Polypeptide combination for inhibiting tumor invasion and metastasis and application thereof

Abstract

This invention discloses a polypeptide combination for inhibiting tumor invasion and metastasis, belonging to the field of therapeutic technology for metastatic breast cancer. The polypeptide combination comprises two polypeptides, each containing a targeting peptide P1 and a targeting peptide P2, respectively. A membrane-penetrating peptide is attached to the N-terminus of both targeting peptides P1 and P2. Histone-lysine N-methyltransferase EZH2 preferentially methylates the aforementioned polypeptide combination, thereby reducing the methylation of lysine residues at positions 53 and 333 of the SMAD3 protein, thus inhibiting tumor cell invasion and metastasis.

Description

[0001] Cross-reference to related applications

[0002] This application is a divisional application of application number CN202111122070.0, filed on September 24, 2021, entitled "A polypeptide combination for inhibiting tumor invasion and metastasis and its application", the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention belongs to the field of therapeutic technology for metastatic breast cancer, specifically a combination of polypeptides that inhibit tumor invasion and metastasis. Background Technology

[0004] Tumor metastasis is closely related to tumor mortality. During tumor growth and development, epithelial cells gradually lose their polarity and intercellular adhesion ability, thereby acquiring a high capacity for invasion and metastasis. During tumor metastasis, cells detach from their primary lesion, invade lymphatic vessels and blood vessels, evade the body's immune surveillance, and implant ectopically. Currently, treatment options for metastatic tumors are limited both domestically and internationally.

[0005] SMAD3 is a key molecule in the TGF-β / SMADs signaling pathway. Multiple studies have shown that the TGF-β / SMADs signaling pathway is abnormally activated during various tumor metastases. During TGF-β / SMADs activation, the C' terminus of SMAD3 can be phosphorylated, and phosphorylated SMAD3 can transcribe and regulate the expression of a series of downstream genes. Activated TGF-β / SMADs signaling induces tumor cells to lose their original epithelial cell polarity, promoting ectopic tumor cell colonization. Recent studies have shown that within the SMADs family, SMAD3 can promote tumor invasion and metastasis, while SMAD2 can inhibit it. Therefore, developing specific drugs targeting SMAD3 may become a new approach for treating metastatic tumors. Posttranscriptional methylation of proteins can regulate the stability, activity, and protein-protein binding of basal proteins. However, methylation modification of SMAD3 has not yet been reported.

[0006] With the advancement of pharmaceutical technology, the use of functional protein peptides in cancer treatment has seen significant progress. However, improving the survival time of patients with metastatic tumors remains a challenge in cancer treatment. Yet, there are no reports of research into therapeutic peptides targeting the TGF-β / SMADs signaling pathway, which is closely related to tumor invasion and metastasis. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention provides a polypeptide combination that can inhibit the TGF-β / SMADs signaling pathway, offering a new therapeutic possibility for the clinical treatment of patients with metastatic tumors. Specifically, this is achieved through the following techniques.

[0008] A polypeptide combination for inhibiting tumor invasion and metastasis, characterized in that it comprises a polypeptide unit, wherein the polypeptide unit is a first polypeptide unit, the first polypeptide unit comprising a membrane-penetrating peptide and a targeting peptide P2, wherein the amino acid sequence of the targeting peptide P2 is shown in SEQ ID NO. 02.

[0009] Furthermore, the polypeptide assembly further includes a second polypeptide unit, which includes a targeting peptide P1; the amino acid sequence of the targeting peptide P1 is shown in SEQ ID NO.01.

[0010] Furthermore, the present invention provides a polypeptide combination for inhibiting tumor invasion and metastasis, comprising two polypeptides, the first polypeptide comprising a membrane-penetrating peptide I and a targeting peptide P1, and the second polypeptide comprising a membrane-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.

[0011] Based on the process of tumor cell invasion and metastasis, the patent applicant accidentally discovered while studying the activation process of SMAD3 protein molecules in the TGF-β / SMADs signaling pathway of tumor cells that methylation of SMAD3 protein molecules can effectively reduce the C-terminal phosphorylation activation of SMAD3 protein molecules, thereby affecting the expression of the marker protein of epithelial-mesenchymal transition in tumor cells, and ultimately affecting the invasive and metastatic ability of tumor cells.

[0012] Based on this, the patent applicant first identified the methylation sites affecting C-terminal phosphorylation activation of SMAD3 through molecular biology experiments, discovering that methylation of lysine residues at positions 53 and 333 of the SMAD3 protein molecule can achieve the aforementioned function. Then, the histone-lysine N-methyltransferase EZH2 at positions 53 and 333 of SMAD3 was obtained through mass spectrometry analysis, and mouse experiments verified that EZH2 can methylate lysine residues at positions 53 and 333 of SMAD3 in vitro. The present invention provides the aforementioned polypeptide combination, in which EZH2 can bind to and methylate lysine residues on the target peptide of the polypeptide combination, thereby avoiding the methylation of lysine residues at positions 53 and 333 of the SMAD3 protein molecule by EZH2, thus reducing the activation of the SMAD3 protein molecule and the invasive and metastatic ability of tumor cells.

[0013] The polypeptide combination provided by this invention comprises two polypeptides, each containing a membrane-penetrating peptide and a targeting peptide. The amino acid sequences of the two targeting peptides correspond to the amino acid sequences near lysine residues at positions 53 and 333 of the SMAD3 protein molecule, respectively. The membrane-penetrating peptide is attached to the N-terminus of the targeting peptide. The membrane-penetrating peptide can carry the targeting peptide molecule into the cell.

[0014] Preferably, the amino acid sequences of both the membrane-penetrating peptide I and the membrane-penetrating peptide II are as shown in SEQ ID NO. 03. In the two peptides of the polypeptide combination, the amino acid sequences of the membrane-penetrating peptides may be the same or different, as long as the function of the membrane-penetrating peptide is achieved.

[0015] More preferably, the amino acid sequences of the two polypeptides are shown in SEQ ID NO.04 and SEQ ID NO.05, respectively.

[0016] More preferably, histone-lysine N-methyltransferase EZH2 methylates lysine residues on the target peptides P1 and P2, thereby inhibiting the TGF-β / SMADs signaling pathway.

[0017] The above-mentioned combination of peptides that inhibit tumor invasion and metastasis is used in the preparation of drugs that inhibit tumor invasion and metastasis.

[0018] Preferably, the above-mentioned polypeptide combination is used to prepare a drug that inhibits tumor invasion and metastasis, wherein the tumor is a tumor cell of cancers such as lung cancer, colon cancer, cervical cancer, and breast cancer. Any tumor or cancer type that is activated by phosphorylation of lysine residues at positions 53 and 333 of the SMAD3 protein's amino acid sequence, thereby enabling tumor cells to acquire invasive and metastatic capabilities, can be treated using the above-mentioned polypeptide combination or by preparing the polypeptide combination into a drug to reduce tumor invasion and metastasis.

[0019] More preferably, the aforementioned drug is an intravenous injection containing a combination of peptides that inhibit tumor invasion and metastasis. This combination of peptides is primarily formulated as an intravenous injection to exert its effect.

[0020] Compared with the prior art, the advantages of this invention are: This invention provides an amino acid site located on the SMAD3 protein, which, through methylation, can affect its phosphorylation activation and thus influence the expression of a marker protein of epithelial-mesenchymal transition in tumor cells—specifically, the methylation of lysine residues at positions 53 and 333 of the SMAD3 protein. Based on this new discovery, a polypeptide combination for inhibiting tumor cell invasion and metastasis is provided. Attached Figure Description

[0021] Figure 1 This is the result of verifying the biological function of the methylation of lysine at positions 53 and 333 of SMAD3 in Example 1;

[0022] Figure 2 This is a validation result for the SMAD3 knockout cell line;

[0023] Figure 3 This verifies the successful mutation of lysine residues at positions 53 and 333 of SMAD3;

[0024] Figure 4 Photographs of mouse lung tissue stained with hematoxylin and eosin (HE) after injection of wild-type SMAD3 breast cancer tumor cells and SMAD3 lysine-mutated cells at positions 53 and 333, respectively, in Example 2.

[0025] Figure 5 for Figure 2 The number and weight of lung metastatic nodules in mouse lung tissue after HE staining;

[0026] Figure 6 This is the result of EZH2 methylation of lysine at position 53 of SMAD3 in Example 3;

[0027] Figure 7 This is the result of EZH2 methylation of lysine at position 333 of SMAD3 in Example 3;

[0028] Figure 8 This is a schematic diagram of the amino acid sequence structure of the two polypeptides in Example 4;

[0029] Figure 9 The image shows mouse lung tissue stained with hematoxylin and eosin (HE) after injection of breast cancer tumor cells, as shown in Example 5.

[0030] Figure 10 for Figure 7 The number of lung metastatic nodules and the weight of lung tissue in mice after HE staining. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1: Identification and confirmation of methylation sites affecting C-terminal phosphorylation activation of SMAD3 protein

[0033] Molecular biology experiments were used to identify methylation sites that can influence C-terminal phosphorylation activation of the SMAD3 protein. Western blot experiments were employed, and the specific experimental procedures can be found in the 2019 foreign literature "SETDB1-mediated methylation of Akt promotes its K63-linked ubiquitination and activation leading to tumorigenesis" (Guihua Wang, et al. Nature). Cell biology The technical content of the “Immunoprecipitation (IP), immunofluorescence (IF) and immunoblotting (IB)” section on page 13 of 217-225 (2019)

[0034] like Figure 1 As shown, the loss of methylation of lysines at positions 53 and 333 of SMAD3 can inhibit TGF-β-induced activation of SMAD3 and epithelial-mesenchymal transition in tumor cells. This indicates that methylation of lysines at positions 53 and 333 of SMAD3 protein can affect its phosphorylation activation and the expression of its marker protein for epithelial-mesenchymal transition in tumor cells.

[0035] Example 2: The effect of methylation of lysine residues at positions 53 and 333 of the SMAD3 protein on the invasion and metastasis of tumor cells.

[0036] Animal experiments were conducted to verify that methylation of lysines at positions 53 and 333 of the SMAD3 protein can affect the invasive and metastatic ability of tumor cells. SMAD3 knockout cell lines were constructed using MDA-MB-231 cells (purchased from American Type Culture Collection) via conventional crisp / cas9 technology (SMAD3 knockout virus, purchased from Genechem). High-expressing SMAD3 WT and SMAD3 K53 / 333R viruses were then synthesized in vitro using the pLKO-AS3W-HA-SMAD3 WT / pLKO-AS3W-HA-SMAD3 K53 / 333R and MD2-Gand PPAX triple packaging system. Subsequently, these viruses were used to infect wild-type SMAD3 breast cancer cells (SMAD3 WT) and breast cancer cells with point mutations at lysines at positions 53 and 333 of SMAD3 (SMAD3 K53 / 333R).

[0037] For the specific methods of constructing the two breast cancer tumor cells mentioned above, please refer to the foreign language paper "Akt methylation by SETDB1 promotes Akt kinase activity and oncogenic functions" published in 2019 (Jianping Guo, et al. Nature). Cell biology, 226-237 (2019) The technical content of the "shRNAs, sgRNAs and CRISPR / CAS9-mediated knock-in and knockout assay" section on page 11.

[0038] The successful construction of the SMAD3 knockout cell line was verified by Western blot experiments. Figure 2 As shown, the finally constructed SMAD3 wild-type breast cancer cells (SMAD3 WT) were successfully infected with the SMAD3 WT high-expression virus; the constructed SMAD3 breast cancer cells with point mutations at positions 53 and 333 refer to the 53rd and 333rd lysine residues being mutated and replaced with arginine. The verification results of the successful mutation are shown in the figure. Figure 3 As shown.

[0039] 4×10 5 Two SMAD3WT or SMAD3 K53 / 333R breast cancer tumor cells were injected into 4-week-old female mice (balb / c nude) via the tail vein. After two months of normal feeding, the mice were euthanized by cervical dislocation, and lung tissue was extracted for HE staining. The number and weight of lung metastatic nodules were counted. The results are shown in the appendix. Figure 4 , 5 .

[0040] from Figure 4 , Figure 5 As can be seen, the number of metastatic nodules and the weight of lung tissue in mice induced by SMAD3WT breast cancer cells were significantly greater than those in mice induced by SMAD3 K53 / 333R breast cancer cells.

[0041] Example 3: Obtaining histone-lysine N-methyltransferase EZH2

[0042] We obtained the histone-lysine N-methyltransferase EZH2, containing lysine residues at positions 53 and 333 of the SMAD3 protein, using mass spectrometry analysis (machine model: Q Exactive Plus LC-MS / MS mass spectrometer (Thermo), 2kV, RF=40, 320℃). We then verified that EZH2 can methylate the aforementioned protein polypeptide chain in vitro using in vitro methylation assays (specifically, the experimental methods described on page 13 of the foreign literature cited in Example 1, under the section "In vivo methylation assays, in vitro binding assays and ubiquitination assays"). The results are attached. Figure 6 , 7 As shown.

[0043] from Figure 6 As can be seen, EZH2 can methylate the lysine at position K53 of SMAD3 in vitro. From Figure 7 As can be seen, EZH2 can methylate lysine at position K333 of SMAD3 in vitro.

[0044] Example 4: Synthesis of polypeptide combinations

[0045] Based on the specific sequence of the five amino acids to the left and right of lysine residues at positions 53 and 333 of the SMAD3 protein, two targeting peptides containing 11 amino acids were designed, as shown in SEQ ID NO.01 and SEQ ID NO.02. Simultaneously, a membrane-penetrating peptide structure was inserted into the N-segment of both targeting peptides, and the amino acid sequence of the membrane-penetrating peptide is shown in SEQ ID NO.03. The final amino acid sequences of the two polypeptides are shown in SEQ ID NO.04 and SEQ ID NO.05, respectively. A schematic diagram is shown below. Figure 8 As shown.

[0046] Example 5: Verification of the effect of the polypeptide combination obtained in Example 4 on inhibiting tumor invasion and metastasis.

[0047] The complete peptides shown in SEQ ID NO. 04 and SEQ ID NO. 05 were synthesized separately by Wuhan Huamei Biotechnology Co., Ltd. as the experimental group; and the membrane-penetrating peptide shown in SEQ ID NO. 03 was separately synthesized as the control. The two peptides were prepared into an intravenous injection solution of 1 μg / ml using PBS buffer, and the membrane-penetrating peptide was prepared into an intravenous injection solution of 1 μg / ml using PBS buffer separately.

[0048] Animal experiments verified that the synthesized unmethylated protein peptides can inhibit tumor invasion and metastasis: 4×10 5Breast cancer tumor cells (purchased from American Type Culture Collection) were injected intravenously into 4-week-old female mice (balb / c nude) via the tail vein. Starting on the third day after injection, 100 μl of intravenous injection of the transmembrane peptide (control group) and 100 μl of intravenous injection of the complete peptide (experimental group) were injected intravenously via the tail vein every three days. Two months later, the mice were euthanized by cervical dislocation, and lung tissue was extracted for HE staining. The number and weight of lung metastatic nodules in each group were counted. The mice were fed normally throughout the process. Results are attached. Figure 9 , 10 As shown.

[0049] from Figure 9 , Figure 10 As can be seen, compared with the use of the membrane-penetrating peptide shown in SEQ ID NO.03 alone, the use of the peptides shown in SEQ ID NO.04 and SEQ ID NO.05 can significantly inhibit lung metastasis of breast cancer, and the combined use of SEQ ID NO.04 and SEQ ID NO.05 has a synergistic effect on inhibiting lung metastasis of breast cancer.

[0050] Therefore, through the experiments in Examples 1-5 above, it can be found that the polypeptide combination obtained according to the amino acid sequences 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.

Claims

1. A polypeptide that inhibits tumor invasion and metastasis, characterized in that, The polypeptide is composed of a membrane-penetrating peptide II and a targeting peptide P2; the amino acid sequence of the targeting peptide P2 is shown in SEQ ID NO. 02; the amino acid sequence of the membrane-penetrating peptide II is shown in SEQ ID NO.

03.

2. The polypeptide for inhibiting tumor invasion and metastasis according to claim 1, characterized in that, The sequence of the polypeptide is shown in SEQ ID NO.

05.

3. The use of the polypeptide for inhibiting tumor invasion and metastasis as described in claim 1 or 2 in the preparation of a drug for inhibiting tumor invasion and metastasis, characterized in that, The tumors are tumor cells from lung cancer, colon cancer, cervical cancer, and breast cancer.

4. The application according to claim 3, characterized in that, The drug is an intravenous injection containing polypeptides that inhibit tumor invasion and metastasis.

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