Application of PINK1 in regulation and control of ER positive breast cancer tamoxifen drug resistance

Abstract

The invention discloses an application of PINK1 in regulation and control of ER positive breast cancer tamoxifen drug resistance. The invention relates to application of PINK1 as a biomarker in evaluating the tamoxifen resistance of ER positive breast cancer cells. The drug resistance is evaluated by detecting the expression level of PINK1 in a sample and combining mitochondrial autophagy activity. The expression level of the PINK1 comprises an mRNA level and / or a protein level. According to the invention, the drug resistance formation process is systematically analyzed from the perspective of quality control of an organelle of mitochondrial autophagy, and through public database analysis and experimental verification, the overall up-regulation of the mitochondrial autophagy pathway in drug-resistant cells is clearly revealed, and the drug-resistant phenotype is promoted by maintaining the mitochondrial steady state, thereby opening up a new direction for understanding the drug-resistant mechanism.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of PINK1 in regulating tamoxifen resistance in ER-positive breast cancer. Background Technology

[0002] In the current technology, acquired resistance is common in ER-positive breast cancer patients receiving tamoxifen (TAM) treatment. However, it is unclear whether mitophagy is involved in the formation of TAM resistance and its specific role. Therefore, the resistance mechanism cannot be reasonably explained from the perspective of mitochondrial homeostasis regulation.

[0003] The expression changes and functional roles of PINK1 in ER-positive breast cancer TAM-resistant cells are still unclear, especially regarding whether PINK1 affects cell sensitivity to TAM by regulating mitophagy, as there is a lack of systematic experimental evidence.

[0004] Whether PINK1-mediated mitophagy enhances tumor cell survival under drug stress and promotes drug resistance phenotype formation by resisting TAM-induced mitochondrial ROS elevation and maintaining mitochondrial membrane potential stability is not clearly reported in the current technology.

[0005] Currently, there is a lack of technical solutions that target PINK1 and its mediated mitophagy pathway to regulate TAM sensitivity in ER-positive breast cancer cells or to assess drug resistance risk, making it difficult to provide new molecular evidence for reversing clinical drug resistance or predicting efficacy.

[0006] This invention aims to address the aforementioned issues by systematically studying TAM-sensitive / resistant cell models and public databases, elucidating the role and mechanism of PINK1-mediated mitophagy in TAM resistance in ER-positive breast cancer, and proposing potential intervention and evaluation strategies targeting the PINK1–mitochondrial autophagy–ROS / MMP axis. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of PINK1 in regulating tamoxifen resistance in ER-positive breast cancer.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The application of PINK1 as a biomarker in assessing tamoxifen resistance in ER-positive breast cancer cells was investigated by detecting the expression level of PINK1 in samples and combining it with mitochondrial autophagy activity to evaluate drug resistance.

[0009] Preferably, the expression level of PINK1 includes mRNA level and / or protein level.

[0010] Preferably, the mitophagy activity is assessed by at least one of the following methods: (1) The degree of co-localization between mitochondria and lysosomes; (2) Changes in LC3-II levels; (3) Changes in TOM20 levels; (4) LC3-II / TOM20 correlation ratio or joint interpretation results.

[0011] A method for in vitro assessment of tamoxifen resistance in ER-positive breast cancer cells includes the following steps: S1: Detection of PINK1 expression levels in cells; S2: Detection of mitochondrial autophagy activity in cells; S3: Compare the test results with the control standard, where high expression of PINK1 and / or increased mitophagy activity indicate enhanced tamoxifen resistance.

[0012] Preferably, the mitochondrial autophagy activity in S2 is achieved by the degree of co-localization of MitoTracker and LysoSensor double staining and / or the co-localization of TOM20 and LC3 immunofluorescence, and / or the detection of LC3-II and TOM20 by Western blot.

[0013] The use of PINK1 expression inhibition methods in the preparation of intervention agents that enhance the sensitivity of ER-positive breast cancer cells to tamoxifen, wherein the PINK1 expression inhibition method is selected from shRNA, siRNA or other nucleic acid methods that can reduce PINK1 expression or function.

[0014] Use of mitochondrial autophagy inhibitors in the preparation of interventional formulations that enhance the sensitivity of ER-positive breast cancer cells to tamoxifen.

[0015] Preferably, the mitophagy inhibitor is Mdivi-1 or a pharmaceutically acceptable salt thereof.

[0016] A drug / intervention composition comprising tamoxifen with a PINK1 expression inhibitor and / or a mitophagy inhibitor.

[0017] A method for establishing a tamoxifen-resistant cell model of ER-positive breast cancer includes the following steps: S1: Select ER-positive breast cancer cell lines; S2: Long-term induction with incremental concentrations of tamoxifen in a culture medium containing tamoxifen; S3: Screening to obtain cell lines that can grow stably under the target concentration of tamoxifen; S4: Verify the drug resistance phenotype by crystal violet susceptibility testing and / or CCK8 susceptibility testing; S5: Detect the expression level of PINK1 and the activity of mitochondrial autophagy in the cell line to confirm phenotypic characteristics.

[0018] The beneficial effects of this invention are as follows: 1. This invention systematically elucidates the drug resistance formation process from the perspective of quality control of the organelle mitophagy. Through analysis of public databases and experimental verification, it clearly reveals that the mitophagy pathway is upregulated in drug-resistant cells as a whole, and promotes the drug resistance phenotype by maintaining mitochondrial homeostasis, thus opening up new directions for understanding the drug resistance mechanism.

[0019] 2. Existing research largely focuses on receptor mutations and bypass signaling pathways. This invention, for the first time, starts with mitophagy, demonstrating that mitophagy is upregulated overall in TAM-resistant ER-positive breast cancer cells. Furthermore, through functional inhibition (Mdivi-1) experiments, it directly verifies that mitophagy has a substantial promoting effect on the drug-resistant phenotype, rather than merely a correlated alteration. 3. This invention does not stop at a broad association at the pathway level, but precisely identifies PINK1 as the core molecule mediating drug resistance-related mitophagy; through bioinformatics screening combined with cell model verification, it is confirmed that PINK1 is continuously highly expressed in drug-resistant cells, and its high expression is significantly associated with poor patient prognosis, thus pinpointing the drug resistance mechanism to a specific and operable molecular target.

[0020] 4. This invention demonstrates the causal chain between PINK1 and mitophagy and tamoxifen sensitivity through gain- and loss-of-function experiments. Overexpression of PINK1 directly promotes mitophagy and induces drug resistance, while knockdown of PINK1 inhibits autophagy and reverses drug resistance. This establishes a complete chain of evidence that PINK1 upregulation leads to mitophagy activation and TAM resistance.

[0021] 5. This invention reveals the functional basis of PINK1-mediated mitophagy. Under TAM drug stress, this pathway effectively reduces the accumulation of reactive oxygen species in mitochondria by clearing damaged mitochondria and maintains the stability of mitochondrial membrane potential. From the perspective of mitochondrial functional homeostasis, this provides an explanation for how drug-resistant cells survive under drug stress.

[0022] 6. This invention proposes a method for assessing the risk of TAM resistance in patients by detecting PINK1 expression or mitophagy activity; and provides a potential therapeutic strategy to reverse resistance and enhance the efficacy of TAM by inhibiting PINK1 or mitophagy. Attached Figure Description

[0023] Figure 1 Schematic diagram of drug resistance model establishment and mitochondrial autophagy pathway enrichment; Figure 2Schematic diagram of upregulation of mitophagy and the Mdivi-1 reversal effect in drug-resistant cells; Figure 3 Schematic diagram of PINK1 screening and expression characteristics in drug-resistant cells; Figure 4 Schematic diagram of the regulatory role of PINK1 in mitophagy; Figure 5 Schematic diagram of the effect of PINK1 on TAM drug sensitivity; Figure 6 Schematic diagram of the regulation of mtROS and MMP by PINK1 under TAM pressure. Detailed Implementation

[0024] This invention provides a technical solution for regulating tamoxifen (TAM) resistance in ER-positive breast cancer mediated by PINK1-mediated mitophagy. Through public database analysis, in vitro construction of drug-resistant cell models, and a series of functional validation experiments, the role of the PINK1–mitochondrial autophagy–mitochondrial homeostasis axis in TAM resistance formation is systematically demonstrated, providing a basis for subsequent target intervention and drug resistance assessment. The technical solution of this invention is further described in detail below with reference to specific embodiments, but the invention is not limited to these embodiments.

[0025] 3.1 Overview of the Overall Technical Solution This invention first utilizes publicly available databases to perform differential expression and pathway enrichment analysis on TAM-sensitive and drug-resistant breast cancer cells, revealing that mitophagy-related pathways are significantly enriched in drug-resistant cells. Based on this, in vitro TAM-resistant cell models MCF7-R and T47D-R are constructed, and a systematic comparison is made regarding mitophagy levels, mitochondrial functional status, and PINK1 expression. Through gene manipulation such as PINK1 overexpression and knockdown, the regulatory role of PINK1 on mitophagy activity and TAM drug sensitivity is verified. Furthermore, changes in mitochondrial ROS and membrane potential are detected under TAM drug stimulation conditions to elucidate the functional role of PINK1-mediated mitophagy in the formation of drug resistance.

[0026] 3.2 Example 1: Construction of a TAM-resistant cell model and enrichment analysis of the mitochondrial autophagy pathway This embodiment provides a method for constructing an ER-positive breast cancer TAM-resistant cell model and identifying the enrichment of the mitochondrial autophagy pathway, including the following steps: (1) Public database analysis steps: Gene expression data of TAM-sensitive and drug-resistant breast cancer cell lines were downloaded from the GSE67916 dataset. Differential expression analysis was used to screen differentially expressed genes, and pathway enrichment analysis tools such as GO / KEGG were used to functionally enrich the sets of genes with significant differences, focusing on the changes in pathways related to mitochondrial function and autophagy.

[0027] (2) Steps for constructing a drug-resistant cell model: Using MCF7 and T47D cells as parental sensitive cells, long-term incremental concentration induction was performed in a TAM-containing medium. By gradually increasing the TAM concentration, cells that could grow continuously under higher TAM concentrations were screened and named MCF7-R and T47D-R, respectively.

[0028] (3) Steps for verifying drug resistance phenotype: a) Crystal violet drug sensitivity test: Sensitive cells and drug-resistant cells were seeded in multi-well plates. After the cells adhered, they were treated with TAM at the same concentration gradient for a certain period of time, fixed and stained with crystal violet. The number of residual cells and staining intensity of different cells after drug treatment were compared. b) CCK8 susceptibility assay: Using the CCK8 assay reagent, cell viability was detected after treatment with different concentrations of TAM, dose-response curves were plotted, and the IC50 value of each cell was calculated.

[0029] The implementation results show that: Figure 1 As shown in Figure A, enrichment analysis of the GSE67916 dataset revealed significantly enhanced mitochondrial function-related pathways in TAM-resistant breast cancer cells, with a marked enrichment of the mitophagy pathway. Figure 1 B Figure 1 As shown in Figure C, under the same TAM concentration treatment, MCF7-R and T47D-R cells retained significantly more surviving cells than sensitive cells, and the IC50 of drug-resistant cells was significantly increased, with the drug inhibition curve shifting to the right. This indicates that this embodiment successfully constructed a TAM-resistant cell model with stable drug resistance characteristics, and suggests that the mitophagy-related pathway may play an important role in drug resistance formation.

[0030] 3.3 Example 2: Upregulation of mitophagy in ER-positive breast cancer TAM-resistant cells and its effect on drug resistance phenotype This embodiment provides a method for detecting and verifying whether mitophagy is upregulated in TAM-resistant cells and its effect on drug resistance phenotype, including the following steps: (1) Steps for detecting mitophagy levels: a) Mitochondria-lysosome colocalization detection: Mitochondria were labeled with MitoTracker probes and lysosomes were labeled with LysoSensor probes. After incubation, the colocalization of mitochondria and lysosomes was observed under a confocal microscope. The degree of colocalization was calculated by image analysis to reflect the level of mitophagy. b) Transmission electron microscopy observation: Prepare ultrathin sections of sensitive and drug-resistant cells, and use transmission electron microscopy to observe the number of autophagosomes in the cells and the extent to which mitochondria are wrapped by a double membrane structure, so as to determine the mitochondrial autophagy activity in the cells.

[0031] (2) Mitochondrial functional status detection steps: MitoSOXRed was used to detect mitochondrial ROS (mtROS) levels, and TMRE was used to detect mitochondrial membrane potential (MMP). Flow cytometry or fluorescence microscopy were used to compare the differences in mtROS levels and MMP stability between sensitive and drug-resistant cells.

[0032] (3) Mitochondrial autophagy inhibition and drug sensitivity testing steps: Mdivi-1 was used as a mitophagy inhibitor. MCF7-R and T47D-R resistant cells were treated at a predetermined low-toxicity effective concentration. CCK8 susceptibility assays were performed before and after treatment to detect changes in TAMIC50 values, in order to assess the functional contribution of mitophagy to the drug resistance phenotype.

[0033] The implementation results show that: Figure 2 As shown in Figure A, the colocalization signal between mitochondria and lysosomes was significantly enhanced in drug-resistant cells, suggesting that the level of mitophagy was significantly higher than that in sensitive cells; transmission electron microscopy results ( Figure 2 B) shows that the number of autophagosomes and mitochondrial encapsulation structures is significantly increased in drug-resistant cells; such as Figure 2 As shown in C, the mtROS level of drug-resistant cells was lower, and MMP was more stable. Based on this, treatment with Mdivi-1 decreased the level of mitophagy, and the TAM susceptibility assay showed a significant decrease in the IC50 of drug-resistant cells. Figure 2 D、 Figure 2 E). The above results indicate that mitophagy is upregulated in ER-positive breast cancer TAM-resistant cells and promotes the drug-resistant phenotype by maintaining mitochondrial homeostasis.

[0034] 3.4 Example 3: Upregulation of PINK1 in TAM-resistant cells and its relationship with prognosis This embodiment provides a method for verifying the expression characteristics of PINK1 in TAM resistance and its relationship with patient prognosis from public databases and at the cellular level, including the following steps: (1) Steps for gene screening and prognostic analysis in public databases: a) Gene screening: Expression profiles and clinical information of breast cancer samples were obtained from TCGA data. Combined with the set of genes related to mitophagy, a prognostic model was constructed using LASSO regression. The key gene PINK1 (PINK1 is the initiator of mitophagy) which is closely related to prognosis was screened. b) Survival analysis: Kaplan-Meier overall survival (OS) analysis was performed based on PINK1 expression level to assess the relationship between high and low PINK1 expression and patient prognosis.

[0035] (2) Cellular expression verification steps: Total RNA and protein were extracted from MCF7-S / MCF7-R and T47D-S / T47D-R cells. The mRNA and protein expression levels of PINK1 were detected by RT-qPCR and Western blot, and the expression intensity and distribution of PINK1 in cells were observed by immunofluorescence staining.

[0036] The implementation results show that: Figure 3 As shown in Figure A, LASSO analysis identified PINK1 as a key gene with a high weight among mitophagy-related genes; survival analysis results ( Figure 3 B) shows that patients with high PINK1 expression had significantly worse overall survival (OS), suggesting that PINK1 is associated with poor prognosis; Figure 3 As shown in Figure C, the mRNA and protein levels of PINK1 in MCF7-R and T47D-R cells were significantly higher than those in their corresponding sensitive cells, and the immunofluorescence signal was also significantly enhanced. These results indicate that PINK1 is persistently highly expressed in TAM-resistant cells and is closely related to poor prognosis.

[0037] 3.5 Example 4: PINK1 promotes mitophagy in ER-positive breast cancer cells This embodiment provides a method for verifying the role of PINK1 in the regulation of mitophagy by regulating PINK1 expression levels, including the following steps: (1) Steps for constructing the PINK1 overexpression and knockdown model: MCF7 and T47D cells were infected with PINK1-overexpressing lentivirus and shPINK1 lentivirus, respectively, to screen for stable PINK1-overexpressing or knocked-down cell lines. The changes in PINK1 expression were verified by RT-qPCR and Western blot.

[0038] (2) Detection steps for mitophagy-related proteins: Western blot was used to detect the protein levels of LC3-II and TOM20 under different PINK1 expression backgrounds (while simultaneously verifying the corresponding protein expression levels in sensitive and drug-resistant cells). LC3-II was used as a biomarker of autophagy activity, and TOM20 was used as a mitochondrial outer membrane protein to assess changes in the expression of mitochondrial autophagy-related proteins.

[0039] (3) Immunofluorescence and confocal observation procedures: a) Use TOM20 and LC3 antibodies for immunofluorescence double staining, and observe the recruitment of LC3 on TOM20 positive mitochondria under a fluorescence microscope or confocal microscope to assess the degree of mitochondrial autophagy structure formation. b) Combine MitoTracker and LysoSensor double staining to observe the degree of co-localization between mitochondria and lysosomes, so as to reflect the changes in mitochondrial autophagy flux.

[0040] The implementation results show that: Figure 4 As shown in Figure A, PINK1 overexpression significantly increased LC3-II levels and decreased TOM20 protein abundance (the same trend was observed in drug-resistant cells), while PINK1 knockdown decreased LC3-II levels and increased TOM20 accumulation. Figure 4 B Figure 4 As shown in D, immunofluorescence double staining revealed a significant increase in LC3 and TOM20 co-localization structures when PINK1 was upregulated, while a decrease was observed when PINK1 was downregulated; confocal double staining results ( Figure 4 C) shows that PINK1 overexpression significantly enhances the co-localization of mitochondria and lysosomes, while PINK1 knockdown weakens this co-localization. These results indicate that PINK1 can promote mitophagy activation in ER-positive breast cancer cells, including LC3 transport, TOM20 degradation, and mitochondrial-lysosomal fusion.

[0041] 3.6 Example 5: PINK1 promotes resistance of ER-positive breast cancer cells to TAM This embodiment provides a method for verifying the effect of regulating PINK1 expression on TAM drug sensitivity, including the following steps: (1) Effect of PINK1 overexpression on TAM susceptibility: PINK1 overexpression models were constructed in MCF7 and T47D parental sensitive cells. Crystal violet and CCK8 susceptibility assays were performed under TAM treatment at predetermined concentration gradients. The effect of PINK1 upregulation on the TAM inhibitory effect was evaluated by comparing the number of residual cells, staining intensity, and IC50 value between the control group and the PINK1 overexpression group at the same TAM concentration.

[0042] (2) Effect of PINK1 knockdown on TAM susceptibility in drug-resistant cells: In established MCF7-R and T47D-R drug-resistant cells, PINK1 expression was knocked down. The crystal violet and CCK8 drug susceptibility tests were repeated to compare changes in cell survival and IC50 under TAM treatment and to assess the effect of reducing PINK1 on drug susceptibility of drug-resistant cells.

[0043] The implementation results show that: Figure 5 A, Figure 5 As shown in B, in PINK1-overexpressing sensitive cells, TAM treatment resulted in more cell retention and deeper staining with crystal violet, and CCK8 results showed an increased IC50 and a rightward shift in the drug inhibition curve; Figure 5 C Figure 5 As shown in Figure D, knockdown of PINK1 in MCF7-R and T47D-R cells resulted in significantly lighter crystal violet staining under TAM treatment, reduced cell viability, and a significantly decreased IC50. These results demonstrate that PINK1 is a key molecule promoting TAM resistance in ER-positive breast cancer cells, and its expression level is negatively correlated with TAM sensitivity.

[0044] 3.7 Example 6: Regulation of ROS and membrane potential by PINK1-mediated mitophagy under TAM drug stress This embodiment provides a method for verifying PINK1 function from the perspective of mitochondrial oxidative stress and membrane potential under TAM drug stress, including the following steps: (1) Detection of TAM-induced mtROS and membrane potential changes under different PINK1 expression backgrounds: a) PINK1 overexpression models were constructed in MCF7 and T47D cells. After treatment with the same concentration of TAM for a certain period of time, the mtROS level was detected using the MitoSOXRed probe and the mitochondrial membrane potential was detected using the TMRE probe. The results were then quantitatively analyzed by flow cytometry or fluorescence microscopy. b) PINK1 was knocked down in MCF7-R and T47D-R cells, and changes in mtROS and MMP were detected under the same TAM treatment conditions.

[0045] The implementation results show that: Figure 6 As shown in Figure A, in cells overexpressing PINK1, TAM-induced mtROS levels were significantly reduced, while membrane potential remained at a high level; Figure 6 As shown in Figure B, knockdown of PINK1 in drug-resistant cells significantly increased mtROS and decreased membrane potential under TAM treatment. These results indicate that PINK1 promotes mitophagy to clear drug-damaged mitochondria, reduces ROS accumulation, and maintains mitochondrial membrane potential stability, thereby providing mitochondrial functional support for the survival of ER-positive breast cancer cells under TAM drug stress.

[0046] As can be seen from the above embodiments, the technical solution of the present invention establishes a complete technical pathway for PINK1-mediated mitophagy to promote TAM resistance in ER-positive breast cancer from multiple levels, including cell model construction, pathway enrichment analysis, PINK1 expression and function verification, and mitochondrial function detection. It has the characteristics of clear structure, well-defined steps, and strong reproducibility, and can provide an implementable technical foundation for the study of related drug resistance mechanisms and the development of potential drug targets.

[0047] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of PINK1 as a biomarker in assessing tamoxifen resistance in ER-positive breast cancer cells, characterized in that, Drug resistance was assessed by detecting the expression level of PINK1 in the samples and combining it with mitophagy activity.

2. The application according to claim 1, characterized in that, The expression level of PINK1 includes mRNA level and / or protein level.

3. The application according to claim 1, characterized in that, The mitophagy activity was assessed using at least one of the following methods: (1) The degree of co-localization between mitochondria and lysosomes; (2) Changes in LC3-II levels; (3) Changes in TOM20 levels; (4) LC3-II / TOM20 correlation ratio or joint interpretation results.

4. A method for in vitro assessment of tamoxifen resistance in ER-positive breast cancer cells, characterized in that, Includes the following steps: S1: Detection of PINK1 expression levels in cells; S2: Detection of mitochondrial autophagy activity in cells; S3: Compare the test results with the control standard, where high expression of PINK1 and / or increased mitophagy activity indicate enhanced tamoxifen resistance.

5. The method according to claim 4, characterized in that, The activity of mitochondrial autophagy in S2 is achieved by the degree of co-localization of MitoTracker and LysoSensor double staining and / or the co-localization of TOM20 and LC3 immunofluorescence, and / or the detection of LC3-II and TOM20 by Western blot.

6. The use of PINK1 expression inhibition in the preparation of interventional agents that enhance the sensitivity of ER-positive breast cancer cells to tamoxifen, characterized in that, The PINK1 expression inhibition method is selected from shRNA, siRNA, or other nucleic acid methods that can reduce PINK1 expression or function.

7. Use of mitochondrial autophagy inhibitors in the preparation of interventional agents that enhance the sensitivity of ER-positive breast cancer cells to tamoxifen.

8. The use according to claim 7, characterized in that, The mitophagy inhibitor is Mdivi-1 or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical / intervention composition, characterized in that, It includes tamoxifen and PINK1 expression inhibitors and / or mitophagy inhibitors.

10. A method for establishing an ER-positive breast cancer tamoxifen-resistant cell model, characterized in that, Includes the following steps: S1: Select ER-positive breast cancer cell lines; S2: Long-term induction with incremental concentrations of tamoxifen in a culture medium containing tamoxifen; S3: Screening to obtain cell lines that can grow stably under the target concentration of tamoxifen; S4: Verify the drug resistance phenotype by crystal violet susceptibility testing and / or CCK8 susceptibility testing; S5: Detect the expression level of PINK1 and the activity of mitochondrial autophagy in the cell line to confirm phenotypic characteristics.

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