METHOD FOR DIAGNOSIS OF METASTATIC CANCER OR TAXANE-BASED DRUG-RESISTANT CANCER BY USING LMCD1 PROTEIN AND GENE CODING THEREFOR

Abstract

The present disclosure relates to a method of diagnosing metastatic cancer or taxane-based drug-resistant cancer, including: measuring an LMCD1 expression level from a complex formed by bringing, into contact with a sample isolated from a subject, an antibody, peptide, protein, or combination thereof that specifically binds to an LMCD1 protein or a fragment thereof; or a probe, primer, nucleotide, or combination thereof that specifically binds to a nucleotide sequence encoding the LMCD1 protein; and comparing the measured LMCD1 expression level of the sample with a measured LMCD1 expression level of a control, and a kit for diagnosing metastatic cancer or taxane-based drug-resistant cancer, including a composition for diagnosing metastatic cancer or taxane-based drug-resistant cancer.

Claims

1. A method of diagnosing metastatic cancer or taxane-based drug-resistant cancer, the method comprising: measuring an LMCD1 expression level from a complex formed by bringing, into contact with a sample isolated from a subject, an antibody, peptide, protein, or combination thereof that specifically binds to an LMCD1 protein or a fragment thereof; or a probe, primer, nucleotide, or combination thereof that specifically binds to a nucleotide sequence encoding the LMCD1 protein; and comparing the measured LMCD1 expression level of the sample with a measured LMCD1 expression level of a control.

2. The method of claim 1, wherein the LMCD1 protein comprises any one sequence selected from SEQ ID NOS: 1 to 4, and the nucleotide sequence encoding the LMCD1 protein comprises any one sequence selected from SEQ ID NOS: 5 to 8.

3. The method of claim 1, further comprising, before the measuring of the LMCD1 expression level, bringing TGF- into contact with the sample isolated from a subject.

4. The method of claim 1, further comprising measuring a degree of phosphorylation of a Smad3 linker or its carboxy terminus from a complex formed by bringing, into contact with the sample isolated from a subject, an antibody, peptide, protein, or combination thereof that specifically binds to a phosphorylated Smad3 linker, its carboxy-terminus, or a fragment thereof.

5. The method of claim 4, wherein the Smad3 linker or its carboxy-terminus comprise amino acid residues 143-230 or amino acid residues 422-425 in an amino acid sequence of SEQ ID NO: 9.

6. The method of claim 4, wherein the phosphorylation comprises phosphorylation of at least one selected from the group consisting of amino acid residues 179, 204, 208, 213, 422, 423, and 425 in an amino acid sequence of the Smad3 linker or its carboxy terminus.

7. The method of claim 3, further comprising measuring a degree of phosphorylation of a Smad3 linker or its carboxy terminus from a complex formed by bringing, into contact with the sample isolated from a subject, an antibody, peptide, protein, or combination thereof that specifically binds to a phosphorylated Smad3 linker, its carboxy-terminus, or a fragment thereof.

8. The method of claim 7, wherein the Smad3 linker or its carboxy-terminus comprise amino acid residues 143-230 or amino acid residues 422-425 in an amino acid sequence of SEQ ID NO: 9.

9. The method of claim 7, wherein the phosphorylation comprises phosphorylation of at least one selected from the group consisting of amino acid residues 179, 204, 208, 213, 422, 423, and 425 in an amino acid sequence of the Smad3 linker or its carboxy terminus.

10. The method of claim 1, further comprising measuring an expression level of a cancer stemness marker from a complex formed by bringing, into contact with the sample isolated from a subject, an antibody, peptide, protein, or combination thereof that specifically binds to a cancer stemness marker protein or a fragment thereof; or a probe, primer, nucleotide, or combination thereof that specifically binds to a nucleotide sequence encoding the cancer stemness marker protein, wherein the cancer stemness marker is Oct4, Nanog, Sox2, CD44, CD24, ALDH1, CD326 (EpCAM), or a combination thereof.

11. The method of claim 1, wherein the measuring of the LMCD1 expression level is performed using at least one method selected from RT-PCR, RNase protection assay (RPA), Northern blotting, and a DNA CHIP.

12. The method of claim 1, wherein the measuring of the LMCD1 expression level is performed using at least one method selected from Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistostaining, immunoprecipitation assay, complement fixation assay, FACS, and a protein chip.

13. The method of claim 1, wherein the cancer is breast cancer.

14. The method of claim 13, wherein the cancer is metastatic breast cancer.

15. The method of claim 1, wherein the sample isolated from a subject is a cell, an organ, a cell lysate, blood, serum, plasma, lymph fluid, extracellular fluid, body fluid, urine, feces, tissue, bone marrow, saliva, sputum, cerebrospinal fluid, or a combination thereof.

16. The method of claim 1, further comprising administrating anti-cancer drug except taxane-based drug to the subject diagnosed with metastatic cancer or taxane-based drug-resistant cancer.

17. The method of claim 16, wherein the anti-cancer drug except taxane-based drug treatment is Anthracyclines, Platinum-based drugs, Vinorelbine, Capecitabine, Gemcitabine, Ixabepilone, Eribulin or combination thereof.

18. The method of claim 17, wherein the anti-cancer drug except taxane-based drug treatment is platinum-based drugs.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0053] FIGS. 1A and 1B illustrate MTT analysis results of evaluation of the degree of susceptibility to a taxane-based anticancer agent in LMCD1-overexpressing metastatic breast cancer cells, and FIG. 1C illustrates MTT analysis results of evaluation of the degree of susceptibility to a taxane-based anticancer agent according to LMCD1 overexpression in metastatic breast cancer cells.

[0054] FIG. 2A illustrates RT-PCR results showing the degree of LMCD1 expression in metastatic breast cancer cells having resistance to a taxane-based anticancer agent, FIG. 2B illustrates MTT analysis results showing a change in susceptibility to a taxane-based anticancer agent according to the presence or absence of LMCD1 expression, and FIGS. 2C and 2D illustrate immunofluorescence results showing the degree of LMCD1 expression in a subject having resistance to a taxane-based anticancer agent.

[0055] FIGS. 3A-3C illustrate phosphorylated amino acid residues according to a phosphorylation site of the Smad3 protein.

[0056] FIGS. 4A and 4B illustrate MTT analysis results of resistance to a taxane-based anticancer agent after metastatic breast cancer cells were survived from cellular apoptosis mediated by infection with an Smad3 EPSM adenovirus, FIG. 4C illustrates RT-PCR results of measuring the degree of LMCD1 mRNA expression after metastatic breast cancer cells were survived from cellular apoptosis mediated by infection with an Smad3 EPSM adenovirus, FIGS. 4D and 4E illustrate MTT analysis results of evaluation of the degree of resistance to a taxane-based anticancer agent after metastatic breast cancer cells were infected with adenovirus inducing phosphorylation at different Smad3 phosphorylation sites, and FIG. 4F illustrates MTT analysis results of evaluation of a change in susceptibility to a taxane-based anticancer agent according to LMCD1 depletion after metastatic breast cancer cells were survived from cellular apoptosis mediated by infection with an Smad3 EPSM adenovirus.

[0057] FIG. 5A illustrates results of analyzing wound healing of an LMCD1-overexpressing model in metastatic breast cancer cells, FIGS. 5B and 5C illustrate an increase in migration and invasiveness according to LMCD1 overexpression by staining of nuclei of cells having passed through Transwell and Matrigel, FIGS. 5D and 5E illustrate migration inhibition phenomena when LMCD1 was depleted in metastatic breast cancer cells, by staining nuclei of cells having passed through Transwell, FIG. 5F illustrates RT-PCR results of labeled factors when LMCD1 was overexpressed in MCF10CA1a.cl1 metastatic breast cancer cells, FIG. 5G illustrates public data analysis results according to Oncomine Compendium of Expression Array data, and FIG. 5H illustrates immunofluorescent staining results showing the degree of LMCD1 expression in metastasized lung tissues after metastatic breast cancer cells were injected into a mouse lung metastasis model.

[0058] FIGS. 6A and 6B are a set of images showing analysis results of the degree of mammosphere formation after LMCD1 was overexpressed in metastatic breast cancer cells, and FIG. 6C illustrates RT-PCR results of measuring a change in mRNA of a stemness marker after LMCD1 was overexpressed in metastatic breast cancer cells.

[0059] FIGS. 7A and 7B illustrate microarray analysis results according to TGF- treatment after metastatic breast cancer cells were infected with Smad3 wild-type and EPSM adenovirus, FIG. 7C illustrates genes exhibiting significant changes in a TGF-6 treated group after metastatic breast cancer cells were infected with Smad3 EPSM adenovirus, FIG. 7D illustrates results of measuring LMCD1 promoter activity according to TGF- treatment after metastatic breast cancer cells were infected with Smad3 EPSM adenovirus, FIG. 7E illustrates results of measuring LMCD1 mRNA expression levels according to treatment with TGF- and SB431542, which is a TGF- inhibitor, after metastatic breast cancer cells were infected with Smad3 EPSM adenovirus, and FIG. 7F illustrates results of measuring LMCD1 expression levels by TGF- treatment according to phosphorylation inhibition of each Smad3 phosphorylation site.

[0060] FIG. 8A illustrates results of identifying LMCD1 secretion in the conditioned media of metastatic breast cancer cells, FIG. 8B illustrates results of analyzing the interaction between LMCD1 and Annexin-II in metastatic breast cancer cells, FIG. 8C illustrates results of measuring LMCD1 secretion in the absence or presence of TGF- after metastatic breast cancer cells were infected with Smad3 EPSM adenovirus, FIG. 8D illustrates results of measuring the activity of MMP-2 and MMP-9 in metastatic breast cancer cells according to phosphorylation status of each Smad3 phosphorylation site, FIGS. 8E to 8G illustrate an increase in invasiveness by staining of nuclei of cells having passed through Matrigel and in resistance to a taxane-based anticancer agent in benign breast cancer cells according to the treatment of LMCD1-containing conditioned media, FIGS. 8H to 8J illustrate an increase in invasiveness by staining of nuclei of cells having passed through Matrigel and in resistance to a taxane-based anticancer agent in metastatic breast cancer cells according to the treatment of LMCD1-containing conditioned media.

[0061] FIG. 9A illustrates ELISA analysis results of measuring LMCD1 protein level in the serums of healthy volunteers and breast cancer patients, FIG. 9B illustrates ELISA analysis results of measuring LMCD1 protein level in the serums of different subtypes of breast cancer patients.

MODE OF DISCLOSURE

[0062] Hereinafter, the present disclosure will be described in further detail with reference to the following examples. However, these examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure.

Example 1: Verification of Resistance to Taxane-Based Anticancer Agent in LMCD1-Overexpressing Cells Through MTT Assay

[0063] To investigate whether LMCD1 protein-overexpressing breast cancer cells have resistance to a taxane-based anticancer agent, an MTT assay, which is used to evaluate cell viability, was performed on MCF-10CA1a.cl1, which are human MCF-10A-based breast cancer cell lines, and MDA-MB231. The MCF-10CA1a.cl1 and MDA-MB231 cell lines are characterized as malignant breast cancer cell lines with high metastasis.

[0064] In particular, human MCF-10A-based breast cancer cell lines, i.e., MCF-10CA1a.cl1 and MDA-MB231, were maintained in a DMEM culture solution containing 10% fetal bovine serum and 1% penicillin/streptomycin (WeIGENE) in a CO.sub.2 incubator at 37 C.

[0065] To overexpress the LMCD1 protein in cells, LMCD1 was cloned into retroviral vector pLPCX, and LMCD1 retrovirus was obtained using Plat-GP cells. The metastatic breast cancer cells were infected with the LMCD1 retrovirus, LMCD1 protein-overexpressing cells were selected using puromycin, and then the selected cells were incubated in a DMEM culture solution containing 10% fetal bovine serum and 1% penicillin/streptomycin (WeIGENE) in a CO.sub.2 incubator at 37 C.

[0066] The cells were seeded in 96 wells at a density of 3,000 cells per well and incubated for 24 hours. The culture solution was removed and a culture solution containing paclitaxel or docetaxel, which is a taxane-based anticancer agent, was added to each well to a final volume of 100 l/well. After incubation for 72 hours, the culture solution was removed from the culture, and 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well, followed by incubation for 2 hours. Media containing an MTT solution was carefully removed, and 200 l of dimethyl sulfoxide was added to each well, followed by incubation at room temperature for 5 minutes. Absorbance at 580 nm was measured using a 96-well microplate detector (Molecular Devices).

[0067] As a result, as illustrated in FIGS. 1A and 1B, it was confirmed that the LMCD1 protein-overexpressing MDA-MB231 cells exhibited resistance to the taxane-based anticancer agents. In addition, as illustrated in FIG. 1C, it was confirmed that resistance to paclitaxel was increased in the MCF10CA1a.cl1 cells when LMCD1 was overexpressed.

Example 2: Evaluation of LMCD1 Overexpression in Cells with Resistance to Taxane-Based Anticancer Agent

[0068] 2-1. Production of Cells with Resistance to Taxane-Based Anticancer Agent

[0069] To evaluate whether the LMCD1 protein is overexpressed in cells having resistance to a taxane-based anticancer agent, cells having resistance to a taxane-based anticancer agent were produced.

[0070] In particular, parental cells were treated with paclitaxel at a small concentration of 0.5 nM, and alive cells were repeated treated with paclitaxel three or four times while being passaged. After the repeated paclitaxel treatment processes, alive cells were treated with paclitaxel with an increment of 0.5 nM, and then passaging of alive cells was further repeated, thereby producing cells having resistance to paclitaxel that did not die even at an IC.sub.50 value of 50 nM.

[0071] 2.2. Evaluation of LMCD1 Expression Level in Cells with Resistance to Taxane-Based Anticancer Agent

[0072] An expression level of LMCD1 in taxane-based anticancer agent-resistant cells was evaluated at an mRNA level through RT-PCR, and it was evaluated whether susceptibility to the taxane-based anticancer agent was restored when the LMCD1 expression level was reduced by inhibiting LMCD1 expression.

[0073] In particular, total RNA was extracted from MCF10CA1a.cl1 cells (M4) and the paclitaxel-resistant MCF10CA1a.cl1 cells (M4 PTX) prepared according to Example 2-1 using a TRIzol reagent (Invitrogen) in accordance with the manufacturer's protocol. The total RNA was converted into cDNA using M-MLV reverse transcriptase (Promega). The cDNA was synthesized and subjected to RT-PCR using a specific primer pair by using an AccuPower PCR PreMix kit (Bioneer Co.). Gene mRNAs were normalized to GAPDH.

[0074] In addition, an MTT assay was performed to confirm whether susceptibility to a taxane-based anticancer agent is restored according to a decrease in LMCD1 expression level, in terms of cell viability. In particular, the paclitaxel-resistant MCF10CA1a.cl1 cells produced according to Example 2-1. The shRNA against LMCD1 was transfected into early-passage 293T cells, and then a lentivirus for LMCD1 depletion was produced. The paclitaxel-resistant MCF10CA1a.cl1 cells were infected with the lentivirus and then selected using puromycin, thereby producing LMCD1-depleted paclitaxel-resistant MCF10CA1a.cl1 cells (PTX-shLmcd1 #1).

[0075] As a result, as illustrated in FIG. 2A, it was confirmed that the LMCD1 mRNA expression level was increased in the paclitaxel-resistant cells. Also in terms of cell viability, as illustrated in FIG. 2B, susceptibility to a taxane-based drug was reduced in LMCD1-overexpressing or paclitaxel-resistant cells (PTX) compared to a control (LPCX), thus exhibiting comparatively high viability, while being increased again when LMCD1 was depleted in the paclitaxel-resistant cells (PTX) (PTX-shLmcd1 #1).

[0076] 2-3. Evaluation of LMCD1 Expression Level in Taxane-Based Anticancer Agent-Resistant Cells In Vivo

[0077] Animal experiments were conducted to investigate whether LMCD1 expression levels were also increased in a subject with resistance to a taxane-based anticancer agent.

[0078] In particular, female NOD/SCID mice were purchased from Orient Bio Inc. (Seongnam, S. Korea), and 210.sup.6 of paclitaxel-resistant MDA-MB231 cells were transplanted into the left fourth mammary fat pad of each female NOD/SCID mouse to prepare a xenograft model, and 10 weeks after transplantation, the mice were sacrificed for further analysis. All procedures were carried out in accordance with the guidelines provided by the CHA Hospital Animal Care and Use Committee. Xenograft paraffin blocks were prepared and cut into 4-m-thickness sections, and paraffin was removed therefrom in xylene and alcohol. After blocking endogenous peroxidase activity and a retrieving antigen, the sections were blocked with PBS containing 5% BSA at 37 C. for 1 hour. The sections were incubated with anti-LMCD1 (Abcam) overnight at 4 C. Incubation was performed at 1:500 for 1 hour using Alexa Fluor 488-conjugated goat anti-rabbit IgG as a secondary antibody. The sections were placed in a culture solution containing DAPI (Vector Laboratories) and evaluated using a confocal laser scanning microscope (LSM-510; Carl Zeiss).

[0079] As a result, as illustrated in FIGS. 2C and 2D, it was confirmed that LMCD1 expression was increased in tumors derived from paclitaxel-resistant cells, as compared to a control.

Example 3: Verification of LMCD1 Overexpression and the Effect of Different Smad3 Protein Phosphorylation in Paclitaxel-Resistant Cells

[0080] 3-1. Production of Smad3 Phosphorylation Inhibition Model

[0081] To verify whether LMCD1 overexpression and Smad3 linker phosphorylation inhibition are exhibited in paclitaxel-resistant cells, as illustrated in FIGS. 3A-3C, phosphorylation inhibition models according to Smad3 phosphorylation site were produced. GFP, Smad3 wild-type, C-terminal phosphorylation-inhibiting mutant (3SA), Smad3 linker phosphorylation-activating mutant (STD), and a Smad3 linker phosphorylation-inhibiting mutant (EPSM) adenovirus were supplied from Dr. Sushil G. Rane (NIDDK, NIH, Bethesda, Md.). To measure transduction efficiency, GFP adenovirus was used as a control. The cells were transfected with 75 to 100 virus particles/cell in a cell solution, and each transfection procedure was repeated three times.

[0082] 3-2. Evaluation of LMCD1 Expression Level and Smad3 Protein Phosphorylation in Paclitaxel-Resistant Cells

[0083] To confirm whether LMCD1 overexpression and Smad3 linker phosphorylation inhibition occur in paclitaxel-resistant cells, resistance to paclitaxel according to a Smad3 phosphorylation site was evaluated in terms of LMCD1 mRNA expression levels and cell viability.

[0084] In particular, MCF10CA1a.cl1 cells, MDA-MB231 cells, and the cells produced according to Example 3-1 were seeded in 96-wells at a density of 3,000 cells/well and incubated for 24 hours. The culture solution was removed and a culture solution containing paclitaxel or docetaxel was added to each well to a final volume of 100 l/well. After incubation for 72 hours, the culture solution was removed from the culture, and 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well, followed by incubation for 2 hours. Media containing an MTT solution was carefully removed, and 200 l of dimethyl sulfoxide was added to each well, followed by incubation at room temperature for 5 minutes. Absorbance at 580 nm was measured using a 96-well microplate detector (Molecular Devices).

[0085] In addition, total RNA was extracted from GFP virus-infected and Smad3 EPSM virus-infected cells using a TRIzol reagent (Invitrogen) in accordance with the manufacturer's protocol, and the extracted RNA was converted into cDNA using M-MLV reverse transcriptase (Promega). The cDNA was synthesized and subjected to RT-PCR using a specific primer pair by using an AccuPower PCR PreMix kit (Bioneer Co.). mRNAs of various genes were measured in triplicate and normalized to GAPDH.

[0086] As a result, as can be seen from FIGS. 4A, 4B, 4D, and 4E, cell viability was increased compared to a control when Smad3 linker phosphorylation was blocked (EPSM), and the increase in cell viability was significant only when Smad3 linker phosphorylation was blocked (especially EPSM) compared to other phosphorylation sites, from which it was confirmed that paclitaxel resistance was induced. Along with an LMCD1 increase in paclitaxel-resistant cells, as can be seen from FIG. 4C, it was confirmed that an LMCD1 mRNA expression level was increased when Smad3 linker phosphorylation was blocked. This phenomenon indicates that, when LMCD1 is depleted in Smad3 EPSM adenovirus-infected cells (EPSM-SV-shLmcd1 #1), susceptibility to paclitaxel is restored, and thus LMCD1 expression is increased through inhibition of Smad3 linker phosphorylation, thus exhibiting a paclitaxel resistance acquisition phenomenon.

[0087] Thus, it can be seen that Smad3 phosphorylation inhibition (particularly, Smad3 linker phosphorylation inhibition) occurs along with LMCD1 overexpression in taxane-based drug-resistant cells.

Example 4: Increasing Metastasis According to LMCD1 Overexpression

[0088] 4-1. Metastasis Evaluation of Breast Cancer Cells by LMCD1 Overexpression

[0089] To evaluate whether metastasis of breast cancer cells is increased when LMCD1 is overexpressed, wound healing assay and migration and invasion assays were carried out.

[0090] In particular, first, for wound healing assay, MCF10CA1a.cl1 cells were seeded in a 6-well plate and incubated for 24 hours until all cultures reached a confluency suitable for use in experiments. A linear scratch was formed with the tip of a pipette, and the cells were observed and images thereof were acquired using a BX43 Clinical (151 Inverted) microscope (Olympus).

[0091] In addition, for the migration and invasion assays, dissociated MCF10CA1a.cl1 cells (510.sup.4 cells for the migration assay and 110.sup.5 cells for the invasion assay) were plated on upper wells of Transwell and Matrigel invasion chambers (BD Biosciences). For the invasion assay, the upper chambers included DMEM containing 0.1% FBS, and lower chambers included DMEM containing 10% FBS. After incubation for 24 hours, cells migrated through a membrane were fixed with 70% ethanol, and cells having not invaded and migrated were removed with a cotton swab, followed by staining with 0.05% crystal violet. For quantification, the cells were imaged using a microscope in four random fields and quantified using Image J software. In addition, for an LMCD1-depleted cell line, lentivirus for LMCD1 depletion was produced in the same manner as in Example 2-2 to infect the cell line.

[0092] In addition, total RNA was extracted from LMCD1-overexpressing MCF10CA1a.cl1 cells using a TRIzol reagent (Invitrogen) in accordance with the manufacturer's protocol, and the extracted RNA was converted into cDNA using M-MLV reverse transcriptase (Promega). The cDNA was synthesized and subjected to RT-PCR using a specific primer pair by using an AccuPower PCR PreMix kit (Bioneer Co.). mRNAs of various genes were measured in triplicate and normalized to GAPDH.

[0093] As a result, as illustrated in FIG. 5A, cell migration was increased in the case of LMCD1 overexpression, and as illustrated in FIGS. 5B-5C, invasion was also increased, and when LMCD1 expression was inhibited, migration and invasion were reduced again (see FIGS. 5D-5E5C). In addition, as illustrated in FIG. 5F, mRNA expression of EMT marker genes were increased by LMCD1 overexpression. Since the MCF10CA1a.cl1 cells used in these examples is known as a malignant breast cancer cell line with high metastasis, it can be seen from the above results that LMCD1 is involved in metastasis of breast cancer cells such as MCF10CA1a.cl1 cells, and thus characteristics of breast cancer cells, stages of breast cancer progression, post-treatment prognosis observation, or the presence or absence of metastasis and recurrence after complete cure may be monitored by measuring an LMCD1 expression level in a sample of a subject.

[0094] 4-2. Clinical Statistical Evaluation of Presence or Absence of LMCD1 Overexpression in Metastatic Breast Cancer

[0095] To clinically evaluate whether LMCD1 overexpression occurs in metastatic breast cancer, public data analysis was carried out. In particular, LMCD1 expression levels of breast cancer and other types of cancer were analyzed using Oncomine Compendium of Expression Array data (https://www.oncomine.org). The P value for the analytical values was set to be 0.05.

[0096] As a result, it was also confirmed clinically that, when human breast cancer was metastatic (invasive), LMCD1 expression was further increased (see FIG. 5G).

[0097] 4-3. Evaluation of LMCD1 in Smad3 Linker Phosphorylation Inhibition-Induced Lung Metastasis in Breast Cancer

[0098] To evaluate whether LMCD1 protein was present in metastasis acquisition by Smad3 linker phosphorylation inhibition, immunofluorescence staining analysis was performed.

[0099] In particular, 510.sup.5 MCF10CA1a.cl1 cells infected with Smad3 EPSM adenovirus were injected into the tail veins of female NOD/SCID mice obtained from Orient Bio Inc. (Seongnam, S. Korea). 3 weeks after injection, the mice were examined by autopsy to see if there was metastasis in internal organs of each mouse. Microscopic quantification of metastasis was performed on lung-transverse sections. Thereafter, paraffin blocks of lung into which GFP and Smad3 EPSM-infected MCF10CA1a.cl1 cells injected via the tail veins of NOS/SCID mice metastasized were prepared and cut into 4 m-thick sections, and paraffin was removed therefrom in xylene and alcohol. After blocking endogenous peroxidase activity and a retrieving antigen, the sections were blocked with PBS containing 5% BSA at 37 C. for 1 hour. The sections were incubated with anti-LMCD1 (Abcam) overnight at 4 C. Incubation was performed at 1:500 for 1 hour using Alexa Fluor 488-conjugated goat anti-rabbit IgG as a secondary antibody. The sections were placed in a culture solution containing DAPI (Vector Laboratories) and evaluated using a confocal laser scanning microscope (LSM-510; Carl Zeiss).

[0100] As a result, as illustrated in FIG. 5H, it was confirmed that LMCD1 expression was significantly increased in the vicinity of lung metastasized by the Smad3 EPSM-infected MCF10ca1a.cl1 cells. It is also evident that, in the case of metastatic breast cancer, Smad3 linker phosphorylation inhibition and LMCD1 overexpression occur simultaneously.

Example 5: Analysis of Expression Sub-Factors by LMCD1 Overexpression

[0101] 5-1. Increase in Expression of Stemness Markers by LMCD1 Overexpression

[0102] Referring to FIGS. 6A-6B, through analysis of mammosphere formation enabling observation of cancer cells having cancer stem cell-like characteristics, it was observed that cancer stemness of MCF10CA1a.cl1 cells was significantly increased when LMCD1 was overexpressed. Then, it was evaluated in terms of mRNA whether cancer stemness markers can be selected as factors capable of functioning as assay sub-markers for evaluating characteristics (e.g., metastasis, tumor formability, and the like) of breast cancer cells and the presence or absence of resistance to a specific drug, as well as LMCD1 overexpression.

[0103] In particular, MCF10CA1a.cl1 cells were maintained in a DMEM culture solution containing 10% fetal bovine serum and 1% penicillin/streptomycin (WeIGENE) in a CO.sub.2 incubator at 37 C.

[0104] LMCD1 overexpression was performed in the same manner as described above.

[0105] Total RNA was extracted from the control and LMCD1-overexpressing cells using a TRIzol reagent (Invitrogen) in accordance with the manufacturer's protocol. The extracted RNA was converted into cDNA using M-MLV reverse transcriptase (Promega), and the cDNA was synthesized, followed by RT-PCR using a pair of primers specific to Oct4, Nanog, and Sox2 by using an AccuPower PCR PreMix kit (Bioneer Co.). mRNAs of various genes were measured in triplicate and normalized to GAPDH.

[0106] As a result, as illustrated in FIG. 6C, it was confirmed that the expression of Oct4, Nanog, and Sox2, which are cancer stemness markers, was significantly increased according to LMCD1 overexpression. From the above result, it is confirmed that the cancer stemness markers are able to function as assay sub-markers for evaluating characteristics of breast cancer cells and the presence or absence of resistance to a specific drug thereof, along with LMCD1 overexpression.

Example 6: Response to TGF- Treatment on Metastasis or Resistance to Taxanes and Smad3 Phosphorylation Activity

[0107] 6-1. Analysis of Change in Expression Level of Intracellular Factor According to TGF- Treatment and Smad3 Linker Phosphorylation Inhibition

[0108] Microarray assay was performed to investigate a factor exhibiting an in vivo change in expression level in accordance with TGF- treatment and Smad3 linker phosphorylation inhibition.

[0109] In particular, MCF10CA1a.cl1 cells were infected with GFP, Smad3 wild-type, or Smad3 EPSM adenovirus for 48 hours, and then treated with TGF- (5 ng/ml) and incubated for 24 hours, followed by DNA microarray assay.

[0110] As a result, as illustrated in FIGS. 7A-7C, it was confirmed that LMCD1 was overexpressed in a sample treated with TGF- in which Smad3 linker phosphorylation was inhibited.

[0111] 6-2. Analysis of Change in Expression Level of LMCD1 According to TGF- Treatment and Smad3 Linker Phosphorylation Inhibition

[0112] To confirm whether LMCD1 is induced by TGF- treatment and Smad3 linker phosphorylation inhibition in the microarray analysis of Example 6-1, LMCD1 promoter analysis and mRNA change measurement were performed.

[0113] In particular, for the LMCD1 promoter analysis, MCF10CA1a.cl1 cells were infected with GFP and Smad3. EPSM adenovirus for 2 days, and then transiently transfected with an Lmcd1 promoter using FuGENE HD (Promega). 24 hours before harvesting, the cells were stimulated with 3 ng/ml of TGF-. Luciferase activity was analyzed using a Luciferase Assay System kit (Promega) in accordance with the manufacturer's protocol. All assays were performed in triplicate, and all values were normalized to transfection efficiency as determined by -galactosidase activity.

[0114] For the measurement of LMCD1 mRNA, MDA-MB231 cells were infected with GFP and Smad3. EPSM adenovirus for 2 days, and then 2 hours before TGF- treatment, the cells were pre-treated with 10 M of SB431542 and treated with 5 ng/ml of TGF-, followed by incubation for 24 hours. Total RNA was extracted from the cells using a TRIzol reagent (Invitrogen) in accordance with the manufacturer's protocol. The extracted RNA was converted into cDNA using M-MLV reverse transcriptase (Promega). The cDNA was synthesized and subjected to RT-PCR using a specific primer pair by using an AccuPower PCR PreMix kit (Bioneer Co.). Gene mRNAs were normalized to GAPDH.

[0115] As a result, as illustrated in FIGS. 7D and 7E, it was confirmed that LMCD1 transcriptional activity and mRNA expression level were increased by TGF- treatment and Smad3 linker phosphorylation inhibition.

[0116] 6-3. Analysis of Change in LMCD1 Expression Level According to Smad3 Protein Phosphorylation Site

[0117] To confirm whether the LMCD1 overexpression phenomenon as described in Example 6-2 is induced by Smad3 linker phosphorylation inhibition, changes in LMCD1 mRNA in accordance with phosphorylation inhibition of each Smad3 phosphorylation site were measured.

[0118] In particular, GFP, Samd3 wild-type, C-terminal phosphorylation-inhibiting mutant (3SA), Smad3 linker phosphorylation-activated mutant (STD), and Smad3 linker phosphorylation-inhibiting mutant (EPSM) adenoviruses were supplied from Dr. Sushil G. Rane (NIDDK, NIH, Bethesda, Md.). To measure transduction efficiency, a GFP adenovirus was used as a control. The cells were infected with 75 to 100 virus particles/cell in a cell solution and stimulated with 5 ng/ml of TGF- for 24 hours. Total RNA was extracted from the cells using a TRIzol reagent (Invitrogen) in accordance with the manufacturer's protocol. The extracted RNA was converted into cDNA using M-MLV reverse transcriptase (Promega). The cDNA was synthesized and subjected to RT-PCR using a specific primer pair by using an AccuPower PCR PreMix kit (Bioneer Co.). Gene mRNAs were normalized to GAPDH.

[0119] As a result, as illustrated in FIG. 7F, it was confirmed that, when Smad3 linker phosphorylation was specifically inhibited, LMCD1 overexpression by TGF- treatment occurred.

[0120] Referring to Examples 1 to 5, when an LMCD1 intracellular expression level is increased, expression levels of cancer sternness- or metastasis-related factors are increased, and taxane-based drug resistance is increased. In addition, it was confirmed that the LMCD1 expression level was increased in metastatic cancer and taxane-based drug-resistant cancer, and it was actually observed that LMCD1 expression was also increased in metastatic breast cancer in clinical data analyses (see Example 4-2). Thus, it is confirmed that, when LMCD1 is overexpressed by stimulation of cells in a subject isolated from a subject by TGF-, the subject may have metastasis or taxane-based drug resistance, and the inhibition of Smad3 protein phosphorylation (particularly, the inhibition of Smad3 linker phosphorylation) is also observed in the cells in a sample isolated from a subject.

Example 7: Analysis of the Effect of LMCD1 Secretion on EMT and Drug Resistance in Breast Cancer

[0121] It has been reported that LMCD1 is mainly located in the nucleus and the cytoplasm. However, LMCD1 was identified for the first time by proteomics to be secreted to the extracellular space in the human aorta. To examine whether LMCD1 is secreted from breast cancer cells, we examined LMCD1 protein expression in the conditioned media of MCF10CA1a.cl1 cells.

[0122] In particular, for collection of the conditioned media, retrovirus-mediated LPCX (control) and LMCD1-overexpressing MCF10CA1a.cl1 cells were grown in 0.5% FBS-containing DMEM for 48 hours before harvest. The media was collected, centrifuged at 1,000 g for 10 minutes. The supernatant was carefully collected and filtered through Amicon Ultra-4 Centrigufal Filter Units (Millipore) according to the manufacturer's instruction. The biotinyated cell surface fractions were prepared by using Pierce Cell Surface Protein Isolation Kit (Thermo Scientific) according to the manufacturer's instruction. For preparation of total cell lysates, cells were lysed in a buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM EDTA, and protease inhibitor cocktail (Roche) on ice for 20 minutes. For the immunoprecipitation assay, cell extracts were incubated with the indicated primary antibodies overnight at 4 C. Antibody-bound proteins were precipitated with Dynabeads Protein G (Invitrogen) for 2 hours at 4 C., followed by washing four times with the same buffer in which cells were lysed. Samples were separated by SDS-PAGE, followed by electrotransfer to polyvinylidene difluoride membranes (Millipore). The membrane was blocked for 1 hour at room temperature (RT) and incubated overnight at 4 C. with the primary antibody. The primary antibodies used were as follows: LMCD1 (ab121788; Abcam), beta-actin (A5441; Sigma), Flag (F3165; Sigma). Horseradish peroxidase-conjugated anti-mouse/rabbit antibodies (Millipore, Temecula, Calif.) were used as secondary antibodies. The peroxidase reaction products were visualized by WESTZOL (Intron) and Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare Life Sciences). All signals were detected by Amersham Imager 600 (GE Healthcare Life Sciences).

[0123] As a result, as illustrated in FIGS. 8A-8C, LMCD1 expression was confirmed not only in the conditioned media of LMCD1-overexpressing MCF10CA1a.cl1 cells, but also in the biotinylated cell surface fractions (FIG. 8A). The interaction between LMCD1 and Annexin II, which belongs to the annexin superfamily that binds to phospholipids and other proteins on the cell surface in a calcium-dependent manner, also supports that LMCD1 may be secreted to the extracellular space in a calcium-dependent manner (FIG. 8B). In addition, Smad3 EPSM adenovirus-mediated blockade of Smad3 linker phosphorylation induced LMCD1 secretion in the conditioned medium of MCF10CA1a.cl1 breast cancer cells (FIG. 8C).

[0124] Next, to confirm whether secreted LMCD1 induced EMT (epithelial-mesenchymal transition) and paclitaxel resistance of breast cancer cells, gelatin zymography assay, which detects the activity of matrix metalloproteinase (MMP-2 or MMP-9) that have been implicated in tumor cell invasion and metastasis was carried out.

[0125] In particular, MCF10CA1a.cl1 cells were placed in DMEM containing 0.5% FBS and infected with GFP, Smad3 WT, Smad3 EPSM, and Smad3 3SA adenoviruses for 48 hours in the absence or presence of TGF-1 (5 ng/ml for 48 hours). The conditioned media were collected and concentrated as described in 1A. The 5 non-reducing sample buffer (4% SDS, 20% glycerol, 0.01% bromophenol blue, 125 mM Tris-HCl) was added to the concentrated conditioned media, and the complexes were ran on Novex 10% Zymogram Gels (ThermoFisher). The gel was washed with washing buffer (2.5% Triton X-100, 50 mM Tris-HCl, 5 mM CaCl.sub.2), 1 M ZnCl.sub.2) at room temperature for 30 minutes, followed by incubation in a buffer (1% Triton X-100, 50 mM Tris-HCl, 5 mM CaCl.sub.2), 1 M ZnCl.sub.2) for 24 hours at 37 C. Then, the gel was stained with Coomassie blue for 30 minutes to 1 hour at room temperature with agitation, followed by destaining in a solution (40% MeOH, 10% acetic acid) until bands were clearly seen.

[0126] For invasion assay, dissociated 110.sup.5 MCF10CA1a.cl1 cells were plated on upper wells of Matrigel invasion chambers (BD Biosciences). The concentrated conditioned media acquired from LPCX and LMCD1-overexpressing MCF10CA1a.cl1 cells were added to the upper chambers. The lower chambers included DMEM containing 10% FBS. After incubation for 24 hours, cells migrated through a membrane were fixed with 70% ethanol, and cells having not invaded and migrated were removed with a cotton swab, followed by staining with 0.05% crystal violet. For quantification, the cells were imaged using a microscope in four random fields and quantified using Image J software.

[0127] For MTT assay to examine resistance to paclitaxel, the cells were seeded in 96 wells at a density of 3,000 cells per well and incubated for 24 hours. The culture solution was removed, and a culture solution containing the concentrated conditioned media acquired from LPCX and LMCD1-overexpressing MCF10CA1a.cl1 cells was added to each well to a final volume of 100 l/well. After incubation for 72 hours, the culture solution was removed from the culture, and 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well, followed by incubation for 2 hours. PBS containing an MTT solution was carefully removed, and 200 l of dimethyl sulfoxide was added to each well, followed by incubation at room temperature for 5 minutes. Absorbance at 580 nm was measured using a 96-well microplate detector (Molecular Devices).

[0128] As a result, as illustrated in FIG. 8D, MMP-2 and MMP-9 activity was significantly increased in the conditioned media of EPSM-infected MCF10CA1a.cl1 cells (which Smad3 linker phosphorylation is inactive) even in the absence of TGF-. It can be seen from the above results that the secreted LMCD1, which is induced by TGF- and Smad3 linker phosphorylation inhibition, may enhance the metastatic potential of breast cancer cells.

[0129] In addition, as illustrated in FIGS. 8E-8J, it was confirmed that resistance to paclitaxel and invasiveness was significantly increased, when treatment of the conditioned media of LMCD1-overexpressing MCF10CA1a.cl1 cells to the wild-type MCF10CAT1k.cl2 (M-II), which forms benign hyperplastic lesions, and MCF10CA1a.cl1 cells. Taken together, these data indicate that secreted LMCD1 is associated with increased EMT and acquired drug resistance of breast cancer cells.

Example 8: Analysis of LMCD1 Secretion in the Serums of Breast Cancer Patients

[0130] The measured LMCD1 expression level in the serum of breast cancer patients was compared with that of healthy volunteers. In particular, the serums of healthy volunteers and breast cancer patients of different subtypes were provided by the Gangnam Severance Hospital, Yonsei University College of Medicine (Seoul, S. Korea). LMCD1 protein was detected by using LMCD1 Elisa Kit (MyBiosource) according to the manufacturer's instruction.

[0131] As a result, as illustrated in FIGS. 9A and 9B, increased LMCD1 protein level was observed in the serums of breast cancer patients compared to those of healthy volunteers (FIG. 9A). Moreover, higher level of LMCD1 was found in the serums of the patients with luminal B and triple negative breast cancer (TNBC) subtype, which are more invasive and frequently involve metastasis and drug resistance (FIG. 9B). These findings suggest that measurement of LMCD1 level in the serum may serve as a marker that predicts metastatic potential or the subtype of breast cancer.