CN111617247B - Application of EGFR (epidermal growth factor receptor) kinase substrate 8 protein 3 in enhancing curative effect of multi-target kinase inhibitor - Google Patents

Abstract

The application relates to application of an EGFR (epidermal growth factor receptor) kinase substrate type 8 protein 3 in enhancing curative effect of a multi-target kinase inhibitor. Epidermal growth factor receptor kinase substrate type 8 protein 3 (EPS 8L 3) is a novel oncogene for HCC. In vivo and in vitro experiments prove that knocking down EPS8L3 can reduce the growth potential of HCC tumor cells. In addition, cells knocked down EPS8L3 are more sensitive to sorafenib treatment. The technical scheme not only provides an important factor for the progress of the HCC, but also provides a potential therapeutic target for advanced HCC treatment.

Description

Application of EGFR (epidermal growth factor receptor) kinase substrate 8 protein 3 in enhancing curative effect of multi-target kinase inhibitor

Technical Field

The present invention relates to the field of medicine. In particular to the synergistic effect of the EGFR kinase substrate type 8 protein 3 and sorafenib.

Background

Liver cancer is a common tumor, with 780000 newly diagnosed liver cancer patients (Ferlay et al 2015) worldwide each year, of which about three-fourths are primary hepatocellular carcinoma (hepatocellular carcinoma, HCC) (Siegel et al 2018). Hepatitis virus, alcoholism, obesity, diabetes, smoking, and the like are all closely related to the development and progression of HCC (Yang and Roberts 2010). Although some early HCC may be cured by surgical resection or liver transplantation, advanced HCC still lacks better treatments. Indeed, sorafenib, regorafenib and like related multi-target kinase inhibitors are the only therapeutic drugs for patients with advanced HCC. In addition, although there have been ten more drugs in clinical trials for HCC treatment, none of the drugs has reached the end of the third phase clinical trial (Llovet and Hernandez-Gea 2014). Unlike other tumors, which gradually decrease with early surgery or other therapeutic approaches, the mortality rate of HCC continues to rise from 2006 to 2015 (Siegel et al, 2018), suggesting the importance of finding new targets for treatment.

Recent genome-wide studies revealed that HCC is a common relevant mutant gene, including TP53, CTNNB1, LTTR1, EEF1A1, SF3B1, and SMARCA4 (Schulze et al 2015; totoki et al 2014). Despite the high mutation rates of these genes in HCC, the specific molecular mechanisms of HCC progression are always not clearly understood.

In this study, the inventors compared the gene expression profiles of malignant and surrounding normal tissues using the RNA database of Cancer Genome Atlas (TCGA) (Weinstein et al, 2013) and found that EGFR kinase substrate class 8 protein 3 (epidermal growth factor receptor kinase substrate-like protein 3, EPS8L 3) was up-regulated in HCC patients, suggesting that the gene may be associated with tumor progression.

EPS8L3, like EPS8L1 and EPS8L2, belongs to the family of EGFR kinase substrate 8 (EPS 8) -related proteins. Due to the similarity in molecular structure, EPS 8-associated proteins generally have similar cellular functions such as Rac-mediated actin cytoskeletal remodeling (Offenhauser et al, 2004). However, only EPS8L1 and EPS8L2 can activate Rac and localize it to actin rich regions, the function of EPS8L3 remains unknown. The role of EPS8family as a key driving gene of tumor has not been reported so far.

Although some early hepatocellular carcinoma can be cured by surgical excision or liver transplantation, advanced hepatocellular carcinoma still lacks a better therapeutic means. Sorafenib is an inhibitor against tyrosine protein kinases such as VEGFR, PDGFR and Raf family kinases. Sorafenib inhibits tumor growth by mediating cell autophagy, and is one of a few treatments for advanced non-operable HCC patients. However, this treatment provides a median lifetime extension of only months, and is often accompanied by subsequent multidrug resistance due to tyrosine kinase mutations. The side effects of high doses of sorafenib also limit the use of the drug. Thus, there is a need for new treatments that overcome the limitations of sorafenib.

Disclosure of Invention

According to some embodiments of the present disclosure, there is provided the use of EPS8 in the manufacture of a medicament for the treatment of primary hepatocellular carcinoma.

According to some embodiments of the present disclosure, there is provided the use of EPS8 as a target in the manufacture of a medicament for the treatment of primary hepatocellular carcinoma.

According to some embodiments of the present disclosure, there is provided the use of an agent that targets EPS8 in the manufacture of a medicament for the treatment of primary hepatocellular carcinoma.

In some embodiments, the agent that targets EPS8 refers to an agent that is capable of modulating the level or activity of expression of EPS8 at the nucleic acid level or at the protein level.

In some specific embodiments, an agent that targets EPS8 is capable of selectively recognizing and binding to the EPS8 gene or an expression product thereof.

In some embodiments, an agent that targets EPS8 is capable of modulating the level or activity of an EPS8 gene or expression product thereof.

In some specific embodiments, an agent that targets EPS8 is capable of reducing the level or activity of the EPS8 gene or an expression product thereof.

In some specific embodiments, the agent that targets EPS8 is capable of knocking out the EPS8 gene.

In some embodiments, the expression product of the EPS8 gene refers to various forms of molecules of the EPS8 gene in various stages, such as, but not limited to, molecules produced by the EPS8 gene during amplification, replication, transcription, splicing, processing, translation, modification, such as cDNA, mRNA, precursor proteins, mature proteins, and fragments thereof.

In some embodiments, the agent that targets EPS8 is selected from: nucleic acids or polypeptides. In some embodiments, the nucleic acid is selected from DNA, RNA, DNA/RNA; the polypeptide is selected from: an antibody or antigen-binding fragment thereof.

According to some embodiments of the present disclosure, there is provided the use of a combination of a targeting agent that modulates the epidermal growth factor receptor kinase substrate 8 protein family (EPS 8) and a multi-target kinase inhibitor in the manufacture of a medicament. In some embodiments, the modulation is selected from: inhibition, reduction, inactivation, knockout, reduction, or a combination thereof. Thus, the targeting agent inhibits, reduces, inactivates, knocks out, or knocks down the activity or level of EPS 8. In some embodiments, the level is a nucleic acid level. In some embodiments, the level is a protein level.

In specific embodiments, the agent that targets EPS8 is selected from: knockdown reagents, antisense oligonucleotides, siRNA, dsRNA, ribozymes, small interfering RNAs (esirnas) or short hairpin RNAs (shrnas) made by endoribonuclease III.

In some embodiments, the knockout reagent refers to those well known in the art, including but not limited to those methods or reagents mentioned in the guidelines for gene knockout experiments, science publishers, written by Marieke Aarts.

In some specific embodiments, EPS8 targets EPS8 genes or their expression products as targets (e.g., by knockdown, RNA interference) to modulate (reduce) the level or activity of intracellular EPS8 genes or their expression products of primary hepatocellular carcinoma.

In some embodiments, EPS8 is selected from: EPS8L1, EPS8L2, EPS8L3, and combinations thereof. In a specific embodiment, is EPS8L3.

In some embodiments, the multi-target kinase inhibitor is selected from the group consisting of: sorafenib, regorafenib, lenvatinib, apatinib, an Luoti ni.

In some embodiments, the targeting agent and the multi-target kinase inhibitor in combination are used to prepare a medicament for the prevention or treatment of primary hepatocellular carcinoma. In particular embodiments, advanced primary hepatocellular carcinoma is treated.

According to some embodiments of the present disclosure, there is provided a therapeutic composition comprising an EPS8 targeting agent and a multi-target kinase inhibitor.

According to some embodiments of the present disclosure, there is provided the use of an identification agent of the epidermal growth factor receptor kinase substrate 8 protein family in the manufacture of a diagnostic device, wherein the diagnostic device is embodied as a kit or chip; the diagnostic device is used for evaluating prognosis of primary hepatocellular carcinoma. In some embodiments, the identification agent is capable of determining the level of the epidermal growth factor receptor kinase substrate 8 protein family. In a specific embodiment, the level is a nucleic acid level or a protein level, preferably an RNA level.

In particular embodiments, when the RNA level of the epidermal growth factor receptor kinase substrate 8 protein family in the subject is higher than the control level, the progression or worsening of primary hepatocellular carcinoma in the subject is indicated.

In particular embodiments, a poor prognosis in a subject with primary hepatocellular carcinoma is indicated when the level of RNA of the epidermal growth factor receptor kinase substrate 8 protein family in the subject is higher than a control level.

In particular embodiments, a decrease in survival of a subject with primary hepatocellular carcinoma is indicated when the level of RNA of the epidermal growth factor receptor kinase substrate 8 protein family in the subject is greater than a control level.

According to some embodiments of the present disclosure, there is provided the use of a targeting agent of the epidermal growth factor receptor kinase substrate 8 protein family in the manufacture of a medicament, wherein the targeting agent inhibits, reduces, inactivates, knocks out or knocks down the activity or level of EPS 8.

In specific embodiments, the agent inhibits proliferation of, or induces apoptosis of, or reduces metastasis of, primary hepatocellular carcinoma.

According to some embodiments of the present disclosure, there is provided the use of a targeting agent of the epidermal growth factor receptor kinase substrate 8 protein family in the preparation of an animal model of primary hepatocellular carcinoma.

In particular embodiments, the primary hepatocellular carcinoma animal model has sensitivity to drug treatment compared to a control animal; the control animals have unregulated expression levels or activity of the epidermal growth factor receptor kinase substrate 8 protein family.

According to some embodiments of the present disclosure, there is provided the use of an agent that targets EPS8 in the preparation of an animal model of primary hepatocellular carcinoma.

In some specific embodiments, the animal model of the present disclosure has sensitivity to drug treatment compared to a control animal. In some specific embodiments, the level of expression or activity of EPS8 is unregulated in a control animal.

In some specific embodiments, the expression level or activity of EPS8 is modulated at the nucleic acid level or at the protein level in an animal model according to the present application. The modulation is selected from: reduced, inactivated, knocked out, knocked down, modified, or a combination thereof.

In some specific embodiments, the expression level or activity of EPS8 is reduced or inhibited in an animal model according to the present application. In some specific embodiments, the EPS8 is knocked out or knocked down in an animal model according to the present application.

In some embodiments, the animal is a mammal other than a human. In some specific embodiments, the animal is selected from the group consisting of: mice, rats, guinea pigs, rabbits, horses, monkeys, dogs. In some specific embodiments, the animal is a mouse, rat, or guinea pig.

Drawings

Fig. 1A: transcriptome analysis data.

Fig. 1B: the expression of EPS8L3 in tumor tissue was 60-fold higher than in normal tissue.

Fig. 1C: expression of EPS8L3 in grade I to IV tumors.

Fig. 1D to 1E: advanced HCC patients are associated with poor prognosis.

Fig. 1F: parameters unrelated to clinical outcome in advanced HCC patients.

Fig. 1G: higher EPS8L3 levels correlate with lower survival.

Fig. 2A: RNA expression of EPS8L3 in the four liver cancer cells was detected by qRT-PCR.

Fig. 2B to 2C: after knocking down EPS8L3 using shRNA, RNA expression of EPS8L3.

Fig. 2D: protein expression of EPS8L3 after knockdown of EPS8L3 using shRNA.

Fig. 2E: BEL-7404 and HepG2 cells with higher EPS8L3 expression were subjected to microarray sequencing.

Fig. 2F: knock down EPS8L3 effects on the full transcriptome.

Fig. 2G: protein expression was measured by western blotting.

Fig. 2H to 2I: EPS8L3 plays a role in cell proliferation.

Fig. 2J: effect of EPS8L3 in autophagosome formation.

Fig. 3A to 3D: HCC cell proliferation was significantly inhibited after EPS8L3 knockdown.

Fig. 3E to 3F: HCC cell proliferation was significantly inhibited after EPS8L3 knockdown.

Fig. 3G to 3I: knocking down EPS8L3 increases the proportion of cells in G1 phase and decreases the proportion of cells in S phase.

Fig. 3J to 3K: knocking down EPS8L3 resulted in apoptosis of HCC cells.

Fig. 4A and 4B: knocking down EPS8L3 reduced the migration rate of BEL-7404 and HepG2 cells.

Fig. 4C to 4F: EPS8L3 reduced the invasive capacity of HCC cells.

Fig. 5A to 5C: knocking down EPS8L3 delayed tumor growth (NC: control; KD: knocking down).

Fig. 6A to 6C: sorafenib slows down cell growth of BEL-7404.

Fig. 6D to 6F: sorafenib slows cell growth of Hep G2.

Fig. 6G to 6H: the combination of low dose sorafenib and knockdown EPS8L3 acts on HepG2 cells.

Detailed Description

The experimental method comprises the following steps:

1. cell culture:

BEL7404, BEL-7402, hepG2 and SMMC-7721 cells were cultured with 10% fetal bovine serum double antibody RPMI-1640 in 5% CO ₂ Incubated at 37℃and detected by Mycoplasma.

2. Cell transfection and transduction:

cell transfection was performed using Lipofectamine 2000 and virus was generated by co-transfection of 293T cell expression vector (GV 115shRNA vector) using lentiviral packaging system (pmd2. G and psPAS 2). GV115 vector contains the U6 promoter multiple cloning site and purine pyrimidine resistance gene. shRNA sequence for EPS8L 3: 5'-gctacattccaagcaacat-3' (SEQ ID No. 1). 16 hours after transfection, fresh 293T broth was changed, and supernatants were collected after 24h and 48h, respectively, and filtered with 0.45 μm filters. The transduced target cells were mixed with lentivirus and centrifuged at 2000rpm for 2min. 72 hours after transduction, puromycin was added to the medium to screen lentiviral infected cells.

qRT-PCR detection of RNA expression:

trizol extracts RNA and cDNA was synthesized from 2. Mu.g total RNA using M-MLV reverse transcriptase.

Internal reference GAPDH primer for PCR:

upstream 5'-tgacttcaacagcgacaccca-3' (SEQ ID No. 2),

downstream 5'-caccctgttgctgtagccaaa-3' (SEQ ID No. 3).

EPS8L3 primer:

upstream 5'-gccctaccaacccacattctca-3' (SEQ ID No. 4),

downstream 5'-cctcattcttcaccagccacca-3' (SEQ ID No. 5).

Ct value indicates the relative expression level of the PCR product.

Celigo and MTT experiments:

2000 cells were seeded per well in 96-well plates and observed with a Celigo cell imaging analyzer for 3-5 days. Daily cell numbers were plotted against time after correction by cell number before inoculation. Mu.l of 5mg/ml MTT was added to each well, after 4 hours the broth was removed, 150. Mu.l of DMSO was added, and the wells were shaken on a shaker for 2-5 minutes to determine the absorbance (OD) values at 490nm/570nm for each well using a microplate reader.

5. Flow cytometry detects cell cycle distribution:

after washing the cells twice with PBS, 100. Mu. lPI dye (PI, 50. Mu.g/ml; RNase, 100. Mu.g/ml) was added and incubated for 1 min in the dark. And (5) detecting on the machine, and analyzing the result by using cell modification software.

Apoptosis detection by Caspasture 3-7 method:

cells were cultured at 37℃for 3-5 days in 96-well plates and tested for apoptosis according to the Caspase-Glo 3/7 kit. Caspase-Glo 3/7 reagent and sample volume were 1:1 after mixing well, incubating for 1h at 37 ℃. The absorbance at 490nm/520nm was read from each well by the upper machine.

Protein detection by western blotting:

after HCC cells transfected with FLAG-tagged EPS8L3 are lysed in RIPA solution, extracted proteins are transferred to PVDF membrane through SDS-PAGE electrophoresis, and after blocking, the cells are incubated for 1h in a shaking table at room temperature, primary and secondary antibodies are added, and then analysis and determination of protein level expression are carried out.

8. Bioinformatics analysis:

the RNA-seq data of TCGA was downloaded by UCSC Cancer Genome Browser (https:// genome-cancer. Ucsc. Edu /). The assay was performed using an Affymetrix HT HG-U133+PM Array chip using a GeneChip Scanner 3000 platform. The gene expression values were normalized using the RMA algorithm and log2 gene expression fold differences were obtained. Screening conditions: the absolute value is more than or equal to 4, and the change is more than 20 percent.

9. Animal experiment:

all animal handling strictly followed the institutional animal care utilization committee requirements of the national academy of medicine. 2X 10 ⁶ The cell suspension was inoculated subcutaneously on the lateral dorsal aspect of BALB/c in nude mice.

Tumor volume was calculated using the following formula: v=a×b ² 2; v is tumor volume (mm) ³ ) A is the long diameter (mm) of the tumor, and b is the short diameter (mm) of the tumor.

Measurements were taken once a week. Luciferase activity detection tumor growth, sorting BEL-7404 cells transfected with GFP and luciferase, detecting luciferase activity by a small animal living body imaging system, and measuring tumor volume according to average photon flux.

10. The statistical method comprises the following steps:

data comparisons between groups were performed using a double-sided independent sample t-test, with Welch's t test if variance was not uniform. Survival analysis was performed using a Kaplan-Meier curve and a log-rank (Mantel-Cox) test. P less than 0.05 is statistically significant for the differences.

Example 1 up-regulation of EPS8L3 expression in HCC patients and associated with poor prognosis

TCGA databases (The Cancer Genome Atlas, TCGA) provide valuable and abundant genomic and transcriptomic analysis data based on human tumors (Tomczak et al 2015). To determine the genes associated with HCC, the inventors aligned the gene expression of tumors and corresponding paraneoplastic normal tissues using hepatocellular carcinoma RNA data information in the TCGA database.

Based on transcriptomics, the biological coefficients of the respective variables were analyzed (fig. 1A), suggesting the role of oncogenes during tumor transformation. The inventors subsequently compared the gene expression in tumor tissue and normal tissue, assuming that the gene whose expression is up-regulated in HCC specimens is associated with tumor formation, EPS8L3 is one of the most significant genes up-regulated, 60-fold in tumor tissue compared to normal tissue (fig. 1B).

To further investigate the correlation of EPS8L3 with clinical symptoms in HCC patients, the inventors compared RNA expression of EPS8L3 at different stages in HCC patients. The inventors found that EPS8L3 expression was closely related to tumor grading, with advanced HCC patients (grade II/III/IV) being significantly higher than early patients (grade I) (fig. 1C). Moreover, advanced HCC patients are associated with poor prognosis fig. 1D and 1E). Higher EPS8L3 levels correlated with lower survival compared to other parameters not related to clinical outcome (e.g., race, etc.) (fig. 1F), suggesting that EPS8L3 correlates with poor prognosis and may reduce patient survival (fig. 1G).

Example 2 EPS8L3 affects cell proliferation-related Signal pathways

In order to better understand the molecular mechanism of EPS8L3, the inventors used shRNA to knock down EPS8L3 in hepatoma cells for non-deflected transcriptomic analysis. The RNA expression of EPS8L3 in the four liver cancer cells (figure 2A) is detected by qRT-PCR, and the delta Ct value of BEL-7404 (liver cancer tissue from Chinese population) and HepG2 (liver cancer tissue from white men) is lower than that of BEL-7402 and SMMC-7721 (liver cancer tissue from Chinese population), namely the EPS8L3RNA expression level of BEL-7404 and HepG2 is higher.

After knocking down EPS8L3 in BEL-7404 and HepG2 cells using shRNA, the RNA and protein expression of EPS8L3 were reduced (FIGS. 2B to 2D).

BEL-7404 and HepG2 cells with higher EPS8L3 expression were subjected to microarray sequencing. PCA plots revealed (FIG. 2E) that knockdown of EPS8L3 had an effect on the full transcriptome. 331 genes were expressed in the knockdown cells twice as much (FIG. 2F). Among these differentially expressed genes, the inventors selected those whose effect on cell proliferation was unknown, and determined protein expression by western blotting (FIG. 2G), protein levels of cell division promoting genes (CCNA 2, CCNB1, CDK1 and BIRC 5) were significantly reduced, while protein levels of CDKN1A were significantly increased. The biological function of this differentially expressed gene was explained by the representational analysis (ORA) and the Gene Set Enrichment Analysis (GSEA). The significant down-regulation of selected genes in EPS8L3 knockdown cells, a number of pathways, which are clearly associated with cell proliferation, further confirm the importance of EPS8L3 in cell proliferation (fig. 2H, fig. 2I). Among them, the most relevant pathway is associated with autophagosome formation (fig. 2J), a process associated with stress and in some cases cell death, an important marker of autophagy upon autophagosome formation (Anding and Baehrecke, 2015), and knockdown of EPS8L3 may initiate autophagy-mediated cell death. Since uncontrolled cell proliferation is an important feature of tumor cells (Hanahan and Weinberg, 2011), microarray data further support that oncogene function of EPS8L3 may play a role by activating cell division and inhibiting apoptosis.

Example 3 inhibition of cell proliferation and mediation of apoptosis by inhibition of EPS8L3

Celigo cell imaging and MTT method detect BEL-7404 and HepG2 cells (EPS 8L3 is expressed higher) proliferation ability after knocking down EPS8L3, and the result shows that HCC cell proliferation is obviously inhibited after knocking down EPS8L3 (FIG. 3A to FIG. 3D, FIG. 3E and FIG. 3F). The ratio of liver cancer cells with different cell cycles after knocking down EPS8L3 is selected through flow cytometry analysis. In both cell lines, knocking down EPS8L3 significantly increased the proportion of cells in the G1 phase and decreased the proportion of cells in the S phase (fig. 3G to 3I). Since the G1 phase cells do not enter cell division directly, but may also enter resting G0 phase, G1 phase cytosis is detrimental to cell proliferation. And DNA replication in S phase provides sufficient preparation for upcoming cell divisions. Thus, cell cycle changes evidence that inhibition of EPS8L3 slows cell proliferation.

Since resting cells also have an inhibitory effect on the cell cycle, it is further explored whether the increase in the proportion of cells in the G1 phase after EPS8L3 knockout has produced an inhibitory effect by promoting cell death or increasing the proportion of resting cells. To this end, the inventors used the Caspase-3/7 assay to detect apoptosis. The cells were added with a non-fluorogenic substrate and the apoptosis marker Caspase-3/7 was added to bind DNA and present detectable fluorescence.

The results showed that the EPS8L3 knockdown HCC cell caspase3/7 increased more than two times (fig. 3J to 3K), suggesting that the EPS8L3 knockdown resulted in apoptosis of HCC cells. The experiment proves that EPS8L3 plays an important role in promoting tumor cell proliferation and inhibiting tumor cell apoptosis in HCC patients.

The up-regulation of pathways including autophagosome aggregation (autophagosome organization) in EPS8L3 knockdown HCC cells also explains why knockdown of EPS8L3 activates Caspase3/7 and mediates apoptosis. Since no targeted drug for EPS8L3 is currently available, drug inhibition for EPS8L3 is particularly important in HCC patients. However, if autophagosome formation brings about a range of effects that inhibit EPS8L3 and activate apoptosis, compounds that mediate autophagy may be potential means for treating HCC.

Example 4 EPS8L3 enhances HCC cell invasiveness

Over 90% of solid tumor-related deaths are due to tumor metastasis (Gupta and Massague, 2006).

The inventors examined the effect of EPS8L3 on the invasive ability of HCC cells by scratch and Transwell experiments. In the scratch experiments, knocking down EPS8L3 significantly reduced the migration rate of BEL-7404 and HepG2 cells (fig. 4A to 4B).

Since HepG2 cells cannot pass through the permeable membrane, the inventors performed an alternative experiment with BEL-7402. The knockdown BEL-7404 and BEL-7404 cells were significantly reduced in cells entering the lower chamber, and EPS8L3 was seen to significantly reduce the invasive capacity of HCC cells (FIGS. 4C to 4F). Notably, the reduced invasive capacity of BEL-7404 cells was more pronounced than BEL-7402 after knocking down EPS8L3, which may be associated with lower EPS8L3 baseline levels in BEL-7402 cells (FIG. 2A).

Example 5 knockdown of EPS8L3 tumor inhibiting effect in vivo

Tumor volumes and bioluminescence imaging were examined weekly using GFP and luciferase-labeled BEL-7404 cells inoculated subcutaneously into nude mice for tumor formation. Knocking down EPS8L3 can significantly delay tumor growth, suggesting that EPS8L3 plays an important role in tumor growth. (FIGS. 5A, 5B, 5C).

Example 6 synergistic inhibition of HCC growth by EPS8L3 and tyrosine protein kinase

Sorafenib is a tyrosine protein kinase inhibitor against VEGFR, PEGFR and Raf family kinases, and has been FDA approved for the treatment of primary renal cancer, advanced thyroid cancer and advanced liver cancer (Keating, 2017). Sorafenib at 5. Mu.M and 10. Mu.M concentrations slowed cell growth after BEL-7404 (FIGS. 6A-6C) and Hep G2 cells (FIGS. 6D-6F).

High-dose sorafenib is inevitably limited in clinical practice due to drug toxicity etc., and patients can only select lower doses of drug due to pathophysiological reasons (Clare et al, 2017). The inventors have combined low dose sorafenib and knockdown EPS8L3 on HepG2 cells, achieving a similar therapeutic effect as the high dose drug (fig. 6G, fig. 6H).

This suggests that the co-treatment of sorafenib with knockdown EPS8L3 may be a possible treatment for HCC patients.

Although EPS8L3 plays a key role in HCC progression, the role of EPS8L3 in other tumor progression is not yet clear. The reason that the other two members of the EPS8L family, EPS8L1 and EPS8L2, do not have similar promoting effects on HCC, is further explored (Offenhauser et al, 2004).

Reference to the literature

Anding, A.L. et al (2015) Autophagy in Cell Life and Cell Death Current topics in developmental biology 114,67-91.

Clare, K.E. et al (2017) editor: sorafenib toxicity, a biomarker of effect, alimentary pharmacology & therapeutics 45,1469-1470.

Ferlay, J. Et al (2015) Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.International journal of cancer 136,E359-386.

Gupta, G.P. et al (2006) Cancer metastasis, building a frame Cell 127,679-695.

Hanahan, D. Et al (2011) Hallmarks of cancer: the next generation, cell 144,646-674.

Keating,G.M.(2017).Sorafenib:A Review in Hepatocellular Carcinoma.Targeted oncology 12,243-253.

Llovet, J.M et al (2014), hepatocellular carcinoma: reasons for phase III failure and novel perspectives on trial design. Clinical cancer research: an official journal of the American Association for Cancer Research, 20, 2072-2079.

Llovet, J.M. et al (2008) Sorafenib in advanced hepatocellular Carcinoma.the New England journal of medicine 359,378-390.

Offenhauser, N.et al (2004) The eps8family of proteins links growth factor stimulation to actin reorganization generating functional redundancy in The Ras/Rac path. Molecular biology of The cell 15,91-98.

Renna, m. et al (2010) Chemical inducers of autophagy that enhance the clearance of mutant proteins in neurodegenerative diseases.the Journal of biological chemistry 285,11061-11067.

Schulze, K.et al (2015) Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets Nature genetics 47,505-511.

Siegel, r.l. et al (2018), cancer statistics,2018.CA:a cancer journal for clinicians 68,7-30.

Tomczak, K.et al (2015) The Cancer Genome Atlas (TCGA): an immeasurable source of knowledges. Contemporary oncology (Poznan, poland) 19, A68-77.

Totoki, Y.et al (2014), trans-ancestry mutational landscape of hepatocellular carcinoma genome Nature genetics 46,1267-1273.

Weinstein, J.N. et al (2013) The Cancer Genome Atlas Pan-Cancer analysis project Nature genetics 45,1113-1120.

Yang, J.D. et al (2010) Hepatocellular carcinoma: A global view Nature reviews Gastroenterology & hepatology 7,448-458.

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Claims (6)

1. Use of a combination of a targeting agent and a multi-target kinase inhibitor in the manufacture of a medicament, wherein:

the targeting agent inhibits, reduces, inactivates, knocks out or knocks down the activity or level of EPS8L 3; the targeting agent is selected from any one of the following: knockout agents, antisense oligonucleotides, siRNA, shRNA;

the multi-target kinase inhibitor is sorafenib;

the medicament is used for preventing or treating primary hepatocellular carcinoma.

2. The use according to claim 1, for the prevention or treatment of advanced primary hepatocellular carcinoma.

3. The use of claim 1, wherein the level is a nucleic acid level or a protein level.

4. The use of claim 1, wherein the targeting agent is esiRNA.

5. A pharmaceutical composition comprising:

a targeting agent and sorafenib,

wherein the targeting agent inhibits, reduces, inactivates, knocks out or knocks down the activity or level of EPS8L 3; the targeting agent is selected from any one of the following: knockout agents, antisense oligonucleotides, siRNA, shRNA.

6. The pharmaceutical composition of claim 5, wherein the targeting agent is esiRNA.

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