CN117224535A - Application of PAI-1 inhibitor in preparation of drug for enhancing treatment sensitivity of HNSCC patient to cetuximab - Google Patents

Abstract

The invention discloses application of a PAI-1 inhibitor in preparation of a drug for enhancing treatment sensitivity of HNSCC patients to cetuximab. Belongs to the technical field of biological medicine. The research proves that PAI-1 expression inhibits the inhibition effect of cetuximab on HNSCC tumor through a plurality of animal models, and Tiplaxtinin can enhance the sensitivity of cetuximab treatment, thereby providing theoretical basis and experimental basis for the application of PAI-1 inhibitor in HNSCC treatment.

Description

Application of PAI-1 inhibitor in preparation of drug for enhancing treatment sensitivity of HNSCC patient to cetuximab

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a PAI-1 inhibitor in preparation of a drug for enhancing treatment sensitivity of HNSCC patients to cetuximab.

Background

Head and neck squamous cell carcinoma (Head and neck squamous cell carcinoma, HNSCC) is the sixth most common malignancy worldwide, accounting for over 90% of head and neck tumors, with about 89 thousand new diagnosis cases and 45 death cases per year, and the incidence of HNSCC is on a year-by-year trend. HNSCC treatment includes a variety of therapeutic approaches such as surgical treatment, radiation therapy, chemotherapy, targeted therapy, etc., but currently HNSCC patients have a 5-year survival rate of only about 40%. Exploration of new treatment schemes, improvement of HNSCC treatment effects and improvement of HNSCC patient prognosis are one of the important problems to be solved urgently.

Epidermal growth factor receptor (Epidermal growth factor receptor, EGFR). Abnormal expression of EGFR is found in many tumors of epithelial origin, such as HNSCC, colorectal cancer, non-small cell lung cancer, pancreatic cancer, and the like. EGFR expression is elevated in 90% or more of HNSCC patients and is associated with poor prognosis.

Cetuximab is the first EGFR monoclonal antibody approved by the FDA for the treatment of recurrent or metastatic HNSCC, prolonging the overall survival of HNSCC patients. In the exteme phase III clinical trial, cetuximab in combination with the PF regimen prolonged the overall survival of HNSCC patients compared to the PF regimen (cisplatin/carboplatin+5-fluorouracil). The median total survival period extends from 7.4 months to 10.1 months; median progression-free survival was extended from 3.3 months to 5.6 months; the response rate of patients to treatment is increased from 20% to 36%. At the same time, the combination treatment regimen did not exacerbate the adverse effects in HNSCC patients. The exteme clinical trial demonstrated the importance of cetuximab in HNSCC treatment and proposed the combination PF regimen of cetuximab as a first-line treatment regimen for patients with recurrent or metastatic HNSCC. Subsequently, domestic researchers modified the EXTREME regimen, reduced the dosage of drug used, and developed a phase-III clinical trial of CHANGE-2. The results show that the overall survival of the patients in the cetuximab-combined PF regimen treatment group was prolonged from 8.9 months to 11.1 months compared to the PF regimen; median progression-free survival was extended from 4.2 months to 5.5 months; the objective response rate of patients to treatment is improved from 26.6% to 50.0%. The CHANGE-2 clinical trial further demonstrated the conclusion of the exteme clinical trial, highlighting the great potential of cetuximab in HNSCC treatment.

However, although cetuximab is the first-line treatment regimen for patients with recurrent or metastatic HNSCC, some patients are insensitive to cetuximab or develop resistance. Cetuximab sensitivity is an important issue in the course of HNSCC treatment. The specific mechanism of HNSCC on cetuximab resistance is explored, and a new treatment strategy is sought, so that the method is an important way for improving the treatment sensitivity of cetuximab and improving the prognosis of HNSCC patients.

Disclosure of Invention

In view of this, the present invention provides the use of a PAI-1 inhibitor in the manufacture of a medicament for enhancing the therapeutic sensitivity of a patient with HNSCC to cetuximab. According to the invention, through a plurality of animal models, the inhibition effect of PAI-1 expression on HNSCC tumor by inhibiting cetuximab is verified, while Tiplaxtinin can enhance the sensitivity of cetuximab treatment, and a theoretical basis and an experimental basis are provided for the application of PAI-1 inhibitor in HNSCC treatment.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

use of a PAI-1 inhibitor for the manufacture of a medicament for enhancing the therapeutic sensitivity of HNSCC patients to cetuximab.

Further, the PAI-1 inhibitor is Tiplaxtinin.

Further, the PAI-1 inhibitor is useful for inhibiting lymph node metastasis, lung metastasis and HNSCC progression.

Further, the PAI-1 inhibitor is used for inhibiting HNSCC cell activity, inhibiting HNSCC cell proliferation and promoting HNSCC cell apoptosis.

Compared with the prior art, the invention has the beneficial effects that:

(1) The expression characteristics of PAI-1 in HNSCC tissues and blood are described for the first time, the correlation between PAI-1 expression and HNSCC clinical adverse prognosis factors is revealed, and the PAI-1 can be used as an effective biomarker for predicting HNSCC patient prognosis and can be used as a potential HNSCC treatment target.

(2) PAI-1 has therapeutic value as a "point-multiple target" in HNSCC. PAI-1 can simultaneously bind EGFR and ITGA5, respectively activate EGFR/AKT channel and ITGA5/FAK channel, inhibit PAI-1 can simultaneously inhibit EGFR/AKT channel and ITGA5/FAK channel, and enhance cetuximab sensitivity.

(3) The present study demonstrates in vitro and in vivo that the PAI-1 inhibitor tiplastinin can enhance the sensitivity of HNSCC to cetuximab treatment.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the expression of PAI-1 in HNSCC tumor tissues without lymph node metastasis and with lymph node metastasis in example 1 of the present invention, wherein A is the result of immunohistochemical staining (N0 represents without lymph node metastasis, N2 represents with lymph node metastasis), B is the result of statistics (N0 represents without lymph node metastasis, N1-3 represents with lymph node metastasis);

FIG. 2 shows the expression of PAI-1 in HNSCC Primary (Primary) and metastatic (Metastasis) tumor tissues according to example 1 of the present invention, wherein A is immunohistochemical staining result and B is statistical result;

FIG. 3 is a correlation analysis of PAI-1 and OS, PFS in HNSCC in example 1 of the present invention;

FIG. 4 shows cytokine expression in blood samples of healthy subjects and HNSCC patients in example 1 of the present invention;

FIG. 5 is a heat map of PAI-1 levels and clinical profile correlation in HNSCC blood samples in example 1 of the present invention;

FIG. 6 shows the basic information of HNSCC tissue chip in embodiment 1 of the present invention;

FIG. 7 shows the PAI-1 expression in HNSCC tissue chips in example 1 of the present invention;

FIG. 8 shows the correlation analysis of PAI-1 expression and lymph node metastasis, T stage (T1-2 is early, T3-4 is middle and late) in HNSCC tissue chip in example 1 of the present invention, wherein A is the expression of PAI-1 in normal tissue and tumor tissue; b is PAI-1 expression and lymph node metastasis correlation; c is the correlation between PAI-1 expression and T stage;

FIG. 9 shows basal expression and stable cell construction of PAI-1 in HNSCC cells in example 2 of the present invention; wherein A is WB to detect the basal expression of PAI-1 in SCC15, SCC25, UM1, fadu, detroit562 cells; b is ELISA to detect the secretion of PAI-1 in the supernatant of SCC15, SCC25, UM1, fadu and Detroit 562;

FIG. 10 shows the HNSCC stably transformed cell assay in example 2 of the present invention; wherein A is cellular protein WB to verify the effect of PAI-1 knockout in SCC15 cells; b is supernatant ELISA to verify the PAI-1 knockout effect in SCC15 cells; c is supernatant freeze-dried protein WB to verify the PAI-1 knockout effect in SCC15 cells; sg-NC as control, sg-PAI-1#1, sg-PAI-1#2, sg-PAI-1#3 represent 3 cell lines in which PAI-1 was knocked out;

FIG. 11 is a graph showing the activity of cells after treatment with cetuximab tested by CCK8 assay in example 2 of the present invention;

FIG. 12 is a graph showing the ability of the clonogenic assay of example 2 of the present invention to detect cell proliferation following cetuximab treatment;

FIG. 13 is a graph showing the level of apoptosis after flow cytometry detection of cetuximab treatment in example 2 of the present invention;

FIG. 14 is a graph showing the activity of Tiplaxtinin in combination with cetuximab after treatment in CCK8 assay in example 2 of the present invention;

FIG. 15 is a graph showing the proliferation potency of cells after a combination treatment in a colony formation assay in example 2 of the present invention;

FIG. 16 shows apoptosis levels after flow cytometry detection combination treatment in example 2 of the present invention;

FIG. 17 shows the expression and secretion of PAI-1 after transfection of FL and ΔSP plasmids in SCC15 cells from which PAI-1 was knocked out, using the cellular proteins (FIG. 17A), supernatant lyophilized protein WB (FIG. 17A) and ELISA (FIG. 17B) of example 2 of the present invention;

FIG. 18 is a clone formation assay in example 2 of the present invention to examine the effect of PAI-1 secretion function on cetuximab therapy;

FIG. 19 is an alignment of amino acid sequences of PAI-1 proteins of different species in example 2 of the present invention;

FIG. 20 is a verification of glycosylation modification of PAI-1 in HNSCC in example 2 of the present invention; wherein A is the change in PAI-1 protein following treatment with a glycosylation inhibitor; b is the change of PAI-1 protein after glycosidase treatment;

FIG. 21 shows PAI-1 glycoprotein staining and Coomassie brilliant blue staining in HNSCC in example 2 according to the present invention;

FIG. 22 shows that the modification of PAI-1 glycosylation in HNSSC maintains its protein stability in example 2 of the present invention;

FIG. 23 shows the effect of TM in combination with MG132, CQ, conA on the half-life of PAI-1 protein in example 2 of the present invention;

FIG. 24 is a graph showing the verification of PAI-1 over-expression stably transformed cells in example 3 of the present invention; wherein A is cellular protein WB to verify PAI-1 over-expression effect in SCC15 cells; b is supernatant freeze-dried protein WB to verify the PAI-1 over-expression effect in SCC15 cells; c is supernatant ELISA to verify the PAI-1 over-expression effect in SCC15 cells;

FIG. 25 is a graph showing that Co-IP in example 3 of the present invention verifies that PAI-1 and EGFR bind to each other in HNSCC;

FIG. 26 is a graph showing that PLA verifies that PAI-1 and EGFR bind to each other in HNSCC in example 3 of the present invention;

FIG. 27 is a graph showing GST-pulldown demonstrating that PAI-1 and EGFR bind to each other in example 3 of the present invention;

FIG. 28 shows HNSCC immunofluorescence in example 3 of the present invention;

FIG. 29 shows HNSCC tumor tissue immunofluorescence in example 3 of the present invention;

FIG. 30 is a graph showing the Co-IP verification of the existence of mutual association of PAI-1 and EGFR extracellular domain in example 3 of the present invention;

FIG. 31 shows PAI-1 activation EGFR/AKT pathway in HNSCC in example 3 of the present invention; wherein A is Co-IP showing PAI-1 promotes EGFR dimerization; b activates EGFR/AKT pathway for PAI-1, promotes EGFR and AKT phosphorylation;

FIG. 32 is an IHC that evaluates PAI-1, p-EGFR, p-AKT expression in HNSCC in example 3 of the present invention;

FIG. 33 is a correlation analysis of PAI-1 expression and p-EGFR, p-AKT expression in HNSCC in example 3 of the present invention, wherein A is a statistical result; b is a correlation analysis chart;

FIG. 34 is a graph showing the Co-IP authentication of PAI-1 and AKT1 binding to each other in example 3 of the present invention;

FIG. 35 is a graph showing that PAI-1 in HNSCC in example 3 of the present invention directly binds AKT1 to promote its phosphorylation;

FIG. 36 is a graph showing the Co-IP validation of the mutual binding between PAI-1 and ITGA5 (A), ITGA5 and EGFR (B) in example 4 of the present invention;

FIG. 37 is a graph showing that PLA verifies that PAI-1 and ITGA5 bind directly to each other in example 4 of the present invention;

FIG. 38 is a diagram showing the direct association of GST-pulldown validation PAI-1 and ITGA5 in example 4 of the present invention;

FIG. 39 is a graph showing the co-localized expression of HNSCC cellular immunofluorescence assay PAI-1 and ITGA5 in example 4 of the present invention;

FIG. 40 shows the co-localized expression of HNSCC tumor tissue immunofluorescence assay PAI-1 and ITGA5 in example 4 of the present invention;

FIG. 41 is a PAI-1 regulatory ITGA5/FAK signaling pathway in HNSCC in example 4 of the present invention;

FIG. 42 is a graph showing the evaluation of PAI-1 and ITGA5 expression in HNSCC by IHC in example 4 of the present invention; wherein A is PAI-1 and ITGA5 expression in HNSCC tissue; b is the correlation analysis of PAI-1 expression and ITGA5 expression in HNSCC;

FIG. 43 shows the modulation of EMT by ITGA5 of PAI-1 in HNSCC in example 4 of the present invention; wherein A is WB to verify the efficiency of siRNA interfering ITGA5 expression; b is PAI-1, and the EMT process is regulated and controlled through ITGA5, so that E-cadherin and N-cadherin expression are influenced; si-NC, as controls, i-ITGA5#1, si-ITGA5#2, si-ITGA5#3 represent 3 cell lines that interfere with ITGA5 expression;

FIG. 44 is a graph showing the activity of CCK8 assay in example 4 of the present invention after treatment with cetuximab;

FIG. 45 is a graph showing the ability of the clonogenic assay of example 4 of the present invention to detect cell proliferation following cetuximab treatment;

FIG. 46 is a graph showing the level of apoptosis after flow cytometry detection of cetuximab treatment in example 4 of the present invention;

FIG. 47 is a graph showing that PAI-1 in HNSCC promotes focal adhesion kinase complex formation by activating FAK in example 4 of the present invention;

FIG. 48 is a graph showing that PAI-1 promotes HNSCC cell migration and invasion by ITAG5 in example 4 of the present invention; wherein A is scratch experiment observation cell migration capability; b is the migration and invasion capacity of cells observed by a Transwell experiment;

FIG. 49 is a model of HNSCC lymph node metastasis in example 5 of the present invention; wherein A is the size of tumor and lymph node tissue of HNSCC lymph node metastasis model mice; b is a tumor growth curve of HNSCC lymph node metastasis model;

FIG. 50 is a sample of HNSCC lymph node metastasis model mouse popliteal fossa and inguinal lymph node HE staining in example 5 of the present invention;

FIG. 51 is a model of HNSCC lung metastasis in example 5 of the present invention; wherein A is lung metastasis model mouse living body imaging; b is lung tissue imaging of a lung metastasis model mouse; c is HE staining of lung tissue of a lung metastasis model mouse;

FIG. 52 is a graph showing that PAI-1 expression affects the sensitivity of HNSCC tumors to cetuximab treatment in example 5 of the present invention; wherein A is the tumor size of the mice; b is a tumor growth curve;

FIG. 53 is a graph showing that Tiplaxtinin increases the susceptibility of HNSCC to cetuximab treatment in example 5 of the present invention; wherein A-B are the tumor sizes of PDX mice; c is a PDX tumor growth curve.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The required medicament is a conventional experimental medicament and is purchased from a commercial channel; the test methods not mentioned are conventional test methods and will not be described in detail herein.

Plasmids used in the examples of the present invention:

PAI-1 plasmid encoding Full Length (FL) and N-terminal signal peptide point mutations (Δsp);

EGFR overexpression plasmid with Myc label;

EGFR extracellular and intracellular plasmids with Myc tags;

EGFR plasmid with HA tag;

AKT1 plasmid with Myc tag;

an ITGA5 overexpression plasmid with an HA tag;

All are commercial products.

Example 1

PAI-1 high expression and poor prognosis correlation in HNSCC

1.1 objects and methods

1.1.1 subjects

The study selects 95 cases of clinical tissue specimens of HNSCC patients collected and treated in the tumor department of maxillofacial ear nose throat of the tumor hospital of Tianjin medical university, performs immunohistochemical staining, improves clinical information of the patients, carries out follow-up visit on all patients, and collates survival information of the patients. Meanwhile, blood samples of 10 healthy people and 40 HNSCC patients are collected, serum is extracted, cytokine array analysis is carried out, and clinical information is perfected.

1.1.2 Experimental methods

1.1.2.1 immunohistochemical staining (IHC)

The tissue slices to be dyed are put into a 70 ℃ oven in advance, and the slices are baked for 1h. Sequentially placing the mixture into ethanol with dewaxing liquid and concentration gradient for dewaxing. The tissue slices are washed for 3 times, fully soaked in antigen retrieval liquid, placed in an autoclave, heated for 3min and cooled to normal temperature. The tissue sections were washed 3 times, endogenous peroxidase blocking reagent was added dropwise, and the sections were blocked at room temperature for 30min. The tissue sections were washed 3 times, sheep serum was added dropwise, and the mixture was blocked at room temperature for 30min. And (3) dripping diluted target antibody to be dyed, putting the target antibody into a wet box, and incubating the wet box in a refrigerator at 4 ℃ overnight. Rewarming for 30min, and cleaning for 3 times. And (3) dropwise adding a biotin-labeled secondary antibody working solution, and incubating for 30min at room temperature. Washing for 3 times, dripping the streptavidin working solution marked by horseradish enzyme, incubating for 30min at room temperature, and washing for 3 times. Dripping 1 XDAB working solution, observing the color development condition of the tissue under a mirror to determine proper dyeing time, and rapidly washing with PBS buffer solution after the dyeing is finished to terminate the dyeing. And (3) dropwise adding hematoxylin dye, observing the change of cytoplasm under a mirror to determine proper dyeing time, and rapidly washing with PBS buffer solution after the dyeing is finished to terminate the dyeing. And (5) sequentially putting the slices into ethanol with different concentrations and dewaxing liquid in the opposite direction for dehydration. After the sections are dried, the sections are sealed by neutral resin and observed under a microscope. The scores were 0 to 3 according to the staining intensities (negative, weak, medium, strong) and 0 to 4 according to the positive cell rates (0%, 1 to 25%, 25 to 50%, 50 to 75%, 75 to 100%). The positive grade of the section staining is judged according to the multiplication of the integral of the staining intensity and the positive cell rate: less than 6 is weakly expressed, and more than 6 is strongly expressed.

1.1.2.2 cytokine array

To each well 100. Mu.l of sample dilution was added and incubated for 1h with shaking at room temperature. The buffer in each well was discarded, 100. Mu.l of standard solution and sample were added and incubated overnight at 4 ℃. To each well, 250. Mu.l of wash I was added, washed 10 times with shaking, 250. Mu.l of wash II was added, and washed 6 times with shaking. The antibody mixture was tube centrifuged rapidly and 1.4ml of sample dilution was added and mixed well. To each well, 80. Mu.l of the antibody mixture was added and incubated for 2h with shaking at room temperature. The slide is washed. The Cy 3-streptavidin tube was centrifuged rapidly, 1.4ml of sample dilution was added and mixed well. To each well, 80. Mu.l of Cy 3-streptavidin solution was added and incubated for 1h at room temperature in the absence of light. After washing the slide, fluorescence detection is performed.

1.1.2.3 statistical analysis

Statistical analysis and mapping was performed using SPSS and GraphPadPrism 9 software. Classification variables were analyzed using chi-square and fischer exact tests. The survival analysis adopts a Kaplan-Meier method, a single-factor Cox regression analysis and a multi-factor Cox regression analysis. * P <0.05; * P <0.01; * P <0.001; * P <0.0001.

1.2 results

1.2.1PAI-1 expression is associated with HNSCC metastasis

The study performed immunohistochemical staining on clinical tissue specimens of 95 HNSCC patients to evaluate PAI-1 expression. The results showed that there were 54 cases of PAI-1 low-expression HNSCC tumor tissue samples and 41 cases of PAI-1 high-expression HNSCC tumor tissue samples. PAI-1 was expressed more highly in HNSCC tumor tissue with lymph node metastasis (p=0.002) than in HNSCC tumor tissue without lymph node metastasis, while the proportion of PAI-1 highly expressed samples was increased in HNSCC tumor tissue with lymph node metastasis (fig. 1). The expression of PAI-1 in HNSCC primary and metastatic tumor tissues was further evaluated. The results showed that PAI-1 expression was elevated in metastatic tumor tissue (P=0.017) as compared to HNSCC primary foci, while the proportion of PAI-1 highly expressed samples was elevated in HNSCC metastatic tumor tissue (FIG. 2).

The correlation of these clinical features of PAI-1 expression and age, sex, smoking history, drinking history, tumor size, presence or absence of lymph node metastasis, presence or absence of extranodal invasion, tumor infiltration depth, degree of differentiation, T-stage, N-stage, and AJCC stage was further analyzed (table 1). The results showed that PAI-1 expression and lymph node metastasis (p=0.002), extranodal invasion (p=0.014), tumor invasion depth (p=0.012), tumor differentiation degree (P < 0.001) and N-staging (p=0.021) in HNSCC were positively correlated (table 1). Cervical lymph nodes are the most common site of metastasis for HNSCC. These results indicate that PAI-1 expression is associated with HNSCC invasive metastasis.

TABLE 1 PAI-1 expression and clinical characterization correlation analysis in HNSCC

1.2.2PAI-1 overexpression suggests poor HNSCC prognosis

To analyze the relationship between PAI-1 expression and prognosis for HNSCC survival, the study followed all patients, perfecting patient survival information. According to the PAI-1 expression condition in HNSCC tumor tissue specimens, all patients are divided into two groups of PAI-1 high expression and PAI-1 low expression, and survival analysis is carried out by using a Kaplan-Meier method.

Survival analysis results showed (FIG. 3) that total survival (OS) and Progression-free survival (PFS) were shortened in the PAI-1 high expression group compared to the PAI-1 low expression group (OS: P=0.042; PFS: P=0.007).

Next, PAI-1 expression and clinical factors were combined with survival information and single and multi-factor Cox regression analysis was performed (table 2). The results of the single factor Cox regression analysis show that lymph node metastasis, N-staging and PAI-1 expression are important factors affecting the survival prognosis of HNSCC patients. These factors were subjected to multifactorial Cox regression analysis, and the results showed that lymph node metastasis and PAI-1 expression were important factors affecting the OS of HNSCC patients, while PAI-1 expression was an independent predictor affecting PFS of HNSCC patients. The results indicate that PAI-1 expression is negatively correlated with HNSCC patient OS, PFS, which can be used as a poor prognosis factor for HNSCC.

TABLE 2 one-and multifactorial analysis of HNSCC Total and progression free survival

Increased PAI-1 expression levels in the blood of 1.2.3HNSCC patients and related to HNSCC progression

PAI-1 is a secreted protein that can be secreted by cells into the blood. In order to better understand the expression characteristics of PAI-1 in HNSCC patients, the study collects blood samples of 10 healthy people and 40 HNSCC patients who are treated in the tumor hospital maxillofacial ear nose throat oncology department of Tianjin medical university, extracts serum, carries out cytokine array analysis and perfects clinical information. Of the 40 HNSCC patients, 12 early HNSCC patients (stage I-II) and 28 late HNSCC patients (stage III-VIA) were included. Cytokine array analysis showed that PAI-1 secretion levels were elevated in blood of HNSCC patients compared to healthy persons, and furthermore, OPN, IGFBP-2, TGF beta-1 secretion levels were also elevated in HNSCC patients (FIG. 4).

Correlation analysis was performed on cytokine expression levels and clinical features in blood of 40 HNSCC patients. The results showed that PAI-1 expression levels were elevated in the blood of patients with intermediate and late HNSCC and patients with lymph node metastasis (FIG. 5), which is consistent with the analysis results in clinical tissues of HNSCC patients. The correlation of PAI-1 expression with HNSCC development was further confirmed by cytokine array analysis.

External verification of PAI-1 expression in 1.2.4HNSCC tumor tissue

In order to make the results more reliable and representative, the research purchases HNSCC organization chips for external verification. The outsourced tissue chip (Biomax in the united states) included 11 normal tissue specimens, 61 HNSCC primary foci tumor tissue specimens, and 8 HNSCC metastatic foci tumor tissue specimens (fig. 6).

The tissue chips were subjected to IHC staining to evaluate PAI-1 expression (FIG. 7). The results showed that PAI-1 expression was elevated in HNSCC tumor tissue (p=0.008) compared to normal tissue (fig. 8A). We also assessed the expression of PAI-1 in HNSCC without lymph node metastasis and in tumor tissues with lymph node metastasis. The results showed that PAI-1 expression was elevated in HNSCC tumor tissue with lymph node metastasis (P < 0.001) compared to HNSCC tumor tissue without lymph node metastasis (FIG. 8B). Although there were fewer samples of metastatic tumor tissue, it was still observed that PAI-1 expression was elevated in metastatic tumor tissue compared to the primary foci of HNSCC (FIG. 7). Furthermore, PAI-1 expression was elevated in T3-T4 phase HNSCC patient tissue specimens (p=0.030) compared to T1-T2 phase HNSCC patients (fig. 8C). These results further demonstrate the importance of PAI-1 expression in the development of HNSCC.

Example 2

PAI-1 affects HNSCC sensitivity to cetuximab treatment

2.1 objects and methods

2.1.1 subjects

Human tongue squamous carcinoma cell lines SCC15, SCC25, UM1, human pharyngeal squamous carcinoma cell line Fadu, and human pharyngeal head cancer hydrothorax transfer cell line Detroit562 were purchased from the American tissue culture Collection (American Tissue Culture Collection, ATCC).

2.1.2 Experimental methods

2.1.2.1 cells were resuscitated, passaged and cryopreserved

Taking out the cell freezing tube from the liquid nitrogen tank, quickly putting the cell freezing tube into a water bath kettle at 37 ℃ for rewarming, and centrifuging at 800rpm for 4min. Removing supernatant, adding 1ml of complete medium, blowing to resuspension, transferring to sterile cell culture dish, adding 8-10 ml of complete medium, uniformly resuspension, adding 37 deg.C and 5% CO ₂ In a cell incubator. The new cell culture medium is changed every 1-3 days. When the growth density reached around 90%, cell passaging was performed. The culture medium is discarded, the cells are gently washed for 2 to 3 times by using sterile 1 XPBS buffer solution, 1 to 2ml pancreatin is added, the complete culture medium is added to stop digestion when the cell suspension is no longer adherent, the cells are uniformly resuspended, transferred into a 15ml sterile centrifuge tube, centrifuged for 4min at 800rpm, and the supernatant is discarded. According to the experimental requirement, the proper proportion is selected for passage. If freezing is needed, the supernatant is discarded after centrifugation, the complete culture medium, the fetal calf serum and the DMSO are respectively added into the centrifuge tube according to the proportion of 5:4:1, and the mixture is split into sterile cell freezing tubes after being uniformly resuspended and is put into a liquid nitrogen tank for preservation.

Construction of 2.1.2.2 stably transformed cell line

Cells to be transfected were then incubated according to 3X 10 ⁴ Cell mass per well. When the growth density reached 20-30%, the medium was discarded and 1ml of complete medium was added to the six-well plate. According to the MOI of the cells and the virus titer, the corresponding virus amount was added, and 40. Mu.l of 25 Xvirus infection enhancing solution was added, and after 16 hours of culture, the culture was changed to complete medium and cultured for 48 hours. And adding puromycin with proper concentration, screening for 48 hours, and when all the empty cell groups die, continuously screening the cells of the infected group by using the puromycin with the concentration, wherein the surviving cells are stable transfer cells.

2.1.2.3 Western blot experiment (WB)

Sucking 30ug protein sample, adding 5 Xprotein loading buffer solution according to the ratio of 4:1, heating at 100 deg.C for 5-10 min, and selecting proper concentration separating gel and concentrating gel for protein electrophoresis. The PVDF membrane was activated with methanol and transferred. 5% skim milk or BSA solution was blocked and washed with TBST buffer. Membranes were cut and incubated overnight with the corresponding protein antibodies in a shaking table at constant temperature of 4 ℃. The following day the corresponding secondary antibody was added and incubated for 1h at room temperature. The TBST solution was washed and then imaged by exposure to light to analyze protein bands.

2.1.2.4 enzyme-Linked immunosorbent assay (ELISA)

Collecting the culture supernatant of the cells to be tested, sucking 30. Mu.l of the sample to be tested, and adding the sample to 275. Mu.l of the sample diluent for later use. The sample was diluted in multiple ratios and added to the elisa plate. Sealing the ELISA plate by using a sealing plate film, and reacting for 90min at 37 ℃. Then 100. Mu.l of biotin anti-antibody working solution was added thereto for reaction at 37℃for 60 minutes. Then 100. Mu.l of ABC working solution was added and reacted at 37℃for 30 minutes. TMB color development liquid is added, and the reaction is carried out for 30min at 37 ℃ in a dark place. Mu.l of stop solution was added and the OD was measured at 450 nm. And drawing a standard curve, and calculating the concentration of the sample.

Transient transfection of 2.1.2.5 cells

Cells were plated in six well plates. The plasmid (6. Mu.g) and the liposome nucleic acid transfection reagent (7.5. Mu.l) were diluted separately for transfection, incubated at room temperature for 5min, and incubated at room temperature for 20min after mixing. At the same time, 1ml of serum-free medium was added again. And adding the transfection complex into a six-hole plate, culturing for 4-6 h, replacing the complete culture medium, continuing culturing for 48-72 h, and verifying the gene level or the protein level.

2.1.2.6CCK8 experiment

The cell suspension concentration was adjusted to 1.5X10 ⁴ Per ml, 200 μl of suspension was added to each well of a 96-well plate. After the cells are attached, adding medicine for 48-72 h. Cell culture medium containing 10% cck8 solution was added to each well and incubated at 37 ℃ in the dark for 1h. The absorbance at 450nm was measured, and the cell viability was calculated. The calculation formula is as follows: (control OD value-experimental OD value)/control OD value x 100%.

2.1.2.7 apoptosis assay

Collecting 1-5X 10 ⁵ The cells were washed, 1 Xbinding Buffer was added, and then annexin V-FITC and PI Staining Solution were added, and incubated at room temperature for 15min under dark conditions after mixing. Adding 1×binding Buffer, mixing, and detecting on machine.

2.1.2.8 clone formation experiment

100 to 500 cells are inoculated in a six-hole plate for 2 to 3 weeks of culture. Then cleaning, adding 4% paraformaldehyde for fixing for 15min, adding 0.1% crystal violet solution for dyeing for 15min, slowly washing off the dyeing liquid by using running water, and drying. Observation, photographing and counting cloning.

2.1.2.9 Coomassie Brilliant blue staining experiment

After the electrophoresis was completed, the gel was taken out and stained in coomassie brilliant blue staining solution at room temperature for 1 hour. Adding a proper amount of decoloring liquid, slowly shaking for decoloring, and after decoloring, putting the gel into water for storage, photographing and recording for analysis.

2.1.2.10 glycoprotein staining experiment

Taking out SDS-PAGE gel, adding 50% methanol solution for fixing for 30min, adding deionized water for washing, adding oxidizing reagent, adding glycoprotein staining reagent for staining for 15min after washing, adding reducing reagent, and finally adding 3% acetic acid solution until clear glycoprotein red main band can be seen. And (5) photographing, recording and analyzing.

2.1.2.11O Glycosidase Glycosidase treatment

10-20. Mu.g glycoprotein is pipetted, 10 Xglycoprotein denaturation buffer is added, deionized water is added and mixed to 10. Mu.l. The glycoprotein was denatured by heating in a metal bath at 100 ℃. 10 Xglycoside buffer, 10% NP40, neuraminidase, O-Glycosidase were added and mixed to 20. Mu.l with deionized water. The incubation reaction is carried out for 1 to 4 hours at 37 ℃. And (5) performing WB detection.

2.1.2.12PNGaseF glycosidase treatment

10-20. Mu.g glycoprotein is pipetted, 10 Xglycoprotein denaturation buffer is added, deionized water is added and mixed to 10. Mu.l. The glycoprotein was denatured by heating in a metal bath at 100 ℃. 10 Xglycoside buffer, 10% NP40, PNGaseF were added and mixed to 20. Mu.l with deionized water. The reaction was incubated at 37℃for 1h. And (5) performing WB detection.

2.1.2.13EndoH glycosidase treatment

10-20. Mu.g glycoprotein is pipetted, 10 Xglycoprotein denaturation buffer is added, deionized water is added and mixed to 10. Mu.l. The glycoprotein was denatured by heating in a metal bath at 100 ℃. 10 Xglycoside buffer, endoH, and deionized water were added and mixed to 20. Mu.l. The reaction was incubated at 37℃for 1h. And (5) performing WB detection.

2.2 results

2.2.1PAI-1 expression affects the sensitivity of HNSCC to cetuximab

The present study examined the expression of PAI-1 in 5 HNSCC cell lines (SCC 15, SCC25, UM1, fadu, detroit 562). The expression level of PAI-1 in cellular proteins was examined using WB, and the results showed that PAI-1 was most expressed in SCC15 (FIG. 9A). The level of PAI-1 secretion in the culture supernatant was measured using ELISA, and the results showed that PAI-1 was also the highest in SCC15 (FIG. 9B).

The SCC15 cells were selected to construct cells for stable knockout of PAI-1, and the PAI-1 knockout stable transgenic cell line was verified by three forms of cellular protein WB, supernatant ELISA and supernatant lyophilized protein WB (FIGS. 10A-C), which showed that the sg-PAI-1#1SCC15 cells were most efficient (P < 0.0001) compared to sg-NC, and the cells were selected for subsequent experiments.

To investigate the role of PAI-1 in HNSCC sensitivity to cetuximab treatment, this study analyzed the effect of PAI-1 expression on cetuximab treatment by CCK8 experiments, apoptosis experiments, clonogenic experiments. The results show that SCC15 cells were effective in treatment with cetuximab, and that treatment with cetuximab inhibited cell proliferation to some extent (fig. 11, 12), promoted apoptosis (fig. 13), but that when PAI-1 was knocked out in SCC15 cells, the sensitivity of the cells to treatment with cetuximab was increased, and the effects of inhibiting cell viability (P < 0.0001), cell proliferation (p=0.0002), and promoting apoptosis (p=0.0019) were more pronounced. These results indicate that PAI-1 expression affects HNSCC cells' sensitivity to cetuximab treatment.

We further validated the role of PAI-1 in HNSCC cells sensitivity to cetuximab treatment using the PAI-1 inhibitor tiplastinin. The effect of cetuximab single drug (1 mg/ml), tiplastinin single drug (50 uM) and combination on cells was analyzed by CCK8 experiments, apoptosis experiments, clone formation experiments. The results showed that cetuximab and tiplastinin single drug treatment had a certain effect compared to the control group, and can inhibit cell viability (P <0.0001, p=0.0042) and cell proliferation (P <0.0001, p=0.0006) to some extent (fig. 14, fig. 15), promote apoptosis (P <0.0001 ) (fig. 16). However, when cetuximab and tiplasinin are combined, the sensitivity of the cells to cetuximab treatment is improved, and the effects of inhibiting cell viability (P < 0.0001), cell proliferation (p=0.0027) and promoting apoptosis (p=0.0006) are more obvious. These results indicate that tiplastinin can enhance the sensitivity of HNSCC cells to cetuximab treatment.

2.2.2PAI-1 affects HNSCC sensitivity to cetuximab through its secreted form

As a secreted protein, PAI-1 needs to be synthesized in the cell and then secreted outside the cell. Secreted proteins generally function in paracrine or autocrine forms, so we studied whether PAI-1 affects the therapeutic sensitivity of cetuximab in dependence on its secretory function. We constructed PAI-1 plasmids encoding full-length (FL) and N-terminal signal peptide point mutations (DeltaSP), transfected in SCC15 cells knocked out PAI-1, and PAI-1 secretion was detected by WB and ELISA. Cell protein WB results showed that after transfection of the FL and ΔSP PAI-1 plasmids, SCC15 cells restored PAI-1 protein expression (FIG. 17A). The results of freeze-drying the protein WB from the supernatant revealed that cells recovered the expression of PAI-1 protein but lost the secretory function of PAI-1 after transfection of the ΔSP's PAI-1 plasmid (FIG. 17A). Supernatant ELISA results further demonstrated that SCC15 cells recovered PAI-1 protein secretion (P < 0.0001) after transfection of FL plasmid, and loss of PAI-1 secretion (P < 0.0001) from SCC15 cells after transfection of Δsp plasmid (fig. 17B).

Next, cells were treated with cetuximab, and cell proliferation was observed. The results showed that cells were less sensitive to cetuximab after transfection of the FLPAI-1 plasmid and less effective in inhibiting cell proliferation (p=0.0011) compared to SCC15 cells transfected with empty PAI-1 knockdown, whereas cells were unchanged in cetuximab sensitivity (p= 0.9937) after transfection of the Δsp PAI-1 plasmid and less effective in inhibiting cell proliferation (p=0.0103) after stimulation with PAI-1 recombinant protein (commercially available PAI-1 protein). These results indicate that PAI-1 affects HNSCC sensitivity to cetuximab through its secreted form.

2.2.3PAI-1 glycosylation modification maintains its protein stability

When the WB results were analyzed, we found that there was a double band in PAI-1, at a position above and below 50 kDa. According to the antibody specification, the molecular weight of PAI-1 is about 45kDa, so we speculate that there may be some post-translational modification of the PAI-1 protein to change its molecular weight. We predicted the possible post-translational modifications of PAI-1 using the Uniport website (https:// www.uniprot.org), and the results showed that N-linked glycosylation modifications of PAI-1 are possible. The amino acid sequences of the PAI-1 proteins of three species of human, mouse and rat are aligned, and 4 NXT (-Asn-X-Ser/Thr-) motif sites capable of N-linked glycosylation, N232, N288, N352 and N388 sites respectively, are found in the PAI-1 sequences of different species, so that glycosylation modification of the PAI-1 has evolutionary conservation (figure 19).

Tunicamycin (TM) is an antibiotic that effectively inhibits N-linked glycosylation by competitively inhibiting DPAGT1 activity. To verify PAI-1 glycosylation modification, SCC15 cells were treated with TM to observe PAI-1 protein changes. As a result, when the TM concentration was increased to 0.5. Mu.g/ml, the molecular weight of the PAI-1 protein was changed, the double band disappeared, and the molecular weight was reduced to about 45kDa (FIG. 20A). PAI-1 protein changes were observed using different glycosidases, including PNGaseF (N-glycosidase), endoH (N-glycosidase), O-glycosidase (O-glycosidase). The results showed that the PAI-1 protein also changed in molecular weight after PNGaseF treatment, the double band disappeared, the molecular weight was reduced to about 45kDa, while the PAI-1 protein was not significantly changed after O-glucosidase O-glycase treatment (FIG. 20B).

To further verify glycosylation modifications, we collected the purified PAI-1 protein, using PNGase F treatment, glycoprotein staining and coomassie brilliant blue staining. The results showed that the PAI-1 protein appeared to be similar to the positive control protein in the glycoprotein staining with a magenta color, indicating that glycosylation modification was present in the PAI-1 protein; while PNGase F treated PAI-1 protein had a disappearance of its magenta color and a similar background to the negative control protein, indicating a disappearance of PAI-1 protein glycosylation following PNGase F treatment, while Coomassie Brilliant blue staining indicated a decrease in protein molecular weight following PNGase F treated PAI-1 protein (FIG. 21). These results fully demonstrate the presence of N-linked glycosylation modifications on PAI-1 proteins.

Glycosylation plays an important role in maintaining protein stability. To investigate the effect of glycosylation modification on the stability of PAI-1 protein, we used TM to treat SCC15 cells, while the protein synthesis inhibitor Cycloheximide (CHX) was used to treat cells for various times to analyze the effect of glycosylation modification on PAI-1 protein half-life. The results showed that the PAI-1 protein half-life of SCC15 cells was approximately 4-8 h, whereas the PAI-1 protein molecular weight was reduced and protein glycosylation was inhibited after treatment of cells with TM, and the protein half-life was shortened to about 1-2 h, indicating that glycosylation modification maintains the PAI-1 protein stability profile (FIG. 22).

Protein half-life is mainly affected by protein synthesis and protein degradation pathways, which mainly include autophagy-lysosomal and ubiquitin-proteasome pathways. To further investigate the way glycosylation modifications affect the stability of PAI-1 proteins, we used TM in combination with the proteasome inhibitor MG132, autophagy/lysosomal inhibitor Chloroquine (CQ), lysosomal acidification inhibitor canavalin (Con a) to treat cells while CHX was used to treat cells for different times to observe the effect of PAI-1 protein half-life. The results show that PAI-1 protein half-life was restored after inhibition of the autophagy/lysosomal pathway with CQ following modification with TM, whereas MG132 treatment did not extend the PAI-1 protein half-life (FIG. 23). These results indicate that PAI-1 glycosylation modification maintains its protein stability by inhibiting the autophagy-lysosomal pathway.

Example 3

PAI-1 binding EGFR in HNSCC activates EGFR/AKT signaling pathway

3.1 objects and methods

3.1.1 subjects

Human squamous cell carcinoma cell line SCC15 was purchased from the American tissue culture Collection (American Tissue Culture Collection, ATCC).

3.1.2 Experimental methods

Some of the experimental methods are as above, and additional experimental methods are required as follows.

3.1.2.1 Co-immunoprecipitation (Co-IP)

The cell lysates were collected in a 1.5ml EP tube and centrifuged at 13000rpm at 4℃for 15min, and 50. Mu.l were pipetted as Input. 25 μl of the magnetic bead suspension was washed and added to the cell lysate, and the mixture was spun at 360℃for 2h. The beads were discarded, and the corresponding antibodies and cognate control IgG were added separately to the cell lysates and incubated overnight. To the sample, 25. Mu.l of magnetic beads were added and incubated for 2h at room temperature. The beads were collected and washed. 1 Xprotein denaturation buffer was added and heated at 100℃for 10min. And (5) performing WB detection and analyzing the result.

3.1.2.2 protein purification

Melting 50 mu lBL (DE 3) competent cells on ice, adding the empty and target plasmid with GST tag, mixing, placing in ice for 30min, placing in a water bath at 42 ℃ for 90s, rapidly transferring to ice, and placing for 3min. Add 500. Mu.l of liquid LB medium (without antibiotics), shake culture at 37℃and 180rpm for 1h, and resuscitate the relevant resistance marker gene on the plasmid. And (3) coating a proper amount of transformed competent cells on a solid LB culture plate containing corresponding antibiotics, and culturing the competent cells for 12-16 hours at 37 ℃ in an inverted mode. Individual clones were picked up into centrifuge tubes containing 30ml of liquid LB medium (100. Mu.g/ml ampicillin solution), shake-cultured at 37℃and 180rpm for 16h, transferred to conical flasks containing 500ml of LB medium, shake-cultured at 37℃and 225rpm to OD ₆₀₀ About 0.6 to 0.8, IPTG (0.5 mmol/L) of proper concentration is added, and the culture is carried out at 16 ℃ and 225rpm under shaking overnight. Collecting bacterial liquid to a 50ml centrifuge tube, centrifuging at 4 ℃ for 15min at 10000rpm, discarding supernatant, adding pre-cooled PBS for re-suspension, centrifuging at 10000rpm for 15min, and washing for 2 times. Placing the thalli at the temperature of minus 20 ℃, repeatedly freezing and thawing the thalli for 3 times, crushing the thalli, adding 10-20 ml of bacteria lysis buffer into each 500ml of culture solution, and performing ultrasonic crushing until the lysis solution becomes clear. Placing into a centrifuge at 4deg.C, centrifuging at 10000rpm for 15min, collecting lysate, and packaging, and preserving at-80deg.C or on ice for use. Protein purity was checked by coomassie blue staining of WB using protein samples.

3.1.2.3GST-pulldown

Mix the resin, draw 50. Mu.l of resin into the fresh EP tube, add 1ml PBS buffer, mix well, centrifuge at 1000rpm for 5min, discard the supernatant and wash the resin 2 times. At the same time, 1ml of the prepared GST-tagged purified protein was pipetted into a 1.5ml EP tube and 50. Mu.l was pipetted as Input. And adding the pre-washed resin into a protein sample, rotating at a low speed of 360 ℃ in a refrigerator at 4 ℃, and incubating for 2 hours. Centrifuging at 1000rpm at 4deg.C for 5min, discarding supernatant, adding 1ml rinsing buffer, rotating at 360 deg.C at low speed, rinsing at room temperature for 5min, centrifuging at 1000rpm for 5min, discarding supernatant, and washing resin for 3 times to obtain GST tag fusion protein resin. Purified protein solution of the sample to be detected is added into the obtained GST tag fusion protein resin, and the sample is rotated at a low speed of 360 degrees in a refrigerator at the temperature of 4 ℃ and incubated for overnight. Centrifugation at 1000rpm at 4℃for 5min, discarding the supernatant, adding 1ml of rinsing buffer thereto, spinning at 360℃at low speed, rinsing at room temperature for 5min, centrifugation at 1000rpm for 5min, discarding the supernatant, and washing the resin 3 times. Adding 50 μl of elution buffer into each group of resin, vortex shaking for 20s, placing on a mixer, eluting at room temperature for 15min, centrifuging at 12000rpm for 1min at room temperature, and taking the supernatant to a new EP tube to obtain the pulldown product. Protein denaturation treatment, WB and coomassie brilliant blue staining analysis was performed.

3.1.2.4 Adjacent connection experiment (PLA)

Sucking 3X 10 ⁵ The cell suspension is added into a 12-well plate with a glass slide, and is cultured for 16-24 hours at 37 ℃. After PBS washing, 4% paraformaldehyde was fixed. 0.5% TritonX-100 permeabilized, and 40. Mu.l was added dropwise to the slideBlocking Solution, incubated in an incubator at 37℃for 1h. Use->The primary antibody was diluted, added dropwise, and incubated overnight in a refrigerator at 4 ℃.1ml of 1 XWashBufferA was added to the slide glass, and then 40. Mu.l of PLA probe mixed solution was added dropwise thereto, and incubated at a constant temperature of 37℃for 1 hour. After that, 1ml of 1 XWashBufferA was added for washing. The slide was then incubated at 37℃for 30min with a solution of ligase. 1ml of 1 XWashBufferA. The slide glass was added dropwise with the Polymerase solution and incubated at 37℃for 100min in the dark. 1ml1 XWashBufferB wash, 0.01 XWashBufferB wash slide. DAPI counterstain, 1ml of 0.01 x WashBufferB wash, and plate. The results were observed under a fluorescence microscope.

3.1.2.5 cell immunofluorescence

3×10 ⁵ Cells were plated in 12-well plates with slides and incubated at 37℃for 16-24 h. After washing, 4% paraformaldehyde was fixed, PBS0.5% Triton X-100 was permeabilized, and 3% BSA solution was blocked. The primary antibody was added to a refrigerator at 4℃and incubated overnight. Adding the fluorescent secondary antibody, and incubating for 1h at room temperature in a dark place. DAPI counterstain, and sealing after cleaning. The results were observed under a fluorescence microscope.

3.2 results

PAI-1 and EGFR proteins bind directly to each other in 3.2.1HNSCC

Since PAI-1 is a secreted protein, it may act by endocytosis or receptor binding, and EGFR is an important receptor on the surface of tumor cell membranes, we studied whether PAI-1 in HNSCC can bind to EGFR. The stable and transgenic cell line over-expressing PAI-1 was verified by three experiments of cell protein WB, supernatant ELISA and supernatant lyophilized protein WB using Flag-tagged PAI-1 over-expressing lentivirus, and the results showed that the expression of PAI-1 was significantly increased in the Lentiv-PAI-1SCC15 cells, and the secretion level was also significantly increased in the supernatant (P=0.0002) compared with the Lentiv-NC cells, and the subsequent experiments were performed using this cell.

Next, the EGFR overexpressing plasmid with Myc tag was transfected into PAI-1 overexpressing SCC15 cells, and the protein was collected for Co-IP. The results showed that both PAI-1 and EGFR were directly bound to each other, whether binding of EGFR protein on PAI-1 protein was observed or binding of PAI-1 protein on EGFR protein was observed (FIG. 25). The PLA experiment is to identify target proteins through probes, when two target proteins are combined with each other and are close to each other, the distance between the two probes is close to each other, so that a proximity effect is generated, and fluorescence is activated. The results showed that a large amount of fluorescent signal was detected in the PAI-1-EGFR group compared to the PAI-1-IgG control group, indicating that both the PAI-1 protein and EGFR protein bound to each other, were in close proximity, and proximity effects occurred (FIG. 26). To further verify the direct interaction of PAI-1 with EGFR, we used PAI-1 and EGFR plasmids to induce bacteria to produce purified proteins, and performed GST-pull down experiments, which also demonstrated that PAI-1 could bind directly to EGFR (FIG. 27).

Co-localized expression of PAI-1 and EGFR in 3.2.2HNSCC

To further verify the interaction of PAI-1 and EGFR, we analyzed the expression and localization of PAI-1 and EGFR using immunofluorescence of HNSCC cells and tissues. Cellular immunofluorescence showed co-localized expression of PAI-1 and EGFR (fig. 28). PAI-1 expression was elevated following stimulation with PAI-1 recombinant protein (120 min results in the figure) and EGFR expression was not significantly altered, with enhanced co-localization signals of PAI-1 and EGFR (FIG. 28). Tissue immunofluorescence also found co-localized expression of PAI-1 and EGFR (fig. 29). Furthermore, we have found that PAI-1 and AKT also present co-localized expression. The above results fully demonstrate the direct interaction between PAI-1 and EGFR.

PAI-1 binding to EGFR extracellular phase in 3.2.3HNSCC activates EGFR/AKT pathway

As EGFR monoclonal antibodies, cetuximab functions by binding to the extracellular domain of EGFR. Thus, we speculate that PAI-1 may bind as a ligand to the extracellular domain of EGFR, competitively inhibiting cetuximab efficacy. To verify this hypothesis, we constructed EGFR extracellular and intracellular plasmids with Myc tag, transfected EGFR fragment plasmids with Myc tag into PAI-1 overexpressing SCC15 cells, and protein was collected for Co-IP. The results showed that both PAI-1 and EGFR extracellular domains were directly bound to each other, whether binding of EGFR-1 protein fragment protein on PAI-1 protein was observed or binding of PAI-1 protein on EGFR fragment protein was observed (FIG. 30), indicating that PAI-1 in HNSCC could competitively bind to EGFR extracellular domains with cetuximab.

EGFR and ligand bind, dimerize, activating downstream signaling pathways, which are necessary for ligand-dependent EGFR activation. To investigate whether PAI-1 HAs a similar effect as EGF, we constructed an EGFR plasmid with an HA tag, transfected Myc-tagged EGFR plasmid and an EGFR plasmid with an HA tag simultaneously into a control and SCC15 cells overexpressing PAI-1, and collected proteins for Co-IP. The results showed that upon over-expression of PAI-1, myc-tagged EGFR protein and HA-tagged EGFR protein bind more, indicating that PAI-1 can promote EGFR dimerization (FIG. 31A). WB was used to further observe EGFR downstream pathway changes. The results showed that p-EGFR (Y1068) and p-AKT (S473) expression decreased after PAI-1 knockdown in SCC15 cells, and p-EGFR (Y1068) and p-AKT (S473) expression was restored after stimulation with recombinant protein, without significant changes in EGFR and AKT proteins throughout the process (FIG. 31B). The results show that PAI-1 in HNSCC can bind to EGFR extracellular segment, promote EGFR dimerization, activate EGFR/AKT channel and influence cetuximab curative effect.

To further verify the correlation of PAI-1 expression and EGFR/AKT signaling pathway, we performed IHC staining on 95 HNSCC clinical tissue specimens to analyze the correlation of PAI-1 expression with p-EGFR (Y1068), p-AKT (S473) expression (FIG. 32). The results showed that PAI-1 expression and p-EGFR (r=0.013), p-AKT (r < 0.001) expression were positively correlated in HNSCC tumor tissue specimens (FIG. 33).

PAI-1 in 3.2.4HNSCC directly binds AKT1 to promote its phosphorylation

HNSCC tissue immunofluorescence found that in addition to co-localization with EGFR, PAI-1 and AKT also co-localized, so we further validated this result in HNSCC cells. AKT1 is the major subtype of AKT. We constructed an AKT1 plasmid with Myc tag, transfected AKT1 plasmid with Myc tag into SCC15 cell over-expressing PAI-1, and collected protein for Co-IP. The results showed that both PAI-1 and AKT1 were directly bound to each other, whether the binding of AKT1 protein on PAI-1 protein was observed or the binding of PAI-1 protein on AKT1 protein was observed (FIG. 34).

Therefore, we hypothesize whether PAI-1 can directly bind AKT1 to promote its phosphorylation independent of EGFR. To verify this hypothesis, we observed AKT1 changes in SCC15 cells knocked out with PAI-1 by blocking EGFR further with cetuximab and then stimulating the cells with low doses of PAI-1 recombinant protein for a short period of time. The results showed that p-EGFR (Y1068) and p-AKT (S473) expression levels were further reduced after treatment of PAI-1 knockdown SCC15 cells with cetuximab compared to the control group, p-EGFR (Y1068) expression was unchanged and p-AKT (S473) expression was increased after short-time stimulation of cells with low doses of PAI-1 recombinant protein (FIG. 35). The above results indicate that PAI-1 can directly bind AKT1 to promote its phosphorylation in HNSCC cells in an EGFR independent manner, affecting cetuximab therapeutic sensitivity.

Example 4

PAI-1 binding ITGA5 in HNSCC activates ITGA5/FAK signaling pathway

4.1 objects and methods

4.1.1 subjects

Human squamous cell carcinoma cell line SCC15 was purchased from the American tissue culture Collection (American Tissue Culture Collection, ATCC).

4.1.2 Experimental methods

Some of the experimental methods are as above, and additional experimental methods are required as follows.

4.1.2.1 cell scratch assay

1×10 ⁶ Cells are spread in 6-well plates and cultured for 16-24 h at 37 ℃. Scoring was performed with a small gun head. Appropriate time points (e.g., 0h,12h,24 h) were selected, the cells were photographed and recorded, and the migration of the cells was analyzed using software.

4.1.2.2 cell migration and invasion experiments

Adding matrigel into the upper layer of the chamber, and culturing at 37deg.C for 1 hr to solidify matrigel. 100 μl of 1×10 is added to the upper layer of the chamber ⁶ The complete culture medium is added into the lower layer of the cell suspension/ml, and the cell suspension is cultured for 24 to 48 hours at the temperature of 37 ℃. The cells were washed with PBS and 4% paraformaldehyde fixed at room temperature. 0.1% crystal violet solution staining. Cells in the upper chamber were wiped off after washing. And cutting the PET film, sealing the gel with a neutral resin, and observing cells under a microscope.

4.2 results

The PAI-1 and ITGA5 proteins in 4.2.1HNSCC bind directly to each other

To investigate the relationship between PAI-1 and ITGA5 in HNSCC, we transfected the ITGA5 overexpressing plasmid with HA tag into PAI-1 overexpressing SCC15 cells, and collected proteins for Co-IP. The results showed that both PAI-1 and ITGA5 were bound to each other, whether the binding of ITGA5 protein on PAI-1 protein or the binding of PAI-1 protein on ITGA5 protein was observed (FIG. 36A). Previous studies have shown that PAI-1 and EGFR are present in HNSCC directly bound to each other, whereas EGFR and ITGA5 are both cell membrane surface receptors, and thus PAI-1 may indirectly bind to each other through EGFR and ITGA 5. To confirm this hypothesis, we transfected into SCC15 cells the HA-tagged ITGA5 plasmid and Myc-tagged EGFR plasmid, collected the proteins for Co-IP, and observed binding of ITGA5 and EGFR. The results showed that there was no binding between EGFR and ITGA5 (fig. 36B). PLA experiments showed that a large amount of fluorescent signal was detected in the PAI-1-ITGA5 group compared to the PAI-1-IgG control group, indicating that both the PAI-1 protein and the ITGA5 protein bound to each other, in close proximity, and proximity effect occurred (FIG. 37). To further verify the direct interaction of PAI-1 and ITGA5, we performed GST-pull down experiments, which also demonstrated that PAI-1 could bind directly to ITGA5 (FIG. 38).

To further verify the interaction of PAI-1 and ITGA5, we analyzed the expression and localization of PAI-1 and ITGA5 using HNSCC cell and tissue immunofluorescence. Cellular immunofluorescence showed co-localized expression of PAI-1 and ITGA5 (FIG. 39). Tissue immunofluorescence also found that co-localized expression was present for PAI-1 and ITGA5 (FIG. 40). The above results fully demonstrate the direct interaction between PAI-1 and EGFR.

PAI-1 binding ITGA5 in 4.2.2HNSCC activates ITGA5/FAK pathway

Next we studied whether PAI-1 and ITGA5, after binding, activate their downstream signaling pathways. WB results showed that ITGA5 expression levels decreased after PAI-1 knockdown in SCC15 cells. Meanwhile, the FAK level was not significantly changed, the expression of p-FAK (Try 397) was decreased, and the expression of ITGA5 and p-FAK (Try 397) was restored after the stimulation with recombinant protein (FIG. 41). The above results indicate that binding of PAI-1 and ITGA5 in HNSCC may have an important role in its protein stability and may activate ITGA5/FAK signaling pathway.

To further verify the correlation of PAI-1 and ITGA5, we IHC stained 95 HNSCC clinical tissue specimens, evaluated the expression of ITGA5, and analyzed the correlation with PAI-1 expression (FIG. 42A). The results showed that PAI-1 expression and ITGA5 expression were positively correlated in HNSCC tissue (r=0.033) (fig. 42B).

4.2.3PAI-1 influence of HNSCC on treatment sensitivity to cetuximab by ITGA5

The integrin family is associated with the EMT process, an important mechanism for anti-EGFR therapeutic resistance and tumor invasion metastasis. Thus, it was next investigated whether PAI-1 affects HNSCC sensitivity to cetuximab treatment via ITGA 5. We used siRNA to interfere with ITGA5 expression in PAI-1 knockdown SCC15 cells. WB results showed that the interference effect of si-itga5#2 was most pronounced compared to si-NC, and thus si-itga5#2 was selected for subsequent experiments (fig. 43A). In SCC15 cells, after knocking out PAI-1, E-cadherin expression was increased and N-cadherin expression was decreased in addition to ITGA5 expression was decreased. After stimulation with PAI-1 recombinant protein, ITGA5 and N-cadherin expression was restored, while E-cadherin expression was decreased. Stimulation with PAI-1 recombinant protein failed to restore ITGA5 and N-cadherin expression and reduced E-cadherin expression after interference with ITGA5 expression in PAI-1 knockdown SCC15 cells (FIG. 43B). The above results indicate that PAI-1 in HNSCC regulates the EMT process through ITGA 5.

To study the effect of ITGA5 on PAI-1 affecting HNSCC on cetuximab treatment, the effect of ITGA5 expression on PAI-1 function was analyzed by CCK8 experiments, apoptosis experiments, clonogenic experiments. The results showed that cells were reduced in sensitivity to cetuximab treatment after stimulation with PAI-1 recombinant protein in PAI-1 knockdown SCC15 cells, the effects of inhibiting cell viability (P < 0.0001), cell proliferation (p=0.0001) and promoting apoptosis (P < 0.0001) were reduced, but PAI-1 recombinant protein stimulation was reduced after interference with ITGA5 expression in PAI-1 knockdown SCC15 cells, PAI-1 functions were blocked, sensitivity to cetuximab treatment was restored, cell viability (P < 0.0001), cell proliferation (p=0.0001) and apoptosis (p=0.0002) promoting effects were enhanced (fig. 44, 45, 46). These results indicate that PAI-1 can affect HNSCC cell sensitivity to cetuximab treatment by modulating ITGA5 expression.

4.2.4PAI-1 promotes HNSCC cell migration and invasion by ITGA5

EMT is not only the mechanism of resistance to EGFR treatment, but also an important mechanism of tumor invasion and metastasis. In earlier studies, sequencing data found SERPINE1 to be correlated with EMT, and clinical specimen analysis results also indicated positive correlation between PAI-1 expression and lymph node metastasis, N stage. Thus, we further investigated the role of ITGA5 in PAI-1 promotion of HNSCC invasion metastasis. The above results demonstrate that PAI-1 in HNSCC binds ITGA5, activating FAK phosphorylation. FAK is an important component of the focal adhesion kinase complex, and the Paxillin, focal adhesion protein (Vinculin), F-actin (F-actin), and integrin families together regulate the assembly and disassembly of the focal adhesion kinase complex. Focal adhesion kinase complexes are located at the protruding edges of cells and are critical to the motor ability of the cells. We performed immunofluorescence of cells, using p-FAK and F-actin to localize focal adhesion kinase complex, and examined whether PAI-1 in HNSCC affected focal adhesion kinase complex formation. The results showed that the number of focal adhesion kinase complexes at the edges of the SCC15 cells over-expressing PAI-1 was significantly increased compared to the control group (p=0.0018), indicating that PAI-1 activation of FAK in HNSCC by ITGA5 promoted the formation of focal adhesion kinase complex, affecting the motor capacity of the cells (fig. 47).

The effect of PAI-1 on HNSCC cell migration and invasiveness was further observed using scratch experiments and Transwell experiments. The results showed that SCC15 cells overexpressing PAI-1 had enhanced scratch healing capacity (P < 0.0001), increased cell numbers across the Transwell cells (P < 0.0001), confirming the ability of PAI-1 to promote HNSCC cell migration and invasion (fig. 48). Whereas after interfering with ITGA5 expression in PAI-1 overexpressing SCC15 cells, the cell scratch healing capacity was reduced (P < 0.0001), the number of cells passing through the Transwell chamber was reduced (P < 0.0001) (fig. 48). The above results indicate that PAI-1 promotes HNSCC cell migration and invasion by ITAG 5.

Example 5

5.1 objects and methods

5.1.1 subjects

The cells used in the experiment are as above.

5.1.2 Experimental methods

Some of the experimental methods are as above, and additional experimental methods are required as follows.

5.1.2.1 lymph node metastasis model

Female BALB/c nude mice of 4-5 weeks old are purchased, the mice are fixed, and the sole is inoculated with 5 multiplied by 10 tumor cells ⁵ /only. After the mice become tumor, every 1 dayTumor size (volume = longest diameter x square of perpendicular shortest diameter/2) was measured and calculated while recording body weight and survival time of mice. After the experiment is finished, the nude mice are dissected, and the tumors are taken out and formalin-fixed or stored at low temperature.

5.1.2.2 model of pulmonary metastasis

Female NPI severe immunodeficiency mice of 4-5 weeks of age were purchased, fixed mice, and tumor cells were inoculated 1X 10 via tail vein ⁶ /only. Dissolving D-luciferin potassium salt solution to prepare 15mg/ml, filtering, sterilizing, mixing, packaging, and preserving at-80deg.C. Each mouse was injected with 100. Mu. l D-potassium luciferin solution and imaged to analyze tumor growth and metastasis. When the mice show obvious lung metastasis, the experiment is terminated, the nude mice are dissected, lung tissues are taken out, and after formalin fixation, HE staining is carried out.

5.1.2.3 hematoxylin-eosin staining

The tissue sections were baked at 70℃for 1h. And rehydrating the dewaxed gradient concentration alcohol solution. After PBS cleaning, hematoxylin and eosin staining solution are dripped for staining. The slices were dehydrated in reverse order in ethanol and dewaxed solution of varying concentrations. After the sections are dried, the sections are sealed by neutral resin and observed under a microscope.

5.1.2.4 subcutaneous tumor-bearing model

Female BALB/c nude mice of 4-5 weeks old were purchased, mice were fixed, and tumor cells were inoculated subcutaneously 1X 10 ⁶ /only. When the tumor grows to 100mm ³ Cetuximab was administered by intraperitoneal injection, 20mg/kg, once every 2 days for 3 weeks. Mice tumor size was measured every other day. After the experiment is finished, the nude mice are dissected, and the tumors are taken out and formalin-fixed or stored at low temperature.

5.1.2.5HNSCC xenograft model of human tissue (PDX)

Female NPI severe immunodeficiency mice of 4-5 weeks of age were purchased, and fresh tissue of the patient was taken to a size of about 1X 1cm ³ Placing on ice surface, and constructing model within 2 h. A small opening was cut into the right dorsal portion of the mouse and injected about 2X 2mm ³ Tumor tissue mass of a size is small. Tumor size was measured periodically. When the tumor volume is as long as 300-1000 mm ³ Passaging was performed at that time. The method is the same as thatWhen the tumor grows to 100mm ³ The administration is right and left. Mice were divided into: control group, cetuximab group, tiplastinin group, combination treatment group. Cetuximab is administered by intraperitoneal injection, 10mg/kg, once every 2 days; tiplaxtinin is administered by gavage, 20mg/kg, once every 2 days. The administration was for 3 weeks. Mice tumor size was measured every other day. After the experiment is finished, the nude mice are dissected, and the tumors are taken out and formalin-fixed or stored at low temperature.

5.2 results

5.2.1PAI-1 promotes HNSCC lymph node metastasis and lung metastasis

To further verify the role of PAI-1 in HNSCC invasion metastasis, we constructed a mouse lymph node metastasis model and lung metastasis model using control and PAI-1 overexpressing SCC15 cells for in vivo verification. The results of the mouse lymph node metastasis model showed that the tumor growth rate was faster (p=0.0077) in the PAI-1 overexpressing group (n=5) compared to the control group (n=5) (fig. 49). After observing the mice for 3 weeks, the mice were dissected, the popliteal lymph nodes adjacent to the sole and the distal inguinal lymph nodes were removed, HE stained, and lymph node metastasis was observed. The volume of the lymph nodes overexpressing PAI-1 was greater than that of the control (FIG. 49A), the proximal popliteal lymph node metastasis was no different (4/5 vs 5/5), while the distal inguinal lymph node metastasis was different (1/5 vs 5/5) (FIG. 50), indicating that PAI-1 promoted HNSCC lymph node metastasis.

Tumor cells are injected into a mouse body from a tail vein, and the tumor metastasis of the mouse is detected by using a living animal imaging technology. After 3 weeks of observation, the mice showed a pronounced metastasis in the lungs (fig. 51A). Mice were sacrificed under anesthesia, lung tissue was removed after dissection, and HE staining was performed. The results showed that the lung fluorescence values were higher in mice overexpressing PAI-1 compared to the control group (a: p=0.0140, B: p=0.0130), suggesting increased lung metastasis (fig. 51A-B), and significantly higher numbers of lung metastases than in the control group (p=0.0002) (fig. 51C). The above results further demonstrate the role of PAI-1 in HNSCC invasive metastasis.

5.2.2PAI-1 expression inhibition of cetuximab on HNSCC tumor inhibition

To further demonstrate the role of PAI-1 in the sensitivity of cetuximab treatment, the present study used control and PAI-1 overexpressing SCC15 cells to construct a subcutaneous tumor-bearing model, and cetuximab treatment was given to observe its efficacy. The results showed that the control mice received cetuximab treatment, the tumors grew slowly, whereas the PAI-1 overexpressing mice grew rapidly (p=0.0080) and were insensitive to cetuximab treatment (fig. 52).

5.2.3Tiplaxtinin increases the sensitivity of HNSCC to cetuximab treatment

Next, we used the PDX model of HNSCC patients for in vivo validation. Mice were divided into four groups: control group, cetuximab-treated group, tiplastinin-treated group, combination-treated group. The results show that cetuximab (P < 0.0001) and tiplastinin (P < 0.0001) treatment alone have certain effects and can inhibit tumor growth to some extent compared with the control group. However, when cetuximab and tiplasinin were combined, the effect of inhibiting tumor growth was significantly improved (P < 0.0001) (fig. 53). These results indicate that tiplastinin can increase the HNSCC tumor inhibition by cetuximab.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (4)

1. Use of a pai-1 inhibitor for the preparation of a medicament for enhancing the therapeutic sensitivity of HNSCC patients to cetuximab.
2. 2. The use of claim 1, wherein the PAI-1 inhibitor is tiplastinin.
3. 3. The use of claim 1, wherein the PAI-1 inhibitor is for inhibiting lymph node metastasis, lung metastasis and HNSCC progression.
4. 4. The use of claim 1, wherein the PAI-1 inhibitor is for inhibiting HNSCC cell viability, inhibiting HNSCC cell proliferation, and promoting HNSCC cell apoptosis.