A polypeptide combination for inhibiting tumor invasion and metastasis and its application

Abstract

The present invention discloses a polypeptide combination for inhibiting tumor invasion and metastasis, and belongs to the field of treatment technology of metastatic breast cancer. The polypeptide combination includes two polypeptides, and the two polypeptides contain targeting peptide P1 and targeting peptide P2 respectively, and the N-termini of targeting peptide P1 and targeting peptide P2 are respectively connected with transmembrane peptides. Histone-lysine N-methyltransferase EZH2 can preferentially methylate the above-mentioned polypeptide combination, thereby reducing the methylation of lysine at positions 53 and 333 of SMAD3 protein, thereby inhibiting tumor cell invasion and metastasis.

Description

Technical Field

[0001] The present invention belongs to the technical field of treatment of metastatic breast cancer, in particular to a polypeptide combination for inhibiting tumor invasion and metastasis. Background Art

[0002] Tumor metastasis is closely related to the mortality rate of tumors. During the growth and development of tumors, epithelial cells gradually lose their polarity and intercellular adhesion ability, and then acquire high invasion and metastasis capabilities. During tumor metastasis, cells break away from their primary lesions, invade lymphatic vessels and blood vessels, evade the body's immune surveillance, and implant ectopically. At present, there are limited treatments for metastatic tumors at home and abroad.

[0003] The protein SMAD3 is a key molecule in the TGF-β / SMADs signaling pathway. Many studies have shown that the TGF-β / SMADs signaling pathway is abnormally activated during the metastasis of various tumors. During the activation of the TGF-β / SMADs signaling pathway, the C' terminus of SMAD3 can be phosphorylated, and the phosphorylated SMAD3 can transcriptionally regulate the expression of a series of downstream genes. The activated TGF-β / SMADs signaling pathway induces tumor cells to lose their original epithelial cell polarity and promotes the ectopic colonization of tumor cells. Recent studies have shown that in the SMADs family, SMAD3 can promote tumor invasion and metastasis, while SMAD2 can inhibit tumor invasion and metastasis. Therefore, the development of specific drugs targeting SMAD3 may become a new method for treating metastatic tumors. Post-transcriptional methylation modification of proteins can regulate the stability, activity and protein-protein binding of background proteins. However, the methylation modification of SMAD3 has not been reported.

[0004] With the development of current pharmaceutical technology, the use of functional protein peptides to treat tumors has made great progress. How to improve the survival time of patients with metastatic tumors has been a difficult problem in the process of tumor treatment. However, there are no reports on the development of therapeutic peptides targeting the TGF-β / SMADs signaling pathway, which is closely related to tumor invasion and metastasis. Summary of the invention

[0005] In view of the above shortcomings of the prior art, the present invention provides a polypeptide combination that can inhibit the TGF-β / SMADs signaling pathway, providing a new therapeutic drug possibility for the clinical treatment of patients with tumor metastasis. Specifically, it is achieved through the following technologies.

[0006] A polypeptide combination for inhibiting tumor invasion and metastasis comprises two polypeptides, the first polypeptide comprises a cell-penetrating peptide I and a targeting peptide P1, and the second polypeptide comprises a cell-penetrating peptide II and a targeting peptide P2; the amino acid sequence of the targeting peptide P1 is shown in SEQ ID NO.01, and the amino acid sequence of the targeting peptide P2 is shown in SEQ ID NO.02.

[0007] Based on the process of tumor cell invasion and metastasis, the applicant of this patent accidentally discovered that methylation of SMAD3 protein molecules can effectively reduce the C-terminal phosphorylation activation of SMAD3 protein molecules when studying the activation process of SMAD3 protein molecules in the TGF-β / SMADs signaling pathway of tumor cells, thereby affecting the expression of marker proteins of epithelial-mesenchymal transition of tumor cells, and ultimately affecting the invasion and metastasis ability of tumor cells.

[0008] On this basis, the patent applicant first found the methylation site that affects the C-terminal phosphorylation activation of SMAD3 through molecular biology experiments, and found that the methylation of the 53rd and 333rd lysines of the SMAD3 protein molecule can achieve the above functions. Then, the 53rd and 333rd histone-lysine N-methyltransferase EZH2 of SMAD3 was obtained by mass spectrometry, and it was verified by mouse experiments that EZH2 can methylate the 53rd and 333rd lysines of SMAD3 in vitro. The above-mentioned polypeptide combination provided by the present invention, EZH2 can bind to the lysine on the targeting peptide of the polypeptide combination and complete methylation, thereby avoiding the methylation of the 53rd and 333rd lysines of the SMAD3 protein molecule by EZH2, thereby reducing the activation of the SMAD3 protein molecule and the invasion and metastasis ability of tumor cells.

[0009] The polypeptide combination provided by the present invention comprises two polypeptides, both of which contain a cell-penetrating peptide and a targeting peptide, the amino acid sequences of the two targeting peptides correspond to the amino acid sequences near the 53rd lysine and the 333rd lysine of the SMAD3 protein molecule, respectively, and the cell-penetrating peptide is connected to the N-terminus of the targeting peptide. The cell-penetrating peptide can carry the targeting peptide molecule into the cell.

[0010] Preferably, the amino acid sequences of the cell-penetrating peptide I and the cell-penetrating peptide II are both as shown in SEQ ID NO. 03. In the two polypeptides of the polypeptide combination, the amino acid sequences of the cell-penetrating peptides may be the same or different, as long as the function of the cell-penetrating peptide can be achieved.

[0011] More preferably, the amino acid sequences of the two polypeptides are shown as SEQ ID NO.04 and SEQ ID NO.05, respectively.

[0012] More preferably, histone-lysine N-methyltransferase EZH2 methylates the lysine on the targeting peptide P1 and the targeting peptide P2 to inhibit the TGF-β / SMADs signaling pathway.

[0013] The above-mentioned polypeptide combination for inhibiting tumor invasion and metastasis is used in the preparation of a drug for inhibiting tumor invasion and metastasis.

[0014] Preferably, the drug for inhibiting tumor invasion and metastasis prepared by the above polypeptide combination is a tumor cell of cancers such as lung cancer, colon cancer, cervical cancer, breast cancer, etc. As long as the lysine at position 53 and lysine at position 333 of the amino acid sequence of SMAD3 protein are methylated to achieve phosphorylation activation, and cause tumor cells to acquire invasion and metastasis capabilities, the above polypeptide combination can be used or the polypeptide combination can be prepared into a drug for treatment to reduce tumor invasion and metastasis.

[0015] More preferably, the above-mentioned drug is an intravenous injection containing a polypeptide combination that inhibits tumor invasion and metastasis. This polypeptide combination is mainly prepared as an intravenous injection to exert its effect after injection.

[0016] Compared with the prior art, the present invention is beneficial in that: the present invention provides an amino acid site located on the SMAD3 protein, which can affect its phosphorylation activation and affect the expression of the marker protein of epithelial-mesenchymal transition in tumor cells through methylation, namely, the methylation of lysine at positions 53 and 333 of the SMAD3 protein. In view of this new discovery, a polypeptide combination for inhibiting the invasion and metastasis of tumor cells is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of biological function verification of methylation of lysine 53 and 333 of SMAD3 in Example 1;

[0018] Figure 2 Validation results for SMAD3 knock out cell lines;

[0019] Figure 3 This is the verification result of the successful mutation of lysine 53 and 333 of SMAD3;

[0020] Figure 4 These are photos of mouse lung tissues after HE staining after injection of SMAD3 wild-type breast cancer tumor cells and SMAD3 lysine 53 and 333 mutation breast cancer tumor cells in Example 2;

[0021] Figure 5 for Figure 2 The number and weight of lung metastatic nodules in mouse lung tissue after HE staining;

[0022] Figure 6 The result of EZH2 methylating lysine 53 of SMAD3 in Example 3;

[0023] Figure 7 The result of EZH2 methylating lysine 333 of SMAD3 in Example 3;

[0024] Figure 8 The diagram is a schematic diagram of the amino acid sequence structure of two polypeptides in Example 4;

[0025] Fig. 9 These are photos of mouse lung tissues after HE staining after injection of breast cancer tumor cells in Example 5;

[0026] Fig.10 for Figure 7 The number of lung metastatic nodules and the weight of lung tissue of mice after HE staining. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Example 1: Searching and confirming the methylation sites that affect the C-terminal phosphorylation activation of SMAD3 protein

[0029] Through molecular biology experiments, we found the methylation sites that can affect the C-terminal phosphorylation activation of SMAD3 protein. We used western blot experiments. The specific experimental steps can be found in the foreign literature published in 2019, "SETDB1-mediated methylation of Akt promotes its K63-linked ubiquitination and activation leading to tumorigenesis" (Guihua Wang, et al. Nature Cell biology , 217-225 (2019)) on page 13 of the “Immunoprecipitation (IP), immunofluorescence (IF) and immunoblotting (IB)” section.

[0030] like Figure 1As shown, the loss of methylation of lysine at positions 53 and 333 of SMAD3 can inhibit TGF-β-induced SMAD3 activation and epithelial-mesenchymal transition of tumor cells. It was found that methylation of lysine at positions 53 and 333 of SMAD3 protein can affect its phosphorylation activation and the expression of marker proteins of epithelial-mesenchymal transition in tumor cells.

[0031] Example 2: Effect of methylation of lysine 53 and 333 of SMAD3 protein on invasion and metastasis of tumor cells

[0032] Animal experiments verified that methylation of lysine at positions 53 and 333 of SMAD3 protein can affect the invasion and metastasis ability of tumor cells: MDA-MB-231 cells (purchased from American Type Culture Collection) were used to construct SMAD3 knock out cell lines using conventional crisp / cas9 technology (SMAD3 knock out virus, purchased from Genechem), and then SMAD3 WT and SMAD3K53 / 333R high expression viruses were synthesized in vitro using pLKO-AS3W-HA-SMAD3 WT / pLKO-AS3W-HA-SMAD3 K53 / 333R, MD2-Gand PPAX three-packaging system. Subsequently, SMAD3 wild-type breast cancer tumor cells (SMAD3 WT) and breast cancer tumor cells with SMAD3 lysine point mutations at positions 53 and 333 (SMAD3 K53 / 333R) were constructed by in vitro viral infection.

[0033] The specific construction methods of the above two breast cancer tumor cells can be found in the foreign literature published in 2019, "Aktmethylation by SETDB1 promotes Akt kinase activity and oncogenic functions" (Jianping Guo, et al. Nature Cell biology, 226-237(2019) ) Technical content of the section “shRNAs, sgRNAsand CRISPR / CAS9-mediated knock-in and knockout assay” on page 11.

[0034] The construction of SMAD3 knock out cell line was verified by western blot experiment. Figure 2As shown in the figure, the finally constructed SMAD3 wild-type breast cancer tumor cells (SMAD3 WT) were successfully infected with the SMAD3 WT high-expression virus; the constructed SMAD3 breast cancer tumor cells with lysine point mutations at positions 53 and 333 refer to lysine at positions 53 and 333 being mutated and replaced with arginine, and the verification results of successful mutation are shown in the figure. Figure 3 shown.

[0035] 4×10 5 SMAD3WT or SMAD3 K53 / 333R breast cancer tumor cells were injected into 4-week-old female mice (balb / c nude) through the tail vein. After two months of normal feeding, the mice were killed by cervical dislocation. The lung tissues were removed for HE staining, and the number and weight of lung metastatic nodules were counted. The results are shown in the attached Figure 4 , 5 .

[0036] from Figure 4 , Figure 5 It can be seen that the metastatic nodules and lung tissue weight in the lung tissue of mice induced by SMAD3WT breast cancer tumor cells are significantly greater than those induced by SMAD3 K53 / 333R breast cancer tumor cells.

[0037] Example 3: Acquisition of histone-lysine N-methyltransferase EZH2

[0038] By mass spectrometry (machine model: Q Exactive Plus LC-MS / MS mass spectrometer (Thermo), 2kV, RF = 40, 320 ° C), we obtained the histone-lysine N-methyltransferase EZH2 of the lysine at positions 53 and 333 of the SMAD3 protein, and verified that EZH2 can methylate the above-mentioned protein polypeptide chain in vitro through in vitro methylation experiments (the specific experimental method adopts the technical content of the "In vivo methylation assays, in vitro binding assays and ubiquitination assays" section on page 13 of the foreign literature cited in Example 1). The results are shown in the attached Figure 6 , 7 shown.

[0039] from Figure 6 As can be seen in Figure 2, EZH2 can methylate SMAD3 K53 lysine in vitro. Figure 7 As can be seen in Figure 3, EZH2 can methylate SMAD3 K333 lysine in vitro.

[0040] Example 4: Synthesis of polypeptide combinations

[0041] According to the specific sequence of 5 amino acids around lysine at positions 53 and 333 of SMAD3 protein, two amino acid sequences of targeting peptides containing 11 amino acids were designed, as shown in SEQ ID NO.01 and SEQ ID NO.02. At the same time, a transmembrane peptide structure was inserted into the N segment of the two targeting peptides, and the amino acid sequence of the transmembrane peptide was shown in SEQ ID NO.03. The amino acid sequences of the two polypeptides finally obtained were shown in SEQ ID NO.04 and SEQ ID NO.05, respectively, and their schematic diagrams are shown in FIG. Figure 8 shown.

[0042] Example 5: Verification of the effect of the polypeptide combination obtained in Example 4 on inhibiting tumor invasion and metastasis

[0043] The complete polypeptides shown in SEQ ID NO.04 and SEQ ID NO.05 were synthesized by Wuhan Huamei Bioengineering Co., Ltd. as experimental groups, and the cell-penetrating peptide shown in SEQ ID NO.03 was synthesized separately as a control experiment. The two polypeptides were prepared into 1ug / ml intravenous injection solution using PBS buffer, and the cell-penetrating peptide was prepared into 1ug / ml intravenous injection solution using PBS buffer alone.

[0044] Animal experiments have verified that the synthesized non-methylated protein peptides can inhibit tumor invasion and metastasis: 4×10 5 Breast cancer tumor cells (purchased from American Type Culture Collection) were injected into 4-week-old female mice (balb / c nude) through the tail vein; starting from the third day after the injection, 100 μl of intravenous injection of the cell-penetrating peptide in the control group and 100 μl of intravenous injection of the complete polypeptide in the experimental group were injected through the tail vein of the mice every three days; two months later, the mice were killed by cervical dislocation, and the lung tissues were removed for HE staining, and the number and weight of lung metastatic nodules in each group of mice were counted. The mice were fed and raised in the normal way throughout the process. The results are attached Fig. 9 , 10 shown.

[0045] from Fig. 9 , Fig.10 It can be seen that compared with the use of the cell-penetrating peptide shown in SEQ ID NO.03 alone, the use of the polypeptides shown in SEQ ID NO.04 and SEQ ID NO.05 can significantly inhibit breast cancer lung metastasis, and the combined use of SEQ ID NO.04 and SEQ ID NO.05 has a synergistic effect on inhibiting breast cancer lung metastasis.

[0046] Therefore, through the experiments of Examples 1-5 above, it can be found that the polypeptide combination obtained according to the amino acid sequence shown in SEQ ID NO.04 and SEQ ID NO.05 can significantly inhibit the lung invasion and metastasis of breast cancer cells after being injected into the tail vein of mice. Sequence Listing <110> Wuhan Bioinno Biotechnology Co., Ltd. <120> A polypeptide combination for inhibiting tumor invasion and metastasis and its application <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <400> 1 Leu Asp Glu Leu Glu Lys Ala Ile Thr Thr Gln 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <400> 2 Pro Ala Thr Val Cys Lys Ile Pro Pro Gly Cys 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <400> 3 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala 1 5 10 <210> 4 <211> twenty three <212> PRT <213> Artificial Sequence <400> 4 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Leu Asp Glu Leu 1 5 10 15 Glu Lys Ala Ile Thr Thr Gln 20 <210> 5 <211> 23 <212> PRT <213> Artificial Sequence <400> 5 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Ala Thr Val 1 5 10 15 Cys Lys Ile Pro Pro Gly Cys 20

Claims

1. A polypeptide combination for inhibiting tumor invasion and metastasis, characterized in that: It consists of two polypeptides, the first polypeptide consists of a cell-penetrating peptide I and a targeting peptide P1, and the second polypeptide consists of a cell-penetrating peptide II and a targeting peptide P2; the amino acid sequence of the targeting peptide P1 is shown in SEQ ID NO.01, and the amino acid sequence of the targeting peptide P2 is shown in SEQ ID NO.02; the amino acid sequences of the cell-penetrating peptide I and the cell-penetrating peptide II are both shown in SEQ ID NO.

03.

2. The polypeptide combination for inhibiting tumor invasion and metastasis according to claim 1, characterized in that: The amino acid sequences of the two polypeptides are shown in SEQ ID NO.04 and SEQ ID NO.05 respectively.

3. Use of the polypeptide combination for inhibiting tumor invasion and metastasis according to claim 1 in the preparation of a drug for inhibiting tumor invasion and metastasis, characterized in that: The tumor is a tumor cell of lung cancer, colon cancer or breast cancer.

4. Use of the polypeptide combination for inhibiting tumor invasion and metastasis according to claim 3 in the preparation of a drug for inhibiting tumor invasion and metastasis, characterized in that: The medicine is an intravenous injection containing a polypeptide combination that inhibits tumor invasion and metastasis.

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