CN120285196A

CN120285196A - Use of Smad1 palmitoylation regulator in the preparation of drugs for enhancing cisplatin sensitivity in high-grade serous ovarian cancer

Info

Publication number: CN120285196A
Application number: CN202510442205.3A
Authority: CN
Inventors: 冯凤芝; 张卫光; 程全成; 苏昊
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2025-04-09
Filing date: 2025-04-09
Publication date: 2025-07-11

Abstract

The invention discloses application of a Smad1 palmitoylation regulator in preparation of a medicament for enhancing cisplatin sensitivity of high-grade serous ovarian cancer. According to the research, smad1 of the high-grade serous ovarian cancer has palmitoylation state, and the palmitoylation is stimulated to have the effect of enhancing the cisplatin sensitivity of the high-grade serous ovarian cancer. In addition, the specific palmitoyl S-acyltransferase of Smad1 palmitoylation and the palmitoyl removal acyl protein thioesterase 1 are screened, and a molecular mechanism of regulating and controlling cisplatin resistance occurrence of high-grade serous ovarian cancer by palmitoylation modification of Smad1 is clear. The Smad1 palmitoylation regulator can effectively improve the Smad1 palmitoylation level, and further enhance the cisplatin sensitivity of high-grade serous ovarian cancer.

Description

Application of Smad1 palmitoylation regulator in preparation of drugs for enhancing cisplatin sensitivity of high-grade serous ovarian cancer

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to application of a Smad1 palmitoylation regulator in preparation of a medicament for enhancing cisplatin sensitivity of high-grade serosity ovarian cancer.

Background

Ovarian epithelial cancer (EPITHELIAL OVARIAN CANCER, EOC) is one of three common malignant tumors of gynaecology, with incidence inferior to cervical and endometrial cancer, but mortality rate being the leading one. EOC is a very heterogeneous tumor, including various types of serous cancers (High-grade serous and low-grade serous), endometrioid, mucinous and clear cell carcinoma, with High-grade serous ovarian cancer (High-grade serous ovarian cancer, HGSOC) being the most common histological subtype, accounting for about 80%, and intensive research into HGSOC being the primary task to improve overall survival in EOC patients.

However, although researches on HGSOC in terms of surgery, chemotherapy, targeted drugs and the like have been advanced to some extent, a brand new mode of 'surgery+chemotherapy+maintenance' of ovarian cancer treatment is started, and the recent treatment effect of ovarian cancer patients is obviously improved, but the 5-year survival rate of the patients is not obviously improved, and 60% of patients in III phase and 80% of patients in IV phase still survive for less than 5 years, so that the patients are easy to relapse and drug resistance, and the method is still a major problem.

Thus, there is a need to study HGSOC the drug resistance mechanisms to develop novel anti-tumor and potentiate chemotherapeutic sensitive drugs.

Disclosure of Invention

In order to solve at least part of the technical problems in the prior art, the invention provides application of a Smad1 palmitoylation regulator in preparing a medicament for enhancing cisplatin sensitivity of high-grade serosity ovarian cancer. Specifically, the present invention includes the following.

In a first aspect of the invention there is provided the use of a Smad1 palmitoylation modulator in the manufacture of a medicament for enhancing cisplatin sensitivity in high grade serous ovarian cancer, wherein the Smad1 palmitoylation modulator increases the palmitoylation level of Smad1 by direct or indirect means.

In certain embodiments, the Smad1 palmitoylation modulator comprises at least one of a small molecule compound, a nucleic acid, a protein, an antibody, an enzyme, a polypeptide, and a gene editing tool.

In certain embodiments, the use according to the invention, wherein the Smad1 palmitoylation modulator is selected from at least one of:

(1) An activator of specific palmitoyl S-acyltransferase associated with Smad1 palmitoylation;

(2) Inhibitors or antagonists of acyl protein thioesterase 1 associated with Smad1 depalmitoylation;

(3) A substance that enhances binding of Smad1 to its specific palmitoyl S-acyltransferase associated with palmitoylation;

(4) Substances that block binding of Smad1 to its palmitoylation-associated acyl protein thioesterase 1.

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

In certain embodiments, the use according to the invention, wherein the effective amount of Smad1 palmitoylation modulator is from 0.1 to 2000mg/Kg.

In certain embodiments, use according to the invention, wherein the enhancing ovarian cancer cisplatin sensitivity comprises at least one of decreasing the IC50 value of cancer cells for cisplatin, increasing the apoptosis rate of cisplatin-induced cancer cells, decreasing the clonogenic capacity of cancer cells, decreasing the proliferative activity of cancer cells, decreasing the repair of DNA damage to cancer cells, decreasing the expression of DNA repair-related proteins in cancer cells, enhancing the double strand break of DNA induced by cisplatin, decreasing the expression of multi-drug resistance-related proteins, enhancing the accumulation of cisplatin in tumor tissue, decreasing epigenetic changes associated with cisplatin resistance, decreasing the expression of cancer stem cell markers associated with cisplatin resistance, increasing the progression free survival of patients, increasing overall survival, increasing objective remission rate, decreasing recurrence rate, decreasing CA125 levels, increasing the interval between cisplatin sensitivity, decreasing tumor burden assessed by RECIST criteria, improving clinical symptoms, and increasing quality of life scores.

In certain embodiments, the use according to the invention, wherein the Smad1 palmitoylation comprises cysteine palmitoylation at positions 57 and/or 64 of Smad 1.

In a second aspect of the invention, there is provided a method of screening for a drug for enhancing cisplatin sensitivity in high grade serous ovarian cancer comprising:

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

b. contacting the test agent with a cell model or animal model;

c. Measuring the Smad1 palmitoylation level in the model after the Smad1 palmitoylation level is contacted with the drug to be detected, so as to obtain a second measurement value;

d. Comparing the first measured value with a second measured value, screening the drug to be tested as a candidate drug capable of enhancing the cisplatin sensitivity of the high-grade serous ovarian cancer when the second measured value is larger than the first measured value, and screening the drug to be tested as a drug useless for enhancing the cisplatin sensitivity of the high-grade serous ovarian cancer when the second measured value is smaller than or equal to the first measured value.

In certain embodiments, a method of screening for a drug for increasing cisplatin sensitivity in high grade serous ovarian cancer according to the present invention, wherein the cells comprise cisplatin-treated ovarian cancer cell line cells, such as cisplatin-treated high grade serous ovarian cancer cells.

In a third aspect of the invention there is provided the use of a Smad1 palmitoylation modulator in the manufacture of a medicament for the treatment of high grade serous ovarian cancer in combination with another therapeutic agent, wherein the Smad1 palmitoylation modulator increases the palmitoylation level of Smad1 by direct or indirect means.

In a fourth aspect of the invention there is provided the use of a Smad1 palmitoylation modulator in the manufacture of a medicament for enhancing cisplatin sensitivity in high grade serous ovarian cancer in combination with other therapeutic agents wherein the Smad1 palmitoylation modulator increases the palmitoylation level of Smad1 by direct or indirect means.

According to the research, smad1 of the high-grade serous ovarian cancer has palmitoylation state, and the palmitoylation is stimulated to have the effect of enhancing the cisplatin sensitivity of the high-grade serous ovarian cancer. In addition, the specific palmitoyl S-acyltransferase of Smad1 palmitoylation and the palmitoyl removal acyl protein thioesterase 1 are screened, and a molecular mechanism of regulating and controlling cisplatin resistance occurrence of high-grade serous ovarian cancer by palmitoylation modification of Smad1 is clear. The Smad1 palmitoylation regulator can effectively improve the Smad1 palmitoylation level, and further enhance the cisplatin sensitivity of high-grade serous ovarian cancer.

Drawings

FIG. 1 shows the overall palmitoylation level change in HGSOC cancer and paracancerous tissues, where two lanes in each group are parallel samples, respectively.

FIG. 2 shows the results of palmitoylation protein modification groups of HGSOC cisplatin-resistant and sensitive tissue clinical samples, wherein A shows the statistics of the results of the differential modification sites, B shows the volcanic image of the differential modification sites, red dots are the obviously up-regulated differential modification sites, the darker the color is the higher the up-regulated multiple, the blue dots are the obviously down-regulated differential modification sites, the darker the down-regulated multiple is the higher the color is, the gray dots are the non-differential modification sites, C shows the protein KEGG function annotation and enrichment of the differential sites, and the red boxes mark the Hippo signal path.

Figure 3 shows mass spectra peak diagrams of Smad 1C 57 and C64 screened by palmitoylated protein modification group.

FIG. 4 shows the relationship between Smad1 and HGSOC prognosis, wherein A shows the result of Smad1 protein immunohistochemical staining of HGSOC cancer tissue, B shows the expression change of Smad1 in HGSOC cancer tissue, and C shows the relationship between Smad1 mRNA level and prognosis of ovarian cancer patient.

Fig. 5 shows subcellular localization changes of Smad1 in HGSOC cisplatin-resistant and sensitive tissues.

Fig. 6 shows the distribution of palmitoylated Smad1, wherein a shows the distribution of palmitoylated Smad1 in the cell membrane and overall distribution, two rows of Smad1 are experimental results at different exposure times, respectively, B shows the distribution of palmitoylated Smad1 in the nucleus and cytoplasm, and C shows the distribution of depalmitoylated Smad1 in the nucleus and cytoplasm.

FIG. 7 shows the distribution of palmitoylated Smad1 from SKOV3/DDP cells treated with ML-348.

FIG. 8 shows the effect of knockdown SKOV3/DDP cell line background Smad1 expression on cell growth, wherein A shows that the effect of lentivirus transfection on SKOV3/DDP cells is better, B shows that the knockdown effect of shRNA 1-carrying lentivirus is best, and C shows that knockdown Smad1 can effectively inhibit the growth of SKOV3/DDP cells.

FIG. 9 shows the results of a clonogenic assay of three ovarian cancer cell lines after different gene edits, wherein A shows the clonogenic assay of A2789 cells with cisplatin drug (DDP) added, and B shows the effect of transfection of Smad1 in different states on drug resistance after knockdown of SKOV3 and SKOV3/DDP cells background Smad1, each group administered DDP.

FIG. 10 shows the subcutaneous tumor growth of SKOV3/DDP cells in different gene editing states, wherein A shows the growth of in vivo tumors and B shows the tumor size.

FIG. 11 shows the effect of ML-348 on cisplatin resistance.

FIG. 12A, B, C shows mass spectral peak diagrams of the ZDHC family members (ZDHC 3, 5, 6) bound to Smad1, respectively.

FIG. 13 shows that SKOV3 (sensitive cells) and SKOV3/DDP (drug resistant cells) transfected Smad1 with Flag tag, and that Smad 1-bound ZDHC was used, wherein A shows that both ZDHC 5/6/3 were bound to Smad1, B shows that the ZDHC 6 bound to Smad1 in drug resistant cells was significantly reduced but the ZDHC 5/3 change was not apparent compared to sensitive cells, and C shows that SKOV3 cells were transfected into Smad1-WT, smad1-C57A, smad1-C64A, smad1-C57A/C64A, respectively, based on knockdown of Smad1 expression, to examine the effect of simulated depalmitoylation on Smad1 and ZDHC 6 binding capacity.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

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 herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

Use of the same

In one aspect of the invention, there is provided the use of a Smad1 palmitoylation modulator in the manufacture of a medicament for enhancing cisplatin sensitivity in high grade serous ovarian cancer, wherein the Smad1 palmitoylation modulator is capable of increasing the palmitoylation level of Smad1 by direct or indirect means. The invention discovers and verifies that palmitoylated Smad1 can be used as a target for enhancing HGSOC cisplatin sensitivity through researches. Thus, without being bound by any theory, any agent that is capable of modulating Smad1 palmitoylation, thereby up-regulating the level of Smad1 palmitoylation, or achieving palmitoylation of Smad1, by direct or indirect means, is within the scope of the present invention.

In a preferred embodiment of the invention, the Smad1 palmitoylation refers to palmitoylation of cysteines at positions 57 and 64 of Smad 1.

In the present invention, the term "enhancing cisplatin sensitivity of high-grade serous ovarian cancer" refers to increasing cisplatin efficacy in cisplatin treatment of high-grade serous ovarian cancer, with the purpose of increasing cisplatin sensitivity and decreasing drug resistance of tumor cells, such as increasing cisplatin accumulation in tumor cells or increasing apoptosis rate. Thus, the subject matter of the first aspect of the invention may also be referred to as "use of Smad1 palmitoylation modulator in the preparation of a cisplatin sensitizer or sensitization medicament for high grade serous ovarian cancer. In the present invention, the terms "enhance", "increase", "upregulate", "promote" or "improve" are used interchangeably.

In the present invention, enhancing cisplatin sensitivity in ovarian cancer includes at least one of decreasing the IC ₅₀ value of cisplatin in cancer cells, increasing the rate of cisplatin-induced apoptosis in cancer cells, decreasing the clonogenic capacity of cancer cells, decreasing the proliferative activity of cancer cells, decreasing the repair of DNA damage in cancer cells, decreasing the expression of DNA repair-related proteins in cancer cells, enhancing the DNA double strand breaks induced by cisplatin, decreasing the expression of multi-drug resistance-related proteins, increasing the accumulation of cisplatin in tumor tissue, decreasing epigenetic changes associated with cisplatin resistance, decreasing expression of cancer stem cell markers associated with cisplatin resistance, increasing the progression-free survival of patients, increasing overall survival, increasing objective remission rates, decreasing recurrence rates, decreasing CA125 levels, increasing the interval between cisplatin sensitivity, decreasing tumor burden assessed by RECIST criteria, improving clinical symptoms, and increasing quality of life scores.

In the present invention, the assay and assay methods for enhancing cisplatin sensitivity in ovarian cancer may employ techniques known in the art, and are not particularly limited, e.g., such sensitivity enhancement is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% or cisplatin-induced apoptosis rate enhancement is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% or cisplatin accumulation enhancement in tumor tissue is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% as measured by any standard technique as compared to an untreated reference group under equivalent conditions.

In the present invention, beneficial or desired clinical results include, but are not limited to, results that are either detectable or undetectable, including reduction in tumor volume, reduction in tumor marker levels, delay or slowing of tumor progression, improvement or stabilization of disease state (i.e., without worsening), alleviation of symptoms, and diminishment (whether partial or total). "enhancing cisplatin sensitivity in high grade serous ovarian cancer" also includes an extended survival period as compared to the expected progression free survival period and total survival period when not receiving treatment.

In the present invention, smad1 palmitoylation modulator comprises at least one of a small molecule compound, a nucleic acid, a protein, an antibody, an enzyme, a polypeptide, and a gene editing tool.

The present application has experimentally verified that palmitoyl S-acyltransferase (in particular ZDHC 6) and acyl protein thioesterase 1, which are closely related to Smad1 palmitoylation, and thus it is understood that it is within the scope of the present application to increase the level of Smad1 palmitoylation by palmitoyl S-acyltransferase and acyl protein thioesterase 1, examples of which include, but are not limited to, (1) activators of specific palmitoyl S-acyltransferases related to Smad1 palmitoylation, (2) inhibitors or antagonists of acyl protein thioesterase 1 related to Smad1 depalmitoylation, (3) agents that enhance binding of Smad1 to specific palmitoyl S-acyltransferase related to its palmitoylation, (4) agents that block binding of Smad1 to acyl protein thioesterase 1 related to its depalmitoylation.

In a preferred embodiment, the activator (or promoter or agonist) of specific palmitoyl S-acyltransferase associated with Smad1 palmitoylation, or the agent that increases palmitoyl S-acyltransferase activity, may be, for example, but not limited to, a genetically engineered acyltransferase mutant, ACSL1, beta-endorphin, palmitic acid, C75, orlistat, etc.

It will be appreciated by those skilled in the art that palmitoylation (S-palmitoylation) is a reversible post-translational modification of a protein, with palmitoyl transferase catalyzing the covalent attachment of palmitic acid to cysteine residues of the protein, while the removal of palmitoylase is responsible for the removal of palmitoyl groups, both of which are active in dynamically regulating the palmitoylation level of the protein. The depalmitoylation inhibitor reduces removal of the palmitoyl group by inhibiting the activity of the depalmitoylase, resulting in accumulation of palmitoylation modifications. This effect can be determined by direct detection of the level of palmitoylation, or by palmitoylation addition of a blocking control, i.e. a palmitoyl transferase inhibitor (e.g. 2-BP) in combination, if the level of palmitoylation is no longer elevated, indicating that the effect of the inhibitor is dependent on palmitoylation-depalmitoylation equilibrium.

In a preferred embodiment, the palmitoylation modulator comprises 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 (e.g., but not limited to ML348, ML349, palmostatin B, etc.), antibodies that target acyl protein thioesterase 1, nucleic acids that target acyl protein thioesterase 1 (e.g., but not limited to antisense oligonucleotides, siRNA, miRNA, nucleic acid aptamers, decoy oligonucleotides, shRNA, gRNA, etc.), gene editing tools for knockdown or knockdown of acyl protein thioesterase 1 (e.g., 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.

In a preferred embodiment, the palmitoylation modulator comprises a substance that blocks binding of Smad1 to its acyl protein thioesterase 1 associated with depalmitoylation, examples of which include, but are not limited to, antibodies that target acyl protein thioesterase 1, inhibitors of cellular signaling pathways upstream of the depalmitoylation process, chemical modification reagents of acyl protein thioesterase 1, and the like.

In a preferred embodiment, the palmitoylation modulator comprises a substance that enhances binding of Smad1 to its specific palmitoyl S-acyltransferase associated with palmitoylation, examples of which include, but are not limited to, an activator that is involved in the cell signaling pathway upstream of the palmitoylation process, or a chemical modification reagent for a specific palmitoyl S-acyltransferase, and the like.

An effective amount of Smad1 palmitoylation modulator may be administered to a subject in need thereof to achieve the objects of the invention. In a preferred embodiment, the effective amount of Smad1 palmitoylation modulator is from 0.1 to 2000mg/Kg, preferably from 0.5 to 500mg/Kg, still preferably from 0.5 to 400mg/Kg, still preferably from 0.5 to 300mg/Kg, more preferably from 0.5 to 200mg/Kg, still preferably from 0.5 to 150mg/Kg, still preferably from 0.5 to 100mg/Kg, still preferably from 0.5 to 50mg/Kg, still preferably from 0.5 to 40mg/Kg, still preferably from 0.5 to 30mg/Kg, most preferably from 0.5 to 25mg/Kg, for example 0.5mg/Kg、0.75mg/Kg、0.95mg/Kg、1mg/Kg、1.25mg/Kg、1.5mg/Kg、1.75mg/Kg、2mg/Kg、2.5mg/Kg、2.75mg/Kg、3mg/Kg、3.25mg/Kg、3.5mg/Kg、3.75mg/Kg、4mg/Kg、4.25mg/Kg、4.5mg/Kg、4.75mg/Kg、5mg/Kg、5.25mg/Kg、5.5mg/Kg、5.75mg/Kg、6mg/Kg、6.25mg/Kg、6.5mg/Kg、6.75mg/Kg、7mg/Kg、7.25mg/Kg、7.5mg/Kg、7.75mg/Kg、8mg/Kg、8.25mg/Kg、8.5mg/Kg、8.75mg/Kg、9mg/Kg、9.25mg/Kg、9.5mg/Kg、9.75mg/Kg、10mg/Kg、11mg/Kg、12mg/Kg、13mg/Kg、14mg/Kg、15mg/Kg、16mg/Kg、17mg/Kg、18mg/Kg、19mg/Kg、20mg/Kg、21mg/Kg、22mg/Kg、23mg/Kg、24mg/Kg、25mg/Kg., may be administered in a single dose once daily, may be administered in divided doses per day, or may be used at intervals.

Screening method

In one aspect of the invention, a method of screening for a drug for enhancing cisplatin sensitivity in high grade serous ovarian cancer is provided, comprising:

a. measuring Smad1 palmitoylation level in the cell model or animal model to obtain a first measurement;

b. contacting the test agent with a cell model or animal model;

c. Measuring Smad1 palmitoylation level in the model after the model is contacted with the drug to be measured to obtain a second measurement value;

In a preferred embodiment, the cell is an ovarian cancer cell (e.g., without limitation, cisplatin-treated high grade serous ovarian cancer cells).

In another preferred embodiment, the animal model is a mouse, rat, rabbit, sheep, pig, cow, dog, fish, or the like.

Combination therapy

In one aspect of the invention there is provided the use of a Smad1 palmitoylation modulator in the manufacture of a medicament for the treatment of high grade serous ovarian cancer in combination with another therapeutic agent, wherein the Smad1 palmitoylation modulator increases the palmitoylation level of Smad1 by direct or indirect means.

In the present invention, other therapeutic agents are not particularly limited, examples of which include, but are not limited to, platinum-based drugs, anti-angiogenic drugs, immune checkpoint inhibitors, poly (adenosine diphosphate) -ribose polymerase (PARP) inhibitors, endocrine therapy drugs, and experimental platinum resistance reversal agents. Examples of such platinum-based drugs include, but are not limited to, cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, and the like. Examples of such anti-angiogenic drugs include, but are not limited to, bevacizumab, ramucirumab, aflibercept, apatinib, sorafenib, and the like. Examples of such immune checkpoint inhibitors include, but are not limited to, PD-1 inhibitors (e.g., but not limited to, nal Wu Liyou mab, pamphlet Li Zhushan antibody, etc.), PD-L1 inhibitors (e.g., but not limited to, actlizumab, dulcis You Shan antibody, etc.), CTLA-4 inhibitors (e.g., but not limited to, ipilimumab), and the like. Examples of such PARP inhibitors include, but are not limited to, olapari, nilaparib, rupa, valiparib, taprazoparylene, and the like. Examples of the endocrine treatment drug include, but are not limited to, tamoxifen, letrozole, anastrozole, exemestane, and the like. The experimental platinum resistance reversal agent is selected from P-glycoprotein inhibitors (such as but not limited to verapamil, cyclosporin 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 thioflavin sulfoxide imine), ATP7A/B transporter inhibitors (such as but not limited to tetraminopyridine), mitochondrial targeting 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 modulators, etc.

Examples

The role of Smad1 palmitoylation in cisplatin treatment of ovarian cancer is exemplified below.

1. Global palmitoylation level change

The overall palmitoylation level change in HGSOC cancerous and paracancerous tissues was examined. As a result, as shown in FIG. 1, the overall palmitoylation level of the cancer tissue was reduced as compared to the paracancerous tissue, and the overall palmitoylation level of the drug-resistant tissue was reduced as compared to the cisplatin-sensitive tissue.

2. Palmitoylated protein modification histology

Palm acylated protein modified histology was performed on HGSOC cisplatin resistant and sensitive tissue clinical samples. The results are shown in FIG. 2, and statistics of the differential modification sites show that the drug resistant group was down-regulated by 207 and up-regulated by 147 relative to the sensitive group. In the Hippo signal pathway, 5 different sites (Smad 1 comprises two sites) enriched with 4 total proteins of Smad1, BMPR1A, PARD and PARD6B are all relatively sensitive groups, and the palmitoylation level of the drug resistant group is down-regulated. In addition, palmitoylated protein modification panels screened two sites Smad 1C 57 and C64 of Smad1, and mass spectra peak diagrams are shown in fig. 3.

Relationship between Smad1 and HGSOC prognosis

Protein immunohistochemical analysis was performed on HGSOC cancer tissues and paracancerous tissues. As shown in FIG. 4, smad1 expression in cancer tissues is obviously increased compared with paracancerous tissues, but compared with cisplatin sensitive tissues, smad1 expression levels in drug resistant tissues are not significantly different, and survival analysis of two groups of ovarian cancer patients with different expression levels of Smad1 mRNA is not significantly different.

4. Subcellular localization

Immunofluorescent staining of Smad1 using clinical HGSOC samples observed subcellular localization, as shown in fig. 5, the drug resistant group Smad1 was predominantly expressed in the nucleus and the sensitive group Smad1 was predominantly expressed in the cytoplasm.

Smad1 palmitoylation positioning Studies

SKOV3/DDP cells ML-349 (in DMSO,3 mg/mL), ML-348 (in DMSO,20 mg/mL), palmostatin B (in DMSO,50 mM) were each administered at 25. Mu.M, and the control group was given an equivalent amount of DMSO. The expression change of Smad1 is detected after subcellular component separation of SKOV3/DDP with different drug interventions. As shown in fig. 6, the inhibitor ML-348 of acyl protein thioesterase 1 (APT 1) showed the most remarkable effect after cell membrane separation, indicating that the depalmitoylase of Smad1 was APT1, but Smad1 of cell membrane was decreased after inhibition of APT1, indicating that palmitoylated Smad1 was not localized to cell membrane but Smad1 on cell membrane was decreased after palmitoylation, and the total Smad1 expression level of cytoplasm and nucleus was unchanged after palmitoylation. The nuclear separation of SKOV3/DDP cells, after ML348 inhibits APT1, enhances palmitoylation, increases Smad1 in cytoplasm, decreases Smad1 in nucleus, and shows that palmitoylation of Smad1 affects nuclear shuttling. Blocking palmitoylation with the palmitoylation inhibitor 2-BP corresponds to depalmitoylation, increased nuclear Smad1, decreased cytoplasmic Smad1, indicating that membrane localization becomes nuclear localization after depalmitoylation.

Further, subcellular localization of SKOV3/DDP cells treated with ML-348 gave the results shown in FIG. 7, which shows that Smad1 was transferred from the nucleus to the cytoplasm after activation of palmitoylation by ML-348 treatment, indicating that ML-348 had an effect on palmitoylation Smad1 cell localization.

6. Knock down of Smad1 expression effects on cell growth

The SKOV3/DDP cell line was knocked down to background Smad1 expression and cell growth was observed. As shown in FIG. 8, the effect of the lentivirus transfected SKOV3/DDP cells is good, the knocking-down effect of the lentivirus carrying shRNA1 is best, the shRNA1 is adopted for the Smad1 knocking-down operation in the subsequent embodiment, and the SKOV3/DDP cells can be effectively inhibited by the Smad1 knocking-down operation.

7. Gene editing

Clone experiments were performed using three ovarian cancer cell lines with different gene edits. As shown in FIG. 9, when DDP was added to the cells A2789, the number of clones of cells transfected with Smad1-WT was increased as compared with those transfected with Smad1, and the number of clones of cells transfected with Smad1-C57A and Smad1-C64A was increased most significantly. The effect of Smad1 transfected into different states on drug resistance after background Smad1 knockdown of SKOV3 and SKOV3/DDP cells was studied. The results show that after both cells knockdown Smad1, smad1-WT is transfected, and after DDP is added, compared with SKOV3 cells, the cloning number of SKOV3/DDP cells is obviously increased, and the number of clone formation is obviously increased by transfecting SKOV3 cells knockdown Smad1 into Smad1-C57A/C64A, which indicates that the wild type inhibits cancer cell proliferation.

Observing the subcutaneous tumor growth of SKOV3/DDP cells in different gene editing states. The SKOV3/DDP cells knocked down for Smad1 background expression, the cells transfected with Smad1-WT and the cells transfected with Smad1-C57A/C64A were treated by intraperitoneal injection of DDP during growth of tumor cells in nude mice, respectively. As shown in FIG. 10, the SKOV3/DDP cells knocked down for Smad1 background expression had minimal tumor mass, smad1-WT transfected cells had slightly larger tumor mass, and Smad1-C57A/C64A transfected cells had the greatest tumor mass. It was demonstrated that Smad1 knockdown enhanced sensitivity to DDP, whereas depalmitoylation of Smad1 reduced sensitivity to DDP.

Effect of ml-348 treatment on cisplatin resistance

Following knockdown of background SMAD1 expression in SKOV3/DDP resistant cells, SMAD1 (SMAD 1-WT, SMAD1-C57A, SMAD-C64A, SMAD 1-C57A/C64A) was transfected into different gene editing states, and then cell proliferation assays were performed by simultaneous administration of the four cell lines ML-348 (ML-348 dissolved in DMSO, 3 mg/mL) for 12 hours at a dose gradient of 0, 10, 25, 50. Mu.M. As shown in FIG. 11, transfected wild-type SMAD1, after activation of palmitoylation by ML-348 treatment, cell viability (cisplatin resistance) decreased with increasing concentration gradient of ML-348. However, transfection mimics depalmitoylated SMAD1, which did not show the above trend after treatment with ML-348, indicating that ML-348 enhanced cisplatin sensitivity by stimulating SMAD1 palmitoylation.

Smad1 binding assay

After transfection of SKOV3/DDP cells with knockdown background Smad1 expression into Smad1 with Flag tag (Flag-Smad 1), protein Pull down bound to Smad1 was subjected to mass spectrometry using Flag antibody, resulting in mass spectrometry peak patterns of zdhc family members (zdhc 3, 5, 6) bound to Smad1 (fig. 12).

Smad1 with Flag tag was transfected with SKOV3 (sensitive cells) and SKOV3/DDP (drug resistant cells), and ZDHC bound to Smad1 by Flag antibody Pull down was used. As shown in FIG. 13, both ZDHs 5/6/3 bind to Smad1, compared with sensitive cells, the drug-resistant cells have significantly reduced binding capacity of Smad1 to ZDHC 6, but the change of ZDHC 5/3 is not obvious, SKOV3 cells are respectively transfected into Smad1-WT and Smad1-C57A, smad1-C64A, smad1-C57A/C64A on the basis of knockdown of Smad1 expression, the influence of simulated depalmitoylation on the binding capacity of Smad1 to ZDHC 6 is detected, and the binding capacity of Smad1 to ZDHC 6 is significantly reduced after C57 and C64 are simulated depalmitoylation, and meanwhile, the binding capacity of Smad1 to ZDHH C6 is significantly reduced after C57 and C64 are simulated depalmitoylation.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some of the technical features may be equivalently replaced. Such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

Use of a Smad1 palmitoylation modulator in the manufacture of a medicament for enhancing cisplatin sensitivity in high grade serous ovarian cancer, wherein the Smad1 palmitoylation modulator increases the palmitoylation level of Smad1 by direct or indirect means.
2. The use according to claim 1, wherein the Smad1 palmitoylation modulator is selected from at least one of a small molecule compound, a nucleic acid, a protein, an antibody, an enzyme, a polypeptide, and a gene editing tool.
3. The use according to claim 1, wherein the Smad1 palmitoylation modulator is selected from at least one of the following:

(1) An activator of specific palmitoyl S-acyltransferase associated with Smad1 palmitoylation;

(2) Inhibitors or antagonists of acyl protein thioesterase 1 associated with Smad1 depalmitoylation;

(3) A substance that enhances binding of Smad1 to its specific palmitoyl S-acyltransferase associated with palmitoylation;

(4) Substances that block binding of Smad1 to its palmitoylation-associated acyl protein thioesterase 1.
4. The use according to claim 3, wherein 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 Smad1 palmitoylation modulator is between 0.1 and 2000mg/Kg.
6. The use of claim 1, wherein the enhancement of ovarian cancer cisplatin sensitivity comprises at least one of decreasing the IC ₅₀ value of cancer cells for cisplatin, increasing the apoptosis rate of cisplatin-induced cancer cells, decreasing the clonogenic capacity of cancer cells, decreasing the proliferative activity of cancer cells, decreasing the repair of DNA damage to cancer cells, decreasing the expression of DNA repair-related proteins in cancer cells, enhancing cisplatin-induced DNA double strand breaks, decreasing the expression of multi-drug resistance-related proteins, enhancing the accumulation of cisplatin in tumor tissue, decreasing epigenetic changes associated with cisplatin resistance, decreasing expression of cancer stem cell markers associated with cisplatin resistance, increasing progression free survival of patients, increasing overall survival, increasing objective remission rate, decreasing CA125 levels, increasing the interval between cisplatin sensitivity, decreasing tumor burden assessed by RECIST criteria, improving clinical symptoms, and increasing quality of life scores.
7. The use according to claim 1, wherein the Smad1 palmitoylation comprises cysteine palmitoylation at position 57 and/or position 64 of Smad 1.
8. A method of screening for a drug for enhancing cisplatin sensitivity in high grade serous ovarian cancer comprising:

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

b. contacting the test agent with a cell model or animal model;

c. Measuring the Smad1 palmitoylation level in the model after the Smad1 palmitoylation level is contacted with the drug to be detected, so as to obtain a second measurement value;

d. Comparing the first measured value with a second measured value, screening the drug to be tested as a candidate drug capable of enhancing the cisplatin sensitivity of the high-grade serous ovarian cancer when the second measured value is larger than the first measured value, and screening the drug to be tested as a drug useless for enhancing the cisplatin sensitivity of the high-grade serous ovarian cancer when the second measured value is smaller than or equal to the first measured value.
9. The method of screening for a drug for increasing cisplatin sensitivity in high grade serous ovarian cancer as defined by claim 8 wherein the cells comprise cisplatin-treated ovarian cancer cells.
Use of a Smad1 palmitoylation modulator in the manufacture of a medicament for the treatment of high grade serous ovarian cancer in combination with another therapeutic agent, wherein the Smad1 palmitoylation modulator increases the palmitoylation level of Smad1 by direct or indirect means.