WO2018223867A1 - 一种前列腺癌的标志物spop及其在指导肿瘤治疗中的应用 - Google Patents

一种前列腺癌的标志物spop及其在指导肿瘤治疗中的应用 Download PDF

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WO2018223867A1
WO2018223867A1 PCT/CN2018/088802 CN2018088802W WO2018223867A1 WO 2018223867 A1 WO2018223867 A1 WO 2018223867A1 CN 2018088802 W CN2018088802 W CN 2018088802W WO 2018223867 A1 WO2018223867 A1 WO 2018223867A1
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spop
protein
prostate cancer
dna
mrna
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孙颖浩
任善成
黄浩杰
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上海长海医院
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention belongs to the field of cancer guiding treatment, and particularly relates to the application of SPOP as a marker for molecular typing of prostate cancer and for guiding treatment of prostate cancer patients with BET inhibitors.
  • PCa Prostatic Cancer
  • Androgen blockade therapy is the main treatment for advanced prostate cancer, but many researchers have found in clinical practice that although continuous androgen blockade therapy is effective in nearly 90% of patients in the first treatment, almost all patients are It is inevitable to progress to androgen-independent prostate cancer within 2 to 3 years, and the prognosis of patients with the above outcome is poor, with a median survival time of only 18 months. In the late stage, patients with different prostate cancers have different responses to endocrine therapy.
  • the object of the present invention is to provide a method for molecular typing of prostate cancer as a marker and to guide treatment of prostate cancer patients with BET inhibitors, and to improve the success rate of BET inhibitor treatment for prostate cancer by detecting SPOP.
  • the present inventors have found that wild-type SPOP proteins are capable of binding and inducing ubiquitination degradation of the BET protein family (BRD2/3/4) by recognizing a proton phantom.
  • SPOP is a bridge protein that binds to a substrate by Cul3, a member of the ubiquitin ligase E3 family, and promotes their degradation by mediating the ubiquitination of many nuclear proteins, thereby participating in the regulation of various functions of cells.
  • the marker for prostate cancer may be DNA, mRNA or a protein encoded by SPOP.
  • the Pubed Gene ID of the SPOP DNA gene is 8405, the NCBI Reference Sequence in NCBI: NG_041815.1, and the sequence is available at https://www.ncbi.nlm.nih.gov/nuccore/NG_041815.1.
  • the nucleotide sequence of the SPOP mRNA is shown in SEQ ID NO. 1, see https://www.ncbi.nlm.nih.gov/nuccore/AJ000644.1; the SPOP protein sequence is SEQ ID NO. See https://www.ncbi.nlm.nih.gov/protein/NP_003554.1.
  • the present invention it is possible to determine the expression level of a gene by mutation of SPOP DNA, and the expression level of SPOP mRNA or a protein encoded thereby, thereby molecularly typing prostate cancer and guiding BET inhibition in prostate cancer patients.
  • the agent is treated.
  • the invention also provides an application of an amplification primer or a detection reagent of SPOP DNA for preparing a product for molecular typing of prostate cancer, and the detection reagent of the SPOP DNA can be, but is not limited to, a nucleic acid probe for specifically detecting SPOP DNA. .
  • Molecular typing of prostate cancer is performed by quantifying whether SPOP DNA is mutated.
  • the invention also provides an application of an amplification primer or detection reagent for SPOP mRNA in preparing a product for molecular typing of prostate cancer
  • the detection reagent of the SPOP mRNA can be, but is not limited to, a nucleic acid probe for specifically detecting SPOP mRNA.
  • Molecular typing of prostate cancer is performed by quantifying the expression level of the gene SPOP mRNA.
  • the expression level of the mRNA can be determined by the following methods: microarray technology, Northern blotting, and quantitative PCR; the quantitative PCR is real-time quantitative PCR or multiplex PCR or the like.
  • the present invention also provides the use of a detection reagent for SPOP protein in the preparation of a product for molecular typing of prostate cancer, which may be, but is not limited to, an antibody that specifically detects a SPOP protein.
  • Molecular typing of prostate cancer is performed by quantifying the expression level of SPOP protein.
  • the expression level of the SPOP protein can be determined by immunohistochemistry, Western blotting, ELISA, RIA, mass spectrometry, and the like.
  • the present invention also provides a prostate cancer in vitro diagnostic product comprising an agent that specifically detects SPOP DNA, and/or an agent that specifically detects SPOP mRNA, and/or an agent that specifically detects a SPOP protein.
  • the prostate cancer in vitro diagnostic product can be used for molecular typing of prostate cancer.
  • the reagent for specifically detecting SPOP DNA may be, but not limited to, a nucleic acid probe capable of specifically recognizing the SPOP DNA; and the reagent for specifically detecting SPOP mRNA may be, but not limited to, a nucleic acid probe.
  • the nucleic acid probe is capable of specifically recognizing the SPOP mRNA; the agent that specifically detects the SPOP protein can be, but is not limited to, an antibody that specifically recognizes the SPOP protein.
  • the prostate cancer in vitro diagnostic product includes a kit, a gene chip, a solid support, and the like.
  • the solid support comprises an array, a microarray, a protein array, and the like.
  • the invention also provides the use of SPOP in directing BET small molecule inhibitors for the treatment of prostate cancer.
  • the present invention also provides the use of a small molecule inhibitor of the BET protein family. If the SPOP DNA is mutated in a patient with prostate cancer relative to a normal population, it is a DNA mutation group, otherwise it is a normal DNA group; or SPOP mRNA Or the expression level of the protein encoded by the lower one is lower, otherwise it is the normal expression group; the normal DNA group and the normal expression group of mRNA or protein can be treated with BET small molecule inhibitor; and the DNA mutation group Both the mRNA and protein low expression groups are not recommended for treatment with a BET small molecule inhibitor; wherein the BET small molecule inhibitor includes, but is not limited to, JQ1.
  • the present invention also provides a method for molecular typing of a patient diagnosed with prostate cancer and for guiding treatment of a prostate cancer patient with a BET inhibitor, the method comprising the steps of:
  • SPOP DNA is mutated relative to the normal population, it is a DNA mutation group, otherwise it is a normal DNA group; or When the expression level of SPOP mRNA or a protein encoded thereby is decreased, it is a low expression group, otherwise it is a normal expression group.
  • Prostate cancer patients with normal DNA and normal expression groups of mRNA or protein can be treated with BET inhibitors; neither the DNA mutation group nor the mRNA or protein low expression group is recommended for treatment with BET inhibitors.
  • the present invention also provides the use of SPOP as an adaptor protein of E3 ubiquitin ligase CUL-3 in the preparation of a medicament for promoting polyubiquitination of BET protein and protein degradation; wherein the BET protein is preferably BRD2, BRD3, BRD4 protein.
  • the invention also provides the use of SPOP for the preparation of a medicament for lowering the expression level of DEK/ERG/SRC-3 protein.
  • the invention also provides the use of SPOP for the preparation of a medicament for reducing the expression level of RAC1 gene mRNA and protein.
  • the invention also provides the use of the SPOP mutant in the preparation of a medicament for up-regulating the BET protein level; wherein the BET protein is preferably a BRD2, BRD3, BRD4 protein; wherein the SPOP mutant comprises Y87C, Y87N, F102C, S119N , F125V, K129E, W131C, W131G, F133L, F133V and K134N.
  • the mutation sites and mutation information corresponding to Y87C, Y87N, F102C, S119N, F125V, K129E, W131C, W131G, F133L, F133V and K134N have been reported in the previous literature. For public information, see http: //www.pnas.org/content/110/17/6997.full.pdf.
  • the invention also provides the use of SPOP in the preparation of a medicament for reducing the drug resistance of JQ1.
  • the present invention also provides the use of a BRD small molecule inhibitor for the preparation of a medicament for reducing the drug resistance of JQ1; wherein the BET protein comprises a BRD2, BRD3, BRD4 protein, preferably a BRD4 protein.
  • the present invention also provides the use of an inhibitor of USP9X/USP49 for the preparation of a medicament for promoting polyubiquitination of BET protein and protein degradation; wherein the BET protein is preferably a BRD2, BRD3, BRD4 protein.
  • the present invention also provides the use of an inhibitor of USP9X/USP49 for the preparation of a medicament for reducing the drug resistance of JQ1.
  • the present invention also provides a method for reducing the drug resistance of JQ1, which comprises increasing the expression level of mRNA and protein of SPOP, or adding a BRD protein inhibitor, or adding a USP9X/USP49 inhibitor or the like.
  • the beneficial effects of the present invention are the novel marker for guiding prostate cancer treatment proposed by the present invention, namely, the SPOP gene, which can molecularly classify prostate cancer and guide prostate cancer patients to treat with BET inhibitor.
  • Figure 1 shows the BET protein family BRD2/3/4 identified by the yeast two-hybrid system screen: the region indicated by the vertical dotted line is the region where the BET protein binds to SPOP, and the region indicated by the arrow is the SBC motif.
  • Figure 2 shows co-precipitation of anti-BRD2/3/4 antibody by co-immunoprecipitation assay (Co-IP) and found that BRD2/3/4 protein can bind to SPOP protein.
  • Co-IP co-immunoprecipitation assay
  • FIG 3 shows that the expression level of BRD2/3/4 protein was down-regulated after exogenously highly expressed SPOP protein; this phenomenon was reversed by the addition of protein inhibitor (MG132).
  • FIG. 4 shows that BRD2/3/4 protein expression levels are up-regulated after interference with SPOP protein expression: siC: siRNA control negative control, siSPOP: SPOP interfering RNA.
  • Figure 5 shows ubiquitination experiments of 293T cells transfected with wild-type SPOP and SPOP mutant SPOP- ⁇ BTB (BTB domain deletion).
  • Wild-type SPOP can promote the polyubiquitination of BRD2/3/4, and the heterologously expressed SPOP mutant SPOP- ⁇ BTB (BTB domain deletion) can not promote the polyubiquitination of BRD2/3/4 protein.
  • Figure 6 shows the presence of SPOP-binding consensus (SBC) in BRD2/3/4 protein, ie ⁇ - ⁇ -S/TS/TS/T, ⁇ is a non-polar residue, and ⁇ is a polar residue.
  • SBC SPOP-binding consensus
  • Figure 7 shows that immunoprecipitation experiments showed that SPOP only binds to wild-type BRD2/3/4, but does not bind to SBC-deficient mutants.
  • Figure 8 shows plasmids transfected with 293T cells with BRD2/3/4 and BRD2/3/4- ⁇ SBC mutations and found that SPOP does not promote degradation of BRD2/3/4- ⁇ SBC mutants.
  • Figure 9 shows the ubiquitination assay of 293T cells transfected with BRD2/3/4 and BRD2/3/4- ⁇ SBC mutant plasmids. It is thought that SPOP cannot promote the degradation of BRD2/3/4- ⁇ SBC mutants.
  • Figure 10 shows Myc-labeled prostate cancer-associated SPOP mutants, including Y87C, Y87N, F102C, S119N, F125V, K129E, W131C, W131G, F133L, F133V and K134N, and the SPOP after mutation cannot be combined with BRD2/3/4 Reactions; further in vivo ubiquitination assays showed that all SPOP mutants lost the ability to promote polyubiquitination of the BRD2/3/4 protein. Heterologous expression of SPOP leads to an increase in endogenous BRD2/3/4 protein levels, suggesting that prostate cancer-associated SPOP mutations do not promote BRD2/3/4 protein polyubiquitination and protein degradation.
  • SPOP mutants including Y87C, Y87N, F102C, S119N, F125V, K129E, W131C, W131G, F133L, F133V and K134N co-immunoprecipitation experiments;
  • SPOP mutant loss promotes BRD2/3/4 protein polyubiquitin The ability to prime;
  • c heterologously expressed SPOP leads to an increase in endogenous BRD2/3/4 protein levels.
  • Figure 11 shows BRD2/3/4 immunohistochemistry in tumor tissues of wild-type and mutant SPOP prostate cancer patients; SPOP-WT: SPOP wild-type prostate cancer patients, SPOP-MUT: SPOP mutant prostate cancer patients.
  • Figure 12 shows that BRD2/3/4 mRNA levels are not significantly different between wild-type SPOP and mutant SPOP prostate cancer tissue samples.
  • Figure 13 is a demonstration of the role of the SPOP mutant F133V cell line in the use of BRD2/3/4 to interfere with lentivirus and JQ1 inhibitors in prostate cancer.
  • EV-shC no-load control
  • EV-shBRD2/3/4 stable interference BRD2/3/4 control
  • F133V-shC SPOP F133V mutation no-load control
  • F133V-shBRD2/3/4 stable interference BRD2/3/ 4 SPOP F133V control
  • JQ1 BET protein inhibitor.
  • Figure 14 is a Venn diagram of up-regulated JQ1 resistance-associated genes in SPOP-mutated prostate cancer patients.
  • Figure 15 is ChlP-seq data of the BRD4 protein at the RAC1 gene locus of wild-type and SPOP F133V mutant C4-2 cells after addition of JQ1.
  • Figure 16 is a demonstration of the role of SPOP mutant F133V cell line in USP49/9X interfering with lentivirus and JQ1 inhibitor in prostate cancer in vivo.
  • EV-shC no-load control
  • EV-shUSP49/9X stable interference USP49/9X4 control
  • F133V-shC SPOP F133V mutation no-load control
  • F133V-shUSP49/9X stable SUP F133V control of USP49/9X
  • JQ1 BET protein inhibitor.
  • Example 1 SPOP binds and recognizes the SBC motif degradant of BRD2/3/4 protein to promote its ubiquitination degradation
  • the present invention screens the target protein of SPOP by the yeast two-hybrid system, and finds 246 positive clone strains including known DEK and SRC-3, and screens the target protein therein, wherein the highest number of hits is the BET protein family. 52 clones were BRD2, 2 clones were BRD3, and 6 clones were BRD4 (as shown in Figure 1).
  • Co-IP Co-immunoprecipitation
  • the SPOP mutant SPOP- ⁇ BTB (BTB domain deletion) was deleted by deleting the corresponding sequence of SPOP with enzymatic activity.
  • the ubiquitination assay showed that wild-type SPOP can promote the polyubiquitination of BRD2/3/4.
  • the highly expressed SPOP mutant SPOP- ⁇ BTB (BTB domain deletion) failed to promote polyubiquitination of the BRD2/3/4 protein (Fig. 5). The above data suggest that SPOP can promote the polyubiquitination and degradation of BRD2/3/4 protein.
  • SPOP acts as an adaptor of the E3 ubiquitin ligase CUL-3 in different types of cells (including prostate cancer cells) to promote the polyubiquitin of the BRD2/3/4 protein. And protein degradation.
  • the substrate protein must have a specific SBC domain (SBC motif).
  • SBC motif SBC motif
  • the BRD2/3/4 mutant (BRD2- ⁇ SBC, BRD3- ⁇ SBC, BRD4- ⁇ SBC) lacking the SBC domain was transfected into 293T cells, and the Myc-SPOP plasmid was added.
  • the co-immunoprecipitation analysis showed that SPOP was only wild type.
  • BRD2/3/4 binds but does not bind to SBC-deficient mutants ( Figure 7-8).
  • the expression of BRD2/3/4 protein and its mutants in 293T cells was detected by ubiquitination assay. It was found that SPOP could not promote the degradation of BRD2/3/4- ⁇ SBC mutants and only promote wild-type BRD2/3/4 protein. Degradation ( Figure 9).
  • the present invention establishes a series of Myc-labeled prostate cancer-associated SPOP mutants, including Y87C, Y87N, F102C, S119N, F125V, K129E, W131C, W131G, F133L, F133V and K134N.
  • Co-immunoprecipitation experiments showed that in 293T prostate cancer cells, the mutated SPOP could not react with BRD2/3/4 (Fig. 10a).
  • Further in vivo ubiquitination assays showed that all SPOP mutants lost the ability to promote polyubiquitination of the BRD4 protein, whereas only wild-type SPOPs were able to promote the ubiquitination of the BRD4 protein (Fig. 10b).
  • the present invention found that 13 prostate cancer samples contained SPOP mutations by genome-wide sequencing, RNA sequencing, and Sanger sequencing in a total of 99 primary prostate cancer cohorts.
  • Immunohistochemistry (IHC) analysis showed that most of the SPOP mutant prostate tissues were strongly positive for BRD2/3/4 protein staining (BRD2: 85% (11/13), BRD3: 92% (12/13), BRD4 : 85% (11/13)) (Figure 11).
  • BRD2/3/4 protein staining was strongly positive (BRD2: 29% (25/86), BRD3: 34% (29/86), BRD4: 41% (35/86)), most BRD2/3/4 staining was weakly positive or negative (BRD2: 71% (61/86), BRD3: 66% (57/86), BRD4: 59% (51/86) ) ( Figure 11). This indicates that the expression level of BRD2/3/4 protein is up-regulated in tumor tissues of prostate cancer patients with SPOP mutation.
  • the present invention found by RNA sequencing of 32 prostate cancer tissue samples (26 wild-type SPOP, 6 mutant SPOP), BRD2/3/4 mRNA levels in wild-type SPOP and mutant SPOP prostate cancer tissue samples have no significant difference (Fig. 12), indicating that the mRNA expression level of BRD2/3/4 in prostate cancer patients with SPOP mutations did not change.
  • the above results suggest that changes in BRD2/3/4 protein levels in SPOP-mutated prostate cancer occur in Post-translational level of BRD2/3/4 mRNA.
  • prostate cancer-associated SPOP mutations can cause an increase in BET protein content at post-translational levels.
  • BET small molecule inhibitors are currently potential tumor-targeted therapies.
  • the present inventors found that the BET inhibitor JQI treatment experimental group (EV-shC JQ1), the interference BRD2/3/4 expression group (EV-shBRD2/3/4JQ1) and the negative control group (EV-shC Vehicle) were respectively found, and JQ1 was found to be Significant inhibition of tumor growth.
  • the SPOP F133V mutant group F133V-shC JQ1
  • the cell line (F133V-shBRD2/3/4JQ1) after knockdown of BRD2/3/4 in the SPOP F133V mutant group JQ1 significantly inhibited tumor growth (Fig. 13), indicating BRD2/3/ after SPOP mutation. 4
  • the protein expression level is up-regulated, the effect of using JQ1 to inhibit tumor growth is poor, which may be the cause of JQ1 resistance.
  • JQ1 The application of JQ1 can not hinder this process, suggesting that this is a mechanism for drug resistance in patients with SPOP mutations using JQ1.
  • the present inventors have found that the USP9X/USP49 group (EV-shUSP9X/USP49JQ1), the SPOP F133V simple mutation group (F133V-shC JQ1), and the USP9X/USP49 knockdown cell in the SPOP F133V mutant group were treated with the BET inhibitor JQI, respectively.
  • Line (F133V-sh USP9X/USP49JQ1), JQ1 was found to be unable to inhibit tumor growth in the SPOP F133V mutant group, but significantly inhibited tumor growth after interfering with USP9X/USP49 protein expression (Fig.
  • the present inventors have found that a decrease in the level of BRD4 protein can cause cells to be sensitive to JQ1, while SPOP knockdown can cause tolerance of cells to JQ1.
  • BRD4 protein is a key molecule in determining the sensitivity of JQ1 in castration-resistant prostate cancer cells (such as C4-2 cells).
  • the increase in BET protein levels in SPOP mutant cells is related to JQ1 resistance.

Abstract

本发明公开了一种前列腺癌标志物SPOP，所述SPOP标志物包括SPOP的DNA、mRNA或由其编码的蛋白质，所述SPOP DNA序列的Pubmed Gene ID为8405；所述SPOP mRNA的核苷酸序列如SEQ ID NO.1所示；所述SPOP的蛋白质序列如SEQ ID NO.2所示。本发明还公开了所述SPOP标志物在对前列腺癌进行分子分型以及指导BET抑制剂用于治疗前列腺癌中的应用。

Description

一种前列腺癌的标志物SPOP及其在指导肿瘤治疗中的应用 技术领域
本发明属于癌症指导治疗领域，具体涉及SPOP作为标志物对前列腺癌进行分子分型以及指导前列腺癌患者应用BET抑制剂进行治疗的应用。
背景技术
前列腺癌(Prostatic Cancer，PCa)是一种严重威胁男性健康的恶性肿瘤，占全球肿瘤发病率第二位、死亡率第六位。我国PCa的发病率近年来一直处于显著的上升趋势。晚期前列腺癌激素治疗敏感性的差异。雄激素阻断治疗是晚期前列腺癌的主要治疗方式，但是，许多学者在临床实践中发现，尽管在首次治疗时运用持续性雄激素阻断治疗对近90％的患者有效，但几乎所有患者在2～3年内均不可避免地进展为雄激素非依赖性前列腺癌，且发生上述转归的患者预后差，中位生存时间仅为18个月。晚期不同前列腺癌患者对内分泌治疗的反应差别迥异，这种现象临床上称之为前列腺癌对内分泌治疗的敏感性不同，综合国际国内研究，这种敏感性的差异尚无有效方法准确预测，这便导致了众多晚期前列腺癌的患者无奈地接受了不合理，不规范的内分泌治疗。溴结构域与额外末端结构域蛋白(Bromodomain and extraterminal domain，BET)逐渐成为乳腺癌和白血病中新的治疗靶点，应用BET小分子抑制剂可以有效抑制肿瘤的生长。而E3泛素酶亚层基结合配接器斑点型POZ蛋白(E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein，SPOP)在前列腺癌中发生高频的突变。
发明内容
本发明的目的在于提供SPOP作为标志物对前列腺癌进行分子分型以及指导前列腺癌患者应用BET抑制剂进行治疗的应用，通过SPOP的检测，提高对前列腺癌进行BET抑制剂治疗的成功率。本发明发现野生型SPOP蛋白通过识别降解子模体能够结合并诱导BET蛋白家族(BRD2/3/4)发生泛素化降解。SPOP为泛素连接酶E3家族成员Cul3与底物结合的桥梁蛋白(adaptor)，通过介导许多核蛋白的泛素化而促进它们的降解，从而参与调控细胞的多种功能。本发明中，作为前列腺癌的标志物可以是SPOP的DNA、mRNA或由其编码的蛋白质。所述SPOP DNA基因Pubmed Gene ID为8405，在NCBI中的序列号(NCBI Reference Sequence)：NG_041815.1，序列见https：//www.ncbi.nlm.nih.gov/nuccore/NG_041815.1。所述SPOP mRNA的核苷酸序列如SEQ ID NO.1所示，见https：//www.ncbi.nlm.nih.gov/nuccore/AJ000644.1；所述SPOP蛋白质序列如SEQ ID NO.2所示， 见https：//www.ncbi.nlm.nih.gov/protein/NP_003554.1。本发明中，可通过SPOP DNA是否发生突变，以及SPOP的mRNA或由其编码的蛋白质的表达水平变化，确定基因的表达水平变化，从而对前列腺癌进行分子分型以及指导前列腺癌患者应用BET抑制剂进行治疗。
本发明还提供了SPOP DNA的扩增引物或检测试剂在制备对前列腺癌进行分子分型的产品中的应用，所述SPOP DNA的检测试剂可以但不限于是特异性检测SPOP DNA的核酸探针。通过SPOP DNA是否突变进行定量，来对前列腺癌进行分子分型。
本发明还提供了SPOP mRNA的扩增引物或检测试剂在制备对前列腺癌进行分子分型的产品中的应用，所述SPOP mRNA的检测试剂可以但不限于是特异性检测SPOP mRNA的核酸探针。通过对所述基因SPOP mRNA的表达水平进行定量，来对前列腺癌进行分子分型。所述mRNA的表达水平可通过以下方法确定：微阵列技术、RNA印迹和定量PCR；所述定量PCR为实时定量PCR或多重PCR等。
本发明还提供了SPOP蛋白质的检测试剂在制备对前列腺癌进行分子分型的产品中的应用，所述蛋白质的检测试剂可以但不限于是特异性检测SPOP蛋白质的抗体。通过对SPOP蛋白质的表达水平进行定量，来对前列腺癌进行分子分型。所述SPOP蛋白质的表达水平可通过以下方法确定：免疫组织化学、蛋白质印迹、ELISA、RIA和质谱法等。
本发明还提供了一种前列腺癌体外诊断产品，所述体外诊断产品包括特异性检测SPOP DNA的试剂，和/或特异性检测SPOP mRNA的试剂，和/或特异性检测SPOP蛋白质的试剂。所述前列腺癌体外诊断产品可用于对前列腺癌进行分子分型。所述特异性检测SPOP DNA的试剂可以但不限于是核酸探针，所述核酸探针能特异性识别所述SPOP DNA；所述特异性检测SPOP mRNA的试剂可以但不限于是核酸探针，所述核酸探针能特异性识别所述SPOP mRNA；所述特异性检测SPOP蛋白质的试剂可以但不限于是抗体，所述抗体能特异性识别所述SPOP蛋白质。
其中，所述前列腺癌体外诊断产品包括试剂盒、基因芯片、固体支持体等。所述固体支持体包括阵列、微阵列、蛋白质阵列等。
本发明还提供了SPOP在指导BET小分子抑制剂用于前列腺癌治疗中的应用。
本发明还提供了BET蛋白家族小分子抑制剂的应用，若相对于正常人群，患有前列腺癌的患者中所述SPOP DNA发生突变，即为DNA突变组，否则为DNA正常组；或SPOP mRNA或由其编码的蛋白质的表达量降低，则为低表达组，否则为正常表达组；DNA正常组和mRNA或蛋白正常表达组前列腺癌患者可采用BET小分子抑制剂进行治疗；而DNA突变组和mRNA或蛋白低表达组均不推荐应用BET小分子抑制剂进行治疗；其中，所述BET小分子抑制剂包括但不限于JQ1。
本发明还提供了一种对已确诊为前列腺癌的患者进行分子分型以及指导前列腺癌患者应用BET抑制剂进行治疗的方法，所述方法包括以下步骤：
a)检测前列腺癌患者的病理样本中SPOP的DNA拷贝数、或mRNA或由其编码的蛋白质的表达量；
b)将患者分为DNA正常组和突变组，mRNA或蛋白质正常表达组和低表达组，若相对于正常人群，所述SPOP DNA发生突变，即为DNA突变组，否则为DNA正常组；或SPOP mRNA或由其编码的蛋白质的表达量降低，则为低表达组，否则为正常表达组。DNA正常组和mRNA或蛋白正常表达组前列腺癌患者可采用BET抑制剂进行治疗；而DNA突变组和mRNA或蛋白低表达组均不推荐应用BET抑制剂进行治疗。
本发明还提供了SPOP作为E3泛素连接酶CUL-3的衔接蛋白在制备促进BET蛋白的多聚泛素化以及蛋白降解的药物中的应用；其中，所述BET蛋白优选为BRD2、BRD3、BRD4蛋白。
本发明还提供了SPOP在制备降低DEK/ERG/SRC-3蛋白表达水平的药物中的应用。
本发明还提供了SPOP在制备降低RAC1基因mRNA和蛋白表达水平的药物中的应用。
本发明还提供了SPOP突变体在制备上调BET蛋白水平的药物中的应用；其中，所述BET蛋白优选为BRD2、BRD3、BRD4蛋白；其中，所述SPOP突变体包括Y87C，Y87N，F102C，S119N，F125V，K129E，W131C，W131G，F133L，F133V和K134N。本发明中，所述Y87C，Y87N，F102C，S119N，F125V，K129E，W131C，W131G，F133L，F133V和K134N对应的突变位点和突变信息，之前文献均已报道，为公开信息，可参见http：//www.pnas.org/content/110/17/6997.full.pdf。
本发明还提供了SPOP在制备降低JQ1的耐药性的药物中的应用。
本发明还提供了BRD小分子抑制剂在制备降低JQ1的耐药性的药物中的应用；其中所述BET蛋白包括BRD2、BRD3、BRD4蛋白，优选为BRD4蛋白。
本发明还提供了USP9X/USP49的抑制剂在制备促进BET蛋白的多聚泛素化以及蛋白降解的药物中的应用；其中，所述BET蛋白优选为BRD2、BRD3、BRD4蛋白。
本发明还提供了USP9X/USP49的抑制剂在制备降低JQ1的耐药性的药物中的应用。
本发明还提供了一种降低JQ1的耐药性的方法，所述方法包括提高SPOP的mRNA和蛋白质的表达水平，或加入BRD蛋白抑制剂，或加入USP9X/USP49抑制剂等。
本发明的有益效果在于，本发明提出的指导前列腺癌治疗的新标志物，即SPOP基因，该标志物能对前列腺癌进行分子分型以及指导前列腺癌患者应用BET抑制剂进行治疗。
附图说明
图1为酵母双杂交系统筛选鉴别的BET蛋白家族BRD2/3/4：垂直虚线所示区域为BET蛋白与SPOP结合的区域，箭头所示区域为SBC模体。
图2为免疫共沉淀实验(Co-IP)应用抗BRD2/3/4抗体进行共沉淀，发现BRD2/3/4蛋白能够与SPOP蛋白发生结合。
图3为外源性高表达SPOP蛋白后，下调了BRD2/3/4蛋白的表达水平；加入蛋白抑制剂(MG132)后逆转了这一现象。
图4为干扰SPOP蛋白表达后，BRD2/3/4蛋白表达水平发生上调：siC：siRNA control阴性对照，siSPOP：SPOP干扰RNA。
图5为293T细胞转染野生型SPOP和SPOP突变体SPOP-ΔBTB(BTB结构域缺失)进行泛素化实验。野生型SPOP能够促进BRD2/3/4的多聚泛素化，异源性表达的SPOP突变体SPOP-ΔBTB(BTB结构域缺失)均无法促进BRD2/3/4蛋白的多聚泛素化
图6为BRD2/3/4蛋白中存在SPOP结合序列(SPOP-binding consensus，SBC)，即Φ-π-S/T-S/T-S/T，Φ为非极性残基，π为极性残基；a为BRD2/3/4、MacroH2A和DEK的SBC；b为野生型以及SBC缺失型BRD2/3/4蛋白。
图7为免疫共沉淀实验分析显示SPOP只与野生型BRD2/3/4结合，但却不与SBC缺失的突变体结合。
图8为293T细胞转染BRD2/3/4和BRD2/3/4-ΔSBC突变的质粒，发现SPOP不能促进BRD2/3/4-ΔSBC突变体的降解。
图9为293T细胞转染BRD2/3/4和BRD2/3/4-ΔSBC突变的质粒后进行泛素化实验，想SPOP无法促进BRD2/3/4-ΔSBC突变体的降解。
图10为与Myc标记的与前列腺癌相关的SPOP突变体，包括Y87C，Y87N，F102C，S119N，F125V，K129E，W131C，W131G，F133L，F133V和K134N，突变后的SPOP无法与BRD2/3/4反应；进一步体内泛素化分析实验显示，所有SPOP突变体失去促进BRD2/3/4蛋白多聚泛素化的能力。异源性表达的SPOP导致内源性BRD2/3/4蛋白水平上升，提示前列腺癌相关的SPOP突变不能促进BRD2/3/4蛋白多聚泛素化以及蛋白降解。a：SPOP突变体，包括Y87C，Y87N，F102C，S119N，F125V，K129E，W131C，W131G，F133L，F133V和K134N的免疫共沉淀实验；b：SPOP突变体失去促进BRD2/3/4蛋白多聚泛素化的能力；c：异源性表达的SPOP导致内源性BRD2/3/4蛋白水平上升。
图11为野生型及突变型SPOP前列腺癌患者肿瘤组织的BRD2/3/4免疫组化；SPOP-WT：SPOP野生型前列腺癌患者，SPOP-MUT：SPOP突变型前列腺癌患者。
图12为BRD2/3/4mRNA水平在野生型SPOP和突变型SPOP前列腺癌组织样本中无明显差异。
图13为体内实验证实SPOP突变F133V细胞系应用BRD2/3/4干扰慢病毒和JQ1抑制剂在前列腺癌中的作用。EV-shC：空载对照；EV-shBRD2/3/4：稳定干扰BRD2/3/4对照；F133V-shC：SPOP F133V突变空载对照；F133V-shBRD2/3/4：稳定干扰BRD2/3/4的SPOP F133V对照；JQ1：BET蛋白抑制剂。*：P＜0.05；***：P＜0.001；n.s：无统计学意义。
图14为SPOP突变的前列腺癌患者中发生上调的JQ1耐药相关基因的韦恩图。
图15为野生型和SPOP F133V突变型C4-2细胞在加入JQ1后BRD4蛋白在RAC1基因位点的ChlP-seq数据。
图16为体内实验证实SPOP突变F133V细胞系应用USP49/9X干扰慢病毒和JQ1抑制剂在前列腺癌中的作用。EV-shC：空载对照；EV-shUSP49/9X：稳定干扰USP49/9X4对照；F133V-shC：SPOP F133V突变空载对照；F133V-shUSP49/9X：稳定干扰USP49/9X的SPOP F133V对照；JQ1：BET蛋白抑制剂。
具体实施方式
结合以下具体实施例和附图，对本发明作进一步的详细说明。实施本发明的过程、条件、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。
实施例1 SPOP结合并识别BRD2/3/4蛋白的SBC模体降解子进而促进其泛素化降解
(1.1)酵母双杂交系统筛选鉴别SPOP的降解靶蛋白
本发明通过对酵母双杂交系统筛选SPOP的靶蛋白，寻找到包括已知DEK和SRC-3在内的246个阳性克隆菌株，并在其中筛选靶蛋白，其中命中数最高的是BET蛋白家族，52个克隆为BRD2，2个克隆为BRD3，6个克隆为BRD4(如图1所示)。
在LNCaP细胞中进行免疫共沉淀反应(Co-IP)，应用IgG或BRD2/3/4抗体进行Western Blot发现SPOP蛋白能够与BRD2/3/4蛋白发生结合(图2)。因此，SPOP蛋白可与BET蛋白家族成员发生结合。
(1.2)SPOP促进BRD2/3/4蛋白降解
如上所述，SPOP蛋白可与BET蛋白家族成员发生结合，提示BRD2/3/4可能为蛋白降解通路的靶点。通过本发明的研究发现，在293T细胞中通过Myc-SPOP质粒异源性表达SPOP蛋白发现SPOP与BRD2/3/4蛋白发生反应，加入蛋白抑制剂MG132后，发现SPOP对BRD2/3/4蛋白的降解作用消失，BRD2/3/4蛋白水平增加(图3)。在LNCaP、22RV1前列腺癌细胞系和BPH-1前列腺正常细胞系中，应用siRNA敲减SPOP表达水平后发现，相应细胞 系中内源性BRD2/3/4蛋白含量上调，SRC作为已知的SPOP下游靶蛋白也发生了蛋白水平相应的上调(图4)。将SPOP具有酶活性的相应序列进行缺失得到SPOP突变体SPOP-ΔBTB(BTB结构域缺失)，进行泛素化检测发现，野生型SPOP能够促进BRD2/3/4的多聚泛素化，而异源性高表达的SPOP突变体SPOP-ΔBTB(BTB结构域缺失)无法促进BRD2/3/4蛋白的多聚泛素化(图5)。以上数据均提示SPOP能促进BRD2/3/4蛋白的多聚泛素化和降解。
综上所述，本发明的研究表明，在不同类型的细胞中(包括前列腺癌细胞)，SPOP作为E3泛素连接酶CUL-3的衔接蛋白可促进BRD2/3/4蛋白的多聚泛素化以及蛋白降解。
(1.3)SPOP特异性识别结合BRD2/3/4蛋白中的SBC结构域
SPOP若想发挥作用，底物蛋白必须要具备特异的SBC域(SPOP-bing consensus motif，SBC motif)。本发明发现BRD2中的260～313氨基酸，BRD中的3148～572氨基酸以及BRD中的4241～335氨基酸分别为需要与SPOP反应的最小区域，且在这些区域中存在一相同的SBC结构域(图6a)。分别构建了缺失SBC结构域的BRD2/3/4突变体(BRD2-ΔSBC，BRD3-ΔSBC，BRD4-ΔSBC)(图6b)。转染缺失SBC结构域的BRD2/3/4突变体(BRD2-ΔSBC，BRD3-ΔSBC，BRD4-ΔSBC)至293T细胞中，加入Myc-SPOP质粒，应用免疫共沉淀实验分析显示SPOP只与野生型BRD2/3/4结合，但却不与SBC缺失的突变体结合(图7-8)。应用泛素化实验检测293T细胞中BRD2/3/4蛋白及其突变体的表达水平，发现SPOP无法促进BRD2/3/4-ΔSBC突变体的降解，仅能促进野生型BRD2/3/4蛋白降解(图9)。
(1.4)SPOP突变无法促进BRD2/3/4蛋白降解
本发明建立了一系列与Myc标记的与前列腺癌相关的SPOP突变体，包括Y87C，Y87N，F102C，S119N，F125V，K129E，W131C，W131G，F133L，F133V和K134N。免疫共沉淀实验显示，在293T前列腺癌细胞中，突变后的SPOP无法与BRD2/3/4反应(图10a)。进一步体内泛素化分析实验显示，所有SPOP突变体失去促进BRD4蛋白多聚泛素化的能力，而仅有野生型SPOP能够发挥促进BRD4蛋白泛素化的作用(图10b)。慢病毒转染SPOP及相应突变体SPOP-Y87C/F102C/F125V/F133L至前列腺癌细胞系C4-2中，高表达相应SPOP蛋白后发现，野生型SPOP能够降低BRD2/3/4及DEK/ERG/SRC-3蛋白水平，而SPOP突变体蛋白无法发挥相应的作用，BRD2/3/4等蛋白表达水平发生上调(图10C)。上述结果提示，前列腺癌相关的SPOP突变不能促进BRD2/3/4蛋白多聚泛素化以及蛋白降解。
实施例2 SPOP对BET蛋白抑制剂抗肿瘤作用的影响
(2.1)前列腺癌患者肿瘤组织BET蛋白表达水平上调
本发明通过对两组样本含量共为99例原发性前列腺癌队列中进行全基因组测序、RNA测序、Sanger测序后发现，有13例前列腺癌样本包含SPOP突变。免疫组织化学(IHC)分 析实验显示，绝大多数SPOP突变的前列腺组织中BRD2/3/4蛋白染色强阳性(BRD2：85％(11/13)，BRD3：92％(12/13)，BRD4：85％(11/13))(图11)。相反，在野生型SPOP的前列腺癌组织样本中，仅有一部分BRD2/3/4蛋白染色强阳性(BRD2：29％(25/86)，BRD3：34％(29/86)，BRD4：41％(35/86))，大多数BRD2/3/4染色呈弱阳性或阴性(BRD2：71％(61/86)，BRD3：66％(57/86)，BRD4：59％(51/86))(图11)。说明发生SPOP突变的前列腺癌患者肿瘤组织中BRD2/3/4蛋白表达水平发生上调。
(2.2)前列腺癌患者肿瘤组织中BRD2/3/4mRNA水平检测
本发明通过对32例前列腺癌组织样本进行RNA测序发现(26例野生型SPOP，6例突变型SPOP)，BRD2/3/4mRNA水平在野生型SPOP和突变型SPOP前列腺癌组织样本中无明显差异(图12)，说明发生SPOP突变的前列腺癌患者肿瘤组织中BRD2/3/4的mRNA表达水平并未发生变化，上述结果提示SPOP突变的前列腺癌中BRD2/3/4蛋白水平的改变发生在BRD2/3/4mRNA翻译后水平。
综上，前列腺癌相关的SPOP突变可在翻译后水平导致BET蛋白含量上升。
(2.3)SPOP对BET抑制剂应用于抗肿瘤的影响
BET小分子抑制剂是目前潜在的肿瘤靶向治疗方法。本发明发现分别用BET抑制剂JQI处理实验组(EV-shC JQ1)、干扰BRD2/3/4表达组(EV-shBRD2/3/4JQ1)和阴性对照组(EV-shC Vehicle)，发现JQ1可明显抑制肿瘤增长。而SPOP F133V突变组(F133V-shC JQ1)中，JQ1对肿瘤的抑制作用消失。在SPOP F133V突变组对BRD2/3/4进行敲低后的细胞系(F133V-shBRD2/3/4JQ1)中，JQ1又可以显著抑制肿瘤的生长(图13)，说明SPOP突变后BRD2/3/4蛋白表达水平上调时，应用JQ1抑制肿瘤生长的效果较差，可能是产生JQ1耐药的原因。
SPOP突变的前列腺癌患者中发生上调的JQ1耐药相关基因与BRD4ChIP-seq相重合的基因包括RAC1、FDFT1、DHCR24等(图14)，进而在ChIP-seq公开数据对于BRD4蛋白是否作用于其启动子区域进行验证。野生型和SPOP F133V突变型C4-2细胞在加入JQ1后BRD4蛋白在RAC1基因位点的ChIP-seq数据发现BRD4结合到RAC1基因启动子区域，在JQ1耐药的SPOP F133V突变细胞系中RAC1基因mRNA和蛋白表达上调(图15)，说明在SPOP突变后BRD4蛋白能够促进RAC1基因转录，应用JQ1不能阻碍这一进程，提示这是SPOP突变患者应用JQ1产生耐药的机制。本发明发现分别用BET抑制剂JQI处理单纯干扰USP9X/USP49组(EV-shUSP9X/USP49JQ1)、SPOP F133V单纯突变组(F133V-shC JQ1)和对SPOP F133V突变组进行USP9X/USP49敲低后的细胞系(F133V-sh USP9X/USP49JQ1)，发现JQ1在SPOP F133V突变组中不能够抑制肿瘤生长，但在干扰USP9X/USP49蛋白表达 后又可以显著抑制肿瘤的生长(图16)。上述结果提示，BRD4与USP9X/USP49发生相互作用，干扰USP9X/USP49后能够促进BRD4多聚泛素化，能够部分阻断SPOP F133V突变引起的BRD4蛋白水平上升，进而能够逆转F133V引起的JQ1耐药。
此外，本发明研究发现BRD4蛋白水平的下降可以引起细胞对JQ1敏感，同时SPOP敲低后可以引起细胞对JQ1的耐受。
以上研究均提示BRD4蛋白为去势抵抗性前列腺癌细胞(如C4-2细胞)中决定JQ1敏感性的关键性分子，SPOP突变细胞中BET蛋白水平升高与JQ1抵抗性有关。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。
Figure PCTCN2018088802-appb-000001
Figure PCTCN2018088802-appb-000002

Claims (15)

  1. 一种前列腺癌SPOP标志物，其特征在于，所述SPOP标志物包括SPOP的DNA、mRNA或由其编码的蛋白质，所述SPOP的DNA在NCBI中的序列号为NG_041815.1；所述SPOP mRNA的核苷酸序列如SEQ ID NO.1所示；所述SPOP的蛋白质序列如SEQ ID NO.2所示。
  2. 如权利要求1所述的SPOP标志物的检测试剂在制备对前列腺癌进行分子分型以及指导前列腺癌患者应用BET小分子抑制剂进行治疗中的应用，其特征在于，所述SPOP标志物的检测试剂包括特异性检测SPOP的DNA、mRNA或由其编码的蛋白质的试剂。
  3. 一种前列腺癌体外诊断产品，其特征在于，所述体外诊断产品包括特异性检测如权利要求1所述的SPOP DNA的试剂，和/或特异性检测SPOP mRNA的试剂，和/或特异性检测SPOP蛋白质的试剂。
  4. 一种对已确诊为前列腺癌的患者进行分子分型以及指导前列腺癌患者应用BET抑制剂进行治疗的方法，其特征在于，所述方法包括以下步骤：
    a)检测前列腺癌患者的病理样本中SPOP的DNA拷贝数、或mRNA或由其编码的蛋白质的表达量；
    b)通过a)中测定的表达量，将患者分为DNA正常组和突变组，mRNA或蛋白正常表达组和低表达组，若相对于正常人群，所述SPOP的DNA发生突变，即为DNA突变组，否则为DNA正常组；或所述SPOP mRNA或由其编码的蛋白质的表达量降低，则为低表达组，否则为正常表达组；DNA正常组和mRNA或蛋白正常表达组前列腺癌患者可采用BET小分子抑制剂进行治疗；而DNA突变组和mRNA或蛋白低表达组均不推荐应用BET小分子抑制剂进行治疗。
  5. 一种BET小分子抑制剂的应用，其特征在于，若相对于正常人群，患有前列腺癌的患者中如权利要求1所述的SPOP DNA发生突变，即为DNA突变组，否则为DNA正常组；或SPOP mRNA或由其编码的蛋白质的表达量降低，则为低表达组，否则为正常表达组；DNA正常组和mRNA或蛋白正常表达组前列腺癌患者可采用BET小分子抑制剂进行治疗；而DNA突变组和mRNA或蛋白低表达组均不推荐应用BET小分子抑制剂进行治疗。
  6. 如权利要求1所述的SPOP标志物在制备促进BET蛋白的多聚泛素化以及蛋白降解的药物中的应用。
  7. 如权利要求1所述的SPOP标志物在制备降低DEK/ERG/SRC-3蛋白表达水平的药物中的应用。
  8. 如权利要求1所述的SPOP标志物在制备降低RAC1基因mRNA和蛋白表达水平的药物中的应用。
  9. 如权利要求1所述的SPOP标志物在制备降低JQ1的耐药性的药物中的应用。
  10. 如权利要求1所述的SPOP标志物的突变体在制备上调BET蛋白水平的药物中的应用。
  11. 如权利要求9所述的应用，其特征在于，所述SPOP突变体包括Y87C，Y87N，F102C，S119N，F125V，K129E，W131C，W131G，F133L，F133V和K134N。
  12. BRD小分子抑制剂在制备降低JQ1的耐药性的药物中的应用。
  13. USP9X/USP49的抑制剂在制备促进BET蛋白的多聚泛素化以及蛋白降解的药物中的应用。
  14. USP9X/USP49的抑制剂在制备降低JQ1的耐药性的药物中的应用。
  15. 一种降低JQ1的耐药性的方法，其特征在于，所述方法包括提高SPOP的mRNA和蛋白质的表达水平，或加入BRD蛋白抑制剂，或加入USP9X/USP49抑制剂。
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