Application of EDEM1 as anti-tumor drug adriamycin resistance marker and breast cancer prognosis marker

Abstract

The invention relates to the technical field of biomedicine, in particular to application of EDEM1 as a doxorubicin drug resistance marker and a breast cancer prognosis marker. According to the application disclosed by the invention, the triple-negative breast cancer cells are firstly stimulated by an endoplasmic reticulum stress inducer, the EDEM1 content in the stimulated triple-negative breast cancer cells is found to be obviously increased, and a result shows that the EDEM1 plays an important role in an endoplasmic reticulum stress process. EDEM1 is subjected to overexpression or interference expression in triple-negative breast cancer cells, the result shows that EDEM1 can relieve endoplasmic reticulum stress caused by an endoplasmic reticulum stress inducer and adriamycin to the cells, the endoplasmic reticulum stress resistance of the triple-negative breast cancer cells is improved, and meanwhile, IC50 of breast cancer drug-resistant cells with interference expression of EDEM1 is lower than that of a control group; further, the EDEM1 can be proved to be used as a breast cancer adriamycin-resistant molecular target. Meanwhile, the EDEM1 content can be used as an independent prediction factor of the prognosis condition of the triple-negative breast cancer patient, and the EDEM1 content and the independent prediction factor are positively correlated.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of EDEM1 as an adriamycin resistance marker and a breast cancer prognosis marker. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Breast cancer is the most common female malignancy worldwide, and its treatment mainly includes local surgical treatment, radiotherapy and systemic chemotherapy, endocrine therapy, molecular targeted therapy, etc. Patients with triple-negative breast cancer (TNBC) cannot benefit from endocrine therapy and targeted therapy due to the lack of therapeutic targets such as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Therefore, in addition to surgical treatment, the treatment of triple-negative breast cancer is mainly combined chemotherapy with cytotoxic chemotherapy drugs. Doxorubicin (DOX) is an anthracycline cytotoxic chemotherapy drug that can induce remission in about 30% of TNBC patients. However, breast cancer cells can evade the toxicity of chemotherapy drugs through a variety of mechanisms and develop chemotherapy resistance. Therefore, in-depth exploration of the biological behavior and resistance mechanism of triple-negative breast cancer will help to find new targets for targeted treatment of triple-negative breast cancer.

[0004] The endoplasmic reticulum is the basis for protein biosynthesis, modification and transport. However, protein homeostasis in the endoplasmic reticulum is very sensitive to certain stimuli such as oncogenic activation, hypoxia, and nutrient deficiency. These stimuli often destroy the function of the endoplasmic reticulum and lead to the accumulation of misfolded proteins. This phenomenon is called endoplasmic reticulum stress. Excessive endoplasmic reticulum stress may lead to cell death, so excessive endoplasmic reticulum stress can be used to eliminate tumor cells. However, in response to endoplasmic reticulum stress, tumor cells have evolved a complete signaling network to promote protein folding and clearance. Among them, endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR) are two key quality control mechanisms in cells and are also important factors related to tumor cell resistance. Therefore, exploring the molecular mechanism of triple-negative breast cancer resistance from the perspective of endoplasmic reticulum stress is expected to provide new ideas for improving the sensitivity of triple-negative breast cancer chemotherapy. Summary of the invention

[0005] In order to overcome the above problems, the present invention provides the use of EDEM1 as a drug resistance marker for the anti-tumor drug doxorubicin and a prognosis marker for breast cancer.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, there is provided the use of EDEM1 as a drug resistance marker for the anti-tumor drug doxorubicin, wherein the amino acid sequence of the EDEM1 is shown in SEQ ID NO.1.

[0008] In one or more embodiments, tumors for which doxorubicin is used as an anti-tumor drug include: acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchial lung cancer (undifferentiated small cell and non-small cell), ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer or liver cancer, preferably breast cancer, and more preferably triple-negative breast cancer.

[0009] In one or more embodiments, the application method includes:

[0010] Application of EDEM1 content detection reagent in the preparation of reagents or diagnostic equipment for doxorubicin resistance assessment.

[0011] The second aspect of the present invention provides a reagent or diagnostic device for evaluating doxorubicin resistance, including an EDEM1 content detection reagent.

[0012] The above research on the mechanism of doxorubicin resistance provides an executable target for reversing doxorubicin resistance in patients with triple-negative breast cancer.

[0013] The third aspect of the present invention provides the use of EDEM1 as a prognostic marker for breast cancer.

[0014] EDEM1 content can be used as an independent predictive factor for the prognosis of patients with triple-negative breast cancer, and the two are positively correlated, that is, increased EDEM1 content in the patient's body or lesion tissue means worse prognosis and shorter survival time.

[0015] In one or more embodiments, the breast cancer comprises triple-negative breast cancer.

[0016] In one or more embodiments, the application method includes:

[0017] The EDEM1 content detection reagent is used to prepare a prognosis judgment product for breast cancer patients.

[0018] A fourth aspect of the present invention provides a product for predicting the prognosis of breast cancer patients, wherein the product comprises an EDEM1 content detection reagent.

[0019] A fifth aspect of the present invention provides the use of EDEM1 as a target in the development, screening and / or preparation of agents for the treatment or adjuvant treatment of breast cancer.

[0020] The sixth aspect of the present invention provides the use of a substance that inhibits or interferes with EDEM1 expression in at least one of the following or in the preparation of a product having at least one of the following functions:

[0021] (1) Treatment or adjuvant treatment of breast cancer;

[0022] (2) Promote chemotherapy drugs to treat breast cancer;

[0023] (3) Combined with chemotherapy drugs to treat breast cancer.

[0024] In one or more embodiments, the chemotherapeutic drug comprises doxorubicin.

[0025] A seventh aspect of the present invention provides use of substances that inhibit or interfere with EDEM1 expression and chemotherapeutic drugs in the preparation of products for the treatment or adjuvant treatment of breast cancer.

[0026] In one or more embodiments, the chemotherapeutic drug comprises doxorubicin.

[0027] The eighth aspect of the present invention provides a product for treating or assisting in treating breast cancer, which is as follows a) or b):

[0028] a) Substances that inhibit or interfere with EDEM1 expression;

[0029] b) Substances and chemotherapeutic drugs that inhibit or interfere with EDEM1 expression.

[0030] In one or more embodiments, the chemotherapeutic drug comprises doxorubicin.

[0031] The beneficial effects of the present invention are:

[0032] In the present invention, triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468 are first stimulated by an endoplasmic reticulum stress inducer, and it is found that the EDEM1 content in the stimulated triple-negative breast cancer cells is significantly upregulated, and the results suggest that EDEM1 plays an important role in the endoplasmic reticulum stress process. Then, by overexpressing or interfering with the expression of EDEM1 in MDA-MB-231 and MDA-MB-468, the results show that EDEM1 can alleviate the endoplasmic reticulum stress caused by the endoplasmic reticulum stress inducer and doxorubicin to the cells, and increase the endoplasmic reticulum stress resistance of triple-negative breast cancer cells. At the same time, it is found that the IC50 of breast cancer resistant cells interfering with the expression of EDEM1 is lower than that of the control group, which can further prove that EDEM1 is a molecular target for doxorubicin resistance in breast cancer.

[0033] At the same time, EDEM1 content can be used as an independent predictive factor for the prognosis of patients with triple-negative breast cancer, and the two are positively correlated, that is, increased EDEM1 content in the patient's body or lesion tissue means worse prognosis and shorter survival time. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0035] Figure 1 Screening of EDEM1, wherein A is the induction of ER stress by TM, TG and DOX in MDA-MB-231 cells and MDA-MB-468 cells, and the detection of intracellular ROS levels before and after cell induction; B is the detection of the expression of three molecules of the EDEM family, EDEM1, EDEM2 and EDEM3, in MDA-MB-231 cells and MDA-MB-468 cells after induction; C is the detection of protein expression levels of EDEM1 and ER stress molecular marker GRP78 by Western blot;

[0036] Figure 2 Figure 2 is the expression of EDEM1 in breast cancer cells and tissues, where A is the expression of EDEM1 in normal breast epithelial cells MCF-10A and breast cancer cells; B is the expression of EDEM1 in breast cancer tissues in the TCGA database and Metabric database; C is the expression of EDEM1 in breast cancer tissues and paracancerous tissues detected by qRT-PCR; D is the difference in EDEM1 expression between breast cancer tissues and paracancerous tissues detected by immunohistochemistry;

[0037] Figure 3 To enhance the endoplasmic reticulum stress resistance of triple-negative breast cancer by EDEM1, A and B are overexpression vectors of EDEM1 transfected into MDA-MB-231 and MDA-MB-468, and the transfected cells were treated with endoplasmic reticulum stress inducers TM, TG or chemotherapeutic drug doxorubicin to detect the intracellular ROS level; C is a small interference factor for transfecting EDEM1 into the doxorubicin-resistant cell line 231 / DOX cells, and the intracellular ROS level was detected after the transfected cells were treated with the same drugs;

[0038] Figure 4 EDEM1 promotes doxorubicin resistance in triple-negative breast cancer, specifically MDA-MB-231, MDA-MB-468 and the drug-resistant cell line 231 / DOX cells transfected with an EDEM1 overexpressing vector IC 50 Test results;

[0039] Figure 5Analysis of the correlation between EDEM1 and the prognosis of triple-negative breast cancer patients. A is the immunohistochemical staining of pathological sections of 76 TNBC patients and 55 non-TNBC patients and the immunohistochemical scoring; B is the ROC curve of EDEM1 high expression and overall survival (OS) and disease-free survival (DFS) in breast cancer patients (the upper left is the ROC curve of EDEM1 high expression and overall survival (OS) in breast cancer patients; the upper right is the survival curve of EDEM1 high expression and overall survival (OS) in breast cancer patients; the lower left is the ROC curve of EDEM1 high expression and disease-free survival (DFS) in breast cancer patients; the lower right is the survival curve of EDEM1 high expression and disease-free survival (DFS) in breast cancer patients); C is the Cox univariate and multivariate risk regression analysis of clinical information of breast cancer patients and EDEM1 expression;

[0040] Figure 6 Figure 3 Analysis of the correlation between EDEM1 and the prognosis of patients with triple-negative breast cancer. A is the difference in immunohistochemical scores of EDEM1 expression levels in triple-negative breast cancer and non-triple-negative breast cancer tissues; B is the ROC curve of overall survival (OS) and disease-free survival (DFS) of patients with high EDEM1 expression and triple-negative breast cancer (upper left is the ROC curve of overall survival (OS) of patients with high EDEM1 expression and triple-negative breast cancer; upper right is the survival curve of overall survival (OS) of patients with high EDEM1 expression and triple-negative breast cancer; lower left is the ROC curve of disease-free survival (DFS) of patients with high EDEM1 expression and triple-negative breast cancer; lower right is the survival curve of disease-free survival (DFS) of patients with high EDEM1 expression and triple-negative breast cancer); C is the Cox univariate and multivariate risk regression analysis of clinical information of TNBC patients and EDEM1 expression. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0043] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0044] Example 1

[0045] Screening and expression of EDEM1

[0046] 1.1 Screening of EDEM1: Reactive oxygen species (ROS) level is an important marker reflecting the level of endoplasmic reticulum stress.

[0047] The endoplasmic reticulum stress inducers TM (Tunicamycin) and TG (Thapsigargin) and doxorubicin (DOX) were used to induce endoplasmic reticulum stress in TNBC cell lines MDA-MB-231 and MDA-MB-468, and the intracellular ROS levels were detected before and after cell induction. The results showed that the intracellular ROS level increased significantly after drug induction ( Figure 1 A).

[0048] At the same time, the expression changes of three molecules of the EDEM family, EDEM1, EDEM2 and EDEM3, in MDA-MB-231 cells and MDA-MB-468 cells after induction were detected. It was found that the expression of EDEM1, EDEM2 and EDEM3 were all upregulated after induction, and the upregulation of EDEM1 was the most obvious ( Figure 1 B). In combination with its role in the ER stress process, EDEM1 was selected for subsequent research. The protein expression levels of EDEM1 and the ER stress molecular marker GRP78 were detected by Western blot, and it was found that the expression of EDEM1 and GRP78 were upregulated after drug stimulation ( Figure 1 C) These results suggest that EDEM1 plays an important role in the endoplasmic reticulum stress induced by doxorubicin.

[0049] 1.2 Expression of EDEM1 in breast cancer cells and tissues: To verify the expression of EDEM1 in breast cancer, we first compared the expression of EDEM1 in normal breast epithelial cells MCF-10A and breast cancer cells. The results showed that EDEM1 was expressed higher in breast cancer cells than in normal breast epithelial cells, and higher in triple-negative breast cancer cells than in non-triple-negative breast cancer cells ( Figure 2 A). In the TCGA database and Metabric database, it was found that the expression of EDEM1 in breast cancer tissues was higher than that in normal tissues ( Figure 2B). Then, the expression of EDEM1 in 35 pairs of breast cancer tissues and adjacent normal breast tissues was detected by qRT-PCR. The results showed that the expression level of EDEM1 in most breast cancer tissues was higher than that in the corresponding adjacent normal breast tissues ( Figure 2 C). At the same time, immunohistochemistry was used to detect the difference in EDEM1 expression in 25 pairs of breast cancer tissues and adjacent adjacent tissues, and the same results were obtained ( Figure 2 D) This indicates that EDEM1 is highly expressed in most breast cancer cells and patient tumor tissues.

[0050] Example 2

[0051] Verification of the correlation between EDEM1 and endoplasmic reticulum stress resistance and doxorubicin resistance in triple-negative breast cancer cells:

[0052] 2.1 Verification of the correlation between EDEM1 and endoplasmic reticulum stress resistance in triple-negative breast cancer:

[0053] To investigate the effect of EDEM1 on the resistance of TNBC cells to endoplasmic reticulum stress, TNBC cells MDA-MB-231 and MDA-MB-468 were transfected with EDEM1 overexpression vectors using Lipofectamine 2000 liposomes. The transfected cells were treated with endoplasmic reticulum stress inducers TM, TG or chemotherapeutic drug doxorubicin, and the intracellular ROS level was detected to reflect the effect of EDEM1 on the endoplasmic reticulum stress level caused by drugs. The results showed that the intracellular ROS level increased after drug treatment, but the ROS level decreased significantly after overexpression of EDEM1 ( Figure 4 A). The doxorubicin-resistant cell line 231 / DOX cells were transfected with EDEM1 small interfering RNA (wherein the sequence of EDEM1 interfering RNA is: 5'-CCCUCCCUGAGAGAUAUAATTUUAUAUCUCUCAGGGAGGGTT-3'), and the transfected cells were treated with the same drugs and the intracellular ROS level was detected. The results showed that the intracellular ROS level increased after drug treatment, and the ROS level was significantly upregulated after knocking down EDEM1 ( Figure 4 B). The above results indicate that EDEM1 can alleviate the ER stress caused by ER stress inducers and doxorubicin, and increase the ER stress resistance of triple-negative breast cancer cells.

[0054] 2.2 Verification of the correlation between EDEM1 and doxorubicin resistance in triple-negative breast cancer: TNBC cells MDA-MB-231 and MDA-MB-468 were transfected with EDEM1 overexpressing vectors, and IC 50 The results showed that breast cancer cells overexpressing EDEM1 50 Higher than the control group ( Figure 4AB). At the same time, the IC of the drug-resistant cell line 231 / DOX cells transfected with EDEM1 was detected by MTT. 50 The results showed that IC of breast cancer resistant cells with knockdown of EDEM1 50 Lower than the control group ( Figure 4 C).

[0055] Example 3

[0056] Evaluation of the potential of EDEM1 as a prognostic marker in breast cancer patients:

[0057] 3.1 Evaluation of the potential of EDEM1 as a prognostic marker in breast cancer patients

[0058] To further analyze the correlation between EDEM1 expression level and clinicopathological characteristics and overall survival of breast cancer patients, we selected 131 patients diagnosed with breast cancer in the Department of Breast Surgery, Qilu Hospital, Shandong University from March 2007 to January 2017, including 76 TNBC patients and 55 non-TNBC patients. Immunohistochemical staining of the patient's pathological tissue sections was performed and immunohistochemical scoring was performed ( Figure 5 A), and calculated the cut-off value, and divided the patients into EDEM1 high expression group and low expression group. The Kaplan-Meier analysis of the correlation between EDEM1 expression level and the prognosis of breast cancer patients showed that high EDEM1 expression was associated with worse overall survival (OS) (HR=9.905, p<0.0001) and disease-free survival (DFS) (HR=3.949, p=0.0001) in breast cancer patients. The ROC curve was drawn, and it was found that EDEM1 had a good predictive effect on OS (AUC=0.794, p<0.0001) and DFS (AUC=0.700, p=0.0001) in breast cancer patients. Figure 5 B). At the same time, Cox univariate and multivariate risk regression analysis was performed on the clinical information of breast cancer patients and the expression of EDEM1. It was found that the number of lymph node metastases greater than 3 (HR=10.425, p=0.001) and high expression of EDEM1 (HR=12.365, p=0.001) could be independent influencing factors for poor OS of patients ( Figure 5 C); the number of lymph node metastases greater than 3 (HR=12.039, p<0.001), distant metastasis (HR=8.047, p<0.001), and high expression of EDEM1 (HR=11.957, p<0.001) can be independent influencing factors for poor DFS ( Figure 5 C).

[0059] 3.2 Evaluation of the potential of EDEM1 as a prognostic marker in patients with triple-negative breast cancer:

[0060] At the same time, we noticed that the EDEM1 immunohistochemical score of tumor tissues of TNBC patients was significantly higher than that of non-TNBC patients ( Figure 6 A). The Kaplan-Meier analysis of the correlation between the expression level of EDEM1 and the prognosis of TNBC patients showed that high EDEM1 expression was associated with worse OS (HR=20.76, p<0.0001) and DFS (HR=3.794, p=0.0045) in TNBC patients. The ROC curve was drawn and it was found that EDEM1 had a good predictive effect on OS (AUC=0.869, p<0.0001) and DFS (AUC=0.722, p=0.0054) in breast cancer patients. At the same time, Cox univariate and multivariate risk regression analysis of the clinical information of TNBC patients and the expression of EDEM1 showed that the number of lymph node metastases was greater than 3 (HR=19.072, p=0.001) and high EDEM1 expression (HR=25.639, p=0.003) could be used as independent influencing factors for poor OS in TNBC patients ( Figure 6 B); lymph node metastasis number 1-3 (HR=6.739, p=0.016); lymph node metastasis number greater than 3 (HR=28.044, p<0.001) can be used as independent influencing factors for poor DFS in TNBC patients ( Figure 6 C).

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of EDEM1 as a drug resistance marker for the anti-tumor drug doxorubicin, wherein the amino acid sequence of EDEM1 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The application methods include: application of EDEM1 content detection reagent in the preparation of reagents or diagnostic equipment for doxorubicin resistance assessment; Or, tumors for which doxorubicin is used as an anti-tumor drug include: acute leukemia, malignant lymphoma, breast cancer, bronchial lung cancer, ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer or liver cancer, preferably breast cancer, and more preferably triple-negative breast cancer.

3. A reagent or diagnostic device for doxorubicin resistance assessment, characterized in that: Includes EDEM1 content detection reagent.

4. Application of EDEM1 as a prognostic marker for breast cancer.

5. The use according to claim 4, characterized in that The application methods include: using EDEM1 content detection reagents to prepare prognosis judgment products for breast cancer patients; Alternatively, the breast cancer comprises triple-negative breast cancer.

6. A product for predicting the prognosis of breast cancer patients, characterized in that: The product includes an EDEM1 content detection reagent.

7. Use of EDEM1 as a target in the development, screening and / or preparation of agents for the treatment or adjuvant treatment of breast cancer.

8. Use of a substance that inhibits or interferes with EDEM1 expression in at least one of the following or in the preparation of a product having at least one of the following functions: (1) Treatment or adjuvant treatment of breast cancer; (2) Promote chemotherapy drugs to treat breast cancer; (3) Combined with chemotherapy drugs to treat breast cancer.

9. Use of substances that inhibit or interfere with EDEM1 expression and chemotherapy drugs in the preparation of products for the treatment or adjuvant treatment of breast cancer.

10. A product for treating or assisting in the treatment of breast cancer, characterized in that: It is either a) or b) as follows: a) Substances that inhibit or interfere with EDEM1 expression; b) Substances and chemotherapeutic drugs that inhibit or interfere with EDEM1 expression.

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