WO2024065243A1

WO2024065243A1 - Drug for treatment of breast cancer and application thereof

Info

Publication number: WO2024065243A1
Application number: PCT/CN2022/121991
Authority: WO
Inventors: Xiaoling Xu; Jun Xu; Chuxia Deng
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2022-09-28
Filing date: 2022-09-28
Publication date: 2024-04-04
Anticipated expiration: 2025-03-28

Abstract

Provided herein is an application of an anti-PS antibody in combination with docetaxel and/or paclitaxel in a drug for the treatment of a breast cancer, belonging to the technical field of drugs. By means of the combination of the anti-PS antibody and docetaxel and/or paclitaxel, an expression phenotype of ATP11B low/Ptdss2 high is changed to the ATP11B high/Ptdss2 low to effectively overcome the metastasis process, which can not only reduce a primary tumor, but also greatly inhibit the metastasis of the primary tumor. Therefore, a new selective therapeutic strategy and drug are provided to prevent and treat breast cancers and their metastasis.

Description

Drug for Treatment of Breast Cancer and Application Thereof

Technical Field

The present invention belongs to the technical field of drugs, and more particularly, relates to an application of an anti-PS antibody in combination with docetaxel (DOC) and/or paclitaxel (PAC) in the treatment of breast cancers.

Background Art

Breast cancer is the most common type of cancers and also the second leading cause of cancer mortality among women in the world. While the majority of breast cancers occur in women without a clear family history, approximately 5-10% of breast cancers are hereditary. Breast cancer-associated genes include a breast cancer-associated gene 1 (BRCA1), a breast cancer-associated gene 2 (BRCA2), a p53 gene, an ATM gene and the like. Approximately 25-40% of all familial breast cancer cases are related to BRCA1.

Tumor metastasis is the cause of death in about 90% of cancer patients. Studies highlight that the tumor microenvironment (TME) plays very important roles in tumorigenesis and tumor metastasis. Mutations that cause altered gene expression in tumor cells can educate surrounding immune cells to establish or change the TME for the growth and metastasis of tumor cells. In the TME, exposed phosphatidylserine (PS) can be found on tumor cells, secretory microvesicles and tumor endothelial cells. PS is an evolutionarily conserved anti-cancer and immunosuppressive signal that prevents local and systemic immune activation through PS-related signaling. The PS-related signaling is highly dysregulated in the TME. By exploring PS as a non-apoptotic signal that maintains the outer leaflet of the cell membrane and promotes cancer metastasis, it may help to develop selective therapeutic strategies for breast cancer patients to prevent cancer metastasis. However, breast cancer is a tumor that is prone to metastasis. In most cases, metastasis has occurred without obvious clinical symptoms, which seriously affects the quality of life and longevity of patients. Therefore, the corresponding development of drugs for the prevention and treatment of breast cancer metastasis is of great significance to improve the survival rate of patients.

Technical Problem

To overcome the defects of the prior art, the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide an application of an anti-PS antibody in combination with docetaxel (DOC) and/or paclitaxel (PAC) in the treatment of breast cancers.

Technical Solution

To solve the above technical problem, the present invention adopts the following technical solutions:

a drug for the treatment of breast cancer, including at least three of an anti-PS antibody, docetaxel, paclitaxel, and carboplatin;

a drug for the prevention and treatment of breast cancer metastasis, comprising at least three of an anti-PS antibody, docetaxel, paclitaxel, and carboplatin;

an application of an anti-PS antibody in combination with docetaxel and/or paclitaxel in a drug for the treatment of breast cancers;

an application of an anti-PS antibody in combination with docetaxel and/or paclitaxel in a drug for the treatment of breast cancer metastasis; and

an application of an anti-PS antibody in combination with docetaxel and/or paclitaxel in a drug that blocks non-apoptotic phosphatidylserine-induced breast cancer metastasis.

Advantageous Effects

The present invention has the following beneficial effects: it is found in the present invention that no/low expression of ATP11B in conjunction with high expression of PTDSS2, which is negatively regulated by BRCA1, promotes breast cancer metastasis by increasing non-apoptotic PS populations on the outer leaflet of the cell membrane. Using the combination of the anti-PS antibody and docetaxel and/or paclitaxel, an expression phenotype of ATP11B ^low/Ptdss2 ^high is changed to the ATP11B ^high/Ptdss2 ^low to effectively overcome the metastasis process. Therefore, a new selective therapeutic strategy and drug are provided to prevent and treat metastasis in breast cancer patients.

Description of Drawings

FIG. 1 shows a flowchart and a schematic result diagram of genome-wide screening of tumor metastatic suppressors with a CRISPR-Cas9 sgRNA library in Brca1 MT 545 cells, wherein

A show a workflow diagram of screening tumor metastatic suppressors using CRISPR-Cas9 (GeCKOv2-sgRNA) library-infected 545 cells;

B shows representative images of lungs and livers from nude mice implanted with 545 cells only (control: n = 16) or 545 cells that are infected with the GeCKOv2 sgRNA library.

C shows enriched sgRNA reads count (10 and more than 10 for every sample) from primary tumors (n = 32), recurrent tumors (n = 15), and metastatic tumors (n= 16) from the lung, brain, liver, spleen, and kidney compared to the cells infected with the GeCKOv2 sgRNA library.

D shows a total of 117 potential human homologous tumor suppressors obtained from a Venn diagram analysis of 1521 potential human tumor suppressors and 1818 potential tumor metastatic supressors obtained from GeCKOv2 sgRNA library screen in vivo, wherein a total of 1521 genes are obtained from 9386 overlapping genes that have low expression compared to that of healthy donors, with 2881 genes related to poor survival outcomes (P < 0.05) when their expression is lower than that of the same genes in the TCGA-BRCA database;

E shows that the top 50 human homologous genes with sgRNA reads equal to or greater than 10 are found in all 15 recurrent tumors (Rec T), 48 blood samples, and 16 metastatic organ-specific genes, including the lung (Lung T), liver (Liver T), brain (Brain T), kidney (Kidney T), and spleen (Spleen T) in mice by Oncoplot;

FIG. 2 shows the metastasis of 545 cell tumors with the expression of sgATP11b in multiple organs.

A shows Venn diagram analysis of common genes in five metastatic organs, including the lung, liver, brain, spleen, and kidney through screening 117 genes from the GeCKOv2 sgRNA library.

B shows the enrichment of sgAT11b from five different metastatic organs compared with those of GeCKOv2 sgRNA library-transfected cells.

C shows the expression of ATP11b in wild-type mammary gland (WTMG), Brca1-MT mammary gland (MTMG), and tumor-adjacent mammary gland (Tumor Adj. MG) (n = 3 mice/group);

D shows the expression of ATP11b in wild-type tumors (WTTs), primary tumors from Brca1-MSK mice (MTPTs), and lung metastatic tissues (LMTs) from Brca1-MSK mice (n = 3 mice/group);

E shows ATP11B protein levels from the 545 cells without or with the expression of sgATP11b before injection (first two lanes), tumors from Brca1-WT breast tumors (WT) and Brca1-MSK mice (third and fourth lanes, n = 3 pairs of mice), and primary breast tumors (PTs) and metastatic tumors (MTs) from sgATP11b-545 nude mice (fifth and sixth lanes, n =861 3 pairs of mice);

F shows images of primary tumors with or without expression of sgATP11b (n=7 mice/group);

G shows representative images of lungs after 8 weeks fat pad implantation with parental 545 cells (Ctr) (n = 10 mice) in lungs (n = 13 mice), brain (n = 2 mice), spleen (n = 3 mice), liver (n = 4 mice), and kidney (n = 3 mice) from 545 cells expressing sgATP11b after eight weeks fat pad implantation.

H-J show representative images of the lung in the parental cell control group (Ctr-628) (H), sgATP11b-628 cells (I), and ATP11b-E186K-628 cells (J);

K shows a quantification diagram of metastatic nodules in (H-J);

FIG. 3 shows that PS displacement on the outer leaflet of the cell membrane is regulated by ATP11B and BRCA1 in an experimental example.

A shows PS displacement on the cell membrane by FACS with an anti-PS antibody in both WT and Brca1-MT primary mammary epithelial cells (n = 4 pairs), 545, MDA-MB41, and T47D cells without or with an expression of sgATP11B.

B shows quantification for panel in (A);

C shows apoptotic cells in both WT and Brca1 MT primary mammary epithelial cells (n = 4 pairs), 545, MDA-MB-436, and T47D cells without or with an expression of sgATP11B detected by FACS through the APO-BrdUTM Kit.

D shows quantification of apoptotic cells for the panel in (C);

E shows protein levels of cleaved Caspase 3 in WT (B477), Brca1-MT (G600) mammary epithelial cells, and 545 Brca1-MT tumor cells without or with an expression of sgATP11b;

F shows representative images of 628 parental cells, 628 cells with the expression of sgATP11b, overexpression (OE) of ATP11b, and OE Brca1 stained with PS antibody and DAPI imaged by ZEISS LSM 880 high-resolution microscope and Airyscan (n = 3 times);

G-H shows the expression of Brca1, ATP11b, and Ptdss2 in 545 cells without or with the expression of mBrca1 cDNA by qPCR (G) and protein levels by Western blots (n = 3 times) (H);

I-J shows the PS displacement on the cell membrane of 545 cells without or with the expression of mBrca1 cDNA by FACS analysis (I), and quantification (J) for (I) (n = 3 times);

K-L shows the expression of hBRCA1, ATP11B, and PTDSS2 in T47D cells without or with an expression of hBRCA1 cDNA by qPCR (K), or by Western (n = 3 times);

M-N show PS displacement on the cell membrane of T47D cells without or with an expression of HBrCl cDNA by FACS (M) and quantification (N) for (M) (n= 3 times);

FIG. 4 shows that Ptdss2 is negatively regulated by Brca1 in an experimental example of the present invention.

A shows the top 10 biological processes in Brca1-MT mammary tissues annotated by GO analysis by comparing RNA-seq outcomes from MTMG, PT, and lung metastatic tissues (LMT) of Brca1 MSK mice to WTMG (n = 3 mice/group);

B shows Ptdss2 expression in wild-type lung, metastatic and primary tumors (n = 3 mice/group);

C shows the expression of PTDSS2 in human breast cancer patients with or without metastasis compared to healthy donors in TCGA-BRCA database (healthy donors: n = 122, nonmetastatic patients: n = 623, metastatic patients: n = 9);

D shows Ptdss2 expression from bulk RNA-seq in lung, LMT, and PT compared to WT controls (n = 3 mice/group);

E shows protein levels of PTDSS2 in WTMG, MTMG, PT, and LMT by Western blots (n = 3 times);

F-G show protein levels of PTDSS2 in both mammary gland (F) and tumors (G) with age-matched WTMG (n = 3 mice) and MTMG (n = 10 mice), WT tumor (WTT) (n = 3 mice), and Brca1 MSK mice (n = 11 mice) by Western blots (n = 3 times);

H shows Ptdss2 promoter activity assay in G600, B477, and 545 cells without or with an expression of mBrca1 cDNA or mPtdss2 cDNA (n = 3 times);

I-J shows expressions of Brca1 and Ptdss2 by qPCR (I) and Western blots (J) in B477 cells without or with shBrca1 cDNA (n = 3 times);

FIG. 5 shows that overexpression of Ptdss2 and sgATP11b increases PS on the cell membrane in an experimental example of the present invention.

A-C show the distribution of PS under the effects of 545 cells expressing sgATP11b/OE-Ptdss2 (left), sgATP11b/OE-Ptdss2/shPtdss2 (right) in (A), and quantification of (A) in (B) at mRNA levels in (C) at the protein level.

D-F show the distribution of PS under the effects of MDA-MB-436 cells expressing sgATP11b/OE-Ptdss2 (left) and sgATP11b/OE-Ptdss2/shPtdss2 (right), and quantification of (D) in (E) at mRNA levels and in (F) at the protein level.

G-H shows the distribution of PS under the effects of sgATP11b/OE-Ptdss2 (left), sgATP11b/OE-Ptdss2/OE-Ptdss2-R235S (middle) and sgATP11/OE-PT DSS2-R35S (right) by FACS analysis (G) and quantification (H).

FIG. 6 shows that ATP11B ^low/PTDSS2 ^high enhances breast cancer metastasis in an experimental example of the present invention;

A shows representative metastatic images of primary breast tumor, lung, ovary, kidney, and abdominal fat from a 7-month-old Brca1-Trp53-MSK female mouse after intraductal injection of mixed sgATP11b and Ptdss2-GFP lentiviruses (n = 5 mice);

B shows validation results of sgATP11b DNA in primary tumor tissues and metastatic tissues, including the lung, liver, ovary, kidney, and abdominal fat tissues, as determined with specific primers for ATP11b by PCR;

C and D show results of 545 and 628 cells without or with the expression of sgATP11b by FACS analysis with a PS antibody;

E shows results of the protein levels of ATP11B in 545, 628, and MDA-MB-436 cells without or with the expression of sgATP11b by Western blots (n = 3);

F shows results of protein levels of ATP11B and PTDSS2 in 545 and 628 cells without or with expressing sgPtdss2, OE-ATP11b, OE-ATP11b/sgPtdss2, or sgATP11b/OE-Ptdss2 by Western blots (n = 3);

G shows representative images of PS displacement in/on the membranes of 628 cells without or with expressing sgATP11b, OE-Ptdss2, sgATP11b/OE-Ptdss2, or OE-ATP11b/sgPtdss2 by IF staining with an anti-PS antibody and imaged by super-resolution microscopy (n = 3);

H shows representative lung bright field (BF) images, GFP signal, and overlapped images showing the BF and GFP signals in nude mice (WT) three weeks after the mammary fat pad is implanted with 628 parental cells, or 628 cells expressing sgAT11b, or with OE-ATP11b in sgATP11b cell, or sgATP11b/OE-Ptdss2, or OE-ATP11b/sgPtdss2 (n = 3);

I-J show quantification diagrams for lung volumes (I) and GFP intensities (J) from the same cohort of mice shown in (H) (n = 8 mice/group);

FIG. 7 shows that a non-apoptotic PS signal enhances an immunosuppressive signal of Brca1/ATP11b double mutant primary tumor in the experimental example of the present invention;

A-C show the correlation of the expression of CD8 (A), ARG1 (B), and NOS2 (C) in human breast cancer patients with different expression levels of ATP11B and PTDSS2, as indicated in the NCBI-GEO database (170 patients with ATP11B ^high/PTDSS2 ^high expression, 59 patients with ATP11B ^high/PTDSS2 ^low expression, and 35 patients with ATP11B ^low/PTDSS2 ^high expression), and their 51 survival outcomes;

D shows a GSE21653 dataset;

E shows images of sections of primary tumors obtained from 545 Ctr mice and sgATP11b-545 mice stained by H&E and anti-TGF-b antibody (n = 5 mice/group);

F shows representative images of metastatic lungs by H&E, GFP, and TGF-b antibody from sgATP11b-545 mice (n = 3 mice/group);

FIG. 8 shows a schematic diagram of a result in an example of the present invention;

A-B shows the percentage of breast cancer patients with high or low ATP11B (A), and PTDSS2 (B) expression in the non-responder (n = 12) and responder (n = 14) groups;

C shows the percentage of patients with infiltration of tumor cells into lymph nodes from the same cohort of patients in (A);

D shows the protein levels of ATP11B and PTDSS2 in 628 and MDA-MB-436 cells after treatment with paclitaxel (PAC) or docetaxel (DOC) at a concentration of 2.4 nM for five days;

E shows the weights of tumors caused in sgATP11b/OE-Ptdss2-628 cells after treatments with control (Ctr), PS antibody (aPS), carboplatin (CAR), PAC, DOC, CAR+PAC+aPS, and CAR+DOC+aPS at day 21;

F-G shows the quantification of GFP intensities and representative images with BF, GFP intensity, the overlap of BF and GFP, and H&E-stained sections of lungs for mice in E (n = 6-15 mice/group);

H shows a heatmap of the gene expression of ATP11b and Ptdss2 genes in breast tumor and lung tissues from the same cohort of mice in (E-G) (n = 6-15 mice/group);

FIG. 9 shows a schematic diagram of a result in an example of the present invention;

A-B show protein levels of ATP11B and PTDSS2 in breast tumors (A) and lungs (B) treated with PAC in combination with CAR+PAC+aPS antibody (n = 4-5 mice/group); and

C-D shows protein levels of ATP11B and PTDSS2 in breast tumors (C) and lungs (D) treated with DOC in combination with CAR+DOC+aPS antibody (n = 4-5 mice/group).

Mode for Invention

To describe the technical content, implementing objects, and effects of the present invention in detail, the following description is given concerning the embodiments and the accompanying drawings.

The most critical concept of the present invention lies in that: an expression phenotype of ATP11B ^low/Ptdss2 ^high is changed to ATP11B ^high/Ptdss2 ^low to effectively overcome a metastasis process.

It is found in the present invention that no/low expression of ATP11B in conjunction with high expression of PTDSS2, which is negatively regulated by BRCA1, promotes tumor metastasis. Cells with low expression of ATP11B and high expression of PTDSS2 (ATP11B ^low/PTDSS2 ^high) are generally associated with poor prognosis and enhanced metastasis in breast cancer patients. An ATP11B ^low/PTDSS2 ^high phenotype is associated with increased levels of nonapoptotic phosphatidylserine (PS) on the outer leaflet of the cell membrane. This PS increase serves as a global immunosuppressive signal to promote breast cancer metastasis through an enriched tumor microenvironment with the accumulation of myeloid-derived suppressive cells (MDSCs) and reduced activity of cytotoxic T cells.

The present invention adopts the following technical solutions.

A drug for the treatment of breast cancer includes at least three an anti-PS antibodies, docetaxel, paclitaxel, and carboplatin as active ingredients, and other components as accessories.

Preferably, a drug for the treatment of breast cancer includes an anti-PS antibody, carboplatin, and docetaxel, or includes an anti-PS antibody, carboplatin, and paclitaxel.

A drug for the prevention and/or treatment of breast cancer metastasis includes at least three an anti-PS antibodies, docetaxel, paclitaxel, and carboplatin as active ingredients, and other components as accessories.

Preferably, a drug for the prevention and/or treatment of breast cancer metastasis includes an anti-PS antibody, carboplatin, and docetaxel, or includes an anti-PS antibody, carboplatin, and paclitaxel, and other components as accessories.

The above drug is a drug used in combination.

The anti-PS antibody, carboplatin in combination with docetaxel is applied in a drug for the treatment of breast cancer.

The anti-PS antibody, carboplatin in combination with paclitaxel is applied in a drug for the treatment of breast cancer.

The anti-PS antibody, carboplatin in combination with docetaxel and paclitaxel is applied in a drug for the treatment of breast cancer.

The anti-PS antibody, carboplatin in combination with docetaxel is applied in a drug for the prevention of breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with docetaxel is applied in a drug for the treatment of breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with docetaxel is applied in a drug for the prevention and treatment of breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with paclitaxel is applied in a drug for the prevention of breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with paclitaxel is applied in a drug for the treatment of breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with paclitaxel is applied in a drug for the prevention and treatment of breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with docetaxel and paclitaxel is applied in a drug for the prevention, carboplatin of breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with docetaxel and paclitaxel is applied in a drug for the treatment of breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with docetaxel and paclitaxel is applied in a drug for the prevention and treatment of breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with docetaxel is applied in a drug for the prevention of non-apoptotic phosphatidylserine-induced breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with docetaxel is applied in a drug for the treatment of non-apoptotic phosphatidylserine-induced breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with docetaxel is applied in a drug for the prevention and treatment of non-apoptotic phosphatidylserine-induced breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with paclitaxel is applied in a drug that prevents and blocks the non-apoptotic phosphatidylserine-induced breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with paclitaxel is applied in a drug that treats and blocks non-apoptotic phosphatidylserine-induced breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with paclitaxel is applied in a drug that prevents and blocks non-apoptotic phosphatidylserine-induced breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with docetaxel and paclitaxel is applied in a drug that prevents and blocks the non-apoptotic phosphatidylserine-induced breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with docetaxel and paclitaxel is applied in a drug that treats and blocks non-apoptotic phosphatidylserine-induced breast cancer metastasis.

The anti-PS antibody, carboplatin in combination with docetaxel and paclitaxel is applied in a drug for the prevention and treatment of non-apoptotic phosphatidylserine-induced breast cancer metastasis.

The application of the above-mentioned anti-PS antibody and carboplatin in combination with docetaxel and/or paclitaxel in the corresponding drug can be understood as the application of a drug used in combination.

Docetaxel may also be called Taxotere, DEX, or DOC.

In the above applications, the anti-PS antibody and the carboplatin are administrated in combination with the docetaxel and/or the paclitaxel.

Preferably, during the same treatment period, the anti-PS antibody, the carboplatin and the docetaxel and/or paclitaxel are administered on separate days.

Preferably, the anti-PS antibody, the carboplatin, the docetaxel, and the paclitaxel are all administered in the form of injections.

The carboplatin is administered by means of injection; and the carboplatin is diluted in water and administered intravenously at a concentration of 0.5 mg/mL together with 5% dextrose.

PAC is administered by means of injection; the paclitaxel is diluted in a 0.9% sodium chloride injection and administered intravenously at a concentration of 0.3 mg to 1.2 mg/mL.

DOC is administered by means of intraperitoneal injection; and DOC can be dissolved in ethanol and then administered intravenously at a concentration of 10 mg/mL.

As can be seen from the above description, the present invention has the following beneficial effects: it is found in the present invention that no/low expression of ATP11B in conjunction with high expression of PTDSS2, which is negatively regulated by BRCA1, promotes breast cancer metastasis by increasing non-apoptotic PS populations on the outer leaflet of the cell membrane. Using the combination of the anti-PS antibody, carboplatin and docetaxel and/or paclitaxel, an expression phenotype of ATP11B ^low/Ptdss2 ^high is changed to the ATP11B ^high/Ptdss2 ^low to effectively overcome the metastasis process, which can not only reduce a primary tumor but also greatly inhibit the metastasis of the primary tumor. Therefore, a new selective therapeutic strategy and drug are provided to prevent and treat metastasis in breast cancer patients.

Experimental examples

1. Materials

A 545-GFP cell line is derived from Brca1Co/Co;

MMTV-Cre (Brca1-MSK) mice, which develop only primary breast tumors and have minimal metastatic ability, are labeled with GFP.

A 628-GFP cell line is also derived from Brca1-MSK mice, which not only develop primary breast tumors but also develop multiple metastatic tumors in other organs, which are identified by GFP labels.

All the cells above are cultured with high glucose DMEM containing 10% FBS and antibiotics (100 µg/mL streptomycin and 100 U/mL penicillin, Gibco). All cell lines are cultured at 37°C in an atmosphere containing 5% CO ₂. All other human breast cancer cell lines are purchased from ATCC (American Type Culture Collection) company. T47D (Cat# HTB-133), MDA-MB-231 (Cat# CRM-HTB-26), MDA-MB-436 (Cat# HTB-130) and HEK293T (Cat# ACS-4500) cells are purchased from ATCC.

All mouse strains are preserved in the animal facility of the Faculty of Health Sciences, University of Macau.

2. Method

2.1 sgRNA lentiviral library preparation, viral Infection and labeling of GFP cell lines

Lentiviruses are generated according to the 2nd lentivirus package system using a standard procedure.

The titer of the virus is confirmed by the infection of 293T cells.

GeCKOv2 sgRNA from Addgene is packaged into lentiviral particles.

545 cells (minimally invasive metastatic breast tumor cells) and 628 cells (highly metastatic breast tumor cells) are infected with lenti-GFP viruses at an MOI (Multiplicity of Infection) of 10 for 48 hours, and GFP-positive cells are isolated by FACS and continued to grow.

545-GFP cells are infected with the lentiviral GeCKOv2 sgRNA library at an MOI of 0.1 for 48 hours, and the transfected cells are grown in a normal medium (DMEM+10% FBS) for 48 hours after the infected cells are selected with puromycin for 7 days. The transfected cells are then implanted into the mammary fat pads of nude mice with 1x10 ^-6 cells per fat pad, and 5 million cells are saved as a control. Primary breast tumors and metastatic tumors in the lung, liver, kidney, spleen, and brain are collected eight weeks after injection of the cells transfected with the GeCKOv2 sgRNA library.

2.2 Validation of metastatic tumor suppressors

Oligo sequences of candidate genes identified in the NGS analysis are cloned into the Lenti-V2 (vector 2), and the individual oligo sequence for sgRNA is packaged.

545-GFP cells infected with lentiviral particles containing the candidate genes are injected into the mammary fat pads of nude mice after puromycin selection for 7 days.

Primary tumors and metastatic tumors from the lung, liver, brain, kidney, and spleen are harvested several weeks after the injection. DNA from the tumor tissues is extracted, and Sanger sequencing is performed to confirm the indels produced by the specific sgRNAs.

Total RNA is extracted using a TRIzol solution in a Precellys Evolution tissue homogenizer, and the purity and concentration of the RNA are measured by using a NanoDrop™ 8000 spectrophotometer (Thermo Fisher Scientific). cDNA is reversely transcribed from qualified total RNA extracted from tumor tissues by using a QuantiTect Reverse Transcription kit.

Gene expression is determined by using a QuantStudio™ 7 Flex Real-Time PCR system and a SYBR-Green kit (TaKaRa company, Cat# RR820A), and the relative expression of mRNA is calculated by the 2 ^- ^ΔΔ ^Ct method.

2.3 Data and statistics

All the statistical analysis is carried out by R 3.4.3 software. For the comparison of multiple groups, 1-way ANOVA and Bonferroni’s multiple-comparison test are used. Survival of patients in 2 groups are analyzed by R Logrank test. * Equals P < 0.05, ** equals P < 0.01, and *** equals P < 0.001. P value lower than 0.05 is considered as a significance.

The RNA-seq data generated in this study has been deposited in the National Center for Biotechnology Information Sequence Read Archive database under accession: PRJNA753220.

3. Results (Referring to FIGS. 1 to 7)

3.1 Identification of metastatic suppressors using CRISPR-Cas9-mediated genome-wide screening

A cohort of 207 tumor-bearing Brca1-MSK mice are analyzed to find that 47 mice (22%) have developed metastasis in multiple organs. To identify metastatic suppressors, Brca1 mutant (Brca1-MT) 545 cells, which are isolated from a primary breast tumor of a Brca1-MSK mouse, are used. These cells are labeled with luc-GFP and implanted orthotopically into the mammary fat pad of nude mice. No metastasis is detected in these cells when the primary tumors reach approximately 2 cm in diameter eight weeks after implantation into fat pad.

Next, the 545-GFP cells are infected with a CRISPR-Cas9 (GeCKOv2 sgRNA) library containing 130,209 sgRNAs that target 20,611 genes (6 sgRNAs/gene). After selection with puromycin for 7 days, the cells are implanted into the mammary fat pad of 23 nude mice, and samples including primary tumors, recurrent tumors, blood, and metastatic tumors are harvested for next-generation sequencing (NGS). Metastatic signals are detected in the lung of all mice that are implanted with 545-GFP-GeCKOv2 cells, and in several other organs including the brain, liver, spleen, and/or kidney. Upon the removal of the primary tumor, the mice exhibit, on average, 3-fold more metastatic nodules in the lung and some other organs. As a control, the parental 545 cells that are not infected with the library are implanted into the mammary fat pad of 16 mice, and no metastatic GFP signal is observed when their primary tumors reach approximately 2 cm. It is indicated that the action of the sgRNAs in the CRISPR-Cas9 library disrupts metastatic suppressor genes in the genome and that loss of function of these genes converts minimally metastatic cells to highly metastatic cells in different organs.

Next, next-generation sequencing is performed on 111 samples, including 32 primary tumors, 15 recurrent tumors, and 16 metastatic tumors eight weeks after implantation of 545-GFP-GeCKOv2 cells and 48 blood samples obtained from two weeks to eight weeks after implantation. Compared with cells prior to implantation, which contain sgRNA for 14806 genes, sgRNAs representing a total of 2593 genes are identified from 111 samples, including 1818 genes from metastatic tumors, and 162 genes shared among all types of samples (i.e., cells from primary tumors, recurrent tumors, blood samples, and metastatic tumors) with a pattern of gradual reduction in the cells obtained from primary, recurrent tumors, and five targeting organs.

To define human homologous metastatic suppressors in the mouse screening process, tumor suppressors are first identified in a TCGA-BRCA dataset that include data on 1100 breast cancer patients and 112 healthy donors. 1521 genes are found to show not only low expression compared with their expression in mammary tissues from healthy donors (P < 0.05) but also are linked to poor survival outcomes compared to the same genes with relatively high expression (P < 0.05). After comparing the 1521 human genes with the 2593 candidate genes identified across all samples, 160 human homologous genes are found. The top 50 genes that might function in driving both tumorigenesis and metastasis are summarized according to the frequency of sgRNA appearance in reads. 1521 human genes are also compared with the 1818 genes identified from metastatic tumors in screening and 117 human homologous genes that might be involved in metastatic processes are identified. The top 50 potential metastatic genes are ranked based on the frequency of their sgRNA appearance reads in the recurrent cancer, blood, and metastatic organs as determine by OncoPrint. From the analysis performed through two different approaches, it is found that ATP11b is ranked at the top. The analysis of the genome-wide screen shows that some genes appear more frequently among different samples, and other genes exhibit organ-specific patterns; and these differential distribution patterns may reflect the different roles of these genes in cancer metastasis.

3.2 Investigation of the functions of ATP11b in inhibiting tumor metastasis in vivo

ATP11b is a potent metastatic suppressor associated with Brca1 deficiency. The sgRNA reads enriched with 117 human homologous genes in five metastatic organs, including lung, liver, spleen, brain, and kidney are compared. This analysis reveals that ATP11b-sgRNA is the only sgRNA that is highly enriched in all five organs, while some other genes appear in 2-3 organs or only one organ. ATP11b is a flippase that is critical for the asymmetrical distribution of phosphatidylserine (PS) on the inner leaflet cell membrane. Loss of ATP11b function allows PS to flip to the outer leaflet of the cell membrane to generate a global immunosuppressive signal.

To verify the expression of ATP11b in mammary tissues of wild-type (WT) and Brca1-MSK mice, tumor tissues from the WT mouse mammary gland (WTMG), MT mammary gland (MTMG), MT tumor-adjacent mammary gland (Tumor Adj. MG), and tumor tissues from WT, MT primary tumors (PTs), and MT metastatic tumors (MTMTs) are extracted and sequenced. The data shows that the expression level of ATP11b in MTMG is only 25% that in WT mammary glands (WTMGs) and is even lower in tumor-adjacent mammary tissues. ATP11b expression in Brca1-mutant primary tumors (MTPTs) is only 10% that in WT tumors (WTTs), as revealed by qPCR (quantitative polymerase chain reaction) and is much lower in mutant metastatic tumor (MTMT). Consistent with RNA expression, it is further reduced in sgATP11b-545 cells and metastatic tumors (MT) from sgATP11b-545-carrying mice compared to its level in tumors from WT and Trp53+/Co; MMTV-Cre (Trp53-MSK) mice, i.e, Brca1WT, and their respective controls.

Parental 545 cells and sgATP11b-545 cells are implanted into the mammary fat pad of nude mice. The metastatic phenotypes are recapitulated in multiple organs 8 weeks after the implantation of sgATP11b-545 cells, but not parental 545 cells, although there is no obvious difference in the size of the primary tumors. Among the mice with a metastatic phenotype, thirteen have lung metastasis, two have brain metastasis, three have spleen metastasis, three have kidney metastasis, and four have liver metastasis. Sanger sequencing confirms targeted mutation of ATP11b. Consistently, analyzing the GEO (Gene Expression Omnibus) dataset GSE61304 reveals that breast cancer patients with low levels of ATP11B have worse survival outcomes after diagnosis. These data demonstrate that ATP11B is a potent metastatic suppressor and that its disruption enhances tumor metastasis to many organs.

An E186K mutation is generated in mouse ATP11b, which equivalents to human E180K in the catalytic domain of ATP11B, and the effects of sgATP11b and ATP11b-E186K in 545 cells are compared. The fluorescence-activated cell sorting (FACS) analysis with a PS antibody shows that the transfection of sgATP11b or ATP11b-E186K achieves comparable increase of PS displacement on the outer leaflet cell membrane in the transfected cells. Similar finding is observed in another Brca1-MT breast tumor cell line, 628, which is derived from a Brca1-MSK mouse with multiorgan metastasis. This line has higher level of PS than 545 and can form metastatic foci in the lung after four weeks of fat pat implantation. The data also reveals comparable increase of PS in the 628 cells transfected with sgATP11b or ATP11b-E186K.

Next, Ctr-628, sgATP11b-628, and ATP11b-E186K-628 cells are implanted into the fat pad of nude mice (n = 8/group) and the recipient mice are sacrificed 3 weeks after the implantation, when the Ctr-628 generates very few or no visible metastatic nodules in the lung. In contrast, both sgATP11b-628 and ATP11b-E186K-628 significantly enhance lung metastasis. These data suggest that the impaired catalytic function of ATP11b is responsible for the enhanced PS displacement and tumor metastasis. Since ATP11b-E186K-545 and ATP11b-E186K-628 cells still contain WTATP11b protein, over-expressed ATP11b-E186K protein may function to block the function of WT protein in a manner of explicit inactivation.

3.3 ATP11B ^low and PTDSS2 ^high enhance breast cancer metastasis

To explore whether OE-Ptdss2 and sgATP11b have a cooperative effect on tumor metastasis in Brca1-Trp53-MSK mice, intraductal injection of virus expressing sgATP11b, mixed viruses expressing either sgATP11b or Ptdss2-GFP or control lenti-v2 virus is performed on 2-month-old Brca1Co/Co; p53+/Co; MMTV-Cre (Brca1-MSK;Trp53-MSK) mice. Since approximately 20% of these mice develop breast tumors at approximately 9-12 months of age, which serves as a reference for endogenous tumor metastasis, and tumor metastasis is examined 5 months after intraductal infection with lentiviruses with different genotypes. The experimental data reveals that GFP metastatic signals are detected in multiple organs, including the lung, liver, abdominal fat, kidney, ovary, and mammary tissues, in all five primary tumor-bearing mice injected with mixed viruses, whereas only one mouse with lung metastasis is observed among four primary tumor-bearing mice expressing only sgATP11b, and no metastasis is observed in the control lenti-v2-injected mice. Deletions within the ATP11b gene are confirmed by Sanger sequencing of samples obtained from three different metastatic organs, and mutations in ATP11b or deletions in Brca1-MT cells are detected in both primary tumors and metastatic tumors. Lungs in mice injected with a mixture of viruses demonstrate that loss of function of ATP11b together with OE-Ptdss2 in mammary epithelial cells dramatically enhances metastasis.

To further investigate the effects of ATP11b and Ptdss2 on tumor metastasis, GFP-628 cells are used, in which a metastatic GFP signal can be detected in the lung four weeks after orthotopic injection because 628 cells have a significantly higher PS population on the cell membrane than 545 cells, as revealed by FACS analysis, and lower ATP11B protein levels than 545 cells, as determined by Western blotting. The levels of the PS population on the cell membrane are further increased in 628 cells expressing sgATP11b.

Next, the PS location on the cell membrane and metastatic signals in nude mice are monitored as detected in 628-GFP cells with expressions of different combination of ATP11b and Ptdss2 expression, including 628-GFP-Ctr, sgATP11b, OE-Ptdss2, OE-ATP11b, sgATP11b/OE-Ptdss2, and OE-ATP11b/sgPtdss2, both in vitro and in vivo. The PS population location on the outer cell membrane is increased in the 628 cells with the expressions of sgATP11b and OE-Ptdss2 compared to the 628-GFP control cells, and the increase is much more in same cells with the expression of the sgATP11b/OE-Ptdss2. In contrast, the PS displacement to the outer membrane is greatly reduced in the 628-GFP cells with OE-ATP11b/sgPtdss2. GFP metastatic signals in the lung are detected by measuring 628-GFP, and for every group with sgATP11b expression, the GFP intensity is increased, but it is greatly reduced in the group of OE-ATP11b cells three weeks after implantation in the mammary fat pad. Notably, the lung volume in the sgATP11b/OE-Ptdss2 and sgATP11b groups is increased significantly with strong GFP signals compared with other groups, even though there are no obvious difference in the growth of primary tumors in all groups. In contrast, the metastatic signals and lung volumes in the groups with OE-ATP11b are minimized compared to the groups of mice expressing either sgATP11b or sgATP11b/OE-Ptdss2. The same effect of ATP11b on metastasis is also observed in EMT6 cells expressing sgATP11b, demonstrating that increased PS signal on the outer leaflet can also enhance metastasis in Brca1-WT mice.

Next, the inventors investigate if ATP11B ^low/PTDSS2 ^high state can be reversed by over expressing catalytically dead PTDSS2. Using software (https://iupred2a.elte.hu/plot_new), it is found that PSS domain in PTDSS2 may be a potential catalytic domain. The function of PTDSS2-R235S in mouse is then tested, which corresponds to human PTDSS2-R313C. The data reveals that OE-PTDSS2-R235S can override 628 and EMT6 cells. With respect to the potential effect of PTDSS2-R235S in cancer metastasis, the data shows that OE-PTDSS2-R235S completely suppresses the lung metastasis induced by sgATP11b/OE-Ptdss2 in 628 cells. These data demonstrate that constitutive exposure of nonapoptotic PS on the outer leaflet of the cell membrane also requires the catalytic function of PTDSS2, which enhances breast cancer metastasis. See FIG. 1 for results.

Example 1

A cohort of patients taking docetaxel treatment, including 12 non-responders and 14 responders are investigated, and results are shown in FIG. 2-A, FIG. 2-B and FIG. 2-C.

All 14 responders exhibit the ATP11B ^high/PTDSS2 ^low expression patterns, the majority (12/14) shows low expression of N-cadherin, and none shows tumor cell infiltration into their lymph nodes. In contrast, mixed expression patterns of these genes are observed in the non-responders, and most non-responders (11/12) have tumor cell infiltration to their lymph nodes and express either high ATP11B or high N-cadherin protein levels, showing that they are not sensitive to drug treatment. These observations suggest that in the responder group, treatment with these drugs may either reverse the expression of these two genes or that patients with ATP11B ^high/PTDSS2 ^low expression pattern are sensitive to drug treatment.

Example 2

Paclitaxel (PAC) or docetaxel (DOC) having a concentration of 2.4 is used to continuously treat 628 and MDA-MB-436 cells. The protein levels of ATP11B and PTDSS2 in 628 and MDA-MB-436 cells are detected after the treatment for five days. The results are shown in FIG. 2-D.

Example 3

The mice having tumors caused in sgATP11b/OE-Ptdss2-628 cells are respectively treated for 21 days with control (Ctr), aPS antibody (aPS), carboplatin (CAR), PAC, DOC, CAR+PAC+aPS, and CAR+DOC+aPS, each group including 6-15 mice, wherein the weight of each mouse is 20-22g.

For a group treated with a single compound, the drugs are administrated by means of peritoneal injection every other day, wherein the concentration of each drug is 5 mg/kg.

For a group treated with a combination treatment of a plurality of compounds, the drugs are administrated twice a week and a PS antibody is given at day 7 and day 14 (200 µg per mouse).

The carboplatin is administered by means of intraperitoneal injection at a concentration of 5 mg/kg.

The PAC is administered by means of peritoneal injection at a concentration of 5 mg/kg.

The DOC is administered by means of peritoneal injection at a concentration of 5 mg/kg.

At day 21 after treatment, the weights of the tumors are detected, and the results are shown in FIG. 2-E, FIG. 2-F, FIG. 2-G and FIG. 2-H.

Example 4

ATP11b ^low/Ptdss2 ^high model mice in different groups (4-5 mice/group) are subjected to drug treatment with PAC, CAR+PAC+aPS antibody, DOC, and CAR+DOC+aPS antibody. After treatment, the results for breast tumors and lungs of the mice are indicated in protein level testing of ATP11B and PTDSS2. The results are shown in FIG. 3. The administration ways and dosages are the same as those of Example 3.

The experimental results in Examples 1-4 reveal that monotreatment with these four compounds separately does not reduce the weight of primary tumors, while the combination treatment reduces the size of the primary tumors, suggesting that the combination treatment may contribute to reducing and delaying the progression of metastasis.

The GFP metastatic signals in lung are greatly reduced in all the monotreatment groups and the GFP signals are reduced much more by the combined treatment administered to mice with ATP11b ^low/Ptdss2 ^high tumors compared to the nontreatment group which has a widespread strong GFP signal. For all cases, improved lung structures are observed but no apparent effect on body weight or spleen or liver volume is observed.

The expression of ATP11b and Ptdss2 is analyzed by aPCR in mouse models administrated with drug treatment as determined at the gene expression and protein levels. It is found that both the single treatment and the combination treatment reverse the expression of ATP11b ^low/Ptdss2 ^high to ATP11b ^high/Ptdss2 ^low in breast and lung tissues in mice models. This observation is confirmed at the protein level in both the primary tumors and lungs in the same cohort of mice. Data demonstrates that PAC or DOC treatment reduces the gene expression of ATP11b ^low/Ptdss2 ^high and increases that of ATP11b ^high/Ptdss2 ^low at both the mRNA and protein levels. The triple-combination treatment not only reduces the primary tumors but also greatly inhibits their metastasis.

The above are only the examples of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent replacements made by using the contents of the description and drawings of the present invention, or directly or indirectly applied in other related technical fields, are similarly included in the scope of patent protection of the present invention.

Claims

A drug for the treatment of a breast cancer, characterized by comprising at least three of an anti-PS antibody, docetaxel, paclitaxel, and carboplatin.
The drug for the treatment of the breast cancer according to claim 1, characterized by comprising an anti-PS antibody and carboplatin, and docetaxel or paclitaxel.
A drug for the prevention and/or treatment of breast cancer metastasis, characterized by comprising at least three of an anti-PS antibody, docetaxel, paclitaxel, and carboplatin.
The drug for the prevention and/or treatment of breast cancer metastasis according to claim 3, characterized by comprising an anti-PS antibody and carboplatin, and docetaxel or paclitaxel.
An application of an anti-PS antibody and carboplatin in combination with docetaxel and/or paclitaxel in a drug for the treatment of a breast cancer.
An application of an anti-PS antibody and carboplatin in combination with docetaxel and/or paclitaxel in a drug for the prevention and/or treatment of breast cancer metastasis.
An application of an anti-PS antibody and carboplatin in combination with docetaxel and/or paclitaxel in a drug for the prevention and/or treatment of non-apoptotic phosphatidylserine-induced breast cancer metastasis.
The application according to any one of claims 5 to 7, characterized in that the anti-PS antibody and the carboplatin are administrated in combination with docetaxel and/or paclitaxel.
The application according to any one of claims 5 to 7, characterized in that the anti-PS antibody, the carboplatin and the docetaxel and/or paclitaxel are administered on separate days.
The application according to any one of claims 5 to 7, characterized in that the anti-PS antibody, the carboplatin, the docetaxel and/or the paclitaxel are all administered in the form of injections.