FZD6 targeted inhibitor capable of effectively inhibiting progress of gallbladder cancer

Abstract

The invention relates to the field of biological medicines, and discloses a targeted inhibitor capable of effectively inhibiting the progress of gallbladder cancer and remarkably improving the lifetime and prognosis of a patient. Through single-cell transcriptome data of a gallbladder cancer patient, single-cell transcriptome data of gallbladder cancer organoid, multiple immunofluorescence experiments, real-time quantitative PCR of gallbladder cancer cell lines, immunohistochemistry of gallbladder cancer tissues and organoid, gallbladder cancer whole exon data and inhibitor agonist experiments, it is found that compared with para-carcinoma, the single-cell transcriptome data of the gallbladder cancer patient, the single-cell transcriptome data of the gallbladder cancer organoid, the single-cell Differential genes of gallbladder cancer epithelial cells in cancer tissues are enriched to a WNT pathway, crosstalk is generated between the gallbladder cancer epithelial cells mainly through a WNT7A-FZD6 ligand and receptor pair in a WNT signal pathway, and inflammatory fibroblasts and epithelial cells can perform intercellular communication through a WNT5A-FZD6 ligand and receptor pair in an ncWNT signal pathway.

Description

Technical Field

[0001] The present invention relates to the field of targeted inhibitors, and in particular to a targeted inhibitor capable of effectively inhibiting the progression of gallbladder cancer, and its application and medicine. Background Art

[0002] Gallbladder cancer is a highly lethal bile duct cancer that is often diagnosed in the late stage and has an overall poor prognosis. According to cancer statistics for 185 countries in 2020, the number of cases of gallbladder cancer was 115,949 and the number of deaths was 84,695. The incidence of gallbladder cancer is related to geographical location, with the incidence in Asia reaching 70.8% of the world's total. Gallbladder cancer is more common in women, with a global incidence of 1.4 per 100,000 women and 0.89 per 100,000 men. The incidence of gallbladder cancer increases with age and is associated with gallstones, gallbladder polyps, cholecystitis, obesity, metabolic syndrome, etc. Gallbladder cancer is a highly invasive cancer. Gallbladder cancer has an insidious initial onset, strong heterogeneity, high malignancy, strong invasiveness, and is prone to metastasis and spread. Only about 10% to 20% of patients can be diagnosed early, and the 5-year survival rate of patients with advanced gallbladder cancer is only 5% to 15%.

[0003] At present, surgical resection is the most effective strategy for the treatment of gallbladder cancer, but the vast majority of patients have metastases when they seek medical treatment and lose the opportunity for surgery. Whether combined radiotherapy and chemotherapy can benefit patients with advanced gallbladder cancer is still controversial. Gemcitabine combined with platinum is the first-line chemotherapy regimen for the treatment of gallbladder cancer. Although it can improve the prognosis of patients, prolong overall survival and reduce recurrence to a certain extent, it is very easy to develop drug resistance. Although non-specific targeted inhibitors for VEGF and PD1 / PDL1 can inhibit tumors to a certain extent, the overall treatment situation is not ideal. Therefore, it is urgent to study specific targeted inhibitors for gallbladder cancer progression and chemotherapy resistance through the biological behavior of gallbladder cancer and the molecular mechanism of tumor occurrence. The WNT pathway plays an important role in regulating stem cell self-renewal and cell fate. Under normal circumstances, the activation of the WNT pathway is strictly controlled by some tumor suppressor genes. However, most tumors are often accompanied by the inactivation of tumor suppressor genes. The inactivation of tumor suppressor genes leads to the continuous activation of the WNT pathway, which gives the tumor the ability to self-renew, but also leads to the occurrence of treatment resistance. Currently, the regulatory role of the WNT pathway in the development of gallbladder cancer is still unclear, and specific targeted inhibitors need to be studied. Summary of the invention

[0004] The main purpose of the present invention is to solve the problem that the drugs for treating gallbladder cancer progression are limited in the prior art, resulting in short survival and poor prognosis of patients.

[0005] A targeted inhibitor that can effectively inhibit the progression of gallbladder cancer, wherein the targeted inhibitor is an inhibitor targeting the WNT pathway receptor FZD6.

[0006] The targeted inhibitor object FZD6 is a key receptor for WNT and ncWNT, which is highly expressed in gallbladder cancer epithelium.

[0007] The targeted inhibitor object FZD6 is highly expressed in gallbladder cancer organoids and cell lines.

[0008] The targeted inhibitor object, activator of FDZ6, affects the growth of gallbladder cancer organoids.

[0009] Gallbladder cancer is a type of cancer with high mutation in the WNT pathway, and epithelial cells with high expression of WNT7A are enriched in the WNT pathway.

[0010] The present invention also relates to the use of a targeted inhibitor that can effectively inhibit the progress of gallbladder cancer in the preparation of drugs for preventing and treating tumors.

[0011] The dosage form of the drug is any pharmaceutically therapeutically acceptable dosage form.

[0012] The dosage of the drug is any therapeutically acceptable dosage.

[0013] A drug for preventing or treating the progression of gallbladder cancer, comprising a targeted inhibitor and a pharmaceutically acceptable carrier.

[0014] The excellent effects of the present invention are:

[0015] The researchers of the present invention have found through research that FZD6 is a key receptor in the WNT pathway of gallbladder cancer, and gallbladder cancer is a cancer with a high mutation of the WNT pathway gene. FZD6 targeted inhibitors provide a new drug for the treatment of gallbladder cancer, which has a wide range of application prospects and great promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 , A. Cell annotation of single-cell transcriptome of gallbladder cancer, where A. All cell annotations of single-cell transcriptome of gallbladder cancer. B. T_NK cell annotations of single-cell transcriptome of gallbladder cancer. C. Myeloid cell annotations of single-cell transcriptome of gallbladder cancer. D. Fibroblast cell annotations of single-cell transcriptome of gallbladder cancer. E. Epithelial cell annotations of single-cell transcriptome of gallbladder cancer.

[0017] Figure 2 GO enrichment circle plot of upregulated genes in epithelial cell cancer;

[0018] Figure 3, A. Communication heat map of epithelial cells, T cells, macrophages and fibroblasts in cancer and adjacent tissues. Outgoing provides ligands and incoming provides receptors. B. Display of intercellular communication strength of three signaling pathways, WNT, ncWNT and FASLG, in cancer tissues. Thick lines represent strong communication, and thin lines represent weak communication. C. Display of intercellular communication ligands and receptors of three signaling pathways, WNT, ncWNT and FASLG, in cancer tissues.

[0019] Figure 4 In the top row of pictures, the horizontal line represents 50 μm, and in the bottom row of pictures, the horizontal line represents 20 μm.

[0020] Figure 5 A. Dot plot of classical marker genes in published data of gallbladder cancer. B. UMAP plot of cell annotation in published data of gallbladder cancer. C. Dot plot of classical marker genes in epithelial cells in published data of gallbladder cancer

[0021] Fig. D. Annotated UMAP map of epithelial cells in published data of gallbladder cancer. E. Analysis of the expression of 10 FZDs in cancer tissues and adjacent tissues. F. RT-qPCR analysis of the expression of FZDs in three gallbladder cancer cell lines.

[0022] Figure 6 , A. Culture of gallbladder cancer organoids. The horizontal line represents 200μm. B. Sample UMAP of single-cell transcriptome sequencing of gallbladder cancer organoids. C. UAMP of EPCAM and WNT7A expression in gallbladder cancer organoids. D. Cells with high expression of WNT7A are enriched in the WNT pathway in organoids and cancer tissues. E. GSEA analysis of cells with high expression of WNT7A in organoids and cancer tissues.

[0023] Figure 7 , A. GO enrichment circle diagram of genes that are mutated in at least two datasets in the cBioPortal website gallbladder cancer dataset. B. GO enrichment circle diagram of genes that are mutated in common between the cBioPortal website gallbladder cancer dataset and our gallbladder cancer dataset. The mutation display of WNT pathway-related genes in our WES data in the CB diagram. .

[0024] Specific process of invention

[0025] The terms "first", "second", "third", "fourth", etc. (if any) in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "including" or "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0026] In this embodiment, the FZD6 targeted inhibitor is an inhibitor targeting the WNT pathway ligand FZD6.

[0027] FZD6 is a gene encoding a WNT pathway receptor and plays an important role in the activation of the classical and non-classical WNT pathways. The occurrence of tumors is often accompanied by excessive activation of the WNT pathway. Antibodies are proteins produced by the immune system that can specifically bind to antigens. Monoclonal antibodies (mAbs) are antibodies produced by a single cell clone and are highly specific and consistent.

[0028] The researchers of the present invention have found through research that FZD6 is a key receptor in the WNT pathway of gallbladder cancer, and gallbladder cancer is a cancer with a high mutation of the WNT pathway gene. Anti-FZD6 mAb, as a FZD6 targeted inhibitor, provides a new drug for the treatment of gallbladder cancer, which has a wide range of application prospects and great promotion and application value.

[0029] The content of the present invention is described in detail below in conjunction with specific drawings. Experimental methods without specific conditions are usually based on conventional experimental scheme conditions, such as the conditions described in textbooks, experimental guides, and product instructions, or according to the conditions recommended by the manufacturer, which are mastered or easily obtained by researchers of related technologies in this field. The following embodiments are only preferred embodiments of the present invention and do not limit the present invention. For researchers of related technologies in this field, the present invention can have various conditions and schemes for selection and optimization. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0030] The present invention performed scRNA sequencing on 11 samples from 4 patients with gallbladder cancer, including 4 cancer samples (T), 3 paracancerous samples (PT), 3 peripheral blood samples (PB) and 1 lymph node sample (LN). After cell quality control, a total of 90,770 cells were retained for subsequent analysis. After clustering the cells, the scRNA data was defined based on the expression of classic genes of cell types and divided into 11 cell subgroups ( Figure 1 A). Then, T_NK, myeloid cells, fibroblasts, and epithelial cells were reclassified and annotated ( Figure 1 ). When analyzing epithelial cells, it was found that the genes upregulated in MUC5B epithelial and KRT17 epithelial cancer tissues were enriched in pathways such as angiogenesis, epithelial cell differentiation, typical WNT signaling pathway, and cell migration ( Figure 2 ).

[0031] In order to extract the cell communication between important cell types in gallbladder cancer, CellChat software was used to explore the cell crosstalk between epithelial cells and other cells. In addition to the MK signaling pathway showing that epithelial cells and inflammatory fibroblasts can communicate with multiple cell types, three signaling pathways including WNT, ncWNT and FASLG were also present in cancer tissue samples ( Figure 3 A). The WNT signaling pathway mainly appears in MUC5B epithelial cells and KRT17 epithelial cells, the ncWNT signaling pathway mainly appears in epithelial cells and inflammatory fibroblasts, and the FASLG signaling pathway mainly appears in CD8 T cells and inflammatory fibroblasts ( Figure 3 B). We used dot plots to show the specific ligand-receptor pairs that increase in cancer tissues in these three signaling pathways, such as WNT7A-FZD6 in the WNT signaling pathway, WNT5A-FZD6 in the ncWNT signaling pathway, and FASLG-FAS in the FASLG signaling pathway. Figure 3 C). Using multiple immunofluorescence, we verified the expression of the ligand receptors involved in these three signaling pathways in gallbladder cancer tissues ( Figure 4 ), where EPCAM is used to indicate epithelial cells, COL1A1 is used to indicate fibroblasts, and CD8A is used to indicate CD8 T cells.

[0032] To further verify this, we reanalyzed the single-cell transcriptome data of gallbladder cancer that our team had published, and analyzed the expression of classical genes in cell types ( Figure 5 A), cell annotation of published scRNA data ( Figure 5 B). Then, based on the expression of differentially expressed genes and marker genes in clustering ( Figure 5 C), annotated epithelial cells from published data ( Figure 5D). Since the tissue types of published data are only cancer tissues and paracancerous tissues, we integrated our cancer tissue and paracancerous tissue scRNA data with the published data. Then, based on the previous results of epithelial cell subpopulation annotation, we analyzed the expression of 10 human FZDs in cancer and paracancerous tissues MUC5B epithelial cells and KRT17 epithelial cells, and found that FZD6 was highly expressed in cancer tissues ( Figure 5 E). Using RT-qPCR, we analyzed the expression of 10 human FZDs in three gallbladder cancer cell lines (OCUG, NOZ, and GBC) and found that FZD6 was also highly expressed in the cell lines ( Figure 5 F).

[0033] Organoids can retain histological and genetic characteristics that are highly similar to those of organs in the body, and the formed tissues have the potential for stem cell proliferation and differentiation. Tumor-derived organoids can be used for drug screening and have important applications in the field of precision medicine for tumors. Using fresh gallbladder cancer tissue, we successfully cultured gallbladder cancer organoids ( Figure 6 A) and subjected it to single-cell transcriptome sequencing ( Figure 6 B), these cells all express EPCAM and are defined as epithelial cells, and some of them express WNT7A ( Figure 6 C). We analyzed the GSEA enrichment analysis of WNT7A-expressing and -non-expressing epithelial cells in organoids and cancer tissues and found that they could be enriched in the WNT signaling pathway ( Figure 6 D), in addition, they can also be enriched in epithelial-mesenchymal transition, inflammatory response, TNFA signaling and P53 pathway ( Figure 6 E).

[0034] cBioPortal is a cancer genome website that can be used to explore, visualize and analyze cancer genome data. On the cBioPortal website (https: / / www.cbioportal.org / ), search for 'gallbladder cancer' and you will find three datasets on gallbladder cancer mutations. Through analysis, we found that there are 220 genes that have mutations in at least two of the three datasets. Then, we performed GO enrichment analysis on these 220 genes and found that they were enriched in the WNT-related pathway ( Figure 7 A). We then performed WES sequencing on 11 patients with gallbladder cancer and combined it with the WES data previously published by our team, analyzing a total of 19 samples. We intersected the mutated genes in our data with the three cBioPortal datasets and found that 566 genes in our data intersected with them. We performed GO enrichment analysis on these 566 genes and found that they could also be enriched in the WNT pathway, with a total of 22 related genes ( Figure 7B). Using maftools, we plotted the mutations of these 22 genes in our WES data and found that 13 of the 19 samples had mutations. In addition, WNK1 and WNK2 had the highest mutation rates (21%), AXIN2 had a mutation rate of 16%, and CTNNB1 and TCF7L2 had a mutation rate of 11% ( Figure 7 C) These results indicate that gallbladder cancer is a type of cancer with high mutations in the WNT pathway.

Claims

1. A targeted inhibitor that can effectively inhibit the progression of gallbladder cancer, characterized in that: The targeted inhibitor is a targeted inhibitor of FZD6, a key receptor of the WNT pathway and the ncWNT pathway.

2. The targeted inhibitor according to claim 1, characterized in that The invention can be applied to the preparation of drugs for preventing gallbladder cancer and treating tumors.

3. The use according to claim 2, characterized in that The dosage form of the drug is any pharmaceutically therapeutically acceptable dosage form.

4. The use according to claim 3, characterized in that The dosage of the drug is any therapeutically acceptable dosage.

5. A drug for preventing or treating the progression of gallbladder cancer, characterized in that: It comprises the FZD6 targeted inhibitor according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

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