HDGF as a tyrosine kinase inhibitor-resistant target and related applications

Abstract

This invention provides hepatocellular carcinoma-derived growth factor (HDGF) as a target for resistance to tyrosine kinase inhibitors and its related applications. This invention provides the application of HDGF as a target in screening and / or preparing drugs that can improve resistance to tyrosine kinase inhibitors in patients. This invention discovers that high expression of HDGF is one of the mechanisms of resistance to molecularly targeted drugs such as gefitinib and other tyrosine kinase inhibitors. Targeting HDGF can effectively improve resistance to gefitinib in NSCLC and enhance therapeutic efficacy.

Description

Technical Field

[0001] This invention relates to targets associated with resistance to tyrosine kinase inhibitors (TKIs), particularly to liver cancer-derived growth factor (HDGF) as a target for resistance to TKIs such as gefitinib in non-small cell lung cancer and its related applications. Background Technology

[0002] Tyrosine kinases (TKs) are crucial factors in cell signal transduction pathways, participating in the regulation of a series of physiological and biochemical processes, including cell growth, differentiation, and apoptosis. Studies have shown that tyrosine kinases are frequently activated in tumor tissues, subsequently activating downstream signaling pathways, promoting cell proliferation, inhibiting apoptosis, and thus inducing tumor development. Due to the key role of tyrosine kinases in tumorigenesis, tyrosine kinase inhibitors (TKIs), by specifically blocking cell proliferation signals, have become important targeted drugs for cancer treatment.

[0003] Lung cancer is currently the leading cause of cancer-related morbidity and mortality worldwide, with both incidence and mortality rates on the rise globally. Lung cancer is mainly divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with NSCLC accounting for approximately 80% of all lung cancer cases. Currently, the treatment of lung cancer remains one of the most challenging problems in the world. Targeted therapies against lung adenocarcinoma driver genes have become first-line drugs for the treatment of advanced NSCLC. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) can significantly prolong the median survival of patients, and gefitinib is a first-generation TKI widely used in clinical practice in my country. Although TKIs are effective against advanced NSCLC with EGFR-sensitive mutations, drug resistance eventually develops, leading to disease progression. The promising second-generation TKI afatinib and the third-generation TKI AZD9291 also face drug resistance issues. Therefore, drug resistance remains a significant factor limiting the clinical efficacy of TKIs, and overcoming TKI resistance to benefit NSCLC patients is a key scientific question.

[0004] Currently identified TKI resistance mechanisms include: secondary EGFR mutations (T790M) (50-60%); abnormal activation of bypass pathways (c-MET, HGF, AXL) (1-25%); abnormalities in downstream signaling pathways (BRAF, PIK3CA, or K-RAS mutations, PTEN deletion, etc.); transformation of non-small cells into small cells or epithelial-mesenchymal transition (EMT) histopathological transformation (5-10%); and tumor heterogeneity. In addition, the causes of resistance in 20-30% of cases remain unclear. Current salvage therapy after resistance primarily includes: chemotherapy, second- or third-generation TKIs, bypass inhibitors combined with TKIs, and PD-1 / PD-L1 immunotherapy combined with TKIs. While second- or third-generation TKIs have some efficacy against T790M mutation resistance, relapse can occur; the efficacy of the latter two regimens is currently under clinical trials. Overall, most clinical studies using single-agent or combination therapies have not yielded satisfactory results for patients resistant to first-generation TKIs. Therefore, drug resistance remains a problem hindering the clinical efficacy of TKIs. Further research is needed to overcome TKI resistance. Summary of the Invention

[0005] One objective of this invention is to identify the resistance mechanism of tyrosine kinase inhibitors (TKIs) in order to screen for drugs or methods that can alleviate TKI resistance in patients, especially those with non-small cell lung cancer, overcome or delay resistance, and improve the efficacy of TKIs for patients.

[0006] The inventors in this case discovered in their research that the expression level of liver cancer-derived growth factor (HDGF) is associated with resistance to tyrosine kinase inhibitors such as gefitinib. Targeting HDGF can effectively improve the resistance of NSCLC to tyrosine kinase inhibitors such as gefitinib, thereby enhancing the therapeutic effect.

[0007] In some specific embodiments of this invention, it has been found that high HDGF expression is one of the mechanisms of resistance to tyrosine kinase inhibitors such as gefitinib. HDGF expression levels in gefitinib-resistant non-small cell lung cancer (NSCLC) cells are significantly higher than in sensitive cells. High levels of HDGF are not only associated with the malignant phenotype of NSCLC but can also induce gefitinib resistance. Signaling pathways associated with TKI resistance, such as the PI3K / Akt and MEK / ERK pathways, can be bypassed by HDGF in an EGFR-independent manner. Inhibition of HDGF enhances the effect of gefitinib on resistant NSCLC cells. Knockdown of HDGF expression in resistant cells significantly reduces the half-maximal effective concentration (IC50) of gefitinib. 50 It weakens cell colony formation ability, promotes tumor cell apoptosis, and inhibits the expression of drug resistance-related proteins p-Akt and p-ERK. Overexpression of HDGF in gefitinib-sensitive cells significantly increases the IC50 of gefitinib.50 HDGF promotes cell colony formation and scratch healing, and induces the expression of drug resistance-related proteins p-Akt and p-ERK. In vivo studies have shown that overexpression of HDGF promotes the growth of NSCLC tumors and the expression of drug resistance-related proteins. In vitro and in vivo results indicate that high HDGF expression is one of the mechanisms of gefitinib resistance, and knockdown of HDGF expression can improve the response of drug-resistant NSCLC to gefitinib. HDGF is a potential target for overcoming gefitinib resistance.

[0008] Thus, on the one hand, the present invention provides the application of liver cancer-derived growth factor HDGF as a target in screening and / or preparing drugs that improve patients' resistance to tyrosine kinase inhibitors.

[0009] On the other hand, the present invention also provides the application of reagents for detecting the expression level of hepatocellular carcinoma-derived growth factor HDGF in the preparation of a detection system for assessing patients' resistance to tyrosine kinase inhibitors.

[0010] On the other hand, the present invention also provides the use of an antagonist against hepatocellular carcinoma-derived growth factor HDGF in the preparation of a medicament for treating tumors and / or reducing resistance to tyrosine kinase inhibitors in patients.

[0011] In this invention, the detection includes auxiliary detection, and the treatment includes auxiliary treatment.

[0012] The antagonists against hepatocellular carcinoma-derived growth factor (HDGF) described in this invention are reagents that reduce HDGF expression levels and / or antagonize HDGF activity. Reagents with such functions may include, but are not limited to, sgRNA, small molecule inhibitors, natural products, antibodies, or combinations thereof. Such reagents are readily available to those skilled in the art based on existing technology and can be any known antagonist capable of reducing HDGF expression levels and / or antagonizing HDGF activity, or reagents that retain the function of reducing HDGF expression levels and / or antagonizing HDGF activity after modification or restructuring based on the molecular formula. In some specific embodiments of this invention, knocking down HDGF expression using gene technology can effectively inhibit the proliferation, migration, spread, and / or invasion of tumor cells, while simultaneously restoring sensitivity to tyrosine kinase inhibitors such as gefitinib.

[0013] In some specific embodiments of the present invention, reducing tyrosine kinase inhibitor resistance includes reducing the half-maximal effective concentration (IC50) of the tyrosine kinase inhibitor. 50 ).

[0014] In some specific embodiments of the present invention, reducing resistance to tyrosine kinase inhibitors includes weakening the ability of tumor cells to form clones and / or inhibiting the proliferation, migration, spread, and / or invasion of tumor cells.

[0015] In some specific embodiments of the present invention, reducing resistance to tyrosine kinase inhibitors includes promoting tumor cell apoptosis and / or inhibiting tumor growth in vivo.

[0016] In some specific embodiments of the present invention, reducing resistance to tyrosine kinase inhibitors includes inhibiting the expression of resistance-related proteins p-Akt and p-ERK.

[0017] According to another aspect of the present invention, the present invention provides the use of an agent that causes tyrosine kinase inhibitor-sensitive cells to overexpress HDGF in the preparation of a research formulation having at least one of the following effects: increasing the IC50 of the tyrosine kinase inhibitor. 50 It promotes cell colony formation and / or scratch healing; enhances cell proliferation, colony formation, migration, and invasion; and / or induces the expression of resistance-related proteins p-Akt and p-ERK. The described research formulation is of great significance for studying the HDGF-driven resistance mechanisms to tyrosine kinase inhibitors such as gefitinib.

[0018] According to a specific embodiment of the present invention, the tyrosine kinase inhibitor includes, but is not limited to, one or more of gefitinib, icotinib, afatinib, AZD9291, etc.

[0019] According to a specific embodiment of the present invention, the patient is a cancer patient.

[0020] According to a specific embodiment of the present invention, the tumor is lung cancer, such as non-small cell lung cancer.

[0021] In some specific embodiments of this invention, it has been demonstrated that HDGF promotes the occurrence and metastasis of NSCLC. Forced expression of HDGF in H292 and PC-9 cells with relatively low HDGF expression levels enhanced cell proliferation, colony formation, migration, and invasion. Simultaneously, exogenous rhHDGF enhanced the proliferation of H292 and PC-9 cells, confirming the role of HDGF as a growth stimulating factor. The tumor-promoting function of HDGF was verified in PC-9 xenograft mice overexpressing HDGF. Conversely, silencing HDGF via CRISPR suppressed the malignant phenotype of H1975 cells and delayed tumor development in vivo.

[0022] In some specific embodiments of the present invention, the research shows that knockdown of HDGF can increase the sensitivity of NSCLC to gefitinib, while HDGF overexpression reduces the efficacy of gefitinib to some extent. In in vitro and in vivo experiments, knockdown of HDGF inhibited tumor growth in H1975 cells, while overexpression of HDGF in PC-9 cells weakened the effect of gefitinib. Immunohistochemical staining results of Ki-67 and HDGF in mouse tumor tissue sections were consistent with the above results. These data indicate that the expression level of HDGF is related to the efficacy of gefitinib in NSCLC.

[0023] In some specific embodiments of this invention, the research shows that HDGF knockdown inhibits Akt and ERK phosphorylation, while HDGF overexpression has the opposite effect, indicating that it regulates the activation of p-Akt and p-ERK. In gefitinib-sensitive NSCLC cells, HDGF overexpression weakens the ability of gefitinib to inhibit p-Akt and p-ERK; however, in drug-resistant H1975 cells, HDGF knockout significantly inhibits Akt and ERK activation. Akt or ERK inhibitors were used to verify whether HDGF induces cell growth and gefitinib resistance through these two pathways. The above results indicate that HDGF can activate the downstream p-Akt and p-ERK signaling pathways of EGFR, which is associated with gefitinib resistance.

[0024] In some specific embodiments of the present invention, the research results indicate that the expression levels of HDGF and p-EGFR are complementary in HDGF knockdown and overexpression NSCLC cells. Gefitinib can reduce EGFR phosphorylation levels, increase HDGF expression, and abnormally activate the PI3K / Akt and MEK / ERK pathways. However, after HDGF knockdown in H1975 cells, p-EGFR expression increased, and gefitinib could restore HDGF levels while reducing p-EGFR levels, suggesting a delicate balance and complementary relationship between HDGF and p-EGFR in NSCLC cells under gefitinib treatment. Taking all relevant information into account, elevated HDGF levels may act as a bypass survival signal triggering gefitinib resistance, and silencing HDGF can overcome this resistance.

[0025] In summary, this invention reveals that HDGF not only promotes the malignant phenotype of NSCLC cells but also enhances resistance to tyrosine kinase inhibitors. HDGF, acting as a bypass and compensatory signaling pathway, activates the downstream PI3K / Akt and MEK / ERK pathways of EGFR, which are associated with resistance to tyrosine kinase inhibitors such as gefitinib. Targeting HDGF can restore sensitivity to tyrosine kinase inhibitors like gefitinib by maintaining the delicate balance between HDGF expression and EGFR activation, as well as their common downstream molecules. Therefore, this invention confirms that HDGF is a novel target for treating and restoring gefitinib sensitivity in cancer patients and improving the efficacy of TKIs. Interfering with HDGF gene expression or modulating HDGF's function using any technology can effectively alleviate the resistance of cancer patients, such as those with non-small cell lung cancer, to TKIs like gefitinib, and can effectively inhibit the proliferation, spread, migration, and invasion of cancer cells. Attached Figure Description

[0026] Figures 1A to 1J This study demonstrates the results of research on how HDGF promotes the malignant phenotype of NSCLC cells. Among them, Figure 1A and Figure 1B HDGF was knocked down in H1975 cells using the CRISPR / Cas9 system, and overexpressed in PC-9 and H292 cells by transfecting the plenti6-TR plasmid. The expression of HDGF was detected by Western blot and qRT-PCR. Figure 1C and Figure 1D NSCLC cells with different HDGF expression levels and stimulated with 5 ng / mL rhHDGF were used to detect cell proliferation at 24, 48, 72, and 96 h. Figure 1E Colony formation ability of NSCLC cells with different HDGF expression levels. Figure 1F show Figure 1E The analysis results. Figure 1G Transwell assays were used to detect the migration and invasion abilities of H1975 cells after HDGF knockdown. Figure 1H for Figure 1G The analysis results. Figure 1I To detect the migration and invasion abilities of PC-9 and H292 cells overexpressing HDGF. Figure 1J for Figure 1I The analysis results. Compared with the control group, * P<0.05, ** P<0.01, *** P<0.005.

[0027] Figures 2A to 2P This study demonstrates the results of research on HDGF regulation of NSCLC tumor growth and gefitinib resistance-related molecules p-Akt and p-ERK. Among them, Figure 2A Representative image of H1975 xenograft tumors with HDGF knockdown. Figure 2B and Figure 2C The volume and weight of the H1975 xenograft are displayed. Figure 2D Expression of HDGF protein in tumor tissue of H1975 mice. Figure 2E show Figure 2D The analysis results. Figure 2F Representative images of PC-9 xenografts in nude mice with HDGF overexpression. Figure 2G and Figure 2H This shows the volume and weight of the PC-9 xenograft. Figure 2I HDGF protein expression in tumor tissues of PC-9 mice. Figure 2J for Figure 2I The analysis results. Figure 2K and Figure 2L This demonstrates the expression of p-Akt and p-ERK in HDGF-knockdown H1975 cells and HDGF-overexpressing PC-9 cells or mouse tumor tissues. Figure 2O , Figure 2P HDGF overexpression or rhHDGF-induced growth of H292 and PC-9 cells was inhibited or reversed by the Akt inhibitor MK2206 or the ERK1 / 2 inhibitor U0126. Compared with the control group, * P<0.05, ** P<0.01, *** P<0.005.

[0028] Figures 3A to 3O The results of studies on the effects of HDGF knockdown or overexpression on the efficacy of gefitinib in non-small cell lung cancer cell lines. Among them, Figures 3A-3C H1975, PC-9 and H292 cells were treated with 0.001-50 μM gefitinib for 72 h. Figures 3D-3F The ability of non-small cell lung cancer cells to form clones after treatment with gefitinib. Figure 3G-Figure 3I :for Figures 3D-3F The analysis results. Figures 3J-3L The migration and invasion ability of NSCLC cells after 24 hours of gefitinib treatment. Figure 3M-Figure 3O for Figures 3J-3L The analysis results showed that, compared with the control group, *P<0.05, **P<0.01, ***P<0.005.

[0029] Figures 4A to 4N The study results show the correlation between HDGF and gefitinib in vivo efficacy in NSCLC. Among them, Figures 4A-4FNSCLC cells were subcutaneously inoculated into nude mice, and gefitinib was administered continuously after tumor formation. The volume, tumor representative images, and tumor weight of HDGF knockdown H1975 xenografts treated with 50 mg / kg gefitinib were analyzed. Figures 4A-4C Volume, tumor representative images, and tumor weight of HDGF-overexpressing PC-9 xenografts treated with 10 mg / kg gefitinib. Figures 4D-4F ). Figure 4G Expression levels of HDGF and Ki-67 in H1975 and PC-9 tumor tissues (20×). Figure 4H Figure 4I Plasma HDGF concentrations in H1975 and PC-9 tumor-bearing mice. Figure 4J , Figure 4K Plasma HDGF concentrations in NSCLC patients before and after treatment with gefitinib or AZD9291 (third-generation TKI) and after resistance development. Figure 4L , Figure 4M Plasma HDGF levels before and after each patient received gefitinib or AZD9291 treatment. Figure 4N The expression of HDGF in biopsy tissue samples before TKI treatment and after disease progression was detected by IHC. Patient 1 received icotinib treatment, and patient 2 received AZD9291 treatment. Compared with the control group, * P<0.05, ** P<0.01, *** P<0.005.

[0030] Figures 5A to 5D The study results show the mechanism by which HDGF drives gefitinib resistance. Among them, Figure 5A , Figure 5B NSCLC cells treated with gefitinib that exhibited either HDGF silencing or overexpression ( Figure 5A ) or xenograft tumor tissue ( Figure 5B The expression of p-Akt and p-ERK in ). Figure 5C Cell viability of PC-9 cells 24 hours after treatment with 1 μM gefitinib in the presence of rhHDGF, MK2206 (Akt inhibitor, 0.5 μM), or U0126 (ERK1 / 2 inhibitor, 5 μM). Figure 5D The cell viability of PC-9 cells after transfection with the pcDNA3.1-EGFRT790M mutant plasmid and HDGF overexpression vector, alone or in combination, followed by gefitinib treatment for 72 h was compared with the control group. *P<0.05,**P<0.01,***P<0.005.

[0031] Figures 6A to 6J The study results show that HDGF and EGFR have complementary effects in NSCLC. Among them, Figure 6AOverlapping pathways between HDGF and EGFR were retrieved from the GeneCards database. Figure 6B , Figure 6C In NSCLC cells with HDGF knockdown or overexpression ( Figure 6B ) and xenograft tumors ( Figure 6C EGFR expression was detected in [the study]. Figure 6D , Figure 6E : NSCLC cell line ( Figure 6D ) and the expression levels of p-EGFR and HDGF in NSCLC cells after gefitinib treatment ( Figure 6E ). Figure 6G , Figure 6H : Gefitinib-treated NSCLC cells ( Figure 6G xenograft tissues with HDGF knockdown and overexpression ( Figure 6H Changes in the expression of HDGF and p-EGFR in ) Figure 6I , Figure 6J for Figure 6G and Figure 6H The analysis results. Compared with the control group, * P<0.05, ** P<0.01, *** P<0.005.

[0032] Figure 7 This is a schematic diagram illustrating how HDGF induces gefitinib resistance in non-small cell lung cancer by activating downstream molecules of EGFR as a bypass. Detailed Implementation

[0033] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0034] Experimental Materials and Methods

[0035] 1.1 Cell lines and culture media

[0036] Human non-small cell lung cancer cell lines H1975, H520, H157, H460, H292, and PC-9 were purchased from the American Type Culture Collection (Manassas, Virginia, USA). A549 and H1650 cells were purchased from the National Laboratory for Experimental Cell Research (Beijing, China). Cells were cultured in medium containing 10% FBS at an environment of 37°C and 5% CO2.

[0037] 1.2 Reagents

[0038] Gefitinib (Iressa) was purchased from AstraZeneca, dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM, and stored at -20°C. MTE (Marsdenia tenacissima extract, trade name: Xiaoyaping Injection) was purchased from Nanjing Shenghe Pharmaceutical Co., Ltd. Antibodies against Akt (9272), ERK1 / 2 (9102), and p-ERK1 / 2 (T202 / Y204) (9101) were purchased from Cell Signaling Technology (Beverly, MA). HDGF and p-Akt (Ser473) were obtained from Abcam (Cambridge, UK). EGFR and p-EGFR were purchased from ABclonal (Wuhan, China). GAPDH, β-actin, and Ki-67 antibodies were purchased from TDYbio (Beijing, China). Recombinant human HDGF (rhHDGF) was purchased from ProSpec-Tany TechnoGene Ltd (cyt-681-a, Israel).

[0039] 1.3 Identification of Differentially Produced Proteins

[0040] Gefitinib-resistant cell line H1975 was treated with one or both of gefitinib and MTE for 24 hours. Differential spots on two-dimensional gels were digested with trypsin, and peptide mixtures were analyzed by LC-MS / MS. MS / MS data were processed using DataAnalysis 4.0 and searched in the Swissprot protein sequence database using MASCOT V.2.4 (Matrix Science Ltd). The TOF quality analyzer was calibrated using 10 mM NaTFA. Peptide charge states for MS / MS ion retrieval were +2, +3, and +4, with an MS / MS tolerance of ±0.1 Da. Probability-based MASCOT scores were estimated by comparing search results to an estimated total random match population and reported as -10×log(P), where P is the absolute probability. A significance threshold of P < 0.05 was set.

[0041] 1.4 Establishment of stable transfected NSCLS cells

[0042] Recombinant HDGF plasmid and pcDNA3.1-EGFR T790M mutant plasmid were obtained through molecular cloning. The single guide RNA (sgRNA) sequence of HDGF was ligated into LentiCRISPRv2 plasmids (sgRNA1: 5′-GGAGTACAAATGCGGGGACC-3′ (SEQ ID NO:1) and sgRNA2: 5′-ACGTCCACACTTAACTGCGC-3′ (SEQ ID NO:2)). The sequence of the control sgRNA (non-targeting sgRNA-Con) was 5′-TTCTCCGAACGTGTCACGTT-3′ (SEQ ID NO:3). The plasmids were transfected into NSCLC cells, and stable cell lines were screened for subsequent studies.

[0043] 1.5 Quantitative RT-PCR

[0044] Total RNA was extracted using Trizol reagent (Invitrogen, USA). qRT-PCR was performed according to the following literature: Li XH, He XR, Zhou YY, Zhao HY, Zheng WX, Jiang ST, et al. Taraxacum mongolicum extract induced endoplasmic reticulum stress associated-apoptosis in triple-negative breast cancer cells. J Ethnopharmacol. 2017; 206: 55-64.

[0045] 1.6 Cell proliferation experiment

[0046] Cell counting was used to determine the effects of HDGF knockdown or overexpression and rhHDGF induction on NSCLC cell growth. The MTT assay was used to assess cell growth inhibition after gefitinib treatment. Cells were treated with gefitinib (0.001–50 μM) for 72 h, and the optical density at 570 nm was measured. IC50 values ​​were calculated using GraphPad Prism 6.0 software.

[0047] 1.7 Cloning Experiment

[0048] H292, PC-9, H1975 cells and HDGF-overexpressing / knocked cells were seeded in 6cm culture dishes and incubated for 14 days. The cells were then fixed with 4% paraformaldehyde, stained with crystal violet, photographed, counted, and compared.

[0049] 1.8 Transwell Migration and Invasion Experiments

[0050] Use 3×10 with or without Matrigel glue. 4 Cell migration and invasion were analyzed in Transwell chambers at varying densities. Four fields of view were randomly selected for photographing, and cell counts were performed under a microscope.

[0051] 1.9 Immunoblotting assay

[0052] Immunoblotting was performed according to the literature: Han SY, Zhao MB, Zhuang GB, Li PP. Marsdenia tenacissima extract restored gefitinib sensitivity in resistant non-small cell lung cancer cells. Lung Cancer. 2012; 75:30-7. Protein bands were visualized using an enhanced chemiluminescence assay kit, and grayscale values ​​were measured using ImageJ software.

[0053] 1.10 Animal Experiments

[0054] All animals were purchased from Beijing Huafukang Biotechnology Co., Ltd. Cells were subcutaneously inoculated into 6-8 week old male BALB / c nude mice to establish an NSCLC xenograft model. When the tumor volume reached approximately 50-100 mm², the xenograft was established. 3 Mice were randomly grouped using random numbers generated by Excel and processed as follows. After the experiment, mice were euthanized, and plasma and tumor samples were collected for further analysis.

[0055] H1975 cells were divided into three groups: sgRNA control, sgRNA1, and sgRNA2. The remaining mice were inoculated with PC-9 cells overexpressing HDGF or its vector control. The effect of HDGF knockdown on gefitinib efficacy was studied in four groups: sgRNA-control, sgRNA2, sgRNA2 + gefitinib (50 mg / kg), and sgRNA2 + solvent control. In PC-9 cells, the effect of HDGF overexpression on gefitinib efficacy was studied in four groups: vector control, HDGF overexpression + gefitinib (10 mg / kg), and HDGF overexpression + solvent control. Tumor volume data were collected using a blinded method.

[0056] 1.11 ELISA Experiment

[0057] The concentration of HDGF in mouse plasma was detected using a kit manufactured by Enzyme-Linked Biotechnology Co., Ltd. (Shanghai). The level of HDGF in the plasma of NSCLC patients was detected using an Anrc (Tianjin) ELISA kit.

[0058] 1.12 Immunohistochemical Experiment

[0059] Immunohistochemistry (IHC) was performed according to standard procedures. Tissue sections were incubated with HDGF or Ki-67 antibodies, and Jackson's (West Grove, PA) peroxidase-labeled goat anti-rabbit secondary antibody specifically bound to the peroxidase substrate diaminobenzidine (DAB) from Pierce (Rockford, IL).

[0060] 1.13 Statistical Analysis

[0061] Data are expressed as mean ± standard deviation. GraphPad Prism 6.0 software was used to fit the data and determine the IC50 value. The Mann-Whitney U test was used to compare gene expression among different groups, and independent samples t-tests were used for other comparisons. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 18.0.

[0062] Example 1: Interfering with the gene expression of HDGF or antagonizing the effect of HDGF can effectively inhibit the in vitro proliferation, migration and invasion of NSCLC.

[0063] HDGF was identified as one of the differentially expressed proteins in H1975 cells treated with gefitinib or MTE alone or in combination, using 2D gel and LC-MS / MS analysis. Western blot results showed that HDGF was significantly downregulated in H1975 cells after gefitinib and MTE combined treatment compared with treatment alone and the control group.

[0064] This invention investigated the expression of HDGF in seven NSCLC cell lines with varying responses to gefitinib. Gefitinib-sensitive PC-9 and H292 cells showed relatively low HDGF expression levels, while resistant H1975 cells exhibited very high HDGF expression levels. Stable HDGF overexpression cell lines were established in H1975 cells by knocking down HDGF using the CRISPR / Cas9 system and by transfecting PC-9 and H292 cells with the HDGFplenti6-TR plasmid. The efficiency of HDGF overexpression or knockdown was verified by Western blot and qRT-PCR.

[0065] See results Figures 1A to 1J HDGF overexpression or knockdown was verified by Western blot and qRT-PCR. Figure 1A , 1B HDGF sgRNA1 and sgRNA2 significantly inhibited the proliferation of H1975 cells. Figure 1C ), Cloning ( Figure 1E , Figure 1F ), migration and invasion ( Figure 1G , Figure 1H The ability of HDGF knockdown to suppress the malignant phenotype of NSCLC cells was measured (P < 0.01, P < 0.001 vs. control), indicating that HDGF knockdown inhibited the malignant phenotype of NSCLC cells. Conversely, HDGF overexpression (P < 0.001 vs. control) Figure 1C ) or rhHDGF stimulation ( Figure 1D HDGF promotes the proliferation of H292 and PC-9 cells. Simultaneously, HDGF overexpression enhances the clonogenic ability of H292 and PC-9 cells. Figure 1E , Figure 1F ) and migration and invasion capabilities ( Figure 1I , Figure 1J (P < 0.01, P < 0.001 vs. control). Therefore, the above data indicate that HDGF promotes the malignant phenotype of NSCLC cells.

[0066] Example 2: In vivo study of the effect of HDGF expression level on NSCLC tumor growth

[0067] This invention investigated the relationship between HDGF and tumor growth in xenograft mice. Figures 2A to 2P As shown, compared with the control group, both HDGF sgRNAs significantly inhibited tumor growth. Figures 2A-2C Furthermore, the inhibitory effect of the sgRNA2 group was more pronounced, indicating that HDGF knockdown limited tumor development in H1975. Simultaneously, Western blot confirmed that HDGF expression in H1975 tumor tissue was inhibited by both sgRNAs. Figure 2D , Figure 2E Conversely, compared to the control group, HDGF overexpression significantly increased the volume and weight of PC-9 tumors. Figures 2F-2H Western blot analysis also confirmed high levels of HDGF in PC-9 tumor tissue. Figure 2I , Figure 2J The above results indicate that HDGF promotes tumor growth, but HDGF knockdown can inhibit this process.

[0068] This invention demonstrates that the downstream molecules p-Akt and p-ERK of EGFR are abnormally activated in NSCLC patients resistant to gefitinib. In the experiments of this invention, alterations in these two molecules were identified in NSCLC cells and mouse tumor tissues. Compared to the control group, knockdown of HDGF significantly reduced the expression of p-Akt and p-ERK in H1975 cells, with a stronger inhibitory effect from sgRNA2. Conversely, overexpression of HDGF enhanced the levels of p-Akt and p-ERK in H292 and PC-9 cells. Figure 2K , Figure 2M In mouse tumor tissues, changes in p-Akt and p-ERK were consistent with those in cells. Figure 2L , Figure 2N In the presence of the Akt inhibitor MK2206 or the ERK inhibitor U0126, the effects of HDGF overexpression or rhHDGF-induced growth in H292 and PC-9 cells were eliminated or reversed. Figure 2O , Figure 2P ).

[0069] Example 3: Relationship between HDGF expression level and gefitinib efficacy

[0070] This invention further investigated the relationship between HDGF expression and the efficacy of gefitinib. For example... Figure 3A As shown, in H1975 cells, sgRNA knockdown of HDGF elicited a good response to gefitinib, with IC50 values ​​of 2.21 μM and 1.08 μM, respectively, compared to 7.30 μM in the sgRNA control group. Conversely, compared to the gefitinib-sensitive parental H292 and PC-9 cells, HDGF increased the IC50 value of gefitinib by 17 and 20-fold, respectively. Figure 3B , Figure 3C This indicates the development of drug resistance. Gefitinib significantly inhibited the colony formation, migration, and invasion abilities of HDGF-knockdown H1975 cells, but its effect on the control group was very weak. Figure 3D , Figure 3G , Figure 3J , Figure 3M Conversely, gefitinib treatment reduced colony formation in H292 and PC-9 cells. Figure 3E , Figure 3F , Figure 3H , Figure 3I ), migration and invasion capabilities ( Figure 3K , Figure 3L , Figure 3N , Figure 3O However, these inhibitory effects weaken or disappear after HDGF overexpression.

[0071] Example 4: Effect of HDGF knockout or overexpression on the efficacy of gefitinib in vivo

[0072] To evaluate the correlation between HDGF and the in vivo efficacy of gefitinib, this invention subcutaneously seeded NSCLC cells with HDGF knockdown or overexpression in mice. Figures 4A-4C As shown, 50 mg / kg gefitinib did not exhibit significant antitumor activity in H1975 tumor-bearing mice. However, in the HDGF sgRNA2 group, tumors shrank significantly, indicating that HDGF knockdown increased gefitinib sensitivity. Conversely, in gefitinib-sensitive PC-9 tumor-bearing mice, tumors stably expressing HDGF grew rapidly compared to the control group, but the pro-tumor effect of HDGF was weakened after administration of 10 mg / kg gefitinib. Figures 4D-4F However, tumor regression occurred in both the HDGF overexpression group and the control group, which may be related to the relatively high dose of gefitinib used in this experiment, as PC-9 cells are highly sensitive to gefitinib. Nevertheless, tumor growth in the HDGF overexpression group was still faster than in the control group after gefitinib treatment (P < 0.05), suggesting that HDGF overexpression weakens the efficacy of gefitinib to some extent. Figure 4G As shown, the expression of Ki-67 and HDGF in mouse tumor tissues xenografted with H1975 and PC-9 followed a trend consistent with tumor volume and weight.

[0073] HDGF is a secreted protein. This invention detected its concentration in the plasma of NSCLC-bearing mice to investigate whether plasma HDGF levels are related to the efficacy of gefitinib. Figure 4H and Figure 4I As shown, the trend of plasma HDGF concentration in mice was consistent with the efficacy of gefitinib. A preliminary analysis was conducted on plasma HDGF concentrations in 8 NSCLC patients before and after gefitinib monotherapy. Figure 4J and Figure 4K As shown, when drug resistance occurred, the HDGF levels in each case and the mean plasma level were significantly increased compared to before medication.

[0074] Furthermore, this invention measured plasma HDGF concentrations in 5 NSCLC patients before and after receiving the third-generation TKI AZD9291, and the results were similar to those observed in NSCLC patients with acquired resistance to gefitinib. Figure 4L , Figure 4M , Figure 4N HDGF expression was detected in paired biopsy specimens before and after TKI treatment. After TKI resistance, HDGF expression was high in both specimens, suggesting that high HDGF levels may indicate poor TKI efficacy.

[0075] Example 5: Study on the mechanism of HDGF-driven gefitinib resistance

[0076] This invention measures NSCLC cells ( Figure 5A ) and mouse tumor tissue ( Figure 5BGefitinib resistance-related proteins p-Akt and p-ERK were detected in H1975 cells. Knockdown of HDGF significantly reduced the expression of p-Akt and p-ERK. Gefitinib increased the expression of p-Akt and p-ERK in the control group, but still significantly reduced the expression of p-Akt and p-ERK in the HDGF knockdown group. Gefitinib significantly inhibited the expression of p-Akt and p-ERK in H292 and PC-9 cells, but could not reverse the phosphorylation of Akt and ERK caused by HDGF overexpression. These data further support the association between HDGF levels and gefitinib resistance-related signaling pathways.

[0077] Previous studies have shown that HDGF-induced cell growth can be inhibited or reversed by inhibitors of the Akt or ERK pathways. Figure 5C In this study, MK2206 (An Akt inhibitor) or U0126 (An ERK inhibitor) were added to verify whether HDGF promotes gefitinib resistance through these two signaling pathways. The results showed that HDGF did indeed reduce the sensitivity of PC-9 cells to gefitinib, while the promoting effect of HDGF on gefitinib resistance was significantly weakened by MK2206 or U0126, suggesting that HDGF may induce gefitinib resistance through the Akt and ERK signaling pathways.

[0078] EGFR T790M mutation is a major mechanism of gefitinib resistance. The pcDNA3.1-EGFR T790M mutant plasmid and HDGF overexpression vector were introduced alone or in combination into PC-9 cells to investigate whether HDGF is involved in T790M-mediated gefitinib resistance. Figure 5D In this study, the results showed that T790M mutation or HDGF overexpression alone led to gefitinib resistance, with IC50 values ​​of 0.107 μM and 0.181 μM, respectively. Cells with both T790M mutation and HDGF overexpression showed significantly higher resistance to gefitinib (IC50 = 0.253 μM) than cells with only T790M mutation and HDGF overexpression. These results suggest that gefitinib resistance caused by T790M mutation or HDGF overexpression may not share the same mechanism. Therefore, HDGF may not be involved in gefitinib resistance induced by T790M mutation.

[0079] This invention found that silencing or overexpressing HDGF affects the activation of downstream Akt and ERK pathways of EGFR, which is associated with TKI resistance. This invention further investigated whether crosstalk exists between HDGF and EGFR. First, a search of pathcards in the GeneCards database (http: / / www.genecards.org) revealed significant overlap between the HDGF pathway and EGFR, including Akt and ERK signaling pathways. Figure 6ANext, this invention found that in NSCLC cells and tumor tissues, p-EGFR levels were negatively correlated with HDGF knockdown or overexpression. Figure 6B , Figure 6C Except for H157 cells, gefitinib dose-dependently inhibited p-EGFR and enhanced HDGF, while the expression levels of both p-EGFR and HDGF in H157 cells were extremely low. Figure 6D , Figure 6E This indicates that gefitinib-induced HDGF elevation only occurs in EGFR-dependent NSCLC cells. Simultaneously, gefitinib treatment also increased the concentration of secreted HDGF in the supernatant of NSCLC cells other than H157 cells. Figure 6F ).

[0080] This invention further explores whether HDGF and EGFR play complementary roles in the response of HDGF knockdown and overexpression NSCLC cells to gefitinib. Figure 6G and Figure 6I As shown, HDGF knockdown led to a decrease in HDGF expression, and the upregulation of p-EGFR was reversed after gefitinib treatment of H1975 cells. In H292 and PC-9 cells, HDGF overexpression resulted in increased HDGF and inhibition of p-EGFR; gefitinib further inhibited p-EGFR but had no significant effect on HDGF expression. Figure 6H and Figure 6J As shown, gefitinib increased HDGF expression in PC-9 xenografts overexpressing HDGF (P < 0.05), and the changes in HDGF and p-EGFR in tumor tissue were consistent with those observed in cell experiments. Therefore, these data indicate an interaction between HDGF and EGFR in NSCLC, with HDGF potentially acting as a bypass signaling molecule for EGFR, activating downstream molecules Akt and ERK. Furthermore, HDGF and EGFR have complementary roles in maintaining tumor cell survival under gefitinib treatment. The role of HDGF in gefitinib resistance is further discussed below. Figure 7 As shown.

[0081] The embodiments described above should not be construed as limiting the scope of this invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this invention, and these modifications and improvements all fall within the scope of protection of this invention.

Claims

1. The use of liver cancer-derived growth factor HDGF as a target in screening and / or preparing drugs that improve resistance to tyrosine kinase inhibitors in patients with non-small cell lung cancer; wherein the tyrosine kinase inhibitors include one or more of gefitinib, icotinib, and AZD9291.

2. Application of reagents for detecting the expression level of hepatocellular carcinoma-derived growth factor (HDGF) in the preparation of a detection system for assessing the resistance of non-small cell lung cancer patients to tyrosine kinase inhibitors; wherein the tyrosine kinase inhibitors include one or more of gefitinib, icotinib, and AZD9291.

3. The use of an antagonist against hepatocellular carcinoma-derived growth factor (HDGF) in the preparation of a medicament for treating non-small cell lung cancer and / or reducing tyrosine kinase inhibitor resistance in patients with non-small cell lung cancer, wherein the antagonist against HDGF is a reagent that reduces the expression level of HDGF and / or antagonizes the effect of HDGF, and the tyrosine kinase inhibitor includes one or more of gefitinib, icotinib, and AZD9291.

4. The application according to claim 3, wherein, Reducing resistance to tyrosine kinase inhibitors includes lowering the half-maximal effective concentration (IC50) of the tyrosine kinase inhibitor. 50 ).

5. The application according to claim 3, wherein, Reducing resistance to tyrosine kinase inhibitors includes weakening the ability of tumor cells to form colonies and / or inhibiting the proliferation, migration, spread, and / or invasion of tumor cells.

6. The application according to claim 3, wherein, Reducing resistance to tyrosine kinase inhibitors includes promoting tumor cell apoptosis and / or inhibiting tumor growth in vivo.

7. The application according to claim 3, wherein, Reducing resistance to tyrosine kinase inhibitors involves inhibiting the expression of resistance-related proteins p-Akt and p-ERK.

8. The application according to claim 3, wherein, The antagonists against HDGF include sgRNA, small molecule inhibitors, natural products, antibodies, or combinations thereof.

9. The application according to claim 8, wherein, The antagonist of HDGF is sgRNA, and the sequence of the sgRNA is shown in SEQ ID NO:

2.

10. Application of reagents that induce HDGF overexpression in tyrosine kinase inhibitor-sensitive cells in the preparation of research formulations with at least one of the following effects: Increase the IC50 of tyrosine kinase inhibitors 50 ; Promotes cell clone formation and / or scratch healing ability; Enhances cell proliferation, colony formation, migration, and invasion; and / or Inducing the expression of drug resistance-related proteins p-Akt and p-ERK; The cells are non-small cell lung cancer cells; the tyrosine kinase inhibitors include one or more of gefitinib, icotinib, and AZD9291.

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