Application of Smad1 palmitoylation regulator in preparation of medicine for enhancing cis-platinum sensitivity of high-grade serous ovarian cancer

Abstract

The invention discloses application of a Smad1 palmitoylation regulator in preparation of a medicine for enhancing cis-platinum sensitivity of high-grade serous ovarian cancer. Research finds that Smad1 of high-grade serous ovarian cancer has a palmitoylation state, and the effect of enhancing cis-platinum sensitivity of the high-grade serous ovarian cancer by stimulating palmitoylation of Smad1 is achieved. In addition, specific palmitoyl S-acyltransferase subjected to palmitoylation of the Smad1 and acyl protein thioesterase 1 subjected to palmitoylation removal are screened, and a molecular mechanism for regulating cis-platinum drug resistance of the high-grade serous ovarian cancer through palmitoylation modification of the Smad1 is defined. The Smad1 palmitoylation regulator disclosed by the invention can be used for effectively improving the palmitoylation level of Smad1, so that the cis-platinum sensitivity of high-grade serous ovarian cancer is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the use of a Smad1 palmitoylation regulator in the preparation of a drug for enhancing cisplatin sensitivity in high-grade serous ovarian cancer. Background Art

[0002] Epithelial ovarian cancer (EOC) is one of the three common gynecological malignancies. Its incidence rate is second only to cervical cancer and endometrial cancer, but the fatality rate ranks first. EOC is a highly heterogeneous tumor, including various types such as serous carcinoma (high-grade serous carcinoma and low-grade serous carcinoma), endometrioid carcinoma, mucinous carcinoma, and clear cell carcinoma. Among them, high-grade serous ovarian cancer (HGSOC) is the most common histological subtype, accounting for about 80%. In-depth research on HGSOC is the primary task for improving the overall survival of EOC patients.

[0003] However, although certain progress has been made in the research on HGSOC in terms of surgery, chemotherapy, and targeted drugs, opening up a new "surgery + chemotherapy + maintenance" treatment model for ovarian cancer and significantly improving the short-term treatment effect of ovarian cancer patients, the 5-year survival rate of patients has not been significantly improved. There are still 60% of stage III and 80% of stage IV patients who survive less than 5 years, and easy recurrence and drug resistance remain major problems.

[0004] Therefore, there is an urgent need to study the drug resistance mechanism of HGSOC to develop new anti-tumor and chemotherapy-sensitizing drugs. Summary of the Invention

[0005] To solve at least some of the above-mentioned technical problems in the prior art, the present invention provides the use of a Smad1 palmitoylation regulator in the preparation of a drug for enhancing cisplatin sensitivity in high-grade serous ovarian cancer. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, there is provided the use of a Smad1 palmitoylation regulator in the preparation of a drug for enhancing cisplatin sensitivity in high-grade serous ovarian cancer, wherein the Smad1 palmitoylation regulator increases the palmitoylation level of Smad1 directly or indirectly.

[0007] In certain embodiments, according to the use of the present invention, the Smad1 palmitoylation regulator includes at least one of small molecule compounds, nucleic acids, proteins, antibodies, enzymes, polypeptides, and gene editing tools.

[0008] In certain embodiments, according to the use of the present invention, the Smad1 palmitoylation regulator is selected from at least one of the following:

[0009] (1) An activator of a specific palmitoyl - S - acyltransferase related to Smad1 palmitoylation;

[0010] (2) An inhibitor or antagonist of acyl - protein thioesterase 1 related to Smad1 depalmitoylation;

[0011] (3) A substance that enhances the binding of Smad1 to its specific palmitoyl - S - acyltransferase related to palmitoylation;

[0012] (4) A substance that blocks the binding of Smad1 to acyl - protein thioesterase 1 related to its depalmitoylation.

[0013] In certain embodiments, for the use according to the present invention, wherein the acyl - protein thioesterase 1 inhibitor or antagonist is selected from at least one of ML348, ML349, and Palmostatin B.

[0014] In certain embodiments, for the use according to the present invention, wherein the effective amount of the Smad1 palmitoylation regulator is 0.1 - 2000 mg / Kg.

[0015] In certain embodiments, for the use according to the present invention, wherein enhancing the cisplatin sensitivity of ovarian cancer includes at least one of the following: reducing the IC50 value of cancer cells to cisplatin, increasing the apoptosis rate of cisplatin - induced cancer cells, reducing the cancer cell clone - forming ability, reducing the cancer cell proliferation activity, reducing cancer cell DNA damage repair, reducing the expression of DNA repair - related proteins in cancer cells, enhancing cisplatin - induced DNA double - strand breaks, reducing the expression of multidrug - resistance - related proteins, enhancing the accumulation of cisplatin in tumor tissues, reducing epigenetic alterations related to cisplatin resistance, reducing the expression of cancer stem cell markers related to cisplatin resistance, increasing the progression - free survival of patients, increasing the overall survival, increasing the objective response rate, reducing the recurrence rate, reducing the CA125 level, increasing the cisplatin - sensitive interval, reducing the tumor burden evaluated by RECIST criteria, improving clinical symptoms and increasing the quality - of - life score.

[0016] In certain embodiments, for the use according to the present invention, wherein the Smad1 palmitoylation includes palmitoylation of cysteine at position 57 and / or position 64 of Smad1.

[0017] In a second aspect of the present invention, there is provided a method for screening a drug for enhancing the cisplatin sensitivity of high - grade serous ovarian cancer, which includes:

[0018] a. Measuring the Smad1 palmitoylation level in a cell model or an animal model to obtain a first measurement value;

[0019] b. Contact the drug to be tested with a cell model or an animal model;

[0020] c. Measure the Smad1 palmitoylation level in the model after contact with the drug to be tested to obtain a second measurement value;

[0021] d. Compare the first measurement value and the second measurement value. When the second measurement value is greater than the first measurement value, screen the drug to be tested as a candidate drug capable of enhancing the cisplatin sensitivity of high-grade serous ovarian cancer. When the second measurement value is less than or equal to the first measurement value, screen the drug to be tested as a drug useless for enhancing the cisplatin sensitivity of high-grade serous ovarian cancer.

[0022] In certain embodiments, according to the method for screening a drug for enhancing the cisplatin sensitivity of high-grade serous ovarian cancer of the present invention, wherein the cells include cells of a cisplatin-treated ovarian cancer cell line, such as cisplatin-treated high-grade serous ovarian cancer cells.

[0023] In a third aspect of the present invention, there is provided the use of a Smad1 palmitoylation regulator in the preparation of a drug for the combined treatment of high-grade serous ovarian cancer with other therapeutic agents, wherein the Smad1 palmitoylation regulator increases the palmitoylation level of Smad1 directly or indirectly.

[0024] In a fourth aspect of the present invention, there is provided the use of a Smad1 palmitoylation regulator in the preparation of a drug for enhancing the cisplatin sensitivity of high-grade serous ovarian cancer in combination with other therapeutic agents, wherein the Smad1 palmitoylation regulator increases the palmitoylation level of Smad1 directly or indirectly.

[0025] It has been found through research in the present invention that Smad1 in high-grade serous ovarian cancer has a palmitoylation state, and stimulating its palmitoylation has the effect of enhancing the cisplatin sensitivity of high-grade serous ovarian cancer. In addition, the present invention has screened the specific palmitoyl S-acyltransferase for Smad1 palmitoylation and acyl protein thioesterase 1 for depalmitoylation, and clarified the molecular mechanism by which the palmitoylation modification of Smad1 regulates the occurrence of cisplatin resistance in high-grade serous ovarian cancer. The Smad1 palmitoylation regulator of the present invention can effectively increase the palmitoylation level of Smad1, thereby enhancing the cisplatin sensitivity of high-grade serous ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shows the changes in the overall palmitoylation levels of HGSOC cancer tissues and adjacent tissues, where the two lanes in each group are parallel samples.

[0027] Figure 2The palmitoylated protein modification omics results of HGSOC cisplatin-resistant and sensitive tissue clinical samples are shown. Among them, A shows the statistical results of differential modification sites; B shows the volcano plot of differential modification sites. Red dots are significantly up-regulated differential modification sites, and the darker the color, the higher the up-regulation fold. Blue dots are significantly down-regulated differential modification sites, and the darker the color, the higher the down-regulation fold. Gray dots are non-differential modification sites; C shows the protein KEGG functional annotation and enrichment of differential sites, and the Hippo signaling pathway is marked with a red box.

[0028] Figure 3 The mass spectrometry peak maps of Smad1 C57 and C64 screened by palmitoylated protein modification omics are shown.

[0029] Figure 4 The relationship between Smad1 and the prognosis of HGSOC is shown. Among them, A shows the results of immunohistochemical staining of Smad1 protein in HGSOC cancer tissues; B shows the expression changes of Smad1 in HGSOC cancer tissues; C shows the relationship between the level of Smad1 mRNA in ovarian cancer patients and the prognosis.

[0030] Figure 5 The subcellular localization changes of Smad1 in cisplatin-resistant and sensitive tissues of HGSOC are shown.

[0031] Figure 6 The distribution of palmitoylated Smad1 is shown. Among them, A shows the distribution of palmitoylated Smad1 in the cell membrane and in total. The two rows of Smad1 are the experimental results under different exposure times; B shows the distribution of palmitoylated Smad1 in the nucleus and cytoplasm; C shows the distribution of depalmitoylated Smad1 in the nucleus and cytoplasm.

[0032] Figure 7 The distribution of palmitoylated Smad1 in SKOV3 / DDP cells after treatment with ML-348 is shown.

[0033] Figure 8 The effect of knocking down the basal Smad1 expression in the SKOV3 / DDP cell line on cell growth is shown. Among them, A shows that the lentiviral transfection of SKOV3 / DDP cells has a good effect; B shows that the lentivirus carrying shRNA1 has the best knockdown effect; C shows that knocking down Smad1 can effectively inhibit the growth of SKOV3 / DDP cells.

[0034] Figure 9The results of colony formation experiments of three ovarian cancer cell lines after different gene edits are shown. Among them, A shows the colony formation experiment of A2789 cells after adding cisplatin drug (DDP); B shows the effect of transfection of different states of Smad1 on drug resistance after knocking down the background Smad1 of SKOV3 and SKOV3 / DDP cells, and DDP was administered to each group.

[0035] Figure 10 The subcutaneous tumor growth of SKOV3 / DDP cells in different gene edit states is shown. Among them, A shows the growth of tumors in vivo; B shows the size of the tumors.

[0036] Figure 11 The effect of ML-348 on cisplatin resistance is shown.

[0037] Figure 12 A, B, and C of [the relevant content] respectively show the mass spectrometry peak maps of members of the ZDHHC family (ZDHHC3, 5, 6) that bind to Smad1.

[0038] Figure 13 SKOV3 (sensitive cells) and SKOV3 / DDP (drug-resistant cells) were transfected with Flag-tagged Smad1, and Flag antibody was used to pull down ZDHHC that binds to Smad1. Among them, A shows that ZDHHC5 / 6 / 3 all bind to Smad1; B shows that compared with sensitive cells, ZDHHC6 that binds to Smad1 in drug-resistant cells is significantly reduced, but the changes in ZDHHC5 / 3 are not obvious; C shows that on the basis of knocking down the expression of background Smad1 in SKOV3 cells, Smad1-WT, Smad1-C57A, Smad1-C64A, and Smad1-C57A / C64A were transfected respectively to detect the effect of mimicking depalmitoylation on the binding ability of Smad1 to ZDHHC6. Detailed implementation manners

[0039] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Each intermediate value within any stated value or stated range and each smaller range between any other stated value or intermediate value within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] 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 belongs. Although only preferred methods and materials are described in this invention, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0042] Use

[0043] In one aspect of the present invention, there is provided the use of a Smad1 palmitoylation regulator in the preparation of a drug for enhancing the cisplatin sensitivity of high-grade serous ovarian cancer, wherein the Smad1 palmitoylation regulator can increase the palmitoylation level of Smad1 directly or indirectly. Through research, the present invention discovers and verifies that palmitoylated Smad1 can serve as a target for enhancing the cisplatin sensitivity of HGSOC. Therefore, without being bound by any theory, any reagent that can regulate Smad1 palmitoylation directly or indirectly to up-regulate the palmitoylation level of Smad1 or achieve the palmitoylation of Smad1 is within the protection scope of the present invention.

[0044] In a preferred embodiment of the present invention, the Smad1 palmitoylation refers to the palmitoylation of cysteine at positions 57 and 64 of Smad1.

[0045] In the present invention, the term "enhancing the cisplatin sensitivity of high-grade serous ovarian cancer" means improving the efficacy of cisplatin in the cisplatin treatment of high-grade serous ovarian cancer, and its purpose is to enhance the sensitivity of tumor cells to cisplatin and reduce drug resistance, such as increasing the accumulation of cisplatin in tumor cells or increasing the apoptosis rate. Therefore, the subject matter of the first aspect of the present invention can also be referred to as "the use of a Smad1 palmitoylation regulator in the preparation of a cisplatin sensitizer or sensitizing drug for high-grade serous ovarian cancer". In the present invention, the terms "enhance", "increase", "up-regulate", "promote" or "improve" can be used interchangeably.

[0046] In the present invention, enhancing the cisplatin sensitivity of ovarian cancer includes at least one of the following situations: reducing the IC of cancer cells to cisplatin 50Values, increasing the apoptosis rate of cisplatin-induced cancer cells, reducing the cancer cell clone formation ability, reducing the cancer cell proliferation activity, reducing cancer cell DNA damage repair, reducing the expression of DNA repair-related proteins in cancer cells, enhancing cisplatin-induced DNA double-strand breaks, reducing the expression of multidrug resistance-related proteins, enhancing the accumulation of cisplatin in tumor tissues, reducing epigenetic changes related to cisplatin resistance, reducing the expression of cancer stem cell markers related to cisplatin resistance, increasing the progression-free survival of patients, increasing the overall survival, increasing the objective response rate, reducing the recurrence rate, reducing the CA125 level, increasing the cisplatin-sensitive interval, reducing the tumor burden evaluated by the RECIST standard, improving clinical symptoms and increasing the quality of life score.

[0047] In the present invention, the determination indexes and determination methods for enhancing the cisplatin sensitivity of ovarian cancer can adopt the technologies known in the art, and are not particularly limited in this regard. For example, compared with the untreated reference group under the same conditions, measured by any standard technology, the degree of enhancement of this sensitivity is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100%; or, the degree of enhancement of the apoptosis rate of cisplatin-induced cancer cells is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100%; or, the degree of enhancement of the accumulation of cisplatin in tumor tissues is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100%.

[0048] In the present invention, beneficial or desirable clinical outcomes include, but are not limited to, the following outcomes, whether detectable or undetectable, including reduction in tumor volume, reduction in tumor marker levels, delay or slowdown in tumor progression, improvement or stabilization of the disease state (i.e., no worsening), remission of symptoms, and alleviation (whether partial or complete). "Enhancing the cisplatin sensitivity of high-grade serous ovarian cancer" also includes the extended survival period compared with the progression-free survival and overall survival expected when not receiving treatment.

[0049] In the present invention, the Smad1 palmitoylation regulator includes at least one of small molecule compounds, nucleic acids, proteins, antibodies, enzymes, polypeptides, and gene editing tools.

[0050] The present invention has verified through experiments the palmitoyl-S-acyltransferases (especially ZDHHC6) and acyl protein thioesterase 1 that are closely related to the palmitoylation of Smad1. Therefore, it can be understood that substances that increase the palmitoylation level of Smad1 through palmitoyl-S-acyltransferases and acyl protein thioesterase 1 are within the protection scope of this application. Examples thereof include, but are not limited to, (1) activators of specific palmitoyl-S-acyltransferases related to the palmitoylation of Smad1; (2) inhibitors or antagonists of acyl protein thioesterase 1 related to the depalmitoylation of Smad1; (3) substances that enhance the binding of Smad1 to specific palmitoyl-S-acyltransferases related to its palmitoylation; (4) substances that block the binding of Smad1 to acyl protein thioesterase 1 related to its depalmitoylation.

[0051] In a preferred embodiment, the activator (or promoter or agonist) of a specific palmitoyl-S-acyltransferase related to the palmitoylation of Smad1, or a substance that increases the activity of palmitoyl-S-acyltransferase can be, for example but not limited to: acyltransferase mutants modified by genetic engineering, ACSL1, β-endorphin, palmitic acid, C75, orlistat, etc.

[0052] Those skilled in the art can understand that palmitoylation (S-palmitoylation) is a reversible post-translational modification of proteins, in which palmitoyltransferase catalyzes the covalent attachment of palmitic acid to the cysteine residues of proteins, and depalmitoylase is responsible for removing the palmitoyl group. The activities of both dynamically regulate the palmitoylation level of proteins. Depalmitoylation inhibitors reduce the removal of palmitoyl groups by inhibiting the activity of depalmitoylase, resulting in the accumulation of palmitoylation modifications. This effect can be determined by directly detecting the palmitoylation level or by using a palmitoylation addition blockade control, that is, by jointly using a palmitoyltransferase inhibitor (such as 2-BP). If the palmitoylation level no longer increases, it indicates that the effect of the inhibitor depends on the palmitoylation-depalmitoylation balance.

[0053] In a preferred embodiment, the palmitoylation modulator includes an acyl protein thioesterase 1 inhibitor or antagonist, examples of which include but are not limited to small molecule drugs that inhibit acyl protein thioesterase 1 (such as but not limited to ML348, ML349, palmostatin B, etc.), antibodies targeting acyl protein thioesterase 1, nucleic acids targeting acyl protein thioesterase 1 (such as but not limited to antisense oligonucleotides, siRNA, miRNA, aptamers, decoy oligonucleotides, shRNA, gRNA, etc.), gene editing tools for knocking down or knocking out acyl protein thioesterase 1 (such as but not limited to ZFNs, TALENs, CRISPR-Cas9, etc.), and the like. In a preferred embodiment, the palmitoylation modulator is ML348. In another preferred embodiment, the palmitoylation modulator is ML349.

[0054] In a preferred embodiment, the palmitoylation modulator includes a substance that blocks the binding of Smad1 to acyl protein thioesterase 1 related to its depalmitoylation, examples of which include but are not limited to antibodies targeting acyl protein thioesterase 1, inhibitors of the upstream cell signaling pathway of the depalmitoylation process, chemical modification reagents of acyl protein thioesterase 1, and the like.

[0055] In a preferred embodiment, the palmitoylation modulator includes a substance that enhances the binding of Smad1 to the specific palmitoyl-S-acyltransferase related to its palmitoylation, examples of which include but are not limited to activators of the upstream cell signaling pathway involved in this palmitoylation process, or chemical modification reagents of the specific palmitoyl-S-acyltransferase, and the like.

[0056] An effective amount of a Smad1 palmitoylation regulator can be administered to a subject in need to achieve the objectives of the present invention. In a preferred embodiment, the effective amount of the Smad1 palmitoylation regulator is 0.1 - 2000 mg / Kg, preferably 0.5 - 500 mg / Kg, more preferably 0.5 - 400 mg / Kg, further preferably 0.5 - 300 mg / Kg, still more preferably 0.5 - 200 mg / Kg, even more preferably 0.5 - 150 mg / Kg, yet more preferably 0.5 - 100 mg / Kg, still more preferably 0.5 - 50 mg / Kg, even more preferably 0.5 - 40 mg / Kg, yet more preferably 0.5 - 30 mg / Kg, most preferably 0.5 - 25 mg / Kg, such as 0.5 mg / Kg, 0.75 mg / Kg, 0.95 mg / Kg, 1 mg / Kg, 1.25 mg / Kg, 1.5 mg / Kg, 1.75 mg / Kg, 2 mg / Kg, 2.5 mg / Kg, 2.75 mg / Kg, 3 mg / Kg, 3.25 mg / Kg, 3.5 mg / Kg, 3.75 mg / Kg, 4 mg / Kg, 4.25 mg / Kg, 4.5 mg / Kg, 4.75 mg / Kg, 5 mg / Kg, 5.25 mg / Kg, 5.5 mg / Kg, 5.75 mg / Kg, 6 mg / Kg, 6.25 mg / Kg, 6.5 mg / Kg, 6.75 mg / Kg, 7 mg / Kg, 7.25 mg / Kg, 7.5 mg / Kg, 7.75 mg / Kg, 8 mg / Kg, 8.25 mg / Kg, 8.5 mg / Kg, 8.75 mg / Kg, 9 mg / Kg, 9.25 mg / Kg, 9.5 mg / Kg, 9.75 mg / Kg, 10 mg / Kg, 11 mg / Kg, 12 mg / Kg, 13 mg / Kg, 14 mg / Kg, 15 mg / Kg, 16 mg / Kg, 17 mg / Kg, 18 mg / Kg, 19 mg / Kg, 20 mg / Kg, 21 mg / Kg, 22 mg / Kg, 23 mg / Kg, 24 mg / Kg, 25 mg / Kg. It can be administered as a single dose once a day, in multiple doses per day, or at intervals.

[0057] Screening method

[0058] In one aspect of the present invention, a method for screening a drug for enhancing cisplatin sensitivity in high - grade serous ovarian cancer is provided, which includes:

[0059] a. Measuring the Smad1 palmitoylation level in a cell model or an animal model to obtain a first measurement value;

[0060] b. Contacting the drug to be tested with the cell model or the animal model;

[0061] c. Measuring the level of Smad1 palmitoylation in the model after contact with the drug to be tested to obtain a second measurement value;

[0062] d. Comparing the first measurement value and the second measurement value. When the second measurement value is greater than the first measurement value, screening the drug to be tested as a candidate drug capable of enhancing the cisplatin sensitivity of high-grade serous ovarian cancer; when the second measurement value is less than or equal to the first measurement value, screening the drug to be tested as a drug useless for enhancing the cisplatin sensitivity of high-grade serous ovarian cancer.

[0063] In a preferred embodiment, the cell is an ovarian cancer cell (such as but not limited to a high-grade serous ovarian cancer cell treated with cisplatin).

[0064] In another preferred embodiment, the animal model is a mouse, rat, rabbit, sheep, pig, cow, dog, fish, etc.

[0065] Combined treatment

[0066] In one aspect of the present invention, there is provided the use of a Smad1 palmitoylation regulator in the preparation of a drug for the combined treatment of high-grade serous ovarian cancer with other therapeutic agents, wherein the Smad1 palmitoylation regulator increases the level of Smad1 palmitoylation directly or indirectly.

[0067] In the present invention, other therapeutic agents are not particularly limited, and examples thereof include but are not limited to platinum-based drugs, anti-angiogenic drugs, immune checkpoint inhibitors, poly (ADP-ribose) polymerase (PARP) inhibitors, endocrine therapy drugs, and experimental platinum resistance reversal agents. Among them, examples of the platinum-based drugs include but are not limited to cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, etc. Examples of the anti-angiogenic drugs include but are not limited to bevacizumab, ramucirumab, aflibercept, apatinib, sorafenib, etc. Examples of the immune checkpoint inhibitors include but are not limited to PD-1 inhibitors (such as but not limited to nivolumab, pembrolizumab, etc.), PD-L1 inhibitors (such as but not limited to atezolizumab, durvalumab, etc.), CTLA-4 inhibitors (such as but not limited to ipilimumab), etc. Examples of the PARP inhibitors include but are not limited to olaparib, niraparib, rucaparib, veliparib, talazoparib, etc. Examples of the endocrine therapy drugs include but are not limited to tamoxifen, letrozole, anastrozole, exemestane, etc. The experimental platinum resistance reversal agents are selected from P-glycoprotein inhibitors (such as but not limited to verapamil, cyclosporine A, quinidine, etc.), DNA repair inhibitors (such as but not limited to ATM inhibitors, ATR inhibitors, CHK1 / 2 inhibitors, etc.), glutathione synthesis inhibitors (such as but not limited to buthionine sulfoximine), ATP7A / B transporter inhibitors (such as but not limited to tetraaminopyridine), mitochondrion-targeted drugs (such as but not limited to dichloroacetic acid), epigenetic regulators (such as but not limited to DNA methylation inhibitors, histone deacetylase inhibitors, etc.), cancer stem cell pathway inhibitors (such as but not limited to Notch pathway inhibitors, Wnt pathway inhibitors, Hedgehog pathway inhibitors, etc.), apoptosis pathway regulators, etc.

[0068] Examples

[0069] The following exemplarily shows the role of Smad1 palmitoylation in cisplatin treatment of ovarian cancer.

[0070] 1. Changes in overall palmitoylation level

[0071] The changes in the overall palmitoylation level of HGSOC cancer tissues and adjacent tissues were detected. The results are as Figure 1 shown. Compared with adjacent tissues, the overall palmitoylation level of cancer tissues decreased, and moreover, compared with cisplatin-sensitive tissues, the overall palmitoylation level of drug-resistant tissues decreased.

[0072] 2. Palmitoylated protein modification proteomics

[0073] Palmitoylated protein modification proteomics analysis was performed on clinical samples of HGSOC cisplatin-resistant and sensitive tissues. The results are as Figure 2As shown, the statistical results of differential modification sites showed that there were 207 down-regulated and 147 up-regulated sites in the drug-resistant group compared with the sensitive group. In the Hippo signaling pathway, 5 differential sites of 4 proteins, namely Smad1, BMPR1A, PARD3, and PARD6B (Smad1 includes two sites), were enriched. Compared with the sensitive group, the palmitoylation level of the drug-resistant group was down-regulated. In addition, two sites of Smad1, Smad1 C57 and C64, were screened out by palmitoylated protein modification proteomics, and the mass spectrometry peak map is as Figure 3 shown.

[0074] 3. Relationship between Smad1 and the prognosis of HGSOC

[0075] Protein immunohistochemical analysis was performed on HGSOC cancer tissues and adjacent tissues. The results are as Figure 4 shown. Compared with adjacent tissues, the expression of Smad1 in cancer tissues was significantly increased. However, compared with cisplatin-sensitive tissues, there was no significant difference in the expression level of Smad1 in drug-resistant tissues; there was no significant difference in the survival analysis of two groups of ovarian cancer patients with different expression levels of Smad1 mRNA.

[0076] 4. Subcellular localization

[0077] Immunofluorescence staining of Smad1 was performed using clinical HGSOC samples to observe subcellular localization. The results are as Figure 5 shown. In the drug-resistant group, Smad1 was mainly expressed in the nucleus, while in the sensitive group, Smad1 was mainly expressed in the cytoplasm.

[0078] 5. Study on the palmitoylation localization of Smad1

[0079] SKOV3 / DDP cells were respectively treated with ML-349 (dissolved in DMSO, 3 mg / mL), ML-348 (dissolved in DMSO, 20 mg / mL), and palmostatin B (dissolved in DMSO, 50 mM), with a concentration of 25 μM. The control group was given an equal amount of DMSO. After subcellular fractionation of SKOV3 / DDP cells intervened with different drugs, the expression changes of Smad1 were detected. The results are as Figure 6As shown, after separating the cell membrane, the inhibitor ML-348 of acyl protein thioesterase 1 (APT1) has the most significant effect, indicating that the depalmitoylase of Smad1 is APT1. However, after inhibiting APT1, the amount of Smad1 in the cell membrane decreases instead, indicating that palmitoylated Smad1 is not localized to the cell membrane. Instead, the amount of Smad1 on the cell membrane decreases after palmitoylation, while the total Smad1 in the cytoplasm and nucleus remains unchanged in terms of expression level after palmitoylation. After separating the nucleus and cytoplasm of SKOV3 / DDP cells, after ML348 inhibits APT1 and enhances palmitoylation, the Smad1 in the cytoplasm increases and the Smad1 in the nucleus decreases, indicating that the palmitoylation of Smad1 affects its nuclear-cytoplasmic shuttling. Using the palmitoylation inhibitor 2-BP to block palmitoylation, which is equivalent to depalmitoylation, the Smad1 in the nucleus increases and the Smad1 in the cytoplasm decreases, indicating that after depalmitoylation, it changes from membrane localization to nuclear localization.

[0080] Furthermore, the subcellular localization of SKOV3 / DDP cells after treatment with ML-348 was determined. The results are as Figure 7 shown. After treatment with ML-348 and activation of palmitoylation, Smad1 translocates from the nucleus to the cytoplasm, indicating that ML-348 affects the cellular localization of palmitoylated Smad1.

[0081] 6. Effect of knocking down Smad1 expression on cell growth

[0082] The background Smad1 expression of the SKOV3 / DDP cell line was knocked down, and cell growth was observed. The results are as Figure 8 shown. Lentiviral transfection of SKOV3 / DDP cells has a good effect; the lentivirus carrying shRNA1 has the best knockdown effect, and shRNA1 was used for the Smad1 knockdown operation in subsequent implementation schemes; knocking down Smad1 can effectively inhibit the growth of SKOV3 / DDP cells.

[0083] 7. Gene editing

[0084] Clonogenicity assays were performed on three ovarian cancer cell lines after different gene editing. The results are as Figure 9As shown, after adding DDP to A2789 cells, compared with the cells with Smad1 knockdown, the number of cell clones transfected with Smad1-WT increased. The increase in the number of cells transfected with Smad1-C57A and Smad1-C64A was more significant, and the number of cell clones transfected with Smad1-C57A / C64A increased the most. After studying the knockdown of the endogenous Smad1 in SKOV3 and SKOV3 / DDP cells, the effects of transfected Smad1 in different states on drug resistance were investigated. The results showed that after knocking down the endogenous Smad1 in both cell lines and transfecting Smad1-WT, after adding DDP, the number of cell clones in SKOV3 / DDP cells was significantly increased compared with SKOV3 cells. When the SKOV3 cells with endogenous Smad1 knockdown were transfected with Smad1-C57A / C64A, the number of colony formations increased significantly, indicating that the wild type inhibits cancer cell proliferation.

[0085] Observe the growth of subcutaneous tumors in SKOV3 / DDP cells with different gene editing states. The SKOV3 / DDP cells with the knockdown of endogenous Smad1 expression, the cells transfected with Smad1-WT again, and the cells transfected with Smad1-C57A / C64A again were respectively injected intraperitoneally with DDP during the growth of the tumor mass in the subcutaneous tumors of nude mice. The results are as Figure 10 shown. The tumor mass of the SKOV3 / DDP cells with the knockdown of endogenous Smad1 expression was the smallest, the tumor mass of the cells transfected with Smad1-WT was slightly larger, and the tumor mass of the cells transfected with Smad1-C57A / C64A was the largest. It shows that knocking out Smad1 can enhance the sensitivity to DDP, while the depalmitoylation of Smad1 reduces the sensitivity to DDP.

[0086] 8. Effect of ML-348 treatment on cisplatin resistance

[0087] After knocking down the endogenous SMAD1 expression in SKOV3 / DDP drug-resistant cells, they were respectively transfected with SMAD1 in different gene editing states (SMAD1-WT, SMAD1-C57A, SMAD1-C64A, SMAD1-C57A / C64A). Then, the above four cell lines were simultaneously treated with ML-348 (ML-348 was dissolved in DMSO, 3 mg / mL) at a dose gradient of 0, 10, 25, and 50 μM for 12 hours. During this period, cisplatin was given for cell killing experiments and cell proliferation detection. The results are as Figure 11 shown. After transfection with wild-type SMAD1 and treatment with ML-348, after activating palmitoylation, the cell viability (cisplatin resistance) decreased with the increase of the ML-348 concentration gradient. However, after transfection with SMAD1 mimicking depalmitoylation and treatment with ML-348, the above trend was not shown, indicating that ML-348 enhances cisplatin sensitivity by stimulating the palmitoylation of SMAD1.

[0088] 9. Smad1 Binding Analysis

[0089] After knocking down the basal Smad1 expression in SKOV3 / DDP cells and transfecting them with Flag-tagged Smad1 (Flag-Smad1), the proteins bound to Smad1 were pulled down using Flag antibody and then analyzed by mass spectrometry. The mass spectrometry peak maps of the ZDHHC family members (ZDHHC3, 5, 6) bound to Smad1 were obtained ( Figure 12 ).

[0090] SKOV3 (sensitive cells) and SKOV3 / DDP (drug-resistant cells) were transfected with Flag-tagged Smad1, and ZDHHC bound to Smad1 was pulled down using Flag antibody. The results are as Figure 13 shown. ZDHHC5 / 6 / 3 all bind to Smad1. Compared with sensitive cells, the amount of ZDHHC6 bound to Smad1 in drug-resistant cells was significantly reduced, but the changes in ZDHHC5 / 3 were not obvious. On the basis of knocking down the basal Smad1 expression in SKOV3 cells, Smad1-WT, Smad1-C57A, Smad1-C64A, and Smad1-C57A / C64A were transfected respectively, and the effects of simulated depalmitoylation on the binding ability of Smad1 to ZDHHC6 were detected. After C57 and C64 were simulated to be depalmitoylated, the binding ability of Smad1 to ZDHHC6 was significantly reduced. At the same time, after C57 and C64 were simulated to be depalmitoylated, the binding ability of Smad1 to ZDHHC6 was reduced more significantly.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements for some of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a Smad1 palmitoylation regulator in the preparation of a drug for enhancing cisplatin sensitivity in high-grade serous ovarian cancer, characterized in that, The Smad1 palmitoylation regulator increases the palmitoylation level of Smad1 directly or indirectly.

2. The use according to claim 1, wherein The Smad1 palmitoylation regulator is selected from at least one of small molecule compounds, nucleic acids, proteins, antibodies, enzymes, polypeptides, and gene editing tools.

3. The use according to claim 1, wherein, The Smad1 palmitoylation regulator is selected from at least one of the following: (1) An activator of a specific palmitoyl-S-acyltransferase related to Smad1 palmitoylation; (2) An inhibitor or antagonist of acyl protein thioesterase 1 related to Smad1 depalmitoylation; (3) A substance that enhances the binding of Smad1 to its specific palmitoyl-S-acyltransferase related to palmitoylation; (4) A substance that blocks the binding of Smad1 to acyl protein thioesterase 1 related to its depalmitoylation.

4. The use according to claim 3, characterized in that The inhibitor or antagonist of acyl protein thioesterase 1 is selected from at least one of ML348, ML349, and Palmostatin B.

5. The use according to claim 1, characterized in that, The effective amount of the Smad1 palmitoylation regulator is 0.1 - 2000 mg / Kg.

6. The use according to claim 1, characterized in that, The enhancement of cisplatin sensitivity in ovarian cancer includes at least one of the following situations: reducing the IC 50 value of cancer cells to cisplatin, increasing the apoptosis rate of cancer cells induced by cisplatin, reducing the colony formation ability of cancer cells, reducing the proliferation activity of cancer cells, reducing DNA damage repair of cancer cells, reducing the expression of DNA repair-related proteins in cancer cells, enhancing cisplatin-induced DNA double-strand breaks, reducing the expression of multidrug resistance-related proteins, enhancing the accumulation of cisplatin in tumor tissues, reducing epigenetic changes related to cisplatin resistance, reducing the expression of cancer stem cell markers related to cisplatin resistance, increasing the progression-free survival of patients, increasing the overall survival, increasing the objective response rate, reducing the recurrence rate, reducing the CA125 level, increasing the cisplatin-sensitive interval, reducing the tumor burden evaluated by the RECIST standard, improving clinical symptoms and increasing the quality of life score.

7. The use according to claim 1, wherein The Smad1 palmitoylation includes palmitoylation of cysteine at position 57 and / or position 64 of Smad1.

8. A method for screening drugs for enhancing cisplatin sensitivity in high-grade serous ovarian cancer, characterized in that, It includes: a. Measuring the Smad1 palmitoylation level in a cell model or an animal model to obtain a first measurement value; b. Contacting the drug to be tested with the cell model or the animal model; c. Measuring the Smad1 palmitoylation level in the model after contacting with the drug to be tested to obtain a second measurement value; d. Comparing the first measurement value and the second measurement value. When the second measurement value is greater than the first measurement value, the drug to be tested is screened as a candidate drug that can enhance the cisplatin sensitivity of high-grade serous ovarian cancer. When the second measurement value is less than or equal to the first measurement value, the drug to be tested is screened as a drug that is useless for enhancing the cisplatin sensitivity of high-grade serous ovarian cancer.

9. The method for screening a drug for enhancing cisplatin sensitivity in high-grade serous ovarian cancer according to claim 8, characterized in that, The cells include ovarian cancer cells treated with cisplatin.

10. Use of a Smad1 palmitoylation regulator in the preparation of a medicament for treating high-grade serous ovarian cancer in combination with other therapeutic agents, characterized in that, The Smad1 palmitoylation regulator increases the palmitoylation level of Smad1 directly or indirectly.

Images