Anti-tumor combined pharmaceutical composition and application thereof

Abstract

The invention discloses an anti-tumor combined pharmaceutical composition and application thereof, and belongs to the technical field of biological medicine. The combined pharmaceutical composition comprises an OTUD4 inhibitor and a ferroptosis inducer or regorafenib which serve as active ingredients, and the OTUD4 inhibitor inhibits expression of OTUD4 in a targeted mode or causes function deficiency of OTUD4 and promotes occurrence of ferroptosis in tumor cells. The combined pharmaceutical composition provided by the invention more effectively inhibits tumor growth by synergistically increasing the oxidative stress level in cells, thereby playing a stronger anti-tumor effect. The combined treatment scheme provided by the invention provides a new thought for tumor treatment, and particularly in patients ineffective or drug-resistant to single-drug treatment, targeted inhibition of OTUD4 and regorafenib can effectively overcome drug resistance and significantly inhibit tumor growth.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-tumor combined pharmaceutical composition and application thereof. Background Art

[0002] In recent years, tumor drug resistance and tumor cell escape mechanisms have become one of the main challenges in the field of tumor treatment. Traditional chemotherapy drugs or targeted therapies can inhibit tumor growth to a certain extent, but due to the genetic heterogeneity of tumor cells and their adaptability to treatment, the treatment effect is often limited and drug resistance is prone to occur. How to effectively inhibit tumor growth and overcome drug resistance has become a problem that needs to be solved urgently.

[0003] Regorafenib is a targeted drug currently used to treat advanced colorectal cancer, gastric cancer and other tumors. It mainly inhibits tumor angiogenesis and tumor cell proliferation by inhibiting multiple kinases, including VEGFR, PDGFR and other pathways. However, in some drug-resistant tumors, the therapeutic effect of regorafenib alone is limited. How to enhance the efficacy of regorafenib is one of the current research hotspots.

[0004] In recent years, ferroptosis, as a type of programmed cell death that depends on iron, has received widespread attention. Ferroptosis is different from traditional apoptosis in that it causes lipid peroxidation and damages the cell membrane, leading to cell death. Therefore, inducing ferroptosis has become a new approach for anti-tumor therapy. However, tumor cells can escape ferroptosis under a variety of stress conditions and continue to proliferate, especially by regulating ferroptosis inhibitory signals through specific molecules to maintain cell survival (Mitochondria-localized cGAS suppresses ferroptosis to promote cancer progression. Cell research , 2023, 33(4): 299-311.; RBMS1 regulates lung cancer ferroptosis through translational control of SLC7A11. The Journal of Clinical investigation , 2021, 131(22).).

[0005] In the previous research of the research group, OTUD4 (OTU domain-containing protein 4) was found to be a new tumor ferroptosis inhibitory molecule. It inhibits the occurrence of ferroptosis by regulating iron metabolism-related proteins and antioxidant signals, thereby promoting the survival and progression of tumor cells.

[0006] According to existing research reports, OTUD4 plays different roles in different types of tumors. For example, Xiaohui Zhao et al. found that OTUD4 may be a potential molecular target for the diagnosis and treatment of breast cancer, liver cancer and lung cancer. Overexpression of OTUD4 inhibits the proliferation, migration and invasion of human breast cancer, liver cancer and lung cancer cells by promoting cancer cell apoptosis and inhibiting the AKT signaling pathway (OTUD4: A Potential Prognosis Biomarker for Multiple Human Cancers. Cancer Management and Research , 2020,12:1503-1512.). Xiu qing Ma et al. found that the loss of OTUD4 in triple-negative breast cancer (TNBC) cells can significantly inhibit cell cloning ability, migration, invasion and cancer stem cell population in vitro, and also significantly inhibit metastasis in vivo (Deubiquitinating enzyme OTUD4regulates metastasis in triple-negative breast cancer by stabilizingSnail1. Experimental Cell Research , 2024, 434(1): 113864.).

[0007] So far, there have been no reports on OTUD4's role in inhibiting ferroptosis and in the survival and progression of colorectal cancer cells, especially in patients who are ineffective or resistant to regorafenib monotherapy. In-depth research on the related mechanism of action is expected to provide new ideas for tumor treatment. Summary of the invention

[0008] The purpose of the present invention is to provide a drug that can promote ferroptosis in tumor cells, and significantly improve the therapeutic effect of tumors, especially refractory and drug-resistant tumors, through combined use.

[0009] To achieve the above object, the present invention adopts the following technical solution: The present invention provides the use of OTUD4 (OTU domain-containing protein 4) as an inhibitory target in the preparation of a tumor ferroptosis enhancer. Specifically, the enhancer promotes the occurrence of ferroptosis in tumor cells by targeted inhibition of OTUD4 expression or causing OTUD4 functional loss.

[0010] The present invention has found that OTUD4 is a tumor ferroptosis inhibitor molecule. By inhibiting OTUD4, the occurrence of tumor cell ferroptosis can be significantly promoted, and the sensitivity of tumor cells to anti-tumor drugs can be enhanced. Therefore, OTUD4 can be used as a drug target to develop corresponding tumor ferroptosis enhancers.

[0011] The technical means for inhibiting OTUD4 in the present invention can be to downregulate the gene expression by using gene knockout, gene interference and other technologies. OTUD4 Gene expression, or the use of inhibitory compounds to target and inhibit OTUD4 expression or cause OTUD4 loss of function.

[0012] The present invention develops a preparation using OTUD4 of humans or other animals as a target, wherein the amino acid sequence of human OTUD4 is shown in SEQ ID NO.1.

[0013] The present invention also provides an anti-tumor combined pharmaceutical composition, comprising an OTUD4 inhibitor and a ferroptosis inducer as active ingredients, wherein the OTUD4 inhibitor targets and inhibits OTUD4 expression or causes OTUD4 functional loss, thereby promoting the occurrence of ferroptosis in tumor cells.

[0014] The present invention uses OTUD4 inhibitors and ferroptosis inducers in combination, and uses OTUD4 inhibitors to target and inhibit OTUD4 in tumor cells, enhance ferroptosis-related oxidative stress responses, amplify ferroptosis responses, and thereby inhibit the growth of tumor cells. Studies have shown that compared with the monotherapy group, the combination of OTUD4 inhibitors and ferroptosis inducers significantly reduced tumor proliferation rates.

[0015] Furthermore, the OTUD4 inhibitor may be, but is not limited to, targeting OTUD4 Gene knockout reagents.

[0016] Furthermore, the ferroptosis inducing agent may be, but is not limited to, Erastin.

[0017] The present invention also provides an anti-tumor combined pharmaceutical composition, comprising an OTUD4 inhibitor and regorafenib as active ingredients, wherein the OTUD4 inhibitor targets and inhibits OTUD4 expression or causes OTUD4 functional loss, thereby promoting the activation of ferroptosis pathways in tumor cells.

[0018] The present invention combines OTUD4 inhibitors with regorafenib. The OTUD4 inhibitors target and inhibit OTUD4 in tumor cells, which can not only promote the activation of the ferroptosis pathway to inhibit tumor growth, but also significantly enhance the anti-tumor effect of regorafenib through a dual targeting mechanism. Studies have shown that compared with the monotherapy group, the combination of drugs can synergistically increase the level of intracellular oxidative stress and significantly enhance the ferroptosis response of tumor cells, thereby exerting a stronger anti-tumor effect. Mechanism studies have shown that after knocking out OTUD4, on the one hand, it promotes the ubiquitination degradation of GPX4, thereby promoting the activation of ferroptosis, and on the other hand, it promotes the degradation of RHEB, activates autophagy, and then promotes the autophagic degradation of GPX4 and promotes the occurrence of ferroptosis.

[0019] Furthermore, the anti-tumor combination pharmaceutical composition may also contain at least one pharmaceutically acceptable carrier.

[0020] Furthermore, the anti-tumor combination pharmaceutical composition can be prepared into various forms such as oral preparations, and drugs in various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0021] The present invention provides the use of the combined pharmaceutical composition in preparing drugs for treating tumors, wherein the tumor is a solid tumor.

[0022] Furthermore, the tumor may be but is not limited to colorectal cancer.

[0023] The present invention also provides the use of a pharmaceutical composition of an OTUD4 inhibitor and regorafenib in the preparation of a drug for treating regorafenib-resistant tumors. Studies have shown that the expression of OTUD4 is upregulated in regorafenib-resistant tumor cells, and the sensitivity of tumor cells to regorafenib can be enhanced by inhibiting OTUD4 expression.

[0024] Furthermore, the tumor is regorafenib-resistant colorectal cancer.

[0025] The present invention has the following beneficial effects: (1) The present invention discloses for the first time that OTUD4 is a tumor ferroptosis inhibitory molecule, and targeted inhibition of OTUD4 can induce ferroptosis in tumor cells.

[0026] (2) The present invention provides an anti-tumor combination pharmaceutical composition, which is composed of an OTUD4 inhibitor and a ferroptosis inducer or regorafenib, which can more effectively inhibit tumor growth by synergistically increasing the level of intracellular oxidative stress, thereby exerting a stronger anti-tumor effect.

[0027] (3) The combined treatment regimen provided by the present invention provides a new idea for tumor treatment, especially in patients who are ineffective or resistant to monotherapy. Targeted inhibition of OTUD4 combined with regorafenib can effectively overcome drug resistance and significantly inhibit tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Western Blot validation of OTUD4 knockout efficiency in LoVo and RKO cells.

[0029] Figure 2 Comparison of the viability of LoVo cell WT and OTUD4 KO after treatment with different concentrations of the ferroptosis inducer Erastin.

[0030] Figure 3Comparison of the viability of RKO cell WT and OTUD4 KO after treatment with different concentrations of the ferroptosis inducer Erastin.

[0031] Figure 4 Flow cytometry was used to detect the level of LPO in LoVo cells after Erastin treatment.

[0032] Figure 5 Flow cytometry was used to detect the level of LPO in RKO cells after Erastin treatment.

[0033] Figure 6 Flow cytometry was used to detect the level of ROS in LoVo cells after Erastin treatment.

[0034] Figure 7 Flow cytometry was used to detect the level of ROS in RKO cells after Erastin treatment.

[0035] Figure 8 Western Blot was used to detect the expression level of GPX4 after OTUD4 knockout.

[0036] Fig. 9 This is the effect of OTUD4 knockout combined with Erastin treatment on mouse tumors.

[0037] Fig.10 for Fig. 9 Figure 3. Effect of tumor volume and tumor weight on statistical graphs.

[0038] Fig.11 IHC was used to detect Ki-67, a proliferation marker of mouse tumors after OTUD4 knockout and Erastin treatment.

[0039] Fig.12 Comparison of the viability of LoVo cells WT and OTUD4 KO after regorafenib treatment.

[0040] Fig.13 Comparison of the viability of RKO cells WT and OTUD4 KO after regorafenib treatment.

[0041] Fig.14 Flow cytometry was used to detect the level of LPO in LoVo cells after regorafenib treatment.

[0042] Fig.15 Flow cytometry was used to detect the level of LPO in RKO cells after regorafenib treatment.

[0043] Fig.16 Flow cytometry was used to detect the level of ROS in LoVo cells after regorafenib treatment.

[0044] Fig.17 Flow cytometry was used to detect the level of ROS in RKO cells after regorafenib treatment.

[0045] Fig.18 Effects of OTUD4 knockout combined with regorafenib treatment on mouse tumors.

[0046] Fig.19 for Fig.18 Figure 3. Effect of tumor volume and tumor weight on statistical graphs.

[0047] Fig. 20 IHC detection of Ki-67, a proliferation marker of mouse tumors after OTUD4 knockout and regorafenib treatment.

[0048] Fig.21 IC of regorafenib-resistant cell lines 50 .

[0049] Fig. 22 is the expression levels of OTUD4 and GPX4 in regorafenib-resistant cell lines. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0052] Example 1: Preparation and screening of OTUD4 knockout cells 1. Construction of OTUD4 knockout plasmid In order to achieve stable knockout of the OTUD4 gene, a plasmid for OTUD4 gene knockout was designed and constructed. OTUD4 was knocked out in colorectal cancer cells using the CRISPR / Cas9 system. The knockout effect was verified and its effect on ferroptosis of tumor cells was evaluated.

[0053] 1.1 Use online tools (such as CRISPR Design Tool) to design specific sgRNA sequences for the human OTUD4 gene (Gene ID: 54726). Select the target sequence located in the exon region of OTUD4 to ensure high specificity and low off-target effects. Design two different sgRNAs to target two different sites of the OTUD4 gene to ensure effective knockout. Designed sgRNA sequences: sgRNA1: 5'-CACCGCAGTAAGCCGGACGAAGGC-3' (SEQ ID NO. 2); sgRNA2: 5'-CACCGATTCAGAACAGAGATGAAC-3' (SEQ ID NO. 3).

[0054] 1.2 sgRNA insertion into plasmid vector Use BbsI to digest the CRISPR / Cas9 plasmid (such as PX330) to remove the original sgRNA placeholder sequence. Synthesize an oligonucleotide pair containing the designed sgRNA sequence, anneal to generate a double strand, and connect it to the linearized PX330 vector to construct a CRISPR / Cas9 plasmid carrying sgRNA.

[0055] 1.3 Plasmid amplification and identification Transform the constructed plasmid into competent E. coli and screen for positive clones. Pick a single clone strain and sequence it to verify whether the sgRNA is correctly inserted.

[0056] 2. Plasmid transfection and gene knockout 2.1 Cell transfection The constructed CRISPR / Cas9-sgRNA plasmid was transfected into LoVo and RKO cells (LoVo and RKO cells were purchased from ATCC) using Lipofectamine 3000 transfection reagent. 48 hours after transfection, positive clones were screened using a culture medium containing puromycin. After screening, positive cells were diluted to the limit to screen out monoclonal cell lines.

[0057] 2.2 Gene knockout verification Western blot detection: Western blot was further used to verify whether the expression of OTUD4 protein disappeared, thereby confirming the knockout effect of the OTUD4 gene.

[0058] 3. Results Analysis The results are as follows Figure 1 As shown, this example successfully constructed OTUD4 gene knockout colorectal cancer cell lines LoVo and RKO. Western blot detection confirmed that the expression of OTUD4 protein in these two cell lines was completely eliminated, verifying the success of OTUD4 knockout. This provides an effective cell model for subsequent studies on the function of OTUD4 in tumor ferroptosis.

[0059] Example 2: Detection of ferroptosis by targeted inhibition of OTUD4 After confirming OTUD4 knockout, the following method was used to detect the effect of OTUD4 gene knockout on ferroptosis of tumor cells.

[0060] 1. Cell viability assay: Cells were treated with Erastin, an inducer of ferroptosis, to detect cell viability and compare the changes in cell viability after OTUD4 knockout.

[0061] The results are as follows Figure 2 and Figure 3 As shown in the figure, after Erastin treatment, the cell viability of the OTUD4 knockout group was significantly reduced, suggesting that OTUD4 may play a role in inhibiting ferroptosis in tumor cells.

[0062] The interaction coefficient CDI value of OTUD4 knockout combined with Erastin drug treatment was calculated according to the following formula: CDI = AB / A × B, calculated based on the number of viable cells (absorbance value), AB is the ratio of the OTUD4 knockout combined with drug treatment group to the control group, A is the ratio of the OTUD4 knockout group to the control group, and B is the ratio of the drug treatment group alone to the control group. If CDI < 1, it proves that the two drugs act synergistically, and when CDI < 0.7, the synergistic effect of the two drugs is very significant; if CDI = 1, the two drugs act additively; if CDI > 1, the two drugs act antagonistically.

[0063] The calculation results showed that the CDI value in LoVo cells was 0.64; the CDI value in RKO cells was 0.73.

[0064] 2. C11-BODIPY staining: Detect intracellular lipid peroxidation (LPO) levels. Increased LPO levels are one of the hallmarks of ferroptosis.

[0065] The results are as follows Figure 4 and Figure 5 As shown, the lipid peroxidation level in the OTUD4 knockout group cells was significantly increased after Erastin treatment, indicating that the loss of OTUD4 enhanced the oxidative stress response associated with ferroptosis.

[0066] The CDI value was calculated and standardized with the ratio of the AB group to the control group as the control value, that is, the ratio of the AB group, A group and B group to the control group was divided by the value of the AB group, and then the standardized value was used for calculation. The results showed that the CDI value in LoVo cells was 0.63; the CDI value in RKO cells was 0.44.

[0067] 3. Reactive oxygen species (ROS) staining: Detect the level of ROS in cells. Increased ROS level is one of the signs of ferroptosis.

[0068] The results are as follows Figure 6 and Figure 7 As shown, the level of reactive oxygen species in the OTUD4 knockout group cells was significantly increased after erastin treatment, indicating that the loss of OTUD4 enhanced the oxidative stress response associated with ferroptosis.

[0069] The CDI value was calculated and the results showed that the CDI value in LoVo cells was 0.44; the CDI value in RKO cells was 0.40.

[0070] 4. GPX4 expression detection: The protein level of GPX4 was detected by Western blot. GPX4 is a key molecule for ferroptosis inhibition, and its downregulation indicates the occurrence of ferroptosis.

[0071] The results are as follows Figure 8 As shown in the figure, the expression of GPX4 protein in cells was significantly decreased after OTUD4 knockout. GPX4 is a key inhibitory molecule of ferroptosis. This change further confirms the inhibitory role of OTUD4 in the ferroptosis process.

[0072] Taken together, these results indicate that OTUD4 promotes the survival of colorectal cancer cells by inhibiting the ferroptosis pathway, and knocking out OTUD4 can significantly enhance the occurrence of ferroptosis, thereby inhibiting the growth of tumor cells.

[0073] Example 3: Animal Experimental Verification of Targeted Inhibition of OTUD4 The OTUD4 knockout LoVo cells and wild-type cells constructed in Example 1 were used to form tumors subcutaneously in nude mice. After the tumors were formed, they were randomly divided into 2 groups, for a total of 4 groups. One group of wild-type and OTUD4 knockout mice was selected to be treated with Erastin only, and the tumor growth volume was measured regularly. When the experimental endpoint was reached, the mice were killed, the tumor tissue was peeled out and photographed, and the tumor mass was weighed; after fixation with 4% paraformaldehyde, the sections were embedded and Ki-67 staining was performed to evaluate the proliferation of the tumor.

[0074] The results are as follows Figure 9-11 As shown, the tumor volume and mass of the Erastin monotherapy group were significantly reduced, and the Ki-67 immunohistochemistry results showed that the proliferation of tumor cells was significantly inhibited, indicating that Erastin can inhibit tumor proliferation. However, in the OTUD4 inhibition combined with Erastin treatment group, the tumor volume and mass were significantly smaller than those of other control groups and the Erastin monotherapy group. Further Ki-67 staining results showed that the tumor proliferation rate of the combined treatment group was significantly reduced, and the inhibitory effect was significantly better than that of the Erastin monotherapy group, indicating that targeted inhibition of OTUD4 can activate the occurrence of ferroptosis in tumors.

[0075] Example 4: Cell experiment verification of combined treatment regimen 1. Cell Model Human colorectal cancer cell lines LoVo and RKO were selected as experimental models. The cells were divided into the following groups: control group: no treatment, only culture medium was added; regorafenib group: cells were treated with 20 μM regorafenib; OTUD4 inhibition group: OTUD4 knockout cells were used for experiments; combined treatment group: cells were treated with combined OTUD4 knockout and regorafenib.

[0076] 2. Experimental steps 2.1 Cell culture: LoVo and RKO cells were cultured in DMEM medium containing 10% FBS until the logarithmic growth phase. 20 μM Regorafenib or 20 μM Regorafenib plus 10 μM Ferrostatin-1 (ferroptosis inhibitor) were added and incubated for 24 hours.

[0077] 2.2 Cell proliferation detection: CCK8 method was used to detect the proliferation of cells in each group.

[0078] 2.3 Ferroptosis detection: The level of ferroptosis was assessed by C11-BODIPY staining and ROS level determination.

[0079] 3. Results Analysis like Fig.12 and Fig.13 As shown in the figure, in the combined treatment group, the cell death rate increased significantly. Specifically, in LoVo cells, the cell death rate was about 20% when treated with regorafenib alone, while the cell death rate reached 35% after knocking out OTUD4 and treating with regorafenib, and reached about 50% in RKO cells, indicating that OTUD4 knockout can enhance the anti-tumor effect of regorafenib.

[0080] like Figure 14-17 As shown, lipid peroxidation (LPO) and reactive oxygen species (ROS) levels were significantly increased in the combined treatment group, indicating that OTUD4 knockout not only enhanced the anti-tumor effect of regorafenib, but also further promoted tumor cell death by promoting the activation of the ferroptosis pathway.

[0081] The CDI values ​​were calculated. In LoVo cells, the CDI values ​​of lipid peroxidation (LPO) levels were 0.62, and the CDI values ​​of reactive oxygen species (ROS) levels were 0.03; in RKO cells, the CDI values ​​of LPO levels were 0.75, and the CDI values ​​of ROS levels were 0.04.

[0082] These results indicate that the combined use of OTUD4 inhibition and regorafenib can synergistically increase the level of intracellular oxidative stress and significantly enhance the ferroptosis response of tumor cells, thereby exerting a stronger anti-tumor effect.

[0083] Example 5: Animal Experimental Verification of Combined Therapy 1. Animal Model The constructed LoVo OTUD4 knockout cells and wild-type cells were used to form subcutaneous tumors in nude mice, and then the nude mice were divided into a control group and a regorafenib group. Ten days after subcutaneous tumor formation, regorafenib was administered by oral gavage once a day for 10 days, and the tumor volume was measured.

[0084] 2. Immunohistochemical detection of subcutaneous tumors When the experiment endpoint was reached, the mice were killed, the tumor tissues were removed and photographed, and the tumor mass was weighed; the tumors were fixed with 4% paraformaldehyde, embedded and sliced, and Ki-67 staining was performed to evaluate the proliferation of the tumor.

[0085] 3. Results Analysis like Figure 18-Figure 20 As shown in the figure, the tumor volume and mass of the regorafenib monotherapy group were significantly reduced, and the Ki-67 immunohistochemistry results showed that the proliferation of tumor cells was significantly inhibited, indicating that regorafenib has the effect of inhibiting tumor proliferation. However, in the OTUD4 inhibition combined with regorafenib treatment group, the tumor volume and mass were significantly smaller than those of the other control groups, the OTUD4 knockout alone treatment group, and the regorafenib alone treatment group. Further Ki-67 staining results showed that the tumor proliferation rate of the combined treatment group was significantly reduced, and the inhibitory effect was significantly better than that of the regorafenib monotherapy group.

[0086] The above results indicate that the combined treatment of OTUD4 inhibition and regorafenib can more effectively inhibit tumor growth through synergistic effects, thereby exerting a stronger anti-tumor effect.

[0087] Example 6: Experimental verification of drug-resistant cell lines for combined treatment regimen 1. Cell Model The human colorectal cancer cell line LoVo was selected as the experimental model. When the cells in the logarithmic growth phase grew to a confluence of 80-90%, 20 μM regorafenib was added and placed in an incubator at 37°C and 5% CO2 for overnight culture. When the cell density reached 50%, the culture medium was discarded, the cells were rinsed twice with PBS, and the medium without drugs was replaced for continued culture. When the density reached 80% again, the above drug treatment was repeated 5-6 times until the cells could grow stably at the final drug concentration.

[0088] Detection of IC of drug-resistant cell lines 50 , calculate the resistance index (RI), RI = IC of the resistant cell line 50 / IC of parental cell line 50 .

[0089] 2. Western Blot Detection of Drug-Resistant Cell Lines When the drug-resistant cell lines reached the drug resistance requirement, the total proteins of the drug-resistant cells and parental cells were extracted for Western Blot detection of the expression levels of OTUD4 and GPX4 to evaluate the expression level of OTUD4 in the drug-resistant cell lines.

[0090] 3. Results Analysis like Fig.21 As shown in the CCK8 experiment results, the IC 50 It is 13.02 μM, RI=2.2, which meets the requirements of drug-resistant cell lines.

[0091] like Fig. 22 As shown in the figure, the results of Western Blot detection showed that the expression level of OTUD4 in the resistant cell line was significantly higher than that in the parental cells, indicating that OTUD4 may be closely related to the occurrence of regorafenib resistance.

[0092] In summary, the present invention proposes a novel combined treatment scheme for treating tumors by combining targeted inhibition of OTUD4 with regorafenib. Through targeted inhibition of the ferroptosis inhibitor molecule OTUD4, the present invention successfully induced the occurrence of ferroptosis in tumor cells, and significantly enhanced the sensitivity of tumor cells to the third-line treatment drug regorafenib. Especially in colorectal cancer and other tumors resistant to regorafenib, the strategy of combined targeted inhibition of OTUD4 shows great potential for clinical application. In the future, the development of OTUD4 inhibitors and combined treatment with regorafenib can become a new anti-tumor treatment option, not only limited to colorectal cancer, but also extended to other types of solid tumors, providing more accurate and effective treatment options for clinical treatment. This combined strategy provides a new treatment method for refractory and resistant tumors, which is expected to significantly improve the prognosis of tumor patients. This method not only provides a new strategy for cancer treatment, but also lays the foundation for the development of more accurate and effective targeted therapeutic drugs.

Claims

1. The use of OTUD4 as an inhibitory target in the preparation of a tumor ferroptosis enhancer, characterized in that: The enhancer promotes the occurrence of ferroptosis in tumor cells by inhibiting OTUD4 expression or causing OTUD4 functional loss.

2. The use according to claim 1, characterized in that The amino acid sequence of human OTUD4 is shown in SEQ ID NO.

1.

3. An anti-tumor combined pharmaceutical composition, characterized in that: The invention comprises an OTUD4 inhibitor and a ferroptosis inducer as active ingredients. The OTUD4 inhibitor targets and inhibits OTUD4 expression or causes OTUD4 functional loss, thereby promoting the occurrence of ferroptosis in tumor cells.

4. The combined pharmaceutical composition according to claim 3, characterized in that: The ferroptosis inducing agent is Erastin.

5. Use of the combined pharmaceutical composition according to claim 3 or 4 in the preparation of a drug for treating tumors, characterized in that: The tumor is a solid tumor.

6. The use according to claim 5, characterized in that The tumor is colorectal cancer.

7. An anti-tumor combined pharmaceutical composition, characterized in that: The invention comprises an OTUD4 inhibitor and regorafenib as active ingredients. The OTUD4 inhibitor targets and inhibits OTUD4 expression or causes OTUD4 functional loss, thereby promoting the activation of the ferroptosis pathway in tumor cells.

8. Use of the combined pharmaceutical composition according to claim 7 in the preparation of a drug for treating tumors, characterized in that: The tumor is a solid tumor.

9. The use according to claim 8, characterized in that The tumor is colorectal cancer.

10. Use of the combined pharmaceutical composition according to claim 7 in the preparation of a drug for treating regorafenib-resistant tumors.

Images