Application of RNFT1 protein as triple negative breast cancer treatment target

Abstract

The invention discloses application of RNFT1 protein as a triple negative breast cancer treatment target. According to the present invention, the E3 ligase RNFT1 is derived from the IP binding protein spectrum detection result of the membrane protein PDL1, and the influence of the RNFT1 on the PDL1 protein can be significantly observed through the RNFT1 overexpression and knock-down. Through flow and Western Blot, the reliability and importance of RNFT1 as a regulation target spot on the stability of the immune checkpoint PDL1 protein can be judged. Animal experiments show that high expression of the RNFT1 is unfavorable for immunotherapy response, and the curative effect of chemotherapy and immunotherapy is reduced, so that powerful evidence can be provided for evaluating the curative effect of immunotherapy by taking the RNFT1 as a predictive index and applying the RNFT1 as a drug of an immunotherapy target spot. RNFT1 plays an important role in regulating and controlling the stability of PDL1 protein on the surface of a cell membrane, and possibly becomes a specific marker for predicting the immunotherapy effect of triple-negative breast cancer, and the discovery possibly provides a basis for the development of new drugs for clinical diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of RNFT1 protein as a target for the treatment of triple-negative breast cancer. Background Art

[0002] The expression of PDL1 protein plays a key role in predicting the efficacy of PD-1 / PDL1 blocking immunotherapy, and has been used as the basis for screening the benefit stratification of the population suitable for immunotherapy in various solid tumors. The abundance of tumor infiltrating lymphocytes (TILs) is positively correlated with PDL1 expression, that is, the higher the PDL1 expression, the more tumor infiltrating lymphocytes. Patients with high PDL1 expression and high density of TILs respond better to immunotherapy.

[0003] Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, accounting for about 10% to 20% of breast cancers, and is characterized by the loss of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2. Given the lack of clear therapeutic targets, chemotherapy remains the standard treatment for TNBC, and the overall prognosis is poor. Recently, the breakthrough progress of PDL1 immune checkpoint inhibitors (ICI) has completely changed the tumor diagnosis and treatment strategy, and immune checkpoint blockade therapy (ICB) has been approved by the FDA for the treatment of many solid tumors including TNBC. However, only a small number of patients show significant and durable responses and achieve survival benefits. More than 80% of TNBC patients carry low to moderate levels of PDL1 (CPS < 10), and most of them respond poorly to ICI or develop immune tolerance after short-term benefits. The lack of TILs is an important factor that makes it difficult for TNBC patients to benefit from immunotherapy. We speculate that the key to increase PDL1 expression may be the key to promote the transformation of TNBC from "non-responsive" to "responsive" to immunotherapy.

[0004] The stability of membrane proteins affects the effect of targeted therapy. PD-L1 is a type I transmembrane protein that presents a dynamic equilibrium state on the cell membrane surface and is regulated by a variety of post-translational modifications, including glycosylation, palmitoylation, ubiquitination, phosphorylation, etc., which affect multiple biological processes such as PD-L1 protein expression, transportation, cell membrane localization, endocytosis, degradation, recycling and extracellular transport. The abundance of PDL1 expression on the cell membrane surface is positively correlated with the therapeutic response of PDL1 monoclonal antibodies. At the same time, PDL1 on the cell membrane surface is rapidly internalized to lysosomes for degradation after binding to PDL1 monoclonal antibodies, which easily leads to rapid consumption of PDL1 monoclonal antibodies in the blood, resulting in secondary immune tolerance. Previous studies have mainly focused on the transcriptional regulation of PDL1, which has rarely been translated into clinical applications. Therefore, this study intends to conduct in-depth research on the regulation of PD-L1 protein homeostasis.

[0005] A large number of clinical research results show that the combination of immunotherapy and chemotherapy can show excellent anti-tumor efficacy. Chemotherapy combined with PD1 / PDL1 checkpoint inhibitors has been approved by the FDA for the treatment of TNBC. Among them, cisplatin has shown obvious efficacy in sensitizing immunotherapy, and studies have shown that the anti-tumor effect of platinum combined with Atezolizumab (anti-PDL1 monoclonal antibody) is significantly better than other combinations. Cisplatin is also involved in regulating the expression of multiple immune checkpoints. However, whether the mechanism of cisplatin-sensitized immunotherapy in triple-negative breast cancer is related to PDL1 expression is still unknown and deserves further exploration.

[0006] RNFT1 (RING finger and transmembrane domain-containing protein 1) is a member of a family of E3 ligases containing zinc finger domains. It mainly mediates K63 ubiquitination modification of downstream proteins through the RING domain, regulating the transport, localization and lysosomal degradation of downstream proteins. It is well known that ubiquitination is a crucial link in the protein regulation system. According to the lysine site of ubiquitin protein, it is mainly divided into K48, K63, K11, K29 and other ubiquitination modifications, among which K48 and K63 are the most common. K48 ubiquitination mainly regulates the downstream substrates to enter the proteasome for degradation, and K63 ubiquitination mainly mediates the lysosomal degradation pathway of membrane proteins. Summary of the invention

[0007] The purpose of the present invention is to provide an application of RNFT1 in the preparation of a targeted therapeutic drug for maintaining the stability of PDL1 protein and improving the expression level of PDL1, so as to provide a reference for clinical sensitization immunotherapy of triple-negative breast cancer.

[0008] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: application of RNFT1 protein as a target for the treatment of triple-negative breast cancer, the amino acid sequence of RNFT1 protein is shown in SEQ ID NO.1.

[0009] The use of an inhibitor of RNFT1 protein in the preparation of a drug for immunotherapy of triple-negative breast cancer, the amino acid sequence of RNFT1 protein is shown in SEQ ID NO.1.

[0010] Furthermore, the inhibitor is an antibody against RNFT1 protein and / or a binding protein to RNFT1 protein.

[0011] The use of an inhibitor of the RNFT1 gene in the preparation of a drug for immunotherapy of triple-negative breast cancer, the amino acid sequence of the RNFT1 protein is shown in SEQ ID NO.1.

[0012] Furthermore, the drug includes a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an inhibitor of the RNFT1 gene.

[0013] A drug for immunotherapy of triple-negative breast cancer, comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredients: an inhibitor of RNFT1 protein and / or an inhibitor of RNFT1 gene, wherein the amino acid sequence of RNFT1 protein is shown in SEQ ID NO.1.

[0014] Furthermore, the inhibitor of RNFT1 protein is selected from antibodies to RNFT1 protein and / or binding proteins to RNFT1 protein; the inhibitor of RNFT1 gene is selected from one or more of RNFT1 gene-specific RNAi, RNFT1 gene-specific microRNA, or inhibitors that inhibit the RNFT1 gene promoter.

[0015] A reagent for detecting the expression level of RNFT1 protein and / or RNFT1 gene in a sample is used in the preparation of a kit for detecting triple-negative breast cancer, wherein the amino acid sequence of the RNFT1 protein is shown in SEQ ID NO.1.

[0016] The present invention found that cisplatin inhibits the K63 ubiquitination modification of PDL1 on the cell membrane through RNFT1, thereby slowing down the endocytosis-lysosomal degradation of PDL1, maintaining the protein stability of PDL1, and promoting the tumor's response to immunotherapy. However, the potential mechanism of RNFT1 as an important target for maintaining PDL1 protein stability and increasing the expression of PDL1 has not yet been disclosed. Using RNFT1 as a new therapeutic target can improve the tumor's response to ICB, achieve an immune synergistic sensitization effect similar to cisplatin, but without the toxic side effects of cisplatin, and develop new therapies targeting the lysosomal system, which will help overcome the bottleneck of ICB treatment of TNBC and have great clinical significance and application value.

[0017] The E3 ligase RNFT1 of the present invention is derived from the IP binding protein spectrum detection results of the membrane protein PDL1. By overexpressing and knocking down RNFT1, the effect of RNFT1 on the PDL1 protein can be significantly observed. Through flow cytometry and Western Blot, the reliability and importance of RNFT1 as a regulatory target for the stability of the immune checkpoint PDL1 protein can be determined. Through animal experiments, it can be observed that high expression of RNFT1 is not conducive to immunotherapy response and reduces the efficacy of chemotherapy combined with immunotherapy, which can provide strong evidence for the use of RNFT1 as a predictive indicator to evaluate the efficacy of immunotherapy and as a drug target for immunotherapy synergy.

[0018] The present invention uses Western to detect the effect of cisplatin on the expression of PDL1 in triple-negative breast cancer cells MDA-MB-231 and HCC1806, and finds that after cisplatin treatment, the PDL1 protein level is time-dependently upregulated within 12 hours, and there is no obvious change in the RNA transcription level. Protein spectrum detection of PDL1 IP experimental samples found that after cisplatin treatment, the level of PDL1-bound E3 ligase RNFT1 significantly decreased. The effect of RNFT1 on the expression of PDL1 in triple-negative breast cancer cells MDA-MB-231 was detected by flow cytometry and Western, and it was found that RNFT1 was involved in regulating the endocytosis-lysosomal degradation of PDL1 on the cell membrane surface, significantly affecting the protein stability of PDL1. The effect of RNFT1 on the immune killing effect of triple-negative breast cancer cells MDA-MB-231 after PDL1 monoclonal antibody treatment was detected by a tumor-immune co-culture model, and it was found that RNFT1 significantly downregulated the therapeutic effect of ICIs and inhibited T cell killing. Animal experiments detected the effect of RNFT1 on immunotherapy response in immune-competent mice and found that RNFT1 was not conducive to anti-tumor immune response and significantly inhibited the benefits of immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The membrane protein PDL1 increased in a time-dependent manner after cisplatin treatment (Figure A shows the changes in PDL1 at the total protein and membrane protein levels), while there was no obvious change in the RNA level (Figure B).

[0020] Figure 2 After the MDA-MB-231-PDL1-KO cell line was stably transfected with the PDL1-flag lentivirus, the cells were treated with cisplatin, and membrane proteins were extracted for PDL1 IP experiments. A portion of the IP product was silver stained ( Figure 2 A is the silver staining result indicating the location of RNFT1), and a part of it was detected by mass spectrometry. The results showed that the top 10 PDL1 binding proteins that were most significantly downregulated in the cisplatin-treated group included RNFT1 ( Figure 2 B).

[0021] Figure 3 The effect of RNFT1 on the internalization of the cell membrane PDL1 protein ( Figure 3 A: RNFT1 overexpression accelerates PDL1 endocytosis. Figure 3 B: Knockdown of RNFT1 inhibits the internalization of PDL1).

[0022] Figure 4 The effect of RNFT1 on the recycling of PDL1 back to the membrane after endocytosis ( Figure 4 A: RNFT1 overexpression slows down the return of PDL1 to the membrane. Figure 4 B is that promoting RNFT1 will accelerate the return of PDL1 to the membrane).

[0023] Figure 5 The effect of RNFT1 on the degradation rate of cell membrane PDL1 (Figure A shows that overexpression of RNFT1 promotes the degradation of PDL1, and Figure B shows that knocking down RNFT1 inhibits the degradation of PDL1).

[0024] Figure 6 The effect of RNFT1 on T cell killing in tumor immune co-culture.

[0025] Figure 7 This is a schematic diagram of the animal experiment design process.

[0026] Figure 8 It is the tumor volume and tumor size in animal experiments.

[0027] Fig. 9 In animal experiments, RNFT1 changes the proportion of infiltrating lymphocytes in tumors. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0029] It should be noted that the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or apparatuses. Example

[0030] PDL1 protein levels were upregulated in a time-dependent manner after cisplatin treatment The MDA-MB-231 and HCC1806 cells used in this experiment were purchased from Shanghai Institute of Biological and Cytological Sciences, Chinese Academy of Sciences. CCK8 reagent, Trizol reagent, and PCR detection kit were purchased from Nanjing Novozyme Biotechnology Co., Ltd., and DMEM medium was purchased from Jiangsu Keygene Biotechnology Co., Ltd.

[0031] Take the logarithmic growth phase MDA-MB-231 and HCC1806 cells and plate them. When the cell confluence reaches about 80%, add Cisplatin 20uM for 0, 2, 4, and 8 hours (DMSO is used as the control). Then use the cell membrane protein lysis kit to collect membrane proteins for Western Blot detection, and use Trizol to collect RNA for PCR detection. The results are shown in Figure 1 As shown: After cisplatin treatment, the cell membrane PDL1 protein level was upregulated in a time-dependent manner, and there was no obvious change in the transcription level. Example

[0032] Protein profiling: After the MDA-MB-231 PDL1-Knockout cell line was stably transfected with PDL1-myc lentivirus, the stable cells were screened, and the cells in the logarithmic growth phase were treated with cisplatin to collect proteins. The samples were collected by IP experiment using PDL1 antibody and sent to Jiangbei XX Company for proteomic high-throughput detection of PDL1 binding proteins and screening of differentially expressed proteins. It was found that the level of E3 ligase RNFT1 bound by PDL1 was significantly decreased after cisplatin treatment. At the cell level, IP experiments were performed again to verify that cisplatin significantly inhibited the binding of PDL1 and RNFT1. Figure 2 shown. Example

[0033] 3. Cell endocytosis-recycling-degradation experiment 3.1 Cell membrane PDL1 endocytosis experiment MDA-MB-231 RNFT1 overexpression and knockdown cells (RNFT1 / Scramble, shRNFT1 / shNC) in the logarithmic growth phase were taken, digested with Tryple-Express, counted, labeled with PDL1-blocking antibody, incubated on ice for 1 hour, washed 3 times with PBS, incubated in a 37 degree Celsius incubator for the corresponding time, and then the cells were collected and labeled with AF-488 fluorescent secondary antibody and detected by flow cytometry, with IgG as the control. The data were analyzed by FlowJo software, and the results showed that RNFT1 significantly promoted the endocytosis of cell membrane PDL1 (with Scramble as the control), and knocking down shRNFT1 significantly inhibited the endocytosis of cell membrane PDL1 (with shNC as the control).

[0034] 3.2 Cell membrane PDL1 recycling experiment MDA-MB-231 RNFT1 overexpression and knockdown cells (RNFT1 / Scramble, shRNFT1 / shNC) in the logarithmic growth phase were taken, digested with Tryple-Express, counted, labeled with PDL1-blocking antibody, incubated on ice for 1 hour, washed 3 times with PBS, incubated in a 37 degree Celsius incubator for 30 minutes to allow endocytosis, eluted the PDL1 antibody on the membrane surface with acidic elution solution, washed 3 times with PBS, and then put back into the 37 degree Celsius incubator for the corresponding time. After collecting the cells, they were labeled with AF-488 fluorescent secondary antibody and detected by flow cytometry, with IgG as the control. The data were analyzed by FlowJo software, and the results showed that RNFT1 significantly reduced the recycling of cell membrane PDL1 back to the membrane, and knocking down RNFT1 significantly accelerated the recycling of cell membrane PDL1 back to the membrane.

[0035] 3.3 Cell membrane PDL1 degradation experiment MDA-MB-231 RNFT1 overexpression and knockdown cells (RNFT1 / Scramble, shRNFT1 / shNC) in the logarithmic growth phase were taken, digested with Tryple-Express, counted, labeled with PDL1-APC antibody, incubated on ice for 1 hour, washed 3 times with PBS, incubated in a 37 degree Celsius incubator for the corresponding time, and then flow cytometry was performed, with IgG as the control. The data were analyzed by FlowJo software, and the results showed that RNFT1 significantly accelerated the degradation of cell membrane PDL1, while shRNFT1 delayed the degradation of cell membrane PDL1. Example

[0036] T cell killing assay MDA-MB-231 RNFT1 overexpressing and Scramble control cells in the logarithmic growth phase were plated, and PBMC cells were activated with cell stimulatory factors for 24 hours. Then, PMBC cells were added to the upper chamber of the culture dish at a ratio of tumor:immune = 1:10 and co-cultured for 24 hours. Tumor cells were taken for apoptosis flow cytometry detection, and upper PMBC were taken for PD1 flow cytometry detection. The results showed that when RNFT1 was overexpressed, the killing effect of T cells was significantly weakened, and the proportion of PD1+ in the exhausted cell population in PBMC was significantly increased. Example

[0037] Animal experiments The mouse triple-negative breast cancer cell line 4T1 was stably transfected with RNFT1 overexpression and scramble control cell lines by lentiviral screening, and tumors were formed by orthotopic injection into the mammary pad of immunocompetent Balb / C mice. Figure 5The drug was administered and the tumor volume was regularly tested. After reaching the endpoint, the mice were euthanized and the tumor tissues were collected for subsequent experiments. The expression level of PDL1 was detected by immunohistochemistry, and the level of infiltrating lymphocytes was detected by flow cytometry. The results showed that RNFT1 overexpression downregulated the expression level of PDL1 and the abundance of TILs. RNFT1 overexpression was not conducive to immune activation and the effect of chemotherapy combined with immunotherapy.

[0038] Conclusion: High expression of RNFT1 can be used as an indicator to predict poor response to immunotherapy. Targeted interference with RNFT1 expression is beneficial to promote the stability of PDL1 protein and further enhance the anti-tumor killing effect of immunotherapy. It provides new ideas and methods for the precise stratification of target populations for immunotherapy and the targeted development of small molecule inhibitors, which is beneficial to optimize immunotherapy regimens and improve the prognosis of clinical patients.

Claims

1. Application of RNFT1 protein as a therapeutic target for triple-negative breast cancer, characterized in that: The amino acid sequence of the RNFT1 protein is shown in SEQ ID NO.

1.

2. Use of an inhibitor of RNFT1 protein in the preparation of a drug for immunotherapy of triple-negative breast cancer, characterized in that: The amino acid sequence of the RNFT1 protein is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that: The inhibitor is an antibody against RNFT1 protein and / or a binding protein to RNFT1 protein.

4. Use of an inhibitor of the RNFT1 gene in the preparation of a drug for immunotherapy of triple-negative breast cancer, characterized in that: The amino acid sequence of RNFT1 protein is shown in SEQ ID NO.

1.

5. The use according to claim 4, characterized in that: The medicine comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an inhibitor of the RNFT1 gene.

6. A drug for immunotherapy of triple-negative breast cancer, characterized in that: It comprises a pharmaceutically acceptable carrier and an effective amount of the following active ingredients: an inhibitor of RNFT1 protein and / or an inhibitor of RNFT1 gene, characterized in that the amino acid sequence of the RNFT1 protein is as shown in SEQ ID NO.

1.

7. The drug according to claim 6, characterized in that: The inhibitor of RNFT1 protein is selected from antibodies of RNFT1 protein and / or binding proteins of RNFT1 protein; the inhibitor of RNFT1 gene is selected from one or more of RNFT1 gene-specific RNAi, RNFT1 gene-specific microRNA, or inhibitors that inhibit the RNFT1 gene promoter.

8. Use of a reagent for detecting the expression level of RNFT1 protein and / or RNFT1 gene in a sample in the preparation of a kit for detecting triple-negative breast cancer, characterized in that: The amino acid sequence of the RNFT1 protein is shown in SEQ ID NO.1.

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