Application of UGT1A7 in breast cancer drug for Herceptin resistance

Abstract

The present invention provides an application of UGT1A7 in a breast cancer drug that is resistant to Herceptin, and UGT1A7 can reverse the resistance of HER-2 positive breast cancer cell lines to Herceptin on cells; a process of finding that UGT1A7 is involved in Herceptin resistance, characterized in that a BT474 Herceptin-resistant cell line is constructed by a concentration gradient method, and it is found that compared with sensitive parental cells, the expression of UGT1A7 in Herceptin-resistant BT474HR cells is significantly downregulated, and it is found at the cellular level that UGT1A7 participates in regulating the Herceptin resistance process through mitochondrial and endoplasmic reticulum stress and promoting EMT process. The present invention can be applied clinically to help clinicians formulate treatment plans, judge prognosis, and become a target for related drug research and development in the future, improve the efficacy of Herceptin, and has good clinical promotion value.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a method for overexpressing UGT1A7 to reverse the drug resistance of HER-2 positive breast cancer cells to Herceptin. Background Art

[0002] Breast cancer is the most common malignant tumor in women, and its incidence rate is increasing year by year. The World Health Organization's International Agency for Research on Cancer (IARC) has recently released the latest global cancer burden data for 2020, among which the number of new cases of breast cancer worldwide is as high as 2.26 million, exceeding the 2.2 million cases of lung cancer, becoming the world's largest cancer. Among all breast cancer subtypes, HER-2-positive patients account for 20-30%, and these patients have a poor prognosis. Herceptin is a humanized monoclonal antibody targeting the extracellular segment of the receptor tyrosine kinase encoded by HER-2, and is currently the most commonly used drug in clinical treatment of HER-2-positive breast cancer patients. Although Herceptin improves the survival of HER-2-positive breast cancer patients, more than 70% of patients develop drug resistance within 1 year after treatment. Therefore, the drug resistance problem of Herceptin has become an important bottleneck affecting the treatment outcome and survival time of HER-2-positive advanced breast cancer patients.

[0003] At present, many studies on the mechanism of Herceptin resistance have been conducted, most of which focus on the activation of signal pathways after resistance and the changes in the structure of EGFR family receptors. In order to delay Herceptin resistance, there are currently drugs developed for resistance mechanisms in the clinic, such as HER-2 tyrosine kinase inhibitors (TKI) such as lapatinib, pyrotinib, and neratinib, and pertuzumab that blocks the formation of HER-2 / HER-3 heterodimers. These drugs combined with Herceptin can improve the efficacy of Herceptin, but patients will eventually develop resistance, leading to disease progression. Existing studies can only partially explain that the activation of EGFR family-related signal pathways may only represent the tip of the iceberg of Herceptin resistance, but there are few studies on new methods to reverse resistance. Therefore, how to reverse or delay Herceptin resistance can greatly prolong the survival time of HER-2 positive breast cancer patients and save patients' treatment costs, which has important clinical significance and social benefits.

[0004] BT474 is the most common HER2-positive breast cancer cell line. We selected BT474 as the research object and constructed the trastuzumab-resistant cell line BT474HR. In order to further explore the new mechanism of trastuzumab resistance, expression profile chip detection was performed. Combined with bioinformatics analysis and screening, it was found that UGT1A7, a member of the glucuronyltransferase (UGT) family, may be closely related to trastuzumab resistance in breast cancer. The UGT1A7 gene was significantly downregulated in both groups of resistant strains.

[0005] UGT1A7 is a member of the UGT1A subfamily and a glycoprotein distributed in the endoplasmic reticulum. It was previously believed that it could catalyze the binding of glucuronic acid to various glycoside substrates and exert detoxification functions. Currently, research on the UGT1A7 subfamily in the field of tumors is almost all focused on molecular epidemiology: that is, studying the relationship between its genetic polymorphism and the risk of tumor occurrence, as well as the toxic reactions caused by its drug metabolism or the relationship between glucuronidation and personalized medication. Recently, there have also been some studies on the involvement of the UGT1A family in anticancer drug resistance, but because the anticancer drugs studied are limited to chemical drugs, the mechanism mainly involves UGT1A participating in the glucuronidation of anticancer drugs, reducing the toxic effects of the drugs. However, as an antibody molecule, trastuzumab does not have a chemical basis for glucuronidation, and it is clear that this mechanism cannot explain how UGT1A7 downregulation mediates trastuzumab resistance. By performing genetic testing on cells that are sensitive and resistant to Herceptin, the inventors discovered for the first time that UGT1A7 expression is reduced in Herceptin-resistant cells, and that it participates in the process of Herceptin resistance through endoplasmic reticulum stress and the promotion of EMT. This provides a foundation and experimental basis for the formulation of treatment plans for HER-2 positive patients, prognosis judgment, and the future preparation of drugs to reverse Herceptin resistance in breast cancer patients. Summary of the invention

[0006] The purpose of the present invention is to discover a new mechanism in which UGT1A7 participates in Herceptin resistance, and to provide a basis for drug selection, prognosis judgment and preparation of drugs for reversing Herceptin resistance in HER-2 positive patients.

[0007] UGT1A7 is involved in the process of Herceptin resistance, specifically:

[0008] We constructed a Herceptin-resistant BT474 cell line using a concentration gradient method and found that UGT1A7 expression was significantly downregulated in Herceptin-resistant BT474HR cells compared with sensitive parental cells. Therefore, we found at the cellular level that UGT1A7 is involved in regulating the Herceptin resistance process by stressing mitochondria and endoplasmic reticulum and promoting EMT.

[0009] A method for detecting differential gene expression in BT474 and BT474HR cells: BT474 and BT474HR were subjected to microarray analysis and compared with third-party data GSE15043. It was found that the UGT1A7 gene was significantly downregulated in both groups of resistant strains, and further verified in small sample clinical samples.

[0010] A method for detecting BT474, BT474 / HR and BT474 overexpression or shRNA UGT1A7 by CCK8 method, comprising the following steps:

[0011] (1) Cell inoculation: The monolayer cultured cells were digested with 0.25% trypsin, and a single cell suspension was prepared with DMEM culture medium containing 10% fetal bovine serum. 4000 cells were inoculated per well in a 96-well culture plate with a volume of 200 μl per well.

[0012] (2) Cultivating cells: Move the culture plate into a CO2 incubator and culture at 37°C, 5% CO2 and relative humidity for 96 h;

[0013] (3) Color development: After 96 h of culture, add 20 μl of CCK-8 solution to each well and incubate at 37°C for another 4 h.

[0014] (4) Colorimetry: Measure the optical density OD at a wavelength of 490 nm. Perform the experiment three times with 3 replicate wells in each group. Calculate the survival rate. Survival rate = OD value of the experimental group / OD value of the control group × 100%;

[0015] (5) BT474HR overexpressing UGT1A7 and BT474shRNA UGT1A7 were seeded on 96-well plates, with 4000 cells / well. Each group was treated with different concentrations of Herceptin, and cell viability was detected by CCK-8 staining 3 days after administration, and the IC50 of each drug was calculated.

[0016] A method for detecting the sensitivity of UGT1A7 to regulate Herceptin by affecting the structure and membrane potential of mitochondria, specifically comprising the following steps:

[0017] (1) Cell inoculation: Digest the monolayer cultured cells with 0.25% trypsin, prepare a single cell suspension with DMEM culture medium containing 10% fetal bovine serum, and inoculate 1×106 / ml cells in a 12-well culture plate with a volume of 500 ul per well; (2) Cell culture: Move the culture plate into a CO2 incubator and culture for 96 h at 37°C, 5% CO2 and relative humidity;

[0018] (3) Centrifugation: 1000 rpm for 10 min;

[0019] (4) Fixation: After centrifugation, add 1 ml of 2.5% glutaraldehyde fixative along the tube wall and store in a refrigerator at 4°C;

[0020] (5) Image acquisition: A transmission electron microscope JEOL JEM-1010 was used to acquire images at 2uM and 500uM scales. BT474 siRNA VECTOR, BT474 siUGT1A7, BT474HR LV-OE-VECTOR, and BT474HR LV-OE-UGT1A7 were inoculated for 24 hours, and then 10ug / ml Herceptin was added and the cells were digested and resuspended for 72 hours.

[0021] Among breast cancer patients, 20-30% are HER-2 positive, and these patients have a poor prognosis. Herceptin is the cornerstone of current HER-2 targeted therapy, which improves the survival of HER-2 positive breast cancer patients, but more than 70% of patients develop drug resistance within 1 year after treatment. Therefore, the drug resistance of Herceptin has become a difficult problem affecting the treatment and survival of HER-2 positive advanced breast cancer patients. How to reverse or delay Herceptin resistance, prolong the survival time of HER-2 positive breast cancer patients, and save patients' treatment costs has important clinical significance and social benefits.

[0022] The present invention firstly performs a whole genome analysis on cells sensitive / resistant to Herceptin, with a total of 487 probes, passes FDR correction, and finds 453 differentially expressed genes. At the same time, the third-party data (GSE15043) of the expression spectrum chip of BT474 and its trastuzumab-resistant strain BT474HR is obtained. Further, KEGG pathway analysis is performed on genes with differential expression of more than 2 times in the two groups of data. It is found that only the drug metabolism signaling pathway is obtained by the simultaneous analysis of the two groups of data, and only the UGT1A7 gene is significantly downregulated in both groups of resistant strains, and the sensitivity to Herceptin is regulated through the process of mitochondrial and endoplasmic reticulum stress and EMT. Its application in clinical practice can help clinicians formulate treatment plans, judge prognosis, and become a target for the development of related drugs in the future, improve the efficacy of Herceptin, and has good clinical promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of whole-genome analysis and comparison with the TCGA database.

[0024] Figure 2 Schematic diagram of the results of using the CCK8 method to detect BT474, BT474 / HR, and BT474 overexpression or shRNA UGT1A7.

[0025] Figure 3 Schematic diagram of the results of detecting the sensitivity of UGT1A7 to Herceptin by affecting the structure and membrane potential of mitochondria.

[0026] Figure 4 Schematic diagram of the results of detecting the sensitivity of UGT1A7 to Herceptin by regulating EMT through affecting endoplasmic reticulum stress. DETAILED DESCRIPTION

[0027] The present invention is further described below by way of examples, but the present invention is not limited thereto.

[0028] General Notes:

[0029] The experimental methods without specific conditions in the examples are basically carried out according to the conditions and methods described in "Molecular Cloning: A Laboratory Manual, 3rd ed., compiled by Sambrook, J. et al." (Molecular Cloning: A Laboratory Manual, 3rd ed., translated by Huang Peitang et al., Science Press, August 2002) or according to the conditions and methods recommended by the material supplier. Other techniques not described in detail are standard methods well known to those skilled in the art.

[0030] Materials of the present invention: The cell lines, plasmids and culture media mentioned in this application are all commercially available or can be obtained by the public through other channels. They are only examples and are not exclusive to the present invention. They can be replaced by other suitable tools and biological materials.

[0031] Example 1

[0032] Detect the differential gene expression in BT474 and BT474HR cells.

[0033] 1. Methods: Immunohistochemistry, bioinformatics.

[0034] 2. Result determination: Figure 1 As shown, through whole-genome analysis and comparison with the TCGA database, it was found that the UGT1A7 gene was significantly downregulated in both groups of resistant strains, and this was further confirmed in patients with Herceptin resistance, p<0.01.

[0035] 3. Result analysis: BT474 and BT474HR were subjected to microarray analysis and analyzed with the third-party data GSE15043. It was found that the UGT1A7 gene was significantly downregulated in both groups of resistant strains, and further verified in small sample clinical samples.

[0036] Example 2

[0037] CCK8 method was used to detect BT474, BT474 / HR and BT474 overexpression or shRNA UGT1A7. CCK8 cell proliferation assay was used to detect cell proliferation and sensitivity to Herceptin. The sensitivity to Herceptin was observed and the IC50 value was calculated.

[0038] 1) Inoculation of cells: The monolayer cultured cells were digested with 0.25% trypsin, and a single cell suspension was prepared with DMEM culture medium containing 10% fetal bovine serum. 4000 cells were inoculated in each well of a 96-well culture plate, with a volume of 200 ul per well.

[0039] 2) Cultivate cells: Move the culture plate into a CO2 incubator and culture for 96 hours at 37°C, 5% CO2 and relative humidity;

[0040] 3) Color development: After 96 hours of culture, add 20ul of CCK-8 solution to each well and incubate at 37°C for another 4 hours.

[0041] 4) Colorimetry: Colorimetry: Measure the optical density OD at a wavelength of 490 nm. Perform the experiment three times with 3 replicate wells in each group. Calculate the survival rate. Survival rate = OD value of experimental group / OD value of control group × 100%.

[0042] 5) BT474HR overexpressing UGT1A7 and BT474shRNA UGT1A7 were seeded on 96-well plates as described above, with 4000 cells / well. Each group was treated with different concentrations of Herceptin, and cell viability was detected by CCK-8 staining 3 days after administration, and the IC50 of each drug was calculated.

[0043] Result determination: Figure 2 As shown, after BT474, BT474 / HR and BT474 overexpression or shRNA UGT1A7 were detected by CCK8 method, the CCK8 cell proliferation test was used to detect the cell proliferation and sensitivity of Herceptin, the sensitivity to Herceptin was observed, and the IC50 value, resistance multiple and reversal index were calculated.

[0044] 6) Analysis of results: After shRNA UGT1A7 was expressed in BT474 cells, the cells showed obvious resistance to Herceptin. However, after overexpression of UGT1A7 in BT474HR cells, the sensitivity of the cells to Herceptin was increased.

[0045] Example 3

[0046] Detection of UGT1A7 regulating the sensitivity of Herceptin by affecting mitochondrial structure and membrane potential

[0047] 1) Inoculation of cells: The monolayer cultured cells were digested with 0.25% trypsin, and a single cell suspension was prepared with DMEM culture medium containing 10% fetal bovine serum. The cells were inoculated into a 12-well culture plate at 1×106 / ml, with a volume of 500 ul per well.

[0048] 2) Cultivate cells: Move the culture plate into a CO2 incubator and culture at 37°C, 5% CO2 and relative humidity for 96 hours;

[0049] 3) Centrifugation: 1000 rpm for 10 min.

[0050] 4) Fixation: After centrifugation, add 1 ml of 2.5% glutaraldehyde fixative along the tube wall and store in a refrigerator at 4°C;

[0051] 5) Image acquisition: The images were acquired using a transmission electron microscope JEOL JEM-1010 at 2 uM and 500 uM scales.

[0052] BT474 siRNA VECTOR, BT474 siUGT1A7, BT474HR LV-OE-VECTOR and BT474HR LV-OE-UGT1A7 were inoculated as above for 24 hours, and 10ug / ml Herceptin was added, and the cells were digested and resuspended for 72 hours.

[0053] 6) Result determination: Figure 3 As shown, UGT1A7 was detected to regulate the sensitivity to Herceptin by affecting the structure and membrane potential of mitochondria.

[0054] 7) Analysis of results: The mitochondrial cristae of BT474 wild-type cells were clear in shape, intact in appearance, and normal in shape. After administration of 10ug / ml Herceptin, the mitochondrial cristae disappeared, the vesicles swelled, and even ruptured, and the mitochondrial membrane potential decreased significantly by 25.25%; while the mitochondria of the UGT1A7 interference group were normal in shape after administration of 10ug / ml Herceptin, with no significant difference from the negative control group, and the mitochondrial membrane potential decreased slightly; after administration of 10ug / ml Herceptin, the mitochondrial cristae of BT474HR were clear in shape, intact in appearance, and normal in shape.

[0055] The mitochondrial membrane potential was further analyzed by flow cytometry. There was no significant difference in the mitochondrial membrane potential of BT474HR and the negative control group. However, after overexpression of UGT1A7 and treatment with 10ug / ml Herceptin, the mitochondrial vesicles swelled, the cristae became less, and the mitochondrial membrane potential decreased significantly by 16.45%. This indicates that the BT474 resistant strain reduces its sensitivity to Herceptin by affecting the function of mitochondria and downregulating UGT1A7.

[0056] Example 4

[0057] Detection: UGT1A7 regulates sensitivity to Herceptin by affecting endoplasmic reticulum stress and regulating EMT.

[0058] 1. Methods: electron microscopy, Western Blot.

[0059] 2. Result determination: Figure 4 As shown, UGT1A7 was detected to regulate the sensitivity to Herceptin by affecting ER stress and regulating EMT.

[0060] 3. Result analysis: The endoplasmic reticulum of BT474 cells was intact and thin in shape. After interfering with UGT1A7, the endoplasmic reticulum was damaged and became thick. The endoplasmic reticulum of BT474 drug-resistant cells was broken and thick in shape. After overexpressing UGT1A7, the endoplasmic reticulum was thin in shape. After BT474HR and BT474 siUGT1A7, p-AMPK, endoplasmic reticulum-related proteins GRP78, CHOP and Cleaved caspase12 were further activated, indicating that BT474HR cells activated endoplasmic reticulum stress by downregulating UGT1A7. In addition, compared with BT474, the expression of EMT-related proteins, E-cad, decreased in BT474HR, and increased in SNAI and TWIST. After BT474 silenced shUGT1A7, consistent results were obtained. After BT474HR overexpressed UGT1A7, the expression of E-cad increased, and SNAI and TWIST decreased. It can be seen that the reduction of UGTA17 can promote the process of EMT. Furthermore, after interfering with UGT1A7 in BT474 cells, we added the endoplasmic reticulum stress inhibitor 4-PBA, which significantly reduced the stress level and inhibited the process of EMT. Similarly, BT474 silenced UGT1A7 and overexpressed CDH1, which inhibited the process of EMT.

[0061] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only preferred examples of the invention and are not intended to limit the present invention. Without departing from the novel spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Use of an overexpressed UGT1A7 protein in the preparation of a drug for treating Herceptin-resistant breast cancer.

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