WO2013123620A1 - Cul4A在前列腺癌诊断、治疗及预后中的用途 - Google Patents

Cul4A在前列腺癌诊断、治疗及预后中的用途 Download PDF

Info

Publication number
WO2013123620A1
WO2013123620A1 PCT/CN2012/000233 CN2012000233W WO2013123620A1 WO 2013123620 A1 WO2013123620 A1 WO 2013123620A1 CN 2012000233 W CN2012000233 W CN 2012000233W WO 2013123620 A1 WO2013123620 A1 WO 2013123620A1
Authority
WO
WIPO (PCT)
Prior art keywords
cul4a
prostate cancer
cells
treatment
cell
Prior art date
Application number
PCT/CN2012/000233
Other languages
English (en)
French (fr)
Inventor
孙颖浩
任善成
吴荻
Original Assignee
Sun Yinghao
Ren Shancheng
Wu Di
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sun Yinghao, Ren Shancheng, Wu Di filed Critical Sun Yinghao
Priority to PCT/CN2012/000233 priority Critical patent/WO2013123620A1/zh
Publication of WO2013123620A1 publication Critical patent/WO2013123620A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the use of Cul4A for the detection, treatment and prognosis of prostate cancer. current technology
  • Cul4A is a highly conserved member of the Cullin family, which contains seven related proteins (Cull, 2, 3, 4A, 4B, 5, and 7) in humans.
  • the sequence of Cul4A can be found in GenBank BC008308 (SEQ ID NO: 1).
  • Cul4A is known to be amplified or overexpressed in a variety of human cancers such as breast cancer, squamous cell carcinoma, hepatocellular carcinoma, and the like. It is also known that Cul4A resides in a locus that is frequently amplified in prostate cancer (Paris PL et al. (2004) Hum Mol Genet 13: 1303-1313). However, there have been no reports of overexpression of Cul4A in prostate cancer. Brief description of the invention
  • One aspect of the invention relates to the use of Cul4 A for detecting prostate cancer.
  • Cul4A or a reagent capable of detecting Cul4A can be used to prepare the kits used in the assay.
  • CuWA can be used as an index for predicting the therapeutic effect of prostate cancer.
  • the treatment is, for example, hormone therapy, preferably anti-androgen therapy, more preferably Casodex treatment; and/or the treatment is, for example, thalidomide treatment.
  • the prostate cancer preferably has a high level of Cul4A or is hormone independent.
  • CuWA can be used to prepare the kits for which the predictions are to be used. By testing Cul4A in biological samples, patients eligible for appropriate treatment can be screened. To this end, a corresponding screening kit can be prepared.
  • kits may include reagents for detecting Cul4A, such as probes for detecting Cul4A gene, primers, or antibodies or other ligands for detecting Cul4 A protein, and the like.
  • the invention also relates to the use of Cul4A in the treatment of prostate cancer, for example, slowing the growth of prostate cancer cells, reversing prostate cancer cells against drugs (eg, Tolerance, such as anti-androgen), inhibit cancer metastasis, and so on.
  • the prostate cancer is selected from the group consisting of: androgen-resistant prostate cancer prostate cancer (HRPC), hormone-independent prostate cancer.
  • the prostate cancer has a high level of Cul4A.
  • the Cul4A can be used to prepare a medicament for use in the treatment of each of the above aspects.
  • the drug may be a short hairpin RNA (shRNA) directed against the Cul4A gene, an antisense oligonucleotide, a small interfering RNA (siRNA), or the like.
  • the invention relates to the use of Cul4A, or an agent that inhibits or knocks down Cul4A levels, for the preparation of a medicament for reversing hormone sensitivity of hormone-independent prostate cancer, Cul4A as an anti-male drug sensitizer Its use, as well as the use of Cul4A in the preparation of kits for the use of anti-male drugs, the kit for judging the efficacy.
  • the invention also relates to the use of seletonide for the treatment of diseases caused by high levels of Cul4A.
  • the disease is preferably cancer, more preferably prostate cancer.
  • the invention also relates to the use of salidomide for modulating ERK signaling pathways.
  • the invention further relates to the use of Cul4A in combination therapy, for example, in combination with chemotherapy and/or with an androgen receptor (AR) antagonist.
  • the invention relates to a combination of a Cul4A inhibitor in combination with a chemotherapeutic agent and/or a drug with an A antagonist. Accordingly, Cul4A can be used to prepare the above-described combination of drugs.
  • Figure 1 shows CUL4A overexpression in prostate cancer cell lines and tissue samples.
  • Figure 2 shows that high expression of CUL4A is associated with poor prognosis in prostate cancer.
  • Figure 3 shows the proliferation of prostate cells in the case of exogenous expression of Cul4A.
  • Figure 3A shows normal prostate cell PWR-1E;
  • Figure 3B shows prostate cancer cell LNCAP.
  • pBABE is a blank control plasmid.
  • Figure 4 shows that ectopic CUL4A expression transforms normal prostate epithelial cells via the ERK pathway and induces invasion.
  • CUL4A significantly promotes RWPE-1 intrusion, which is eliminated by U0126.
  • CUL4 A significantly promotes RWPE-1 intrusion, which is eliminated by U0126. Matrigel invasion assay of RWPE-1 cells stably transfected with CUL4A-expressing virus or control.
  • FIG. 5 shows that Cul4A shRNA inhibits proliferation of prostate cancer cell line 22RV1,
  • CUL4A knockdown inhibits prostate cell growth and induces apoptosis in vivo and in vitro. All P-values are from the t-test.
  • shRNA down-regulates CUL4A protein levels.
  • the 22RV1 cells with shCUL4A showed a significant decrease in proliferation compared to 22RV1 cells with shCtrl. Mean and standard deviation (SD) were calculated from three tests for each trial.
  • Knockdown CUL4A induces apoptosis in 22RV1 cells. Apoptosis was detected by the TU EL test.
  • Figure 7 shows the growth curves of LNCAP and LNCAP-R in ⁇ Casodex medium.
  • Figure 8 shows the growth curves of 22RV1-Cul4A shRNA and 22RV1-pSUPER. .
  • Figure 8A is a growth curve in FBS + different concentrations of Casodex;
  • Figure 8B is a growth curve in CSS + different concentrations of Casodex.
  • pSUPER is a blank control plasmid.
  • Figure 9 is an electrophoresis photograph showing the interaction between Cul4A and the androgen receptor (AR) in LNCAP cells and CWR22 cells, and its effect on cell proliferation.
  • GAPDH glyceraldehyde-3-brate dehydrogenase.
  • Figure 10 is an electrophoresis photograph showing the regulation of Cul4A expression by the androgen-AR pathway in AR-positive and negative CWR22 cells.
  • GAPDH glyceraldehyde-3-lein dehydrogenase
  • DHT dihydrotestosterone.
  • CUL4A expression levels determine the sensitivity of prostate cancer cells to seletonide.
  • Ectopic CUL4A expression enhances sensitivity to seletonide treatment in seletonide-tolerant PC-3 cells.
  • (c) Down-regulation with specific shRNA CUL4A confers tolerance to treatment with seletonide in thalitrimine-sensitive 22RV1 cells.
  • Thalidomide reduces CUL4A and p-ERK in a dose- and dependent manner.
  • Thalidomide induces death in sensitive LNCAP cells and is not induced in tolerant PC-3 cells. LNCAP and PC-3 cells were exposed to thalidomide (0, 5, 20, 200 ug/ml) for 48 h, and cell lysates were analyzed by Western blot for CUL4A and PARP.
  • thalidomide (0, 5, 20, 200 ug/ml) for 48 h, and cell lysates were analyzed by Western blot for CUL4A and PARP.
  • thalidomide (0, 5, 20, 200 ug/ml)
  • cell lysates were analyzed by Western blot for CUL4A and PARP.
  • f Down-regulation of specific shRNA CUL4A reduces cell death caused by seletonide treatment in thalidomide-sensitive 22RV1 cells.
  • Ectopic CUL4A expression induces death due to treatment with seleton
  • FIG. 1 Loss of CUL4A expression confers secondary serotonin tolerance.
  • C4-2 salinamide-resistant cells C4-2-R were established by treating C4-2 cells with seletonide for 3 months.
  • CUL4A protein levels in C4-2-R cells are lower than in C4-2 cells. Summary of the invention
  • the inventors first discovered that CuWA is in a palace such as prostate cancer, kidney cancer, bladder cancer, It is overexpressed in various cancers such as cervical cancer, endometrial cancer and breast cancer, suggesting that Cul4A is a potential oncogene in many endocrine-reproductive system tumors mentioned above.
  • the present application provides in vitro and in vivo evidence that CUL4A is an oncogene that causes prostate cancer.
  • CUL4A is very common in prostate cancer cell lines and primary premalignant tumors.
  • the protein and mRNA levels of Cul4A were significantly elevated in prostate cancer cells compared to normal prostate epithelial cells.
  • Normal prostate epithelial cells were overexpressed by virus overexpression of Cul4A in vitro, and it was surprisingly found that only this one gene change can convert normal prostate epithelial cells into prostate cancer cells and is invasive.
  • CUL4 A activates the ERK pathway and thus causes prostate cancer progression.
  • Overexpression of CUL4A greatly increased ERK protein, while knockdown of CUL4A significantly reduced ERK.
  • the results of the present application also unexpectedly show that the regulation of ERK by CUL4A is achieved by transcription rather than by ubiquitination.
  • CUL4A is known to regulate histone thiolation.
  • the present application further shows that CUL4A upregulates ERK by enrichment of trimethylated H3K4 at the ERK1 and ERK2 promoters.
  • kits for testing the prognosis of prostate cancer can be prepared, which comprises an agent for detecting the expression of CUL4A, such as an antibody for testing its protein level or a reagent for testing its mRNA level. The inventors have found that the level of Cul4A affects the growth of prostate cells.
  • Down-regulation of Cul4A inhibits proliferation and cell cycle of prostate cancer cells in vitro, reduces colony formation, and induces apoptosis; inhibits tumor formation or slows prostate cancer growth in vivo.
  • Down-regulation of Cul4A can be designed to knock down Cul4A shRNA, siRNA, Antisense oligonucleotides and the like are implemented. It is known that shRNA, a short hairpin RNA, is an RNA sequence capable of forming a tight hairpin transition and can be used to silence gene expression via RNA interference. The shRNA hairpin structure is cleaved into siRNA by a cellular mechanism.
  • siRNAs are generally double-stranded RNA molecules (dsRNA) with short tails, including sense RNA (which has a sequence homologous to the target gene mRNA) and antisense RNA (which has a sequence complementary to the sense RNA).
  • dsRNA double-stranded RNA molecules
  • antisense RNA which has a sequence complementary to the sense RNA.
  • the siRNA binds to the RNA-induced silencing complex (RISC), and after the matched mRNA is bound by the siRNA, the complex attached to the siRNA degrades the mRNA, thereby inhibiting the expression of the target gene.
  • RISC RNA-induced silencing complex
  • the present invention also demonstrates that seletonide can also down-regulate Cul4A, suggesting that seletonide can be used to treat diseases caused by overexpression of Cul4A, such as cancer, especially prostate cancer, for example. Therefore, seletonide can be used for the preparation of a medicament for treating a disease caused by overexpression of Cul4A, such as cancer, especially prostate cancer.
  • Cul4A promotes cancer progression by activating the ERK pathway in prostate cancer, suggesting that seletonide can also be used to modulate the ERK pathway via Cul4A, thereby achieving the purpose of treating diseases. Therefore, salidomide can also be used to prepare drugs that modulate the ERK pathway via Cul4A.
  • down-regulating Cul4A with shRNA effectively reverses the drug resistance of a hormone-sensitive cell line (eg, H PC cell line 22RV1), making it re-sensitive to anti-male drugs, which not only indicates that treatment for Cul4A It can be reversed to indicate that Cul4A leads to the formation of androgen-resistant prostate cancer (HRPC), and HRPC can be effectively treated by regulating Cul4A levels.
  • Cul4A can also be used as an indicator for predicting the effects of hormone therapy.
  • Cul4A inhibitors for example, can knock down Cul4A shRNA, siRNA, antisense oligonucleotides, and/or salidomide.
  • AR antagonists such as anti-androgen drugs.
  • Common anti-male drugs are well known in the art and a variety of mature products are commercially available.
  • Casodex Sigma-Aldrich, Cas No.: 90357-06-5
  • Flutamide Schering-Plough Corp, Kenilworth, NJ
  • Cul4A In view of the treatment of Cul4A, such as lowering Cul4A, it can reverse the hormone sensitivity of hormone-independent prostate cancer.
  • CuWA can be regarded as a potential anti-androgenic drug sensitizer, as well as an anti-male drug and an indicator for judging the efficacy.
  • thalidomide has clinically proven activity in hematological tumors such as multiple sclerosis and mantle cell lymphoma, its use in clinical solid tumors has been slow and the results are not optimistic.
  • Attempts have been made to treat lung cancer and prostate cancer in combination with seletonide and cytotoxic agents, but have failed to show an advantage in survival (Buiris HA, et al. (2010) Cancer Invest 28: 408-412; Lee SM, et al (2009) J Clin Oncol 27: 5248-5254; and Pacheco AV, et al. (2008) Urol Oncol 26: 610-015).
  • Cul4A can be used as a new mechanism for the treatment of hormone-independent prostate cancer with thalidomide.
  • the present application also found that different prostate cancer cell lines have different responses to seletonide treatment, which is significantly associated with CUL4A water.
  • CUL4A High levels of prostate cancer cells are particularly sensitive to seletonide.
  • the present application also demonstrates that the apoptotic effect of salidomide in prostate cancer also depends on high levels of CUL4A.
  • CUL4A overexpression in salitonide-tolerant cells increased cell sensitivity and apoptosis to the drug, while saliride sensitivity CUL4A knockdown in cells results in decreased tolerance to the drug and decreased levels of apoptosis.
  • CUL4A protein levels are decreased in secondary salidomide-tolerant cells obtained by long-term treatment of sensitive cells.
  • thalidomide can reduce the expression of Cul4A in a time-dependent and dose-dependent manner.
  • salidomide can inhibit prostate cancer cells.
  • Cul4A is a direct target of thalidomide, which plays a decisive role in the induction of apoptosis by thalidomide.
  • CUL4A expression is an important marker for predicting the efficacy of thalidomide in the treatment of prostate cancer. It is also confirmed in this paper that Cul4A is a key factor in prostate cancer metastasis. Inhibition of Cul4A can reduce metastasis of prostate cancer. Targeting Cul4A combined with chemotherapy is a more effective treatment for metastatic prostate cancer.
  • the primers are as follows:
  • the first antibodies are: CUL4A (#ab84382; Abeam), ki-67 (#ZM-0166). All staining was assessed by pathologists who were unclear about the source of the sample and the consequences of the patient, using the EnVision+ system (Dako). The intensity and range of staining were examined using the widely accepted German semi-quantitative scoring system.
  • the intensity score of the stained cells is multiplied by the range score to obtain the final immune response score from 0 (minimum score) to 12 (maximum score).
  • the final immune response score of 6 is low expression and >6 is high expression.
  • CUL4A of prostate cancer was strongly stained, while BPH and normal prostate were weakly stained or even stained (Fig. 2).
  • CUL4A is expressed in a group of prostate cancer cell lines (LNCaP, C4-2, 22RV1, CWR22R, DU145 and PC3), and two immortalized non-tumorigenic human prostate epithelial cell lines (RWPE-1 and PWR-1E) In the test. All cells were purchased from the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110-2209) and cultured according to ATCC recommendations.
  • ATCC American Type Culture Collection
  • Proteins were extracted with RIPA reagent (Biomiga), subjected to SDS-PAGE and Western blotting, and Cul4A protein levels in these cells were detected by the following primary antibodies: anti-phospho-Ser473 ERK (#4370; Cell Signaling Technology), anti-total-ERK (#4695; Cell Signaling Technology), anti-CUL4A (#2699; Cell Signaling Technology), anti-PA P (#9542; Cell Signaling Technology), anti-GAPDH (#470661; bioworld), using GAPDH protein as a photo .
  • the immortalized non-tumorigenic human prostate epithelial cell RWPE-1 was modified to stably express CUL4A.
  • Western blot analysis showed that CUL4A-transfected clone (RWPE-1 - CUL4A) had a higher level of CUL4A protein than the pBabe-blank transfected control (RWPE-1 - Ctrl) (Fig. 4a).
  • RWPE-1-CUL4A cells grew rapidly and formed large round colonies on soft agar (CytoSelectTM; 96-Well Cell Transformation Assay) within 10 days.
  • RWPE-1-Ctrl cells did not form macroscopic colonies under the soft agar conditions tested (Fig. 4a). This indicates that CUL4A overexpression converts RWPE-1 cells into malignant.
  • 2.3 Cul4A overexpression promotes prostate cancer metastasis
  • 0.1 mL of serum-free DMEM medium containing 5 x 10 5 normal cells RWPE-1 or prostate cancer cell PC-3 was added to a Matrig-coated Boyden chamber (Costar) with a pore size of 8 ⁇ M.
  • the lower chamber was added to serum-free keratinocyte medium (GIBICO, 17005-042).
  • GEBICO serum-free keratinocyte medium
  • the Boyden Chambers were then fixed in 4% furfural for 20 minutes at room temperature, then stained with 0.1% crystal violet for 30 minutes and finally washed with water. Count the cells under the membrane to assess changes in cell invasive ability.
  • the Cul4A shRNA was cloned into the pSUPER.retro.puro vector (Oligoengine, Seattle, WA, USA) according to the instructions.
  • the Cul4A shRNA was annealed and ligated into the Bglll and Hindlll cleavage sites of the pSUPER.retro.puro vector.
  • This vector was then transfected into One Shot® TOP 10 chemically competent E. coli cells (Invitrogen, Carlsbad, CA, USA). Single clones were sequenced in LB medium containing 50 ug/ml ampicillin to verify successful transfection.
  • Cul4A shRNA was transfected into prostate cancer cell 22RV1 to establish a stable cell line with low expression of Cul4A, 22RV1-shRNA-Cul4a. The cell growth and cycle progression changes of this cell line compared to the control were observed. The results are shown in Figure 5.
  • Stable transfectants were generated in 22RV1 cells using the above Cul4A shRNA.
  • the shRNA effectively reduced CUL4A protein expression in the 22RV1 cell line (Fig. 6a).
  • the proliferation of 22RV 1 transfected with shCUL4A continued to decrease, and on day 6 the cells transfected with shCtrl were reduced by 35% (Fig. 6a).
  • TdT-mediated dUTP was performed using the ApopTag Fluoresin In situ Apoptosis Detection Kit (Intergen Company, Purchase, NY) according to the manufacturer's instructions. Notch end marker (TUNEL) test.
  • 22RV1 and 22RV1-shRNA-cul4a were injected into 7 mice (BALB/c-nu rats, purchased from Shanghai Institute of Zoology) subcutaneously, and the tumors of the mice were measured twice a week. The volume of the tumor was measured one month later. The results showed that the tumor volume and total amount in the 22RV1-shRNA-cul4a group were significantly smaller than those in the 22RV1 group.
  • CUL4A immunohistochemical staining was performed in these xenograft tumors. As a result, it was found that the CUL4A protein was significantly reduced. Consistent with in vitro data, the xenograft tumor carrying shCUL4A showed a significant decrease in the proliferation index Ki67 compared to the shSh-bearing tumor, suggesting that CUL4A knockdown does inhibit the proliferation of prostate cancer cells in vivo.
  • CUL4A plays a key role in tumorigenesis of prostate cancer, and that knockdown of CUL4A inhibits tumor growth both in vitro and in vivo.
  • Example 4 CUL4A Activates ERK Signaling via Transcriptional Regulation
  • Ras/ERK signaling pathway is frequently activated in many types of human cancers, including prostate cancer. It has been reported that RWPE-1 cells undergo malignant transformation by overexpressing v-Ki-Ras. To this end, it was investigated whether CUL4A contributes to prostate cancer progression via signaling.
  • Chromatin Immunoprecipitation (ChIP) analysis was performed as described, for example, in Shang Y, Myers M, Brown M (2002). Mol Cell 9: 601-610. Preparation of soluble staining shields with anti-triMeK4H3 or non-immune IgG as a control Precipitation.
  • Real-time ChIP qPCR was performed using SYBR green Champion ChIP qPCR assay kit (SABiosciences), and the samples were amplified with ABI Prism 7000 Sequence Detector (Applied Biosystems). The primers used were as follows:
  • the hormone-dependent prostate cancer cell line LNCAP (purchased from ATCC) was cultured in Casodex (SIGMA, USA) (37 ° C, 5% carbon dioxide) for 2 months, and the hormone-resistant subtype cell line LNCAP-R was successfully established. (See Figure 7), this cell mimics the pathological process of hormone-sensitive to tolerant patients in prostate cancer in vitro: using the anti-androgant drug Casodex The amount of testosterone in the cell microenvironment gradually reached the castration level. At the beginning of the cell, the growth was inhibited and even some cells were apoptotic. After a while, the viable cells continued to grow, indicating that the cell growth has not depended on the presence of androgen, and the cells have been Hormone sensitivity progresses to hormonal tolerance.
  • Cul4A up-regulates androgen receptor (AR) and enhances its transcriptional activity (see Example 2) ( Figure 9). Similarly, AR also promotes the expression of Cul4A protein and mRNA (Fig. 10). Sexual feedback, and the vicious circle between the two oncogenes stimulates prostate cancer cells to proliferate.
  • Example 7. CUL4A determines the response to seletonide in prostate cancer. The growth inhibitory effect of thalidomide in a group of prostate cancer cell lines with different endogenous CUL4A expression was first evaluated (Fig. 11a and b). . After 72 hours of treatment with salidomide, cell viability was determined by MTT assay.
  • CUL4A was ectopically expressed in salbutaline-tolerant PC-3 and RWPE-1 cells, resulting in increased sensitivity to the drug ( Figure lib) And llh).
  • shRNA was used to down-regulate CUL4A expression levels in seletonide-sensitive 22RV1 cells, resulting in tolerance to the drug (Fig. 11).
  • C4-2-R Secondary salivary tolerant C4-2 cells
  • Fig. 12a Western blot analysis showed that C4-2-R expressed very low levels of CUL4A compared to its parental cells (Fig. 12b), indicating a loss of CUL4A during the tolerance to salvianide.
  • CUL4 A down-regulation of CUL4 A is a major determinant of salinamide-induced apoptosis in prostate cancer cells, whereas CUL4 A basal levels are biomarkers of reactivity to seletonide treatment.

Abstract

提供了滞蛋白家族基因Cul4A作为潜在的癌基因在前列腺癌的诊断、治疗、以及预后方面的用途。

Description

Cul4A在前列腺癌诊断、 治疗及预后中的用途 技术领域
本发明涉及 Cul4A在前列腺癌的检测、 治疗和预后方面的用途。 现有技术
Cul4A属于滞蛋白（Cullin)家族中高度保守的成员，该家族在人类 中包含 7个相关蛋白（Cull, 2, 3, 4A, 4B, 5， 和 7)。 Cul4A的序列可参 见 GenBank BC008308 (SEQ ID NO: 1)。 已知 Cul4A在诸如乳腺癌、 鳞状细胞癌、 肝细胞癌等多种人类癌症中扩增或过表达。 还已知 Cul4A居于前列腺癌中频繁扩增的基因座中（Paris PL等 （2004) Hum Mol Genet 13: 1303-1313)。但迄今未有 Cul4A在前列腺癌中过表达的 报道。 发明简述
本发明一方面涉及， Cul4 A在检测前列腺癌方面的用途。 为此， Cul4A或者能检测 Cul4A 的试剂可用于制备所述检测中用到的试剂 盒
在本发明中， CuWA可以作为预测前列腺癌治疗效果的指标。 所 述治疗例如是激素治疗， 优选抗雄激素治疗， 更优选 Casodex治疗； 和 /或所述治疗例如是沙度利胺 (thalidomide)治疗。 所述前列腺癌优选 具有高水平 Cul4A或为激素非依赖性。 为此， CuWA可用于制备所述 预测要用到的试剂盒。 通过检测生物样品中的 Cul4A， 可以对适合相 应治疗的患者进行筛选。 为此， 可以制备相应的筛选试剂盒。
上述多方面的试剂盒中可以包括检测 Cul4A 的试剂， 例如检测 Cul4A基因的探针、 引物， 或检测 Cul4 A蛋白的抗体或其它配体， 等 等。
本发明还涉及 Cul4A 在治疗前列腺癌方面的用途， 所述治疗例 如是， 减緩前列腺癌细胞的生长， 逆转前列腺癌细胞对药物（例如激 素， 如抗雄激素）的耐受性， 抑制癌转移， 等等。 优选地， 所述前列 腺癌选自： 雄激素耐受性前列腺癌前列腺癌 (HRPC)、 激素非依赖性 前列腺癌。优选所述前列腺癌具有高水平的 Cul4A。为此，所述 Cul4A 可用于制备上述各方面治疗所用的药物。 例如， 所述药物可以是针对 Cul4A基因的短发夹 RNA (shRNA)，反义寡核苷酸，小分子干扰 RNA (siRNA)等。
在优选实施方案中， 本发明涉及 Cul4A、 或者抑制或敲低 Cul4A 水平的试剂，在制备用于逆转激素非依赖性前列腺癌的激素敏感性的 药物方面的用途， Cul4A作为抗雄药物增敏剂的用途， 以及 Cul4A在 制备选用抗雄药物的试剂盒、 判断疗效的试剂盒中的用途。
本发明还涉及沙度利胺在治疗由 Cul4A 高水平引起的疾病方面 的用途。 所述疾病优选癌症， 更优选前列腺癌。 本发明还涉及沙度利 胺在调节 ERK信号传递途径方面的用途。
本发明还涉及， Cul4A在联合治疗中的用途， 所述联合治疗例如 是， 与化疗联合和 /或与雄激素受体 (AR)拮抗剂进行联合。 为此， 本 发明涉及 Cul4A抑制剂与化疗药组合和 /或与 A 拮抗剂的药物组合。 相应地， Cul4A可用于制备上述的药物组合。 附图说明
图 1 显示前列腺癌细胞系和组织样品中的 CUL4A过表达。 (a) PWR-1E, RWPE-1, LNCAP, C4-2, CWR22, 22RV1, PC-3和 DU-145中 CUL4A蛋白水平的 Western印迹分析。 （b)用 qRT-PCR分析上述细胞 系中的 CUL4A mRNA水平。
图 2显示 CUL4A高表达与前列腺癌预后不良相关。组织芯片上， 具有不同的 Gleason分值 (G4, G6, G8和 G10)的正常、 良性前列腺增 生 BPH), 前列腺上皮内肿瘤 (PIN)， 和前列腺癌中 CUL4A表达的免 疫组化染色。
图 3显示， 在外源性表达 Cul4A的情况下， 前列腺细胞的增殖。 图 3A为正常前列腺细胞 PWR-1E; 图 3B为前列腺癌细胞 LNCAP。 各为 3次独立实验的平均值。 pBABE为空白对照质粒。 图 4显示异位 CUL4A表达经由 ERK途径转化正常的前列腺上 皮细胞， 并诱导侵入。 （a)稳定转染的细胞 RWPE-1 在软琼脂上的集 落形成， 表示为每铺板 1,000个细胞的平均集落数 ± SD; n =3份生物 学样品。 （b) CUL4A显著促进 RWPE-1侵入， 该侵入被 U0126消除。 被表达 CUL4A的病毒或对照稳定转染的 RWPE-1细胞的 Matrigel侵 入试验。 （c)敲低 CUL4A显著降低 22RV1中的总 ERK水平， 而异位 CUL4A表达在 RWPE-1中增加总 ERK。（d)敲低 CUL4A在 22RV1 中 显著降低 ERK1和 的 mRNA水平， 而异位 CUL4A表达在 RWPE-1 中增加 ERK1 和 2 的 mRNA水平。 （e)制备可溶性染色质， 用来自 PC-3-Ctrl和 PC-3-CUL4A细胞的抗 -triMeK4H3或对照非免疫 IgG沉 淀。 计算相对于起始量的倍数富集。 结果表示为每份样品三个值的平 均值士 SD。 （f) CUL4 A显著促进 RWPE-1侵入， 该侵入被 U0126消 除。被表达 CUL4A的病毒或对照稳定转染的 RWPE-1细胞的 Matrigel 侵入试验。
图 5显示， Cul4A shRNA抑制前列腺癌细胞 22RV1的增殖， 为
3次独立实验的平均值。
图 6. CUL4A敲低在体内和体外抑制前列腺细胞生长， 并诱导凋 亡。 所有 P-值均来自 t-检验。 （a) shRNA下调 CUL4A蛋白水平。 带 有 shCUL4A的 22RV1细胞与带有 shCtrl的 22RV1细胞相比， 增殖 显著减少。从每一项试验的三次测试计算平均值和标准偏差 (SD)。 (b) 敲低 CUL4A在 22RV1细胞中诱导凋亡。 凋亡用 TU EL试验检测。 (c) 带有 shCUL4A和 shCtrl的 22RV1细胞的软琼脂集落形成试验， 显示敲低 CUL4A减少集落数并使集落大小减小。 从平行三份生物学 样品计算每铺板 1000个细胞的集落平均数和大小以及 SD。上图显示 这些集落的相应相差显微镜图像。 （d)下调 CUL4A 体内抑制 22RV1 细胞生长。 示出了每一组的平均肿瘤体积士 SD (n = 7只小鼠 /组)。 (e) 2RVl-shCUL4A形成的肿瘤的大小和重量都远小于 22RVl-shCtrl 形 成的肿瘤。
图 7显示 LNCAP和 LNCAP-R在 ΙΟμΜ Casodex培养基中的生 长曲线。 图 8显示 22RVl-Cul4A shRNA和 22RVl-pSUPER的生长曲线。。 图 8A为在 FBS+不同浓度 Casodex中的生长曲线； 图 8B为在 CSS+ 不同浓度 Casodex中的生长曲线。 pSUPER为空白对照质粒。
图 9为电泳照片，显示在 LNCAP细胞和 CWR22细胞中， Cul4A 与雄激素受体 (AR)的相互影响， 及其对细胞增殖的影响。 GAPDH: 甘油醛 -3-磚酸脱氢酶。
图 10为电泳照片， 显示 AR阳性和阴性的 CWR22细胞中， 雄 激素 -AR通路对 Cul4A表达的调控。 GAPDH:甘油醛 -3-碑酸脱氢酶； DHT: 双氢睾酮。
图 11. CUL4A表达水平决定了前列腺癌细胞对沙度利胺的敏感 性。 （a) 有高水平 CUL4A 的前列腺细胞 (LNCAP， C4-2, CWR22, 22RV1)对沙度利胺敏感，有低水平 CUL4A的细胞 (RWPE-1, PWR-1E, PC-3, DU-145)对该药物耐受。 点代表均值 (n=3), 柱代表 SD。 （b)异 位 CUL4A表达在沙度利胺耐受性 PC-3细胞中提高沙度利胺治疗敏 感性。 （c)用特异性 shRNA下调 CUL4A在沙度利胺敏感性 22RV1细 胞中赋予对沙度利胺治疗的耐受。 （d)沙度利胺以剂量和依赖方式降 低 CUL4A和 p-ERK。 （e) 沙度利胺在敏感性 LNCAP细胞中诱导死 亡， 在耐受性 PC-3细胞中不诱导。 LNCAP和 PC-3细胞暴露于沙度 利胺 (0, 5, 20, 200ug/ml) 48 h， 细胞裂解物用 Western 印迹分析 CUL4A和 PARP。 （f) 用特异性 shRNA下调 CUL4A在沙度利胺敏感 性 22RV1细胞中减少沙度利胺治疗引起的细胞死亡。（g) 异位 CUL4A 表达在沙度利胺耐受性 PC-3 细胞中诱导因沙度利胺治疗引起的死 亡。（h)沙度利胺耐受性 RWPE-1细胞中的 CUL4A异位表达提高对该 治疗的敏感性。
图 12. CUL4A表达的损失赋予继发性沙度利胺耐受。 （a) C4-2沙 度利胺耐受性细胞 (C4-2-R)通过用沙度利胺处理 C4-2 细胞 3个月来 建立。 （b) C4-2-R细胞中的 CUL4A蛋白水平比 C4-2细胞中的降低。 发明内容
本发明人首次发现， CuWA在诸如前列腺癌、 肾癌、 膀胱癌、 宫 颈癌、 子宫内膜癌和乳腺癌等多种癌症中均过表达， 提示 Cul4A 在 上述多种内分泌 -生殖系统肿瘤中为一潜在的癌基因。 本申请提供了体外和体内证据表明， CUL4A是引起前列腺癌的 致癌基因。
在前列腺癌细胞系和原发性前^腺肿瘤中， CUL4A 的过表达非 常常见。 与正常前列腺上皮细胞相比， Cul4A的蛋白和 mRNA水平 在前列腺癌细胞中均显著升高。
将正常前列腺上皮细胞在体外通过病毒转染过表达 Cul4A， 结果 惊讶的发现，仅仅这一个基因的变化可将正常前列腺上皮细胞转化为 前列腺癌细胞， 并且侵入性增强。
机制研究显示， CUL4 A激活 ERK途径并因此引起前列腺癌进展。 过表达 CUL4A大大增加 ERK蛋白， 而敲低 CUL4A显著减少 ERK。 本申请的结果还意外显示， CUL4A对 ERK的调节是通过转录而非通 过泛素化来实现。 已知 CUL4A调节组蛋白曱基化。 本申请进一步显 示， CUL4A通过富集 ERK1和 ERK2启动子处的三曱基化 H3K4而 上调 ERK。 重要的是， CUL4A-诱导的恶性表现可以被 ERK抑制剂 U0126部分地逆转， 表明 CUL4A在前列腺癌中通过上调 ERK而发 挥致癌作用。 本申请人发现， CUL4A 的过表达与前列腺癌患者的高 PSA 和 Gleason分值，局部侵入和不良的总体存活显著相关。这表明， CUL4A 水平是前列腺癌患者临床后果不良的预后性生物标志物。可以制作测 试前列腺癌预后的试剂盒， 其中包含检测 CUL4A表达的试剂， 例如 测试其蛋白水平的抗体或测试其 mRNA水平的试剂等。 本发明人发现， Cul4A 的水平影响前列腺细胞的生长。 下调 Cul4A， 在体外实验中抑制了前列腺癌细胞的增殖和细胞周期， 能减 少集落的形成， 并能诱导凋亡； 在体内则抑制肿瘤形成或减緩前列腺 癌的生长。下调 Cul4A可以通过设计能敲低 Cul4A的 shRNA、 siRNA、 反义寡核苷酸等来实现。 已知 shRNA即短发夹 RNA， 是能形成紧密 发夹转折的 RNA序列， 可用于经由 RNA干扰作用使基因表达沉默。 shRNA发夹结构经细胞机制切割成 siRNA。 siRNA —般是带有短尾 的双链 RNA分子 (dsRNA), 包括有义 RNA (它具有与靶基因 mRNA 同源的序列）和反义 RNA (它具有与有义 RNA互补的序列）。 siRNA与 RNA-诱导的沉默复合物 (RISC)结合， 通过 siRNA结合匹配的 mRNA 后， 附在 siRNA上的复合物对 mRNA进行降解， 从而使靶基因表达 被抑制
本发明还证实， 沙度利胺也可下调 Cul4A， 提示沙度利胺可以用 于治疗因 Cul4A过表达所致的疾病， 如癌症， 尤其例如前列腺癌等。 故可将沙度利胺用于制备治疗因 Cul4A 过表达所致疾病， 如癌症， 尤其例如前列腺癌等的药物。 另外， 经本申请证实， Cul4A在前列腺 癌中通过激活 ERK途径而弓 I起癌症进展， 提示沙度利胺也可以用于 经由 Cul4A调节 ERK途径， 从而达到治疗疾病的目的。 所以， 沙度 利胺也可以用于制备经由 Cul4A调节 ERK途径的药物。
在具体实施方案中， 用 shRNA下调 Cul4A, 有效逆转了激素敏 感型细胞系（例如 H PC细胞系 22RV1)的耐药性， 使其对抗雄药物重 新变得敏感， 这不仅说明， 针对 Cul4A 的治疗可以逆转反过来表明 Cul4A导致形成雄激素耐受性前列腺癌 (HRPC), 通过调节 Cul4A水 平可有效治疗 HRPC, Cul4A还可作为预测激素治疗效果的指标。
机制研究揭示， CuWA可上调雄激素受体 (AR)、 并增强其转录活 性， 而 AR也可促进 Cul4A的蛋白和 mRNA的表达， 两者形成正反 馈。 两个癌基因之间的恶性循环刺激前列腺癌细胞不断增殖。 可见， 靶向联合 CuWA和 AR能更有效的治疗前列腺癌， 尤其是 HRPC。 例 如， 可以使用 Cul4A抑制剂和 AR拮抗剂的药物组合。
Cul4A抑制剂例如能敲低 Cul4A的 shRNA、 siRNA,反义寡核苷 酸等， 和 /或沙度利胺。
AR拮抗剂例如抗雄激素药物。常用的抗雄药物是本领域熟知的， 并且市场上已有多种成熟商品在售。例如 Casodex (Sigma- Aldrich, Cas No.: 90357-06-5), Flutamide (Schering-Plough Corp, Kenilworth, NJ) 等等。
鉴于针对 Cul4A的治疗， 例如降低 Cul4A， 可以逆转激素非依赖 性前列腺癌的激素敏感性， 可以将 CuWA视为潜在的抗雄药物增敏 剂， 以及选用抗雄药物及判断疗效的指示因子。 虽然沙度利胺对诸如多发性硬化和套细胞淋巴瘤等血液系统肿 瘤有经过临床验证的活性， 但在临床实体瘤中的应用则进展緩慢， 结 果不容乐观。有人尝试用沙度利胺和细胞毒制剂联合治疗肺癌和前列 腺癌， 但未能显示存活率方面的优势 (Buiris HA, et al. (2010) Cancer Invest 28: 408-412; Lee SM, et al. (2009) J Clin Oncol 27: 5248-5254; 和 Pacheco AV, et al. (2008) Urol Oncol 26: 610-015)。
本申请发现， Cul4A可作为沙利度胺治疗激素非依赖性前列腺癌 的新机制。
本申请还发现，不同的前列腺癌细胞系对沙度利胺处理有不同反 应， 这与 CUL4A水.平显著关联。 CUL4A 高水平的前列腺癌细胞对 沙度利胺特别敏感。本申请还证实， 沙度利胺在前列腺癌中的凋亡效 应也依赖于高水平的 CUL4A。 通过用过表达和敲低策略操作前列腺 癌细胞中的 CUL4A表达， 发现沙度利胺耐受性细胞中的 CUL4A过 表达增加细胞对该药物的敏感性和凋亡，而沙度利胺敏感性细胞中的 CUL4A敲低导致对该药物的耐受和凋亡水平下降。 本申请还发现， 通过长期处理敏感细胞得到的继发沙度利胺耐受性细胞中， CUL4A 蛋白水平下降。 而且， 沙利度胺可以时间依赖性地和剂量依赖性地降 低 Cul4A的表达。 进一步地， 通过抑制 Cul4A， 沙度利胺可以抑制前 列腺癌细胞。 总之， Cul4A是沙利度胺的直接靶标， 其对沙度利胺诱 导的凋亡起决定性作用， CUL4A表达是预测沙度利胺治疗前列腺癌 的效果的一个重要标志。 本文中还证实， Cul4A是前列腺癌转移的关键因子， 抑制 Cul4A 可减少前列腺癌的转移， 靶向 Cul4A联合化疗是转移性前列腺癌的 更有效的治疗方法。 实施例 实施例 1 Cul4A在前列腺癌中的表达
5 . 1.1 原发性前列腺癌中的 CUL4A表达
定量 RT-PCR (qRT-PCR)
从 17例经临床和病理学诊断为前列腺癌的病例取肿瘤组织和临 近的正常前列腺组织。 用 TRIZol (Invitrogen， 货号： 15596-026)从前 列腺癌和癌旁组织提取总 RNA, 用 SYBR PrimeScript RT-PCR Kit l o (Takara)进行 qRT-PCR， 以 β-肌动蛋白做内部参照， 检测 Cul4 Α表达 量。 引物如下：
CUL4A
正向: 5，-TGCTCCTCATGTTCAACGAG-3， (SEQ ID NO:2) 反向： 5，-TTCTGACGATAGCAGCATCAA-3，（SEQ ID NO:3)
15 月几动蛋白
正向： 5，-CGCGAGAAGATGCCCAGATC-3， (SEQ ID NO:4) 反向： 5，-TCACCGGAGTCCATCACGA-3， (SEQ ID NO:5) 发现 58.8%的前列腺癌病例（ 10/17)有 CUL4 A过表达。
在另一组的 13例良性前列腺增生 (benign prostatic hyperplasia, 0 BPH)和 13例前列腺癌样品中， 同样地进行检测， 发现前列腺癌中的 CUL4A mRNA水平远远高于 BPH (p=0.0044)。
免疫组化
用福尔马林固定且石蜡包埋的组织样品制作组织芯片 , 检测 CUL4A蛋白水平： 正常前列腺 (n=8)， BPH (n=13), 前列腺癌 (n=121)。 5 第一抗体是： CUL4A (#ab84382; Abeam), ki-67 (#ZM-0166)。 所 有染色由不清楚样品来源和患者后果的病理学人员进行评估， 使用 EnVision+系统 (Dako)。 用已被广泛接受的 German半定量评分系统来 考察染色强度和区域范围。 每份标本根据细胞核、 细胞质、 和 /或细 胞膜的染色强度打分 (无染色 = 0; 弱染色 = 1 , 中度染色 = 2， 强染 0 色 = 3)， 还根据被染色的细胞所占范围打分 (0% = 0, 1-24% = 1, 25-49% = 2, 50-74% = 3, 75-100% = 4)。将被染色的细胞的强度分值与 范围分值相乘，得到最终的从 0 (最小分值)至 12(最大分值)的免疫反应 分值。 最终的免疫反应分值 6为低表达， 〉6为高表达。 结果， 前列 腺癌的 CUL4A为强染色， 而 BPH和正常前列腺中为弱染色甚至无染 色 (图 2)。
这些数据提示， CUL4A在前列腺癌中频繁地过表达。 1.2 前列腺癌细胞系中的 CUL4A表达
CUL4A 的表达在一组前列腺癌细胞系（LNCaP, C4-2, 22RV1, CWR22R, DU145 和 PC3)、 以及两种永生化的非肿瘤发生性人类前 列腺上皮细胞系（RWPE-1 和 PWR-1E)中检测。 所有细胞都购自美国 '典型培养物保藏中心 (ATCC, 10801 University Boulevard, Manassas, VA 20110-2209), 并按照 ATCC的建议进行培养。
用 RIPA试剂 (Biomiga)提取蛋白， 进行 SDS-PAGE和 Western印 迹， 用 下列一抗检测这些细胞中 的 Cul4A 蛋白 水平： anti-phospho-Ser473 ERK (#4370; Cell Signaling Technology)， anti-total-ERK (#4695; Cell Signaling Technology), anti-CUL4A (#2699; Cell Signaling Technology) , anti-PA P (#9542; Cell Signaling Technology), anti-GAPDH (#470661; bioworld), 以 GAPDH蛋白作为 于照。
结果如图 la, b所示， CUL4A的 m NA和蛋白水平在这些人类 前列腺癌细胞系中虽然各不相同，但都比在非肿瘤发生性人类前列腺 上皮细胞系中大大升高。 而且， CUL4A蛋白水平与其 mRNA水平相 关联。 实施例 2 Cul4A过表达
2.1 Cul4A过表达促进正常前列腺细胞生长和细胞周期进展 将 pBabe-puro 逆转录病毒载体用于转染 CuMA 基因。 用 Superscript One-Step RT-PCR kit (Invitrogen)扩增野生型 Cul4A 的 cDNA后， 将 Cul4A通过 EcoRV和 Xhol的限制性酶切位点克隆进 pcDNA3.1+/myc-His vector (Invitrogen)。 然后将 EcoRV和 Pmel片段 插入 pBabe-puro的 SnaBI位点 ώ 将所得质粒转染入正常前列腺细胞， 观察其与对照相比的细胞生长和周期进展变化。 结果见图 3 和 2.2 Cul4A过表达使正常前列腺细胞转化为肿瘤细胞
对永生化的非肿瘤发生性人类前列腺上皮细胞 RWPE-1 进行改 造， 以稳定表达 CUL4A。 Western印迹分析显示， CUL4A-转染的克 隆 (RWPE- 1 -CUL4A)比 pBabe-空白转染的对照 (RWPE- 1 -Ctrl)有更高 水平的 CUL4A蛋白（图 4a)。 RWPE-1-CUL4A细胞生长迅猛， 10天 内就在软琼脂 (CytoSelect™; 96-Well Cell Transformation Assay)上形 成了大的圆形集落。 相比之下， RWPE-1-Ctrl细胞在所测试的软琼脂 条件下， 不能形成肉眼可见的集落 (图 4a)。 这表明， CUL4A过表达 将 RWPE-1细胞转化为恶性。 2.3 Cul4A过表达促进前列腺癌细胞转移
锚着依赖性生长和侵入是肿瘤形成和进展的两个关键事件，本发 明人发现， 它们都可以被 CUL4A过表达诱导。
将 0.1 mL 含有 5 x 105 个正常细胞 RWPE-1 或前列腺癌细胞 PC-3的无血清 DMEM培养基加入有 8 μιη膜孔的涂基质胶的 Boyden chamber (Costar)。 下室加入无血清角质细胞培养基 （GIBICO, 17005-042)。 48 小时后用棉签刮除膜上面的细胞。 之后将 Boyden Chambers室温在 4%的曱醛中固定 20分钟， 接着用 0.1%的结晶紫染 色 30分钟， 最后用水清洗。 计数膜下面的细胞， 评估细胞侵袭能力 的变化。
结果显示， 体外过表达 Cul4A使正常细胞 RWPE-1 和前列腺癌 细胞 PC-3 发生上皮细胞间质化 (这是所有肿瘤转移的重要步骤）。 Boyden小室实验还显示， CUL4A过表达显著增加 RWPE-1细胞迁移 (图 4b和 4f)。 说明 Cul4A促进前列腺癌的转移， 抑制 Cul4A可减少 前列腺癌的转移。 实施例 3 Cu!4A下调
3.1 下调 Cul4A使前列腺癌细胞的增殖和细胞周期被抑制
设计短发夹 RNA (shR A)以针对 CUL4A (shCUL4A)。 Cul4A sh NA序列来自（Ambion, Austin, TX, USA)。
正向 5 '-GATCCCCGGTTTATCCACGGTAAAGATTCAAGAGA TCTTTACCGTGGATAAACCTTTTTGGAAA-3' (SEQ ID NO:6)
反向 5'-AGCTTTTCCAAAAAGGTTTATCCACGGTAAAGATCT CTTGAATGTTTA CCGTGGATAAACCGGG-3' (SEQ ID NO:7)
按说明书将该 Cul4A shRNA 克隆入 pSUPER.retro.puro 载体 (Oligoengine, Seattle, WA, USA)。 Cul4A shRNA 退火后连接到 pSUPER.retro.puro载体的 Bglll和 Hindlll酶切位点。 然后将该载体 转染入 One Shot® TOP 10 chemically competent E. coli cells (Invitrogen, Carlsbad, CA， USA)。在含 50ug/ml氨苄青審素的 LB培养基中挑选单 克隆送测序验证转染成功。
将 Cul4A shRNA转染入前列腺癌细胞 22RV1， 建立稳定的低表 达 Cul4A的细胞系， 22RVl-shRNA-Cul4a。 观察该细胞系与对照相比 的细胞生长和周期进展变化。 结果见图 5。
用上述 Cul4A shRNA在 22RV1细胞中产生稳定的转染子。 用乱序 (scrambled) shRNA作对照 (shCtrl)。所述 shRNA在 22RV1细胞系中有效 降低了 CUL4A蛋白表达 (图 6a)。 在整个测试过程中， 转染了 shCUL4A 的 22RV 1的细胞增殖持续降低， 在第 6天时比 shCtrl转染的细胞降低 35% (图 6a)。
此前有研究表明， CUL4A缺陷在造血细胞中引起凋亡。 为了调 查 CUL4A敲低引起的 22RV1细胞增殖的抑制是否归因于凋亡， 用 ApopTag荧光素原位凋亡检测试剂盒（Intergen Company, Purchase, NY)， 按照厂家说明， 进行 TdT-介导的 dUTP缺口末端标记 (TUNEL) 试验。
结果，转染了 shCUL4A的 22RV1细胞的死亡明显有被诱导的迹象 (图 6b)。 CUL4A敲低还明显减少 22RV1在软琼脂上形成的菌落的个数 和大小（图 6c)。 3.2 敲低 CUL4 A在体内抑制前列腺癌细胞生长
将 5 X 106 22RV1 和 22RVl-shRNA-cul4a分别注射到 7 只小鼠 (BALB/c-nu棵鼠， 购自上海中科院动物所)皮下， 每周测量小鼠肿瘤 2次。 一个月后测量肿瘤的体积。 结果显示， 22RVl-shRNA-cul4a组 肿瘤体积和总量显著小于 22RV1组。
Figure imgf000013_0001
单位： mm3。
从肿瘤体积来评估， 带有 shCUL4A的 22RV1细胞的生长大大慢 于对照细胞（图 6d)。 而且， CUL4A敲低导致肿瘤重量和大小相比于 带 shCtrl的细胞都明显降低 (图 6e)。
为证实 CUL4A在体内确实被 shRNA敲低， 在这些异种移植肿 瘤中进行了 CUL4A免疫组化染色。结果发现 CUL4A蛋白显著减少。 与体外数据一致的是， 携带 shCUL4A的异种移植肿瘤与携带 shCtrl 的肿瘤相比， 增殖指数 Ki67显著降低， 表明 CUL4A敲低确实在体 内抑制前列腺癌细胞增殖。
22RVl-shCUL4A和 22RVl-shCtrl的冰冻样品经 TU EL试验显 示， 敲低 CUL4A在体内诱导凋亡。
这些数据总体上说明， CUL4A在前列腺癌的肿瘤发生中起关键 作用， 而敲低 CUL4A在体外和体内都抑制肿瘤生长。 实施例 4 CUL4A经转录调节激活 ERK信号途径
Ras/ERK信号途径在多种类型的人类癌症包括前列腺癌中常常 被激活。 有报道称 RWPE-1细胞通过过表达 v-Ki-Ras而进行恶性转化。 为此， 研究了 CUL4A是否经由信号传递途径而对前列腺癌进展作出 贡献。
按照例如 Shang Y， Myers M, Brown M (2002). Mol Cell 9: 601-610 所述， 进 ^亍染色质免疫沉淀 (Chromatin Immunoprecipitation, ChIP)分 析。 制备可溶性染色盾， 并用抗 -triMeK4H3或作为对照的非免疫 IgG 进 ^"沉淀。 用 SYBR green Champion ChIP qPCR assay kit (SABiosciences)进行实时 ChIP qPCR, 样品用 ABI Prism7000 Sequence Detector (Applied Biosystems)扩增。 所用引物如下：
ERK1 :
正向 5，- GCCGAACCTCCCGGTGACCT-3 ' (SEQ JD NO:8) 反向 5，-GGGCCTGGAGCTGTCACGTG-3，（SEQ ID NO:9) ERK-2:
正向 5，-TGGGTCCTGCTTCATGGGCTAAAT-3， (SEQ ID NO: 10) 反向 5，-TTCAAGACTAGCCTGGGCAACACA-3，(SEQ ID NO: l l) 如图 4c所示， 两种 ERK蛋白在过表达 CUL4A的 RWPE-1细胞中大 大增加， 而在 22RV1细胞中， 用 sh NA敲低 CUL4A大大降低了两种 ERK蛋白的总水平（图 4c)。 还发现 服7和 2的转录水平随 CUL4A水平 而改变（图 4d)， 表明 CUL4A经由转录调节而激活 ERK信号传递途径。
此前研究显示， CUL4A在 H3K4调节组蛋白曱基化。 故， 发明人 认为， CUL4 A通过在 ERK启动子处富集 H3K4的三曱基化而转录激活 ERK。 结果显示， RWPE-1 -CUL4A与 RWPE-1-Ctr相比， 表达较高水 平的 H3K4三甲基化。 ChIP显示， 在 ERK1和 ERK2处的 H3K4三甲基化 在 RWPE-1-CUL4A中显著高于在 RWPE-1-Ctr中（图 4e)。 接着， 用 ERK 抑制剂 U0126处理 RWPE- 1 -CUL4A和 RWPE- 1 -Ctrl细胞， 然后监控 CUL4A-介导的恶性表现改变。 象预计的一样， CUL4 A-介导的侵入被 ERK抑制剂 U0126减弱（图 4b和图 4f)。 这些结果总体上表明， ERK是 CUL4 A-介导的恶性转化的下游效应物。 实施例 5 CUL4A与前列腺癌预后
用组织芯片法研究一组前列腺癌患者的 CUL4A蛋白水平与多种 临床因素和结局的关联性。 CUL4A水平与下列各因素都显著相关： PSA 高水平（p=0.019) , Gleason 高分值（p=0.033)， 淋巴结转移 (p=0.038), 淋巴结阳性 (p=0.05)， 精囊阳性 (ρ=0·029) (图 2a , 表 1) 表 1 :
临床变量 I CUL4A I P 低 高
<10 18 14
PSA 0.019
〉=10 28 60
<=5 12 9
6 16 17
Gleason分值 0.033
7 15 37
8-10 3 12
2 22 30 临床期 3 23 35 0.11
4 1 10 阴性 27 35 外膜 0.26 阳性 19 40 阴性 45 65 淋巴结 0.05 阳性 1 10 阴性 28 36 神经 0.19 阳性 18 39 阴性 44 73 膀胱 0.63 阳性 2 2 阴性 40 51 精嚢 0.029 阳性 6 24 外科手术 阴性 35 52
0.53 边界 阳性 11 23
Kaplan-Meier log-rank分析显示 , 在总共 121例肿瘤患者中， 具 有 CUL4A高表达的肿瘤的患者 (n = 75)具有显著缩短的总体存活期 (P
= 0.034)。.
综上， 这些数据提示， CUL4A水平是前列腺癌患者临床后果不 良的预后性生物标志物。 实施例 6 Cul4A与抗雄药物
6.1 建立激素耐受性前列腺癌细胞系
将激素依赖性前列腺癌细胞 LNCAP (购自 ATCC)置于 Casodex (美国 SIGMA公司）中培养 (37°C， 5%二氧化碳） 2个月， 成功建立了 激素耐受的亚型细胞系 LNCAP-R (见图 7)，这一细胞在体外模拟了前 列腺癌患者由激素敏感到耐受的病理过程：使用抗雄药物 Casodex使 得细胞微环境睾酮量逐渐达到去势水平，细胞开始时生长受抑制甚至 出现一部分细胞凋亡， 一段时间后， 存活细胞仍继续生长， 说明细胞 生长已经不依赖于雄激素的存在，细胞已经由激素敏感进展为激素耐 受。
6.2 检测 Cul4A的表达
WESTERN印迹显示， CuW A表达在 LNCAP-R中要明显高于在 LNCAP中， 提示 Cul4A可能与雄激素耐受性前列腺癌的形成相关。
6.3 Cul4A下调的影响
用 shRNA下调 Cul4A (见实施例 2),有效逆转 HRPC细胞系 22RV1 (购自 ATCC)的耐药性， 使其对抗雄药物 Casodex重新变得敏感 (见图 8A和 8B)。 这有力的证实， CuWA是形成雄激素耐受性 HRPC的一 种机制， 并且 Cul4A是 HRPC治疗的有效靶标， 可能是预测激素治 疗效果的指标。
6.4 Cul4A上调的影响
Cul4A可上调雄激素受体 (AR)并增强其转录活性 (见实施例 2)见 (图 9)， 同样 AR也可促进 Cul4A的蛋白和 mRNA的表达 (图 10)， 可 见， 两者形成正性反馈， 且两个癌基因之间的恶性循环刺激前列腺癌 细胞不断增殖。 实施例 7、 CUL4A在前列腺癌中决定了对沙度利胺的反应 先评估了一组有不同的内源 CUL4A表达的前列腺癌细胞系中沙度利 胺的生长抑制效应（图 11a和 b)。 沙度利胺处理 72小时后， 用 MTT实验 测定细胞活力。 有趣的是， 具有高水平 CUL4A的细胞 (LNCAP, C4-2, CWR22R, 22RV1)对沙度利胺特别敏感， CUL4A低水平的细胞 (PC-3, DU-145, RWPE-1和 PWR-1E)显示耐受（图 lla)。
为建立 CUL4A与沙度利胺敏感性之间的直接联系， 在沙度利胺 耐受性 PC-3和 RWPE-1细胞中异位表达了 CUL4A, 导致对该药的敏感 性增加（图 lib和 llh)。 在修正的实验中， 用 shRNA下调沙度利胺敏感 性 22RV1细胞中的 CUL4A表达水平， 导致对该药耐受（图 llc)。 接着， 为了研究 CUL4A与对沙 WJ^ i
先研究了沙度利胺是否影响 CUL4A表达。 结果发现， 沙度利胺在 22RV1细胞中以剂量依赖性方式下调 CUL4 A及其下游 ERK(图 11 d)。
此前多项研究表明，沙度利胺的生长抑制作用通过诱导凋亡来介 导。 本发明发现， 沙度利胺仅在敏感性细胞系 LNCAP中以剂量依赖 性方式诱导凋亡（图 lle)。 在耐受细胞 PC-3中， 所测试的所有浓度都 无凋亡证据（图 lle)。 而且， 在敏感性细胞 22RV1中， 敲低 CUL4A阻 断了由沙度利胺诱导的凋亡 (图 llf)。 相反， 在耐受性 PC-3中， 异位过 表达 CUL4A促进沙度利胺诱导的凋亡（图 llg)。这些结果提示， CUL4A 是前列腺癌细胞中沙度利胺诱导的凋亡的关键性决定分子。
构建了继发性沙度利胺耐受性 C4-2细胞 (C4-2-R), 方法是将这些 细胞用沙度利胺处理 3个月（图 12a)。 免疫印迹分析显示， C4-2-R与其 亲代细胞相比， 表达非常低水平的 CUL4A (图 12b)， 表明在获得沙度 利胺耐受性期间， CUL4A损失。
总之， 下调 CUL4 A在前列腺癌细胞中是沙度利胺诱导的凋亡的 主要决定分子， 而 CUL4 A基础水平是对沙度利胺治疗的反应性的生 物标志物。

Claims

权 利 要 求 书
1. 治疗前列腺癌的方法， 包括对患者施用抑制或敲低 Cul4A 的试剂。
2. 权利要求 1 的方法， 其中所述前列腺癌选自： 雄激素耐受 性前列腺癌前列腺癌 (H PC) , 激素非依赖性前列腺癌， 和 /或具有高水平 Cul4A的前列腺癌。
3. 权利要求 1的方法， 其中所述抑制或敲低 Cul4A的试剂选 自下列之一或多项： shR A， siRNA, 反义寡核苷酸和沙 度利胺。
4. 权利要求 1 的方法， 还包括给患者施用 A 拮抗剂如抗雄 激素药物。
5. Cul4A基因或其编码的蛋白在检测前列腺癌方面的用途。
6. Cul4A基因或其编码的蛋白作为预测前列腺癌治疗效果的 指标的用途。
7. 权利要求 7的用途， 其中所述治疗选自 AR拮抗剂如抗雄 激素药物的治疗和沙度利胺治疗。
8. 用于治疗前列腺癌的药物组合， 包括 Cul4 A抑制剂和 A 拮抗剂。
9. 权利要求 8的药物组合， 其中所述 Cul4A抑制剂选自针对 Cul4A的 shRNA和沙度利胺。
10. 评估前列腺癌患者预后的方法， 包括检测患者的 Cul4A基 因或蛋白水平。
11. 筛选适于接受抗雄激素药物治疗和 /或沙度利胺治疗的前 列腺癌患者的方法， 包括检测患者的 Cul4 A基因或蛋白水 平。
PCT/CN2012/000233 2012-02-24 2012-02-24 Cul4A在前列腺癌诊断、治疗及预后中的用途 WO2013123620A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2012/000233 WO2013123620A1 (zh) 2012-02-24 2012-02-24 Cul4A在前列腺癌诊断、治疗及预后中的用途

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2012/000233 WO2013123620A1 (zh) 2012-02-24 2012-02-24 Cul4A在前列腺癌诊断、治疗及预后中的用途

Publications (1)

Publication Number Publication Date
WO2013123620A1 true WO2013123620A1 (zh) 2013-08-29

Family

ID=49004905

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/000233 WO2013123620A1 (zh) 2012-02-24 2012-02-24 Cul4A在前列腺癌诊断、治疗及预后中的用途

Country Status (1)

Country Link
WO (1) WO2013123620A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1901911A (zh) * 2003-11-06 2007-01-24 细胞基因公司 使用沙利度胺治疗和控制癌症和其它疾病的组合物和方法
CN101863806A (zh) * 2010-03-18 2010-10-20 湖北省医药工业研究院有限公司 抗前列腺癌药物(r)-比卡鲁胺的制备方法
CN102282162A (zh) * 2008-04-29 2011-12-14 康奈尔大学 用于增强dna修复的物质和组合物及使用方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1901911A (zh) * 2003-11-06 2007-01-24 细胞基因公司 使用沙利度胺治疗和控制癌症和其它疾病的组合物和方法
CN102282162A (zh) * 2008-04-29 2011-12-14 康奈尔大学 用于增强dna修复的物质和组合物及使用方法
CN101863806A (zh) * 2010-03-18 2010-10-20 湖北省医药工业研究院有限公司 抗前列腺癌药物(r)-比卡鲁胺的制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CUI, ZILIAN ET AL.: "Cul4A and hormone-resistant prostate cancer", JOURNAL OF UROLOGY FOR CLINICIAN (ELECTRONIC VERSION), vol. 3, no. 2, 30 June 2011 (2011-06-30), pages 37 - 39 *
LI, XUESONG ET AL.: "Advances of Therapeutic Approaches for Hormone Refractory Prostate Cancer", CHINA CANCER, vol. 12, no. 12, 25 December 2003 (2003-12-25), pages 699 - 703 *

Similar Documents

Publication Publication Date Title
Wu et al. Exosomes from the tumour-adipocyte interplay stimulate beige/brown differentiation and reprogram metabolism in stromal adipocytes to promote tumour progression
Li et al. microRNA-141-3p fosters the growth, invasion, and tumorigenesis of cervical cancer cells by targeting FOXA2
Watson et al. GSK3β and β-catenin modulate radiation cytotoxicity in pancreatic cancer
Fang et al. Overexpression of a chromatin remodeling factor, RSF-1/HBXAP, correlates with aggressive oral squamous cell carcinoma
Zhao et al. HOTTIP predicts poor survival in gastric cancer patients and contributes to cisplatin resistance by sponging miR-216a-5p
Tian et al. The miR-218/GAB2 axis regulates proliferation, invasion and EMT via the PI3K/AKT/GSK-3β pathway in prostate cancer
Chen et al. MiR-299-3p functions as a tumor suppressor in thyroid cancer by regulating SHOC2.
JP5590693B2 (ja) がん患者の予後推定用バイオマーカー及びその利用
Liu et al. Μolecular impact of bone morphogenetic protein 7, on lung cancer cells and its clinical significance
Ye et al. 17‐AAG suppresses growth and invasion of lung adenocarcinoma cells via regulation of the LATS 1/YAP pathway
US7902166B2 (en) Compositions comprising inhibitors of RNA binding proteins and methods of producing and using same
Hao et al. Eukaryotic initiation factor 3C silencing inhibits cell proliferation and promotes apoptosis in human glioma
Wang et al. PIK3CA is regulated by CUX1, promotes cell growth and metastasis in bladder cancer via activating epithelial-mesenchymal transition
Cho et al. Puf-A promotes cancer progression by interacting with nucleophosmin in nucleolus
Haughian et al. Protein kinase C alpha (PKCα) regulates growth and invasion of endometrial cancer cells
JP5611953B2 (ja) 癌の処置における治療標的としてのチロシンキナーゼ受容体ｔｙｒｏ３
US20120076788A1 (en) C-cbl and antagonists thereof for the treatment and diagnosis of cancer
Liu et al. KLHL22 promotes malignant melanoma growth in vitro and in vivo by activating the PI3K/Akt/mTOR signaling pathway.
Lei et al. Inhibition of zinc finger protein 367 exerts a tumor suppressive role in colorectal cancer by affecting the activation of oncogenic YAP1 signaling
WO2013123620A1 (zh) Cul4A在前列腺癌诊断、治疗及预后中的用途
Ren et al. Inhibition of non-small cell lung cancer cell proliferation and tumor growth by vector-based small interfering RNAs targeting HER2/neu
CA2656577A1 (en) Method for evaluation of a cancer
Zhao et al. LncRNA FOXD3-AS1/miR-128-3p axis-mediated IGF2BP3 in glioma stimulates cancer angiogenesis and progression
He et al. HOXC10 promotes esophageal squamous cell carcinoma progression by targeting FOXA3 and indicates poor survival outcome
JP7137183B2 (ja) がんの治療又は予防用医薬および癌のバイオマーカー

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12869360

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12869360

Country of ref document: EP

Kind code of ref document: A1