CN116966308A - Agents and uses for treating neuroendocrine prostate cancer - Google Patents

Agents and uses for treating neuroendocrine prostate cancer Download PDF

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CN116966308A
CN116966308A CN202210431313.7A CN202210431313A CN116966308A CN 116966308 A CN116966308 A CN 116966308A CN 202210431313 A CN202210431313 A CN 202210431313A CN 116966308 A CN116966308 A CN 116966308A
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kit
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高栋
李飞
韩铭
张晔晗
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Center for Excellence in Molecular Cell Science of CAS
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Abstract

The invention relates to an agent for treating neuroendocrine prostate and application thereof, in particular to application of the agent in preparing a medicament for treating or preventing neuroendocrine prostate cancer, wherein the agent comprises one or more of the following components: (1) a FOXA2 inhibitor, (2) a FOXA1 promoter, and (3) a KIT response signaling pathway inhibitor. The invention discovers a novel target for treating or preventing neuroendocrine prostate cancer.

Description

Agents and uses for treating neuroendocrine prostate cancer
Technical Field
The invention relates to the field of biological medicine, in particular to a tyrosine kinase receptor inhibitor for treating neuroendocrine prostate.
Background
Prostate cancer is a malignancy with first and second mortality rates in the european countries (Siegel, r.l., miller, k.d., fuchs, h.e., and Jemal, a. (2021) Cancer Statistics,2021.CA Cancer J Clin 71,7-33), whose incidence rates also appear to increase year by year in china (Fu, z.t., guo, x.l., zhang, s.w., zheng, r.s., zen, h.m., chen, r., wang, s.m., sun, k.x., wei, w., and He, j. (2020) [ Statistical analysis of incidence and mortality of prostate cancer in China,2015]. Zhonghua Zhong Liu Za Zhi, 718-722). With the advancement of modern progress in China, westernization of dietary structures and further deepening of population aging degree, prostate cancer will develop into one of important malignant tumors affecting the quality of life and life health of men in China. Adult normal prostate epithelium is composed of Luminal (Luminal), basal (Basal) and very rare neuroendocrine (Neuroendocrine cell) cells. Unlike normal prostate epithelial formation, primary prostate cancer exhibits a pathological feature that is a massive expansion of luminal cells. In luminal cells, the androgen receptor (Androgen receptor, AR) exerts a gene regulatory function of transcription factors into the nucleus by binding to androgens, which is critical for cell survival and proliferation (Huggins, c., and Hodges, c.v. (1972) Studies on prostatic cancer.i. the effect of castration, of estrogen and androgen injection on serum phosphatases in metastatic carcinoma of the program.ca Cancer J Clin 22, 232-240). Thus, clinically treating primary prostate cancer is performed by chemically or surgically depriving androgens and inhibiting the transcriptional activity of AR (Androgen-deprivation therapy, ADT). Although most primary prostate cancer shows a very significant therapeutic effect when treated for ADT at the beginning of the treatment, a significant fraction of prostate cancer patients develop Castration Resistant Prostate Cancer (CRPC) after a period of treatment that is resistant to the treatment. Among them, the tumor type with the fastest tumor progression and worst prognosis is neuroendocrine prostate cancer (Neuroendocrine prostate cancer, NEPC), which has unique neuroendocrine pathological features. However, the exact molecular mechanisms that regulate this adeno-neuroendocrine lineage shift remain unclear, and more serious, clinically effective and reliable drugs are emerging for treatment of NEPC. Therefore, the discovery of NEPC therapeutic targets and the rapid development of effective medicaments will provide a more tamped theoretical basis and guiding suggestions with reference value for clinically treating NEPC.
Disclosure of Invention
In view of the above problems, the present invention first provides a use of an agent for the preparation of a medicament for the treatment or prevention of neuroendocrine prostate cancer, the agent comprising one or more selected from the group consisting of:
(1) A FOXA2 inhibitor,
(2) FOXA1 promoter, and
(3) KIT responds to inhibitors of signaling pathway.
In one or more embodiments, FOXA2 inhibitors include inhibitory molecules that interfere with transcription and/or expression of genes encoding FOXA2 proteins, or down-regulate FOXA2 protein activity. The inhibition molecules take protein or encoding genes or transcripts thereof as inhibition targets. In one or more embodiments, the inhibitory molecule is selected from the group consisting of: (1) A small molecule compound, an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, lncRNA, a specific antibody or ligand, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1). Preferably, the inhibitor molecule is an siRNA or construct thereof that uses the FOXA2 protein-encoding gene or transcript thereof as an inhibition target.
In one or more embodiments, the FOXA2 inhibitor is an agent that knocks out or knocks down the FOXA2 gene, e.g., sgRNA, using a technique selected from ZFN, TALEN, and CRISPR.
The FOXA1 promoter is an agent that upregulates FOXA1 expression or activity. In one or more embodiments, the FOXA1 promoter is selected from the group consisting of: small molecule compounds, nucleic acid molecules, or combinations thereof. Preferably, the nucleic acid molecule is a nucleic acid construct comprising a FOXA1 coding sequence.
In one or more embodiments, the amino acid sequence of FOXA1 is selected from one or more of the following: (a) a polypeptide having a sequence as set forth in SEQ ID NO. 1 or 2; (b) A polypeptide derived from (a) having the function of (a) and formed by substitution, deletion or addition of one or more (e.g., 1 to 20; preferably 1 to 10; more preferably 1 to 5) amino acid residues to the sequence shown in SEQ ID NO. 1 or 2; or (c) a polypeptide derived from (a) having more than 90% (preferably 93%; more preferably 95% or 98%) homology to the polypeptide sequence of (a) and having the function of the polypeptide of (a).
In one or more embodiments, the KIT response signaling pathway inhibitor comprises a KIT inhibitor.
In one or more embodiments, KIT inhibitors include inhibitory molecules that interfere with transcription and/or expression of a gene encoding a KIT protein, or down-regulate KIT protein activity. The inhibition molecules take protein or encoding genes or transcripts thereof as inhibition targets. In one or more embodiments, the inhibitory molecule is selected from the group consisting of: (1) A small molecule compound, an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, lncRNA, a specific antibody or ligand, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1). Preferably, the inhibitory molecule is an siRNA or construct thereof that targets the KIT protein encoding gene or transcript thereof for inhibition.
In one or more embodiments, the KIT inhibitor is an agent, such as sgRNA, that knocks out or knocks down a KIT gene using a technique selected from ZFN, TALEN, and CRISPR.
In one or more embodiments, the KIT inhibitor is a drug against other tumors that have KIT mutants.
In one or more embodiments, the KIT inhibitor is a tyrosine kinase receptor inhibitor, preferably Imatinib (Imatinib), sorafenib (Sorafenib), sunitinib (Sunitinib), and Cabozantinib (Cabozantinib).
The invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises one or more selected from the following:
(1) A FOXA2 inhibitor,
(2) FOXA1 promoter, and
(3) KIT responds to inhibitors of signaling pathway.
In one or more embodiments, FOXA2 inhibitors include inhibitory molecules that interfere with transcription and/or expression of genes encoding FOXA2 proteins, or down-regulate FOXA2 protein activity. The inhibition molecules take protein or encoding genes or transcripts thereof as inhibition targets. In one or more embodiments, the inhibitory molecule is selected from the group consisting of: (1) A small molecule compound, an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, lncRNA, a specific antibody or ligand, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1). Preferably, the inhibitor molecule is an siRNA or construct thereof that uses the FOXA2 protein-encoding gene or transcript thereof as an inhibition target.
The FOXA1 promoter is an agent that upregulates FOXA1 expression or activity. In one or more embodiments, the FOXA1 promoter is selected from the group consisting of: small molecule compounds, nucleic acid molecules, or combinations thereof. Preferably, the nucleic acid molecule is a nucleic acid construct comprising a FOXA1 coding sequence.
In one or more embodiments, the amino acid sequence of FOXA1 is selected from one or more of the following: (a) a polypeptide having a sequence as set forth in SEQ ID NO. 1 or 2; (b) A polypeptide derived from (a) having the function of (a) and formed by substitution, deletion or addition of one or more (e.g., 1 to 20; preferably 1 to 10; more preferably 1 to 5) amino acid residues to the sequence shown in SEQ ID NO. 1 or 2; or (c) a polypeptide derived from (a) having more than 90% (preferably 93%; more preferably 95% or 98%) homology to the polypeptide sequence of (a) and having the function of the polypeptide of (a).
In one or more embodiments, the KIT response signaling pathway inhibitor comprises a KIT inhibitor.
In one or more embodiments, KIT inhibitors include inhibitory molecules that interfere with transcription and/or expression of a gene encoding a KIT protein, or down-regulate KIT protein activity. The inhibition molecules take protein or encoding genes or transcripts thereof as inhibition targets. In one or more embodiments, the inhibitory molecule is selected from the group consisting of: (1) A small molecule compound, an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, lncRNA, a specific antibody or ligand, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1). Preferably, the inhibitory molecule is an siRNA or construct thereof that targets the KIT protein encoding gene or transcript thereof for inhibition.
In one or more embodiments, the KIT inhibitor is a drug against other tumors that have KIT mutants.
In one or more embodiments, the KIT inhibitor is a tyrosine kinase receptor inhibitor, preferably Imatinib (Imatinib), sorafenib (Sorafenib), and Sunitinib (Sunitinib).
The invention also provides application of the FOXA1 gene or protein, the FOXA2 gene or protein or the gene or protein of the KIT response signal pathway as a target spot in screening medicaments for treating or preventing animal neuroendocrine prostate cancer or as a molecular index in clinic.
In one or more embodiments, the animal is a mammal, preferably a mouse, rabbit or human.
In one or more embodiments, the gene of the KIT response signal pathway comprises a KIT gene.
In one or more embodiments, the drug is a FOXA1 promoter, FOXA2 inhibitor, or KIT inhibitor.
Also provided herein is the use of a reagent for detecting a FOXA1 gene or protein, a FOXA2 gene or protein, or a gene or protein of the KIT response signal pathway in the preparation of a KIT for diagnosing neuroendocrine prostate cancer.
Also provided herein is a detection KIT comprising reagents for detecting a FOXA1 gene or protein, a FOXA2 gene or protein, or a gene or protein of a KIT response signal pathway, such as primers, probes, or antibodies specific for the detection of the gene.
In one or more embodiments, the detection kit further comprises nucleic acid detection reagents or immunological detection reagents, e.g., reagents required for PCR, reagents required for ELISA.
Drawings
FIG. 1 is a single cell, multi-pack landscape of NEPC progression. (A) Immunofluorescence staining patterns of Syp, trp63, krt8 and Ar in TPPRC tumors at different stages of NEPC progression. The scale bar is 50. Mu.m. (B) Schematic of experimental design of single-cell multicellular multi-group chemical sequencing of TPPRC tumors. (C) Schematic representation of single-cell multicellular sequencing of ATAC and RNA from the same cell. (D) UMAP diagrams based on RNA, ATAC or WNN, respectively. Cell annotation was performed from WNN analysis based on representative lineage markers. (E) RNA expression level of representative marker genes in the cell annotation population, hetmap. (F) Dot plots of RNA expression levels of representative marker genes in the cell annotation population. (G) Heatmap of chromatin accessibility level of representative marker genes in the cell annotation population. (H) Chromatin accessibility visualization of representative marker gene loci for each cell population.
FIG. 2 shows the analysis of cellular heterogeneity during NEPC progression. (A) WNN analysis calculates UMAP maps of prostate lumen and neuroendocrine cells. (B) RNA expression level Feature map of Ar in all cell populations. (C) RNA expression level Feature map of Chga in all cell populations. (D) cell composition map of NEPC progression at different stages. (E) RNA expression level hetmap of top 10 marker genes in each cell population is annotated. Representative marker genes for each cell population were labeled with different colors. (F) The pathway of each cell population is enriched and its associated network. (G) RNA expression levels Violin plots for Tff3, clu, mki67, ascl1 and Nfib in 5 cell populations based on single cell RNA-seq. (H) RNA expression levels of Tff3, clu, mki67, ascl1 and Nfib in 5 cell populations based on single cell RNA-seq. (I) immunofluorescence staining patterns of Syp, ki67 and Ascl 1. Verification of NEPC-M (Syp) + /Ki67 high )、NEPC-A(Syp + /Ascl1 high ) And NEPC-N (Syp) + /Ascl1 low ) Is represented by a light purple circle, an orange circle and a green circle, respectively. The scale bar is 50. Mu.m.
FIG. 3 is a graph depicting the identification of Foxa2 as a precursor transcription factor for neuroendocrine lineage regulation. (a) wnumap graph for pseudo-time analysis. (B) Violin plot of pseudo-time for 5 cell populations. (C) Violin plot of pseudo-time for NEPC progression through different phases. (D) wnumap map of two tumor progression tracks. (E) process trace wnnmap graph of lineage 1. (F) process trace wnnmap graph of lineage 2. (G) NEPC pseudo time progression of dynamically changing chromatin accessibility heatmap. (H) RNA expression heat map of dynamically changing genes in NEPC pseudo-time progression. (I) Motifs are enriched to identify potential precursor transcription factors early in NEPC progression. (J) RNA expression level map of Foxa1 with NEPC progress. (K) Feature plots of RNA expression levels of Foxa1 based on wnumap. The (L) motif is enriched to identify potential precursor transcription factors in advanced stages of NEPC progression. (M) RNA expression level map of Foxa2 with NEPC progression. (N) Feature map of RNA expression level of Foxa2 based on wnUMAP. (O) immunofluorescence staining patterns of Syp, foxa1 and Foxa2 in TPPRC tumors at different stages of NEPC progression. The scale bar is 50. Mu.m. Box plot of RNA expression level of Foxa2 in (P) FHCRC. (Q) Box plot of RNA expression levels of Foxa2 in WCM. (R) Box plot of RNA expression level of Foxa2 in UW. (S) Fenature plots of UMAP-based RNA expression levels of Foxa2 and Foxa1, indicating mutually exclusive expression patterns of Foxa2 and Foxa 1. Red dashed lines represent luminal cells and blue dashed lines represent neuroendocrine cells. (T) Violin plot of RNA expression levels of Foxa1 in all cell types. (U) Violin plot of RNA expression levels of Foxa2 in all cell types. The P value is calculated according to a two-tailed test.
FIG. 4 shows that Foxa2 regulates neuroendocrine differentiation and promotes Kit-mediated cell proliferation. (A) Heatm ap plots based on Foxa1 ChIP intensities at early and late stages of NEPC progression. (B) Heatmap based on Foxa2 ChIP intensity of NEPC early and late progression. (C) The number of Foxa 1-bound target genes was visualized in four groups, respectively. IGV plots show peak intensities of representative target gene sites. Sox2 and Fkbp5 are target genes for Foxa 1. Sox2 is a NEPC signature gene whose expression is inversely related to Foxa1 expression. Fkbp5 is the ARPC trait gene whose expression is positively correlated with Foxa1 expression. (D) The number of Foxa 2-bound target genes was visualized in four groups, respectively. IGV plots show peak intensities of representative target gene sites. Syt1 and Trim36 are target genes for Foxa 2. Syt1 is a NEPC trait gene whose expression is positively correlated with Foxa2 expression. Trim36, in turn, is an ARPC trait gene whose expression is inversely related to Foxa2 expression. (E) Venn plot of number of Foxa1 specific peaks, foxa2 specific peaks and overlapping peaks. (F) A hetmap of ChIP intensities of Foxa1 and Foxa2 based on Foxa1 specific binding site, foxa2 specific binding site and co-binding site. (G) A Heatmap of chromatin accessibility of Foxa1 specific binding sites, foxa2 specific binding sites and common binding sites. (H) IGV plots of representative peaks for three binding sites. Including Foxa1 specificity, foxa2 specificity, and Foxa1 and Foxa2 co-binding. (I) And (3) a Dot diagram of a pathway enrichment analysis result of the GO-BP-based Foxa2 target gene. The dot size indicates the number of target genes in each lane, and the color indicates the adjust. P value. (J) Dot plots were based on Pearson-related analysis of 28407 prostate cancer cells. The first 10 20 genes with the largest or smallest correlation coefficients were selected for display. (K) Pearson correlation analysis of RNA expression levels for Foxa2 and Kit. (L) RNA and protein expression level profile of Kit with NEPC progression. The scale bar is 50. Mu.m. (M) IGV plots of specific binding of Foxa2 at the Kit site and the level of chromatin accessibility of the Kit site at early (W2) or late (M6) progression of NEPC. (N) QRT-PCR results of relative RNA expression levels of Foxa2 in ARPC cells overexpressing PCDH-EV or PCDH-Foxa 2. Double tail t-test, mean ± SEM, n=3. (O) QRT-PCR results of relative RNA expression levels of Kit in ARPC cells overexpressing PCDH-EV or PCDH-Foxa 2. Double tail t-test, mean ± SEM, n=3.
FIG. 5 shows that the intercellular interactions reveal that the Kit signaling pathway is a neuroendocrine-specific communication. (A) Cyclic graph of the strength of interaction between each pair of cell types during NEPC progression. The line thickness indicates the interaction strength and the arrow indicates the cell type as the received signal. (B) Dot plot of the intensity of communication for all cell types. The dot size represents the communication strength. (C) an Allluval plot of the target cell receiving signal pattern. (D) Dot plot of target cell reception signal pattern. The red dashed box marks neuroendocrine specific pathways. (E) Dot plot of neuroendocrine specific reception signal patterns. The color represents the signal contribution. (F) showing a Kit signal path network. Arrows indicate target cells. (G) Kit signal network. The edge width represents the probability of communication. (H) Heatmap of Kit pathway communication probability in each pair of cell types. The variables on the X-axis or Y-axis represent secretory cells or target cells, respectively. (I) Feature map based on the expression level of Kit RNA of wnUMAP. The red dotted circle marks the neuroendocrine cell. (J) Feature map of Kitl RNA expression level based on wnUMAP. The red dotted circle marks the neuroendocrine cell. (K) Violin plots of the RNA expression levels of Kit and Kitl in all cell types. The red dashed box marks neuroendocrine cells.
Figure 6 shows that clinical grade inhibitors of Kit signaling pathway significantly slowed NEPC progression. (A) The viability assay of the mouse neuroendocrine cancer cells was treated with vehicle, 5. Mu.M, 7.5. Mu.M, 10. Mu.M imatinib, or 5. Mu.M enzalutamide, respectively. Cell viability was measured on day 2 and 4 after drug treatment, respectively. One-way ANOVA and Tukey test, mean ± SEM, n=5. (B) NEPC organogenesis assays on day 0, day 2 and day 4 post drug treatment. The scale bar is 50. Mu.m. (C) vitality determination results of neuroendocrine cancer cells of mice treated with vehicle, 0.1. Mu.M, 1. Mu.M, 3. Mu.M, 7. Mu.M sorafenib. (D) NEPC organogenesis assays on day 0, day 2 and day 4 post drug treatment. The scale bar is 50. Mu.m. (E) vitality measurement results of mouse neuroendocrine cancer cells were treated with vehicle, 0.1. Mu.M, 1. Mu.M, and 5. Mu.M sunitinib, respectively. (F) NEPC organogenesis assays on day 0, day 2 and day 4 post drug treatment. The scale bar is 50. Mu.m. (G) Growth curves of NEPC tumors in mice were treated with PBS, imatinib (50 mg/kg), sorafenib (50 mg/kg), sunitinib (40 mg/kg) and enzalutamide (10 mg/kg), respectively. One-way ANOVA and Tukey test, mean ± SEM, n=8-16. (H) Box plot of CHGA and Kit RNA expression levels in three data sets FHCRC, WCM and UW. (I) Dot plot of Kit expression levels. (J) RNA expression levels of signature genes and GSVA calculated NEPC and ARPC scoring heatmaps. (K) cell viability assay of human NEPC organoids ST 88. Cell viability after 4 days and 8 days with 0.1 μm, 1 μm, 5 μm or 10 μm Imatinib drug, respectively. One-way ANOVA and Tukey test, mean ± SEM, n=5. (l) IC50 values of BM1 and ST88 cells. Cell viability was measured 4 days after treatment with 0.1 μm, 1 μm, 5 μm, 10 μm or 50 μm imatinib drug, respectively, n=3. (M) cell viability values of BM1 and ST 88. Cell viability was measured 2 days, 4 days and 8 days after 5 μ Mimatinib treatment. Double tail t-test, mean ± SEM, n=3.
Detailed Description
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.
In this context, "treatment" and the like refer to any action that provides a benefit to a patient suffering from NEPC, including amelioration of the condition by alleviation or inhibition of at least one of symptoms and delay of disease progression. As used herein, "preventing" refers to providing beneficial behavior to a patient at risk of NEPC, including avoiding discomfort or disease progression or reducing one or more symptoms of the disease in the event of a disease. Family history may cause the patient to be at risk. The terms "subject" or "patient" in reference to treatment, management, diagnosis as used herein are animals, typically mammals, e.g. mice (e.g. mice), rabbits, humans, at risk of developing NEPC.
The inventors found that Kit signaling pathway is critical in mediating cell interactions by neuroendocrine tumor cells, tyrosine kinase receptor Kit inhibitors are capable of significantly slowing down tumor progression of NEPC. Therefore, kit can be used as a drug target for NEPC treatment. The inventors have also found that Foxa2 directly regulates Kit expression, regulates differentiation of the neuroendocrine lineage; foxa2 exhibited a negative correlation with Foxa1 gene expression level and motif activity, and Foxa1 DNA binding activity decreased as NEPC tumors progressed.
FOXA2 inhibitors
The invention relates to the treatment or prevention of NEPC by taking FOXA2 (mouse: NCBI Entrez Gene:15376, amino acid sequence shown as SEQ ID NO: 3; human: NCBI Entrez Gene:3170, amino acid sequence shown as SEQ ID NO: 4) as a target. In particular, the present invention relates to the use of FOXA2 inhibitors in the manufacture of a medicament for the prevention, alleviation, treatment of neuroendocrine prostate cancer or regression of said disease.
FOXA2 inhibitors include inhibitory molecules that interfere with transcription and/or expression of the gene encoding FOXA2 protein, or down regulate FOXA2 protein activity. The inhibition molecules take protein or encoding genes or transcripts thereof as inhibition targets. In one or more embodiments, the inhibitory molecule is selected from the group consisting of: (1) A small molecule compound, an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, lncRNA, a specific antibody or ligand, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1). Preferably, the inhibitor molecule is an siRNA or construct thereof that uses the FOXA2 protein-encoding gene or transcript thereof as an inhibition target.
The FOXA2 inhibitor may also be an agent that knocks out or knocks down the FOXA2 gene using a technique selected from ZFN, TALEN and CRISPR, for example sgRNA.
FOXA1 promoter
The invention relates to the treatment or prevention of NEPC by taking FOXA1 (mouse: NCBI Entrez Gene:15375, amino acid sequence shown as SEQ ID NO: 1; human: NCBI Entrez Gene:3169, amino acid sequence shown as SEQ ID NO: 2) as a target. In particular, the invention relates to the use of FOXA1 promoters in the manufacture of a medicament for preventing, slowing, treating neuroendocrine prostate cancer or causing regression of said disease.
The FOXA1 promoter is an agent that upregulates FOXA1 expression or activity. Herein, FOXA1 promoter is selected from the group consisting of: small molecule compounds, nucleic acid molecules, or combinations thereof. Preferably, the nucleic acid molecule is a nucleic acid construct comprising a FOXA1 coding sequence. The amino acid sequence of FOXA1 is selected from one or more of the following: (a) a polypeptide having the sequence shown in SEQ ID NO. 1; (b) A polypeptide derived from (a) having the polypeptide function of (a) and formed by substitution, deletion or addition of one or more (e.g., 1 to 20; preferably 1 to 10; more preferably 1 to 5) amino acid residues to the sequence shown in SEQ ID NO. 1; or (c) a polypeptide derived from (a) having more than 90% (preferably 93%; more preferably 95% or 98%) homology to the polypeptide sequence of (a) and having the function of the polypeptide of (a).
KIT response signaling pathway inhibitors
The present invention relates to the treatment or prevention of NEPC with KIT responsive signal pathway genes and proteins as targets. In particular, the invention relates to the use of a KIT response signaling pathway inhibitor in the manufacture of a medicament for preventing, slowing, treating, or causing regression of neuroendocrine prostate cancer.
The inventors have found that by inhibiting KIT response-related signaling pathways, the growth of neuroendocrine prostate tumor cells can be inhibited. Accordingly, the present invention provides a method of treating neuroendocrine prostate cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agent that targets an agent that inhibits expression or activity of a KIT response-associated signaling pathway protein.
The KIT response signaling pathway inhibitor may be a KIT protein (mouse: NCBI Entrez Gene:16590, amino acid sequence shown in SEQ ID NO: 5; human: NCBI Entrez Gene:3815, amino acid sequence shown in SEQ ID NO: 6). KIT inhibitors include inhibitory molecules that interfere with transcription and/or expression of a gene encoding a KIT protein, or that down-regulate KIT protein activity. The inhibition molecules take protein or encoding genes or transcripts thereof as inhibition targets. In one or more embodiments, the inhibitory molecule is selected from the group consisting of: (1) A small molecule compound, an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, lncRNA, a specific antibody or ligand, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1). Preferably, the inhibitory molecule is an siRNA or construct thereof that targets the KIT protein encoding gene or transcript thereof for inhibition.
KIT inhibitors are agents that can also knock out or knock down KIT genes, such as sgrnas, using techniques selected from ZFNs, TALENs, and CRISPRs.
In addition, KIT inhibitors may also be drugs against other tumors with KIT mutants. In one or more embodiments, the KIT inhibitor is a tyrosine kinase receptor inhibitor, preferably Imatinib (Imatinib), sorafenib (Sorafenib), and Sunitinib (Sunitinib).
Drug screening
The invention also provides application of the FOXA1 gene or protein, the FOXA2 gene or protein or a gene (such as a KIT gene) or protein of a KIT response signal pathway as a target in screening medicines for treating or preventing neuroendocrine prostate cancer or as a molecular index for clinically treating the neuroendocrine prostate cancer. The medicine is FOXA1 promoter, FOXA2 inhibitor or KIT inhibitor.
The invention also provides a method of screening for a drug comprising: (1) Contacting a candidate agent with a FOXA1 protein, FOXA2 protein, or KIT responsive signal pathway protein (e.g., KIT) or a nucleic acid molecule encoding the same or a system containing the same, and (2) detecting a change in expression or activity of the protein or nucleic acid molecule encoding the same, can be indicative of the candidate agent as the drug of interest. If the candidate agent increases the expression or activity of the FOXA1 protein, FOXA2 protein, or KIT responsive signal pathway protein (e.g., KIT) or a nucleic acid molecule encoding the same, the candidate agent is indicated to be a potential agent for preventing or treating NEPC. In a preferred embodiment, step (1) comprises: in the test group, adding the candidate substance to a system of FOXA1 protein, FOXA2 protein or KIT protein; and/or step (2) comprises: detecting the expression or activity of FOXA1 protein, FOXA2 protein or KIT protein in the system of the test group, and comparing with a control group, wherein the control group is a system expressing FOXA1 protein, FOXA2 protein or KIT protein without adding the candidate substance. If the expression of FOXA1 protein in the test group is statistically higher (preferably significantly higher, e.g., 20% higher, preferably 50% higher, more preferably 80% higher) than that in the control group, this candidate is indicative of a potential agent for preventing or treating NEPC. If the expression of FOXA2 protein or KIT protein in the test group is statistically lower (preferably significantly lower, e.g., more than 20% lower, preferably more than 50% lower, more preferably more than 80% lower) than that in the control group, this candidate is indicative of a potential agent for preventing or treating NEPC. The system is selected from the group consisting of: a cell system (e.g., a cell of FOXA1 protein, FOXA2 protein, or KIT protein) (or a cell culture system), a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
Drug administration and formulation
The invention also provides a pharmaceutical composition comprising an effective amount of a FOXA2 inhibitor, FOXA1 promoter, or KIT response signaling pathway inhibitor as an active ingredient, in combination with pharmaceutically acceptable excipients.
The pharmaceutical composition of the invention comprises one or more active ingredients and one or more pharmaceutically acceptable auxiliary materials. The term "pharmaceutically acceptable excipients" refers to excipients used in the administration of therapeutic agents, including various excipients, diluents and carriers. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutically acceptable carrier may be solid or liquid. Suitable pharmaceutically acceptable carriers are known in the art and include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides or polyethylene glycol 400, hydrogenated castor oil, cyclodextrin and the like. Pharmaceutical formulations are generally adapted to the mode of administration, for example by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions are preferably manufactured under sterile conditions.
The medicament or the pharmaceutical composition of the invention can be any suitable dosage form, including powder, tablets, dispersible granules, capsules, cachets, suppositories, solutions, suspensions, transdermal agents, injections, sustained release agents, emulsions and the like.
Examples
Experimental materials
All mouse studies were approved by the animal management committee of the superior innovation center of molecular cell science in the national academy of sciences (institute of biochemistry and cell biology in the raw Shanghai) and were conducted with feeding and genetic manipulations strictly in accordance with animal regulations related to the Shanghai laboratory animal center. Rosa26 EYFP/+ Mice were purchased from jackson laboratory (Jackson Laboratory). NOD/SCID mice were purchased from Ling Biotech. Chga tdTomato/+ Mice were constructed by the Shanghai, southwest model biological research center. Tmpfss 2 CreERT2/+ Mice were bred by the mouse genetics core facility of the Stoneketjen cancer souvenir center (MSKCC). Pten flox/flox From p.paolo Pandolfi laboratories. Trp53 flox/flox 、Rb1 flox/flox Mice were given away by Ji Gong investigators. TPPRC mice were injected with tamoxifen (Sigma, catalog number T5648-5G) at 8 weeks of age.
Experimental method
Prostate isolation and digestion in mice
Immediately taking out the prostate tissue (normal prostate tissue or prostate tumor tissue) of the mice, placing the prostate tissue in precooled PBS, removing redundant tissues such as fat, blood vessels and the like by using ophthalmic scissors and ophthalmic forceps, placing each separated prostate leaf in a centrifugal tube cover of 1.5mL, cutting the prostate leaf by using scissors, and then placing the prostate leaf in enzymolysis liquid for digestion. Parameters of the constant temperature mixing instrument are set as follows: the temperature was 37℃and the rotational speed was 1500rpm. The prostate tissue digestion liquid is put on a constant temperature mixer for digestion for 30 minutes, and is used for full digestion under the condition of shaking 70 up and down at intervals of 10 minutes during the digestion period, and the prostate is observed to be changed into a finger-like structure from small blocks.
Subsequently, after adding 750. Mu.L of TrypLE pre-warmed at 37℃it was centrifuged at 1700rpm for 5 minutes. The supernatant was aspirated, 750. Mu.L of TrypLE was added and digestion was continued on a thermostatic mixer for 15 minutes, during which time 1mL of pipette was blown against the bottom of the centrifuge tube for 30 minutes. The digestion medium was stopped by adding 750. Mu.L of DMEM and centrifuged at 1700rpm for 5 minutes, and this step was repeated once. After the supernatant was aspirated, 200 μl of mouse prostate organoid medium was added for resuspension and counting.
Single cell multiunit chemical library construction
The prostate of TPPRC mice was isolated and minced with scissors. Isolated prostate cells were resuspended in erythrocyte lysis solution (Miltenyi Biotec, cat# 130-094-183), and after incubation at room temperature for 2 minutes, dead cells were removed using a dead cell removal kit (Miltenyi Biotec, cat# 130-090-101) and cell counted by a Countess II FL automatic cell counter (Thermo Fisher Scientific). The cells were then lysed on ice for 5 minutes. The separated nuclei were transferred and loaded onto Chromium Next GEM chips. The chip containing the prostate cells is then loaded onto the controller. Subsequent library creation was performed according to the manufacturer's instructions.
Library construction of ATAC-seq
The library construction step of the ATAC-seq was performed with reference to the method of Omni-ATAC (cores et al, 2017), the specific steps are as follows: pre-cooling the centrifugal machine to 4 ℃ in advance, and preparing a Wash buffer and a Lysis buffer, wherein the ratio of the two buffers is as follows.
ATAC Wash buffer component
ATAC Lysis buffer component
After cell counting was completed using countless ii, 8 ten thousand cells were aspirated into 1mL PBS pre-chilled at 4 ℃. Cells were collected by centrifugation at 500g for 5 minutes at 4 ℃. The supernatant was discarded, 50. Mu.L of pre-chilled Lysis buffer was added, and the cells were gently pipetted for 5 times and lysed on ice for 3 minutes. To the cell lysate, 1ml of Wash buffer was added, and the mixture was centrifuged at 500g at 4℃for 10 minutes to collect nuclei. The subsequent steps were all performed with the Norwegian TD 501.
Library construction of ChIP-seq
After counting the digested cells, 1000 ten thousand cells were taken as the starting cells for ChIP-seq. After the cells were resuspended in 10mL of PBS at room temperature, 278. Mu.L of 37% formaldehyde was added to fix the cells by crosslinking, and the cells were placed on a rotary mixer and fixed at room temperature for 10 minutes. Then 541. Mu.L of 2.5M glycine was added to terminate the crosslinking reaction, and the mixture was placed on a rotary mixer and terminated at room temperature for 5 minutes. At 2500g, the supernatant was pipetted off and washed once with 5mL PBS.
Then 500. Mu.L of Pre-Lysis Buffer at 4deg.C was added and the mixture was placed on a rotary mixer and subjected to pyrolysis at 4deg.C for 30 minutes. At 2500g, the supernatant was removed by centrifugation at 4℃for 5 minutes, and 500. Mu.L of Pre-chilled Pre-Lysis Buffer at 4℃was added for one wash. 880. Mu.L of pre-chilled Nuclear Lysis Buffer at 4℃was added to resuspend the cells and the cells were transferred to 1mL AFA Millitube for sonication. The following parameters were set on a Covaris S220 sonicator for sonication. After completion of sonication, the mixture was centrifuged at 15000g for 15 minutes at 4℃to leave 40. Mu.L of the supernatant as 5% input, and the remaining supernatant was used in the subsequent experiments.
Ultrasound parameter setting
The above supernatant was transferred to two 1.5mL centrifuge tubes, 400. Mu.L per tube, and 750. Mu. LChIP Dilution Buffer was added to each tube for dilution of SDS. Pipette 30. Mu. L Protein G beads, add 500. Mu. L ChIP Dilution Buffer, gently blow 10 times with a pipette, leave it on a magnetic rack for 1 minute after 3 minutes at room temperature, discard the supernatant, add 30. Mu. L ChIP Dilution Buffer and resuspend the beads. To each tube of supernatant was added 15 mu L ChIP Dilution Buffer resuspended beads and placed on a rotary mixer and spun at 4℃for 1 hour. The mixture was allowed to stand on a magnetic rack for 1 minute, and the supernatant was retained. An appropriate amount of antibody was added to the supernatant of each tube, and the mixture was placed on a rotary mixer and rotated overnight at 4 ℃. The next day, 100 μ L Protein G beads was aspirated, added to 500 μ L ChIP Dilution Buffer, gently pipetted 10 times, left standing at room temperature for 3 minutes, left standing on a magnetic rack for 1 minute, the supernatant discarded, and 100 μ L ChIP Dilution Buffer resuspended Protein G beads added. 50 mu L ChIP Dilution Buffer resuspended Protein G beads were added to each tube and placed on a rotary mixer and spun at 4℃for 3 hours.
The mixture was allowed to stand on a magnetic rack for 1 minute, and the supernatant was aspirated off. The beads were resuspended by adding 500. Mu. L Low Salt Wash Buffer, at which time the beads of the two tubes were pooled together, then allowed to stand at room temperature for 1 minute, allowed to stand on a magnetic rack for 1 minute, the supernatant discarded, and the procedure repeated once. The beads were washed twice each with High Salt Wash Buffer and LiCl Wash Buffer references in sequence. Add 100. Mu. L DNA Elution Buffer resuspended beads, mix on vortex at room temperature for 10 minutes at low speed, then transfer to a constant temperature mixer, elute at 37℃for 10 minutes at 1500rpm, place on magnetic rack for 1 minute, collect supernatant into a new 1.5ml EP tube. The above procedure was repeated 1 time, and the total of the collected eluate supernatants was 200. Mu.L. To the eluate, 10. Mu.L of proteinase K was added and digested at 65℃for 4 hours at 1500 rpm. Subsequently, DNA was recovered and purified using DNA Clean & Concentrator-5 kit, and DNA was eluted by adding 15. Mu.L of nuclease-free water.
In vitro organoid growth experiments
Imatinib (S1026, seleck) was dissolved in PBS, sorafenib (seleck, S1040) and sunitinib (seleck, S1042) were dissolved in DMSO. 10000 NEPC cells were mixed with Matrigel uniformly and inoculated in 96-well suspension plates, and 100ul of medium was added after Matrigel solidified. The next day, 100 μl of prostate organoid medium containing placebo or inhibitors at different concentrations was added. Organoid growth experiments were performed on ST88 and BM1 cells with an initial cell number of 4500. After a certain period of drug treatment, cell viability measurements were performed using CellTiter Glo 3D (Promega).
In vivo mouse tumor transplantation experiments
Imatinib (seleck, S1026) was dissolved in PBS at a concentration of 50mg/150 μl before use. Enzalutamide was dissolved in dmso containing 1%carboxymethyl cellulose, 0.1% tween 80 and 5%. TPPRC tumors were transplanted onto SCID mice by subcutaneous injection. When the tumor volume reaches 200-300mm 3 At this time, mice were randomly assigned to each treatment group and were treated with imatinib (50 mg/kg) or PBS by intraperitoneal injection twice daily; sorafenib is administered by once daily intraperitoneal injection (seleck, S7397) (50 mg/kg); enzalutamide (10 mg/kg) and sunitinib (Selleck, S7781) (40 mg/kg) were administered by gavage once daily.
Example 1: lineage plasticity in prostate cancer progression
To explore the specific mechanism of lineage plasticity in prostate cancer progression, a gene specific to the luminal cell Tmprss2 was first established CreERT2 Mouse model of induced driven Pten, trp53 and Rb1 deletions. Furthermore, based on this construction strategy, tdTomato was placed downstream of the endogenous gene Chga promoter and translation-fixed stop element (LSL), a mouse model called TPPRC (Tmprss 2 CreERT2/+ ；Pten flox/flox ；p53 flox/flox ；Rb1 flox/flox ；Chga LSL -tdTomato/+ ). The mouse model not only ensures that NEPC originates from luminal cells, but expression of tdmamto can be directly indicative of neuroendocrine differentiation.
Prostate tumors of TPPRC mice at various time points were analyzed, including 2 weeks, 1 month, 2.5 months, 3.5 months, 4.5 months, and 6 months after tamoxifen injectionAnd (5) month. Syp is observed 1 month after tamoxifen injection + /tdTomato + Neuroendocrine cancer cells, and in line with expectations, the percentage of neuroendocrine cancer cells gradually increased over time (fig. 1, a). Furthermore, the pattern of simultaneous expression of tdTomato and Syp demonstrates that tdTomato is able to accurately indicate neuroendocrine cancer cell differentiation (FIG. 1, a).
Next the progression track of NEPC was systematically resolved based on successful establishment of TPPRC model for the transition from luminal to neuroendocrine cell lineage. Live cells were isolated from prostate tumors at different stages of progression and single cell multiunit sequencing was performed (FIG. 1, b), a technique that enabled a combined analysis of chromatin accessibility and gene expression in the same cell (FIG. 1, c). Finally 107201 high quality cells were obtained, and after the technical batch effect was excluded, 13 cell populations from different lineages, mainly including prostate epithelial cells, seminal vesicle cells, mesenchymal cells, endothelial cells, immune cells and neurons were identified using weighted nearest neighbor analysis (WNN) to integrate RNA expression and chromatin accessibility data (fig. 1 d). The results of these clusters were then mapped onto sc-RNA-seq and sc-ATAC-seq, and consistency was found between the two maps (FIG. 1, d). Furthermore, lineage marker genes of all cell populations have unique RNA expression and corresponding chromatin accessibility (fig. 1, e-h). The prostate epithelial cells were further annotated as Krt8 positive luminal cells, krt5 positive basal cells, and Chga positive neuroendocrine cells. The single-cell multi-group landscape of NEPC progress is drawn, and abundant resources are provided for elucidating the molecular mechanism of lineage transformation and providing corresponding treatment strategies.
Example 2: dissection of prostate tumor cell heterogeneity
After determining the transition of the lumen to the neuroendocrine lineage during NEPC progression, the heterogeneity of prostate tumor cells was next further dissected. For this purpose, the luminal and neuroendocrine cells were specifically extracted for subsequent analysis and re-clustered according to RNA expression and chromatin accessibility, ultimately yielding 5 different finesCell populations (FIG. 2, a). The RNA expression patterns of Ar and Chga clearly indicate that cell populations 1 and 2 are characterized by luminal cells, while cell populations 3, 4 and 5 are characterized by neuroendocrine cells (FIG. 2, b-c), which are consistent with changes in cell composition during tumor progression (FIG. 2, d). Next, pathway enrichment analysis was performed on the highly expressed genes in cell populations 1 and 2, which were found to be associated with epithelial differentiation and morphogenesis in prostate cancer (FIG. 2, e-f). While cell populations 3, 4 and 5 were relatively enriched for neuronal development and function related signaling pathways consistent with specific expression of Chga (fig. 2, f). Interestingly, cell population 3 significantly highly expressed cell cycle related genes, such as Cenpf, top2a, and Mki67 (fig. 2, e-f), indicating that cell population 3 has a highly proliferative character. In addition, cell population 5 expressed either low or no Ascl1, but high levels of Nfib and Sox6 (fig. 2, e). We further defined cell populations 1, 2, 3, 4, 5 as ARPC-T (Tff 3), ARPC-C (Clu), NEPC-M (Mki 67), NEPC-se:Sub>A (Ascl 1) and NEPC-N (Nfib) cells, respectively, based on lineage markers of these cell populations (fig. 2, e-h). The presence of these three different types of neuroendocrine cells was further verified by immunofluorescence analysis. Consistent with the results of single cell multiunit, syp was once again confirmed + /Ki67 + NEPC-M cells, syp + /Ascl1 high NEPC-A cells and Syp + /Ascl1 low NEPC-N cells (FIG. 2, i). And it was also observed that NEPC-A cells and NEPC-N cells each exhibited se:Sub>A centrally distributed pattern, which may indicate that both cells were in their respective terminally differentiated states (FIG. 2, i).
Example 3: foxa2 is a precursor transcription factor that regulates NEPC lineage plasticity
Because of the diversity of cells found in the luminal to neuroendocrine tumor transition, pseudo-temporal analysis was performed to further delineate the differentiation trajectories of the cells. The results indicated that ARPC-T cells and ARPC-C cells were mainly in the early pseudo-time phase and NEPC-M, NEPC-A and NEPC-N cells were in the late pseudo-time phase (FIG. 3, a). This analysis was in close agreement with the true tumor progression of TPPRC mice, further confirming the reliability and rationality of the pseudo-temporal analysis (fig. 3, b-c).
Further analysis of the differentiation trajectories found that NEPC progression was accompanied by two different differentiation trajectories which shared the same differentiation pattern in the early stages (FIG. 3, d-f) and reached NEPC-A and NEPC-N cells, respectively, in the latter stages. To study the mechanism of the luminal to neuroendocrine differentiation pathway, dynamic changes in gene expression and chromatin accessibility during tumor progression were analyzed. Differentiation trajectories 1 and 2 had similar chromatin opening patterns at early stages of progression (fig. 3, g). In addition, dynamic changes in gene expression also showed consistent patterns (FIG. 3, h).
Dynamic processes that regulate gene expression during lineage differentiation require the participation and regulation of precursor transcription factors. Thus, to identify precursor transcription factors active at different stages of tumor progression, an integration analysis based on motif enrichment and expression levels was performed. As a result, foxa1 and AP1 family members (Fosl 2, junb, atf3, fosl1, jun) were found to be precursor transcription factors for early stages of prostate cancer progression (FIG. 3,l-k). Interestingly, in addition to the discovery of one previously reported transcription factor Sox2 for NEPC differentiation, we also found that Foxa2 could act as a precursor transcription factor regulating NEPC progression (fig. 3,l-n). In agreement therewith, in general, foxa2 + The percentage of cells gradually increased with tumor progression, while Foxa1 + The percentage of cells gradually decreased (fig. 3, o). To verify these results in human NEPC, analysis of three publicly published human NEPC-based RNA expression profiles found that NEPC had higher Foxa2 expression levels compared to ARPC, consistent with the results of the mouse model (fig. 3, p-r). Furthermore, we also performed comprehensive analysis of two common single cell RNA-seq data. Similarly, consistent with the results of the mouse model, foxa2 was specifically expressed in neuroendocrine cancer cells, while Foxa1 was highly expressed in luminal cells (fig. 3, s-u). Taken together, our results indicate that Foxa2 is a precursor transcription factor that regulates the plasticity of the NEPC lineage, and has an important role in the differentiation of neuroendocrine tumors, which exhibits a negative correlation with the gene expression level and motif activity of Foxa 1.
Example 4: foxa2 regulates differentiation of prostate cancer toward neuroendocrine cancer and promotes Kit-mediated cell proliferation
Based on previous experimental results, it was hypothesized that the balance between Foxa1 and Foxa2 controls the lineage conversion of prostate cancer. To explore regulatory elements of Foxa1 and Foxa2 at the whole genome level, foxa1 and Foxa2 chromatin immunoprecipitation sequencing analysis (Chromatin immunoprecipitation sequencing, chIP-seq) was performed on prostate tumor cells in the early (2 weeks after tamoxifen injection) and late (6 months after tamoxifen injection) phases of NEPC progression, respectively. Consistent with the results of single cell multiunit inference, as NEPC tumor progressed, the DNA binding activity of Foxa1 decreased (fig. 4, a), while that of Foxa2 gradually increased (fig. 4, b).
Next, the association between Foxa 2-driven regulatory elements and prostate cancer lineage shift was explored. Based on sc-RNA-seq data of 28,407 prostate cancer cells, pearson-related analysis was performed on the expression levels of Foxa2 and its target gene, to define the role of Foxa2 in regulating NEPC or ARPC. Based on this analysis, 305 NEPC signature genes, such as Syt1, that bind directly to Foxa2, are defined, which are often positively regulated by Foxa 2. 97 ARPC trait genes, such as Trim36, were also identified that were negatively regulated by Foxa2 (FIG. 4, d). These results indicate that Foxa2 tends to promote differentiation of the neuroendocrine lineage while inhibiting differentiation of the luminal lineage. Consistent with the findings of previous studies, that Foxa1 is a cofactor for AR and promotes differentiation of the luminal lineages, it was also demonstrated that Foxa1 expression level was positively correlated with the expression level of ARPC trait gene (e.g., fkbp 5), but negatively correlated with the expression level of NEPC trait gene, e.g., sox2 (fig. 4, c). Further analysis found that Foxa1 and Foxa2 bound together at only 11288 sites, whereas Foxa1 bound specifically at 42467 sites and Foxa2 bound specifically at 22100 sites (FIG. 4, e-h), this result demonstrated that Foxa1 and Foxa2 had different patterns in regulating prostate cancer lineage plasticity.
The function of lineage precursor transcription factors is closely related to chromatin remodeling involved in gene regulation. To determine if binding of Foxa1 and Foxa2 is associated with chromatin status, an ATAC-seq analysis was next performed on prostate tumor cells in early and late stages of NEPC progression. Notably, foxa1 reduced the level of chromatin accessibility occupied at the late stage compared to the early stage, while Foxa2 significantly increased the level of chromatin accessibility occupied (fig. 4, f-g). Above, it has been demonstrated that Foxa2 can act as a precursor transcription factor controlling the differentiation of neuroendocrine cells, and the specific biological processes involved in Foxa2 have been explored next. In addition to neuronal differentiation and epithelial development, signal pathways associated with cell proliferation were also found using pathway enrichment analysis (fig. 4, i). Further Pearson correlation analysis ranked the genes in this pathway to evaluate the correlation between their expression and Foxa2 expression, and found that Kit was a highly correlated gene (FIG. 4,j-m). Single cell RNA-seq clearly revealed high expression levels of Kit in neuroendocrine cancer cells but not in luminal cancer cells (fig. 4, l). To verify whether Foxa2 directly regulated Kit expression, we found that Kit expression levels were also significantly increased after Foxa2 was overexpressed in ARPC organoids (fig. 4, n-o). Overall, we demonstrate that Foxa2 regulates differentiation of the neuroendocrine lineage, is functionally associated with chromatin remodeling, and can bind directly to Kit to promote NEPC progression.
Example 5: kit pathway is a specific communication mode of neuroendocrine cancer cells
The c-Kit encoded by the Kit gene is mainly activated by secreted stem cell factor encoded by Kit for further transduction of intracellular signals. Thus, this signal interaction through soluble factors and membrane-bound receptors is critical for regulating a variety of biological processes including cell growth or death, migration and differentiation, and the like.
To explore intercellular communication in NEPC, the strength of interaction between cell types was calculated two by two based on three main communication types (secretion signal, ECM receptor and cell-cell contact). In general, the signals were found to be mainly emitted by non-parenchymal cells (such as mesenchymal and endothelial cells) as well as neurons (fig. 5, a-b). There is a difference between the intensity of the signals received between different cell types, for example, the overall intensity of the signals received by neuroendocrine cells is relatively low compared to luminal cells (fig. 5, b). Attempts to study the communication pattern of signals received by all cell types resulted in the determination of 6 different, lineage-associated patterns (FIG. 5, c). For example, the manner in which immune cells receive signals is primarily pattern 4, which represents a variety of immune-related pathways, including but not limited to CCL1, IL1 and TNF (FIG. 5, c-d).
Although communication is generally similar between prostate lumen cells, basal cells and even seminal vesicle cells, neuroendocrine cells have relatively unique patterns of signal reception (fig. 5, c). Next 7 neuroendocrine cell-specific signaling patterns were found. Among them, the Kit pathway had the highest signal intensity (fig. 5,e), consistent with its role in the established promotion of NEPC proliferation (fig. 4,j). By specifically analyzing the information flow of the Kit pathway, neuroendocrine cells were found to be the primary targets for receiving Kit ligand, kitl, secreted by different cell types (FIG. 5, f-h). Consistent with this result, high expression of Kit was observed only in neuroendocrine cells, while Kit was highly expressed in various cell types including neuroendocrine cells (FIG. 5,i-k). These results indicate that the neuroendocrine lineage specific Kit pathway may function in an autocrine and paracrine manner. Overall, systematic analysis of intercellular interactions revealed unique communication patterns of neuroendocrine cells, again validating the potential role of Kit in promoting NEPC progression.
Example 6: inhibition of tyrosine kinase receptor Kit pathways may be an effective means of potentially treating NEPC
The above experimental data indicate that the Kit signaling pathway is capable of mediating specific communication in neuroendocrine cells, and next it is desirable to investigate whether inhibition of the Kit pathway would slow down the progression of NEPC. Imatinib (Imatinib, also known as "glifehrin") is a tyrosine kinase inhibitor and is currently the only drug recommended in the united states national integrated cancer network clinical practice guidelines for melanoma with Kit mutants. To further evaluate the effect of imatinib on NEPC progression, prostate tumors from TPPRC mice injected with tamoxifen for 4.5 months were digested for primary tumor organoid construction. Notably, using an in vitro organoid culture system, imatinib was found to significantly inhibit the growth of neuroendocrine tumor cells in a concentration-dependent manner, whereas the inhibitor enzalutamide of AR did not have significant efficacy (fig. 6, a-b). In addition, the effect of the other two Kit inhibitors Sorafenib (Sorafenib) and Sunitinib (Sunitinib) was evaluated using the tumor organoid culture system. It was found that sorafenib and sunitinib, like imatinib, were also able to significantly inhibit the growth of neuroendocrine tumor cells under in vitro conditions, and that the inhibition pattern was somewhat concentration dependent (fig. 6, c-f). To further evaluate the efficacy of Kit inhibitors in vivo, freshly isolated NEPC tumors were transplanted into SCID mice by subcutaneous injection. Consistent with the results of in vitro organoid culture experiments, all three Kit inhibitors consistently showed significant effects in inhibiting NEPC tumor progression (fig. 6, g).
The important role of Kit in the development of mouse NEPC prompted us to next explore whether Kit also has similar function in human NEPC. By analyzing the common data for multiple prostate cancer-based, NEPC was found to have significantly higher Kit expression levels in all 3 data sets compared to ARPC (AR-positive prostate cancer) (fig. 6,h). In agreement with this, CRPC with higher Kit expression levels had a greater likelihood of exhibiting endocrine differentiation (figure 6,i). Based on this, we next investigated whether imatinib was able to limit the growth of human NEPC tumor cells. For this purpose, the effect of imatinib was evaluated using laboratory pre-constructed NEPC organoids ST88 and found to have a significant inhibitory effect on ST88 growth (FIG. 6,j). In addition, the sensitivity differences of ST88 and another non-NEPC organoid BM1 to imatinib treatment were compared and ST88 was found to be more sensitive to imatinib than BM1 (fig. 6,k-l). In conclusion, the Kit inhibitor can effectively slow down the development of NEPC and has a certain tumor type specificity through an in vitro organoid culture system and an in vivo transplantation experiment of human and mice.
By combining a plurality of bioinformatics technical means such as single cell multiunit, chIP-seq, ATAC-seq and the like, time series system analysis is carried out on transcriptome, apparent group and tumor microenvironment in NEPC progress, the important role of a tyrosine kinase receptor Kit signal pathway in mediating cell interaction carried out by neuroendocrine tumor cells is identified, and Kit is provided as a drug target point of NEPC treatment. In addition, in NEPC, the ligand Kitl of Kit is found to be widely expressed in various types of cells such as epithelial cells, mesenchymal cells and immune cells, and the Kit is specifically and highly expressed in neuroendocrine tumor cells, so that the great potential of Kit as a specific drug target is further proved.
Through in vitro organoid models of human and mice and in vivo tumor implantation experiments, it is proved that various tyrosine kinase receptor Kit inhibitors including imatinib, sorafenib, sunitinib and the like can remarkably slow down the tumor progress of NEPC, and the inhibition effect has a certain tumor type selectivity. It is also important that imatinib, sorafenib and sunitinib have all been approved by the FDA for the treatment of other various types of tumors, so that these inhibitors have various advantages of clear pharmacokinetic characteristics and high safety, and to a great extent ensure that they can enter the clinic rapidly through the "old drug new use" mode for evaluation of the drug efficacy.
Sequence listing
<110> China academy of sciences molecular cell science Excellent innovation center
<120> agents and uses for treating neuroendocrine prostate cancer
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Ser Tyr Tyr Ala Asp Thr Gln Glu Ala Tyr Ser Ser Val Pro Val Ser
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Asn Met Asn Ser Gly Leu Gly Ser Met Asn Ser Met Asn Thr Tyr Met
35 40 45
Thr Met Asn Thr Met Thr Thr Ser Gly Asn Met Thr Pro Ala Ser Phe
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Asn Met Ser Tyr Ala Asn Thr Gly Leu Gly Ala Gly Leu Ser Pro Gly
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Ala Val Ala Gly Met Pro Gly Ala Ser Ala Gly Ala Met Asn Ser Met
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Thr Ala Ala Gly Val Thr Ala Met Gly Thr Ala Leu Ser Pro Gly Gly
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Met Gly Ser Met Gly Ala Gln Pro Ala Thr Ser Met Asn Gly Leu Gly
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Pro Tyr Ala Ala Ala Met Asn Pro Cys Met Ser Pro Met Ala Tyr Ala
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Pro Ser Asn Leu Gly Arg Ser Arg Ala Gly Gly Gly Gly Asp Ala Lys
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Thr Phe Lys Arg Ser Tyr Pro His Ala Lys Pro Pro Tyr Ser Tyr Ile
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Ser Leu Ile Thr Met Ala Ile Gln Gln Ala Pro Ser Lys Met Leu Thr
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Leu Ser Glu Ile Tyr Gln Trp Ile Met Asp Leu Phe Pro Tyr Tyr Arg
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Gln Asn Gln Gln Arg Trp Gln Asn Ser Ile Arg His Ser Leu Ser Phe
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Asn Asp Cys Phe Val Lys Val Ala Arg Ser Pro Asp Lys Pro Gly Lys
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Gly Ser Tyr Trp Thr Leu His Pro Asp Ser Gly Asn Met Phe Glu Asn
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Gly Cys Tyr Leu Arg Arg Gln Lys Arg Phe Lys Cys Glu Lys Gln Pro
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Gly Ala Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Gly Gly Pro Glu
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Ser Arg Lys Asp Pro Ser Gly Pro Gly Asn Pro Ser Ala Glu Ser Pro
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Leu His Arg Gly Val His Gly Lys Ala Ser Gln Leu Glu Gly Ala Pro
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Ala Pro Gly Pro Ala Ala Ser Pro Gln Thr Leu Asp His Ser Gly Ala
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Thr Ala Thr Gly Gly Ala Ser Glu Leu Lys Ser Pro Ala Ser Ser Ser
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Ala Pro Pro Ile Ser Ser Gly Pro Gly Ala Leu Ala Ser Val Pro Pro
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Ser His Pro Ala His Gly Leu Ala Pro His Glu Ser Gln Leu His Leu
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Lys Gly Asp Pro His Tyr Ser Phe Asn His Pro Phe Ser Ile Asn Asn
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Leu Met Ser Ser Ser Glu Gln Gln His Lys Leu Asp Phe Lys Ala Tyr
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Glu Gln Ala Leu Gln Tyr Ser Pro Tyr Gly Ala Thr Leu Pro Ala Ser
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Leu Pro Leu Gly Ser Ala Ser Val Ala Thr Arg Ser Pro Ile Glu Pro
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Ser Ala Leu Glu Pro Ala Tyr Tyr Gln Gly Val Tyr Ser Arg Pro Val
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Leu Asn Thr Ser
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Ser Tyr Tyr Ala Asp Thr Gln Glu Ala Tyr Ser Ser Val Pro Val Ser
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Asn Met Asn Ser Gly Leu Gly Ser Met Asn Ser Met Asn Thr Tyr Met
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Thr Met Asn Thr Met Thr Thr Ser Gly Asn Met Thr Pro Ala Ser Phe
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Asn Met Ser Tyr Ala Asn Pro Gly Leu Gly Ala Gly Leu Ser Pro Gly
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Ala Val Ala Gly Met Pro Gly Gly Ser Ala Gly Ala Met Asn Ser Met
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Thr Ala Ala Gly Val Thr Ala Met Gly Thr Ala Leu Ser Pro Ser Gly
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Met Gly Ala Met Gly Ala Gln Gln Ala Ala Ser Met Asn Gly Leu Gly
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Pro Tyr Ala Ala Ala Met Asn Pro Cys Met Ser Pro Met Ala Tyr Ala
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Pro Ser Asn Leu Gly Arg Ser Arg Ala Gly Gly Gly Gly Asp Ala Lys
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Thr Phe Lys Arg Ser Tyr Pro His Ala Lys Pro Pro Tyr Ser Tyr Ile
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Ser Leu Ile Thr Met Ala Ile Gln Gln Ala Pro Ser Lys Met Leu Thr
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Leu Ser Glu Ile Tyr Gln Trp Ile Met Asp Leu Phe Pro Tyr Tyr Arg
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Gln Asn Gln Gln Arg Trp Gln Asn Ser Ile Arg His Ser Leu Ser Phe
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Asn Asp Cys Phe Val Lys Val Ala Arg Ser Pro Asp Lys Pro Gly Lys
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Gly Ser Tyr Trp Thr Leu His Pro Asp Ser Gly Asn Met Phe Glu Asn
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Gly Cys Tyr Leu Arg Arg Gln Lys Arg Phe Lys Cys Glu Lys Gln Pro
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Gly Ala Gly Gly Gly Gly Gly Ser Gly Ser Gly Gly Ser Gly Ala Lys
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Gly Gly Pro Glu Ser Arg Lys Asp Pro Ser Gly Ala Ser Asn Pro Ser
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Ala Asp Ser Pro Leu His Arg Gly Val His Gly Lys Thr Gly Gln Leu
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Glu Gly Ala Pro Ala Pro Gly Pro Ala Ala Ser Pro Gln Thr Leu Asp
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His Ser Gly Ala Thr Ala Thr Gly Gly Ala Ser Glu Leu Lys Thr Pro
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Ala Ser Ser Thr Ala Pro Pro Ile Ser Ser Gly Pro Gly Ala Leu Ala
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Ser Val Pro Ala Ser His Pro Ala His Gly Leu Ala Pro His Glu Ser
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Gln Leu His Leu Lys Gly Asp Pro His Tyr Ser Phe Asn His Pro Phe
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Ser Ile Asn Asn Leu Met Ser Ser Ser Glu Gln Gln His Lys Leu Asp
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Phe Lys Ala Tyr Glu Gln Ala Leu Gln Tyr Ser Pro Tyr Gly Ser Thr
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Leu Pro Ala Ser Leu Pro Leu Gly Ser Ala Ser Val Thr Thr Arg Ser
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Pro Ile Glu Pro Ser Ala Leu Glu Pro Ala Tyr Tyr Gln Gly Val Tyr
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Ser Arg Pro Val Leu Asn Thr Ser
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Glu Pro Ser Asp Trp Ser Ser Tyr Tyr Ala Glu Pro Glu Gly Tyr Ser
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Ser Val Ser Asn Met Asn Ala Gly Leu Gly Met Asn Gly Met Asn Thr
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Tyr Met Ser Met Ser Ala Ala Ala Met Gly Gly Gly Ser Gly Asn Met
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Ser Ala Gly Ser Met Asn Met Ser Ser Tyr Val Gly Ala Gly Met Ser
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Pro Ser Leu Ala Gly Met Ser Pro Gly Ala Gly Ala Met Ala Gly Met
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Ser Gly Ser Ala Gly Ala Ala Gly Val Ala Gly Met Gly Pro His Leu
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Ser Pro Ser Leu Ser Pro Leu Gly Gly Gln Ala Ala Gly Ala Met Gly
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Gly Leu Ala Pro Tyr Ala Asn Met Asn Ser Met Ser Pro Met Tyr Gly
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Gln Ala Gly Leu Ser Arg Ala Arg Asp Pro Lys Thr Tyr Arg Arg Ser
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Tyr Thr His Ala Lys Pro Pro Tyr Ser Tyr Ile Ser Leu Ile Thr Met
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Ala Ile Gln Gln Ser Pro Asn Lys Met Leu Thr Leu Ser Glu Ile Tyr
180 185 190
Gln Trp Ile Met Asp Leu Phe Pro Phe Tyr Arg Gln Asn Gln Gln Arg
195 200 205
Trp Gln Asn Ser Ile Arg His Ser Leu Ser Phe Asn Asp Cys Phe Leu
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Lys Val Pro Arg Ser Pro Asp Lys Pro Gly Lys Gly Ser Phe Trp Thr
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Leu His Pro Asp Ser Gly Asn Met Phe Glu Asn Gly Cys Tyr Leu Arg
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Arg Gln Lys Arg Phe Lys Cys Glu Lys Gln Leu Ala Leu Lys Glu Ala
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Ala Gly Ala Ala Ser Ser Gly Gly Lys Lys Thr Ala Pro Gly Ser Gln
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Ala Ser Gln Ala Gln Leu Gly Glu Ala Ala Gly Ser Ala Ser Glu Thr
290 295 300
Pro Ala Gly Thr Glu Ser Pro His Ser Ser Ala Ser Pro Cys Gln Glu
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His Lys Arg Gly Gly Leu Ser Glu Leu Lys Gly Ala Pro Ala Ser Ala
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Leu Ser Pro Pro Glu Pro Ala Pro Ser Pro Gly Gln Gln Gln Gln Ala
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Ala Ala His Leu Leu Gly Pro Pro His His Pro Gly Leu Pro Pro Glu
355 360 365
Ala His Leu Lys Pro Glu His His Tyr Ala Phe Asn His Pro Phe Ser
370 375 380
Ile Asn Asn Leu Met Ser Ser Glu Gln Gln His His His Ser His His
385 390 395 400
His His Gln Pro His Lys Met Asp Leu Lys Ala Tyr Glu Gln Val Met
405 410 415
His Tyr Pro Gly Gly Tyr Gly Ser Pro Met Pro Gly Ser Leu Ala Met
420 425 430
Gly Pro Val Thr Asn Lys Ala Gly Leu Asp Ala Ser Pro Leu Ala Ala
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Asp Thr Ser Tyr Tyr Gln Gly Val Tyr Ser Arg Pro Ile Met Asn Ser
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Ser
465
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Met His Ser Ala Ser Ser Met Leu Gly Ala Val Lys Met Glu Gly His
1 5 10 15
Glu Pro Ser Asp Trp Ser Ser Tyr Tyr Ala Glu Pro Glu Gly Tyr Ser
20 25 30
Ser Val Ser Asn Met Asn Ala Gly Leu Gly Met Asn Gly Met Asn Thr
35 40 45
Tyr Met Ser Met Ser Ala Ala Ala Met Gly Ser Gly Ser Gly Asn Met
50 55 60
Ser Ala Gly Ser Met Asn Met Ser Ser Tyr Val Gly Ala Gly Met Ser
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Pro Ser Leu Ala Gly Met Ser Pro Gly Ala Gly Ala Met Ala Gly Met
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Gly Gly Ser Ala Gly Ala Ala Gly Val Ala Gly Met Gly Pro His Leu
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Ser Pro Ser Leu Ser Pro Leu Gly Gly Gln Ala Ala Gly Ala Met Gly
115 120 125
Gly Leu Ala Pro Tyr Ala Asn Met Asn Ser Met Ser Pro Met Tyr Gly
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Gln Ala Gly Leu Ser Arg Ala Arg Asp Pro Lys Thr Tyr Arg Arg Ser
145 150 155 160
Tyr Thr His Ala Lys Pro Pro Tyr Ser Tyr Ile Ser Leu Ile Thr Met
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Ala Ile Gln Gln Ser Pro Asn Lys Met Leu Thr Leu Ser Glu Ile Tyr
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Gln Trp Ile Met Asp Leu Phe Pro Phe Tyr Arg Gln Asn Gln Gln Arg
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Trp Gln Asn Ser Ile Arg His Ser Leu Ser Phe Asn Asp Cys Phe Leu
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Lys Val Pro Arg Ser Pro Asp Lys Pro Gly Lys Gly Ser Phe Trp Thr
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Leu His Pro Asp Ser Gly Asn Met Phe Glu Asn Gly Cys Tyr Leu Arg
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Arg Gln Lys Arg Phe Lys Cys Glu Lys Gln Leu Ala Leu Lys Glu Ala
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Ala Gly Ala Ala Gly Ser Gly Lys Lys Ala Ala Ala Gly Ala Gln Ala
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Ser Gln Ala Gln Leu Gly Glu Ala Ala Gly Pro Ala Ser Glu Thr Pro
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Ala Gly Thr Glu Ser Pro His Ser Ser Ala Ser Pro Cys Gln Glu His
305 310 315 320
Lys Arg Gly Gly Leu Gly Glu Leu Lys Gly Thr Pro Ala Ala Ala Leu
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Ser Pro Pro Glu Pro Ala Pro Ser Pro Gly Gln Gln Gln Gln Ala Ala
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Ala His Leu Leu Gly Pro Pro His His Pro Gly Leu Pro Pro Glu Ala
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His Leu Lys Pro Glu His His Tyr Ala Phe Asn His Pro Phe Ser Ile
370 375 380
Asn Asn Leu Met Ser Ser Glu Gln Gln His His His Ser His His His
385 390 395 400
His Gln Pro His Lys Met Asp Leu Lys Ala Tyr Glu Gln Val Met His
405 410 415
Tyr Pro Gly Tyr Gly Ser Pro Met Pro Gly Ser Leu Ala Met Gly Pro
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Val Thr Asn Lys Thr Gly Leu Asp Ala Ser Pro Leu Ala Ala Asp Thr
435 440 445
Ser Tyr Tyr Gln Gly Val Tyr Ser Arg Pro Ile Met Asn Ser Ser
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Met Arg Gly Ala Arg Gly Ala Trp Asp Leu Leu Cys Val Leu Leu Val
1 5 10 15
Leu Leu Arg Gly Gln Thr Ala Thr Ser Gln Pro Ser Ala Ser Pro Gly
20 25 30
Glu Pro Ser Pro Pro Ser Ile His Pro Ala Gln Ser Glu Leu Ile Val
35 40 45
Glu Ala Gly Asp Thr Leu Ser Leu Thr Cys Ile Asp Pro Asp Phe Val
50 55 60
Arg Trp Thr Phe Lys Thr Tyr Phe Asn Glu Met Val Glu Asn Lys Lys
65 70 75 80
Asn Glu Trp Ile Gln Glu Lys Ala Glu Ala Thr Arg Thr Gly Thr Tyr
85 90 95
Thr Cys Ser Asn Ser Asn Gly Leu Thr Ser Ser Ile Tyr Val Phe Val
100 105 110
Arg Asp Pro Ala Lys Leu Phe Leu Val Gly Leu Pro Leu Phe Gly Lys
115 120 125
Glu Asp Ser Asp Ala Leu Val Arg Cys Pro Leu Thr Asp Pro Gln Val
130 135 140
Ser Asn Tyr Ser Leu Ile Glu Cys Asp Gly Lys Ser Leu Pro Thr Asp
145 150 155 160
Leu Thr Phe Val Pro Asn Pro Lys Ala Gly Ile Thr Ile Lys Asn Val
165 170 175
Lys Arg Ala Tyr His Arg Leu Cys Val Arg Cys Ala Ala Gln Arg Asp
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Gly Thr Trp Leu His Ser Asp Lys Phe Thr Leu Lys Val Arg Ala Ala
195 200 205
Ile Lys Ala Ile Pro Val Val Ser Val Pro Glu Thr Ser His Leu Leu
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Lys Lys Gly Asp Thr Phe Thr Val Val Cys Thr Ile Lys Asp Val Ser
225 230 235 240
Thr Ser Val Asn Ser Met Trp Leu Lys Met Asn Pro Gln Pro Gln His
245 250 255
Ile Ala Gln Val Lys His Asn Ser Trp His Arg Gly Asp Phe Asn Tyr
260 265 270
Glu Arg Gln Glu Thr Leu Thr Ile Ser Ser Ala Arg Val Asp Asp Ser
275 280 285
Gly Val Phe Met Cys Tyr Ala Asn Asn Thr Phe Gly Ser Ala Asn Val
290 295 300
Thr Thr Thr Leu Lys Val Val Glu Lys Gly Phe Ile Asn Ile Ser Pro
305 310 315 320
Val Lys Asn Thr Thr Val Phe Val Thr Asp Gly Glu Asn Val Asp Leu
325 330 335
Val Val Glu Tyr Glu Ala Tyr Pro Lys Pro Glu His Gln Gln Trp Ile
340 345 350
Tyr Met Asn Arg Thr Ser Ala Asn Lys Gly Lys Asp Tyr Val Lys Ser
355 360 365
Asp Asn Lys Ser Asn Ile Arg Tyr Val Asn Gln Leu Arg Leu Thr Arg
370 375 380
Leu Lys Gly Thr Glu Gly Gly Thr Tyr Thr Phe Leu Val Ser Asn Ser
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Asp Ala Ser Ala Ser Val Thr Phe Asn Val Tyr Val Asn Thr Lys Pro
405 410 415
Glu Ile Leu Thr Tyr Asp Arg Leu Ile Asn Gly Met Leu Gln Cys Val
420 425 430
Ala Glu Gly Phe Pro Glu Pro Thr Ile Asp Trp Tyr Phe Cys Thr Gly
435 440 445
Ala Glu Gln Arg Cys Thr Thr Pro Val Ser Pro Val Asp Val Gln Val
450 455 460
Gln Asn Val Ser Val Ser Pro Phe Gly Lys Leu Val Val Gln Ser Ser
465 470 475 480
Ile Asp Ser Ser Val Phe Arg His Asn Gly Thr Val Glu Cys Lys Ala
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Ser Asn Asp Val Gly Lys Ser Ser Ala Phe Phe Asn Phe Ala Phe Lys
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Gly Asn Asn Lys Glu Gln Ile Gln Ala His Thr Leu Phe Thr Pro Leu
515 520 525
Leu Ile Gly Phe Val Val Ala Ala Gly Ala Met Gly Ile Ile Val Met
530 535 540
Val Leu Thr Tyr Lys Tyr Leu Gln Lys Pro Met Tyr Glu Val Gln Trp
545 550 555 560
Lys Val Val Glu Glu Ile Asn Gly Asn Asn Tyr Val Tyr Ile Asp Pro
565 570 575
Thr Gln Leu Pro Tyr Asp His Lys Trp Glu Phe Pro Arg Asn Arg Leu
580 585 590
Ser Phe Gly Lys Thr Leu Gly Ala Gly Ala Phe Gly Lys Val Val Glu
595 600 605
Ala Thr Ala Tyr Gly Leu Ile Lys Ser Asp Ala Ala Met Thr Val Ala
610 615 620
Val Lys Met Leu Lys Pro Ser Ala His Leu Thr Glu Arg Glu Ala Leu
625 630 635 640
Met Ser Glu Leu Lys Val Leu Ser Tyr Leu Gly Asn His Met Asn Ile
645 650 655
Val Asn Leu Leu Gly Ala Cys Thr Val Gly Gly Pro Thr Leu Val Ile
660 665 670
Thr Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Phe Leu Arg Arg Lys
675 680 685
Arg Asp Ser Phe Ile Phe Ser Lys Gln Glu Glu Gln Ala Glu Ala Ala
690 695 700
Leu Tyr Lys Asn Leu Leu His Ser Thr Glu Pro Ser Cys Asp Ser Ser
705 710 715 720
Asn Glu Tyr Met Asp Met Lys Pro Gly Val Ser Tyr Val Val Pro Thr
725 730 735
Lys Thr Asp Lys Arg Arg Ser Ala Arg Ile Asp Ser Tyr Ile Glu Arg
740 745 750
Asp Val Thr Pro Ala Ile Met Glu Asp Asp Glu Leu Ala Leu Asp Leu
755 760 765
Asp Asp Leu Leu Ser Phe Ser Tyr Gln Val Ala Lys Gly Met Ala Phe
770 775 780
Leu Ala Ser Lys Asn Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile
785 790 795 800
Leu Leu Thr His Gly Arg Ile Thr Lys Ile Cys Asp Phe Gly Leu Ala
805 810 815
Arg Asp Ile Arg Asn Asp Ser Asn Tyr Val Val Lys Gly Asn Ala Arg
820 825 830
Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Ser Cys Val Tyr
835 840 845
Thr Phe Glu Ser Asp Val Trp Ser Tyr Gly Ile Phe Leu Trp Glu Leu
850 855 860
Phe Ser Leu Gly Ser Ser Pro Tyr Pro Gly Met Pro Val Asp Ser Lys
865 870 875 880
Phe Tyr Lys Met Ile Lys Glu Gly Phe Arg Met Val Ser Pro Glu His
885 890 895
Ala Pro Ala Glu Met Tyr Asp Val Met Lys Thr Cys Trp Asp Ala Asp
900 905 910
Pro Leu Lys Arg Pro Thr Phe Lys Gln Val Val Gln Leu Ile Glu Lys
915 920 925
Gln Ile Ser Asp Ser Thr Lys His Ile Tyr Ser Asn Leu Ala Asn Cys
930 935 940
Asn Pro Asn Pro Glu Asn Pro Val Val Val Asp His Ser Val Arg Val
945 950 955 960
Asn Ser Val Gly Ser Ser Ala Ser Ser Thr Gln Pro Leu Leu Val His
965 970 975
Glu Asp Ala
<210> 6
<211> 976
<212> PRT
<213> Homo sapiens
<400> 6
Met Arg Gly Ala Arg Gly Ala Trp Asp Phe Leu Cys Val Leu Leu Leu
1 5 10 15
Leu Leu Arg Val Gln Thr Gly Ser Ser Gln Pro Ser Val Ser Pro Gly
20 25 30
Glu Pro Ser Pro Pro Ser Ile His Pro Gly Lys Ser Asp Leu Ile Val
35 40 45
Arg Val Gly Asp Glu Ile Arg Leu Leu Cys Thr Asp Pro Gly Phe Val
50 55 60
Lys Trp Thr Phe Glu Ile Leu Asp Glu Thr Asn Glu Asn Lys Gln Asn
65 70 75 80
Glu Trp Ile Thr Glu Lys Ala Glu Ala Thr Asn Thr Gly Lys Tyr Thr
85 90 95
Cys Thr Asn Lys His Gly Leu Ser Asn Ser Ile Tyr Val Phe Val Arg
100 105 110
Asp Pro Ala Lys Leu Phe Leu Val Asp Arg Ser Leu Tyr Gly Lys Glu
115 120 125
Asp Asn Asp Thr Leu Val Arg Cys Pro Leu Thr Asp Pro Glu Val Thr
130 135 140
Asn Tyr Ser Leu Lys Gly Cys Gln Gly Lys Pro Leu Pro Lys Asp Leu
145 150 155 160
Arg Phe Ile Pro Asp Pro Lys Ala Gly Ile Met Ile Lys Ser Val Lys
165 170 175
Arg Ala Tyr His Arg Leu Cys Leu His Cys Ser Val Asp Gln Glu Gly
180 185 190
Lys Ser Val Leu Ser Glu Lys Phe Ile Leu Lys Val Arg Pro Ala Phe
195 200 205
Lys Ala Val Pro Val Val Ser Val Ser Lys Ala Ser Tyr Leu Leu Arg
210 215 220
Glu Gly Glu Glu Phe Thr Val Thr Cys Thr Ile Lys Asp Val Ser Ser
225 230 235 240
Ser Val Tyr Ser Thr Trp Lys Arg Glu Asn Ser Gln Thr Lys Leu Gln
245 250 255
Glu Lys Tyr Asn Ser Trp His His Gly Asp Phe Asn Tyr Glu Arg Gln
260 265 270
Ala Thr Leu Thr Ile Ser Ser Ala Arg Val Asn Asp Ser Gly Val Phe
275 280 285
Met Cys Tyr Ala Asn Asn Thr Phe Gly Ser Ala Asn Val Thr Thr Thr
290 295 300
Leu Glu Val Val Asp Lys Gly Phe Ile Asn Ile Phe Pro Met Ile Asn
305 310 315 320
Thr Thr Val Phe Val Asn Asp Gly Glu Asn Val Asp Leu Ile Val Glu
325 330 335
Tyr Glu Ala Phe Pro Lys Pro Glu His Gln Gln Trp Ile Tyr Met Asn
340 345 350
Arg Thr Phe Thr Asp Lys Trp Glu Asp Tyr Pro Lys Ser Glu Asn Glu
355 360 365
Ser Asn Ile Arg Tyr Val Ser Glu Leu His Leu Thr Arg Leu Lys Gly
370 375 380
Thr Glu Gly Gly Thr Tyr Thr Phe Leu Val Ser Asn Ser Asp Val Asn
385 390 395 400
Ala Ala Ile Ala Phe Asn Val Tyr Val Asn Thr Lys Pro Glu Ile Leu
405 410 415
Thr Tyr Asp Arg Leu Val Asn Gly Met Leu Gln Cys Val Ala Ala Gly
420 425 430
Phe Pro Glu Pro Thr Ile Asp Trp Tyr Phe Cys Pro Gly Thr Glu Gln
435 440 445
Arg Cys Ser Ala Ser Val Leu Pro Val Asp Val Gln Thr Leu Asn Ser
450 455 460
Ser Gly Pro Pro Phe Gly Lys Leu Val Val Gln Ser Ser Ile Asp Ser
465 470 475 480
Ser Ala Phe Lys His Asn Gly Thr Val Glu Cys Lys Ala Tyr Asn Asp
485 490 495
Val Gly Lys Thr Ser Ala Tyr Phe Asn Phe Ala Phe Lys Gly Asn Asn
500 505 510
Lys Glu Gln Ile His Pro His Thr Leu Phe Thr Pro Leu Leu Ile Gly
515 520 525
Phe Val Ile Val Ala Gly Met Met Cys Ile Ile Val Met Ile Leu Thr
530 535 540
Tyr Lys Tyr Leu Gln Lys Pro Met Tyr Glu Val Gln Trp Lys Val Val
545 550 555 560
Glu Glu Ile Asn Gly Asn Asn Tyr Val Tyr Ile Asp Pro Thr Gln Leu
565 570 575
Pro Tyr Asp His Lys Trp Glu Phe Pro Arg Asn Arg Leu Ser Phe Gly
580 585 590
Lys Thr Leu Gly Ala Gly Ala Phe Gly Lys Val Val Glu Ala Thr Ala
595 600 605
Tyr Gly Leu Ile Lys Ser Asp Ala Ala Met Thr Val Ala Val Lys Met
610 615 620
Leu Lys Pro Ser Ala His Leu Thr Glu Arg Glu Ala Leu Met Ser Glu
625 630 635 640
Leu Lys Val Leu Ser Tyr Leu Gly Asn His Met Asn Ile Val Asn Leu
645 650 655
Leu Gly Ala Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Thr Glu Tyr
660 665 670
Cys Cys Tyr Gly Asp Leu Leu Asn Phe Leu Arg Arg Lys Arg Asp Ser
675 680 685
Phe Ile Cys Ser Lys Gln Glu Asp His Ala Glu Ala Ala Leu Tyr Lys
690 695 700
Asn Leu Leu His Ser Lys Glu Ser Ser Cys Ser Asp Ser Thr Asn Glu
705 710 715 720
Tyr Met Asp Met Lys Pro Gly Val Ser Tyr Val Val Pro Thr Lys Ala
725 730 735
Asp Lys Arg Arg Ser Val Arg Ile Gly Ser Tyr Ile Glu Arg Asp Val
740 745 750
Thr Pro Ala Ile Met Glu Asp Asp Glu Leu Ala Leu Asp Leu Glu Asp
755 760 765
Leu Leu Ser Phe Ser Tyr Gln Val Ala Lys Gly Met Ala Phe Leu Ala
770 775 780
Ser Lys Asn Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu
785 790 795 800
Thr His Gly Arg Ile Thr Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp
805 810 815
Ile Lys Asn Asp Ser Asn Tyr Val Val Lys Gly Asn Ala Arg Leu Pro
820 825 830
Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asn Cys Val Tyr Thr Phe
835 840 845
Glu Ser Asp Val Trp Ser Tyr Gly Ile Phe Leu Trp Glu Leu Phe Ser
850 855 860
Leu Gly Ser Ser Pro Tyr Pro Gly Met Pro Val Asp Ser Lys Phe Tyr
865 870 875 880
Lys Met Ile Lys Glu Gly Phe Arg Met Leu Ser Pro Glu His Ala Pro
885 890 895
Ala Glu Met Tyr Asp Ile Met Lys Thr Cys Trp Asp Ala Asp Pro Leu
900 905 910
Lys Arg Pro Thr Phe Lys Gln Ile Val Gln Leu Ile Glu Lys Gln Ile
915 920 925
Ser Glu Ser Thr Asn His Ile Tyr Ser Asn Leu Ala Asn Cys Ser Pro
930 935 940
Asn Arg Gln Lys Pro Val Val Asp His Ser Val Arg Ile Asn Ser Val
945 950 955 960
Gly Ser Thr Ala Ser Ser Ser Gln Pro Leu Leu Val His Asp Asp Val
965 970 975

Claims (10)

1. Use of an agent comprising one or more selected from the group consisting of:
(1) A FOXA2 inhibitor,
(2) FOXA1 promoter, and
(3) KIT responds to inhibitors of signaling pathway.
2. The use according to claim 1, wherein the FOXA2 inhibitor comprises an inhibitory molecule that interferes with transcription and/or expression of a gene encoding FOXA2 protein, or down regulates FOXA2 protein activity,
preferably, the inhibitory molecule is selected from the group consisting of: (1) A small molecule compound, an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, lncRNA, a specific antibody or ligand, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1),
more preferably, the FOXA2 inhibitor is an siRNA or construct thereof that targets the FOXA2 protein-encoding gene or transcript thereof as an inhibition, or the FOXA2 inhibitor is an sgRNA knocked out or knocked down of the FOXA2 gene using a technique selected from ZFN, TALEN and CRISPR.
3. The use according to claim 1, wherein the FOXA1 promoter is an agent that upregulates FOXA1 expression or activity,
Preferably, the FOXA1 promoter is selected from the group consisting of: a small molecule compound, a nucleic acid molecule, or a combination thereof,
more preferably, the nucleic acid molecule is a nucleic acid construct comprising a coding sequence for FOXA1, wherein the amino acid sequence of FOXA1 is selected from one or more of the following: (a) a polypeptide having a sequence as set forth in SEQ ID NO. 1 or 2; (b) A polypeptide which is formed by substitution, deletion or addition of one or more amino acid residues to the sequence shown in SEQ ID NO. 1 or 2 and has the function of the polypeptide (a) and is derived from the polypeptide (a); or (c) a polypeptide derived from (a) which has more than 90% homology with the polypeptide sequence of (a) and has the function of the polypeptide of (a).
4. The use of claim 1, wherein the KIT response signaling pathway inhibitor comprises a KIT inhibitor,
preferably, the KIT inhibitor comprises an inhibitory molecule that interferes with transcription and/or expression of a gene encoding a KIT protein, or down-regulates KIT protein activity, said inhibitory molecule being selected from the group consisting of: (1) A small molecule compound, an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, lncRNA, a specific antibody or ligand, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1),
more preferably, the KIT inhibitor is an siRNA or construct thereof that has a KIT protein encoding gene or transcript thereof as an inhibition target; alternatively, the KIT inhibitor is an sgRNA knocked out or knocked down of a KIT gene using a technique selected from ZFN, TALEN, and CRISPR; alternatively, the KIT inhibitor is a tyrosine kinase receptor inhibitor, such as imatinib, sorafenib, sunitinib, and cabitinib.
5. A pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, the active ingredient comprising one or more selected from the group consisting of:
(1) A FOXA2 inhibitor,
(2) FOXA1 promoter, and
(3) KIT responds to inhibitors of signaling pathway.
6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition has a characteristic selected from any one of the following (1) - (3):
(1) The FOXA2 inhibitor includes an inhibitory molecule that interferes with transcription and/or expression of a gene encoding FOXA2 protein, or down regulates the activity of FOXA2 protein,
preferably, the inhibitory molecule is selected from the group consisting of: (a) A small molecule compound, an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, lncRNA, a specific antibody or ligand, or a combination thereof, and (b) a nucleic acid construct capable of expressing or forming (a),
more preferably, the FOXA2 inhibitor is an siRNA or construct thereof that targets the FOXA2 protein-encoding gene or transcript thereof as an inhibition target, or the FOXA2 inhibitor is an sgRNA that knocks out or knocks down the FOXA2 gene using a technique selected from ZFN, TALEN and CRISPR,
(2) The FOXA1 promoter is an agent that upregulates FOXA1 expression or activity,
preferably, the FOXA1 promoter is selected from the group consisting of: a small molecule compound, a nucleic acid molecule, or a combination thereof,
More preferably, the nucleic acid molecule is a nucleic acid construct comprising a coding sequence for FOXA1, wherein the amino acid sequence of FOXA1 is selected from one or more of the following: (a) a polypeptide having a sequence as set forth in SEQ ID NO. 1 or 2; (b) A polypeptide which is formed by substitution, deletion or addition of one or more amino acid residues to the sequence shown in SEQ ID NO. 1 or 2 and has the function of the polypeptide (a) and is derived from the polypeptide (a); or (c) a polypeptide derived from (a) which has more than 90% homology with the polypeptide sequence of (a) and has the function of the polypeptide of (a),
(3) The KIT response signaling pathway inhibitor comprises a KIT inhibitor,
preferably, the KIT inhibitor comprises an inhibitory molecule that interferes with transcription and/or expression of a gene encoding a KIT protein, or down-regulates KIT protein activity, said inhibitory molecule being selected from the group consisting of: (1) A small molecule compound, an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, lncRNA, a specific antibody or ligand, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1),
more preferably, the KIT inhibitor is an siRNA or construct thereof that has a KIT protein encoding gene or transcript thereof as an inhibition target; alternatively, the KIT inhibitor is an sgRNA knocked out or knocked down of a KIT gene using a technique selected from ZFN, TALEN, and CRISPR; alternatively, the KIT inhibitor is a tyrosine kinase receptor inhibitor, such as imatinib, sorafenib, sunitinib, and cabitinib.
The application of FOXA1 gene or protein, FOXA2 gene or protein or gene or protein of KIT response signal path as target spot in screening medicine for treating or preventing neuroendocrine prostate cancer or as molecular index for clinical application in neuroendocrine prostate cancer,
preferably, the gene of the KIT response signal pathway comprises a KIT gene.
8. The use according to claim 7, wherein the medicament is a FOXA1 promoter, FOXA2 inhibitor or KIT inhibitor.
9. The application of a reagent for detecting the FOXA1 gene or protein, the FOXA2 gene or protein or the gene or protein of a KIT response signal pathway in preparing a KIT for diagnosing neuroendocrine prostate cancer,
preferably, the reagent is a primer, probe, or protein-specific antibody for detecting a gene,
preferably, the detection kit further comprises a nucleic acid detection reagent or an immunological detection reagent.
10. A detection KIT comprising a reagent for detecting a FOXA1 gene or protein, a FOXA2 gene or protein, or a gene or protein of a KIT response signal pathway,
preferably, the reagent is a primer, probe, or protein-specific antibody for detecting a gene,
Preferably, the detection kit further comprises a nucleic acid detection reagent or an immunological detection reagent.
CN202210431313.7A 2022-04-22 2022-04-22 Agents and uses for treating neuroendocrine prostate cancer Pending CN116966308A (en)

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