KR102181813B1 - Therapeutic compositions for breast cancer containing protein kinase D1 - Google Patents

Abstract

One of the characteristics of human breast cancer is cancer stem cellity, which is characterized by self-modifying ability and drug resistance. Protein kinase D1 (PRKD1) functions as a major regulator of many cellular processes and is downregulated in invasive breast cancer cells. The present invention revealed that PRKD1 is upregulated in MCF-7-ADR human breast cancer cells with drug resistance characteristics. In addition, the present inventors revealed that PRKD1 expression is negatively regulated by binding of miR-34a to PRKD1 3'-UTR. PRKD1 expression in MCF-7-ADR cells increased after performing tumor cell formation assay. The present inventors also revealed that the reduction of PRKD1 by translocation miR-34a expression or PRKD1 siRNA treatment in breast cancer stem cells inhibits the self-remodeling ability, furthermore, PRKD1 inhibitor CRT0066101 reduces phosphorylated PKD/PKCμ, GSK3/β- It was confirmed that breast cancer stem cells were inhibited through catenin signaling. In MCF-7-ADR cells, PRKD1 inhibition also influenced the onset of apoptosis. Tumor growth, proliferation, and induced apoptosis were inhibited in tumors of nude mice treated with miR-34a or CRT0066101. These results demonstrate that PRKD1 regulation, a target of the novel miR-34a, will contribute to overcoming cancer cell stemness and drug resistance in human breast cancer.

Description

Therapeutic compositions for breast cancer containing protein kinase D1, comprising a protein kinase D1 inhibitor

The present invention relates to a composition for inhibiting breast cancer stem cells comprising a protein kinase D1 (protein kinase D1) inhibitor and an anticancer adjuvant for preventing recurrence of breast cancer.

Breast cancer is the most common tumor and is the number one cause of tumor-related deaths in women worldwide [1]. Despite efforts to improve patient survival, problems with breast cancer treatment still exist, including tumor metastasis and drug resistance [2,3]. Tumors are composed of tumor stem cells (CSCs) and non-tumorigenic cells that form tumor mass [4]. Tumor stem cells are thought to be the cause of tumor, tumor metastasis, drug resistance and tumor recurrence [5]. In particular, breast cancer tumor stem cells (BCSCs) exhibit stem cell characteristics and are characterized by the expression of the cell surface marker CD44+/CD24- [6]. Different miRNAs are involved in the formation and regulation of human breast cancer stem cells [7], and according to previous studies, the translocation expression of miR-34c (ectopic expression) inhibits epithelial-mesenchymal cell transition and self-renewal in human breast cancer stem cells. Decreases ability [8].

Serine/threonine-protein kinase D1 (PRKD1) mediates stimulatory activity by acting as an effector of diacylglycerol and protein kinase C (PKC) [9]. PKD/PKCμ-related cellular processes were activated by two phosphorylations through PKC-dependent phosphorylation (Ser744/Ser748) and PKC-independent autophosphorylation (Ser910) [10-13]. Therefore, PRKD1 is thought to be a major regulator of several cellular processes, including the regulation of NF-kB signaling pathways, cell cycle progression and DNA synthesis, and other pathogenic conditions [14-16].

In breast cancer, microRNAs regulate apoptosis, tumorigenesis and angiogenesis. The major regulator of tumor suppression, miR-34, is a direct transcriptional target of the tumor suppressor p53, and the miR-34a promoter portion contains a p53-binding site [17]. In breast cancer studies, miR-34a plays a role in inhibiting cell survival by upregulating p53 after irradiation after DNA damage [18]. In addition, miR-34a promoted tumor cell apoptosis by targeting Bcl-2 and SIRT1 [19]. Therefore, miR-34a is associated with targets that induce breast cancer.

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The present invention aims to provide a more effective pharmaceutical composition for inhibiting breast cancer proliferation and metastasis.

In addition, the present invention aims to provide an adjuvant that more effectively restores the drug sensitivity of anticancer drug-resistant breast cancer.

In addition, the present invention aims to more effectively provide a pharmaceutical composition for inhibiting recurrence of breast cancer.

The present inventors revealed that PRKD1 overexpressed in MCF-7-ADR cells was inhibited by miR-34a. In addition, PRKD1 activated self-renewal ability in breast cancer stem cells through glycogen synthase kinase 3 (GSK3)/β-catenin signaling, and contributed to eliminating drug resistance. These results suggest that PRKD1 as a new target of miR-34a may play an important role in the treatment of human breast cancer.

The present invention relates to a pharmaceutical composition for inhibiting the growth of cancer stem cells containing an inhibitor of the expression or activity of protein kinase D1 (protein kinase D1) as an active ingredient.

In addition, the protein kinase D1 of the present invention is characterized by having an amino acid sequence described in SEQ ID NO: 1, and the protein kinase D1 is not limited to the amino acid sequence described in SEQ ID NO: 1, including analogs thereof. It is characterized.

In addition, the protein kinase D1 expression inhibitor of the present invention is a group consisting of antisense nucleotides complementary to the mRNA of the protein kinase D1 gene, short interfering RNA, short hairpin RNA, and miR-34a. It relates to a pharmaceutical composition for inhibiting cancer stem cell growth, characterized in that any one selected from.

In addition, the protein kinase D1 activity inhibitor of the present invention is any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies and CRT0066101 that specifically bind to protein kinase D1 protein. It relates to a pharmaceutical composition for inhibiting cell growth.

In addition, the cancer of the present invention relates to a pharmaceutical composition for inhibiting cancer stem cell growth, characterized in that selected with the cancer stem cell markers CD44+ and CD24-.

In addition, the cancer of the present invention is one selected from the group consisting of breast cancer, liver cancer, colon cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer, and pancreatic cancer. Preferred, and more preferably breast cancer, but is not limited thereto.

In addition, in order to avoid cancer cell recurrence and metastasis and completely eliminate cancer, it is necessary to remove cancer stem cells by targeting cancer stem cells that have the characteristics of stem cells beyond the limitations of current cancer treatment methods that only attack cancer cells. And, the present invention is characterized by inhibiting the growth of the cancer stem cells, in particular, breast cancer stem cells, by inhibiting the cancer stem cells, it is possible to prevent cancer recurrence.

The present invention relates to a pharmaceutical composition for inhibiting breast cancer proliferation and metastasis, comprising an inhibitor of protein kinase D1 (protein kinase D1) expression or activity as an active ingredient.

In addition, the present invention relates to a composition for inhibiting breast cancer proliferation and metastasis, characterized in that the protein kinase D1 inhibitor inhibits stemability of breast cancer cells.

The present invention relates to an anticancer adjuvant for preventing recurrence of breast cancer containing an inhibitor of the expression or activity of protein kinase D1 as an active ingredient.

In addition, the composition of the present invention significantly inhibits the growth of breast cancer stem cells, and thus relates to an anticancer adjuvant capable of inhibiting recurrence of breast cancer.

The present invention provides a method for inhibiting breast cancer stem cells comprising the step of treating an individual with an inhibitor of the expression or activity of protein kinase D1.

In addition, the present invention provides a method for measuring breast cancer prognosis comprising the step of determining whether or not breast cancer recurs by checking whether protein kinase D1 (protein kinase D1) is expressed or active in an individual.

In breast cancer stem cells, protein kinase D1 is expressed, and when it is inhibited, breast cancer stem cell growth is significantly inhibited. Therefore, an inhibitor of protein kinase D1 expression or activity can be usefully used to inhibit breast cancer stem cells. Since there is a risk of recurrence of breast cancer due to cells, the prognosis of breast cancer can be measured by measuring the expression or activity of protein kinase D1.

The composition of the present invention can be used alone or in combination with radiation therapy, chemotherapy, and a method of using a biological response modifier.

The composition of the present invention comprises 0.0001 to 50% by weight based on the total weight of the therapeutic composition.

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions. The composition of the present invention can be administered orally or parenterally during clinical administration, and can be used in the form of a general pharmaceutical formulation.

The composition of the present invention may be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrin, glycerol, ethanol, as well as combinations thereof. Such compositions may be formulated to provide rapid release, or sustained or delayed release of the active ingredient after administration.

When the inhibitor against the protein of the present invention is an antibody, the pharmaceutically acceptable carrier may be composed of a minimum amount of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers that increase the shelf life or effectiveness of the binding protein.

The composition of the present invention may contain a pharmaceutically suitable and physiologically acceptable adjuvant, and such adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, lubricants or solubilizing agents. .

In addition, the composition of the present invention can be preferably formulated into a pharmaceutical composition, including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.

Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterilized and suitable for living organisms, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and these One or more of the components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, and the like, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical formulation form of the composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of active compounds. .

The composition of the present invention is administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. can do.

"Effective amount" of the present invention means an amount required to achieve the effect of inhibiting breast cancer proliferation, metastasis, and stem cell growth. Therefore, the "effective amount" of the active ingredient of the present invention is the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, weight, general health condition, sex, and It can be adjusted according to various factors including diet, administration time, administration route and secretion rate of the composition, treatment period, and drugs used simultaneously. In the case of adults, the gene or protein inhibitor is administered once to several times a day, for siRNA 0.01 ng/kg to 10 mg/kg, for antisense oligonucleotides for the mRNA of the gene 0.01 ng/kg to 10 It is preferable to administer at a dose of 0.1 ng/kg to 10 mg/kg, for a compound, and 0.1 ng/kg to 10 mg/kg for a monoclonal antibody against the protein.

In addition, for those of ordinary skill in the art to which the present invention belongs, it is self-evident to prepare siRNA targeting PRKD1, an antibody thereof, etc. as follows.

Antisense nucleotide

Antisense nucleotides are those that bind (hybridize) to the complementary sequence of DNA, immature-mRNA or mature mRNA, as defined by Watson-Click base pair, and interfere with the flow of genetic information from DNA to protein. The nature of antisense nucleotides that are specific to the target sequence makes them exceptionally multifunctional. Because antisense nucleotides are long chains of monomer units, they can be easily synthesized against the target RNA sequence. Many recent studies have demonstrated the usefulness of antisense nucleotides as a biochemical means for studying target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81:1539-1544, 1999). The use of antisense nucleotides can be considered as a new type of inhibitor, as there have been many recent advances in the field of oligonucleotide chemistry and nucleotide synthesis showing improved cell line adsorption, target binding affinity and nuclease resistance.

Peptide Minetics

Peptide Minetics is a peptide or non-peptide that inhibits the binding domain of PRKD1 protein that leads to PRKD1 activity. The main residues of non-hydrolyzable peptide analogs include β-turn dipeptide core (Nagai et al. Tetrahedron Lett 26:647, 1985), keto-methylene pseudopeptides (Ewenson et al. J Med chem 29:295, 1986; And Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce chemical co.Rockland, IL, 1985), azepine (Huffman et al. in Peptides: chemistry and Biology, GR Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), benzodiazepines (Freidinger et al. in Peptides; chemistry and Biology, GR Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), β-amino alcohols (Gordon et al. Biochem Biophys Res) commun 126:419 1985) and substituted gamma lactam ring (Garvey et al. in Peptides: chemistry and Biology, GR Marshell ed., EScOM Publisher: Leiden, Netherlands, 1988).

siRNA molecule

It is preferable that the sense RNA and the antisense RNA form a double-stranded RNA molecule, wherein the sense RNA is an siRNA molecule containing the same nucleic acid sequence as the target sequence of the contiguous nucleotides of some of the PRKD1 mRNA. The siRNA molecule is preferably composed of a sense sequence consisting of 10 to 30 bases selected within the nucleotide sequence of the PRKD1 gene and an antisense sequence complementary to the sense sequence, but is not limited thereto. Any double-stranded RNA molecule having a sense sequence capable of complementarily binding to a base sequence can be used. Most preferably, the antisense sequence has a sequence complementary to the sense sequence.

Antibody

The PRKD1 antibody may be prepared through injection of PRKD1 or purchased commercially. In addition, the antibodies include polyclonal antibodies, monoclonal antibodies, and fragments capable of binding to epitopes.

Polyclonal antibodies can be produced by a conventional method in which the PRKD1 is injected into an animal, blood is collected from the animal, and serum containing the antibody is obtained. Such polyclonal antibodies can be purified by any method known in the art, and can be made from any animal species host, such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, etc.

Monoclonal antibodies can be prepared using any technique that provides the production of antibody molecules through cultivation of continuous cell lines. Such techniques include, but are not limited to, hybridoma technology, human B-cell line hybridoma technology, and EBV-hybridoma technology (Kohler G et al., Nature 256:495-497, 1975; Kozbor. D et al., J Immunol Methods 81:31-42, 1985; cote RJ et al., Proc Natl ACad Sci 80:2026-2030, 1983; and cole SP et al., Mol cell Biol 62:109-120, 1984).

In addition, an antibody fragment containing a specific binding site for PRKD1 can be prepared. For example, but not limited to these, the F(ab') ₂ fragment can be prepared by digesting the antibody molecule with pepsin, and the Fab fragment can be prepared by reducing the disulfide bridge of the F(ab') ₂ fragment. As another method, a monoclonal Fab fragment having a desired specificity can be quickly and easily identified by reducing the Fab expression library (Huse WD et al., Science 254: 1275-1281, 1989).

The antibody may be bound to a solid substrate to facilitate subsequent steps such as washing or separation of the complex. Solid substrates include, for example, synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres and fine beads. In addition, the synthetic resin includes polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF and nylon.

Aptamer

Aptamer is a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure by itself and can bind to a target molecule with high affinity and specificity. Aptamer has been developed since the first development of an aptamer discovery technology called SELEX (Systematic Evolution of Ligands by EXponential enrichment) (Ellington, AD and Szostak, JW., Nature 346:818-822, 1990), small molecule organic matter, peptide, membrane protein. Many aptamers capable of binding to various target molecules have been continuously discovered. Aptamer is compared with a single antibody because of its inherent high affinity (usually pM level) and specificity that it can bind to a target molecule, and has a high potential as an alternative antibody, especially as a "chemical antibody".

Advantages and features of the present invention, and a method of achieving them will become apparent with reference to embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, and only these embodiments are intended to complete the disclosure of the present invention, and provide common knowledge in the technical field to which the present invention pertains. It is provided to completely inform the scope of the invention to those who have it, and the invention is only defined by the scope of the claims.

According to the present invention, inhibition of PRKD1 expression and/or activity inhibited stem cell formation of breast cancer cells. Therefore, if an inhibitor for the expression or activity of PRKD1 is used, it can be used for suppressing proliferation, metastasis or recurrence due to stem cell formation of breast cancer, and can restore the drug sensitivity of breast cancer.

1A to 1C reveal that PRKD1 is a new target of miR-34a. Figure 1a: PRKD1 mRNA expression and miR-34a expression in various human breast cancer cell lines were quantified by qRT-PCR. Fig. 1b: Protein, mRNA and total RNA obtained 48 hours after transfection of miRNA-34 variant. Western blots are representative of three independent experiments. β-actin was used as a loading control, and qRT-PCR was performed to confirm the expression of PRKD1 mRNA and miR-34 mutant. The expression levels of miR-34a, b and c were detected after translocation expression of miR-34a, b and c respectively. Figure 1c: miR-34a binding site and reporter construct predicted from wild-type/mutant PRKD1 3'-UTR. The activity of the 3'-UTR reporter construct was normalized against the activity of the coat transfected phRL-Luc vector. The graph is expressed as the mean value ± standard deviation obtained from three independent experiments. * p <0.05; ** p <0.001; *** p <0.0001.
FIG. 2 shows the effect of PKD/PKCμ lowering regulation on breast cancer stem cell properties through GSK3/β-catenin signaling in MCF-7-ADR cells. All results were obtained from at least five independent experiments. 2A and 2B: miR-34a precursors and PRKD1 siRNAs (15 nM) were transfected into MCF-7-ADR cells, and miR-34a and PRKD1 expressions were confirmed by qRT-PCR. GSK3/β-catenin signaling was analyzed by Western blot. β-actin was used as a loading control. Figure 2c: Shows the basic phosphorylation and expression of PKD/PKCμ in tumor cells derived from MCF-7-ADR cells compared to two-dimensional cultured MCF-7 cells. Figure 2d: Using Olympus IX71 at a magnification of 400×, a representative image of tumor sphere formation confocal was captured. The size bar is 50 μm. Figure 2e: Analysis of cell surface expression of breast cancer stem cell markers in MCF-7-ADR cultured cells. The histogram shows the results of five independent experiments. Percentage represents the number of cells in each quartile. Bars represent each sample performed three times, and error bars represent ± standard deviation. * p <0.05; ** p <0.001; *** p <0.0001.
3a to 3c show the effect of CRT0066101 on breast cancer stem cell properties through GSK3/β-catenin signaling in MCF-7-ADR cell lines. Figure 3a: After 0.1-10 μM of CRT0066101 treatment, GSK3/β-catenin signaling was analyzed by Western blot. The blot represents five independent experiments. β-actin was used as a loading control. Figure 3B: Tumor cells were treated with distilled water (control) or 1 μM or 5 μM CRT0066101. Representative confocal images of tumor formation were taken at 400× magnification using Olympus IX71. Size bars represent 50 μm. The graph represents the number of tumor cells per 2000 cells. 3C: Analysis of CD44+/CD24- expression on the surface of MCF-7-ADR cells after treatment with CRT0066101. The histogram shows the results of three independent experiments. Percentage represents the number of cells in each quartile. * p <0.001; ** p <0.0001; *** p <0.0001.
4A to 4E show the inhibitory effect of PKCμ on MCF-&-ADR cell apoptosis. Figure 4a: After transfection of MCF-7-ADR cells with a control vector or PRKD1 siRNA, cell viability was detected. After knocking down PRKD1 expression, doxorubicin was treated at different doses and cell viability was measured. Figure 4b: The level of casphase-3 activity after PRKD1 downregulation and the level of casphase-3 activity after doxorubicin treatment. Figure 4c: WST-8 analysis results after 70 hours treatment (0.1-5 μM) of CRT0066101 in MCF-7-ADR cells. The optical density was measured at 450 nm. Figure 4d: After treatment with 1 μM/2 μM CRT0066101 or 10 μM doxorubicin, Casphase-3 activity was measured. Relative casphase-3 activity was measured at 405 nm. Figure 4e: Annexin V/PI-stained cell photos were taken with a confocal microscope at 200 times magnification. Size bar: 50 μm. Data are expressed as mean ± standard deviation. * p <0.05; ** p <0.001.
5A to 5G show that PKCμ lowering control inhibits tumor formation in a xenograft model. Figure 5a: PRKD1 mRNA expression was measured by qRTPCR in the control and miR-34a overexpressing tumors. Figure 5b: Immunohistochemistry was performed to detect PKCμ and PCNA expression in the control and miR-34a overexpressing tumors. Magnification: 200X; Size bar: 10 μm. Figure 5c: TUNEL analysis and DAPI staining were performed. Magnification: 400x; Size bar: 20 μm. 5D: CRT0066101 (65 mg/kg) was administered orally to tumors established in nude mice every day for 4 weeks. These are the results of three representative xenografts. 5E: The volume of the tumor treated with CRT0066101 was decreased compared to the control tumor. However, no change was detected in the mouse weight. Figure 5f: Western blot analysis of phosphorylated PRKD1 and GSK3/β-catenin signaling. β-actin was used as a loading control. 5G: Immunofluorescence photographs showing TUNEL and Ki67 in control and CRT0066101 treated tumors. Data are expressed as mean ± standard deviation. * p <0.05, ** p <0.001.
6 is a schematic diagram of the hypothesis of the present invention. miR-34a directly inhibits PRKD1 and CRT0066101 inhibits self-phosphorylated PKD/PKCμ. The two directions represent different regulation of self-renewal ability in breast cancer stem cells, and drug resistance in MCF-7-ADR cells through GSK3/β-catenin signaling.
Figure 7 shows that there is no correlation between the expression of miR-34b or miR-34c and PRKD1 expression. A and B show the expression levels of miR-34b and miR-34c in various breast cancer cell lines, respectively, confirmed by qRT-PCR.
8 is a comparison of the efficiency of three different PRKD1 siRNA.
9 shows the effect of suppressed miR-34a expression in MCF-7 cells. A: After transfection with miR-34a inhibitor in MCF-7 cells, the expression level of miR-34a was confirmed by qRT-PCR. B: GSK3/β-catenin signaling was measured by Western blot analysis. β-actin was used as a loading control.
10 shows the expression level of cancer cell stemming markers in MCF-7-ADR cells. A, B: OCT4 and SOX2 expression levels were confirmed by qRT-PCR.
11 shows the expression levels of miR-34a and PRKD1 in MCF-7 cells and MCF-7-ADR breast cancer stem cells. A: miR-34a was highly expressed in MCF-7 cells in a spherical state, and PRKD1 was highly expressed in B: MCF-7-ADR breast cancer stem cells.
12 shows that there was no change in GSK3/β-catenin signaling and cell viability when treated with CRT0066101 in MCF-7 cells. A: As a result of Western blot analysis, the effect of CRT0066101 was shown in MCF-7 cells. B: Results of WST-8 analysis after CRT0066101 treatment (0.1-5 μM) in MCF-7 cells.
13 shows the correlation between upregulated PRKD1 expression and poor prognosis in breast cancer patients. A: There is no correlation between miR-34a and PRKD1 expression in various cell lines. B: Overall survival rate according to PRKD1 expression level in TCGA clinical data set.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those of ordinary skill in the art that the scope of the present invention is not limited only to the description of the examples.

Chemicals and reagents

CRT0066101 was purchased from R&D Systems (Minneapolis, MN, USA); This drug was resuspended in sterilized distilled water and used for in vivo studies. To treat CRT0066101, MCF-7-ADR cells (American Type Culture Collection, Manassas, VA, USA) were seeded, 0.1-10 μM CRT0066101 was added, and then cultured for one hour. WST-8 is manufactured by Enzo Life Sciences, Inc. (Farmingdale, NY, USA). PRKD1 siRNA and scrambled siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were transfected using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA).

Cell culture and transfection

Human breast adenocarcinoma MCF7, MCF-7-ADR and MDAMB-231 cell lines (American Type Culture Collection) were cultured in DMEM (Dulbecco's modified Eagle's Medium; Welgene, Daejeon, South Korea) and in a humid incubator at 37°C. Supplemented with 10% FBS (Welgene) and 1% penicillin, streptomycin under 5% CO ₂ . For RNAi transfection, MCF-7-ADR cells were seeded in a medium containing no antibiotics in a 10-cm plate. After 24 hours, cells were transfected with PRKD1 siRNA using Lipofectamine RNAiMAX (Invitrogen). After 48 hours, the cells were collected and subjected to Western blot analysis or resuspended in breast cancer stem cell (mammosphere) medium. For microRNA transfection, MCF-7-ADR cells were mixed with miRNA precursors (miR-34a/b/c) for 48 hours using siPORT NeoFX Transfection Agent (Ambion; Thermo Fisher, St. Louis, MO, USA). Seed while. The miRNA precursor and negative control precursor were purchased from Thermo Fisher.

qRT-PCR

qRT-PCR (Quantitative reverse-transcription PCR) was performed according to the manufacturer's instructions in a SYBR Green-based method using an RG3000 device (Corbett Robotics, San Francisco, CA, USA). The ABI-7500 device (Thermo Fisher) was used to evaluate PRKD1 expression in various breast cancer cell lines. All oligonucleotide primers were designed with DNASTAR (Madison, WI, USA). All qRT-PCR graphs were obtained using relative C _t (ΔΔC _t ) values.

Western blotting and antibodies

A total of 30 µg of protein extract was separated by 8% SDS-PAGE, and the protein was electromigrated to a PVDF membrane. The primary antibodies used were phosphorylated PKD/PKCμ (Ser916), GSK3β, phosphorylated GSK3α (Ser21)/β (Ser9), and β-catenin, and these antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). And, PKD/PKCμ antibody was purchased from Santa Cruz Biotechnology. β-actin (Bethyl Laboratories, Montgomery, TX, USA) was used as a loading control. The membrane was washed with 1×PBS/0.1% Tween 20, and the bound protein was detected with an enhanced chemiluminescent reagent (Amersham Pharmacia Biotech, Parsippany, NJ, USA).

Luciferase analysis method

The 3'-UTR reporter construct of PRKD1 was cloned into a pGL3-control vector, and 3'-UTRs of PRKD1 were amplified from genomic DNA of HEK293T cells. The miR-34 seed sequence from PRKD1 w was mutated by a PCR-based method, and the reporter construct was confirmed by sequencing. Using Lipofectamine 2000 (Invitrogen), HEK293T cells were transiently transfected with a 3′-UTR reporter construct (1.5 μg per well in a 6-well plate) and 15 nM of miR-34 family precursor (Ambion). The activity of the 3'-UTR reporter construct was normalized to the activity of coat transfected pCMV-hRL (40 ng per well, Promega in 6 well plate). After culturing for 24 hours, cells were lysed with 1× passive lysis buffer, and the activity was measured according to the manufacturer's instructions using a Dual Luciferase Assay kit (Promega).

Tumorsphere formation assay (TSA)

For tumor cell culture, cells (2000 cells/mL) were added to 1% penicillin, B27 (1:50; Gibco; Thermo Fisher), 20 ng/mL epidermal growth factor (Prospec, East Brunswick, NJ, USA), 5 mg/mL. Insulin (Sigma-Aldrich, St. Louis, MO, USA), and 0.4% bovine serum albumin (Sigma-Aldrich) was suspended in serum-free DMEM/F12 (welGENE) containing and cultured. After about 10 days, the plate was analyzed to confirm the formation of tumor cells and quantified under a microscope (Olympus IX71; Olympus, Tokyo, Japan). To count tumor cells, MCF-7-ADR cells were filtered and quantified with a 70 μm pore strainer (BD Biosciences, East Rutherford, NJ, USA). CRT0066101 treatment was performed on the 6th and 8th days of culture.

Surface marker analysis using flow cytometry

After transfection with RNAi of PRKD1 or treatment with CRT0066101, cells were collected and evaluated for CD44+/CD24- surface marker expression. Cells were washed with 2% FBS, stained with anti-CD44 (APC-conjugated; BD Biosciences) and anti-CD24 (PEconjugated; BD Biosciences) in PBS containing 2% FBS, and placed on ice in the dark for 30 minutes. . After washing the cells with cold PBS buffer again, >10,000 cells were loaded into the BD CantoII flow cytometer (BD Biosciences), and analyzed by flow cytometry using FACSDiVa software (BD Biosciences).

Cell viability analysis

MCF-7-ADR cells were placed in a 24-well plate and cultured for 72 hours with CRT0066101 at various concentrations (0.1, 0.5, 1, 5, 10 μM). Cell viability was analyzed by WST-8 assay (Sigma-Aldrich), and optical density was measured at 450 nm using a microplate reader.

Fluorescent Immunohistochemistry

Control or miR-32a overexpressing tumors and carriers or CRT0066101-treated tumors were cut and paraffin-treated slides were used. Paraffin was removed from the slide, rehydrated 3-4 times in Histoclear, and then passed through ethanol of different concentrations (100%, 95%, 80%, 70%) in order. Antigen restoration was performed by immersing the section in a 0.01M citric acid solution (pH 6.0) and boiling in a microwave for 15 minutes. In the case of TUNEL analysis, an in situ apoptosis detection kit, apoptosis positive cells labeled with a fluorescent substance (Roche, Indianapolis, USA) and Ki-67 primary antibody (Vector Lab, USA) were applied to the section and overnight at 4°C. Cultured. Then, the slides were incubated with DAPI and secondary antibody for two hours. Finally, it was treated with a mounting solution (Dako) and a picture was taken with a confocal microscope (Zeiss).

Manufacture of breast cancer xenograft mice

All studies including the use of nude mice were approved by the Animal Protection and Use Committee of Yonsei University College of Medicine (2015-0087), and were carried out in a facility free of specific pathogens and under conditions in accordance with the guidelines of the committee. Mice were anesthetized with 150 µl of saline/zoletil/rompun (7:1:1) on the outer side of each femur, and 1.5×10 ⁶ MCF-7-ADR cells were injected subcutaneously. Six mice were randomly assigned to one group, and treatment was started from day 10 after tumor implantation. CRT0066101 was administered orally to tumor-bearing animals, and was administered for 4 weeks, 5 times per week, and 1.6mg/kg each time. From palpable tumor formation to completion, tumor size was measured every 3 to 4 days using a caliper, and tumor volume was calculated by the formula of length X width ² X 0.5236. Mice were sacrificed in a 7.5% CO ₂ chamber, and tumors were isolated and used for immunohistochemistry and other analyses.

Results 1: miR-34a inhibits PRKD1 in MCF-7-ADR cells.

PRKD1 expression was evaluated in breast cancer cell lines including MCF-10A, MCF-7, ZR-75-1, MCF-7-ADR, SK-BR-3, MDA-MB-231 and MDAMB-468. As a result, the level of PRKD1 expression was increased in MCF-7-ADR cells (Fig. 1A). The present inventors use the microRNA prediction online database [miRanda (http://www.microrna.org/ microrna/home.do) and TargetScan (http://www.targetscan. org/)] to control PRKD1 miRNA Was determined. Considering that miR-34 is a candidate regulator, PRKD1 mRNA expression and protein translation levels were measured after ectopic expression of miR-34a, miR-34b and miR-34c. Although miR-34a, miR-34b, and miR-34c have the same seed sequence, the results showed that PKD/PKCμ was reduced-regulated only by miR-34a (FIG. 1B). In order to confirm whether miR-34a binds to PRKD1 3′-UTR, the miR-34a expected binding site on PRKD1 3′-UTR was mutated, and the mutant sequence was inserted into the pGL3-control vector (Fig. 1C). As shown in Figure 1C, miR-34a overexpression inhibited the luciferase activity of the PRKD1 wild-type sequence, but the mutation in MCF-7-ADR cells did not. We screened the expression level of miR-34a in breast cancer cell lines, and as a result, miR-34a was lowered and regulated in MCF-7-ADR cells, consistent with the results of FIG. 1A. These results suggest that miR-34a negatively regulates PRKD1 (Fig. 1A).

Expression levels of miR-34b and miR-34c were also detected in MCF-7-ADR cells, but significant lowering control was not observed (Figs. 7A and B). These results suggest that PRKD1 is downregulated by miR-34a in MCF-7-ADR cell line.

Results 2: PRKD1 promotes breast cancer stemming through GSK3/β-catenin signaling.

In order to examine the PRKD1 inhibitory effect on tumor stem cells, MCF-7-ADR cells were transfected with miR-34a precursors and PRKD1 siRNAs. After transfection, the expression level of miR-34a increased and the level of PKD/PKCμ decreased compared to the negative control (FIG. 2A ). Compared to the level observed with respect to the control siRNA transfection, the PRKD1 expression level was also decreased, which is due to the PRKD1 siRNA transfection. Interestingly, the PKD/PKCμ level also decreased with PRKD1 siRNA transfection (FIG. 2B ). In order to exclude non-specific effects, the present inventors checked the efficiency of three different PRKD1 siRNAs and selected PRKD1 siRNA #1 because this inhibited PRKD1 better than others (FIG. 8 ).

PRKD1 phosphorylation of β-catenin at Thr112/Thr120 may be critical for cell-cell junctions in prostate cancer cells [20]. Furthermore, the complex of CDC42, PAR6 and PKCζ binds to GSK3β and inhibits GSK3β by catalyzing Ser9 phosphorylation [21]. To correlate PRKD1 to GSK3/β-catenin signaling, Western blot analysis was performed. The results showed that PKD/PKCμ reduction inhibited β-catenin expression and GSK3α and GSKβ phosphorylation (FIG. 2A ). These results were confirmed in the control and PRKD1 siRNA-treated cells (Fig. 2B). In addition, the present inventors confirmed that the miR-34a expression level was decreased by changing the GSK3/β-catenin signaling after transfection with miR-34a in MCF-7 cells as a control (Fig. 9A, B).

PRKD1 expression and GSK3/β-catenin signaling were upregulated in MCF-7-ADR cells forming tumor cells (FIG. 2C ). Tumor stem-like markers such as OCT4 and SOX2 were highly expressed in MCF-7-ADR cells in a tumorigenic state (Fig. 10A, B). In addition, it was observed that the expression of miR-34a was low and the expression of PRKD1 was high in MCF-7-ADR breast cancer stem cells compared to MCF-7 breast cancer stem cells (Figs. 11 A and B). In order to investigate the effect of PRKD1 knockdown on breast cancer cell stemability, tumor cell formation assay was performed. PRKD1 knockdown and PRKD1 siRNA by the miR-34a precursor significantly reduced the number of tumor cells (>70 μm) compared to the control (FIG. 2D ). In addition, the number of breast cancer stem cells (BCSC) was analyzed by fluorescence-activated cell sorting by staining the breast cancer stem cell marker CD44+/CD24-. CD44+/CD24- number (Q1) was also reduced by PRKD1 knockdown (FIG. 2E ). Taken together, PRKD1 can regulate cancer cell stemability by altering GSK3/β-catenin signaling in MCF-7-ADR cells.

Results 3: Inhibition of PKD/PKCμ phosphorylation decreases breast cancer stem cell self-renewal ability.

The PKD/PKCμ phosphorylation-related process was found to have two possible active pathways. One is protein kinase C (PKC)-dependent phosphorylation (Ser744/Ser748) and the other is autophosphoryation (Ser916). For sufficient activation, self-phosphorylation must occur immediately after PKC-dependent phosphorylation [10, 11]. CRT0066101 is an inhibitor targeting PKD autophosphorylation [16]. To examine the role of PKD/PKCμ autophosphorylation, MCF-7-ADR cells were treated with 1 μM or 5 μM CRT0066101. Western blotting results, CRT0066101 inhibited the phosphorylation of PKD/PKCμ and GSK3/β-catenin in MCF-7-ADR cells (FIG. 3A). However, GSK3/β-catenin signaling in MCF-7 cells was not affected by CRT0066101 treatment (Fig. 12A).

After CRT0066101 treatment (1 μM or 5 μM), the number of tumor cells (>70 μm) decreased in a dose-dependent manner compared to the control group (FIG. 3B ). As expected, the number of CD44+/CD24- (Q1) also decreased after 1 μM CRT0066101 treatment (FIG. 3C ). These results suggest that PKD/PKCμ autophosphorylation through GSK3/β-catenin signaling is necessary for the regulation of breast cancer cell stemability.

Results 4: PRKD1 restores drug resistance.

Previous research results reported that PKD/PKCμ is associated with apoptosis through casphase-3 inhibition [22]. Therefore, the present inventors investigated whether PRKD1 inhibition activates apoptosis in MCF-7-ADR cells. As a result, it was found that the reduction reaction occurred more in breast cancer stem cells. As shown in FIG. 4A, when MCF-7-ADR cells were exposed to doxorubicin (DOX), the cell viability was decreased in a dose-dependent manner. Importantly, PRKD1 knockdown increased the level of apoptosis compared to the control. In order to determine whether the decrease in cell viability was due to apoptosis, casphase-3 activation was measured. As a result of inhibition of PRKD1, higher casphase-3 activity was observed compared to the control group. In addition, knockdown of PRKD1 after doxorubicin treatment increased casphase-3 activity compared to the control group treated with doxorubicin (FIG. 4B). In addition, PRKD1 inhibition by 0.1-5 μM CRT0066101 treatment in MCF-7-ADR cells dose-dependently reduced cell viability (Fig. 4C), but in MCF-7 cells, cell viability did not decrease compared to CRT0066101 treatment ( Fig. 12 B). Combination of CRT0066101-treatment and doxorubicin-induced apoptosis significantly lowered cell viability compared to treatment with CRT0066101 alone (Fig. 4D). In addition, we performed Annexin V and propidium iodide (PI) staining to confirm that PRKD1 knockdown or inhibition of PKD/PKCμ phosphorylation enhances apoptosis. As a result, it was confirmed that the initiation of apoptosis was increased in miR-34a precursor-treated cells, PRKD1 siRNA-treated cells, and doxorubicin-treated cells compared to the control group. Interestingly, when CRT0066101 was treated with doxorubicin-treated cells, the apoptosis level was increased (FIG. 4E). Taken together, these data suggest that PRKD1 depression or inhibition of PKD/PKCμ autophosphorylation in MCF-7-ADR cells restores drug resistance.

Results 5: In a xenograft model, inhibition or reduction of PKCμ function inhibits tumor growth.

In our previous study, the present inventors confirmed that miR-34a inhibits tumor formation by inhibiting NOTCH1 expression in nude mice [5]. In the present invention, it was studied whether the PRKD1 lowering regulation by miR-34a inhibits tumor growth in a xenograft model. Compared to the control tumor, the expression level of PRKD1 in the miR-34a overexpressing tumor was lowered and regulated (FIG. 5A ). As a result of immunohistochemistry (IHC) staining, PKCμ and proliferating cell nuclear antigen (PCNA) were decreased in miR-34a overexpressing tumors (FIG. 5B). In order to determine whether PRKD1 inhibition by miR-34a inhibits tumor growth through apoptosis, the present inventors performed TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) analysis. As a result, the miR-34a overexpressing tumor had more self-killing cells than the control tumor (Fig. 5C).

In order to further evaluate the inhibitory effect of PKD/PKCμ, mice with tumors established by xenograft with MCF-7-ADR cells were treated with 65 mg/kg CRT0066101 daily for 4 weeks. In the mice treated with CRT0066101, the tumor size was decreased compared to the untreated control group (Fig. 5D). As expected, the tumor weight of the mice treated with CRT0066101 decreased compared to the control tumor. CRT0066101 treatment in all animals did not cause significant signs of toxicity and side effects such as weight loss (FIG. 5E ). In addition, the reduction of phosphorylated PKD/PKCμ through GSK3/β-catenin signaling was also confirmed by Western blot analysis (FIG. 5F). Finally, the present inventors performed TUNEL analysis and Ki-67 staining to confirm whether CRT0066101 enhances apoptosis and inhibits proliferation. As a result, the CRT0066101-treated tumor increased apoptosis and decreased proliferation compared to the control group (FIG. 5G ). In conclusion, PKCμ inhibition by miR-34a or CRT0066101 plays a role in reducing tumor growth through initiation of apoptosis in vivo.

PRKD1 is involved in cell proliferation, apoptosis, cell conjugation, invasion and vesicle trafficking [23]. Interestingly, PRKD1 expression exhibits different patterns in a variety of tumor cells and performs dual functions as tumor cells or tumor suppressors [24]. PRKD1 expression is downregulated in invasive human breast cancer compared to normal breast tissue [25]. Similar expression patterns were identified in microarray analysis and invasive cell models such as SK-BR-3, T-47D and MDA-MB-231 [25, 26]. Furthermore, reversing PRKD1 promoter methylation blocks breast cancer cell invasion and metastasis [27]. Experimental results of the present inventors showed that the PRKD1 expression pattern in the MCF-7-ADR cell line increases drug resistance. PRKD1 was highly expressed in drug-resistant cell lines including doxorubicin-resistant MCF-7-ADR cells, tamoxifen-resistant LCC2 cells, and tamoxifen and fluvestrans-resistant LCC9 cells. Therefore, we conclude that PRKD1 expression is associated with drug resistance. The present inventors studied miR-34a and PRKD1 expression in the TCGA data set (Fig. 13A). Because PRKD1 was highly expressed in drug-resistant breast cancer cells, we could not find an inverse correlation between miR-34a and PRKD1 expression in the TCGA database. The present inventors further confirmed the overall survival rate according to the expression level of PRKD1 in the TCGA clinical data set (FIG. 13B). This graph shows that patients with high PRKD1 expression have a lower survival rate than those with low expression. Although the inverse correlation between miR-34a and PRKD1 expression in all breast cancer samples could not be confirmed, the relationship between PRKD1 expression and breast cancer patients with poor prognosis was elicited. In addition, we confirmed that the reduced-regulated PRKD1 alters the apoptosis signal. Thus, it is suggested that PRKD1 may be a potential option to restore drug sensitivity in breast cancer cells.

microRNA miR-34a plays an important role in tumor suppression. Previous studies have reported that miR-34a inhibits tumor stem cells in various tumors, including prostate cancer [28], pancreatic cancer [29], medulloblastomas [30] and glioblastomas [31]. have. This molecule also inhibits targets associated with cell cycle, differentiation and apoptosis, while inhibiting tumor cell survival, tumor stemness, metastasis and resistance to chemical drugs [17]. In the present invention, it was confirmed that miR-34a negatively regulates PRKD1 in MCF-7-ADR cells. In addition, PRKD1 is a new target of miR-34a, and it was found that miR-34a binds to PRKD1 3'-UTR. Furthermore, the present inventors have found that miR-34a-PRKD1 interaction plays an important role in overcoming tumor stem cell properties and drug resistance in breast cancer cell lines. Previous studies have reported that PRKD1 phosphorylates β-catenin at Thr112/Thr120 and that PRKD1 overexpression inhibits β-catenin mediated transcriptional activity [32]. β-catenin phosphorylation occurs via GSK3, which targets β-catenin as part of the Wnt-signaling protein complex [33]. In addition, GSK3β is a kinase involved in prostate cancer cellularity and migration through a Wnt-independent mechanism [34]. In the present invention, it was observed that the reduced PRKD1 inhibits the self-remodeling ability of breast cancer stem cells through alteration of GSK3/β-catenin signaling. Therefore, these results suggest that PRKD1 activates breast cancer stemness through GSK3/β-catenin signaling.

Harikumar et al. discovered CRT0066101 as a specific inhibitor for all PKD isoforms [16], and found that CRT0066101 blocks pancreatic cancer growth by inhibiting PRKD1 autophosphorylation [16]. The present inventors blocked PRKD1 activation by treating breast cancer cell lines and xenograft animal models with CRT0066101. These results suggest that CRT0066101 could be a promising treatment for breast cancer patients.

In the present invention, PRKD1 overexpression in MCF-7-ADR cell line showed a negative correlation with miR-34a overexpression. The present inventors found that miR-34a inhibits cancer cell stemability in breast cancer stem cells through the GSK3/β-catenin signaling pathway by binding with PRKD1 3'-UTR. Furthermore, the present inventors revealed that CRT0066101, known as a PRKD1 inhibitor, affects the reduction of breast cancer stem cells and drug resistance through the GSK3/β-catenin signaling pathway (FIG. 6). In addition, we observed that the ectopic expression of miR-34a and CRT0066101 treatment inhibited cancer growth in the xenograft model. In conclusion, PRKD1 was regulated in a negative relationship by miR-34a to suppress cancer cell stemability and drug resistance in breast cancer cell lines. These results suggest that PRKD1 is a major molecule that activates breast cancer stem cellity and drug resistance, and promotes it as a potential therapeutic target in breast cancer.

<110> Industry academic cooperation foundation sookmyoung women university <120> Theraputic compositions for breast cancer containing protein kinase D1 <130> PB2019-213 <150> KR 10-2016-071126 <151> 2016-06-08 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 912 <212> PRT <213> Homo sapiens <400> 1 Met Ser Ala Pro Pro Val Leu Arg Pro Pro Ser Pro Leu Leu Pro Val 1 5 10 15 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Leu Val Pro Gly Ser Gly 20 25 30 Pro Gly Pro Ala Pro Phe Leu Ala Pro Val Ala Ala Pro Val Gly Gly 35 40 45 Ile Ser Phe His Leu Gln Ile Gly Leu Ser Arg Glu Pro Val Leu Leu 50 55 60 Leu Gln Asp Ser Ser Gly Asp Tyr Ser Leu Ala His Val Arg Glu Met 65 70 75 80 Ala Cys Ser Ile Val Asp Gln Lys Phe Pro Glu Cys Gly Phe Tyr Gly 85 90 95 Met Tyr Asp Lys Ile Leu Leu Phe Arg His Asp Pro Thr Ser Glu Asn 100 105 110 Ile Leu Gln Leu Val Lys Ala Ala Ser Asp Ile Gln Glu Gly Asp Leu 115 120 125 Ile Glu Val Val Leu Ser Ala Ser Ala Thr Phe Glu Asp Phe Gln Ile 130 135 140 Arg Pro His Ala Leu Phe Val His Ser Tyr Arg Ala Pro Ala Phe Cys 145 150 155 160 Asp His Cys Gly Glu Met Leu Trp Gly Leu Val Arg Gln Gly Leu Lys 165 170 175 Cys Glu Gly Cys Gly Leu Asn Tyr His Lys Arg Cys Ala Phe Lys Ile 180 185 190 Pro Asn Asn Cys Ser Gly Val Arg Arg Arg Arg Leu Ser Asn Val Ser 195 200 205 Leu Thr Gly Val Ser Thr Ile Arg Thr Ser Ser Ala Glu Leu Ser Thr 210 215 220 Ser Ala Pro Asp Glu Pro Leu Leu Gln Lys Ser Pro Ser Glu Ser Phe 225 230 235 240 Ile Gly Arg Glu Lys Arg Ser Asn Ser Gln Ser Tyr Ile Gly Arg Pro 245 250 255 Ile His Leu Asp Lys Ile Leu Met Ser Lys Val Lys Val Pro His Thr 260 265 270 Phe Val Ile His Ser Tyr Thr Arg Pro Thr Val Cys Gln Tyr Cys Lys 275 280 285 Lys Leu Leu Lys Gly Leu Phe Arg Gln Gly Leu Gln Cys Lys Asp Cys 290 295 300 Arg Phe Asn Cys His Lys Arg Cys Ala Pro Lys Val Pro Asn Asn Cys 305 310 315 320 Leu Gly Glu Val Thr Ile Asn Gly Asp Leu Leu Ser Pro Gly Ala Glu 325 330 335 Ser Asp Val Val Met Glu Glu Gly Ser Asp Asp Asn Asp Ser Glu Arg 340 345 350 Asn Ser Gly Leu Met Asp Asp Met Glu Glu Ala Met Val Gln Asp Ala 355 360 365 Glu Met Ala Met Ala Glu Cys Gln Asn Asp Ser Gly Glu Met Gln Asp 370 375 380 Pro Asp Pro Asp His Glu Asp Ala Asn Arg Thr Ile Ser Pro Ser Thr 385 390 395 400 Ser Asn Asn Ile Pro Leu Met Arg Val Val Gln Ser Val Lys His Thr 405 410 415 Lys Arg Lys Ser Ser Thr Val Met Lys Glu Gly Trp Met Val His Tyr 420 425 430 Thr Ser Lys Asp Thr Leu Arg Lys Arg His Tyr Trp Arg Leu Asp Ser 435 440 445 Lys Cys Ile Thr Leu Phe Gln Asn Asp Thr Gly Ser Arg Tyr Tyr Lys 450 455 460 Glu Ile Pro Leu Ser Glu Ile Leu Ser Leu Glu Pro Val Lys Thr Ser 465 470 475 480 Ala Leu Ile Pro Asn Gly Ala Asn Pro His Cys Phe Glu Ile Thr Thr 485 490 495 Ala Asn Val Val Tyr Tyr Val Gly Glu Asn Val Val Asn Pro Ser Ser 500 505 510 Pro Ser Pro Asn Asn Ser Val Leu Thr Ser Gly Val Gly Ala Asp Val 515 520 525 Ala Arg Met Trp Glu Ile Ala Ile Gln His Ala Leu Met Pro Val Ile 530 535 540 Pro Lys Gly Ser Ser Val Gly Thr Gly Thr Asn Leu His Arg Asp Ile 545 550 555 560 Ser Val Ser Ile Ser Val Ser Asn Cys Gln Ile Gln Glu Asn Val Asp 565 570 575 Ile Ser Thr Val Tyr Gln Ile Phe Pro Asp Glu Val Leu Gly Ser Gly 580 585 590 Gln Phe Gly Ile Val Tyr Gly Gly Lys His Arg Lys Thr Gly Arg Asp 595 600 605 Val Ala Ile Lys Ile Ile Asp Lys Leu Arg Phe Pro Thr Lys Gln Glu 610 615 620 Ser Gln Leu Arg Asn Glu Val Ala Ile Leu Gln Asn Leu His His Pro 625 630 635 640 Gly Val Val Asn Leu Glu Cys Met Phe Glu Thr Pro Glu Arg Val Phe 645 650 655 Val Val Met Glu Lys Leu His Gly Asp Met Leu Glu Met Ile Leu Ser 660 665 670 Ser Glu Lys Gly Arg Leu Pro Glu His Ile Thr Lys Phe Leu Ile Thr 675 680 685 Gln Ile Leu Val Ala Leu Arg His Leu His Phe Lys Asn Ile Val His 690 695 700 Cys Asp Leu Lys Pro Glu Asn Val Leu Leu Ala Ser Ala Asp Pro Phe 705 710 715 720 Pro Gln Val Lys Leu Cys Asp Phe Gly Phe Ala Arg Ile Ile Gly Glu 725 730 735 Lys Ser Phe Arg Arg Ser Val Val Gly Thr Pro Ala Tyr Leu Ala Pro 740 745 750 Glu Val Leu Arg Asn Lys Gly Tyr Asn Arg Ser Leu Asp Met Trp Ser 755 760 765 Val Gly Val Ile Ile Tyr Val Ser Leu Ser Gly Thr Phe Pro Phe Asn 770 775 780 Glu Asp Glu Asp Ile His Asp Gln Ile Gln Asn Ala Ala Phe Met Tyr 785 790 795 800 Pro Pro Asn Pro Trp Lys Glu Ile Ser His Glu Ala Ile Asp Leu Ile 805 810 815 Asn Asn Leu Leu Gln Val Lys Met Arg Lys Arg Tyr Ser Val Asp Lys 820 825 830 Thr Leu Ser His Pro Trp Leu Gln Asp Tyr Gln Thr Trp Leu Asp Leu 835 840 845 Arg Glu Leu Glu Cys Lys Ile Gly Glu Arg Tyr Ile Thr His Glu Ser 850 855 860 Asp Asp Leu Arg Trp Glu Lys Tyr Ala Gly Glu Gln Gly Leu Gln Tyr 865 870 875 880 Pro Thr His Leu Ile Asn Pro Ser Ala Ser His Ser Asp Thr Pro Glu 885 890 895 Thr Glu Glu Thr Glu Met Lys Ala Leu Gly Glu Arg Val Ser Ile Leu 900 905 910

Claims (7)

It contains an inhibitor of the expression or activity of protein kinase D1 as an active ingredient,
The expression inhibitor is any one selected from the group consisting of an antisense nucleotide complementarily binding to the mRNA of the protein kinase D1 gene, a short interfering RNA (RNA), and a short hairpin RNA (RNA),
The inhibitor of activity is CRT0066101,
As a pharmaceutical composition for inhibiting the growth of drug-resistant breast cancer stem cells,
The drug-resistant breast cancer is a drug-resistant breast cancer having resistance to a drug selected from the group consisting of doxorubicin, tamoxifen and fluvestrans, the composition.
The pharmaceutical composition according to claim 1, wherein the protein kinase D1 has an amino acid sequence represented by SEQ ID NO: 1.
According to claim 1, wherein the breast cancer is cancer stem cell markers CD44+ and CD24-, characterized in that the selection, drug-resistant breast cancer stem cell growth inhibition pharmaceutical composition.
The pharmaceutical composition of claim 1, wherein the protein kinase D1 expression or activity inhibitor suppresses β-catenin expression and inhibits GSK3α and GSKβ phosphorylation.
It contains an inhibitor of the expression or activity of protein kinase D1 as an active ingredient,
The expression inhibitor is any one selected from the group consisting of an antisense nucleotide complementarily binding to the mRNA of the protein kinase D1 gene, a short interfering RNA (RNA), and a short hairpin RNA (RNA),
The inhibitor of activity is CRT0066101,
As an anticancer adjuvant for preventing recurrence of drug-resistant breast cancer,
The drug-resistant breast cancer is a drug-resistant breast cancer having resistance to a drug selected from the group consisting of doxorubicin, tamoxifen and fluvestrans, anticancer adjuvant.
The anticancer adjuvant for preventing recurrence of drug-resistant breast cancer according to claim 5, wherein the protein kinase D1 has an amino acid sequence represented by SEQ ID NO: 1.
As a method for measuring protein kinase D1 expression or activity for providing information on prognosis of drug-resistant breast cancer comprising the step of measuring the expression or activity of protein kinase D1 in cells or tissues isolated from an individual with breast cancer,
If the expression or activity of the protein kinase D1 is higher than that of the normal control, it is determined as a drug-resistant breast cancer,
The drug-resistant breast cancer is a drug-resistant breast cancer having resistance to a drug selected from the group consisting of doxorubicin, tamoxifen, and fluvestrans.

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