Application of SHC3 as diagnosis and prognosis marker and treatment target of lung metastasis of breast cancer

Abstract

The invention discloses application of SHC3 as a diagnosis and prognosis marker and a treatment target of lung metastasis of breast cancer. The research shows that the expression of SHC3 in TNBC lung metastatic cells is remarkably up-regulated, and the up-regulation is closely related to the prognosis of TNBC patients; the increase of SHC3 expression is positively correlated with the migration ability of the TNBC cells, which proves that the SHC3 gene is obviously up-regulated in the TNBC cells of pneumophilic metastasis and can be used as a biomarker of lung metastasis of breast cancer; researches show that silence of SHC3 can significantly weaken the metastasis ability of TNBC and reduce the number of pulmonary metastasis nodules, overexpression of SHC3 can enhance the metastasis activity of TNBC, and SHC3 can be used as a therapeutic target of breast cancer pulmonary metastasis; the invention provides a new marker for prognosis of breast cancer and diagnosis of lung metastasis of breast cancer, and provides a new target and treatment strategy and direction for treatment of lung metastasis of breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of SHC3 as a diagnostic and prognostic marker and therapeutic target for breast cancer lung metastasis. Background Art

[0002] Breast cancer (BC) is the most common malignant tumor in women worldwide. Among breast cancer subtypes, triple-negative breast cancer (TNBC) is the most aggressive and has the worst prognosis. Over 90% of deaths in TNBC patients are related to organ metastasis, with the lungs being a common target. However, the mechanisms of lung metastasis in TNBC, particularly the key factors driving this process, remain largely unknown.

[0003] Src Homology 2 Domain-Containing Transforming Protein 3 (SHC3) is an adaptor protein of the SHC family. As an adaptor protein, SHC3 plays a key role in multiple signal transduction networks through its N-terminal phosphotyrosine-binding domain and C-terminal SH2 domain. However, the role of SHC3 in TNBC lung metastasis has been rarely reported.

[0004] During tumor progression, the metabolic characteristics and preferences of tumor tissue undergo reprogramming, involving changes in multiple metabolic pathways to adapt to the rapid growth and proliferation needs of tumor cells. In in situ tumors, the most classic metabolic pathways are glycolysis (Warburg effect) and glycogen metabolism. These metabolic reprogramming processes provide energy and biosynthetic precursors for cancer cell proliferation and survival. Glycolysis (Warburg effect) is a core characteristic of cancer cell metabolism. It refers to the fact that even in the presence of sufficient oxygen, cancer cells still preferentially use glycolysis to rapidly break down glucose to produce lactate, rather than relying on mitochondrial oxidative phosphorylation. Its mechanisms include: rapid production of ATP to meet the energy needs of proliferation; provision of intermediate metabolites (such as glucose-6-phosphate) for nucleic acid and lipid synthesis; and lactate accumulation that acidifies the microenvironment, inhibiting immune cell function and promoting angiogenesis. This metabolic reprogramming confers a growth advantage on cancer cells, making it an important target for tumor diagnosis (such as FDG-PET imaging) and treatment.

[0005] Metastatic tumor cells have metabolic characteristics distinct from those of primary tumor cells. Metabolic signatures of metastatic tumor cells include increased glycolysis and altered glutamine and fatty acid metabolism. These changes support rapid tumor growth and metastasis. Therefore, identifying metabolic nodes in tumor cells is a potential avenue for targeted therapy in patients with malignant tumors. Metabolites play multiple supporting roles in tumor growth, which can be summarized as follows: First, metabolites provide tumor cells with energy and biosynthetic precursors. Through metabolic pathways such as glycolysis and the TCA cycle, tumor cells generate adenosine triphosphate (ATP), nicotinamide adenine dinucleotide phosphate (NADPH), and precursors necessary for the biosynthesis of nucleotides, lipids, and amino acids, meeting the energy and material requirements for their rapid proliferation. For example, pyruvate is converted to acetyl-CoA by pyruvate dehydrogenase, which then participates in the TCA cycle, providing tumor cells with energy and metabolic intermediates. Second, metabolites help maintain redox homeostasis in tumor cells. For example, glutathione produced by glutamine metabolism can replenish the mitochondrial NADPH pool and inhibit the generation of reactive oxygen species (ROS), thereby enhancing the resistance of tumor cells to oxidative stress and promoting their survival and proliferation. Furthermore, some metabolites act as signaling molecules to regulate gene expression. Metabolites such as α-KG and acetyl-CoA can act as signaling molecules to regulate gene expression patterns by affecting epigenetic regulatory mechanisms such as histone modification, thereby promoting the proliferation and survival of tumor cells. Finally, metabolites are also involved in promoting the immune escape of tumor cells. For example, lactic acid acidifies the tumor microenvironment and inhibits the functional activity of immune cells, thereby helping tumor cells evade the body's immune surveillance.

[0006] Existing studies have shown that metastatic tumor foci often exhibit higher metabolic activity, with an enhanced ability to uptake nutrients such as glucose, amino acids, and fatty acids compared to primary tumors. This is because metastatic tumor cells need to adapt to the new microenvironment and rapidly proliferate to establish new tumor lesions. Furthermore, metastatic tumor cells exhibit increased metabolic flexibility, enabling them to switch metabolic pathways based on the availability of surrounding nutrients, prioritizing the use of the most abundant energy sources. The TCA cycle, a core pathway of cellular energy metabolism, exhibits particularly pronounced variations in metastatic tumor foci, making it a hot topic in current tumor metabolism research. Compared to primary tumors or normal tissues, metastatic tumor cells often exhibit enhanced TCA cycle flux to accommodate the higher energy and biosynthetic demands of their rapid proliferation and migration. This enhanced flux may be attributed to upregulated expression or activity of key enzymes, such as citrate synthase and isocitrate dehydrogenase, which play a crucial role in regulating TCA cycle rate. Furthermore, the accumulation of TCA cycle intermediates is also a significant phenomenon in metastatic tumors. For example, the specific accumulation of intermediates such as succinate and fumarate not only disrupts the normal functioning of the TCA cycle but also acts as a key signaling molecule, regulating various biological behaviors of tumor cells. Succinate accumulation, in particular, can inhibit prolyl hydroxylase activity and stabilize hypoxia-inducible factor, thereby promoting the expression of genes associated with tumor angiogenesis and metastasis, providing strong support for tumor malignant progression. Furthermore, the coupling between the TCA cycle and other metabolic pathways in metastatic tumors has become increasingly close and complex. On the one hand, the TCA cycle's connection with glutamine metabolism is significantly strengthened. Glutamine is converted to α-KG through transamination and enters the TCA cycle, providing tumor cells with an additional energy source and biosynthetic precursors. On the other hand, the interaction between the TCA cycle and fatty acid metabolism also changes during tumor metastasis. Acetyl-CoA produced by fatty acid oxidation enters the TCA cycle more frequently, providing essential energy and biochemical support for tumor cell growth and migration. In summary, the differential expression of the TCA cycle in metastatic tumors and its biological significance cannot be ignored. In-depth exploration of these differences and the molecular mechanisms behind them will not only help us better understand the complexity of tumor metabolic regulation, but also provide new ideas and targets for developing effective treatment strategies for metastatic tumors.

[0007] Adenosine Monophosphate Activated Protein Kinase α (AMPKα) is a key energy sensor that can keenly sense changes in intracellular energy status, primarily relying on relative changes in the intracellular concentrations of adenosine phosphates (AMP, ADP, and ATP). Under normal conditions of sufficient energy, intracellular ATP levels are high, while AMP and ADP levels are relatively low. However, when cells experience energy stress, such as hypoxia, nutrient deprivation, or exercise, ATP consumption increases while its production is restricted, leading to a rapid increase in intracellular AMP and ADP concentrations. AMPKα is a heterotrimer composed of three subunits: α, β, and γ. The γ subunit contains four cystathionine-β-synthase (CBS) domains that can specifically bind AMP, ADP, or ATP. When intracellular AMP levels rise, AMP binds to the CBS domain of the γ subunit, causing a conformational change in the γ subunit, which in turn exposes the threonine 172 (Thr172) site of the α subunit. The exposed Thr172 site becomes a target for upstream kinases, creating conditions for the activation of AMPKα. This mechanism of regulating the conformation and activity of AMPKα through changes in adenylate concentration allows cells to respond to energy status in a timely manner.

[0008] Activation of AMPKα has significant effects on multiple metabolic pathways, primarily through phosphorylation of downstream effector molecules. Studies have shown that AMPKα promotes the formation of the TSC1 / TSC2 complex by phosphorylating TSC2, thereby inhibiting mTORC1 activity, reducing protein synthesis and lowering cellular energy expenditure. In fatty acid metabolism, AMPKα phosphorylates acetyl-CoA carboxylase (ACC), inhibiting fatty acid synthesis and promoting fatty acid oxidation to increase ATP production. AMPKα also regulates glucose metabolism by promoting glucose uptake and degradation while inhibiting glucose synthesis. During protein synthesis, AMPKα phosphorylates translation initiation factors such as eEF2, further inhibiting protein synthesis and thus conserving energy. Breast cancer studies have shown that activation of AMPKα is associated with poor metastasis-free survival in breast cancer patients. Studies have also shown that AMPK-mediated phosphorylation of pyruvate dehydrogenase α (PDHA) enables cancer cells to adapt to the adverse microenvironment at metastatic sites by rewiring cellular metabolism within the TCA cycle. These findings suggest that AMPK is also intricately involved in metabolic reprogramming. Summary of the Invention

[0009] The purpose of the present invention is to address the above problems and provide an application of SHC3 as a diagnostic and prognostic marker and therapeutic target for breast cancer lung metastasis.

[0010] In order to achieve its purpose, the present invention adopts the following technical solutions:

[0011] A first aspect of the present invention provides use of a reagent for detecting the expression of the biomarker SHC3 in the preparation of a product for auxiliary diagnosis or diagnosis of breast cancer lung metastasis in a subject.

[0012] The diagnostic method comprises: obtaining a test sample from a subject suspected of having breast cancer lung metastasis, and determining the expression level of SHC3 in the test sample; if SHC3 expression is higher than that of a normal control, it indicates that the subject is a high-risk group for breast cancer lung metastasis or a patient with breast cancer lung metastasis.

[0013] A second aspect of the present invention provides use of a reagent for detecting the expression of the biomarker SHC3 in the preparation of a product for prognosis of breast cancer patients.

[0014] The prognosis method comprises: obtaining a test sample from a subject suffering from breast cancer, and determining the expression level of SHC3 in the test sample. High expression of SHC3 indicates a poor prognosis for the patient.

[0015] The third aspect of the present invention provides the use of SHC3 as a target in screening drugs for treating breast cancer lung metastasis.

[0016] The drug inhibits the expression of SHC3, thereby inhibiting breast cancer lung metastasis.

[0017] A fourth aspect of the present invention provides use of an SHC3 expression inhibitor in the preparation of a medicament for treating breast cancer lung metastasis.

[0018] The expression inhibitors include nucleic acid molecules, proteins, and compounds.

[0019] Preferably, the nucleic acid molecule comprises siRNA or shRNA;

[0020] It is further preferred that the sequence of the siRNA is any one of SEQ ID NO.25, SEQ ID NO.26, and SEQ ID NO.27, and the sequence of the shRNA is SEQ ID NO.26.

[0021] In the above-mentioned application technology solution, the breast cancer is triple-negative breast cancer.

[0022] The beneficial effects of the present invention are:

[0023] The present study showed that SHC3 expression was significantly upregulated in TNBC lung metastatic cells, and this upregulation was closely related to the prognosis of TNBC patients; increased SHC3 expression was positively correlated with the migration ability of TNBC cells, suggesting that SHC3 plays a key role in the metastasis of TNBC cells; it was demonstrated that the SHC3 gene was significantly upregulated in TNBC cells with lung metastasis, suggesting its key role in promoting lung metastasis of triple-negative breast cancer.

[0024] Both in vivo and in vitro experiments confirmed that silencing SHC3 could significantly weaken the metastatic ability of TNBC and reduce the number of lung metastatic nodules, while overexpression of SHC3 enhanced its metastatic activity, indicating that SHC3 can serve as a therapeutic target for TNBC lung metastasis.

[0025] SHC3, highly expressed in TNBC cells, has been found to profoundly influence the expression and phosphorylation status of AMPKα, a key metabolic regulator. This regulation, in turn, promotes the progression of the TCA cycle, which is crucial for energy production and biosynthesis during cancer cell metastasis. Ultimately, this enhanced TCA cycle activity leads to the lung-specific metastasis of TNBC cells. Mechanistically, SHC3 modulates the phosphorylation status of AMPKα, enhancing the TCA cycle and, in turn, promoting cancer cell metabolism and lung metastasis. The present study further elucidates the SHC3-AMPKα-IDH2 signaling axis, which enhances the potential of TNBC cells to metastasize to the lung by increasing α-KG concentrations. This finding highlights that TNBC lung metastatic cells have metabolic characteristics that differ significantly from primary TNBC tumors, providing new insights and avenues for a deeper understanding of the metastatic mechanisms of TNBC and the design of therapeutic interventions.

[0026] The present invention provides a new marker for breast cancer prognosis and diagnosis of breast cancer lung metastasis, and provides a new target, treatment strategy and direction for the treatment of breast cancer lung metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Figure 3: SHC3 is highly expressed in lung-metastatic TNBC cells and is associated with the prognosis of TNBC: (A) Heat map showing the RNA-seq results of differentially expressed genes between lung metastatic cells (MDA-MB-231-LM3) and parental cells (MDA-MB-231-P); (B) Heat map showing the RNA-seq results of differentially expressed genes between another lung metastatic cell line (HCC1806-LM3) and its parental cell line (HCC1806-P); (C) UMAP diagram showing the cell type differences between lung metastases and primary tumors, and the heat map showing the differences between primary tumors and lung metastases. scRNA-seq results for differentially expressed genes in the T1T2 cell cluster within the tumor; (D) Venn diagram showing the intersection of the three datasets; (E) Kyoto Encyclopedia of Genes and Genomes analysis of 11 candidate genes in signaling pathways associated with tumorigenesis and metastasis; (F) Relative mRNA levels of 11 candidate genes between MDA-MB-231-LM3 and MDA-MB-231-P; (G) Kaplan-Meier analysis showing the survival of patients with high and low SHC3 expression in TNBC; (H) SHC3 expression in breast cancer based on major subtypes (including TNBC types). Data are expressed as mean ± SD. **P < 0.01, ***P < 0.001.

[0028] Figure 2 The results showed that silencing SHC3 could inhibit the migration ability of triple-negative breast cancer (TNBC) cells: qRT-PCR (A) and Western blotting (B) were used to detect the silencing efficiency of SHC3 by transfected siRNA; the data are expressed as mean ± standard deviation, **P < 0.01, ***P < 0.001.

[0029] Figure 3 Showing that SHC3 promotes the migration of lung metastatic breast cancer cells: (AB) qRT-PCR (A) and Western blotting (B) were used to detect the efficiency of SHC3 overexpression or knockdown; (CD) Transwell invasion assay (C) and scratch healing assay (D) were used to evaluate the effect of SHC3 overexpression or knockdown on the invasive ability of specific TNBC cells (scale bar, 100 μm); data are expressed as mean ± SD, **P < 0.01, ***P < 0.001.

[0030] Figure 4 Showing that SHC3 promotes lung metastasis of TNBC cells: (A) Schematic diagram of mouse treatment regimen and representative bioluminescence imaging images; (B) Representative images of lung tissue, H&E staining, and statistical results and analysis of mouse lung breast cancer metastases (n = 7 mice per group, arrows indicate metastatic nodules, scale bar, 200 μm); Data are expressed as mean ± SD, ***P < 0.001.

[0031] Figure 5 Figure 3: Effect of SHC3 on the TCA cycle in lung metastatic TNBC cells: (A) Heat map showing differentially expressed metabolites in TNBC cells ectopically overexpressing SHC3; (B, D) Metabolic signaling pathway enrichment analysis (B), volcano plot drawing (C), and KEGG enrichment analysis (D) were performed based on these differentially expressed metabolites; (E) Schematic diagram of the TCA cycle; (F, G) The levels of citrate (F) and α-KG (G) in specific TNBC cells ectopically overexpressing or knocking down SHC3 were detected using the corresponding kits; (H) IDH2 enzyme activity in TNBC cells overexpressing or silencing SHC3 was detected using the kit; Data are expressed as mean ± standard deviation, *P < 0.05, **P < 0.01, ***P < 0.001.

[0032] Figure 6 Results show that SHC3 activates IDH2 by promoting AMPKα phosphorylation: (A) Western blotting was used to detect the expression levels of AMPKα and phosphorylated AMPKα (p-AMPKα) in the specified TNBC cells; (B-C) qRT-PCR (B) and western blotting (C) were used to determine the relative expression of AMPKα in cells transfected with AMPKα siRNA; (D) Western blotting was used to detect the expression levels of AMPKα, p-AMPKα, and IDH2 in TNBC cells under specific conditions; (E-G) The enzyme activity of IDH2 (E), citric acid concentration (F), and α-KG concentration (G) in TNBC cells under specific conditions were evaluated using corresponding kits; data are expressed as mean ± SD, **P < 0.01, ***P < 0.001.

[0033] Figure 7 Showing that SHC3 is involved in the lung metastasis of TNBC cells through the AMPKα-IDH2 axis: (A) Representative images show immunohistochemical staining of AMPKα and phosphorylated AMPKα (p-AMPKα) (scale bar, 200 μm); (B, D) IDH2 enzyme activity (B), citric acid concentration (C), and α-KG concentration (D) in lung tissues were assessed using corresponding kits (n = 7 mice per group); Data are expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0036] Example 1 SHC3 is highly expressed in TNBC cell lung metastases and is associated with TNBC prognosis

[0037] 1 Materials and Methods

[0038] 1.1 Cell lines and experimental animals

[0039] 1.1.1 Cell lines

[0040] The human TNBC cell lines MDA-MB-231 and HCC1806 were obtained from the American Type Culture Collection. Luciferase (Luc) and green fluorescent protein (GFP) dual-labeled Luc-GFP MDA-MB-231 and Luc-GFP HCC1806 cells were obtained from our research group.

[0041] 1.1.2 Experimental animals

[0042] The experimental animals used in this study, including female Balb / c mice and female athymic nude BALB / c-nu mice (age range: 4 to 6 weeks), were purchased from Chongqing Tengxin Biotechnology Co., Ltd. through reputable channels and maintained at the professionally qualified Animal Experiment Center of Chongqing Medical University. During the animal husbandry and experimental procedures, we strictly adhered to national and international animal ethics guidelines and the relevant provisions of the "Regulations on the Administration of Laboratory Animals" to ensure that the welfare and rights of experimental animals were fully protected. All experimental protocols involving animals were approved by the Ethics Committee of Chongqing Medical University (approval number 2021084) and performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0043] 1.2 Main experimental reagents and consumables

[0044]

[0045] The primer sequences used for qRT-PCR analysis are as follows:

[0046]

[0047]

[0048] 2 Experimental studies

[0049] 2.1 Establishment of a breast cancer organotropic metastasis mouse model and generation of a highly lung-tropic metastatic cell line

[0050] To establish a more precise experimental metastatic model of TNBC, we selected the luciferase- and green fluorescent protein-dual-labeled TNBC cell lines HCC1806 and MDA-MB-231 as the basis for our study. Six to eight weeks after orthotopic fat pad injection of these two cell lines, in vivo imaging revealed the presence of cancer cells in the mammary glands and lungs of mice. Metastatic tissue was removed and tumor cells isolated. First-generation lung metastatic cells were isolated through primary culture and reintroduced into mice. Through repeated fat pad injections, combined with a meticulous selection and expansion process for in vivo metastatic clones, we successfully generated organ-specific TNBC metastatic cell lines. These derived cell lines are systematically named based on their original organ of origin and the number of experimental passages to ensure the accuracy and traceability of our findings. The lung-specific metastatic potential of lung metastatic cells increased with increasing passage number, with the proportion of specific lung metastases increasing from 0.1-0.2% to 3-4%. Notably, no significant changes were observed in the ability of lung metastatic cancer cells to metastasize to organs such as the brain, liver, spleen, and kidney. Based on the above observations, it can be confirmed that we have successfully established a lung-tropic metastatic cell model for breast cancer.

[0051] To further analyze gene expression signatures closely associated with lung metastasis, we selected two pairs of specific lung metastatic cell lines: HCC1806-LM3 and its parental cell line, HCC1806-P, and MDA-MB-231-LM3 and its parental cell line, MDA-MB-231-P. We performed transcriptome sequencing (RNA-Seq) on each pair. Furthermore, single-cell sequencing was performed on orthotopic and lung metastatic tumor tissues formed by MDA-MB-231 in mice. Bioinformatics analysis revealed three cell subpopulations: T1, T2, and T3. The T1 and T2 cell subpopulations were found to have a specific propensity for lung metastasis.

[0052] 2.2 Single-cell sequencing combined with multi-omics reveals a unique expression profile of lung-metastatic breast cancer cells

[0053] RNA-Seq data of HCC1806-LM3 and MDA-MB-231-LM3 and their respective parental cells, HCC1806-P and MDA-MB-231-P, were analyzed. By setting the screening thresholds of |log2FoldChange| ≥ 1 and P < 0.05, the results showed that 382 genes were upregulated and 269 genes were downregulated in MDA-MB-231-LM3 compared with their respective parental cells; while 1001 genes were upregulated and 967 genes were downregulated in HCC1806-LM3 ( Figure 1 AB).

[0054] At the same time, single-cell sequencing analysis was performed on the primary tumor and lung metastasis tissues formed by MDA-MB-231. The results showed that both primary tumors and lung metastases were rich in T1 and T2 cell clusters ( Figure 1 C, left panel). Further analysis of differentially expressed genes between these two cell clusters revealed that, under the screening conditions of |log2FoldChange|≥0.58 and P<0.05, we identified a total of 1652 genes that were differentially expressed in the T1 and T2 cell clusters in lung metastases and primary tumors ( Figure 1 C, left panel).

[0055] By performing intersection analysis on these three datasets, we found that a total of 50 genes were abnormally expressed in all three datasets ( Figure 1 D). Next, we used KEGG to analyze these 50 differentially expressed genes and identified 11 differentially expressed candidate genes involved in major signaling pathways, including FGF1, FGF2, FGF23, GRPR, NRG1, NRG2, PDGFB, PDGFC, PDGFD, PDGFRB, and SHC3 ( Figure 1 E). The expression levels of these 11 differential candidate genes in the sequencing data are shown in Table 1.

[0056] To verify the expression of these candidate genes, we used qRT-PCR to compare the expression of these genes in MDA-MB-231-LM3 cells and MDA-MB-231-P cells. The analysis results showed that ( Figure 1 F) Expression levels of FGF23, PDGFB, and PDGFRB were extremely low, while PDGFC expression showed no statistical difference between the two cell groups. Furthermore, the expression trends of FGF2, GRPR, NRG1, and NRG2 were opposite to those in the sequencing results. After excluding these genes, FGF1, PDGFD, and SHC3 were included in subsequent analyses.

[0057] Using the online database Kaplan-Meier and TCGA analysis, it was found that high expression of SHC3 was associated with poor prognosis in TNBC patients ( Figure 1 GH). Taken together, these data suggest that SHC3 is not only closely associated with the prognosis of TNBC but may also be involved in the lung metastasis process of TNBC.

[0058] Table 1. Expression differences of 11 candidate genes in three datasets

[0059]

[0060] 3 Analysis

[0061] In this study, we successfully constructed TNBC cell lines HCC1806-LM3 and MDA-MB-231-LM3 with pneumophilic metastasis. Using sequencing technology combined with bioinformatics analysis, we identified 11 differentially expressed candidate genes associated with TNBC pneumophilic metastasis. Further investigation revealed that the SHC3 gene was significantly upregulated during TNBC lung-specific metastasis. Comparative analysis across online databases confirmed that SHC3 has a specific expression pattern in TNBC, and its expression level is closely associated with poor prognosis in cancer patients. However, although high SHC3 expression is associated with TNBC pneumophilic metastasis, its specific regulatory mechanism remains to be further explored and verified in future studies.

[0062] Example 2: SHC3 promotes lung metastasis of TNBC

[0063] 1 Materials and Methods

[0064] 1.1 Cell lines

[0065] The cells were the same as in Example 1.

[0066] 1.2 Main experimental reagents and consumables

[0067]

[0068]

[0069] In the SHC3 knockdown and overexpression viruses, the NCBI Gene ID number of SHC3 is: 53358.

[0070] The siRNA sequence of the SHC3 gene is as follows:

[0071]

[0072] The sequence of SHC3 shRNA is shown as SEQ ID NO.26.

[0073] 2 Experimental studies

[0074] 2.1 Verification of SHC3 overexpression lentivirus and shRNA lentivirus knockout efficiency

[0075] To further clarify the role of SHC3 as a key regulatory factor in TNBC lung metastasis, this study used specific siRNA to silence the SHC3 gene in MDA-MB-231-LM3 cells. First, the transfection efficiency of three SHC3 siRNAs was verified by qRT-PCR and Western blotting. The results showed that all three siRNAs could effectively silence SHC3 gene expression, with siRNA-2 having the best effect ( Figure 2Based on the above results, this study selected si-2 to construct SHC3 shRNA lentivirus, which was used to stably knock down SHC3 expression in MDA-MB-231-LM3 cells. At the same time, wild-type MDA-MB-231 (MDA-MB-231-WT) cells were infected with the lentivirus carrying the SHC3 gene to achieve SHC3 overexpression. qRT-PCR and Western blotting were used again to evaluate the SHC3 knockdown efficiency in MDA-MB-231-LM3 cells and the SHC3 overexpression efficiency in MDA-MB-231-WT cells. Figure 3 AB), the results showed that the SHC3 overexpression and silencing cell models were successfully constructed.

[0076] 2.2SHC3 promotes the metastatic ability of TNBC cells

[0077] The results of Transwell migration assay and wound healing assay further showed that knockdown of SHC3 in highly metastatic MDA-MB-231-LM3 cells could inhibit their metastatic ability, while ectopic overexpression of SHC3 in wild-type MDA-MB-231-WT cells could enhance their metastatic ability ( Figure 3 CD). These results strongly suggest that SHC3 has a positive effect on promoting the metastatic ability of TNBC cells.

[0078] 2.3SHC3 promotes lung metastasis of TNBC in vivo

[0079] To verify whether SHC3 can promote lung metastasis of TNBC in vivo, this study used tail vein injection to detect the metastatic potential of SHC3-overexpressing MDA-MB-231-WT and SHC3-knockdown MDA-MB-231-LM3 cells in mice ( Figure 4 A). Bioluminescence imaging and lung tissue HE staining results showed that silencing SHC3 in MDA-MB-231-LM3 cells significantly reduced the number of lung metastatic nodules ( Figure 4 AB). In contrast, overexpression of SHC3 in MDA-MB-231-WT cells resulted in a stronger metastatic activity ( Figure 4 AB).

[0080] 3 Analysis

[0081] Both in vitro and in vivo experiments confirmed that silencing SHC3 significantly impaired the metastatic capacity of TNBC and reduced the number of lung metastatic nodules, while overexpressing SHC3 enhanced its metastatic activity. These experimental results fully demonstrate the role of SHC3 in promoting the lung metastasis of TNBC.

[0082] Example 3: SHC3 promotes AMPK phosphorylation and activates IDH2 to drive the TCA cycle and participate in TNBC lung metastasis

[0083] 1 Materials and Methods

[0084] 1.1 Cell lines

[0085] Same as Example 1.

[0086] 1.2 Main experimental reagents and consumables

[0087]

[0088] The sequence of the siRNA targeting AMPKa is as follows:

[0089]

[0090] 2 Experimental studies

[0091] 2.1 Overexpression of SHC3 enhances tumor metabolic reprogramming in TNBC cells

[0092] To investigate whether SHC3 is involved in regulating tumor metabolic reprogramming, this study used GC / LC–MS technology to identify metabolites in MDA-MB-231-WT cells overexpressing SHC3 and control cells. The results showed that MDA-MB-231-WT cells overexpressing SHC3 exhibited unique metabolic characteristics, with significantly increased levels of α-KG (α-ketoglutarate), xanthine, and oxypurinol ( Figure 5 AC). KEGG analysis showed that TCA cycle metabolism in SHC3 overexpressing cells was significantly different ( Figure 5 DE). Recent studies have found that TCA cycle flux in lung metastases is higher than in primary tumors. Given that SHC3 is associated with breast cancer lung metastasis, we hypothesized that SHC3 could enhance TCA cycle flux in TNBC cells.

[0093] To this end, we regulated the expression of SHC3 in TNBC cells and examined the changes in α-KG and citric acid, two key metabolites in the TCA cycle. Figure 5 FG). The results showed that with the reduction of SHC3 in MDA-MB-231-LM3 cells, the concentration of citric acid increased significantly, while the accumulation of α-KG decreased significantly. In contrast, enhancing the expression of SHC3 in MDA-MB-231-WT cells inhibited the concentration of citric acid and promoted the accumulation of α-KG. We then detected the enzyme activity of IDH2 (isocitrate dehydrogenase), a key enzyme that catalyzes the conversion of citric acid to α-KG, and found that after overexpression of SHC3, the enzyme activity of IDH2 in MDA-MB-231-WT cells was significantly increased, while the activity of this enzyme was significantly reduced when SHC3 was silenced ( Figure 5H). In conclusion, during TNBC lung metastasis, high expression of SHC3 promotes the enhancement of TCA cycle.

[0094] 2.2SHC3 regulates the TCA cycle by promoting AMPK phosphorylation and activating IDH2 enzyme activity

[0095] Previous studies have reported that the SHC family can regulate the activity of adenylate-activated protein kinase α (AMPKα), which in turn promotes the TCA cycle. However, it is still unclear whether SHC3 promotes the lung metastasis of TNBC cells by activating AMPKα and then restarting the TCA cycle. First, we verified the changes in AMPKα expression levels in SHC3 knockdown or overexpression cells and found that the expression level and phosphorylation level of AMPKα were positively correlated with SHC3 ( Figure 6 A).

[0096] To further establish SHC3 as a regulatory factor of AMPKα, we silenced AMPKα in MDA-MB-231-LM3 cells using siRNA. qRT-PCR and Western blotting results showed that AMPKα siRNA could effectively inhibit the expression level of AMPKα ( Figure 6 BC). Through rescue experiments, we found that SHC3 affects IDH2 protease activity rather than protein expression level through AMPKα ( Figure 6 DE). After silencing AMPKα, even increasing SHC3 expression cannot activate the enzymatic activity of IDH2 protein, resulting in increased citric acid concentration and reduced α-KG accumulation ( Figure 6 FG). Taken together, these data indicate that SHC3 regulates the enzymatic activity of IDH2 through AMPKα, thereby initiating the TCA cycle in TNBC cells.

[0097] 2.3SHC3 participates in the lung metastasis of TNBC cells through the AMPKα-IDH2 axis

[0098] We detected the expression of AMPKα and p-AMPKα in lung metastatic nodules with SHC3 overexpression and SHC3 deficiency by immunohistochemical staining ( Figure 7 A). Next, we measured the enzyme activity of isocitrate dehydrogenase 2 (IDH2) and the contents of citrate and α-KG in the lung metastasis tissues of mice ( Figure 7 BD). The trends in protein expression, IDH2 enzyme activity, and metabolite expression were consistent with the results in the cell model. Taken together, these data suggest that SHC3 promotes the TCA cycle through AMPKα and plays a key role in the lung metastasis of TNBC cells.

[0099] 3 Analysis

[0100] The above study revealed that MDA-MB-231 cells overexpressing SHC3 exhibited unique metabolic characteristics, suggesting that SHC3 exerts metabolic remodeling and metabolic adaptation in the specific tissue microenvironment of lung tissue. Further in vitro and in vivo mechanistic exploration revealed that SHC3 regulates IDH2 enzymatic activity through AMPKα, thereby promoting the TCA cycle and α-KG accumulation in TNBC cell lung metastasis.

[0101] In summary, this study reveals a mechanism by which upregulated SHC3 expression activates AMPK, thereby promoting lung metastasis in triple-negative breast cancer. A key aspect of this process involves IDH2, ultimately leading to significant accumulation of α-KG. Notably, previous studies have clearly established a close relationship between high wild-type IDH2 expression and lymphovascular invasion in breast cancer, identifying it as an independent prognostic factor for shortened breast cancer-specific survival. Furthermore, wild-type IDH2 occupies a crucial position in the TCA cycle, suggesting it as a potential therapeutic target for TNBC. IDH2, as the rate-limiting enzyme in the TCA cycle, catalyzes the oxidative decarboxylation of isocitrate to produce α-KG, while simultaneously releasing carbon dioxide. It is currently believed that increasing the concentration of α-KG in tumor cells can inhibit tumor growth. However, intriguing findings in radioresistant lung cancer cells suggest that elevated α-KG levels may trigger activation of the AhR signaling pathway, thereby inducing cancer stem-like characteristics. In the context of breast cancer lung metastasis, α-KG promotes mTORC1 signaling in tumor cells, paving the way for metastasis. Our results further elucidate that the SHC3-AMPK-IDH2 signaling axis enhances the lung metastatic potential of TNBC cells by increasing the concentration of α-KG. This finding emphasizes that TNBC lung metastatic cells exhibit a metabolic signature that is significantly different from primary TNBC tumors, providing new insights and avenues for understanding the metastatic mechanism of TNBC and designing therapeutic interventions.

Claims

1. Use of a reagent for detecting the expression of the biomarker SHC3 in the preparation of a product for auxiliary diagnosis or diagnosis of breast cancer lung metastasis in a subject.

2. The use according to claim 1, characterized in that The diagnostic method comprises: obtaining a test sample from a subject suspected of having breast cancer lung metastasis, and determining the expression level of SHC3 in the test sample; if the expression level of SHC3 is higher than that of a normal control, it indicates that the subject is a high-risk group for breast cancer lung metastasis or a patient with breast cancer lung metastasis.

3. Use of a reagent for detecting the expression of the biomarker SHC3 in the preparation of a product for the prognosis of breast cancer patients.

4. The use according to claim 3, characterized in that The prognosis method comprises: obtaining a test sample from a subject suffering from breast cancer, and determining the expression level of SHC3 in the test sample. High expression of SHC3 indicates a poor prognosis for the patient.

5. Application of SHC3 as a target in screening drugs for the treatment of breast cancer lung metastasis.

6. The use according to claim 5, characterized in that: The drug inhibits the expression of SHC3, thereby inhibiting breast cancer lung metastasis.

7. Use of SHC3 expression inhibitors in the preparation of drugs for the treatment of breast cancer lung metastasis.

8. The use according to claim 7, characterized in that: The expression inhibitors include nucleic acid molecules, proteins, and compounds.

9. The use according to claim 8, characterized in that: The nucleic acid molecule includes siRNA or shRNA; Preferably, the sequence of the siRNA is shown in any one of SEQ ID NO.25, SEQ ID NO.26, and SEQ ID NO.27, and the sequence of the shRNA is shown in SEQ ID NO.

26.

10. The use according to any one of claims 1 to 9, characterized in that: The breast cancer is triple-negative breast cancer.

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