes a method for producing an animal model of lung cancer disease except human, comprising knocking out the epithelial membrane protein 2 (EMP2) gene, and an EMP 2 knockout animal model and cancer prepared by the method. It provides a method for producing a lung cancer disease animal model other than human, comprising the step of crossing the disease animal model

Epipital membrane protein 2 knocked out in the present invention (Epithelial membrane protein 2; EMP 2) is a membrane protein of GAS3 / PMP22 (growth arrest specfic gene 3 / peripheral myelin protein-22) series, its expression is elevated in breast cancer, uterine cancer It is known that the expression is high in normal lung tissue, but the relationship in lung cancer disease is not known. Accordingly, the present inventors have attempted to newly establish the relationship between the EMP2 gene and the onset and metastatic ability of lung cancer.

As used herein, the term "knock-out" means partial, substantial, complete deletion, silencing, inactivation or down-regulation of a gene, and by the method the EMP2 gene Mutants knocked out also include hetero mutants (+/-), thereby providing an animal model in which lung cancer is induced.

In addition, "lung cancer", a disease caused by the method of the present invention, is a malignant tumor occurring in lung tissue, and is classified into primary lung cancer, a malignant tumor originating from the lung, and metastatic cancer of the lung that has metastasized to the lung from other organs. The animal model provides a method for producing an animal model of lung cancer disease metastasized from other organs as well as primary lung cancer.

The present invention also provides a lung cancer disease animal model produced by the method. The animal may be a mouse, a rat, a cow, a horse, a pig, a monkey, a duck, a dog, a cat, or the like, and is preferably a mouse, but is not limited thereto.

In addition, the cancer disease animal model in the present invention is preferably MMTV-PyMT (murine mammary tumor virus-polyoma middle T) mouse or K-Ras gene mutant mouse (KRAS ^G12D ), but is not limited thereto.

As used herein, the term "metastasis" refers to extracellular matrix (ECM), such as interstitial stroma and basal membrane (BM), from which metastatic cancer cells are separated from early tumor cells. Invasion enters blood vessels or lymphatic vessels, and then attaches to capillary walls of other target tissues, leaches out of capillaries through the substrate and basement membrane of cells, and proliferates in new tissues. In the target tissue means lung tissue, the initial tumor cells may be preferably breast cancer, gastric cancer, colon cancer, or pancreatic cancer cells, but is not limited thereto.

In one embodiment of the present invention, compared to the control group in the mouse knocked out EMP2 gene was confirmed excellent lung cancer-inducing effect and cancer metastasis (see Examples 1 and 2), the knockout mouse and cancer disease mouse (MMTV- PyMT mice) and K-Ras gene mutant mice (KRAS ^G12D ), respectively, cross- ^linked tumor cells in lung tissue and nodules significantly increased in animal models. It was confirmed that it can be usefully used for the treatment and research of (see Example 3).

Accordingly, the present invention provides a method for screening a drug for treating lung cancer using an animal model prepared by the above method.

The screening method comprises the steps of: a) treating a test substance to the animal model; b) measuring the growth of lung cancer cells in the animal model treated with the test substance; And c) selecting a substance that inhibits the growth of lung cancer cells as a lung cancer treatment substance, as compared with the non-treated group, wherein the therapeutic substance is a natural compound, a synthetic compound, RNA, DNA, polypeptide, enzyme, protein Is preferably any one selected from the group consisting of ligands, antibodies, antigens, bacterial or fungal metabolites and bioactive molecules, but is not limited thereto.

In another aspect, the present invention provides an epithelial membrane protein 2 (EMP2) protein or a gene encoding the protein as an active ingredient, a pharmaceutical composition for inhibiting cancer metastasis and a method for inhibiting cancer metastasis using the same do.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example  One. EMP2 On knockout  Preparation and Validation of Lung Cancer Disease Mice

In this example, in order to investigate the association between the EMP2 gene and the development or metastasis of lung cancer, a transgenic mouse using a vector knocked out of the EMP2 gene was prepared.

More specifically, as shown in Figure 1, EMP2 gene is Exon 1 (SEQ ID NO: 1), Exon 2 (SEQ ID NO: 2), Exon 3 (SEQ ID NO: 3), Exon 4 (SEQ ID NO: 4), and Exon 5 ( SEQ ID NO: 5), and a targeting vector was prepared for the preparation of knockout mice in which a third exon consisting of SEQ ID NO: 3 was deleted. Subsequently, a target vector was introduced into mouse embryonic stem cells (E14Tg2A) using electroporation, and 100 embryonic stem cell clones selectively surviving puromycin drugs were obtained, and homologous recombination occurred. Mouse embryonic stem cell clones with targeted locus were found. Thereafter, genomic DNA was cut with BamHI enzyme and Southern blot analysis was performed using Southern blot probe. As shown in FIG. 2, a signal (DNA band) was observed at a 12.4 kb position in the wild-type and a signal at 9.6 bk in the case of a targeted locus. The mouse embryonic stem cell clones (Emp2 (+/-)) identified with mutations were injected into blastocysts and transplanted into surrogate mothers to obtain chimera mice 20 days later. Among mice born when the chimeric mice were crossed with C57BL / 6 mice, Emp2 (+/−) mice were distinguished by genetic testing. And Emp2 (+/-) mouse male and female were crossed to obtain Emp2 (-/-).

 In addition, as shown in Figure 3, compared with the control group (WT (+ / +) MEF), it was confirmed that EMP2 expression is suppressed in EMP2 knockout mice (EMP2 (-/-) MEF), thereby in Example 1 The prepared EMP2 knockout mice were verified.

Example  2. EMP2 Knockout  Lung cancer induced and excellent using mouse Metastatic power  Confirm

In this example, the association between lung cancer induction and metastasis was analyzed using EMP2 knockout mice prepared in Example 1. Twenty six-week-old EMP2 (-/-) and EMP2 (+ / +) mice were treated with urethane for 10 weeks to induce lung cancer. Afterwards, the degree of lung cancer induction between EMP2 knockout mice and wild-type control group was compared through histopathologic examination of lungs, distribution of lung tumor cells, and tumor cell numbers. In addition, 2 × 10 ⁵ Lewuis lung carcinoma cells were injected into the tail vein of EMP2 knockout mice and wild-type controls to induce cancer metastasis to the lungs. After 30 days, the mice were euthanized to reduce the degree of cancer metastasis to the lungs. Lung nodule due to gross observation, distribution of lung tumor cells and metastasis was compared.

As a result, as shown in Figures 4 to 6, compared to wild-type control, EMP2 knockout mice confirmed pathological histological examination, infiltration and vigorous cell division of undifferentiated cells, significant tumor cell distribution in lung tissue And increase in cell number.

In addition, as shown in Figures 7 to 9, the EMP2 knockout mice compared with the wild-type control, as a result of visual observation, confirmed the swelling of tumor cells in lung tissue due to metastasis, significant tumor cells in lung tissue An increase in distribution and nodules was confirmed.

The results indicate that by knocking out EMP2, not only can promote lung cancer induction, but also can promote cancer metastasis to the lung.

Example  3. EMP2 Knockout  Preparation and Validation of Lung Cancer Mice Using Mice

3-1. K- Ras  Confirmation of Lung Cancer Promoting Effect by Crossing with Gene Mutant Mice

In this example, a lung cancer disease model was prepared by crossing ^EMP2 knockout mice and mice with a K-Ras gene mutation known as a tumor gene (KRAS ^G12D ).

In addition, in the prepared mouse, in order to confirm the induction of lung cancer, lung pathology of the lung cancer disease mouse (K-Ras / EMP (-/-)) and the control K-Ras / EMP (+ / +) mice Histological examination was performed and the distribution of lung tumor cells and the number of nodules were compared. Specifically, animal breeding was performed in a facility set at a temperature of 22 ° C., a relative humidity of 50%, an illumination time of 12 hours (lighted at 8 am-turned off at 8 pm) and illuminance 200-300 lux. After the period of purification, each of them was placed in a polycarbonate breeding box and fed freely with tap water equipped with a solid feed and water purifier for experimental animals. Five mice were set as one experimental group, and all experimental animals were treated according to the 'Guide for the Care and Use of Laboratory Animal' (1996, USA) of the Institute of Laboratory Animal Resources. After 12-week-old experimental animals were sacrificed with CO ₂ gas, lungs were extracted and fixed in 10% neutral buffered formalin (NBF) fixative for 24 hours. Thereafter, the number of cancerous tissues on the surface of the lung was measured and embedded in paraffin after alcohol washing and dehydration according to a conventional method. Embedded lung tissue was prepared by using a thin sectioning machine to prepare 4 μm-thick serial sections, hematoxylin-eosin (H & E) staining, and then encapsulated in permount to make a permanent specimen, followed by histopathological analysis.

As a result, as shown in Figure 10, compared with the K-Ras / EMP (+ / +) mice, the area of tumor cells infiltrated into lung tissue in lung cancer disease mice (K-Ras / EMP (-/-)) (See Fig. 10a) and nodule (nodule) was significantly increased (see Fig. 10b), and as shown in Figure 11, lung cancer disease in the lung tissue increased lung cancer in mice was confirmed through a microscope.

Given that mutations in the K-Ras gene are closely associated with gastric cancer, colorectal cancer, and pancreatic cancer, the results were obtained by crossing EMP2 knockout mice and K-ras gene mutant mice, thereby preventing from gastric cancer, colorectal cancer, or pancreatic cancer. Animal models of metastatic lung cancer disease can also be produced effectively.

3-2. MMTV - PyMT  Confirmation of promoting cancer metastasis through mating with mouse

In this embodiment, MMTV-PyMT (Murine mammary tumor virus-polyoma middle T) mouse is a breast cancer-induced animal model, to develop an animal model that promotes metastasis from breast cancer to lung, based on the experimental results of Example 2 above. , EMP2 knockout mice and MMTV-PyMT mice were crossed to prepare animal models of lung cancer disease caused by cancer metastasis.

As shown in Table 1, first, Emp2 (+/-) MMTV-PyMT mice were prepared, followed by Emp2 (-/-); MMTV-PyMT mouse females were prepared. To prepare Emp2 (+/-) MMTV-PyMT mice, genetic tests were performed on pups obtained by crossing MMTV-PyMT mice with Emp2 (-/-) mice, of which Emp2 (+/-); MMTV Mice with PyMT genotype were selected. Subsequently, Emp2 (+/-) mice and Emp2 (+/-); MMTV-PyMT mice obtained in step 1 were crossed to obtain pups, and among them, Emp2 (+ / +); MMTV-PyMT , Emp2 (-/-); MMTV-PyMT mice were distinguished and each was used as a control and experimental group.

In addition, in the prepared mouse, in order to confirm the metastatic capacity from breast cancer to lung, lung cancer diseased mouse (EMP (-/-) MMTV-PyMT) by the cancer metastasis and control EMP (+ / +) MMTV-PyMT mouse We performed gross observation and histopathologic examination of the lungs and compared the distribution of lung tumor cells and the number of nodules due to metastasis.

As shown in FIG. 12, the development of breast cancer in lung cancer diseased mice (EMP (-/-) MMTV-PyMT) and control EMP (+ / +) MMTV-PyMT mice was confirmed by pathological histological examination. EMP2 (+ / +) / MMTV-PyMT It was confirmed that the development of breast cancer was significant in the lung cancer disease mouse (EMP (-/-) MMTV-PyMT) while the incidence of cancer was minimal at 6 weeks of age. As shown in Figure 14, compared with the control EMP (+ / +) MMTV-PyMT mice, it was confirmed that nodules in lung tissue due to breast cancer metastasis significantly increased in lung cancer disease mice, The increase in the area of infiltrated tumor cells was confirmed by electron microscopy.

In addition, as shown in Fig. 15 and 16, it was confirmed by numerical comparison that the number of tumor cells and nodules in lung tissue significantly increased in lung cancer diseased mice.

The above results indicate that by crossing the EMP2 knockout mouse and MMTV-PyMT mouse, which is an animal model of breast cancer, an animal model of lung cancer disease metastasized from breast cancer can be effectively produced.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (9)

1. A method for producing an animal model of lung cancer disease, except human, comprising knocking out the epithelial membrane protein 2 (EMP2) gene.
2. A method for preparing an animal model of lung cancer disease except human, comprising the following steps:

   a) preparing an animal model knocked out of epithelial membrane protein 2 (EMP2) gene; And

   b) crossing said animal model with a cancer disease animal model.
3. The method of claim 2,

   The cancer disease animal model is characterized in that the MMTV-PyMT (Murine mammary tumor virus-polyoma middle T) mouse or K-Ras gene mutant mouse (KRAS ^G12D ).
4. The method of claim 2,

   The cancer is characterized in that any one selected from the group consisting of breast cancer, stomach cancer, colon cancer, and pancreatic cancer.
5. A lung cancer disease animal model prepared by the method of claim 1.
6. A method for screening a lung cancer therapeutic substance, comprising the following steps:

   a) treating the test substance to the animal model of claim 5;

   b) measuring the growth of lung cancer cells in the animal model treated with the test substance; And

   c) selecting a substance that inhibits the growth of lung cancer cells as a lung cancer treatment material as compared to the non-treated group.
7. Epithelial membrane protein 2 (Epithelial membrane protein 2; EMP2) protein or a gene comprising the gene encoding the protein as an active ingredient, inhibiting cancer metastasis pharmaceutical composition.
8. A method of inhibiting cancer metastasis, comprising administering the pharmaceutical composition of claim 7 to a subject.
9. Use of epithelial membrane protein 2 (EMP2) or the gene encoding the protein to inhibit cancer metastasis.

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