A fam189b polypeptide and its use in the preparation of an anti-tumor drug

Abstract

The application discloses a FAM189B polypeptide and application thereof in preparation of an anti-tumor drug, and belongs to the technical field of biological medicines, wherein the sequence of the FAM189B polypeptide is shown as SEQ ID NO. 1 or SEQ ID NO. 2. Based on the discovery that FAM189B protein can interact with YAP protein and then stabilize YAP protein in the cytoplasm, the application develops FAM189B polypeptide which can effectively block the connection between FAM189B protein and YAP protein in cells by analyzing the direct contact area of the two. The FAM189B polypeptide can inhibit the proliferation of cancer cells and the like by regulating the activity of YAP and a downstream path of YAP, thereby inhibiting the growth of tumors in the body. The FAM189B polypeptide provided by the application has a good application prospect in the development of anti-tumor drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a FAM189B polypeptide and its application in the preparation of antitumor drugs. Background Technology

[0002] Tumorigenesis and development is a complex, multi-step process involving the abnormal activation or inhibition of multiple signaling pathways. Among these, the Hippo signaling pathway is a recently discovered pathway playing a crucial role in organ size control and tumor suppression. This pathway controls cell proliferation and apoptosis by regulating the activity of the transcriptional coactivators YAP (Yes1 Associated Transcriptional Regulator) and TAZ (Transcriptional Coactivator with PDZ-binding Motif). In the Hippo pathway, MST1 / 2 kinases activate and phosphorylate LATS1 / 2, thereby phosphorylating YAP / TAZ. This causes them to bind to the 14-3-3 protein and become isolated in the cytoplasm, thus inhibiting their transcriptional activation function. When the Hippo pathway is inhibited, YAP / TAZ translocates to the nucleus, promoting the expression of cell proliferation-related genes, thereby driving tumor development.

[0003] YAP proteins not only function in the Hippo pathway, but also perform intracellular functions through multiple mechanisms. For example, YAP can bind to TEAD (TEA Domain Transcription Factor) family transcription factors independently of the Hippo pathway, regulating gene expression. Furthermore, YAP is involved in processes such as cytoskeleton remodeling, cell migration, and immune escape, making it a key oncogenic factor in various tumor types.

[0004] FAM189B (Family with Sequence Similarity 189 Member B) has been found to be abnormally elevated in various tumors, including liver cancer, lung cancer, and breast cancer, but the function of FAM189B is not yet fully understood. Summary of the Invention

[0005] Through multi-sample clinical results and biological methods, the inventors discovered that the FAM189B protein can directly interact with the YAP protein, thereby stabilizing the YAP protein in the cytoplasm. Based on this new discovery, the inventors identified the protein regions where the FAM189B and YAP proteins interact and obtained a polypeptide that can block the intracellular interaction between FAM189B and YAP. Experimental results showed that this polypeptide molecule can significantly inhibit the growth of liver cancer cells.

[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a FAM189B polypeptide, the sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0007] The experiments of this invention demonstrate that the FAM189B protein interacts with the YAP1 protein through PPxY motifs. The FAM189B protein contains three PPxY motifs in its amino acid sequence; specifically, FAM189B peptides can be designed targeting the first and third PPxY motifs. The resulting FAM189B peptides can block the interaction between the FAM189B protein and the YAP1 protein, thereby affecting the degradation of the YAP1 protein.

[0008] Secondly, this invention provides a FAM189B polypeptide derivative comprising the FAM189B polypeptide and a cell-penetrating peptide sequence. Cell-penetrating peptides are short peptides composed of 5-30 amino acids, capable of carrying proteins, nucleic acids, liposomes, and other substances into cells via non-classical endocytosis mechanisms. Therefore, there are no specific limitations on the cell-penetrating peptide used for the FAM189B polypeptide, as long as it can carry a peptide segment with a sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2 into the cell. For example, in some embodiments of this invention, the cell-penetrating peptide is a TAT peptide, with an amino acid sequence as shown in SEQ ID NO. 3. It should be understood that, in addition to delivery using cell-penetrating peptides, those skilled in the art can also deliver the FAM189B polypeptide into cells using other methods (such as using transfection reagents).

[0009] Preferably, in the above-mentioned FAM189B polypeptide derivative, the cell-penetrating peptide sequence is attached to the N-terminus of the sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0010] Preferably, in the above-mentioned FAM189B polypeptide derivative, the N-terminus is acetylated and the C-terminus is amidated.

[0011] Thirdly, the present invention provides the application of the above-mentioned FAM189B polypeptide in the preparation of antitumor drugs.

[0012] FAM189B peptide inhibits tumor cell proliferation, migration, and invasion by blocking the interaction between FAM189B protein and YAP protein, thereby suppressing the nuclear accumulation and activation of YAP protein. For example, in the treatment of liver cancer, FAM189B peptide has a significant inhibitory effect on the viability and proliferation of liver cancer cells, and can inhibit the development of liver cancer.

[0013] Fourthly, the present invention provides an antitumor drug with FAM189B polypeptide as its active ingredient.

[0014] Preferably, the above-mentioned antitumor drugs are used to treat liver cancer.

[0015] Among the aforementioned antitumor drugs, FAM189B peptide can be the sole active ingredient or used in combination with other active ingredients to fight tumors. For example, in the treatment of liver cancer, FAM189B peptide alone can significantly inhibit the development of liver cancer, but when FAM189B peptide is used in combination with sorafenib or lenvatinib, it can significantly improve the anticancer effect.

[0016] Preferably, the aforementioned antitumor drugs also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to pharmaceutical excipients widely used in the pharmaceutical manufacturing field, specifically including one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0017] For the aforementioned antitumor drugs, there are no specific restrictions on the dosage form, which can be a solid dosage form, a semi-solid dosage form, or a liquid dosage form, including but not limited to tablets, capsules, powders, pills, granules, suppositories, injections, etc.; there are also no specific restrictions on the method of drug administration, as long as an effective dose of the drug is administered to an individual, such as in some embodiments of the present invention, where mice are administered the drug by injection.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention develops a FAM189B peptide based on the direct contact region between FAM189B and YAP proteins. This peptide effectively blocks the binding of FAM189B and YAP proteins, thereby influencing tumor progression by affecting the degradation of YAP protein. Taking liver cancer as an example, data from this invention show that the FAM189B peptide can inhibit the activity and proliferation of liver cancer cells, thus exerting a significant anti-tumor function. Furthermore, the FAM189B peptide can be used in combination with other anti-tumor drugs to further enhance the anti-tumor effect. This invention provides a new candidate drug for tumor treatment and offers new insights into the development of anti-tumor drugs. Attached Figure Description

[0019] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0020] Figure 1This is a diagram showing the results of using YAP protein to fish for FAM189B protein in Example 1; Figure 2 This is a diagram showing the results of using FAM189B protein to fish for YAP protein in Example 1. Figure 3 This is a diagram showing the results of the direct interaction between the YAP protein and the FAM189B protein in Example 1; Figure 4 This is a graph showing the degradation results of YAP1 in the FAM189B overexpressing cell line at a specified time point in Example 2; Figure 5 This is a graph showing the degradation results of YAP1 in the FAM189B knockdown cell line at a specified time point in Example 2; Figure 6 This is a schematic diagram illustrating the truncated expression of the YAP1 protein in Example 3; Figure 7 This is a schematic diagram illustrating the truncated expression of the FAM189B protein in Example 3; Figure 8 This is a screenshot showing the screening results of the region where the YAP protein binds to the full-length FAM189B protein in Example 3. Figure 9 This is a graph showing the screening results of the region where the FAM189B protein binds to the full-length YAP protein in Example 3; Figure 10 This is a graph showing the screening results of the FAM189B protein PPxY motif mutant that interacts with the YAP1 protein in Example 3; Figure 11 Analysis of YAP1 protein expression level in HepG2 liver cancer cells treated with FAM189B peptide for 24 hours in Example 4; Figure 12 The figure shows the cell viability analysis of hepatocellular carcinoma 97H cells after treatment with different concentrations of FAM189B peptide in Example 4. represent P <0.0001; Figure 13 The figure shows the cell proliferation capacity analysis of liver cancer 97H cells after treatment with FAM189B peptide in Example 4. represent P <0.01; Figure 14Example 5 illustrates the effect of FAM189B peptide alone in treating mouse liver cancer. A shows a schematic diagram of the hydrodynamic injection protocol for C57BL / 6 mice using the indicated plasmid; B shows bioluminescence imaging of representative mice hydrodynamically injected with the myr-AKT plasmid, images taken 2 weeks after injection (n=5 mice per group); C shows quantitative analysis of bioluminescence intensity in hydrodynamically injected mice (n=5), with each data point representing a single mouse, and data expressed as mean ± standard deviation; D shows bioluminescence in representative mice hydrodynamically injected with the myr-AKT plasmid. Bioluminescence imaging was performed on mice 4 weeks after drug administration (n=5 mice per group); E shows the quantitative analysis of bioluminescence intensity in hydrodynamically injected mice (n=5), with each data point representing a single mouse, and data expressed as mean ± standard deviation; F shows representative macroscopic morphological images of the liver of mice at the endpoint of the hydrodynamic oncogene-driven HCC model; G shows the detection of serum AST / ALT levels in the hydrodynamic oncogene-driven HCC mouse model (n=5), with serum collected 6 weeks after hydrodynamic injection and measured by ELISA; ns in the figure represents... P >0.05, represent P <0.05, represent P <0.01; Figure 15 This section describes the efficacy of sorafenib / lenvatinib combined with FAM189B peptide in treating mouse liver cancer in Example 5. A shows a schematic diagram of the hydrodynamic injection C57BL / 6 mouse model established using the indicated plasmid; B shows bioluminescence imaging of representative mice after hydrodynamic injection of the myr-AKT plasmid, taken 2 weeks after injection (n=6 mice per group); C shows quantitative analysis of bioluminescence intensity in hydrodynamically injected mice (n=5), with each data point representing a single mouse, and data expressed as mean ± standard deviation; D shows the bioluminescence intensity of mice after hydrodynamic injection of the myr-AKT plasmid. Phenotypic bioluminescence imaging in mice, images taken 4 weeks after drug administration (n=6 mice per group); E shows quantitative analysis of bioluminescence intensity in hydrodynamically injected mice (n=5), with each data point representing a single mouse, and data expressed as mean ± standard deviation; F shows representative macroscopic morphological images of the liver in mice at the endpoint of the hydrodynamic oncogene-driven HCC model; G shows the detection of serum AST / ALT levels in the hydrodynamic oncogene-driven HCC mouse model (n=3), with serum collected 6 weeks after hydrodynamic injection and measured by ELISA; ns in the figure represents P >0.05, represent P <0.05, represent P <0.01. Detailed Implementation

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0022] Although FAM189B The gene has been found to be abnormally elevated in various tumors, but its function is not fully understood, and current research has not revealed a direct interaction between the FAM189B and YAP proteins. In the inventors' previous research, they discovered that the FAM189B protein can directly interact with the YAP protein, thereby stabilizing YAP in the cytoplasm, indicating that… FAM189B It is possible that YAP participates in tumor progression by influencing its degradation, meaning that it exists in various tumor types. FAM189B Abnormal gene expression may lead to abnormal activation of YAP and TAZ, which in turn promotes the proliferation and survival of tumor cells.

[0023] Based on the aforementioned new findings and speculations, the inventors, by analyzing the direct contact region between FAM189B and YAP proteins, developed a FAM189B peptide capable of effectively blocking the connection between these proteins. The FAM189B peptide inhibits tumor cell proliferation by blocking the interaction between intracellular FAM189B and YAP proteins, thereby reducing intracellular YAP protein levels.

[0024] This invention provides a FAM189B polypeptide or polypeptide derivative, specifically in the following two scenarios: Case 1: The FAM189B polypeptide or polypeptide derivative contains the sequence shown in SEQ ID NO.1; Case 2: The FAM189B polypeptide or polypeptide derivative contains the sequence shown in SEQ ID NO.2.

[0025] The FAM189B protein interacts with the YAP1 protein through the first and third PPxY motifs. Therefore, the two FAM189B peptides mentioned above were designed by targeting the sequences of the first and third PPxY motifs, respectively.

[0026] Furthermore, in some embodiments of the present invention, the polypeptide derivative is composed of FAM189B polypeptide and cell-penetrating peptide sequence, so that FAM189B polypeptide can effectively enter the cell, thereby blocking the interaction between intracellular FAM189B protein and YAP protein.

[0027] Furthermore, the cell-penetrating peptide is a TAT peptide with the amino acid sequence shown in SEQ ID NO.3, which can be linked to the N-terminus of the sequence shown in SEQ ID NO.1 or SEQ ID NO.2. The TAT peptide is derived from the TAT protein of human immunodeficiency virus and can efficiently deliver drugs into cells.

[0028] Furthermore, in some embodiments of the present invention, the N-terminus of the FAM189B peptide is acetylated, while its C-terminus is amidated. These modifications enhance the in vivo stability of the peptide, ensuring that it is not degraded by enzymes in the intercellular matrix or cytoplasm before reaching its intracellular target (YAP / FAM189B), thereby guaranteeing its effective function in vivo.

[0029] This invention also provides the application of FAM189B peptide in anti-tumor therapy.

[0030] Liver cancer is one of the most common malignant tumors, and FAM189B The expression level of FAM189B peptide is abnormally elevated in liver cancer. Therefore, this example uses liver cancer as an example to test the anti-tumor effect of FAM189B peptide through cell experiments and in vivo animal experiments. Specifically, cell experiments showed that FAM189B peptide not only significantly inhibits the activity of liver cancer cells but also significantly inhibits their proliferation. Further animal experiments showed that FAM189B peptide can inhibit tumor growth and slow down the development of liver cancer.

[0031] Surprisingly, the FAM189B peptide, when used in combination with other anti-liver cancer drugs, exhibits superior therapeutic effects for liver cancer. For example, sorafenib and lenvatinib are both commonly used targeted therapies for liver cancer. In the examples provided, the combination of FAM189B peptide with sorafenib / lenvatinib for treating liver cancer showed significantly better therapeutic effects than either FAM189B peptide alone or sorafenib / lenvatinib alone.

[0032] Based on the discovery that the FAM189B protein can directly interact with the YAP protein, the FAM189B peptide developed in this invention can effectively exert anti-tumor functions by inhibiting cancer cell proliferation, thus providing a new candidate drug for tumor treatment. Furthermore, when preparing anti-tumor drugs using the FAM189B peptide, the FAM189B peptide can be either the sole active ingredient or one of the active ingredients. Moreover, in the preparation of anti-tumor drugs containing the FAM189B peptide, a pharmaceutically acceptable carrier can be added to improve drug properties (such as stability and bioavailability).

[0033] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used in the embodiments where the manufacturer is not specified are all conventional products that can be obtained commercially.

[0034] Immunoprecipitation is a common method for verifying protein-protein interactions within cells based on the specific binding between antigens and antibodies. Western blotting is a method for detecting target proteins by using antibodies to recognize proteins. Both are existing technologies, so the specific procedures of immunoprecipitation and western blotting experiments will not be described in detail in the following examples.

[0035] Example 1 This study investigated the interaction between FAM189B and YAP proteins using co-immunoprecipitation and GST-pull-down assays. The methods and results are as follows: (1) Use YAP antibody to fish for FAM189B protein.

[0036] For HEK293T cells that had grown to 90% confluence in culture dishes, YAP antibody was used for immunoprecipitation to extract FAM189B protein. For example... Figure 1 As shown, the YAP protein successfully fished out the FAM189B protein, indicating that FAM189B and YAP interact.

[0037] (2) Use FAM189B antibody to fish for YAP protein.

[0038] For HEK293T cells that had grown to 90% confluence in culture dishes, YAP protein was extracted using an immunoprecipitation assay with FAM189B antibody. Figure 2 As shown, the FAM189B protein successfully hooked the YAP protein, indicating that FAM189B and YAP interact.

[0039] (3) The direct interaction between YAP protein and FAM189B protein was detected by the GST-pull down experiment.

[0040] GST and GST fusion protein particles were transformed into BL21 competent cells, respectively. After normal bacterial selection and activation, and once the specified OD value was reached, IPTG was added to induce protein expression. The cells were then incubated overnight at 20°C in a shaker. The next day, the cells were centrifuged at 4°C, and the precipitate was collected. Phosphate-buffered saline (PBS) and a protease inhibitor were added to resuspend the cells, which were then transferred to 10 mL centrifuge tubes. The cells were sonicated until clear. The clarified cells were centrifuged at 4°C, and the supernatant was collected. Pre-washed Glutathione Sepharose was added to the supernatant, and the cells were incubated at 4°C for 2 hours to allow the GST-tagged fusion protein to fully adsorb and bind to the resin beads. The precipitate was washed eight times with the corresponding washing buffer to remove unbound protein. Coomassie brilliant blue staining was performed to confirm successful protein purification.

[0041] Beads containing GST and GST-YAP proteins were mixed with cell lysis buffer overexpressing GFP-FAM189B protein. After washing, the resin bead samples were subjected to Western blotting. The results are as follows: Figure 3 The results show that FAM189B does indeed interact directly with YAP.

[0042] Example 2 This example further explores the interaction mechanism between YAP protein and FAM189B protein. The specific experiments and results are as follows: CHX, or actinomycin, is a common protein synthesis inhibitor that can be used to detect protein degradation. Therefore, this example uses CHX time-gradient treatment of cells for validation. Figure 4 As shown, in HepG2 cells stably transfected with FAM189B, the addition of CHX (10 μg / mL) for treatment times of 0, 4, 8, and 12 h revealed that FAM189B significantly inhibited the degradation of YAP1 protein. Similarly, as... Figure 5 As shown, in HCCLM3 cells stably transfected with FAM189B knockdown, the degradation of YAP1 protein was accelerated. These results collectively suggest that FAM189B may alter the protein level of YAP1 by regulating its degradation process.

[0043] Example 3 Based on the fact that FAM189B protein can directly interact with YAP protein, this example obtained the sequence of a polypeptide that can block the interaction between FAM189B protein and YAP protein using the following method.

[0044] (1) Construct a vector expressing the truncated form using PCR technology.

[0045] like Figure 6As shown, the YAP1 protein was truncated (amino acid sequence as shown in SEQ ID NO.4) into four parts: 1-171 aa, 1-263 aa, 263-504 aa, and 171-504 aa. Figure 7 As shown, the FAM189B protein (amino acid sequence as shown in SEQ ID NO. 5) was truncated into two parts: 1-200 aa and 201-668 aa. Expression plasmids were constructed for each of these truncated parts. When constructing the expression plasmids, the primers contained protective bases, restriction enzyme sites, and the target fragment. The annealing temperature during amplification was approximately 60°C. The primers, upon specific binding to the template, replaced the original bases and recognized the substituted bases.

[0046] The specific process of plasmid construction is as follows: ① PCR amplification of the target fragment: Use a plasmid containing the full length of the gene as a template, and amplify the length of the target fragment. Mix all reagents thoroughly before use.

[0047] ②The PCR product was recovered using a gel extraction kit and finally eluted with 43 μL ddH2O.

[0048] ③ The purified target fragment and plasmid empty vector are digested with enzymes to form complementary sticky ends so that they can be ligated to obtain a fusion expression vector.

[0049] ④ After enzyme digestion, the enzyme digestion product should be recovered according to the enzyme digestion product kit and eluted with 30 μL ddH2O.

[0050] ⑤ Usually, the purified target fragment and the empty vector fragment are mixed at a ratio of empty vector: fragment = 1:3 (concentration ratio), and then the enzyme is ligated at 16℃ for 6 h.

[0051] ⑥ Finally, the purified product obtained in step ⑤ is transformed into E. coli. After picking a single colony and shaking it, it is directly sequenced. Strains with correct sequencing results are preserved by adding glycerol or by directly expanding the culture to extract plasmids for preservation, so as to be used in subsequent experiments.

[0052] (2) Determination of the interaction region between FAM189B protein and YAP protein.

[0053] Each truncated variant obtained in step (1) was co-transfected with the corresponding full-length plasmid into HEK293T cells. The binding interactions between each truncated variant of FAM189B protein and each truncated variant of YAP protein were identified by immunoprecipitation and Western blotting.

[0054] Figure 8The results show the interaction between the truncated versions of YAP1 protein and the full-length FAM189B protein. The results indicate that the 1-263 aa region and the 171-504 aa region of YAP1 protein can bind to the full-length FAM189B protein. Figure 9 The results show the interaction between the truncated forms of FAM189B and the full-length YAP1 protein. These results indicate that only the 201-668 aa region of the FAM189B protein can bind to the full-length YAP1 protein. Therefore, the results suggest that the FAM189B protein binds to the WW domain of the YAP1 protein through the 201-668 aa region.

[0055] (3) Construct vectors for expressing mutants using PCR technology.

[0056] The WW domain is a modular protein structure that recognizes proline-rich Pro-Pro-x-Tyr (PPxY) motifs in specific target proteins. In the Hippo / YAP signaling pathway, multiple complexes exist that interact through the WW domain and PPxY motifs. Analysis of the amino acid sequence of the FAM189B protein revealed three PPxY motifs; therefore, the inventors mutated them to AAXA to disrupt the PPxY motifs.

[0057] When mutating the FAM189B protein, primers should be designed around the mutated amino acid, with approximately 15 bp added before and after it, for a total length of about 33 bp. A complementary pair of primers should also be synthesized. The annealing temperature during amplification is approximately 60℃. When the primers specifically bind to the template, they replace the original bases, recognizing the substituted bases and forming a complete circular plasmid. Because the DNA obtained from PCR is unmethylated, while the plasmid extracted from *E. coli* is methylated, the original template plasmid can be digested using the methylation digestive enzyme DpnI, leaving only the successfully mutated plasmid.

[0058] The construction process of the mutant plasmid is as follows: ① PCR amplification of mutant plasmids: Using the successfully constructed plasmid as a template, the amplification length is the total length of the target fragment and the vector. Because the time is relatively long, additional mutations are likely to occur. Therefore, a high-fidelity enzyme should be selected for the amplification experiment to prevent the generation of new mutations.

[0059] ②Recovery of PCR amplified products: The products were recovered using a gel extraction kit and finally eluted with 43 μL ddH2O.

[0060] ③ The recovered DNA product was digested with DpnI enzyme for 1 h in a 37°C water bath. The enzyme digestion system used is shown in Table 1.

[0061] Table 1 Composition of the enzyme digestion system

[0062] ④ After enzyme digestion, the enzyme digestion product was recovered using an enzyme digestion product kit and eluted with 30 μL ddH2O.

[0063] ⑤ Transform the purified product obtained in step ④ into E. coli, pick a single colony, shake it, and then directly sequence it. For the successfully mutated strains with correct sequencing results, preserve them by adding glycerol or by directly expanding the culture to extract plasmids for preservation, so as to use them in subsequent experiments.

[0064] (4) Determination of polypeptide sequence.

[0065] The mutants obtained in step (3) were co-transfected with the YAP1 expression vector into HEK293T cells, and the binding of each FAM189B mutant to the YAP1 protein was identified by immunoprecipitation and western blotting.

[0066] The results of the detection of the interaction between the various PPxY motif mutants of FAM189B protein and the full-length YAP1 protein are as follows: Figure 10 As shown, this result indicates that the FAM189B protein interacts with the YAP1 protein through the first and third PPxY motifs.

[0067] Based on the FAM189B protein sequence, the sequences of its first and third PPxY motifs can be obtained, thereby enabling the acquisition of a polypeptide (i.e., the FAM189B polypeptide) capable of blocking the interaction between the FAM189B protein and the YAP protein. Specifically, this polypeptide contains an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0068] Example 4 This example demonstrates the antitumor function of the FAM189B peptide through cell experiments. The experiments included the following: (1) Modification of FAM189B polypeptide.

[0069] Based on the sequence of the FAM189B polypeptide provided in Example 1, the following processes were performed sequentially: ① The sequence of a cell-penetrating peptide is attached to the N-terminus, and the sequence of the cell-penetrating peptide is YGRKKRRQRRR (SEQ ID NO.3). ② The peptide is acetylated at the N-terminus and amidated at the C-terminus.

[0070] The two short peptides obtained were named FAM189B 244-258 aa and FAM189B 279-292 aa, respectively.

[0071] (2) Effect of FAM189B peptide on YAP expression.

[0072] HepG2 liver cancer cells were treated with the short peptide obtained in step (1) for 24 h, with a final concentration of 1 μM. A control group (containing cells and DMEM medium with 10% FBS) and an experimental group (containing cells, DMEM medium with 10% FBS and the short peptide drug) were set up. Cell lysates were collected and Western blotting was performed to detect the expression levels of YAP protein and its downstream CTGF protein.

[0073] The expression level of YAP protein in HepG2 liver cancer cells after 24 h of short peptide treatment is as follows: Figure 11 As shown, this result indicates that, compared with the control group, short peptide treatment can significantly reduce the expression levels of YAP protein and its downstream target proteins, suggesting that short peptides can affect the degradation of YAP protein.

[0074] (3) Effect of FAM189B polypeptide on the viability of liver cancer cells.

[0075] Taking hepatocellular carcinoma 97H cells as an example, the effect of the short peptide obtained in step (1) on the viability of hepatocellular carcinoma cells was tested.

[0076] Healthy hepatocellular carcinoma 97H cells were collected and placed in a 1.5 mL sterile centrifuge tube. After washing and resuspending with phosphate-buffered saline (PBS), 10 μL of the cell suspension was used for cell counting on a counting chamber. The cell density was adjusted to 1 × 10⁻⁶ cells / mL. 5 cells / mL, at 10 4 Cells were seeded per well in 96-well plates, 100 μL per well. Experimental setups included a background group (no cells, no drugs, DMEM medium containing 10% FBS), a control group (cells, sorafenib and lenvatinib, DMEM medium containing 10% FBS, no short peptide drugs), and a short peptide drug group (cells, sorafenib and lenvatinib, DMEM medium containing 10% FBS, short peptide drugs (0.04 μM, 1 μM, or 25 μM)). Each group had 6 replicates. Cells were incubated at 37°C in a 5% CO2 incubator. After 72 h of culture, the CCK8 assay was performed: 10 μL of CCK8 reagent was added to each well (10 μL of CCK8 per 100 μL of medium). The cells were incubated at 37°C in a 5% CO2 incubator for 1 h, and the absorbance (OD450) at 450 nm was measured using a microplate reader.

[0077] The results of the detection of the effect of short peptides on the viability of 97H liver cancer cells are as follows: Figure 12 As shown, compared with the control group, the cell survival rate was significantly reduced, indicating that the short peptide can significantly inhibit the viability of liver cancer 97H cells.

[0078] (4) Effect of FAM189B polypeptide on the proliferation of liver cancer cells.

[0079] Using the same 97H hepatocellular carcinoma cells, the effect of the short peptide obtained in step (1) on the viability of hepatocellular carcinoma cells was tested.

[0080] Log-phase hepatocellular carcinoma 97H cells were harvested, digested with 0.25% trypsin, and pipetted to form single cells. The cells were then suspended in complete culture medium (DMEM medium with 10% FBS) for later use. 300 cells were seeded into each well of a 12-well plate pre-warmed to 37°C and cultured in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. Drug stimulation was administered 48-72 hours after cell adhesion, without changing the medium. Experimental setups included a control group (containing cells, sorafenib and lenvatinib, DMEM medium with 10% FBS, but no short-peptide drug) and a short-peptide drug group (containing cells, sorafenib and lenvatinib, DMEM medium with 10% FBS, and 1 μM of the short-peptide drug). Both groups were cultured routinely at 37°C, 5% CO2. After 10 days of culture, the original culture medium was discarded, and the cells were fixed with 5% paraformaldehyde for 15 min. After fixation, wash with PBS, add an appropriate amount of 1% crystal violet staining solution and stain for 10 min, then slowly wash away the staining solution with PBS and air dry. Invert the petri dish and take a picture.

[0081] Figure 13 The results showed that the proliferation capacity of 97H hepatocellular carcinoma cells after short peptide treatment was significantly reduced compared with the control group, with a decrease in the number of clones (P<0.01), indicating that the short peptide drug significantly inhibited cell proliferation.

[0082] Example 5 This example demonstrates the antitumor function of the FAM189B peptide in vivo by establishing a mouse model of liver cancer, as detailed below: (1) FAM189B polypeptide as a single treatment for liver cancer.

[0083] Seven-week-old male black mice were injected intravenously via tail vein with SB transposase plasmid, myr-AKT plasmid, and Myc plasmid to establish a hepatocellular carcinoma model. Two weeks later, when hepatocellular carcinoma tumors were established, mice were intraperitoneally injected three times a week with the vector (physiological saline), FAM189B 244-258 aa (25 mg / kg), and FAM189B 279-292 aa (25 mg / kg). After three weeks of administration, the hepatocellular carcinoma was observed by in vivo imaging, and the number of tumors and liver mass were recorded.

[0084] The therapeutic effects of two FAM189B peptides on liver cancer are as follows: Figure 14As shown, tumor size was inhibited and liver weight ratio decreased in the treatment group. These results indicate that the two FAM189B peptides can inhibit the development of liver cancer.

[0085] (2) Sorafenib / lenvatinib combined with FAM189B peptide for the treatment of liver cancer.

[0086] Seven-week-old male black mice were injected intravenously via tail vein with SB transposase plasmid, myr-AKT plasmid, and Myc plasmid to establish a hepatocellular carcinoma model. Two weeks later, when hepatocellular carcinoma tumors were established, mice were intraperitoneally injected three times a week with the following medications: a vector (saline), lenvatinib (20 mg / kg), sorafenib (40 mg / kg), FAM189B 244-258 aa (25 mg / kg), and FAM189B279-292 aa (25 mg / kg). After three weeks of administration, the hepatocellular carcinoma was observed using in vivo imaging, and the number of tumors and liver mass were recorded.

[0087] The therapeutic effects of sorafenib / lenvatinib combined with FAM189B peptide on liver cancer are as follows: Figure 15 As shown, compared with the control group, the tumor size in the experimental group was significantly inhibited, and the liver weight ratio in mice decreased. These results indicate that combined drug therapy can significantly inhibit the development of liver cancer.

[0088] In summary, the FAM189B peptide provided by this invention blocks the connection between FAM189B and YAP proteins by acting on the direct contact region between FAM189B and YAP proteins, thereby regulating the activity of YAP and its downstream pathways. Moreover, experiments have shown that the FAM189B peptide can inhibit the proliferation and migration of liver cancer cells, and in vivo animal experiments have shown that it can inhibit the growth of liver cancer tumors. It is evident that the FAM189B peptide has great application potential in the preparation of anti-tumor drugs.

[0089] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A FAM189B polypeptide derivative, characterized in that, The product contains a FAM189B polypeptide and a cell-penetrating peptide sequence, wherein the sequence of the FAM189B polypeptide is shown in SEQ ID NO.1 or SEQ ID NO.2, and the cell-penetrating peptide sequence is the TAT peptide shown in SEQ ID NO.

3. The cell-penetrating peptide sequence is attached to the N-terminus of the sequence shown in SEQ ID NO.1 or SEQ ID NO.

2. The N-terminus of the FAM189B polypeptide derivative is acetylated and the C-terminus is amidated.

2. The use of the FAM189B polypeptide derivative as described in claim 1 in the preparation of anti-liver cancer drugs.

3. An antitumor drug, characterized in that, The antitumor drug contains the FAM189B polypeptide derivative as described in claim 1, and the tumor is liver cancer.

4. The antitumor drug according to claim 3, characterized in that, The antitumor drugs include sorafenib and / or lenvatinib.

5. The antitumor drug according to claim 3, characterized in that, The antitumor drug contains a pharmaceutically acceptable carrier.

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