An anti-tumor combined pharmaceutical composition and its application

Abstract

The present invention discloses an anti-tumor combination pharmaceutical composition and its application, belonging to the field of biomedicine technology. The combination pharmaceutical composition comprises an OTUD4 inhibitor and a ferroptosis inducer or regorafenib 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. The combination pharmaceutical composition provided by the present invention more effectively inhibits tumor growth by synergistically increasing the level of intracellular oxidative stress, thereby exerting a stronger anti-tumor effect. The combined treatment regimen provided by the present invention provides a new approach to 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.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, 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 major challenges in cancer 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, treatment effectiveness is often limited and drug resistance is prone to occur. How to effectively inhibit tumor growth and overcome drug resistance has become a pressing issue.

[0003] Regorafenib is a targeted drug currently used to treat advanced colorectal and gastric cancers. It inhibits multiple kinases, including VEGFR and PDGFR, to suppress angiogenesis and tumor cell proliferation. However, in some drug-resistant tumors, regorafenib alone has limited therapeutic efficacy. Potentially enhancing the efficacy of regorafenib is a hot topic in current research.

[0004] In recent years, ferroptosis, an iron-dependent programmed cell death, has garnered widespread attention. Unlike traditional apoptosis, ferroptosis triggers lipid peroxidation, destroying the cell membrane and leading to cell death. Therefore, inducing ferroptosis has become a new avenue for anti-tumor therapy. However, tumor cells can evade ferroptosis under various stress conditions, allowing them to continue proliferating. In particular, ferroptosis-inhibiting signals are regulated by specific molecules to maintain cell survival (mitochondrial-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 research group's previous studies, OTUD4 (OTU domain-containing protein 4) was discovered as 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] Existing research reports indicate that OTUD4 plays different roles in different tumor types. 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 apoptosis and inhibiting the AKT signaling pathway (OTUD4: A Potential Prognosis Biomarker for Multiple Human Cancers). Cancer Management and Research , 2020,12:1503-1512.). Xiuqing Ma et al. found that the loss of OTUD4 in triple-negative breast cancer (TNBC) cells significantly inhibited cell clonogenicity, migration, invasion, and cancer stem cell populations in vitro and also significantly suppressed metastasis in vivo (Deubiquitinating enzyme OTUD4 regulates metastasis in triple-negative breast cancer by stabilizing Snail1). Experimental Cell Research , 2024, 434(1): 113864.).

[0007] Currently, there are no reports on the role of OTUD4 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 relevant 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 solutions:

[0010] This 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 ferroptosis in tumor cells by targetedly inhibiting OTUD4 expression or causing OTUD4 functional loss.

[0011] This study discovered that OTUD4 is a tumor ferroptosis inhibitor. Inhibiting OTUD4 can significantly promote ferroptosis in tumor cells and enhance their sensitivity to anti-tumor drugs. Therefore, OTUD4 could serve as a drug target for the development of tumor ferroptosis enhancers.

[0012] The technical means of 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 use inhibitory compounds to target and inhibit OTUD4 expression or cause OTUD4 loss of function.

[0013] 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.

[0014] The present invention also provides an anti-tumor combination pharmaceutical composition, comprising 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.

[0015] This study combines an OTUD4 inhibitor with a ferroptosis inducer. The OTUD4 inhibitor targets OTUD4 in tumor cells, enhancing the oxidative stress response associated with ferroptosis and amplifying the ferroptosis response, thereby inhibiting tumor cell growth. Studies have shown that the combination of an OTUD4 inhibitor and a ferroptosis inducer significantly reduces tumor proliferation compared to monotherapy.

[0016] Furthermore, the OTUD4 inhibitor may be, but is not limited to, targeting OTUD4 Gene knockout reagents.

[0017] Furthermore, the ferroptosis inducer may be, but is not limited to, Erastin.

[0018] The present invention also provides an anti-tumor combination pharmaceutical composition, comprising 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.

[0019] The present invention combines an OTUD4 inhibitor with regorafenib. The OTUD4 inhibitor targets and inhibits OTUD4 in tumor cells, not only promoting the activation of the ferroptosis pathway to inhibit tumor growth, but also significantly enhancing the anti-tumor effect of regorafenib through a dual-targeting mechanism. Studies have shown that compared with the monotherapy group, the combination therapy can synergistically increase intracellular oxidative stress levels and significantly enhance the ferroptosis response of tumor cells, thereby exerting a stronger anti-tumor effect. Mechanistic studies have shown that knocking out OTUD4 promotes the ubiquitination and degradation of GPX4, thereby promoting the activation of ferroptosis, and also promotes the degradation of RHEB, activating autophagy, which in turn promotes the autophagic degradation of GPX4 and promotes the occurrence of ferroptosis.

[0020] Furthermore, the anti-tumor combination pharmaceutical composition may further contain at least one pharmaceutically acceptable carrier.

[0021] 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.

[0022] The present invention provides the use of the combined pharmaceutical composition in preparing a drug for treating tumors, wherein the tumor is a solid tumor.

[0023] Furthermore, the tumor may be but is not limited to colorectal cancer.

[0024] The present invention also provides the use of a pharmaceutical composition combining an OTUD4 inhibitor and regorafenib in the preparation of a medicament for treating regorafenib-resistant tumors. Studies have shown that OTUD4 expression is upregulated in regorafenib-resistant tumor cells, and inhibiting OTUD4 expression can enhance tumor cell sensitivity to regorafenib.

[0025] Furthermore, the tumor is regorafenib-resistant colorectal cancer.

[0026] The present invention has the following beneficial effects:

[0027] (1) The present invention discloses for the first time that OTUD4 is a tumor ferroptosis inhibitory molecule. By targeting and inhibiting OTUD4, ferroptosis in tumor cells can be induced.

[0028] (2) The present invention provides an anti-tumor combination pharmaceutical composition, which is composed of an OTUD4 inhibitor and a ferroptosis inducer or regorafenib. By synergistically increasing the level of intracellular oxidative stress, it can more effectively inhibit tumor growth, thereby exerting a stronger anti-tumor effect.

[0029] (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

[0030] Figure 1 Western Blot verification of OTUD4 knockout efficiency in LoVo and RKO cells.

[0031] Figure 2 Comparison of the viability of LoVo cells WT and OTUD4 KO after treatment with different concentrations of the ferroptosis inducer Erastin.

[0032] Figure 3Comparison of the viability of RKO cells WT and OTUD4 KO after treatment with different concentrations of the ferroptosis inducer Erastin.

[0033] Figure 4 Flow cytometry was used to detect the level of LPO in LoVo cells after treatment with Erastin.

[0034] Figure 5 Flow cytometry was used to detect the level of LPO in RKO cells after erastin treatment.

[0035] Figure 6 Flow cytometry was used to detect the ROS level in LoVo cells after Erastin treatment.

[0036] Figure 7 Flow cytometry was used to detect the ROS levels in RKO cells after Erastin treatment.

[0037] Figure 8 Western Blot was used to detect the expression level of GPX4 after OTUD4 knockout.

[0038] Figure 9 This is the effect of OTUD4 knockout combined with Erastin treatment on mouse tumors.

[0039] Figure 10 for Figure 9 Figure 2. Effect of tumor volume and tumor weight on statistical graphs.

[0040] Figure 11 IHC was used to detect Ki-67, a proliferation marker of mouse tumors after OTUD4 knockout and Erastin treatment.

[0041] Figure 12 Comparison of the viability of LoVo cells WT and OTUD4 KO after regorafenib treatment.

[0042] Figure 13 Comparison of the viability of RKO cells WT and OTUD4 KO after regorafenib treatment.

[0043] Figure 14 Flow cytometry was used to detect the level of LPO in LoVo cells after regorafenib treatment.

[0044] Figure 15 Flow cytometry was used to detect the LPO level in RKO cells after regorafenib treatment.

[0045] Figure 16 Flow cytometry was used to detect the ROS levels in LoVo cells after regorafenib treatment.

[0046] Figure 17 Flow cytometry was used to detect the ROS levels in RKO cells after regorafenib treatment.

[0047] Figure 18 Effects of OTUD4 knockout combined with regorafenib treatment on mouse tumors.

[0048] Figure 19 for Figure 18 Figure 2. Effect of tumor volume and tumor weight on statistical graphs.

[0049] Figure 20 IHC detection of Ki-67, a proliferation marker of mouse tumors after OTUD4 knockout and regorafenib treatment.

[0050] Figure 21 IC of regorafenib-resistant cell lines 50 .

[0051] Figure 22 represents the expression levels of OTUD4 and GPX4 in regorafenib-resistant cell lines. DETAILED DESCRIPTION

[0052] The present invention will be further described below in conjunction with specific examples. The following examples 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 fall within the scope of the present invention.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0054] Example 1: Preparation and screening of OTUD4 knockout cells

[0055] 1. Construction of OTUD4 knockout plasmid

[0056] 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 impact on ferroptosis of tumor cells was evaluated.

[0057] 1.1 Use online tools (such as the CRISPR Design Tool) to design specific sgRNA sequences for the human OTUD4 gene (Gene ID: 54726). Select target sequences located in the exon region of OTUD4 to ensure high specificity and low off-target effects. Design two different sgRNAs, one targeting each of the two different sites in the OTUD4 gene to ensure effective knockout. Designed sgRNA sequences:

[0058] sgRNA1: 5'-CACCGCAGTAAGCCGGACGAAGGC-3' (SEQ ID NO. 2);

[0059] sgRNA2: 5'-CACCGATTCAGAACAGAGATGAAC-3' (SEQ ID NO. 3).

[0060] 1.2 sgRNA insertion into plasmid vector

[0061] Digest the CRISPR / Cas9 plasmid (e.g., PX330) with BbsI to remove the pre-existing sgRNA placeholder sequence. Synthesize an oligonucleotide pair containing the designed sgRNA sequence, anneal to create a double-stranded oligonucleotide, and ligate it into the linearized PX330 vector to construct the sgRNA-carrying CRISPR / Cas9 plasmid.

[0062] 1.3 Plasmid amplification and identification

[0063] Transform the constructed plasmid into competent E. coli and screen for positive clones. Pick a single clone and perform sequencing to verify the correct insertion of the sgRNA.

[0064] 2. Plasmid transfection and gene knockout

[0065] 2.1 Cell transfection

[0066] The constructed CRISPR / Cas9-sgRNA plasmid was transfected into LoVo and RKO cells (both purchased from ATCC) using Lipofectamine 3000 transfection reagent. Forty-eight hours after transfection, positive clones were screened using puromycin-containing medium. Following screening, positive cells were subjected to limiting dilution to isolate single clones.

[0067] 2.2 Gene knockout verification

[0068] Western blot analysis: Western blot was further used to verify whether OTUD4 protein expression disappeared, thereby confirming the knockout effect of the OTUD4 gene.

[0069] 3. Results Analysis

[0070] The results are as follows Figure 1 As shown in this example, OTUD4 gene knockout colorectal cancer cell lines LoVo and RKO were successfully constructed. Western blot analysis confirmed the complete loss of OTUD4 protein expression in both cell lines, confirming the successful OTUD4 knockout. This provides an effective cell model for subsequent studies on the function of OTUD4 in tumor ferroptosis.

[0071] Example 2: Detection of Ferroptosis by Targeted Inhibition of OTUD4

[0072] After confirming OTUD4 knockout, the following method was used to detect the effect of OTUD4 gene knockout on ferroptosis of tumor cells.

[0073] 1. Cell viability assay: Cells were treated with the ferroptosis inducer Erastin to detect cell viability and compare the changes in cell viability after OTUD4 knockout.

[0074] The results are as follows Figure 2 and Figure 3 As shown in Figure 3, 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.

[0075] The interaction coefficient (CDI) for OTUD4 knockout combined with erastin treatment was calculated using the following formula: CDI = AB / A × B. Calculated based on the number of viable cells (absorbance), 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-only treatment group to the control group. A CDI < 1 indicates synergistic interaction between the two drugs, while a CDI < 0.7 indicates highly significant synergistic interaction. A CDI = 1 indicates additive interaction between the two drugs, and a CDI > 1 indicates antagonism.

[0076] The calculation results showed that the CDI value in LoVo cells was 0.64; the CDI value in RKO cells was 0.73.

[0077] 2. C11-BODIPY staining: Detect intracellular lipid peroxidation (LPO) levels. Increased LPO levels are one of the hallmarks of ferroptosis.

[0078] The results are as follows Figure 4 and Figure 5 As shown, the lipid peroxidation level in OTUD4 knockout cells was significantly increased after erastin treatment, indicating that the loss of OTUD4 enhanced the oxidative stress response associated with ferroptosis.

[0079] The CDI values ​​were calculated and normalized using the ratio of groups AB to the control group. That is, the ratios of groups AB, A, and B to the control group were divided by the value of group AB, and the normalized values ​​were used for calculation. The results showed that the CDI value in LoVo cells was 0.63, and the CDI value in RKO cells was 0.44.

[0080] 3. Reactive oxygen species (ROS) staining: Detect the level of ROS in cells. Elevated ROS levels are one of the hallmarks of ferroptosis.

[0081] The results are as follows Figure 6 and Figure 7 As shown, the level of reactive oxygen species in OTUD4 knockout cells was significantly increased after erastin treatment, indicating that the loss of OTUD4 enhanced the oxidative stress response associated with ferroptosis.

[0082] The CDI values ​​were calculated and the results showed that the CDI value in LoVo cells was 0.44; the CDI value in RKO cells was 0.40.

[0083] 4. GPX4 expression detection: Western blot was used to detect the protein level of GPX4. GPX4 is a key molecule for ferroptosis inhibition, and its downregulation indicates the occurrence of ferroptosis.

[0084] The results are as follows Figure 8 As shown in the results, 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.

[0085] Taken together, these results indicate that OTUD4 promotes the survival of colorectal cancer cells by inhibiting the ferroptosis pathway. Knocking out OTUD4 can significantly enhance the occurrence of ferroptosis, thereby inhibiting the growth of tumor cells.

[0086] Example 3: Animal Experimental Verification of Targeted Inhibition of OTUD4

[0087] The OTUD4 knockout LoVo cells and wild-type cells constructed in Example 1 were used to establish subcutaneous tumors in nude mice. After tumor formation, the mice were randomly divided into two groups, for a total of four groups. One group, each of the wild-type and OTUD4 knockout cells, was treated with erastin alone. Tumor growth volume was measured regularly. At the experimental endpoint, the mice were sacrificed, and the tumor tissue was removed, photographed, and weighed. After fixation with 4% paraformaldehyde, the tumors were embedded and sectioned, and Ki-67 staining was performed to assess tumor proliferation.

[0088] The results are as follows Figures 9-11As shown, tumor volume and mass were significantly reduced in the erastin-only treatment group, and Ki-67 immunohistochemistry revealed that tumor cell proliferation was significantly suppressed, indicating that erastin can inhibit tumor growth. However, in the OTUD4 inhibition combined with erastin treatment group, tumor volume and mass were significantly smaller than those in the other control groups and the erastin-only treatment group. Further Ki-67 staining revealed that the tumor proliferation rate in this combination treatment group was significantly reduced, with a significantly better inhibitory effect than that in the erastin-only treatment group, indicating that targeted inhibition of OTUD4 can activate ferroptosis in tumors.

[0089] Example 4: Cell experiment verification of combined treatment regimen

[0090] 1. Cell Model

[0091] Human colorectal cancer cell lines LoVo and RKO were used as experimental models. The cells were divided into the following groups: control group (no treatment, culture medium only); regorafenib group (20 μM regorafenib treatment); OTUD4 inhibition group (OTUD4 knockout cells); and combined treatment group (OTUD4 knockout and regorafenib combined treatment).

[0092] 2. Experimental steps

[0093] 2.1 Cell Culture: LoVo and RKO cells were cultured in DMEM supplemented with 10% FBS until the logarithmic growth phase. 20 μM regorafenib or 20 μM regorafenib plus 10 μM ferrostatin-1 (a ferroptosis inhibitor) was added and incubated for 24 hours.

[0094] 2.2 Cell proliferation detection: CCK8 method was used to detect the proliferation of cells in each group.

[0095] 2.3 Ferroptosis detection: Ferroptosis levels were assessed by C11-BODIPY staining and ROS level determination.

[0096] 3. Results Analysis

[0097] like Figure 12 and Figure 13 As shown, the combination treatment group showed a significant increase in cell death rate. Specifically, in LoVo cells, regorafenib alone resulted in approximately 20% cell death rate, while OTUD4 knockout and regorafenib treatment increased cell death rate to 35%, and in RKO cells, it reached approximately 50%, indicating that OTUD4 knockout can enhance the anti-tumor effect of regorafenib.

[0098] like Figure 14-17As shown, the levels of lipid peroxidation (LPO) and reactive oxygen species (ROS) were significantly increased in the combined treatment group, suggesting 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.

[0099] 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.

[0100] 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.

[0101] Example 5: Animal Experimental Verification of Combined Therapy

[0102] 1. Animal Model

[0103] Subcutaneous tumors were established in nude mice using LoVo OTUD4 knockout cells and wild-type cells. The mice were then divided into a control group and a regorafenib group. Ten days after subcutaneous tumor formation, regorafenib was administered via oral gavage once daily for 10 days, and tumor size was measured.

[0104] 2. Immunohistochemistry detection of subcutaneous tumors

[0105] When the experiment reached its end point, 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 in sections, and Ki-67 staining was performed to evaluate tumor proliferation.

[0106] 3. Results Analysis

[0107] like Figures 18-20 As shown, tumor volume and mass were significantly reduced in the regorafenib monotherapy group, and Ki-67 immunohistochemistry revealed a significant suppression of tumor cell proliferation, indicating that regorafenib inhibits tumor growth. However, in the OTUD4 inhibition combined with regorafenib treatment group, tumor volume and mass were significantly smaller than those in the other control groups, the OTUD4 knockout alone group, and the regorafenib monotherapy group. Further Ki-67 staining revealed a significant reduction in tumor proliferation in the combined treatment group, demonstrating a significantly superior inhibitory effect compared to the regorafenib monotherapy group.

[0108] 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.

[0109] Example 6: Experimental verification of drug-resistant cell lines for combined treatment regimen

[0110] 1. Cell Model

[0111] The human colorectal cancer cell line LoVo was used as an experimental model. When cells in the logarithmic growth phase reached 80-90% confluence, 20 μM regorafenib was added and the cells were cultured overnight in a 37°C, 5% CO2 incubator. When the cell density reached 50%, the culture medium was discarded, the cells were rinsed twice with PBS, and then replaced with drug-free medium for continued culture. When the cell density again reached 80%, the drug treatment was repeated 5-6 times until the cells could stably grow at the final drug concentration.

[0112] 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 .

[0113] 2. Western Blot Detection of Drug-Resistant Cell Lines

[0114] 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.

[0115] 3. Results Analysis

[0116] like Figure 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.

[0117] like Figure 22 As shown in the figure, Western Blot detection results 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.

[0118] In summary, the present invention proposes a novel combined therapy for the treatment of tumors by targeting the inhibition of OTUD4 in combination with regorafenib. Through targeted inhibition of the ferroptosis inhibitory molecule OTUD4, the present invention successfully induced ferroptosis in tumor cells while significantly enhancing the sensitivity of tumor cells to the third-line treatment drug regorafenib. The combined strategy of targeted inhibition of OTUD4 has shown great potential for clinical application, especially in colorectal cancer and other tumors resistant to regorafenib. In the future, the development of OTUD4 inhibitors and combined treatment with regorafenib could become new anti-tumor treatment options, not only limited to colorectal cancer, but also extended to other types of solid tumors, providing more precise and effective treatment options for clinical treatment. This combined strategy provides a new treatment approach for refractory and drug-resistant tumors and is expected to significantly improve the prognosis of cancer patients. This approach not only provides a new strategy for cancer treatment but also lays the foundation for the development of more precise and effective targeted therapeutic drugs.

Claims

1. The use of OTUD4 as a substance that inhibits target sites in the preparation of a tumor ferroptosis enhancer, characterized in that: The substance that targets OTUD4 as an inhibitory target is targeted OTUD4 A gene knockout reagent for a gene; the gene knockout reagent is a CRISPR / Cas9 reagent, wherein the sgRNA sequence is: 5'-CACCGCAGTAAGCCGGACGAAGGC-3'; 5'-CACCGATTCAGAACAGAGATGAAC-3'; the synergist promotes the occurrence of ferroptosis in tumor cells by targeted inhibition of OTUD4 expression or causing loss of OTUD4 function; the tumor cells are colorectal cancer cell lines LoVo or RKO.

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: Includes targeted OTUD4 A gene knockout reagent and a ferroptosis inducer for a gene; the gene knockout reagent is a CRISPR / Cas9 reagent, wherein the sgRNA sequence is: 5'-CACCGCAGTAAGCCGGACGAAGGC-3'; 5'-CACCGATTCAGAACAGAGATGAAC-3'; the ferroptosis inducer is Erastin; the tumor is colorectal cancer, and the cell line is LoVo or RKO.

4. An anti-tumor combined pharmaceutical composition, characterized in that: Includes targeted OTUD4 A gene knockout reagent and regorafenib; the gene knockout reagent is a CRISPR / Cas9 reagent, wherein the sgRNA sequence is: 5'-CACCGCAGTAAGCCGGACGAAGGC-3'; 5'-CACCGATTCAGAACAGAGATGAAC-3'; the tumor is colorectal cancer, and the cell line is LoVo or RKO.

5. Use of the combined pharmaceutical composition according to claim 4 in the preparation of a drug for treating tumors, characterized in that: The tumor is colorectal cancer, and the cell line is LoVo or RKO.

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