Application of TEAD4 as a predictive marker for metastasis and chemotherapy sensitivity in nasopharyngeal carcinoma

Abstract

The present invention discloses the application of TEAD4 as a marker for predicting nasopharyngeal carcinoma metastasis and chemotherapy sensitivity. The inventors have found through research that TEAD4 is associated with the malignant progression of nasopharyngeal carcinoma cells, and is highly expressed in nasopharyngeal carcinoma cells and tissues, and high expression is associated with poor patient prognosis. By quantifying the amount of TEAD4 in tumor sample tissues, the risk of nasopharyngeal carcinoma metastasis and chemotherapy sensitivity can be well determined, which is convenient for guiding personalized treatment of nasopharyngeal carcinoma patients, improving the effectiveness of treatment, and reducing ineffective treatment.

Description

Technical Field

[0001] The present invention belongs to the medical field, and specifically relates to the application of TEAD4 as a marker for predicting nasopharyngeal carcinoma metastasis and chemotherapy sensitivity. Background Art

[0002] Nasopharyngeal carcinoma is a head and neck tumor with a tendency to metastasize. Its prevalence distribution is extremely uneven, and it is highly prevalent in East Asia, especially in southern China. In recent years, a large number of studies on chemoradiotherapy strategies (for example, cisplatin-based induction chemotherapy, which can reduce distant metastasis) and the biological mechanisms of nasopharyngeal carcinoma have given the inventors a deeper understanding of nasopharyngeal carcinoma (such as fewer gene mutations, but a large number of epigenetic abnormalities). According to current guidelines, chemotherapy combined with radiotherapy is the main treatment for high-risk nasopharyngeal carcinoma. Almost all newly diagnosed nasopharyngeal carcinoma patients receive empirical cytotoxic chemoradiotherapy, but about 20% of them still develop distant metastases. Therefore, further elucidating the mechanism of nasopharyngeal carcinoma progression is of great value in guiding the treatment of nasopharyngeal carcinoma.

[0003] Tumor master regulators are transcription factor proteins that define and regulate the transcriptional properties of tumors. Abnormal activity of master regulators is essential for maintaining the state of tumor cells; therefore, systematic identification and characterization of tumor master regulators will help to elucidate the plasticity reprogramming mechanisms of different cell states and provide potential therapeutic and prognostic predictive markers. The development of systems biology has made it possible to study transcriptional regulatory network models, which in turn has paved the way for the identification of master regulators in human malignancies.

[0004] TEA domain transcription factor 4 (TEAD4) belongs to the TEADs protein family and is a downstream transcription factor of the Hippo signaling pathway. Studies have found that abnormalities in the Hippo signaling pathway play an important role in tumorigenesis and metastasis. TEAD4 is usually activated by two transcriptional co-stimulatory factors, YAP and TAZ, to promote downstream gene transcription. Previous studies have mostly focused on the important role of YAP and TAZ in promoting cancer through the Hippo signaling pathway. In recent years, studies have found that TEAD4 can promote tumor progression independently of YAP / TAZ and is a potential therapeutic target for intervening in the Hippo signaling pathway. However, there are no studies reporting whether TEAD4 plays an important role in the metastasis and chemotherapy resistance of nasopharyngeal carcinoma, and whether its expression can predict prognosis and guide treatment. Summary of the invention

[0005] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide the use of TEAD4 as a marker for predicting nasopharyngeal carcinoma metastasis and chemotherapy sensitivity.

[0006] The technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides:

[0008] Application of reagents for quantifying TEAD4 in the preparation of reagents for predicting the metastasis risk of nasopharyngeal carcinoma.

[0009] In some application examples, the sample detected by the reagent for quantifying TEAD4 is a tumor sample tissue.

[0010] In some application examples, the reagent for quantifying TEAD4 is selected from an immunohistochemical reagent or a microscopic evaluation system.

[0011] The second aspect of the present invention provides:

[0012] Application of reagents for quantifying TEAD4 in the preparation of reagents for predicting chemotherapy sensitivity of nasopharyngeal carcinoma.

[0013] In some application examples, the sample detected by the reagent for quantifying TEAD4 is a tumor sample tissue.

[0014] In some application examples, the reagent for quantifying TEAD4 is selected from an immunohistochemical reagent or a microscopic evaluation system.

[0015] In some application examples, the chemotherapy is induction chemotherapy based on platinum-based chemotherapy drugs.

[0016] In some application examples, the platinum chemotherapy drug is cisplatin.

[0017] The third aspect of the present invention provides:

[0018] A system for predicting the risk of metastasis or chemotherapy sensitivity of nasopharyngeal carcinoma, comprising:

[0019] A sample TEAD4 quantification device, used for quantifying the content of TEAD4 in a sample;

[0020] An analytical device for determining the risk of metastasis or chemotherapy sensitivity of nasopharyngeal carcinoma based on the amount of TEAD4 in a sample;

[0021] The result output device outputs the analysis result of the analysis device.

[0022] In some examples of the systems, the level of TEAD4 is determined using an IRS scoring device.

[0023] In some examples of the systems, the risk of NPC metastasis is determined based on a Kaplan–Meier curve or a Cox proportional hazards model.

[0024] In some systemic examples, high TEAD4 expression indicates a high risk of metastasis and poor chemotherapy sensitivity in NPC.

[0025] In some examples of the system, the sample TEAD4 quantification device is selected from an immunohistochemistry analysis device.

[0026] In some embodiments of the system, the sample is a tumor tissue specimen from a patient.

[0027] In some systemic examples, the chemotherapy is a platinum-based induction chemotherapy.

[0028] In some embodiments, the platinum-based chemotherapy drug is cisplatin.

[0029] The beneficial effects of the present invention are:

[0030] The inventors found through research that TEAD4 is associated with the malignant progression of nasopharyngeal carcinoma cells. It is highly expressed in nasopharyngeal carcinoma cells and tissues, and high expression is associated with poor prognosis in patients. By quantifying the amount of TEAD4 in tumor sample tissues, the metastasis risk and chemotherapy sensitivity of nasopharyngeal carcinoma can be well determined, which is convenient for guiding personalized treatment of nasopharyngeal carcinoma patients, improving the effectiveness of treatment, and reducing ineffective treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Figure 3 is the expression of TEAD4 in NPC tissues and cell lines; the mRNA expression level (A) and protein expression level (B) of TEAD4 in normal NPC tissues and NPC; the expression level of TEAD4 in immortalized NPC epithelial and NPC cell lines (C).

[0032] Figure 2 This is the experimental result that knocking down TEAD4 inhibits the invasion and migration of nasopharyngeal carcinoma.

[0033] Figure 3 This is the experimental result that knocking down TEAD4 promotes apoptosis of nasopharyngeal carcinoma.

[0034] Figure 4 Representative images of TEAD4 immunohistochemical staining in NPC tissues (A); IRS score distribution of TEAD4 expression levels in 219 patients (B).

[0035] Figure 5 Kaplan–Meier curve analysis of disease progression-free survival (A), distant metastasis-free survival (B), and overall survival (C) of NPC patients in the TEAD4 high expression group and TEAD4 low expression group.

[0036] Figure 6 Figure 2 Kaplan–Meier curves analyzing the progression-free survival of NPC patients who received cisplatin-based induction chemotherapy and those who did not receive cisplatin-based induction chemotherapy in the TEAD4 low expression group (A) and TEAD4 high expression group (B), respectively. DETAILED DESCRIPTION

[0037] The first aspect of the present invention provides:

[0038] Application of reagents for quantifying TEAD4 in the preparation of reagents for predicting the metastasis risk of nasopharyngeal carcinoma.

[0039] In some application examples, the sample detected by the reagent for quantifying TEAD4 is a tumor sample tissue.

[0040] In some application examples, the reagent for quantifying TEAD4 is selected from an immunohistochemical reagent or a microscopic evaluation system.

[0041] The second aspect of the present invention provides:

[0042] Application of reagents for quantifying TEAD4 in the preparation of reagents for predicting chemotherapy sensitivity of nasopharyngeal carcinoma.

[0043] In some application examples, the sample detected by the reagent for quantifying TEAD4 is a tumor sample tissue.

[0044] Existing methods can be used to quantify the TEAD4 content in tumor sample tissue. In some application examples, the reagent for quantifying TEAD4 is selected from an immunohistochemical reagent or a microscopic evaluation system.

[0045] In some application examples, the chemotherapy is an induction chemotherapy based on platinum-based chemotherapy drugs. Studies have shown that based on the expression of TEAD4 in tumor sample tissue, the sensitivity of induction chemotherapy based on platinum-based chemotherapy drugs can be well determined.

[0046] In some application examples, the platinum chemotherapy drug is cisplatin.

[0047] The third aspect of the present invention provides:

[0048] A system for predicting the risk of metastasis or chemotherapy sensitivity of nasopharyngeal carcinoma, comprising:

[0049] A sample TEAD4 quantification device, used for quantifying the content of TEAD4 in a sample;

[0050] An analytical device for determining the risk of metastasis or chemotherapy sensitivity of nasopharyngeal carcinoma based on the amount of TEAD4 in a sample;

[0051] The result output device outputs the analysis result of the analysis device.

[0052] In some examples of the system, the content of TEAD4 is determined by an IRS scoring device. The use of the IRS scoring device can better quantify the content of TEAD4 in tumor sample tissue.

[0053] In some systematic examples, the risk of NPC metastasis is determined based on the Kaplan–Meier curve or the Cox proportional hazards model. These methods are classic risk determination methods and can evaluate the risk well.

[0054] In some system examples, high expression of TEAD4 indicates a high risk of metastasis of nasopharyngeal carcinoma and poor chemotherapy sensitivity. It can be determined whether it is highly expressed according to known methods. For example, in a group of known samples, an expression level higher than the median is defined as high expression, and vice versa, it is defined as low expression.

[0055] Based on the research samples of the present invention, in some system examples, high expression of TEAD4 refers to an IRS score greater than 6. Considering the different sources of samples, the specific IRS score of high expression of TEAD4 may vary, but is basically around 6.

[0056] In some examples of the system, the sample TEAD4 quantification device is selected from an immunohistochemistry analysis device.

[0057] In some embodiments of the system, the sample is a tumor tissue specimen from a patient.

[0058] In some systemic examples, the chemotherapy is a platinum-based induction chemotherapy.

[0059] In some embodiments, the platinum-based chemotherapy drug is cisplatin.

[0060] The inventors discovered 13 major regulators in nasopharyngeal carcinoma through Algorithm for the Reconstruction of Accurate Cellular Networks through Adaptive Partitioning (ARACNe-AP) and Virtual Inference of Protein Activity by Enriched Regulon (VIPER) algorithms, and further found that TEAD4 is associated with cell malignant progression. It is highly expressed in nasopharyngeal carcinoma cells and tissues, and high expression is associated with poor prognosis of patients.

[0061] Gene set enrichment analysis (GSEA) showed that metastasis and cisplatin resistance were enriched in the high TEAD4 group. Transwell, CCK8 and apoptosis experiments showed that knocking down or overexpressing TEAD4 can inhibit or promote NPC migration, invasion and chemotherapy resistance. Mechanism: ChIP-seq, ChIP-qPCR, luciferase reporter gene and WB experiments showed that TEAD4 can promote BZW2 transcription and activate the AKT signaling pathway to promote NPC metastasis and chemotherapy resistance. Subsequently, the in vivo lymph node metastasis model, lung metastasis model and subcutaneous transplant tumor model showed that knocking down TEAD4 can inhibit NPC metastasis and chemotherapy resistance.

[0062] Furthermore, the inventors selected paraffin tissue specimens of 219 patients with NPC who were initially diagnosed. During the subsequent treatment and follow-up, 40 patients (18.3%) died. Immunohistochemistry (antibody: ab58310, Abcam) was used to stain tissue specimens of NPC patients, and then the staining intensity was determined under a microscope: 0: no staining; 1: weak, light yellow; 2,: medium, yellow-brown and 3: strong brown. Positive cell ratio: 1, <10%; 2, 10–35%; 3, 36–70%; and 4, >70%. The IRS (immunoreactive score) was then calculated, which was obtained by multiplying the staining intensity by the positive cell ratio. According to the median IRS score of the patients, the 219 NPC patients were divided into TEAD4 high expression group (IRS = 7-12) and low expression group (IRS = 0-6) according to the median IRS.

[0063] Combined with the prognostic data, the inventors found that high TEAD4 expression was associated with high disease progression, distant metastasis and death in patients with NPC. Kaplan–Meier survival and multivariate analysis showed that TEAD4 was a poor prognostic factor for disease progression-free survival, distant metastasis-free survival and overall survival in patients with NPC. In addition, the inventors analyzed and found that in the TEAD4 low expression group, the disease control rate of NPC patients who received platinum-based induction chemotherapy was significantly better than that of patients who did not receive platinum-based induction chemotherapy. In the TEAD4 high expression group, platinum-based induction chemotherapy failed to benefit NPC patients.

[0064] The technical solution of the present invention is further explained below in combination with experiments and experimental results.

[0065] 1. Cell Culture

[0066] Immortalized normal human nasopharyngeal epithelial cell lines (NP69 and N2-Tert) were cultured in Keratinocyte SFM medium (Invitgen, Carlsad, CA, USA) containing bovine pituitary extract (BPE) and human epidermal growth factor. Bovine pituitary extract (BD Biosciences, San Jose, CA, USA) was also added. Human nasopharyngeal carcinoma cell lines (HNE1, HONE-1, SUNE-1, 5-8F, 6-10B, CNE1, and CNE2) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). All cells were placed in a 5% CO 2 in a 37 °C cell culture incubator.

[0067] 2. RNA Extraction and Reverse Transcription Real-time Quantitative PCR (RT-qPCR)

[0068] Total RNA was extracted from cells using TRIzol reagent (Invitgen), and the quality and content of RNA were determined using NanoDrop 2000 software. Reverse transcription was performed using the GoScript reverse transcription system (Promega, Madison, WI, USA). Next, qRT-PCR was performed using SYBR Green qPCR reagent (Invitgen) on a CFX96 Touch Sequence Detection System (Bio-Rad, Hercules, CA, USA).

[0069] 3. Immunoblotting

[0070] Cells were collected and lysed in RIPA buffer (Merck Millipol, Billerica, MA, USA) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). The cells were incubated in a metal bath at 95°C for 10 minutes, centrifuged at 12,000 rpm for 5 minutes, and the protein concentration was determined using a BCA protein assay kit (Thermo Fisher Science). Equal amounts of protein lysates were separated using 4-12% gel and then transferred to a PVDF membrane (Merck Milliporte). The membrane was blocked with 5% skim milk, incubated with the primary antibody overnight at 4°C, and exposed to the secondary antibody after incubation at room temperature for 1 hour.

[0071] 4. Migration and Invasion Assays

[0072] (1) Add Matrigel to ice-cold serum-free culture medium at a ratio of 1:9, pipette and mix thoroughly, then add 50 μl of the mixture to the bottom of each Transwell chamber and place in an incubator for half an hour to solidify;

[0073] (2) Take out the transiently or stably transfected nasopharyngeal carcinoma cells, digest them, count them, wash them with PBS, centrifuge them, and add 500 μl of culture medium containing 10% fetal bovine serum to the lower chamber of the Transwell. In the migration experiment, the cells were prepared with serum-free culture medium (SUNE-1: 5×10 4 / 200μl; HONE-1: 3.5×10 4 In the invasion assay, the cell suspension was prepared as (SUNE-1: 1×10 5 / 200μl; HONE-1: 7×10 4 Add 200 μl of the prepared cell suspension to the upper chamber (without gel for migration experiments and with Matrigel for invasion experiments) and incubate at 37°C and 5% CO 2 The cells were cultured for 16 h (migration) or 22 h (invasion) under the conditions of

[0074] (3) Take out the Transwell plate from the incubator, wash it twice with PBS, and then gently wipe the gel and cells on the inner membrane of the chamber with a cotton swab;

[0075] (4) After fixing with methanol for 30 min, rinse with clean water and spin dry;

[0076] (5) Hematoxylin staining for 2 h, rinse with clean water, and spin dry;

[0077] (6) Place the chamber under a microscope to observe the migrating or invading NPC cells, take pictures and count them for further analysis.

[0078] 5. Flow Cytometry Analysis of Cell Apoptosis

[0079] After 30 h of cisplatin treatment, the cells were digested with 0.25% trypsin (without EDTA). Each sample was resuspended in 300 μL of binding buffer and incubated with 2.5 μL of Annexin V-APC and 3 μL of 7AAD fluorescent dye. The cell apoptosis rate was detected by CytExpert flow cytometer and analyzed by FlowJo software. APC+ / 7AAD+ cells are late apoptotic or dead cells, APC+ / 7AAD- cells are early apoptotic cells, and APC- / 7AAD- cells are surviving cells.

[0080] 6. Immunohistochemistry

[0081] (1) Dewaxing and hydration: Place the paraffin sections in an oven at 65°C for 30 minutes, then place them in xylene for 5 minutes, anhydrous ethanol for 2 minutes, 95% ethanol for 2 minutes, and 75% ethanol for 2 minutes.

[0082] (2) High temperature and high pressure antigen repair: Prepare 3% hydrogen peroxide solution, put it in a pressure cooker and boil it, then put the slide in and continue heating for 150 seconds; after stopping heating, open the lid and let it cool naturally to room temperature;

[0083] (3) Immunohistochemistry blocking solution, incubate at room temperature for 30 minutes;

[0084] (4) Discard the blocking solution, prepare the primary antibody in the appropriate proportion, and incubate at 4°C overnight;

[0085] (5) Biotin-labeled secondary antibody, incubated at 37°C for 25 min;

[0086] (6) DAB color development: freshly prepared DAB color development solution is used for color development;

[0087] (7) Rinse with tap water, counterstain with hematoxylin for 2-5 min, and rinse with tap water;

[0088] (8) Dehydration: Place the sections in 75% ethanol for 2 min, 5% ethanol for 2 min, and anhydrous ethanol for 2 min (air dry).

[0089] (9) Evaluation: The tissue was scored immunohistochemically based on the proportion and degree of positivity.

[0090] 7. Data Analysis

[0091] The survival curves were drawn using the Kaplan-Meier method, and the differences were compared using the log-rank test. The Cox proportional hazards model (CPH) model was used for multivariate analysis to calculate the hazard ratio (HR) and determine independent prognostic factors. The CPH model was further used for covariate interaction analysis to evaluate whether there was a difference in the therapeutic effect of induction chemotherapy in the high and low expression TEAD4 groups. The data are expressed as mean ± SD, and P values ​​< 0.05 were considered statistically significant.

[0092] The experimental results are as follows:

[0093] The inventors detected the expression of TEAD4 in NPC tissues and cell lines and found that compared with normal NPC tissues, TEAD4 in NPC tissues was significantly upregulated at both mRNA and protein levels. Similarly, the expression of TEAD4 in NPC cell lines (HNE-1, HONE-1, SUNE1, 5-8F, 6-10B, CNE-1 and CNE-2) was significantly higher than that in immortalized NPC epithelial cells ( Figure 1 ).

[0094] The inventors established SUNE-1 and HONE-1 cell lines with stable knockdown of TEAD4. Transwell migration and invasion experiments showed that TEAD4 gene knockdown significantly inhibited the migration and invasion ability of nasopharyngeal carcinoma cells ( Figure 2 ). Flow cytometry apoptosis experiments showed that knocking down TEAD4 could significantly enhance the apoptosis rate of nasopharyngeal carcinoma cells SUNE-1 and HONE-1 after cisplatin treatment ( Figure 3 ).

[0095] Furthermore, the inventors selected 219 biopsy paraffin tissue specimens from patients initially diagnosed with nasopharyngeal carcinoma. The tissue specimens of nasopharyngeal carcinoma patients were stained using immunohistochemistry (antibody: ab58310, Abcam), and the staining intensity was then determined under a microscope: 0: no staining; 1: weak, light yellow; 2,: medium, yellow-brown and 3: strong brown. Positive cell ratio: 1, <10%; 2, 10–35%; 3, 36–70%; and 4, >70%. The IRS (immunoreactive score) was then calculated, which was obtained by multiplying the staining intensity by the positive cell ratio. According to the median IRS score of the patients, the 219 nasopharyngeal carcinoma patients were divided into TEAD4 high expression group (IRS = 7-12) and low expression group (IRS = 0-6) according to the median IRS ( Figure 4 ).

[0096] Combined with the prognostic data, the inventors found that NPC patients with high TEAD4 expression had a higher risk of disease progression, distant metastasis, and death ( Figure 5 ). Kaplan–Meier survival and multivariate analysis showed that TEAD4 was a negative prognostic factor for progression-free survival, distant metastasis-free survival, and overall survival in patients with NPC (Table 1). In addition, the inventors found that in the TEAD4 low expression group, the disease control rate of NPC patients who received platinum-based induction chemotherapy was significantly better than that of patients who did not receive platinum-based induction chemotherapy. In the TEAD4 high expression group, platinum-based induction chemotherapy failed to benefit NPC patients ( Figure 6 In addition, interaction analysis revealed a significant interaction between treatment (with or without cisplatin-based induction chemotherapy) and TEAD4 expression level (high vs. low) in terms of survival (Table 2).

[0097] Table 1 Multivariate analysis of TEAD4 expression in nasopharyngeal carcinoma patients

[0098]

[0099]

[0100] Table 2

[0101]

[0102] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. Application of the quantitative TEAD4 reagent in the preparation of a nasopharyngeal carcinoma metastasis risk prediction reagent. The sample detected by the quantitative TEAD4 reagent is a tumor sample tissue.

2. The use according to claim 1, It is characterized in that The reagent for quantifying TEAD4 is selected from immunohistochemical reagents.

3. Application of a reagent for quantifying TEAD4 in the preparation of a reagent for predicting the chemotherapy sensitivity of nasopharyngeal carcinoma. The sample detected by the reagent for quantifying TEAD4 is a tumor sample tissue, and the chemotherapy is an induction chemotherapy based on platinum-based chemotherapy drugs.

4. The use according to claim 3, It is characterized in that The reagent for quantifying TEAD4 is selected from immunohistochemical reagents.

5. A system for predicting the risk of metastasis or chemotherapy sensitivity of nasopharyngeal carcinoma, wherein the chemotherapy is induction chemotherapy based on platinum-based chemotherapy drugs, It is characterized in that include: A sample TEAD4 quantification device, used for quantifying the content of TEAD4 in a sample, wherein the sample is a tumor sample tissue; An analytical device for determining the risk of metastasis or chemotherapy sensitivity of nasopharyngeal carcinoma based on the amount of TEAD4 in a sample; The result output device outputs the analysis result of the analysis device.

6. The system according to claim 5, It is characterized in that High expression of TEAD4 indicates a high risk of metastasis and poor chemotherapy sensitivity in NPC.

7. The system according to claim 5 or 6, It is characterized in that The sample TEAD4 quantification device is selected from the immunohistochemistry analysis device.

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