Application of LACTB in preparation of medicine for treating liver cancer

Abstract

The invention discloses application of LACTB in preparation of a medicine for treating liver cancer. LACTB promotes ferroptosis by adjusting a p53 / HSPA8 axis and inhibits the progress of liver cancer, and the LACTB can also improve the sensitivity of liver cancer cells to the lenvatinib. And a new strategy can be provided for evaluating liver cancer treatment prognosis, checking drug adaptability and treating liver cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer treatment drugs, and particularly to drugs for treating liver cancer. Background Art

[0002] Liver cancer is the sixth most commonly diagnosed cancer globally and the third leading cause of cancer-related deaths. Due to the lack of specific clinical manifestations in the early stage, most patients are diagnosed with advanced liver cancer. Only 5%-15% of patients are eligible for surgical resection, and chemotherapy and immunotherapy are the best treatment options for most patients.

[0003] Lenvatinib is an oral small molecule tyrosine kinase inhibitor that can be used as a first-line targeted therapy for unresectable liver cancer instead of sorafenib. Lenvatinib exerts its anti-tumor effect by targeting multiple molecules (such as vascular endothelial growth factor receptors and members of the epidermal growth factor receptor family). Although lenvatinib has shown excellent efficacy in the clinical treatment of liver cancer, drug resistance is inevitable in cancer targeted therapy. Therefore, elucidating the key molecular events driving liver cancer progression will provide new targets for clinical treatment and may potentially develop combination therapies to improve the efficacy of lenvatinib.

[0004] Ferroptosis is a new type of regulated cell death form, which is different from apoptosis, necrosis, heat denaturation, and autophagy. It is an iron-dependent cell death form caused by the accumulation of lipid peroxides mediated by reactive oxygen species. Iron balance in the body is strictly regulated. Intracellular iron is stored in ferritin, and the selective cargo receptor NCOA4 can directly bind to ferritin, promoting the autophagy-lysosomal degradation of ferritin and releasing iron simultaneously. This process is called ferroptophagy. The ferroptophagy mediated by NCOA4 is necessary to maintain intracellular and systemic iron balance. Excessive free iron will trigger the Fenton reaction, promoting the production of lipid peroxidation and thus initiating ferroptosis. There are multiple antioxidant systems in cells to resist iron poisoning, among which the SLC7A11 (usually also called xCT) / glutathione (GSH) / GPX4 axis has been the most widely studied. SLC7A11 on the cell membrane transports extracellular cystine into the cell, then reduces it to cysteine for the synthesis of GSH. Subsequently, GPX4 uses GSH as a cofactor to detoxify lipid peroxides into lipid alcohols, thereby inhibiting ferroptosis. The SLC7A11 / GSH / GPX4 antioxidant axis is often dysregulated and is crucial for tumor development.

[0005] Serine beta-lactamase-like protein (LACTB) is an active-site serine protease that has recently attracted attention due to its potential roles in lipid metabolism and tumorigenesis. Its role as a tumor suppressor or oncogene depends on the context. Low expression of LACTB has been observed in glioblastoma, melanoma, ovarian cancer, breast cancer, and colorectal cancer. Overexpression of LACTB inhibits the proliferation, migration, and invasion of cancer cells. In contrast, LACTB acts as an oncogene in pancreatic adenocarcinoma and nasopharyngeal carcinoma by regulating different signaling pathways. Notably, a study has proposed that LACTB is downregulated in liver cancer, which is associated with a poor prognosis of liver cancer; however, the mechanism of action of LACTB has not been elucidated. In addition, it has been reported that LACTB can promote erastin-induced ferroptosis and mitochondrial dysfunction in bladder cancer cells, but it is currently unclear whether LACTB is an intrinsic trigger for ferroptosis. Summary of the Invention

[0006] The object of the present invention is to provide the application of LACTB in the preparation of a drug for treating liver cancer, so as to provide a new drug for treating liver cancer.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] Application of LACTB in the preparation of a drug for treating tumors.

[0009] Application of LACTB in the preparation of a drug for treating tumors with the characteristic of LACTB downregulation.

[0010] Application of LACTB in the preparation of a drug for treating liver cancer.

[0011] Application of LACTB in the preparation of a drug for treating liver cancer with the characteristic of LACTB downregulation.

[0012] Application of LACTB in the preparation of a kit for evaluating the prognosis of liver cancer treatment.

[0013] Application of LACTB in the preparation of a kit for evaluating the adaptability of lenvatinib in treating liver cancer.

[0014] The advantages of the present invention are as follows: LACTB promotes ferroptosis by regulating the p53 / HSPA8 axis, inhibits the progression of liver cancer, and LACTB can also improve the sensitivity of liver cancer cells to lenvatinib. It can provide new strategies for evaluating the prognosis of liver cancer treatment, drug adaptability examination, and treating liver cancer. Brief Description of the Drawings

[0015] The drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and do not constitute an improper limitation of the present invention. In the drawings:

[0016] Figure 1-1 It is one of the experimental figures showing LACTB inhibiting the progression of liver cancer;

[0017] Figure 1-2 It is the second experimental figure showing LACTB inhibiting the progression of liver cancer;

[0018] Figure 1-3 It is the third experimental figure showing LACTB inhibiting the progression of liver cancer;

[0019] Figure 2-1 It is the fourth experimental figure showing LACTB inhibiting the progression of liver cancer;

[0020] Figure 2-2 It is the fifth experimental figure showing LACTB inhibiting the progression of liver cancer;

[0021] Figure 3-1 It is one of the experimental figures showing LACTB as a driver of ferroptosis;

[0022] Figure 3-2 It is the second experimental figure showing LACTB as a driver of ferroptosis;

[0023] Figure 3-3 It is the third experimental figure showing LACTB as a driver of ferroptosis;

[0024] Figure 3-4 It is the fourth experimental figure showing LACTB as a driver of ferroptosis;

[0025] Figure 3-5 It is the fifth experimental figure showing LACTB as a driver of ferroptosis;

[0026] Figure 3-6 It is the sixth experimental figure showing LACTB as a driver of ferroptosis;

[0027] Figure 3-7 It is the seventh experimental figure showing LACTB as a driver of ferroptosis;

[0028] Figure 3-8 It is the eighth experimental figure showing LACTB as a driver of ferroptosis;

[0029] Figure 4-1 It is one of the experiments showing LACTB inducing ferroptosis;

[0030] Figure 4-2 It is the second experiment showing LACTB inducing ferroptosis;

[0031] Figure 4-3 It is the third experiment showing LACTB inducing ferroptosis;

[0032] Figure 4-4 It is the fourth experiment showing LACTB inducing ferroptosis;

[0033] Figure 4-5 This is the fifth experiment on LACTB-induced ferroptosis;

[0034] Figure 4-6 This is the sixth experiment on LACTB-induced ferroptosis;

[0035] Figure 5-1 This is one of the experimental figures showing that HSPA8 is a target of LACTB;

[0036] Figure 5-2 This is the second experimental figure showing that HSPA8 is a target of LACTB;

[0037] Figure 5-3 This is the third experimental figure showing that HSPA8 is a target of LACTB;

[0038] Figure 5-4 This is the fourth experimental figure showing that HSPA8 is a target of LACTB;

[0039] Figure 5-5 This is the fifth experimental figure showing that HSPA8 is a target of LACTB;

[0040] Figure 6-1 This is one of the experimental figures showing that LACTB regulates HSPA8;

[0041] Figure 6-2 This is the second experimental figure showing that LACTB regulates HSPA8;

[0042] Figure 6-3 This is the third experimental figure showing that LACTB regulates HSPA8;

[0043] Figure 7-1 This is one of the experimental figures showing that LACTB regulates HSPA8 through p53;

[0044] Figure 7-2 This is the second experimental figure showing that LACTB regulates HSPA8 through p53;

[0045] Figure 7-3 This is the third experimental figure showing that LACTB regulates HSPA8 through p53;

[0046] Figure 7-4 This is the fourth experimental figure showing that LACTB regulates HSPA8 through p53;

[0047] Figure 8-1 This is one of the experimental figures showing that LACTB drives ferroptosis by regulating the p53 / HSPA8 axis;

[0048] Figure 8-2 This is the second experimental figure showing that LACTB drives ferroptosis by regulating the p53 / HSPA8 axis;

[0049] Figure 8-3 It is the third experimental figure showing that LACTB drives ferroptosis by regulating the p53 / HSPA8 axis;

[0050] Figure 8-4 It is the fourth experimental figure showing that LACTB drives ferroptosis by regulating the p53 / HSPA8 axis;

[0051] Figure 8-5 It is the fifth experimental figure showing that LACTB drives ferroptosis by regulating the p53 / HSPA8 axis;

[0052] Figure 9-1 It is one of the experimental figures showing that LACTB enhances the response of cancer to cabozantinib;

[0053] Figure 9-2 It is the second experimental figure showing that LACTB enhances the response of cancer to cabozantinib;

[0054] Figure 9-3 It is the third experimental figure showing that LACTB enhances the response of cancer to cabozantinib;

[0055] Figure 9-4 It is the fourth experimental figure showing that LACTB enhances the response of cancer to cabozantinib;

[0056] Figure 9-5 It is the fifth experimental figure showing that LACTB enhances the response of cancer to cabozantinib;

[0057] Figure 10 It is the experimental figure showing the ferroptosis-promoting effect of LACTB on cabozantinib. Detailed implementation mode

[0058] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Here, the schematic embodiments and descriptions of the present invention are used to explain the present invention, but do not limit the present invention.

[0059] 1. LACTB is a tumor suppressor in liver cancer

[0060] The expression of LACTB in 10 pairs of fresh frozen liver cancer tissues and adjacent normal tissues was detected. The results are as Figure 1-1 shown in A (N is Normal, T is Tumor). It was found that the LACTB protein in liver cancer tissues was significantly downregulated compared with adjacent normal tissues. In addition, paraffin-embedded tissues were collected for IHC staining. The results are as Figure 1-1 shown in B, and the expression of LACTB was low in liver cancer tissues. As Figure 1-1 shown in C, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database also showed a decrease in the expression level of LACTB protein in liver cancer tissues. As Figure 1-1 D, Figure 2-1As shown in A, SK-HEP-1 and HepG2 cell lines expressing LACTB were constructed, and the results are as Figure 1-2 E, Figure 2-1 B, Figure 1-2 F show that ectopic expression of LACTB significantly inhibits cell viability and colony formation. As Figure 1-2 G, Figure 2-1 C shows, the LACTB- / - cell line was constructed using the CRISPR / Cas9 gene editing technology. The results are as Figure 1-2 H, Figure 2-1 D, Figure 1-2 I show that after knocking out LACTB, cell viability and colony formation were significantly enhanced. To verify the role of LACTB in vivo, we used a xenograft tumor model. HepG2 cells were subcutaneously injected into mice, and no tumor formation was observed after one month. Therefore, SK-HEP-1 cells were used instead of HepG2 cells, and the results are as Figure 1-3 J show that overexpression of LACTB leads to a reduction in tumor volume and weight, while as Figure 2-2 E shows, knocking out LACTB has the opposite effect.

[0061] 2. LACTB Induces Ferroptosis

[0062] As Figure 3-1 A shows, RNA sequencing was performed on SK-HEP-1 cells expressing LACTB, and the results are as Figure 3-2 B shows that a group of genes (841 up-regulated, 318 down-regulated) are related to LACTB, as Figure 3-2 C shows, and are involved in key pathways such as metabolic pathways, ferroptosis, and p53 signaling pathways. As Figure 3-3 D shows, gene set enrichment analysis (GSEA) indicates that high LACTB is significantly positively correlated with ferroptosis. It is inferred that LACTB inhibits cancer through ferroptosis. As Figure 3-3 E, Figure 3-3 F, Figure 4-1 A, Figure 3-4 G, Figure 3-4 H show that overexpression of LACTB significantly increased the levels of Fe 2+ , lipid peroxidation, and MDA, but decreased the GSH level. As Figure 3-4 I, Figure 3-4 J, Figure 4-1 B show that after overexpression of LACTB, the ferroptosis markers PTGS2 and 4HNE were significantly increased. On the contrary, as Figure 3-4 K, Figure 3-5 L, Figure 3-5 M, Figure 4-2 C, Figure 4-2 D-F show that compared with control cells, the levels of Fe in LACTB- / - cells 2+Levels of lipid peroxidation, PTGS2, MDA, and 4HNE were significantly reduced, while the content of GSH increased.

[0063] As Figure 4-3 Shown in G-H, the establishment of a mouse model confirmed the role of LACTB in promoting ferroptosis in vivo. As Figure 3-6 N-O, Figure 4-3 I, Figure 4-4 J shown, overexpression of LACTB made HepG2 and SK-HEP-1 cells more sensitive to ferroptosis inducers Erastin and RSL3, while as Figure 3-6 P-Q, Figure 4-4 K, Figure 4-5 L, Figure 4-5 M shown, knockdown of LACTB had the opposite effect. As Figure 3-7 R shown, the reduction in cell viability caused by LACTB was effectively rescued by ferrostatin-1 / liproxstatin-1 (an inhibitor of ferroptosis), deferoxamine (an iron chelator), and N-acetylcysteine (a ROS scavenger), but not by Z-VAD-FMK (a pan-caspase inhibitor) or Necrostatin-1 (another inhibitor of necroptosis). As Figure 4-6 As shown in N, the proportion of apoptotic cells increased slightly after overexpression of LACTB.

[0064] To make the results more clinically relevant, we established a PDX model and then injected an adeno-associated virus expression vector or LACTB into the tumor. The results are shown in Figure 3-7 S, overexpression of LACTB significantly inhibited tumor growth, and at the same time, as Figure 3-7 Shown in T-V, 3-8W, accompanied by an increase in Fe 2+ , PTGS2, and 4HNE and a downregulation of GSH.

[0065] 3. LACTB induces ferroptosis by inhibiting HSPA8

[0066] As Figure 3-1 Shown in B, the mRNA abundance of HSPA8 decreased most significantly after overexpression of LACTB. Given that HSPA8 blocks ferritin deposition by promoting SLC7A11 / GSH / GPX4 signaling and inhibiting NCOA4-mediated ferritinophagy, we speculated that HSPA8 might be a downstream target of LACTB. As Figure 5-1 Shown in A, overexpression and knockdown of LACTB decreased and increased the expression of HSPA8 mRNA, respectively. As Figure 5-1 B, Figure 6-1As shown in A, in HepG2 and SK-HEP-1 cells expressing LACTB, the protein levels of HSPA8, SLC7A11, GPX4, and FTH1 were decreased, while the protein level of NCOA4 was increased. Knockout of LACTB produced the opposite effect; however, as Figure 5-1 C, Figure 6-1 B shows, re-expression of LACTB in LACTB- / - cells eliminated these effects. As Figure 5-1 D, Figure 6-1 C shows, overexpression of HSPA8 blocked the decrease in the levels of SLC7A11, GPX4, and FTH1 and the increase in the level of NCOA4 caused by overexpression of LACTB. As Figure 5-1 E, Figure 6-1 D shows, after silencing HSPA8, the increase in SLC7A11, GPX4, and FTH1 and the decrease in NCOA4 induced by LACTB knockout were counteracted. As Figure 5-2 F-G, Figure 5-3 H-J, Figure 5-4 K, Figure 6-2 E-H show, the ferroptosis phenotypes caused by overexpression and knockout of LACTB were significantly eliminated by overexpression and silencing of HSPA8, respectively. As Figure 6-2 I shows, the TCGA and CPTAC databases showed that the levels of HSPA8 mRNA and protein were significantly increased in cancer tissues compared with normal tissues. As Figure 5-4 L shows, as shown by the Kaplan-Meier Plotter database, high expression of HSPA8 was associated with poor overall survival. Importantly, as Figure 5-5 M, Figure 6-3 J shows, overexpression of HSPA8 significantly affected the reduction in tumor volume and the increase in the levels of Fe 2+ , PTGS2, and 4HNE caused by overexpression of LACTB.

[0067] 4. LACTB inhibits HSPA8 and requires wild-type p53

[0068] As Figure 7-1 A-D show, in p53-mutant Huh7 and p53-deficient Hep3B cells, overexpression of LACTB did not affect Fe 2+ , lipid peroxidation, PTGS2, or erastin sensitivity. LACTB inhibits the progression of colorectal cancer in a wild-type p53-dependent manner; LACTB can directly bind to the p53 protein and inhibit p53 ubiquitination and degradation by preventing the interaction between MDM2 and p53. Therefore, it is hypothesized in this paper that the expression of LACTB in cancer is the same. In Huh7 and Hep3B cells expressing LACTB, as Figure 8-1 A, Figure 7-2As shown in E - F, there were no changes in HSPA8 mRNA and protein levels; as Figure 7-2 shown in G, knockout of endogenous p53 had no effect on the expression of HSPA8 in Huh7 cells. However, as Figure 8-1 shown in B - C, Figure 7-2 H, after p53 silencing, the decrease in HSPA8 caused by LACTB overexpression in HepG2 and SK - HEP - 1 cells carrying wild - type p53 was significantly improved, indicating that wild - type p53 is required for LACTB - mediated regulation of HSPA8. As Figure 8-1 shown in D, Figure 7-2 I, after LACTB overexpression in HepG2 and SK - HEP - 1 cells, the half - life of p53 was significantly prolonged, and at the same time, as Figure 8-1 shown in E, Figure 7-2 J, p53 ubiquitination decreased, which was consistent with our previous findings. As Figure 7-3 shown in K, we introduced p53 into p53 - deficient Hep3B cells and found that as Figure 8-1 shown in F, LACTB decreased the expression of HSPA8 in a dose - dependent manner.

[0069] To understand how HSPA8 mRNA is regulated by the LACTB / p53 axis, the promoter activity of HSPA8 was tested. The results of luciferase reporter gene assays showed that as Figure 8-2 shown in G, LACTB overexpression significantly inhibited HSPA8 promoter activity, while p53 knockout blocked this effect. By analyzing the sequence of the HSPA8 promoter, as Figure 8-2 shown in H, a highly conserved p53 - binding motif was found at - 2386 to - 2372. As Figure 8-2 shown in I, we designed a series of primers for chromatin immunoprecipitation (ChIP) assays, and the results as Figure 8-2 shown in J, Figure 7-3 L indicated that p53 directly binds to the HSPA8 promoter (p21, a well - known target of p53, was used as a positive control). As Figure 8-2 shown in K, Figure 7-3 M, DNA pull - down analysis showed that wild - type HSPA8 promoter probes significantly enriched p53 protein, but mutant probes (with a “CWWG” core mutation) did not enrich p53 protein. As Figure 7-3 shown in N - Figure 7-4 O, p53 overexpression significantly inhibited the transcription of the wild - type HSPA8 promoter, but did not affect the transcription of the mutant promoter. As Figure 8-3As shown in , we used single-stranded oligodeoxynucleotides (ssODNs) for CRISPR / Cas9 genome editing and generated SK-HEP-1 cells with endogenous mutations in the p53-binding site within the HSPA8 promoter (a four-nucleotide mutation from ACTGGCTTTGACAAG to AATGTCTTGATAAT). As Figure 8-3 M- Figure 8-4 As shown in , compared with wild-type cells, the mutant cells were less sensitive to the ferroptosis inducers erastin and RSL3, with lower levels of lipid peroxidation and Fe 2+ levels. As Figure 7-4 As shown in , p53 did not bind to the HSPA8 promoter in the mutant cells. As Figure 7-4 Q, Figure 8-4 As shown in , overexpression of p53 or LACTB had no effect on HSPA8 expression. As Figure 7-4 As shown in , the ferroptosis-promoting effect of LACTB was greatly attenuated in the mutant cells. Finally, we detected the expression of LACTB, wild-type p53, and HSPA8 using a tissue microarray containing 71 cancer cases. The results Figure 8-5 are shown in , indicating that LACTB was positively and negatively correlated with wild-type p53 (r = 0.545) and HSPA8 (r = -0.402), respectively.

[0070] 5. LACTB enhances the response of liver cancer to lenvatinib

[0071] The tyrosine kinase inhibitor lenvatinib is a potential ferroptosis inducer, and the above data suggest that LACTB induces ferroptosis. Next, we investigated whether there is a connection between LACTB and lenvatinib. As Figure 9-1 As shown in A– Figure 9-2 B, lenvatinib increased the levels of Fe 2+ and lipid peroxidation in a dose-dependent manner, indicating that lenvatinib can induce ferroptosis. As Figure 9-2 As shown in , the protein level of LACTB increased after treatment of HepG2 and SK-HEP-1 cells with lenvatinib. As Figure 9-3 As shown in D-G, Figure 9-4 H, the increases in Fe 2+ and lipid peroxidation levels and the decreases in cell viability and colony formation caused by lenvatinib were more obvious when LACTB was overexpressed, while LACTB knockdown blocked these effects.

[0072] Next, we evaluated the in vivo effect of LACTB on lenvatinib using a xenograft tumor model. When the subcutaneous tumor volume reached approximately 0.05–0.1 cm 3 , mice were treated with lenvatinib and injected with adeno-associated virus expressing LACTB or shLACTB intratumorally. The results Figure 9-4 H- Figure 9-4 I showed that overexpression and knockout significantly enhanced and weakened the anti-tumor efficacy of lenvatinib, respectively. As Figure 9-4 J, Figure 10 shown, compared with the control group, LACTB, p53, NCOA4, Fe 2+ , PTGS2 and 4HNE increased in the lenvatinib group, while as Figure 9-4 shown in J, HSPA8, SLC7A11, GPX4 and FTH1 decreased; as Figure 9-4 J, Figure 10 shown, these phenomena became obvious and weakened in the lenvatinib + LACTB and lenvatinib + sh LACTB groups, respectively.

[0073] In summary, compared with non-tumor cells, tumor cells have a higher demand for iron, which makes tumor cells more vulnerable to iron-catalyzed death, namely ferroptosis. Therefore, appropriately manipulating ferroptosis is considered a promising approach to combat cancer and improve efficacy, and it is particularly important to clarify the regulatory network of ferroptosis. The present invention identified an unknown intrinsic driver of cancer ferroptosis. As Figure 9-5 shown in K, LACTB stabilizes the p53 protein to inhibit HSPA8 transcription, thereby activating NCOA4-mediated ferritinophagy and inhibiting SLC7A11 / GSH / GPX4 signaling, thereby triggering ferroptosis and inhibiting liver tumorigenesis. The present invention demonstrates that LACTB is an intrinsic inducer of ferroptosis, and restoring LACTB may be a potentially effective method for treating cancer. In addition, LACTB promotes iron-deficient lung disease by regulating HSPA8-mediated ferroptosis-related signaling.

[0074] Lenvatinib induces ferroptosis. Lenvatinib increases the expression of LACTB in a dose-dependent manner. In a mouse model, adeno-associated virus-mediated overexpression of LACTB significantly enhanced the tumor-suppressive effect of lenvatinib, indicating that the combination of lenvatinib and LACTB may be helpful for treating cancer patients. LACTB is important in driving ferroptosis. Targeting the newly identified LACTB / p3 / HSPA8 axis may be an effective treatment strategy for cancer patients.

[0075] The technical solutions provided by the embodiments of the present invention have been introduced in detail above. Specific examples are used in this article to elaborate on the principles and implementation manners of the embodiments of the present invention. The descriptions of the above embodiments are only applicable to help understand the principles of the embodiments of the present invention; at the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. Use of LACTB in the preparation of a drug for treating tumors.

2. Use of LACTB in the preparation of a drug for treating tumors characterized by down - regulation of LACTB.

3. Use of LACTB in the preparation of a drug for treating liver cancer.

4. Use of LACTB in the preparation of a drug for treating liver cancer characterized by down - regulation of LACTB.

5. Use of LACTB in the preparation of a kit for evaluating the prognosis of liver cancer treatment.

6. Use of LACTB in the preparation of a kit for evaluating the adaptability of lenvatinib in treating liver cancer.

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