WO2019184306A1 - 一种肿瘤转移药物治疗的靶标及其应用 - Google Patents

一种肿瘤转移药物治疗的靶标及其应用 Download PDF

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WO2019184306A1
WO2019184306A1 PCT/CN2018/110767 CN2018110767W WO2019184306A1 WO 2019184306 A1 WO2019184306 A1 WO 2019184306A1 CN 2018110767 W CN2018110767 W CN 2018110767W WO 2019184306 A1 WO2019184306 A1 WO 2019184306A1
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prak
medicament
expression
benefit
tumor
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张毓
王巍
王羽晴
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北京大学
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Priority to EP18912072.8A priority patent/EP3785768A4/en
Priority to KR1020207029270A priority patent/KR102591642B1/ko
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Definitions

  • the present invention relates to the field of biotechnology and medicine, and in particular to the use of PRAK as a drug target for screening drugs for inhibiting or preventing tumor cell metastasis.
  • Malignant tumors are prone to metastasis in four ways: 1. Direct spread to adjacent sites; 2. Lymphatic metastasis: Primary cancer cells are transferred to lymph nodes and distant sites such as lung, liver, bone, and brain with lymphatic drainage. Waiting for the formation of secondary tumors; 3, blood transfer: cancer cells enter the blood vessels, in the blood vessels, or with the blood flow to distant sites such as lung, liver, bone, brain, etc., forming secondary tumors; 4 Planting: Tumor cells are detached and planted in another part, such as visceral cancer, which is planted on the peritoneum or pleura. Malignant tumor metastasis has a major impact on the outcome of the disease.
  • P38-regulated/activated protein kinase is a member of the mitogen-activated protein kinase (MAPK) family.
  • MAPK mitogen-activated protein kinase
  • PRAK catalyzes phosphorylation of Rheb, resulting in inhibition of mTORC1 activity, thereby regulating cell metabolism.
  • PRAK inhibits tumorigenesis by promoting cell senescence on the one hand, and accelerates the development of already formed tumors by inducing angiogenesis on the other hand.
  • its role in tumor metastasis is still unknown.
  • the present invention first provides the use of PRAK as a drug target for screening drugs for inhibiting or preventing tumor cell metastasis.
  • the present invention provides the use of an inhibitor of PRAK for the preparation of a medicament for inhibiting or preventing tumor cell metastasis.
  • the inhibitor appears to reduce PRAK expression levels or inhibit PRAK biological activity.
  • the inhibitor includes, but is not limited to, an inhibitor that specifically or non-specifically reduces the expression level of PRAK or inactivates PRAK biological activity. That is, it is within the scope of the present invention to use a drug that achieves inhibition or prevention of tumor cell metastasis by reducing the expression of PRAK or reducing the biological activity of PRAK.
  • the inhibitor may be selected from the group consisting of a chemical drug, a biological macromolecule, a polypeptide, a single chain antibody, an antisense oligonucleotide, a small hairpin RNA, a small interfering RNA, and a gene editing system.
  • the chemical agent comprises a compound which reduces the expression level of PRAK or inhibits the biological activity of PRAK or a pharmaceutically acceptable salt thereof.
  • CN102036997A which are useful as inhibitors of PRAK, It inhibits the biological activity of PRAK, reduces the migration ability of tumor cells, and regulates the expression and function of HIF1 ⁇ , MMP2 and EMT series molecules, and achieves inhibition or prevention of tumor cell metastasis. That is, the present invention provides a novel use of the compound and its pharmaceutical composition for the inhibition or prevention of tumor cell metastasis.
  • the present invention finds that the inhibitor inhibits the biological activity of PRAK, reduces the migration ability of tumor cells, and regulates the expression and/or function of HIF1 ⁇ , MMP2 and/or EMT series molecules by a large number of objective experiments to explore the mechanism. Inhibition or prevention of cell metastasis.
  • the inhibitor reduces the expression of HIF1 ⁇ protein by inhibiting PRAK biological activity and modulating the ability of mTOR to promote protein translation.
  • the tumor cells of the present invention include, but are not limited to, melanoma cells, breast cancer cells, and the like. According to the exemplary description given in the specific embodiments of the present invention, those skilled in the art can infer from conventional knowledge that the tumor may further include brain tumor, lung cancer, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer.
  • head and neck cancer cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, esophageal cancer, gallbladder cancer, non-Hodgkin's lymphoma, prostate cancer, thyroid cancer, female genital cancer, lymphoma, bone Cancer, skin cancer, colon cancer, testicular cancer, etc.
  • the present invention provides a drug which inhibits or prevents tumor cell metastasis based on the above research results, and the active ingredient of the drug inhibits PRAK biological activity.
  • the present invention also provides a medicament for reducing tumor cell migration ability, regulating expression and/or function of HIF1 ⁇ , MMP2 and/or EMT series molecules, and a medicament for regulating mTOR activity,
  • the active ingredient of the drug inhibits the biological activity of PRAK.
  • PRAK biological activity and "mTOR activity”, and "expression and/or function of HIF1 ⁇ , MMP2 and/or EMT series molecules" by research, and therefore, The use of PRAK as a target inhibitor to modulate the activity of mTOR, or to regulate the expression and/or function of HIF1 ⁇ , MMP2 and/or EMT series molecules, is also within the scope of the present invention.
  • the experimental means used in the present invention are (1) small molecule compound inhibitors; (2) gene level intervention, specifically: PRAK knockout mice (PRAK knockout); B16 (mouse melanoma) cell lines using Cas9 PRAK (PRAK knockout) was knocked out; A375 (human melanoma)/MDA-MB-231 (human breast cancer cell line) cell line was knocked down with PRAK by shRNA transfection.
  • Transwell chamber test results and scratch test results of B16 (mouse melanoma), A375 (human melanoma) and MDA-MB-231 (human breast cancer cell line) demonstrate that PRAK molecular deletion or expression levels will significantly reduce tumor cells in Migration ability in vitro, but does not affect tumor cell proliferation (MTS test) and apoptosis (annexin V-7-AAD combined staining).
  • the B16 tail vein injection model (i.v.) results show that the use of PRAK knockout/knockdown or inhibitor will greatly reduce the lung metastasis rate of this model with a dose-dependent effect. Moreover, this intervention is more significant in the early stage of tumor cell metastasis.
  • the inhibitory effect on the distal metastasis of tumor cells can be achieved by using PRAK inhibitors in the first 5 days after the injection of tumor cells, and the effect is almost equivalent to the effect of the whole injection inhibitor. . If the time window is missed and the inhibitor is used, it is almost ineffective.
  • the use of shPRAK and PRAK inhibitors in A375 human melanoma has a similar effect.
  • C) MDA-MB-231 in vivo imaging model A method similar to the B16 tail vein injection model was obtained by in vivo fluorescence detection. That is, the use of shPRAK/PRAK inhibitors will strongly inhibit the colonization of lung cells in tumor cells, and the monitoring results of 14 days/21 days (the time of obtaining and sacrificing mice) show that the fluorescence intensity of the lungs in the inhibitor group is much lower. In the group where no inhibitor is used.
  • MMTV-PyMT mouse spontaneous breast cancer model MMTV-PyMT spontaneous breast cancer mice (almost 100% onset, onset time 8-12 weeks, commercially available, for example, available from Jackson Lab (JAX)) Hybridization with PRAK knockout mice yielded wild-type and PRAK knockout mice in the MMTV-PyMT background. The results showed that the use of PRAK knockout can significantly reduce the spontaneous lung metastasis rate of PyMT mice. Only one (1/19) of the PRAK knockout PyMT mice found a metastasis in the lungs. Thus, PRAK knockout can be seen. Significantly increase the metastasis inhibition rate of cancer cells.
  • the biological characteristics of the tumor cells in situ in the mouse are close to the results of in vitro experiments, that is, the deletion of PRAK only affects the effect of distal metastasis, and its proliferation And apoptosis is not affected. Similar metastatic inhibition effects were also observed with PRAK inhibitors.
  • RNA-seq big data analysis PRAK expression is closely related to hypoxia and redox-related pathways
  • PRAK can regulate the expression and function of HIF1 ⁇ , MMP2 and EMT series molecules
  • PRAK can regulate the synthesis of HIF1 ⁇ protein by regulating mTOR activity.
  • the invention provides a target PRAK which can effectively inhibit tumor cell metastasis, and explores its mechanism of action, and finds that by reducing the enzyme activity of PRAK or lowering the expression level of PRAK, the tumor cell migration ability can be reduced, and mTOR activity can be regulated, Expression and/or function of HIF1 ⁇ , MMP2 and/or EMT series molecules to achieve inhibition or prevention of tumor cell metastasis.
  • the present invention also verified the characteristics of PRAK inhibitors different from existing tumor chemotherapy drugs through experimental studies.
  • Chemotherapy drugs usually exert anti-tumor effects by affecting the growth and survival of tumor cells, and toxicity and drug resistance to normal tissues are almost inevitable.
  • the knockout/knockdown of PRAK or the use of inhibitors does not affect the growth and survival of tumor cells in situ, but only acts on the metastasis to prevent the occurrence of distant metastasis. This intervention is more important in the early stage of tumor cell metastasis.
  • the inhibitory effect on the distant metastasis of tumor cells can be achieved by using PRAK inhibitors in the first 5 days of intravenous injection of tumor cells, and the lung colonization of tumor cells can be strongly inhibited.
  • Figure 1 shows the effect of PRAK knockout and PRAK inhibitors on B16-F10 proliferation.
  • Figure 2 shows the effect of PRAK knockout and PRAK inhibitors on B16-F10 apoptosis.
  • Figure 3 shows the effect of PRAK knockout and PRAK inhibitor on B16-F10 migration.
  • Figure 4 shows the effect of PRAK knockout and PRAK inhibitor on B16-F10 invasion.
  • Figure 5 shows the effect of PRAK knockout and PRAK inhibitors on in situ growth of B16-F10 tumors.
  • Figure 6 shows the effect of PRAK knockout and PRAK inhibitors on B16-F10 lung colonization ability.
  • Figure 7 shows the effect of treatment with different time and three different PRAK inhibitors on distant metastasis of B16-F10 tumor cells.
  • Figure 8 shows the effect of PRAK knockdown and PRAK inhibitors on the invasive ability of A375.
  • Figure 9 shows the effect of PRAK knockdown and PRAK inhibitors on A375 lung colonization ability.
  • Figure 10 shows the effect of PRAK knockdown and PRAK inhibitors on the invasive ability of MDA-MB-231.
  • Figure 11 shows the effect of PRAK knockdown and PRAK inhibitors on MDA-MB-231 lung colonization ability.
  • Figure 12 is a graph showing the effect of PRAK knockout and PRAK inhibitor on spontaneous mammary tumorigenesis in MMTV-PyMT mice.
  • Figure 13 shows the effect of PRAK knockout and PRAK inhibitor on lung metastasis of spontaneous breast tumors in MMTV-PyMT mice.
  • Figure 14 is a graph showing the effect of PRAK knockdown on the proliferation of MMTV-PyMT mouse tumor cells in vitro.
  • Figure 15 is a graph showing the effect of PRAK knockdown on apoptosis of MMTV-PyMT mouse tumor cells in vitro.
  • Figure 16 is a distribution of differentially expressed genes in RNA Seq results of wild type and PRAK knockout B16-F10 cells.
  • Figure 17 is a GO pathway enrichment of wild-type and PRAK knockout B16-F10 cell differentially expressed genes.
  • Figure 18 shows that PRAK knockout and PRAK inhibitors significantly reduced protein expression levels of B16-F10 cell lines HIF-1 ⁇ and MMP2.
  • Figure 19 is a graph showing the effect of PRAK knockdown on the expression of EMT-related molecules in the B16-F10 cell line.
  • Figure 20 is a graph showing changes in phosphorylation levels of mTOR pathway-associated molecules induced by PRAK knockdown and PRAK inhibitors.
  • Figure 21 shows the detection of PRAK mRNA levels in lung cancer patients without distant metastases and distant metastases.
  • Figure 22 is a correlation analysis of PRAK and MMP2 mRNA expression levels in tumor samples of lung cancer patients.
  • Figure 23 is a graph showing the survival of patients with high and low expression of PRAK in the GEPIA database.
  • the PRAK inhibitor used in the following examples of the invention is selected from the following compounds:
  • This example uses mouse melanoma as an example to illustrate the effect of PRAK on B16-F10 proliferation, in vitro survival, invasion ability, tumor in situ growth and early lung colonization.
  • MTS The same number of PRAK WT and KO cells were plated into 96-well plates.
  • Cell Titer 96 Aqueous cell proliferation assay solution was added at different time points after adherence. After incubating for 1 to 4 hours at 37 ° C, absorbance values were measured at 490 nm, and cell proliferation curves were drawn. .
  • AnnexinV/7-AAD staining The cells were plated into 24-well plates, and the cells were treated with PRAK inhibitor at 0.1 ⁇ M and 1 ⁇ M. After 24 hours, the cells were stained with Annexin V/7-AAD, and the apoptosis was analyzed by flow cytometry.
  • mice 6-8 weeks of C57BL/6 female mice (commercially available, for example, available from Vitalivic) were selected for the experiment, and the B16-F10 cells in the logarithmic growth phase were digested and washed twice with PBS. After counting, the PBS was resuspended to adjust the cell suspension concentration to 1.5 ⁇ 10 6 /ml.
  • Tumor cells were subcutaneously inoculated into the flank of the mouse, 200 ⁇ L of cell suspension/only, containing 3 ⁇ 10 5 cells.
  • the drug-administered group was intraperitoneally injected with a PRAK inhibitor of 2 mg/kg/d.
  • the tumor long diameter (L) and short diameter (S) were measured with vernier calipers every two days, and the tumor size was calculated by the formula L ⁇ S ⁇ S ⁇ 0.5, and the tumor growth curve was drawn.
  • mice 6-8 weeks old C57BL/6 female mice were selected for the experiment.
  • the B16-F10 cells in the logarithmic growth phase were digested and washed twice with PBS. After counting, the concentration of the cell suspension was adjusted to 5 ⁇ by PBS. 10 5 /ml.
  • Tumor cells were injected into the mice via tail vein injection, and each was injected with 200 ⁇ L of cell suspension, 1 ⁇ 10 5 cells. After 15 days, the mice were sacrificed, the lungs were removed, and the number of tumor nodules on the surface of the lungs was counted.
  • the administration component is administered for 0-4 days or 5-15 days by intraperitoneal injection of a PRAK inhibitor of 2 mg/kg/d.
  • PRAK knockout and PRAK inhibitors significantly inhibited the invasive ability of B16-F10.
  • PRAK knockout and PRAK inhibitors had no significant effect on the in situ growth of subcutaneously inoculated B16-F10 tumors.
  • PRAK knockout and PRAK inhibitors significantly inhibited the lung colonization ability of intravenous B16-F10 cells.
  • PRAK knockout and PRAK inhibitors have no significant effect on the proliferation and in situ growth of B16-F10, but can significantly inhibit the invasion ability and lung colonization ability of B16-F10.
  • This example uses human melanoma as an example to illustrate the effect of PRAK on A375 invasive ability and lung colonization ability.
  • Example 2 In the same manner as in Example 1, B16-F10 was replaced with A375, PRAK knockdown was transfected with shRNA, and the in vivo vaccinated recipient mouse was Scid-Beige (commercially available, for example, from Vitalia).
  • PRAK knockdown and PRAK inhibitors significantly inhibited the invasive ability of A375.
  • PRAK knockdown and PRAK inhibitors significantly inhibited the lung colonization ability of A375.
  • PRAK knockdown and PRAK inhibitors can significantly inhibit the invasive ability and lung colonization ability of A375.
  • This example uses a human breast cancer cell line as an example to illustrate the effect of PRAK on MDA-MB-231 invasive ability and lung colonization ability.
  • mice 6-8 weeks of SCID Beige female mice were selected for the experiment, and the wild-type or PRAK shRNA transfected MDA-MB-231 cells in logarithmic growth phase were digested, washed twice with PBS, counted, and PBS was weighed. The suspension was adjusted to a cell suspension concentration of 2.5 x 10 6 /ml. Tumor cells were injected into the mice via the tail vein, and each was injected with 200 ⁇ L of cell suspension, 5 ⁇ 10 5 cells. Mice lung tumor cell growth was monitored using an IVIS Spectrum small animal in vivo imaging system. After 21 days, the mice were sacrificed, the lungs were removed, and the number of tumor nodules on the lung surface was counted. The administration group was intraperitoneally injected with a PRAK inhibitor of 2 mg/kg/d for the first five days.
  • PRAK knockdown and PRAK inhibitors significantly inhibited the invasive ability of MDA-MB-231.
  • PRAK knockdown and PRAK inhibitors significantly inhibited the lung colonization ability of MDA-MB-231.
  • PRAK knockdown and PRAK inhibitors can significantly inhibit the invasive ability and lung colonization ability of MDA-MB-231.
  • This example uses MMTV-PyMT spontaneous breast cancer mice as an example to illustrate the effect of PRAK on spontaneous lung cancer metastasis.
  • MMTV-PyMT mice were crossed with PRAK mice to obtain MMTV-PRAK WT and MMTV-PRAK knockout mice, and tumors in the breast site were observed from 8-10 weeks, and another group of MMTV-PRAK WT mice from 12 From week onwards, PRAK inhibitor (1 mg/kg) was administered every other day. At 15 weeks, the mice were sacrificed, the breast tumor nodules were counted, and the weight was weighed, while the lung tumor nodules were counted.
  • mice mammary gland tumor was removed, shredded, and the tumor cells were isolated by DNase and collagenase digestion and density gradient centrifugation for MTS analysis and Annexin V/7-AAD staining.
  • PRAK knockout and PRAK inhibitors had no significant effect on the incidence and growth of spontaneous tumors in the breast.
  • PRAK knockout and PRAK inhibitors significantly inhibited lung metastasis of spontaneous mammary tumors in mice.
  • PRAK knockout had no significant effect on the proliferation of tumor cells isolated from mouse spontaneous breast tumors in vitro.
  • PRAK knockout had no significant effect on the in vitro apoptosis level of tumor cells isolated from mouse spontaneous breast tumors.
  • This example is intended to illustrate the effect of PRAK knockdown on the transcriptional profile of the B16-F10 cell line gene.
  • RNA of PR16 WT and PRAK knockout B16-F10 cells were extracted, RNA Seq was performed, and the differentially expressed genes were subjected to GO enrichment.
  • This example is intended to illustrate the effect of PRAK on the expression and function of HIF1 ⁇ , MMP2 and/or EMT series molecules.
  • the Western Blot method was used to detect changes in protein levels of each molecule after PRAK knockout and PRAK inhibitor treatment.
  • PRAK knockout and PRAK inhibitors significantly reduced the expression levels of HIF-1 ⁇ and MMP2 in the B16-F10 cell line.
  • PRAK knockdown significantly reduced the expression of N-cadherin in the B16-F10 cell line and promoted the expression of E-cadherin.
  • This example is intended to illustrate the effect of PRAK on the expression of mTOR-related molecules.
  • phosphorylation levels of mTOR pathway-associated molecules were down-regulated after PRAK knockout and PRAK inhibitor treatment.
  • This example is used to illustrate the correlation between PRAK and tumor metastasis ability in lung cancer patients.
  • Examples 2-9 were tested with PRAK inhibitor-23 as a PRAK inhibitor, and repeated experiments were performed by replacing PRAK inhibitor-22 and PRAK inhibitor-29. The functions of the three inhibitors were verified by experiments. The same (both can achieve the same suppression effect).
  • the present invention discloses the use of PRAK as a drug target for screening drugs for inhibiting or preventing tumor cell metastasis.
  • PRAK is a tumor cell metastasis target, and further provides a drug capable of inhibiting or preventing tumor cell metastasis, wherein the active ingredient of the drug can reduce PRAK expression level or inhibit PRAK biological activity by regulating cell migration, HIF1 ⁇ ,
  • MMP2 and/or EMT series molecules can inhibit or prevent tumor cell metastasis, and has good economic value and application prospect.

Abstract

PRAK作为药物靶标在筛选抑制或预防肿瘤细胞转移的药物中的应用。基于对PRAK作为肿瘤细胞转移靶标的发现，进一步提供了可抑制或预防肿瘤细胞转移的药物，所述药物的活性成分可降低PRAK表达水平或抑制PRAK生物活性，通过调控细胞迁移、HIF1α、MMP2和/或EMT系列分子的表达和/或功能，实现对肿瘤细胞转移的抑制或预防。

Description

一种肿瘤转移药物治疗的靶标及其应用
交叉引用
本申请要求2018年3月27日提交的专利名称为“一种肿瘤转移药物治疗的靶标及其应用”的第201810259369.2号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及生物技术和医药领域，具体地说，涉及PRAK作为药物靶标在筛选抑制或预防肿瘤细胞转移的药物中的应用。
背景技术
恶性肿瘤容易发生转移，其方式有四种：1、直接蔓延到邻近部位；2、淋巴转移：原发癌的细胞随淋巴引流转移到各级***及远隔部位如肺、肝、骨、脑等处，形成继发性肿瘤；3、血行转移：癌细胞进入血管，在血管内，或随血流转移至远隔部位如肺、肝、骨、脑等处，形成继发性肿瘤；4、种植：瘤细胞脱落后种植到另一部位，如内脏的癌播种到腹膜或胸膜上。恶性肿瘤转移对疾病转归有重大影响。
据估计，90％肿瘤病人的死亡是源于转移。因此，人们一直致力于研究如何抑制/预防肿瘤细胞的转移。
P38-调节/活化蛋白激酶(PRAK，也称MK5)是丝裂原活化蛋白激酶(MAPK)家族成员之一。它作为p38MAPK下游底物，本身也是一种激酶，能催化多种底物，如HSP27、ERK3/4、14-3-3ε、p53、FOXO3、Rheb等的磷酸化，参与调控细胞应激、代谢、运动、生长、衰老等生命过程。例如，在能量耗竭等状态下，p38被激活，进而活化PRAK，后者催化Rheb磷酸化，导致mTORC1的活性被抑制，从而调控细胞代谢。关于其在肿瘤形成中的作用，现有研究表明，PRAK一方面通过促进细胞衰老抑制肿瘤的发生，另一方面又通过诱导血管形成加快业已形成的肿瘤的发展。但其在肿瘤转移中的作用还不得而知。
发明内容
为了解决现有技术中存在的问题，本发明的目的是提供一种肿瘤转移药物治疗的靶标及其应用。
为了实现本发明目的，本发明的技术方案如下：
本发明首先提供了PRAK作为药物靶标在筛选抑制或预防肿瘤细胞转移药物中的应用。
进一步地，本发明提供了PRAK的抑制剂在制备抑制或预防肿瘤细胞转移药物中的应用。
所述抑制剂表现为降低PRAK表达水平或抑制PRAK生物活性。其中，所述抑制剂包括但不限于特异性或非特异性降低PRAK的表达水平或使PRAK生物活性失活的抑制剂。即只要利用降低PRAK的表达或使PRAK生物活性降低而实现肿瘤细胞转移的抑制或预防的药物，均属于本发明的保护范围。
可选地，所述抑制剂可选自化学药物、生物大分子、多肽、单链抗体、反义寡聚核苷酸、小发夹RNA、小干扰RNA、基因编辑体系。
其中，所述化学药物包括可降低PRAK表达水平或抑制PRAK生物活性的化合物或其药学上可接受的盐。
本发明在研究过程中，在现有技术中发现了一种用于治疗变性和炎性疾病的[1.2.4]***并[1.5-a]吡嗪化合物或其可药用盐、溶剂化物或前药以及其立体异构体、同位素变体和互变异构体(由公开号为CN101454326A的专利申请记载)，以及一种用于治疗变性和炎性疾病的咪唑并[1.2-a]吡嗪化合物或其可药用盐、溶剂化物或前药以及其立体异构体、同位素变体和互变异构体(由公开号为CN102036997A的专利申请记载)，可作为PRAK的抑制剂，抑制PRAK的生物活性，降低肿瘤细胞迁移能力，并调控HIF1α、MMP2以及EMT系列分子的表达和功能，实现对肿瘤细胞转移的抑制或预防。即本发明提供了该化合物及其药用组合物的新用途，将其用于肿瘤细胞转移的抑制或预防。
进一步地，本发明通过大量客观试验探究机理，发现所述抑制剂通过抑制PRAK生物活性，降低肿瘤细胞迁移能力，并调控HIF1α、MMP2和/或EMT系列分子的表达和/或功能，实现对肿瘤细胞转移的抑制或预防。
更进一步地，所述抑制剂通过抑制PRAK生物活性，调控mTOR促进蛋白质翻译的能力来降低HIF1α蛋白的表达。
由于PRAK表达于多种肿瘤细胞，而HIF1α蛋白是肿瘤转移的主调控因子，因此，本发明所述的肿瘤细胞包括但不限于黑色素瘤细胞、乳腺癌细胞等。依据本发明在具体实施方式中给出的示例性说明，本领域技术人员可以依据常规知识推知，所述的肿瘤还可包括脑瘤，肺癌，膀胱癌，胃癌，卵巢癌，腹膜癌，胰腺癌，头颈癌，子***，子宫内膜癌，结直肠癌，肝癌，肾癌，食管癌，胆囊癌，非霍奇金淋巴瘤，***癌，甲状腺癌，雌性生殖道癌，淋巴瘤，骨癌，皮肤癌，结肠癌，睾丸癌等。
因此，本发明基于上述研究成果，提供了一种可抑制或预防肿瘤细胞转移的药物，所述药物的活性成分可抑制PRAK生物活性。
进一步地，基于上述机理，本发明还提供了一种降低肿瘤细胞迁移能力、调控HIF1α、MMP2和/或EMT系列分子的表达和/或功能的药物，以及一种调控mTOR活性的药物，所述药物的活性成分可抑制PRAK生物活性。
需要说明的是，本发明通过研究发现了PRAK生物活性与“mTOR活性”，以及与“HIF1α、MMP2和/或EMT系列分子的表达和/或功能”之间的关系，因此，通过利用针对以PRAK作为靶标的抑制剂，来调控mTOR的活性、或调控HIF1α、MMP2和/或EMT系列分子的表达和/或功能，而实现的衍生应用，也属于本发明的保护范围。
本发明所采用的实验手段为(1)小分子化合物抑制剂；(2)基因水平干预，具体包括：PRAK基因敲除小鼠(PRAK敲除)；B16(小鼠黑色素瘤)细胞系采用Cas9敲除PRAK(PRAK敲除)；A375(人黑色素 瘤)/MDA-MB-231(人乳腺癌细胞系)细胞系采用shRNA转染敲低PRAK。
在制备了PRAK抑制剂、PRAK敲低小鼠和各种敲除/敲低的细胞系后，用之干预黑色素瘤和乳腺癌细胞系的体外功能及其荷瘤小鼠和自发乳腺癌小鼠，得到实验结果如下：
(1)体外实验：
B16(小鼠黑色素瘤)，A375(人黑色素瘤)以及MDA-MB-231(人乳腺癌细胞系)的transwell小室实验结果和划痕实验结果证明PRAK分子缺失或表达水平将大幅降低肿瘤细胞在体外的迁移能力，但并不影响肿瘤细胞的增殖(MTS测试)和凋亡(annexin V-7-AAD联合染色)。
(2)体内实验：
A)B16皮下注射模型(s.c.)结果显示PRAK敲除/敲低或抑制剂的使用不影响原位肿瘤细胞的生长，其肿瘤生长曲线和最终肿瘤大小/重量与野生型组或未使用抑制剂组无明显差别。
B)B16尾静脉注射模型(i.v.)结果显示PRAK敲除/敲低或抑制剂的使用将极大地降低该模型的肺转移率，有剂量依赖效应。且该项干预在肿瘤细胞转移早期意义更为重大，在肿瘤细胞注射后头5天使用PRAK抑制物即可完成对肿瘤细胞远端转移的抑制作用，其作用效果几乎等同于全程注射抑制剂的效果。若错失了该时间窗口，再使用抑制剂，则几乎无效。A375人黑色素瘤中应用shPRAK和PRAK抑制物有类似效果。
C)MDA-MB-231活体成像模型：采用活体荧光检测的方法，得到与B16尾静脉注射模型类似的结果。即shPRAK/PRAK抑制物的使用，将强烈抑制肿瘤细胞的肺部定植，14天/21天(最后获取并牺牲小鼠的时间)的监测结果均显示抑制剂组别的肺部荧光强度远低于未使用抑制剂的组别。
D)MMTV-PyMT小鼠自发乳腺癌模型：MMTV-PyMT自发乳腺癌小鼠(几乎100％发病，起病时间为8-12周，市售可得，例如可购自Jackson  Lab(JAX))与PRAK敲除小鼠杂交，得到MMTV-PyMT背景下的野生型和PRAK敲除小鼠。结果显示，若使用PRAK敲除可大幅降低PyMT小鼠的自发肺转移率，PRAK敲除PyMT小鼠中仅有一只(1/19)肺部发现一个转移灶，由此可见，PRAK敲除可显著提高癌细胞的转移抑制率。并且该小鼠原位的肿瘤细胞生物学特性(将原位肿留细胞直接研磨消化后进行测试)接近体外实验的结果，即PRAK的缺失只会影响其远端转移的效应，而对其增殖和凋亡不受影响。使用PRAK抑制剂也有类似的转移抑制效果。
(3)PRAK调控肿瘤转移的潜在机制：
A)RNA-seq大数据分析，PRAK的表达和缺氧以及氧化还原相关途径密切相关；
B)PRAK可以调控HIF1α、MMP2以及EMT系列分子的表达和功能；
C)PRAK可以通过调控mTOR活性来调控HIF1α蛋白的合成。
(4)PRAK表达与肺腺癌/鳞癌患者转移的相关性：对比发生肿瘤转移的病人和未发生肿瘤转移的病人的原位肿瘤标本,发生肿瘤转移的病人表达更高水平的PRAK(p<0.005)。且PRAK的表达与MMP2呈正相关(r(Spearman)＝0.5050238(p＝3.651e-05))。
(5)对PRAK的RNA水平和肺癌患者的生存率的大数据筛查显示，PRAK的表达高低与患者的生存率呈明显负相关。
在符合本领域常识的基础上，上述各优选条件，可以相互组合，得到具体实施方式。
本发明的有益效果在于：
本发明提供了一种可有效抑制肿瘤细胞转移的作用靶标PRAK，并探究了其作用机制，发现通过降低PRAK的酶活性或使PRAK表达水平降低，可降低肿瘤细胞迁移能力，并调控mTOR活性、HIF1α、MMP2和/或EMT系列分子的表达和/或功能，从而实现对肿瘤细胞转移的抑制或预 防。
另一方面，本发明还通过实验研究验证了PRAK抑制剂不同于现有肿瘤化疗药物的特性。化疗药物通常通过影响肿瘤细胞的生长和存活发挥抗肿瘤效应，对正常组织的毒性和耐药性几乎是无可避免的。而PRAK的敲除/敲低或抑制剂的使用不影响原位肿瘤细胞的生长和存活，而只是作用于转移环节，防止远端转移的发生。该干预在肿瘤细胞转移早期意义更为重大，在肿瘤细胞静脉注射头5天使用PRAK抑制物即可完成对肿瘤细胞远端转移的抑制作用，并可强烈抑制肿瘤细胞的肺部定植。
附图说明
图1为PRAK敲除以及PRAK抑制剂对B16-F10增殖的影响。
图2为PRAK敲除以及PRAK抑制剂对B16-F10凋亡的影响。
图3为PRAK敲除以及PRAK抑制剂对B16-F10迁移的影响。
图4为PRAK敲除以及PRAK抑制剂对B16-F10侵袭的影响。
图5为PRAK敲除以及PRAK抑制剂对B16-F10肿瘤原位生长的影响。
图6为PRAK敲除以及PRAK抑制剂对B16-F10肺定植能力的影响。
图7为不同时间和三种不同的PRAK抑制剂处理对B16-F10肿瘤细胞远端转移的影响。
图8为PRAK敲低以及PRAK抑制剂对A375侵袭能力的影响。
图9为PRAK敲低以及PRAK抑制剂对A375肺定植能力的影响。
图10为PRAK敲低以及PRAK抑制剂对MDA-MB-231侵袭能力的影响。
图11为PRAK敲低以及PRAK抑制剂对MDA-MB-231肺定植能力的影响。
图12为PRAK敲除以及PRAK抑制剂对MMTV-PyMT小鼠自发乳腺肿瘤发生的影响。
图13为PRAK敲除及PRAK抑制剂对MMTV-PyMT小鼠自发乳腺 肿瘤肺转移的影响。
图14为PRAK敲除对MMTV-PyMT小鼠肿瘤细胞体外增殖的影响。
图15为PRAK敲除对MMTV-PyMT小鼠肿瘤细胞体外凋亡的影响。
图16为野生型和PRAK敲除的B16-F10细胞的RNA Seq结果差异表达基因的分布。
图17为野生型和PRAK敲除的B16-F10细胞差异表达基因的GO通路富集。
图18为PRAK敲除以及PRAK抑制剂显著降低B16-F10细胞系HIF-1α及MMP2的蛋白表达水平。
图19为PRAK敲除对B16-F10细胞系中EMT相关分子表达的影响。
图20为PRAK敲除以及PRAK抑制剂诱导的mTOR通路相关分子的磷酸化水平的改变。
图21为未发生远端转移与发生远端转移的肺癌患者肿瘤样本PRAK mRNA水平的检测。
图22为肺癌患者肿瘤样本PRAK及MMP2mRNA表达水平的相关性分析。
图23为GEPIA数据库中PRAK高表达与低表达肺癌患者的生存期分析。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明下述实施例中所使用的PRAK抑制剂选自如下化合物：
Figure PCTCN2018110767-appb-000001
实施例1
本实施例以小鼠黑色素瘤为例，用于说明PRAK对B16-F10体外增殖、体外存活、侵袭能力、肿瘤原位生长及早期肺定植的影响。
一、实验方法：
1、对体外增殖与存活的影响
实验步骤：
MTS：将相同数目的PRAK WT及KO细胞铺至96孔板，贴壁后不同时间点加入Cell Titer 96 Aqueous细胞增殖检测溶液，37℃孵育1～4h后，490nm检测吸光度值，绘制细胞增殖曲线。
AnnexinV/7-AAD染色：将细胞铺至24孔板，贴壁后加入PRAK抑 制剂0.1μM和1μM处理，24h后收细胞进行AnnexinV/7-AAD染色，流式分析细胞凋亡情况。
2、对迁移及侵袭能力的影响
实验步骤：
划痕实验：将相同数目的PRAK WT及KO细胞铺至12孔板，贴壁后用枪尖在孔板中央进行划痕，并换用0.1％FBS的培基培养，24h比对划痕愈合情况。类似地，还可以比较不同浓度PRAK抑制剂对划痕愈合的影响。
侵袭实验：将Matrigel包被的Transwell小室37℃预先再水化，2h后，将0.5～2×10 5的细胞重悬于无血清培基，置于上室，下室加入10％FBS的培养基，12～20小时后取出上室，将液体吸干，置于提前预冷的甲醇中，4℃固定15min。将甲醇吸干，用棉签将室内膜表面擦干，将小室置于结晶紫中避光染色20min。PBS洗3次，每次5min。将室内膜表面擦干，封片，镜下计数。
3、对肿瘤原位生长的影响
实验步骤：选取6-8周的C57BL/6雌性小鼠(市售可得，例如可购自维通丽华)进行实验，将处于对数生长期的B16-F10细胞消化，PBS洗两遍，计数后，PBS重悬调整细胞悬液浓度为1.5×10 6/ml。将肿瘤细胞皮下接种至小鼠侧肋部，200μL细胞悬液/只，含3×10 5个细胞。给药组腹腔注射PRAK抑制剂2mg/kg/d。
每两天用游标卡尺测量肿瘤长径(L)及短径(S)，用公式L×S×S×0.5计算肿瘤大小，绘制肿瘤生长曲线。
4、对早期肺定植的影响
实验步骤：选取6-8周的C57BL/6雌性小鼠进行实验，将处于对数生长期的B16-F10细胞消化，PBS洗两遍，计数后，PBS重悬调整细胞悬液浓度为5×10 5/ml。将肿瘤细胞经尾静脉注射打入小鼠体内，每只注入200μL细胞悬液，1×10 5个细胞。15天后，将小鼠处死，取出肺脏，计数 肺表面肿瘤结节数目。给药组分为0-4天或5-15天给药，给药方式为腹腔注射PRAK抑制剂2mg/kg/d。
二、实验结果：
1、如图1所示，PRAK敲除以及PRAK抑制剂对B16-F10的增殖没有明显影响。
2、如图2所示，PRAK敲除以及PRAK抑制剂对B16-F10的凋亡没有明显影响。
3、如图3所示，PRAK敲除以及PRAK抑制剂会明显抑制B16-F10的迁移能力。
4、如图4所示，PRAK敲除以及PRAK抑制剂会明显抑制B16-F10的侵袭能力。
5、如图5所示，PRAK敲除以及PRAK抑制剂对皮下接种的B16-F10肿瘤的原位生长没有明显影响。
6、如图6所示，PRAK敲除以及PRAK抑制剂会明显抑制静脉注射B16-F10细胞的肺定植能力。
7、如图7所示，细胞静脉注射后第0-4天使用PRAK抑制剂可明显抑制B16-F10肿瘤细胞远端转移。第5-15天使用PRAK抑制剂无明显抑制效果。不仅如此，该实验结果还可体现出，三种不同的PRAK抑制剂均有类似的抑制效果。
由此可以说明，PRAK敲除以及PRAK抑制剂对B16-F10的增殖及原位生长没有明显影响，但可以显著抑制B16-F10的侵袭能力和肺定植能力。
实施例2
本实施例以人黑色素瘤为例，用于说明PRAK对A375侵袭能力和肺定植能力的影响。
一、实验方法：
同实施例1，将B16-F10替换为A375，PRAK敲低使用shRNA转染， 体内接种的受体鼠为Scid-Beige(市售可得，例如可购自维通丽华)。
二、实验结果：
1、如图8所示，PRAK敲低以及PRAK抑制剂会明显抑制A375的侵袭能力。
2、如图9所示，PRAK敲低以及PRAK抑制剂会明显抑制A375的肺定植能力。
由此可以说明，PRAK敲低以及PRAK抑制剂可以显著抑制A375的侵袭能力和肺定植能力。
实施例3
本实施例以人乳腺癌细胞系为例，用于说明PRAK对MDA-MB-231侵袭能力和肺定植能力的影响。
一、实验方法：
实验步骤：选取6-8周的SCID Beige雌性小鼠进行实验，将处于对数生长期的野生型或PRAK shRNA转染的MDA-MB-231细胞消化，PBS洗两遍，计数后，PBS重悬调整细胞悬液浓度为2.5×10 6/ml。将肿瘤细胞经尾静脉注射打入小鼠体内，每只注入200μL细胞悬液，5×10 5个细胞。使用IVIS Spectrum小动物活体成像***监测小鼠肺部肿瘤细胞生长情况，21天后，将小鼠处死，取出肺脏，计数肺表面肿瘤结节数目。给药组在最初五天腹腔注射PRAK抑制剂2mg/kg/d。
二、实验结果：
1、如图10所示，PRAK敲低以及PRAK抑制剂会明显抑制MDA-MB-231的侵袭能力。
2、如图11所示，PRAK敲低以及PRAK抑制剂会明显抑制MDA-MB-231的肺定植能力。
由此可以说明，PRAK敲低以及PRAK抑制剂可以显著抑制MDA-MB-231的侵袭能力和肺定植能力。
实施例4
本实例以MMTV-PyMT自发乳腺癌小鼠为例，用于说明PRAK对自发乳腺癌肺转移的影响。
一、实验方法：
1、对小鼠原发乳腺肿瘤发生及生长及的影响
将MMTV-PyMT小鼠与PRAK小鼠杂交，获得MMTV-PRAK WT及MMTV-PRAK敲除的小鼠，8-10周起观察乳腺部位肿瘤发生情况，另有一组MMTV-PRAK WT小鼠自12周起，隔天给予PRAK抑制剂(1mg/kg)。至15周，处死小鼠，计数乳腺肿瘤结节，并称量重量，同时计数肺部肿瘤结节。
2、对小鼠原发肿瘤体外增殖及凋亡的影响
实验步骤：将小鼠乳腺原位肿瘤取出，剪碎，通过DNA酶及胶原酶消化处理、密度梯度离心的方式分离出肿瘤细胞，进行MTS分析以及AnnexinV/7-AAD染色。
二、实验结果：
1、如图12所示，PRAK敲除以及PRAK抑制剂对乳腺自发肿瘤发生率及生长无明显影响。
1、如图13所示，PRAK敲除以及PRAK抑制剂明显抑制了小鼠乳腺自发肿瘤的肺转移。
2、如图14所示，PRAK敲除对自小鼠自发乳腺肿瘤分离获取的肿瘤细胞在体外的增殖能力无明显影响。
3、如图15所示，PRAK敲除对自小鼠自发乳腺肿瘤分离获取的肿瘤细胞体外凋亡水平无明显影响。
实施例5
本实施例用于说明PRAK敲除对B16-F10细胞系基因转录谱的影响。
一、实验方法：
1、分别提取PRAK WT和PRAK敲除的B16-F10细胞RNA，进行 RNA Seq，并对得到的差异表达基因进行GO富集。
二、实验结果：
1、如图16所示，与野生型相比，PRAK基因敲除后出现的差异表达基因大部分为下调，小部分表达上调。
2、如图17所示，PRAK WT与PRAK敲除差异表达基因的功能富集显示，PRAK敲除后下调的基因很多与乏氧反应相关。
实施例6
本实施例用于说明PRAK对HIF1α、MMP2和/或EMT系列分子的表达和功能的影响。
一、实验方法：
Western Blot方法检测PRAK敲除以及PRAK抑制剂处理后各分子蛋白水平的变化。
二、实验结果：
1、如图18所示，PRAK敲除以及PRAK抑制剂会显著降低B16-F10细胞系HIF-1α及MMP2的表达水平。
2、如图19所示，PRAK敲除显著降低B16-F10细胞系N-cadherin表达，同时促进E-cadherin的表达。
实施例7
本实施例用于说明PRAK对mTOR相关分子表达的影响。
一、实验方法：
Western Blot方法检测PRAK敲除以及PRAK抑制剂处理后各分子蛋白磷酸化水平的变化。
二、实验结果：
如图20所示，PRAK敲除以及PRAK抑制剂处理后，mTOR通路相关分子的磷酸化水平下调。
实施例8
本实施例用于说明PRAK与肺癌患者肿瘤转移能力的相关性。
一、实验方法：
1、收集肺腺癌及肺鳞癌患者的肿瘤标本，对PRAK表达进行mRNA水平的检测，并根据术后随访分为未发生远端转移组(N)和发生远端转移组(T)，对结果进行统计学分析。
2、对上述患者样本的PRAK和MMP2进行mRNA水平检测，并对结果进行相关性分析。
3、利用GEPIA网站数据库分析PRAK表达水平对肺癌患者生存期的影响。
二、实验结果：
1、如图21所示，相对于未发生远端转移的患者，发生转移的患者其肿瘤样本中PRAK表达较高。
2、如图22所示，肿瘤患者样本中PRAK和MMP2的表达水平呈正相关。
3、如图23所示，肺癌患者生存期与PRAK基因具有显著相关性，PRAK高表达的患者较PRAK低表达的患者生存期更短。
统一说明：实施例2-9均在采用PRAK抑制剂-23作为PRAK抑制剂进行实验后，替换PRAK抑制剂-22、PRAK抑制剂-29进行重复实验，经实验验证，三种抑制剂的功能相同(均能实现相同的抑制效果)。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明公开了PRAK作为药物靶标在筛选抑制或预防肿瘤细胞转移的药物中的应用。本发明基于对PRAK作为肿瘤细胞转移靶标的发现，进一步提供了可抑制或预防肿瘤细胞转移的药物，所述药物的活性成分可降低PRAK表达水平或抑制PRAK生物活性，通过调控细胞迁移、HIF1α、 MMP2和/或EMT系列分子的表达和/或功能，实现对肿瘤细胞转移的抑制或预防，具有较好的经济价值和应用前景。

Claims (10)

  1. PRAK作为药物靶标在筛选抑制或预防肿瘤细胞转移药物中的应用。
  2. PRAK的抑制剂在制备抑制或预防肿瘤细胞转移药物中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述抑制剂通过降低PRAK表达水平或抑制PRAK生物活性，调控细胞迁移、HIF1α、MMP2和/或EMT系列分子的表达和/或功能，实现对肿瘤细胞转移的抑制或预防。
  4. 根据权利要求3所述的应用，其特征在于，所述抑制剂通过降低PRAK表达水平或抑制PRAK生物活性，调控mTOR及其底物的表达，进一步来调控HIF1α蛋白的表达。
  5. 一种抑制或预防肿瘤细胞转移的药物，其特征在于，所述药物的活性成分可降低PRAK表达水平或抑制PRAK生物活性。
  6. 根据权利要求5所述的药物，其特征在于，所述药物可为化学药物、生物大分子、多肽、单链抗体、反义寡聚核苷酸、小发夹RNA、小干扰RNA、基因编辑体系。
  7. 根据权利要求6所述的药物，其特征在于，所述药物包括[1.2.4]***并[1，5-a]吡嗪化合物或其可药用盐、溶剂化物或前药以及其立体异构体、同位素变体和互变异构体。
  8. 根据权利要求6所述的药物，其特征在于，所述药物包括咪唑并[1.2-a]吡嗪化合物或其可药用盐、溶剂化物或前药以及其立体异构体、同位素变体和互变异构体。
  9. 一种调控HIF1α、MMP2和/或EMT系列分子的表达和/或功能的药物，其特征在于，所述药物的活性成分可降低PRAK表达水平或抑制PRAK生物活性。
  10. 一种调控mTOR活性的药物，其特征在于，所述药物的活性成分可降低PRAK表达水平或抑制PRAK生物活性。
PCT/CN2018/110767 2018-03-27 2018-10-18 一种肿瘤转移药物治疗的靶标及其应用 WO2019184306A1 (zh)

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