JP2020128349A - Cancer metastasis-inhibiting agents and methods for selecting candidate substances of cancer metastasis-inhibiting agents - Google Patents

Abstract

To provide cancer metastasis-inhibiting agents and methods for selecting candidate substances of cancer metastasis-inhibiting agents on the basis of a novel mechanism.SOLUTION: Provided is a cancer metastasis-inhibiting agent comprising an inhibitor of an enzyme that phosphorylates the 322nd serine from the N-terminal of Tron-2, as the effective ingredient. The inhibiting agent is preferably an inhibitor of protein kinase Cα and protein kinase Cδ. Also provided is a method for selecting candidate substances having a cancer metastasis-inhibiting agent for selecting substances having cancer metastasis-inhibiting action from the test samples by using the activity to inhibit the phosphorylation of the 322nd serine from the N-terminal of Trop-2 as an index.SELECTED DRAWING: Figure 17

Description

The present invention relates to a cancer metastasis inhibitor and a method for selecting a candidate substance for a cancer metastasis inhibitor.

A major challenge in cancer treatment is the development of technology to prevent cancer metastasis. That is, cancer metastasis is a major factor that threatens the life of cancer patients, and how to prevent or suppress cancer metastasis is an important issue. Regarding the cancer metastasis suppressor, for example, Patent Document 1 describes a cancer metastasis suppressor containing N,N'-propylenedinicotinamide or a pharmaceutically acceptable salt thereof as an active ingredient. Patent Document 2 describes an antitumor agent that suppresses the function of STK24 protein, and describes its application to a tumor metastasis inhibitor. Patent Document 3 describes a drug for suppressing or preventing metastasis of a malignant tumor, which contains a non-peptide angiotensin type 2 receptor agonist as an active ingredient.

By the way, most of the cancer cells affected by humans are derived from epithelial cells. Cells in normal epithelial tissues are connected by three characteristic types, namely, tight junctions, adherens junctions, and desmosome-like adhesive structures. After carcinogenesis, this intercellular bond must be disrupted in order for the cancer cells to form a tumor mass or dissociate for metastasis. However, this location in epithelial tissue and the cascade of time-dependent collapse processes are not well defined.

JP, 2017-218380, A JP, 2010-6730, A International Publication No. 2015/152393

It is hard to say that the development of cancer metastasis suppressors is sufficiently advanced compared to anticancer drugs. Therefore, it is an object of the present invention to provide a cancer metastasis inhibitor based on a new mechanism and a method for selecting a candidate substance for a cancer metastasis inhibitor.

The present inventor has focused on Trop-2 (trophoblast cell surface antigen 2) as a membrane protein that may be involved in the early stage of the above-described cascade of cell-cell bond disruption processes. Then, it was found that phosphorylation of a specific amino acid among the amino acids constituting Trop-2 significantly affects the adhesiveness and migration ability of cancer cells. Then, they found that by inhibiting phosphorylation of the specific amino acid, metastasis of cancer cells can be suppressed.

One aspect of the present invention is a cancer metastasis inhibitor, which comprises an inhibitor of an enzyme that phosphorylates the serine at the 322nd position from the N-terminal of Trop-2 as an active ingredient.

Preferably, the inhibitor is an inhibitor of protein kinase Cα and protein kinase Cδ.

Another aspect of the present invention is characterized by selecting a substance having a cancer metastasis-inhibiting action from a test substance by using as an index the action of inhibiting phosphorylation of serine at the 322nd position from the N-terminus of Trop-2. This is a method for selecting a candidate substance for a cancer metastasis inhibitor.

Preferably, in the selection method, the action of inhibiting phosphorylation by protein kinase Cα and protein kinase Cδ is used as an index.

ADVANTAGE OF THE INVENTION According to this invention, the cancer metastasis inhibitor based on a new mechanism and the selection method of the cancer metastasis inhibitor candidate substance can be provided.

It is a graph showing the result of having used HCT116/M and HCT116/WT for flow cytometry. It is a photograph showing the result of subjecting cell extracts of HCT116/M and HCT116/WT to immunoblotting. It is a photograph showing the result of subjecting an N-glycanase-treated product of a cell extract of HCT116/WT to immunoblotting. It is a microscope picture of HCT116/M and HCT116/WT. It is a microscope picture showing the result of the migration assay of HCT116/M and HCT116/WT. It is a graph which digitized the result of FIG. It is a photograph showing the result of subjecting an N-glycanase-treated product of a cell extract of HCT116/WT to Phos-tag SDS-PAGE followed by immunoblotting. 8 is a photograph showing the results of performing immunoblotting similar to that of FIG. 7 on HCT116/WT, HCT116/S303A, and HCT116/S322A. It is the graph which digitized the result of FIG. It is a photograph showing the result of subjecting PMA-treated HCT116/WT cell extract to immunoblotting. It is a microscope picture of HCT116/S303A, HCT116/S322A, HCT116/S322E. It is a microscope picture showing the result of the migration assay of HCT116/S303A, HCT116/S322A, and HCT116/S322E. It is a graph which digitized the result of FIG. It is a microscope picture showing the result of the migration assay of PANC-1/M, PANC-1/WT, PANC-1/S322A, and PANC-1/S322E. It is the graph which digitized the result of FIG. It is a photograph showing a result of subjecting an extract of a lung of a mouse transplanted with each cancer cell subcutaneously to immunoblotting. 3 is a photograph showing the appearance of a mouse with lung metastasis and a mouse without lung metastasis. It is a graph showing the result of DNA microarray analysis. It is a photograph showing the result of immunoblotting which investigated the effect of various inhibitors. It is a graph which digitized the result of FIG. It is a photograph showing the result of subjecting siRNA-treated HCT116/WT cell extract to immunoblotting. 22 is a graph in which the result of FIG. 21 is digitized. It is a microscope picture showing the result of the migration assay of HCT116/WT which knocked down PKCα and PKCδ. It is a graph which digitized the result of FIG. 3 is a micrograph showing the results of migration assay of HCT116/WT treated with PKC inhibitor. It is a graph which digitized the result of FIG.

First, Trap-2 will be described.
Trop-2 is a membrane glycoprotein with a molecular weight of 36 kDa (which can be 50 to 75 kDa by the addition of sugar chains) that is expressed in many epithelial cancer cells. Trap-2 belongs to the Tumor-associated calcium signal transducer (TACSTD) family. It is known that the expression of Trop-2 increases with malignant transformation of tumor, and it has been reported to be involved in the malignant transformation of tumor. On the other hand, it has been reported that loss of the expression of Trop-2 promotes carcinogenesis and epithelial-mesenchymal transition and has a cancer suppressive effect. For example, it is known that the causative gene of Gelatinous drop-like corneal dystrophy (GDLD), which is an abnormal disease of epithelial tissue of the cornea, is Trop-2. In this disease, the expression level of Claudin-1 and Claudin-7, which are the membrane proteins of tight junction, was decreased and the distribution was changed, and Trop-2 interacts with these membrane proteins. It is said to be involved in maintaining their stability. Since the function of tight junction regulates the polarity of epithelial cells, that is, the distribution of membrane proteins in epithelial cells, the abnormality of this molecule is also expected to be the origin of epithelial tissue collapse. In fact, decreased expression of claudin-7 has been reported in many epithelial cancer cells. Therefore, it is considered that Trap-2 is involved in maintaining the structure and function of tight junctions through the interaction with Claudin-1 and 7.
The amino acid sequence of human Trop-2 is shown in SEQ ID NO:1.

The cancer metastasis inhibitor of the present invention contains an inhibitor of an enzyme that phosphorylates serine at the 322nd position from the N-terminal of Trop-2 (hereinafter sometimes abbreviated as "Ser322" etc.) as an active ingredient. is there. That is, the present invention shows that when Ser322 of Trop-2 in a cancer cell is phosphorylated, the adhesion of the cancer cell is decreased and the migration ability is improved, while when the phosphorylation of Ser322 is blocked, the adhesion of the cancer cell is decreased. It is based on the new finding that

The inhibitor is not particularly limited as long as it can inhibit phosphorylation of Ser322 of Trap-2. In a preferred embodiment, the inhibitor is an inhibitor of protein kinase Cα (PKCα) and protein kinase Cδ (PKCδ). That is, as shown in Examples described later, both protein kinase Cα and protein kinase Cδ have an action of phosphorylating Ser322 of Trap-2. Specific examples of the inhibitor include Go6983 (CAS number: 133053-19-7), bisindolylmaleimide 1 (Bisindolylmaleimide 1, BIM-1) (CAS number: 133052-90-1), and these compounds. Examples include derivatives. Further included are pharmaceutically acceptable salts thereof.

The cancer targeted by the cancer metastasis inhibitor of the present invention is not particularly limited, and may be a primary cancer, metastatic cancer, or the like. The type of cancer cell is also not particularly limited, and it is colon cancer cell, pancreatic cancer cell, liver cancer cell, lung cancer cell, gastric cancer cell, esophageal cancer cell, kidney cancer cell, prostate cancer cell, breast cancer cell, ovarian cancer cell, skin cancer. Cancers derived from all organs/sites such as cells can be targeted.

The administration method of the cancer metastasis inhibitor of the present invention, for example, the administration route, the dose, and the administration time can be appropriately selected according to the type of active ingredient, the age, body weight, condition of the patient and the like. The administration route may be either an oral route or a parenteral route. Parenteral routes include intravenous, subcutaneous, intramuscular, intraarterial, rectal, buccal and the like.
The dosage form of the cancer metastasis inhibitor of the present invention can be appropriately selected depending on the administration method and the like.

In addition, the above-mentioned inhibitors which are active ingredients, for example, inhibitors of PKCα and PKCδ can be used in the form of a conjugate with an anti-Trop-2 antibody. According to this embodiment, a more effective cancer metastasis suppressing effect can be expected.

The present invention provides a cancer metastasis inhibitor candidate substance for selecting a substance having a cancer metastasis inhibitory action from a test substance by using as an index the action of inhibiting phosphorylation of serine (Ser322) at the 322nd position from the N-terminal of Trop-2. Including selection methods. For example, the action of inhibiting phosphorylation by protein kinase Cα and protein kinase Cδ can be used as an index. For example, a cancer metastasis inhibitor candidate substance can be selected by searching for a compound having an action of inhibiting the phosphorylation activity of protein kinase Cα and protein kinase Cδ in vitro.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.

1. Experimental method 1-(1) Construction of expression plasmid Total RNA was prepared from human ovarian cancer cell OVCAR3, and reference 1 (Akita, K. et al., Eur. J. Cell Biol. 92, 257-263 (2013)). CDNA synthesis was performed by the method described. PCR was performed using the synthesized cDNA and primers (SEQ ID NOs: 2 and 3) to amplify the human Trop-2 gene. The PCR product was cloned into the pCR2.1-TOPO TA vector (Invitrogen). After confirming the sequence, this vector was treated with EcoO65I and the ends were blunted with a Blunting Kination Ligation Kit (Takara Bio). The blunt-ended vector was treated with BamHI and subcloned into the blunted KpnI site and overhanging BamHI site of the pFLAG-CMV-3 expression vector. As a result, the wild-type Trop-2 (WT-Trop-2) gene was subcloned.

Site-directed mutagenesis was performed using the QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene) to obtain three mutant Trop-2 genes. Specifically, a mutation that replaces Ser303 with Ala was introduced using the primers of SEQ ID NOS: 4 and 5. A mutation that replaces Ser322 with Ala was introduced using the primers of SEQ ID NOs: 6 and 7. A mutation that replaces Ser322 with Glu was introduced using the primers of SEQ ID NOs: 8 and 9. Glu can be regarded as equivalent to phosphorylated Ser.

1-(2) Transfection of plasmid and cell culture Human colon cancer cell HCT116 and human pancreatic cancer cell PANC-1 were obtained from ATCC. Using FuGENE HD Transfection Reagent (Promega), cells were transfected with the above expression vector or an empty vector, and stable transformants were selected in the presence of 600 μg/mL G418.

<Prepared transformant>
・Mock cells (HCT116/M, PANC-1/M)
-FLAG-tagged wild-type Trop-2 expressing cells (HCT116/WT, PANC-1/WT)
FLAG-tagged mutant Trop-2 expressing cells (HCT116/S303A) in which Ser303 was replaced with Ala
FLAG-tagged mutant Trop-2 expressing cells (HCT116/S322A, PANC-1/S322A) in which Ser322 was replaced with Ala.
FLAG-tagged mutant Trop-2 expressing cells (HCT116/S322E, PANC-1/S322E) in which Ser322 was replaced with Glu.

These cells were cultured with DMEM containing 10% FBS (heat inactivated), 4 mM glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin.

1-(3) Preparation of Cell Extract and Tissue Extract Cells grown to subconfluence were treated with 25 mM Tris-HCl, pH 7.5, 150 containing solubilization buffer (Protease and phosphatase inhibitor cocktails (Nacalai Tesque)). It was dissolved with mM NaCl, 5 mM EDTA, 1% Triton X-100 (TX-100)). After solubilization, centrifugation (13,000 rpm) was performed at 4° C. for 10 minutes, and the supernatant was collected and used as a cell extract.

The liver and lungs excised from the mouse were cut into small pieces and washed with PBS. The tissue pellet was solubilized in the same manner as above, and then centrifuged at 4° C. for 30 minutes (13,000 rpm) to recover the supernatant, which was used as a tissue extract.

1-(4) Treatment with PKC/PKD inhibitor, treatment with PMA or 4αPMA The cells were treated with the following PKC inhibitor, PKD inhibitor, or DMSO for 1 hour.
BIM-1 (pan-PKC (α/βI/βII/γ/δ/ε) inhibitor, 1 μM, Cell Signaling Technology Co., Ltd.)
-Go6983 (pan-PKC (α/βI/βII/γ/δ/ζ) inhibitor, 1 μM, Sigma-Aldrich)
・Go6976 (PKCα/βI inhibitor, 1 μM, Cayman Chemical Co., Ltd.)
・Hispidin (PKCβI/βII inhibitor, 10 μM, Merck Millipore)
・HBDDE (PKCα/γ inhibitor, 100 μM, Enzo Life Science)
・CID755673 (PKD inhibitor, 10 μM, Merck Millipore)

The cells were treated with 100 ng/mL PMA or 4α-PMA for a predetermined time.

1-(5) Immunoprecipitation
Anti-FLAG FLAG-tagged Trop-2 was immunoprecipitated from the cell extract using M2 Magnetic Beads (Sigma-Aldrich).
Claudin-7 was immunoprecipitated from the cell extract using mouse anti-claudin-7 antibody (Invitrogen) and Protein G Sepharose 4 Fast Flow (GE Healthcare).
Trop-2 was immunoprecipitated from the tissue extract using goat anti-Trop-2 antibody (R&D Systems) and Protein G Sepharose 4 Fast Flow.

1-(6) Immunoblotting The cell extract or tissue extract prepared above or an immunoprecipitate thereof was subjected to SDS-PAGE for immunoblotting.
After incubating with any of the following primary antibodies and horseradish peroxidase (HRP)-labeled secondary antibody, the bands were detected by chemiluminescence, and the bands were quantified by Image J Software.

<Primary antibody>
-HRP-labeled mouse anti-FLAG M2 antibody (Sigma-Aldrich)
・Mouse anti-claudin-7 antibody (Cell Signaling Technology)
・Mouse anti-β-actin antibody (Sigma-Aldrich)
・Rabbit anti-PKCα antibody (Abcam)
・Rabbit anti-PKCδ antibody (Abcam)
・Rabbit anti-phosphorylated Trop-2 antibody (self-made product)

1-(7) Preparation of anti-phosphorylated Trop-2 antibody, preparation of anti-non-phosphorylated Trop-2 antibody, specificity evaluation Ser322 phosphorus of keyhole limpet hemocyanin (KLH)-binding peptide (Trop-2 intracellular domain) The oxide and the non-phosphorus oxide were obtained from Hokkaido System Science Co. These peptides were emulsified with Freund's complete (first) and incomplete (second) adjuvant (Difco Laboratories) and subcutaneously injected into 12-week-old female New Zealand white rabbits 5 times. did. Blood was collected after the fifth immunization, and an IgG fraction was prepared from the serum using a protein A column.
The IgG fraction containing antibodies against phosphorylated Trop-2 was subjected to KLH-binding non-phosphorylated peptide CNBr activated Sepharose 4 Fast Flow. The flow-through fraction was collected to obtain anti-phosphorylated Trop-2 antibody.
The IgG fraction containing the antibody against non-phosphorylated Trop-2 was subjected to KLH-conjugated CNBr-activated Sepharose 4 Fast Flow. The flow-through fraction was collected to obtain anti-non-phosphorylated Trop-2 antibody.

The following plate assay was performed to examine the specificity of each antibody.
Phosphorylated or non-phosphorylated Trop-2 intracellular peptide was immobilized on a 96-well MaxiSorp plate (Nunc) overnight at 4°C. Serially diluted anti-phosphorylated Trop-2 antibody or anti-non-phosphorylated Trop-2 antibody (0-1 μg/mL) was placed in each well and incubated. 1-Step Ultra TMB-ELISA Substrate (Thermo Fisher Scientific) was added and the absorbance at 450 nm was measured to detect the immune complex. In each plate assay, experiments were performed in quadruplicate.

1-(8) Flow cytometry Cells were incubated with fluorescein isothiocyanate (FITC)-labeled mouse anti-FLAG M2 antibody (Sigma-Aldrich) at 4°C for 2 hours. After washing with 1% BSA/PBS, the expression of Trap-2 was detected using FACS Calibur (BD Biosciences).

1-(9) DNA microarray analysis DNA microarray analysis was basically performed by the method of Reference 2 (Mori, Y. et al., J. Biol. Chem. 289, 35193-35204 (2014)).
Total RNA was isolated using ISOGEN (Nippon Gene). Cyanine-3-labeled cRNA was prepared from the total RNA using a Low Input Quick Amp Labeling Kit (Agilent Technologies). After purification and fragmentation, the labeled cRNA was hybridized with SurePrint G3 Human Gene Expression 8×60K v3 Microarray (Agilent Technologies). After washing, array slides were scanned with an Agilent SureScan Microarray Scanner (G2600D).

1-(10) Migration Assay, MTT Assay Migration assay and MTT assay were basically performed by the method described in the above-mentioned Document 1.
The cells (4×10 ⁴ cells) were suspended in a serum-free medium containing 0.2% BSA, and seeded in the upper chamber of a transwell (24-well culture plate, pore size 8.0 μm, Corning) coated with fibronectin. Some PKC inhibitors or DMSO, or PMA or 4α-PMA were added as needed. The bottom plate was filled with serum-free medium and the cells were incubated for 20 hours. The chamber was fixed and stained with hematoxylin, after which non-migrating cells were removed. The migration of cells was quantified by counting at a magnification of 100 times in 5 randomly selected fields.

An MTT assay was performed to examine the effect of cell proliferation on the cell migration assay.
Cells (4×10 ⁴ cells) were cultured in a 96-well plate for 18 hours in the same medium as the migration assay. MTT reagent (Nacalai Tesque) was added and incubated for 2 hours, and the absorbance at 570 nm was measured with a reference of 630 nm. The MTT assay was run in at least duplicate.

1-(11)Phos-tag SDS-PAGE analysis FLAG-tag-bound Trop-2 immunoprecipitates were treated with glycopeptidase F (Takara Bio Inc.). Desalting and concentration were performed using Nanosep Centrifugal Device with a 10 kDa cutoff (Pole Life Sciences). The obtained sample was subjected to Zn ²⁺ -Phos-tag SDS-PAGE and immunoblotting was performed. Phosphorylated and non-phosphorylated Trop-2 were detected using HRP-labeled mouse anti-FLAG M2 antibody.

1-(12) siRNA treatment
By reverse transfection using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), cells were transfected with Silencer Select siRNA of PKCα (5 nM) and/or PKCδ (10 nM), or Silencer Select Negative Control #1 siRNA, Incubated for 3 days. The following were used as target-specific siRNAs.
-PKCα siRNA #1 (SEQ ID NOS: 10 and 11), #2 (SEQ ID NOS: 12 and 13)
-PKC delta siRNA #1 (sequence number 14, 15), #2 (sequence number 16, 17)

1-(13) Metastasis 6-week-old BALB/c nu/nu male mice were intraperitoneally or subcutaneously injected with cells (5×10 ⁶ cells). Lungs and livers were removed 4 weeks after intraperitoneal injection and 8 weeks after subcutaneous injection. These were used for biochemical detection of FLAG-bound Trop-2 and histological observation of tumor tissues by H&E staining.
To examine the effect of Go6983 on metastasis, nude mice inoculated intraperitoneally with HCT116/WT cells were given daily intraperitoneal injection of Go6983 (0.25 mg/mouse/day).

1-(14) Statistical analysis Two groups were compared by the two-tailed Student's t-test. Differences between more than two groups were assessed by ANOVA, Tukey-Kramer, or Dunnett's test. In all the analyzes, there was a significant difference at p<0.05.

2. Results 2-(1) Study on malignant transformation of tumor associated with expression of Trop-2 The expression of Trop-2 in HCT116/WT (FLAG-tagged wild-type Trop-2 expressing cells) was confirmed by flow cytometry (Fig. 1). In FIG. 1, “M” indicates mock cells (HCT116/M), and “WT” indicates HCT116/WT (2 clones). It was confirmed that HCT116/WT expressed Trop-2.

Immunoprecipitates of HCT116/M and HCT116/WT extracts were subjected to SDS-PAGE and immunoblotting was performed (FIG. 2). As a result, a broad band of 50-75 kDa was detected from HCT116/WT. This suggested that Trop-2 may have been modified with a sugar chain.

The immunoprecipitate of the extract of HCT116/WT was treated with N-glycanase, and the same SDS-PAGE and immunoblotting were performed (FIG. 3). As a result, in the N-glycanase-treated sample, a band was detected near 40 kDa. From this, it was shown that Trop-2 was subjected to sugar chain modification.

When the population of each cell was observed under a microscope, HCT116/WT appeared to have looser cell-cell adhesion as compared with mock cells (FIG. 4).

A migration assay using a Transwell was performed. FIG. 5 is a photograph showing the results of microscopic observation, and FIG. 6 is a graph showing the results in numerical form. That is, HCT116/WT had high mobility.
As a result of the MTT assay, there was no difference in the proliferation ability between the mock cells and HCT116/WT.

2-(2) Identification of phosphorylation site of Trop-2 In order to distinguish phosphorylated Trop-2 and non-phosphorylated Trop-2, immunoprecipitates of HCT116/WT lysate treated with N-glycanase were subjected to Phos-tag SDS. -Subjected to PAGE and immunoblotting was performed (Fig. 7). As a result, two bands were detected, and it was found that a part of Trop-2 was phosphorylated. No band was detected with the anti-phosphotyrosine antibody.

Similar immunoblotting was performed using HCT116/WT (2 clones), HCT116/S303A (2 clones), and HCT116/S322A (2 clones). FIG. 8 is a photograph showing the result of immunoblotting, and FIG. 9 is a graph quantifying the result. As shown in FIG. 8, two bands were detected in HCT116/S303A as in HCT116/WT. On the other hand, in HCT116/S322A, only the band of non-phosphorylated Trop-2 was detected. That is, phosphorylation of Trop-2 was observed only for the 322nd Ser (Ser322).

Similar immunoblotting was performed using the immunoprecipitate of HCT116/WT lysate when PKC was activated by PMA treatment (FIG. 10). As a result, the phosphorylated Trop-2 band was only darkened, and no new band appeared. This indicated that only the 322nd Ser (Ser322) was phosphorylated.

2-(3) Different Morphology and Mobility of Wild-type and Mutant-type Trop-2-expressing Cells HCT116/S303A, HCT116/S322A, and HCT116/S322E were observed under a microscope (FIG. 11). As a result, HCT116/S303A appeared to have loose adhesion between cells, as in HCT116/WT shown in FIG. On the other hand, in HCT116/S322A, the cells were strongly bound to each other. HCT116/S322E appeared to have weaker cell-cell adhesion than HCT116/WT. As described above, Glu(E) can be regarded as equivalent to phosphorylated Ser(S).

For HCT116/S303A, HCT116/S322A, and HCT116/S322E, migration assay using a Transwell was performed. FIG. 12 is a photograph showing the results of microscopic observation, and FIG. 13 is a graph showing the results in numerical form. That is, the mobility of HCT116/S322E was the highest, followed by that of HCT116/S303A. On the other hand, the mobility of HCT116/S322A was low. As a result of the MTT assay, there was no difference in the proliferation ability of these three types of cells.

Migration assays were similarly performed for PANC-1/M, PANC-1/WT, PANC-1/S322A, and PANC-1/S322E. FIG. 14 is a photograph showing the result of microscopic observation, and FIG. 15 is a graph showing the result numerically. The results were the same as when HCT116 was used, and PANC-1/WT and PANC-1/S322E had high mobility and PANC-1/S322A had low mobility. As a result of the MTT assay, there was no difference in the proliferative ability of these four types of cells.

From the above results, it was suggested that phosphorylation of Ser (Ser322) at the 322nd position regulates cell migration ability.

Next, HCT116/M, HCT116/WT, HCT116/S322A, and HCT116/S322E were examined for in vivo metastasis by the method 1-(15). That is, each cell was subcutaneously transplanted into a nude mouse (N=5), and 8 weeks later, the lung and liver were taken out. Each organ was solubilized, and Trop-2 was immunoprecipitated using an anti-FLAG antibody. FLAG-tagged Trop-2 was detected by SDS-PAGE and immunoblotting. Results using lungs are shown in FIG. The β-actin band is the control. That is, HCT116/WT (wild type) and HCT116/S322E (Ser322→Glu substitution) were detected in the band of Trop-2 (around 75 kDa), and metastasis was observed (WT: 3 out of 5 individuals, S322E: 5). 1 of the individuals). On the other hand, no band was detected in HCT116/S322A (Ser322→Ala substitution), and no metastasis was observed (zero in 5 individuals).
No metastasis to the liver was observed in any of the cells.

Similarly, each cell was intraperitoneally transplanted, and 4 weeks later, the lung and liver were taken out. The same SDS-PAGE and immunoblotting were performed. As a result, liver metastasis (2 out of 5 individuals) and lung metastasis (4 out of 5 individuals) were observed in HCT116/WT (wild type). HCT116/S322E (Ser322→Glu substitution) also showed liver metastasis (2 out of 4 individuals) and lung metastasis (3 out of 4 individuals). On the other hand, in HCT116/S322A (Ser322→Ala substitution), neither liver metastasis nor lung metastasis was observed.

FIG. 17 shows a photograph of a mouse with lung metastasis and a mouse with no lung metastasis. From the left, mice transplanted with HCT116/M (mock), HCT116/WT (wild type), HCT116/S322A (mutant: Ser322→Ala substitution), and HCT116/S322E (mutant: Ser322→Glu substitution) cells. Is. Mice transplanted with HCT116/M, HCT116/WT, or HCT116/S322E have lung metastases and have abnormal appearance. Mice transplanted with HCT116/S322A have no lung metastases and have a normal appearance.

2-(4) Phosphorylation of Trop-2 by PKCα and PKCδ PKC is conventional (conventional or classical: α, βI, βII, γ) or novel (novel: novel) depending on its structure, activation mechanism and physiological function. δ, ε, η, θ), and atypical: ζ, λ/ι). Therefore, an attempt was made to identify PKC that phosphorylates Trop-2.

PKC isozymes present in HCT116/WT cells were examined by DNA microarray analysis (FIG. 18). As a result, there were one conventional type (PKCα), three new types (PKCδ, ε, η), and two atypical types (PKCζ, ι).

According to the method of 1-(4), HCT116/WT was treated for 1 hour in the presence or absence of a PKC inhibitor or a PKD inhibitor. Subsequently, it was treated with PMA or 4α-PMA for 2 hours. Each cell lysate was subjected to SDS-PAGE and immunoblotting (Fig. 19). The upper row of FIG. 19 shows the results of detection with anti-phosphorylated Trop-2 antibody, and the lower row shows the results of detection with anti-FLAG antibody. FIG. 20 is a graph in which the results of FIG. 19 are quantified, and the ratio of phosphorylated Trop-2 to total Trop-2 (p-Trop-2/FLAG) when the case of 4α-PMA treatment is set to 1. Represents. As a result, it was found that BIM-1 and Go6983 strongly inhibit phosphorylation. Since PKCs which both inhibit in common are PKCα and PKCδ, it was suggested that the phosphorylation of Trop-2 is either or both of PKCα and PKCδ.

By the siRNA treatment described in 1-(12), either or both PKCα and PKCδ of HCT116/WT cells were knocked down. As a control, it was treated with siRNA for control. After each cell was treated with PMA or 4α-PMA, the cell lysate was subjected to SDS-PAGE and immunoblotting (FIG. 21). In FIG. 21, “Cont.”, “siPKCα”, “siPKCδ”, and “siPKCα+δ” represent control, PKCα knockdown, PKCδ knockdown, and both PKCα and PKCδ knockdown, respectively. As a result, when both PKCα and PKCδ were knocked down (the rightmost lane), the phosphorylated Trop-2 band disappeared, and the phosphorylation of Trop-2 was almost completely blocked. From this, it was suggested that both PKCα and PKCδ are involved in the phosphorylation of Trop-2.

FIG. 22 is a numerical representation of the results of FIG. 21, showing the relative amount of phosphorylated Trop-2.

A migration assay using a Transwell was performed on cells in which both PKCα and PKCδ were knocked down. FIG. 23 is a photograph showing the result of microscopic observation, and FIG. 24 is a graph showing the result numerically. That is, in the control cells, the mobility was increased by the PMA treatment, but in the cells in which PKCα and PKCδ were knocked down, the mobility was not increased.
As a result of the MTT assay, there was no difference in proliferative ability in all cells.

2-(5) Inhibitory Effect of PKC Inhibitor on Migration Ability of Wild-Type Cells in Vitro and Metastasis in Vivo PMA-treated HCT116/WT cells were treated with a PKC inhibitor, and migration assay using Transwell was performed. It was FIG. 25 is a photograph showing the result of microscopic observation, and FIG. 26 is a graph showing the result numerically. That is, the migration ability of the cells treated with BIM-1 and the cells treated with Go6983 was strongly inhibited. As a result of the MTT assay, there was no difference in the proliferative ability among these cells.

HCT116/WT cells were intraperitoneally injected into nude mice according to the method of 1-(13). From day 1 of injection, Go6983 was intraperitoneally administered daily (0.25 mg/mouse). DMSO was administered as a control. Four weeks later, the lung and liver were removed. Each organ was solubilized, and Trop-2 was immunoprecipitated using an anti-FLAG antibody. FLAG-tagged Trop-2 was detected by SDS-PAGE and immunoblotting.

As a result, liver metastasis was detected in all 9 control mice. In contrast, liver metastasis was detected only in 1 out of 9 mice in the Go6983-administered mice. Lung metastases were detected in 7 out of 9 control mice. In contrast, lung metastasis was detected only in 3 out of 9 mice treated with Go6983.
From the above, it was possible to suppress the metastasis of cancer by administering Go6983, which is an inhibitor of PKCα/δ, to mice.

Claims (4)

A cancer metastasis inhibitor, which comprises, as an active ingredient, an inhibitor of an enzyme that phosphorylates the serine at the 322nd position from the N terminus of Trop-2. The cancer metastasis inhibitor according to claim 1, wherein the inhibitor is an inhibitor of protein kinase Cα and protein kinase Cδ. Selection of a candidate substance for a cancer metastasis inhibitor, which is characterized by selecting a substance having a cancer metastasis inhibitory action from a test substance using an action of inhibiting phosphorylation of serine at the 322nd position from the N-terminal of Trop-2 as an index Method. The method for selecting a candidate substance for a cancer metastasis inhibitor according to claim 3, wherein the activity of inhibiting phosphorylation by protein kinase Cα and protein kinase Cδ is used as an index.