Application of 5, 6-EET as predictive marker of triple-negative breast cancer lung metastasis

Abstract

The invention provides an application of 5, 6-EET as a predictive marker of triple negative breast cancer pulmonary metastasis, and 5, 6-EET promotes triple negative breast cancer pulmonary metastasis. And 5, 6-EET promotes lung metastasis of triple-negative breast cancer by enhancing the permeability of pulmonary vessels. The 5, 6-EET promotes TNBC cells to generate pulmonary parenchyma metastasis by enhancing the permeability of mouse pulmonary vessels, and the discovery helps people to understand the scientific significance of arachidonic acid metabolism reprogramming in the TNBC pulmonary metastasis process, clarifies the molecular mechanism of 5, 6-EET metabolism in the TNBC pulmonary metastasis process, and provides a potential reference index for predicting the pulmonary metastasis risk.

Description

Technical Field

[0001] This invention belongs to the field of medicine, and in particular relates to the application of 5,6-EET as a predictive biomarker for lung metastasis in triple-negative breast cancer. Background Technology

[0002] Triple-negative breast cancer (TNBC) is the most aggressive and deadliest subtype of breast cancer, accounting for 15-20% of newly diagnosed cases. TNBC typically has a higher histopathological grade, is diagnosed at a younger age, progresses more rapidly, and has a worse prognosis. Metastatic TNBC (also known as stage IV) spreading beyond the breast to the lungs, bones, liver, or brain is a leading cause of death in TNBC patients. Like other tumors, breast cancer metastasis begins with cancer cells invading adjacent tissues, followed by dissemination; each step is a rate-limiting step in the successful formation of metastatic lesions. Compared to the permeability of the primary tumor's blood vessels, the capillaries in the lungs are much denser. Therefore, when TNBC cells become stagnant in the lungs, one obstacle is the endothelial structure of the pulmonary vessels. TNBC cells must actively disrupt the tight junctions between pulmonary endothelial cells to extravasate into the lung parenchyma. This process is mediated by multiple proteins; for example, angiopoietin-like 4 (ANGPTL4) has been shown to cause breast cancer cells to extravasate into the lung parenchyma by disrupting the endothelial layer. On the other hand, numerous studies have shown that metastatic breast cancer cells differ from primary breast cancer cells in their metabolic and molecular characteristics, a process known as metabolic reprogramming, including glucose metabolism, lipid metabolism, and amino acid metabolism. Furthermore, different metabolomes have been used to study organ-specific metastases. This is because once metastatic cancer cells reach secondary organs, they must adapt to and counteract the specific environment of each organ; therefore, the levels of these metabolites can serve as indicators for predicting the risk of metastasis to a specific organ.

[0003] Arachidonic acid (AA), also known as eicosapentaenoic acid (C20:4, ω-6), is a polyunsaturated fatty acid found in human cell membranes. It is typically esterified into glycerides or glycerophospholipids to maintain cell membrane structure and function. The synthesis of free AA involves phospholipase A2 (PLA2), which can be activated by neural effectors such as norepinephrine, angiotensin II, and bradykinin. AA metabolites can act as paracrine factors and second messengers, regulating lung, kidney, and cardiovascular function and inflammatory responses. AA produces various bioactive metabolites through three different enzymatic pathways: cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP). (1) The two main enzymes in the COX pathway, COX-1 and COX-2, generate five important prostanoids (PGs), such as PGD2, PGE2, PGF2, PGI2 and thromboxane (TXs), which play an important role in inflammation, pain and tumor progression. (2) The LOX pathway mainly involves 5-LOX, 8-LOX, 12-LOX and 15-LOX catalyzing the oxidation of AA to generate hydroperoxy-eicosatetraenoic acids (HPETEs), which are then converted into various leukotrienes (LTs), lipotoxins (LXs), hepoxillins (HOs) and hydroxyeicosatetraenoic acids (HETEs), causing inflammation, allergic reactions, bronchoconstriction and vasoconstriction. (3) The CYP pathway is an enzymatic pathway divided into two sub-pathways: the cyclooxygenase (EPO) pathway and the ω-hydroxylase (ω-Hydroxylase) pathway. The ω-Hydroxylase pathway produces 19-,20-hydroxyeicosatetraenoic acid (19-,20-HETE), which causes inflammation, vasoconstriction, and vascular remodeling. The EPO pathway mainly catalyzes the metabolism of endogenous amino acids into 5,6-,8,9-,11,12-, and 14,15-EET, and this reaction is mainly catalyzed by members of the P450 CYP2C gene subfamily. Among them, 11,12-EET has been identified as a pro-angiogenic molecule and, together with 14,15-EET, has been confirmed as a potent mitogen.However, the invasive and / or angiogenic and permeability properties of 5,6- and 8,9-EET and their potential mechanisms of action remain unclear.

[0004] TRPV4 channels on the plasma membrane have been reported to participate in maintaining pulmonary vascular endothelial calcium. 2+ TRPV4 channels maintain homeostasis and are activated by a variety of stimuli, including physical (voltage, temperature, and mechanical) and chemical (second messenger and neurohumoral) stimuli. Different research models indicate that due to the structural and functional differences between pulmonary capillary endothelium and pulmonary artery endothelium, the downstream targets and regulatory mechanisms involved in TRPV4 channels differ between the two structures. In pulmonary arterioles, TRPV4 channels mediate Ca2+... 2+ Influx occurs through mechanisms such as selective activation of endothelial nitric oxide synthase (eNOS) and promotion of NO release, dilating pulmonary arterioles and reducing resting pulmonary artery pressure. In pulmonary capillary endothelial cells, the activity of TRPV4 channels is primarily related to endothelial hyperpermeability, playing a crucial role in the alveolar-capillary barrier and leading to pulmonary edema and lung injury. The pulmonary microcirculatory endothelium is an important barrier protecting lung tissue from invasion by tumor cells, toxins, and pathogens in the bloodstream. However, when lung tissue is attacked by tumor cells, increased endothelial cell permeability promotes the migration of tumor cells and immune cells from the blood into the alveoli. Ultimately, tumor cells and protein-rich fluid accumulate in the alveolar interstitium, leading to life-threatening pulmonary space-occupying lesions and pulmonary edema. Therefore, activation of TRPV4 channels plays a vital role in the pulmonary arrest and extravasation of tumor cells into the lung parenchyma.

[0005] In 2017, researchers used a combination of molecular docking techniques and molecular dynamics simulations to identify an EET-binding pocket in the TRPV4 molecule that specifically mediates the binding of 5,6-EET to TRPV4. Furthermore, they structurally resolved that the K535 site of TRPV4 plays a crucial role in the activation of TRPV4 by 5,6-EET. The mutant TRPV4-K535A lost its ability to bind to 5,6-EET, while its response to GSK1016790A, 4α-phorbol 12,13-didecanoate, and thermal stimulation remained unaffected. Therefore, 5,6-EET-induced TRPV4 activation is due to its direct binding to the cleft of the S1-S4 helical segment of the TRPV4 subunit, while the K535 residue located on the S2-S3 linker plays a key role in stabilizing the ligand position. Summary of the Invention

[0006] In view of this, the present invention aims to overcome the deficiencies in existing diagnostic and treatment technologies and proposes the application of a lipid substance 5,6-EET as a predictive biomarker for lung metastasis in triple-negative breast cancer.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows: This invention provides the application of 5,6-EET in the preparation of predictive biomarkers for lung metastasis in triple-negative breast cancer. 5,6-EET promotes lung metastasis in triple-negative breast cancer; 5,6-EET promotes lung metastasis in triple-negative breast cancer by enhancing pulmonary vascular permeability.

[0008] Furthermore, 5,6-EET targets TRPV4 to enhance pulmonary vascular permeability.

[0009] Furthermore, 5,6-EET enhances the permeability of pulmonary vascular endothelial cells by binding to TRPV4 and activating its calcium ion channel activity.

[0010] Furthermore, 5,6-EET enhances the permeability of pulmonary vascular endothelial cells through the TRPV4-FAK / AKT-mTOR axis; 5,6-EET also enhances the permeability of pulmonary vascular endothelial cells by disrupting cell junctions and the normal distribution and function of cytoskeletal proteins through the TRPV4-FAK / AKT-mTOR axis.

[0011] Furthermore, the imbalance between PLA2 and EPHX2 levels promotes lung metastasis in triple-negative breast cancer by mediating the abnormal accumulation of 5,6-EET.

[0012] Furthermore, PLA2 knockout inhibited lung metastasis in triple-negative breast cancer by reducing 5,6-EET levels; EPHX2 overexpression inhibited lung metastasis in triple-negative breast cancer by reducing 5,6-EET levels.

[0013] Furthermore, the application of the 5,6-EET in the preparation of a predictive kit for lung metastasis in triple-negative breast cancer.

[0014] The present invention also provides the application of 5,6-EET in the preparation of a drug for treating lung metastases of triple-negative breast cancer.

[0015] This invention also provides the application of a TRPV4 inhibitor in the preparation of reagents for the treatment or prevention of lung metastasis in triple-negative breast cancer, wherein the TRPV4 inhibitor is GSK2798745.

[0016] This invention also provides the application of a TRPV4 inhibitor in the preparation of a drug for treating lung metastases of triple-negative breast cancer, wherein the TRPV4 inhibitor is GSK2798745, which inhibits lung metastases of triple-negative breast cancer.

[0017] Compared with the prior art, the present invention has the following advantages: The 5,6-EET described in this invention promotes lung parenchymal metastasis of TNBC cells by enhancing the permeability of pulmonary blood vessels in mice. 5,6-EET targets TRPV4 to enhance the permeability of pulmonary vascular endothelial cells and disrupts the tight / adhesive junction functional complex of endothelial cells through the TRPV4-FAK / AKT-mTOR axis. These findings help us to: 1) understand the scientific significance of arachidonic acid metabolic reprogramming during TNBC lung metastasis and elucidate the molecular mechanism of 5,6-EET metabolism in TNBC lung metastasis; 2) provide potential reference indicators for predicting lung metastasis risk; 3) utilize gene-level methods to regulate the levels of 5,6-EET-related enzymes to reduce 5,6-EET levels in breast cancer, providing potential technical reference for the prevention and treatment of breast cancer lung metastasis; and 4) utilize TRPV4 inhibitors to provide potential drug references for the treatment and prevention of breast cancer lung metastasis. Attached Figure Description

[0018] Figure 1 The LuM cell line described in this embodiment of the invention tends to metastasize to the lungs in vivo: Figure a shows the lung metastasis signal acquisition of mice after in situ mammary gland inoculation with LuM cell line; Figure b shows the statistical bar chart of fluorescence signal intensity in the chest of mice; Figure c shows that LuM cells mainly metastasize to the lungs of mice; Figure d shows that the lung surface nodules of mice in the LuM cell group are significantly increased, accompanied by pulmonary congestion and pulmonary edema; Figure e shows that the survival time of LuM mice is significantly shortened. Figure 2 The arachidonic acid metabolite derivatives described in this embodiment of the invention are associated with TNBC lung metastasis: Figure a shows the detection of LuM and MDA-MB-231 cell lines by high-resolution mass spectrometry metabolomics. 10 7 Cells were cultured in DMEM without phenol red and with 10% dialyzed serum. After 8 h, cells were collected, and metabolites were extracted using the -80℃ frozen methanol method and detected by LC-MS. Z-scores were calculated and heatmaps were plotted for differential metabolites (P-value < 0.05). Figure b shows the KEGG enrichment analysis of differential metabolites on the MetaboAnalyst website, arranged in descending order of significance. Figure c shows the comparison between LuM and MDA-MB-231 cell lines, and a horizontal volcano plot was plotted using metabolite peaks and P-values ​​(Log2FC threshold: 1, P-value < 0.05). Figure d shows the fold comparison of differential metabolites with elevated levels in LuM and MDA-MB-231, arranged in descending order. Figure 3 The elevated 5,6-EET synthase and decreased hydrolytic enzyme levels in LuM cells, as described in this embodiment of the invention, are associated with poor prognosis in TNBC: LuM or MDA-MB-231 cell lines were lysed with Trizol (10... 6The following data were compared using reference transcriptomics. Figure a shows a volcano plot combining adjusted p-value and fold change. Figure b is a schematic diagram of 5,6-EET as a downstream metabolite of arachidonic acid and related metabolic enzymes. Figure c shows the mRNA levels of PLA2G4A and EPHX2 in LuM and MDA-MB-231 cell lines detected by RT-PCR (*, P < 0.05). Figure d shows the protein levels of PLA2G4A and EPHX2 in LuM and MDA-MB-231 cell lines detected by Western blotting. Figure e shows the expression of PLA2G4A and EPHX2 in different breast cancer subtypes analyzed using the GOBO database. Figure f shows the relationship between PLA2G4A and EPHX2 expression and TNBC (Basal) recurrence-free survival rate analyzed using Kaplan-Meier online survival curve analysis software. Figure g shows the analysis of TCGA + GTE (Normal) and TCGA (Breast) recurrence-free survival rates. The database of cancer was used, and the expression correlation between PLA2G4A and EPHX2 was analyzed using the Pearson method. Figure h shows the targeted LC-MS detection of 5,6-EET in the plasma of TNBC patients and normal control women. Figure 4 The knockout of PLA2G4A or overexpression of EPHX2 described in this embodiment of the invention can inhibit LuM lung metastasis: Figure a shows the detection of PLA2G4A knockout in LuM cells by Western blotting; Figure b shows the detection of corresponding intracellular metabolite levels by targeted metabolic mass spectrometry; Figure c shows the orthotopic injection of PLA2G4A knockout LuM cell lines into the mammary glands of NCG mice, followed by tumor harvesting for weighing and diameter measurement; Figure d shows the observation and H&E staining of mouse lung tissue under a microscope; Figure e shows the microscopic counting of the number of lung metastases; Figure f shows the detection of EPHX2 overexpression in stable screening cell lines by Western blotting; Figure g shows the detection of corresponding intracellular metabolite levels by targeted metabolic mass spectrometry; Figure h shows the orthotopic injection of stable EPHX2 overexpressing LuM cell lines into the mammary glands of NCG mice, followed by tumor harvesting for weighing and diameter measurement; Figure i shows the observation and H&E staining of mouse lung tissue. Stained microscopic images were taken. Figure j shows the number of lung metastases in LuM after EPHX2 overexpression, as counted under the microscope. Figure k shows the survival curves of mice. Figure 5The following is an embodiment of the invention illustrating how 5,6-EET promotes TNBC lung metastasis in vivo: Figure a shows mice with orthotopic tumor cells 14 days after cell line formation, with plasma collected for LC-MS detection of 5,6-EET levels; Figure b shows MDA-MB-231 orthotopic tumor mice after intraperitoneal injection of 5,6-EET 2 h later, with plasma 5,6-EET levels detected by LC-MS; Figure c shows MDA-MB-231 tumor mice after intraperitoneal injection of 5,6-EET every other day, with in vivo imaging of the small animals; Figure d shows the statistical analysis of tumor fluorescence intensity; Figure e shows the removal of mouse tumors for weighing and diameter measurement; Figure f shows the statistical analysis of fluorescence intensity in the mouse chest; Figure g shows the removal of mouse lungs, liver, and spleen for fluorescence collection; Figure h shows the statistical analysis of fluorescence signal intensity in lung tissue; Figure i shows the observation of mouse lung tissue and H&E staining under a microscope; Figure j shows the weighing of the lungs; Figure k shows the microscopic counting of the number of metastatic lesions in the lungs; Figure l shows the plotting of mouse survival curves. Figure 6 The following is an example of 5,6-EET-induced pulmonary vascular permeability in mice with MDA-MB-231 cells undergoing orthotopic mammary tumor formation, followed by rhodamine-dextran injection via tail vein and fluorescence acquisition. Figure b shows in vivo red fluorescence imaging of mice at different time points after rhodamine-dextran tail vein injection. Figure c shows the statistical analysis of red fluorescence signal intensity in the chest. Figure d shows red fluorescence imaging of excised lung tissue. Figure e shows the fluorescence intensity of rhodamine-dextran in the lung parenchyma observed in frozen sections. Figure f shows the statistical analysis of fluorescence intensity in mouse lung tissue. Figure g shows the vascular endothelial region indicated by CD34 in lung tissue, where the intensity of the cell tight junction core protein ZO-1 is significantly weakened. Figure h shows the comparison of the relative fluorescence intensity of ZO-1 to CD34. Figure 7 To illustrate the enhancement of pulmonary vascular endothelial cell permeability by targeting TRPV4 with 5,6-EET as described in this embodiment of the invention: Figure a shows a schematic diagram and result analysis of a red fluorescent dextran penetration assay performed after direct incubation with 1 μM 5,6-EET, or after incubating a monolayer of HULEC-5 cells with TNBC cells in culture medium for 48 hours; Figure b shows the Western blotting detection of TRPV4 protein levels in endothelial cells after knockout and TRPV4 replenishment, performed after direct incubation of HULEC-5 cells with 1 μM 5,6-EET; Figure c shows the immunofluorescence assay detecting changes in TRPV4 localization (DAPI: cell nucleus); Figure d shows the detection of intracellular Ca2+. 2+ Concentration changes (expressed as normalized Fluo-8,AM ratios): Figure e shows the green fluorescence intensity of Fluo-8,AM observed under a microscope at 3 seconds; Figure f shows the red fluorescent dextran penetration assay detecting the permeability of TRPV4-mutated monolayer endothelial cells, performed after endothelial cells were treated with 5,6-EET or TNBC cells for 48 hours; Figure g shows intracellular Ca...2+ Concentration changes (expressed as normalized Fluo-8,AM ratio), Figure h shows the green fluorescence intensity of Fluo-8,AM in cells observed under a microscope at 3 seconds, and Figure i shows the red fluorescent dextran penetration experiment; Figure 8 The 5,6-EET described in this embodiment of the invention disrupts the tight / adhesive junctions between pulmonary vascular endothelial cells via the TRPV4-FAK / AKT-mTOR axis: Figure a shows 5,6-EET. The results of phosphorylated proteomics microarrays after treating pulmonary vascular endothelial cells are as follows: Figure b shows the intensity of the microarray sites corresponding to FAK / AKT-mTOR-P70S6K axis-related proteins; Figure c shows the statistical comparison of the phosphorylation intensity of corresponding proteins in the pathway; Figure d shows the Western blot (WB) experiment using specific antibodies; Figure e shows the WB experiment performed after incubating endothelial cells with 5,6-EET or different tumor cell sources for 48 hours and then incubating for 24 hours; Figure f shows the detection of TRPV4 point mutation in endothelial cells; Figure g shows the immunofluorescence detection of F-actin labeled with phalloidin; Figure h shows that treatment of endothelial cells with the TRPV4 inhibitor GSK2798745 weakened the effect of 5,6-EET on FAK / AKT-mTOR-P70S6K axis activation and the reduction of connective protein levels; Figure i shows that GSK2798745 inhibited the disruptive effect of 5,6-EET on the normal distribution of F-actin. Figure 9 To illustrate the inhibition of TNBC lung metastasis by the TRPV4 inhibitor GSK2798745 described in this embodiment of the invention: Figure a shows the administration of GSK2798745 to LuM orthotopic tumor-bearing mice via gavage every other day, followed by in vivo imaging of the small animals 45 days later; Figure b shows the statistical analysis of tumor fluorescence intensity; Figure c shows the statistical analysis of chest fluorescence intensity; Figure d shows the observation of fluorescence in the lungs, liver, and spleen of mice; Figure e shows the statistical analysis of lung tissue fluorescence intensity; Figure f shows the observation and H&E staining of mouse lungs; Figure g shows the statistical analysis of the number of metastatic lesions in the lungs; and Figure h shows the plotting of mouse survival curves. Detailed Implementation

[0019] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0020] The present invention will be described in detail below with reference to the embodiments.

[0021] Example 1: LuM tends to metastasize to the lungs in vivo. To further explore the molecular mechanisms of lung metastasis in TNBC, we introduced a TNBC-prone cell line, LuM-Luciferase (LuM), and its original cell line, ATCC MDA-MB-231-Luciferase (MDA-MB-231). We first validated the lung metastasis characteristics of this cell line. Severely immunodeficient mice (NOD / ShiltJGpt-Prkdcem26Cd52Il2rgem26Cd22 / Gpt, NCG) were used as animal models for in situ mammary tumorigenesis experiments. The above cell lines were injected into the right mammary fat pad of the fourth pair of female NCG mice at 6 weeks of age. Every 10 days, the growth and metastasis of mammary tumors were detected using an IVIS small animal in vivo imaging system. It was observed that, compared with the original MDA-MB-231 cells, the lung metastasis signal in LuM cell line mice gradually increased with prolonged cell seeding time. Figure 1 a- Figure 1 (b) The growth rate of the orthotopic tumor also increased slightly. Fluorescence imaging of various mouse organs confirmed that the original MDA-MB-231 cells could metastasize to multiple organs, including the lungs, liver, and intestines, while LuM cells mainly metastasized to the mouse lungs (b). Figure 1 c). Statistical analysis of metastatic nodules on the lung surface confirmed a significant increase in lung surface nodules in the LuM cell group mice, accompanied by pulmonary congestion and pulmonary edema. Figure 1 d), compared with the original MDA-MB-231 cell line, the lifespan of LuM mice was significantly shortened ( Figure 1 e).

[0022] Example 2: Arachidonic acid metabolites are associated with TNBC lung metastasis. To further investigate the metabolic reprogramming mechanism during lung metastasis, we used high-resolution mass spectrometry metabolomics to compare the LuM cell line with MDA-MB-231, detecting 37 upregulated and 25 downregulated metabolites. Figure 2 a). KEGG enrichment analysis of differential metabolites revealed that the arachidonic acid metabolic pathway showed the most significant changes. Figure 2 b). Simultaneously, a significance analysis of the changes in differentially metabolites revealed significantly elevated levels of four metabolites in the arachidonic acid pathway: arachidonic acid (AA), prostaglandin E2 (PGE2), 5,6-epoxyeicosatrienoic acid (5,6-EET), and 11(R)-hydroxyeicosatetraenoic acid (11(R)-HETE). Figure 2 c). We compared the abundance of metabolites with MDA-MB-231 and found that PGE2, 5,6-EET, and 11(R)-HETE were the top three metabolites in terms of fold increase. Figure 2 d). The above results confirm that the arachidonic acid metabolic pathway differs significantly between lung metastases of LuM cell lines and their original cells, and that four metabolites are significantly accumulated.

[0023] Example 3: Elevated 5,6-EET synthase and decreased hydrolase levels are associated with poor prognosis in TNBC. To further investigate the regulatory mechanism of this abnormal accumulation, we first performed a transcriptomic comparison between the two cell lines. We found that compared to the original MDA-MB-231 cell line, the mRNA level of arachidonic acid synthase-phospholipase G4A (Group IVA phospholipase A 2, PLA2G4A, also known as PLA2) was significantly increased in LuM cells, while the level of the 5,6-EET downstream metabolic enzyme epoxide hydrolase 2 (EPHX2) was significantly decreased. Figure 3 a- Figure 3 b).

[0024] At the same time, the levels of the PGE2 synthases, prostaglandin-endoperoxide synthase 2 (PTGS2) and prostaglandin E synthase (PTGES), were also increased. Figure 3 a- Figure 3 (b) Since the function of PGE2 has been previously confirmed by researchers through numerous experiments—activating receptors such as EP2 / 4 and EGFR, and downstream pathways such as MAPK / Erk and mTORC1, thereby promoting angiogenesis, tumor cell proliferation, and metastasis—we focused our attention on the pro-lung metastasis mechanism of 5,6-EET.

[0025] We used RT-PCR and WB experiments to confirm that, at both the mRNA and protein levels, PLA2G4A levels were increased and EPHX2 levels were significantly decreased in LuM cells. Figure 3 c- Figure 3 d). The above results confirm that during TNBC lung metastasis, the expression of 5,6-EET synthase is enhanced, while the expression of downstream hydrolases is reduced.

[0026] We used clinical databases to conduct a comprehensive analysis of the relationship between the expression of these two enzymes and breast cancer, particularly TNBC. Using the Gene Expression-based Outcome for Breast Cancer Online (GOBO) tool, we analyzed four breast cancer subtypes and found that PLA2G4A expression was highest in the Basal-like (TNBC) subtype, while EPHX2 expression was lowest. Figure 3 e). Furthermore, analysis of the TCGA database using the Kaplan-Meier website revealed that in the Basal-like (TNBC) subtype, high expression of PLA2G4A and low expression of EPHX2 were associated with poor patient prognosis. Figure 3 f). More importantly, using the Gene Expression Profiling Interactive Analysis (GEPIA) website, we performed expression correlation analysis on the two genes in the TCGA and GTEx databases. We found that PLA2G4A and EPHX2 expression were positively correlated in normal breast tissue, while they were negatively correlated in breast cancer tissue. Figure 3 g). This result suggests from a molecular functional perspective that in normal breast tissue, PLA2G4A and EPHX2 work synergistically to mediate the normal synthesis and metabolism of 5,6-EET. However, in breast cancer, their functions are imbalanced, leading to 5,6-EET metabolic disorders, which ultimately play an important role in the malignant progression of breast cancer. Finally, targeted liquid chromatography-tandem mass spectrometry (LC–MS / MS) metabolomics analysis of human plasma samples showed that the 5,6-EET level in patients with triple-negative breast cancer (TNBC) was significantly higher than that in the control group of healthy women. Figure 3 This confirms a clinical association between the accumulation of 5,6-EET and the progression of triple-negative breast cancer.

[0027] Example 4: Knockout of PLA2G4A or overexpression of EPHX2 both inhibited LuM lung metastasis. To further investigate the effect of 5,6-EET on lung metastasis in TNBC cells, we constructed LuM cell lines with PLA2G4A knockout or stable overexpression of EPHX2. In vitro tumorigenesis experiments were performed on these cell lines, and the results confirmed that PLA2G4A knockout not only reduced the levels of PGE2, 5,6-EET, and 11(R)-HETE, but also slowed the rate of TNBC in situ tumorigenesis and inhibited lung metastasis. Figure 4 a- Figure 4 e). After overexpression of EPHX2, the intracellular level of 5,6-EET was significantly reduced ( Figure 4 f- Figure 4g). In mice, although it had no significant effect on the in situ tumor growth rate ( Figure 4 However, it significantly inhibited the metastasis of LuM cells to the lungs, and the number of lung metastatic lesions was significantly reduced (h). Figure 4 i- Figure 4 j). Compared with the empty control cells, the survival time of mice overexpressing EPHX2 was significantly prolonged ( Figure 4 The above experiments confirm that knockout of PLA2G4A or overexpression of EPHX2 both reduce cellular 5,6-EET levels and significantly inhibit TNBC lung metastasis.

[0028] Example 5: 5,6-EET promotes TNBC lung metastasis in vivo To directly confirm the lung metastasis-promoting effect of 5,6-EET, we used a standard to quantify the level of 5,6-EET in mouse plasma by mass spectrometry. Figure 5 a) MDA-MB-231 cell orthotopic tumor mice were injected intraperitoneally with 5,6-EET ( Figure 5 b). After 40 days, it was confirmed that 5,6-EET significantly enhanced the lung metastasis ability of cells. Figure 5 c), but it had no significant effect on the in situ growth rate of the tumor. Figure 5 d- Figure 5 e). Significantly enhanced fluorescence signal in the lungs ( Figure 5 c, Figure 5 f- Figure 5 h) The weight of the lungs increased significantly. Figure 5 j), the number of lung metastases increased significantly ( Figure 5 i, Figure 5 k). Ultimately, the lifespan of the mice was significantly shortened ( Figure 5 l). The above experiments confirm that 5,6-EET levels can significantly promote TNBC lung metastasis.

[0029] Example 6: 5,6-EET-induced pulmonary vascular hyperpermeability Since 5,6-EET has been reported to be structurally capable of binding and promoting Ca2+, 2+ The displacement and activation of the TRPV4 channel were investigated, and TRPV4 is involved in increasing vascular permeability in pulmonary capillary endothelial cells, promoting pulmonary edema and lung injury. Therefore, we hypothesize that TNBC cell 5,6-EET can enhance pulmonary microvascular permeability and promote TNBC cell lung metastasis by activating TRPV4 in pulmonary microvascular endothelial cells.

[0030] To verify this hypothesis, we first examined the effect of 5,6-EET on pulmonary vascular endothelial permeability. Using red fluorescent dextran, we measured the fluorescence intensity and reaction of 5,6-EET into the lung parenchyma, and determined the permeability of pulmonary vascular endothelium. Mice with 231 cell orthotopic tumors were injected with 5,6-EET for 14 days. Figure 6 a). The results confirmed that vascular permeability was generally increased throughout the mice, especially in the lungs ( Figure 6 b- Figure 6 c). A significant increase in red fluorescence was observed in the lungs, particularly in the lung parenchyma. Figure 6 d- Figure 6 f). Furthermore, in lung tissue, the strength of the cell tight junction core protein ZO-1 was significantly weakened in the CD34-indicated vascular endothelial region. Figure 6 g- Figure 6 This result confirms that 5,6-EET can enhance pulmonary vascular permeability.

[0031] Example 7: 5,6-EET targets TRPV4 to enhance the permeability of pulmonary vascular endothelial cells. We first seeded endothelial cells into a monolayer, added red fluorescent dextran to the chambers, and detected the fluorescence intensity as it penetrated the intercellular spaces into the lower chambers, reflecting the permeability of substances between endothelial cells. Figure 7 a). The results confirmed that 5,6-EET enhanced glucan permeability over time. LuM also enhanced glucan permeability compared to primitive cells. Figure 7 a). The above results indicate that 5,6-EET can promote the permeability of substances by reducing the connections between endothelial cells.

[0032] We used gene knockout and protein replenishment techniques to construct a mutant endothelial cell line TRPV4-K535A with a 5,6-EET binding site to TRPV4. Figure 7 b). After adding 5,6-EET to the cell culture medium for 5 minutes, the localization of TRPV4 was detected. It was found that WT-type TRPV4 showed a shift towards the cell membrane, while the TRPV4 binding site mutant did not show a significant shift. Figure 7 c). Detection of Ca in endothelial cells 2+ The flow was found to produce a Ca within one minute of adding 5,6-EET. 2+ The peak value of the flow, while TRPV4 showed no significant change after being mutated ( Figure 7 d- Figure 7 e). A chamber red fluorescent dextran penetration assay confirmed that after a mutation in the binding site of TRPV4 to 5,6-EET, 5,6-EET lost its enhancing effect on endothelial cell permeability. Figure 7 f).

[0033] As of 2020, more than 20 TRPV4 antagonists have been used to treat cardiogenic respiratory diseases, gastrointestinal disorders, pain, and tumors. Among them, GSK2798745 entered Phase I clinical trials in 2019. Our treatment of endothelial cells with GSK2798745 also showed that it could neutralize the activating effect of 5,6-EET on TRPV4. Figure 7 g- Figure 7 h), and at the same time, it also loses its effect of enhancing endothelial cell permeability ( Figure 7 i).

[0034] The results above indicate that 5,6-EET must bind to TRPV4 in endothelial cells to activate their calcium ion channel activity in order to exert its effect on promoting endothelial cell permeability.

[0035] Example 8: 5,6-EET disrupts the tight / adhesive junction function of endothelial cells via the TRPV4-FAK / AKT-mTOR axis. Since TRPV4 acts as a calcium ion channel, it primarily mediates the influx of Ca2+. 2+ As a second messenger, it initiates specific cascade signals, thereby regulating complex physiological processes such as gene expression, cell proliferation, and differentiation. To further clarify the specific molecular mechanism by which 5,6-EET activates TRPV4 and regulates endothelial permeability, we performed a phosphorylated protein antibody microarray screening. The results showed that after endothelial cells were treated with 5,6-EET, FAK / AKT-mTOR-P70S6K axis-related proteins were activated (…). Figure 8 a- Figure 8 c). Western blotting experiments using specific antibodies confirmed elevated phosphorylation levels of these proteins, and decreased levels of tight junction marker ZO-1 and adhesion junction marker VE-cadherin, with this change being 5,6-EET-dependent. Figure 8 d). After incubating endothelial cells with 5,6-EET or different tumor cell sources for 48 hours, Western blot experiments confirmed that these proteins were activated accordingly. Figure 8 e), and endothelial cells incubated with culture medium containing LuM cells overexpressing EPHX2 showed decreased phosphorylation levels (e). Figure 8 e). The above results indicate that 5,6-EET can activate the FAK / AKT-mTOR-P70S6K signaling axis in endothelial cells and reduce the levels of tight / adhesion junction-related proteins. When we used TRPV4 point-mutated endothelial cells for testing, we found that neither 5,6-EET nor tumor cell-derived culture media could activate this pathway. Figure 8 f).

[0036] Stably polymerized F-actin forms functional complexes with tight junctions (TJs) and adhesion junctions (AJs), creating linearly distributed "intercellular junctional zones" in the marginal contact regions between vascular endothelial cells. These complexes collectively maintain vascular barrier function and overall stability, preventing vascular leakage. Using phalloidin-labeled F-actin for immunofluorescence detection, we found that 5,6-EET disrupts the normal distribution of F-actin, while TRPV4 point mutations eliminate this function. Figure 8 g). Similarly, treatment of endothelial cells with the TRPV4 inhibitor GSK2798745 not only weakened the effect of 5,6-EET on FAK / AKT-mTOR-P70S6K axis activation and the reduction of connexin levels ( Figure 8 h), and also inhibited the disruptive effect of 5,6-EET on the normal distribution of F-actin ( Figure 8 i).

[0037] The above experiments demonstrate that 5,6-EET activates TRPV4, which in turn activates the downstream FAK / AKT-mTOR-P70S6K cascade signaling, disrupting the normal distribution and function of cell junctions, cytoskeletal proteins, etc., and ultimately damaging the barrier function of pulmonary vascular endothelial cells, leading to increased vascular permeability. This elucidates the molecular mechanism by which 5,6-EET promotes TNBC lung metastasis.

[0038] Example 9: TRPV4 inhibitor GSK2798745 inhibits TNBC lung metastases To further explore the potential application value of TRPV4 inhibitors in the lung metastasis process of triple-negative breast cancer, we used a TNBC orthotopic tumorigenesis mouse model to test the therapeutic effect of the inhibitor. The results confirmed that oral administration of the inhibitor significantly reduced the TNBC lung metastasis rate. Figure 9 a), but it has no significant effect on the growth of tumors in situ ( Figure 9 b). The intensity of lung fluorescence was significantly reduced ( Figure 9 c- Figure 9 e), the number of lung metastases was significantly reduced ( Figure 9 f- Figure 9 g). Ultimately, the lifespan of the mice was also extended to some extent ( Figure 9 h). The above results indicate that TRPV4 inhibitors have a potential inhibitory effect on lung metastasis in triple-negative breast cancer.

[0039] In summary, during TNBC lung metastasis, not only do tumor cells themselves undergo metabolic reprogramming to adapt to the different microenvironments at each stage, but corresponding changes also occur when lung tissue is invaded, ultimately leading to successful tumor cell metastasis. Therefore, to further explore the metabolic changes during TNBC lung metastasis and whether differentially metabolized metastases have potential value in assessing lung metastasis risk, we introduced a lung metastasis TNBC cell line, LuM-4175-Luciferase (LuM), and its original cell line. We found that arachidonic acid metabolism was significantly altered in LuM cells, and 5,6-EET levels were significantly elevated. We also found significantly elevated 5,6-EET levels in the plasma of TNBC patients compared to normal female plasma. The mRNA and protein levels of the synthase PLA2G4A were significantly increased in LuM cells; while the level of the 5,6-EET hydrolase EPHX2 was significantly decreased. Unlike normal breast tissue, PLA2G4A and EPHX2 expression are negatively correlated in breast cancer, and their levels have a completely opposite relationship with TNBC prognosis. This corroborates our findings that TNBC lung metastasis is closely related to the accumulation of 5,6-EET levels caused by abnormal arachidonic acid metabolism. Knocking out PLA2G4A or overexpressing EPHX2 in LuM cells reduced intracellular and extracellular 5,6-EET levels, significantly inhibiting the lung metastasis tendency of LuM cells in vivo and prolonging mouse survival. More importantly, 5,6-EET not only promoted the translocation and activation of TRPV4 to the cell membrane in human lung microvascular endothelial cells HULEC-5a in vitro, but also reduced the tight junctions and transendothelial activity of HULEC-5a cells. Furthermore, in TNBC model mice, 5,6-EET enhanced pulmonary vascular permeability and promoted lung parenchymal metastasis of TNBC cells. These findings help us understand the scientific significance of arachidonic acid metabolic reprogramming during TNBC lung metastasis, elucidate the molecular mechanism of 5,6-EET metabolism in TNBC lung metastasis, and provide a potential reference indicator for predicting the risk of lung metastasis.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

The application of 1,5,6-EET in the preparation of predictive biomarkers for lung metastasis in triple-negative breast cancer, characterized by: 5,6-EET promotes lung metastasis in triple-negative breast cancer; 5,6-EET promotes lung metastasis in triple-negative breast cancer by enhancing pulmonary vascular permeability.

2. The application of 5,6-EET according to claim 1 in the preparation of predictive biomarkers for lung metastasis in triple-negative breast cancer, characterized in that: 5,6-EET targets TRPV4 to enhance pulmonary vascular permeability.

3. The application of 5,6-EET according to claim 2 in the preparation of predictive biomarkers for lung metastasis in triple-negative breast cancer, characterized in that: 5,6-EET enhances the permeability of pulmonary vascular endothelial cells by binding to TRPV4 and activating its calcium ion channel activity.

4. The application of 5,6-EET according to claim 1 in the preparation of predictive biomarkers for lung metastasis in triple-negative breast cancer, characterized in that: 5,6-EET enhances the permeability of pulmonary vascular endothelial cells through the TRPV4-FAK / AKT-mTOR axis; 5,6-EET also enhances the permeability of pulmonary vascular endothelial cells by disrupting cell junctions and the normal distribution and function of cytoskeletal proteins through the TRPV4-FAK / AKT-mTOR axis.

5. The application of 5,6-EET according to claim 1 in the preparation of predictive biomarkers for lung metastasis in triple-negative breast cancer, characterized in that: Imbalances in PLA2 and EPHX2 levels promote lung metastasis in triple-negative breast cancer by mediating abnormal accumulation of 5,6-EET.

6. The application of 5,6-EET according to claim 5 in the preparation of predictive biomarkers for lung metastasis in triple-negative breast cancer, characterized in that: Knocking out PLA2 inhibits lung metastasis in triple-negative breast cancer by reducing 5,6-EET levels; overexpression of EPHX2 inhibits lung metastasis in triple-negative breast cancer by reducing 5,6-EET levels.

7. The application of 5,6-EET according to claim 1 in the preparation of predictive biomarkers for lung metastasis in triple-negative breast cancer, characterized in that: The application of the 5,6-EET in the preparation of a predictive kit for lung metastasis in triple-negative breast cancer. Application of 8,5,6-EET in the preparation of drugs for treating lung metastases of triple-negative breast cancer.

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