阅读说明：本技术 luminal型和HER2型乳腺癌诊断及预后的标志物、治疗用PPARγ抑制剂 (Diagnostic and prognostic marker for luminal and HER2 breast cancers, and therapeutic PPAR γ inhibitor ) 是由 程金科 郑铨 曹颖 屠俊 王田实 贺兼理 周炜 于 2020-04-16 设计创作，主要内容包括：本发明公开了成纤维细胞生长因子21FGF21或其编码基因、SENP2蛋白或其编码基因、SENP2蛋白第123位的丝氨酸、和/或脂肪酸转运受体CD36蛋白或其编码基因在作为诊断乳腺癌及其迁移或转移能力,预测乳腺癌的迁移或转移能力、以及乳腺癌病人生存率的标志物中的应用。luminal型和HER2型乳腺癌病人血清中FGF21的浓度与乳腺癌的转移成正相关性,所述FGF21通过ERK1/2磷酸化激活SENP2,促进乳腺癌细胞的脂质代谢过程,导致乳腺癌细胞侵袭和转移的能力增强。本发明利用中和抗体降低FGF21的浓度或者抑制乳腺癌细胞中SENP2的磷酸化激活等干预措施,可以有效抑制乳腺癌细胞的侵袭和转移。本发明利用PPARγ的抑制剂可以显著地抑制乳腺癌细胞的侵袭、迁移和转移。(The invention discloses application of fibroblast growth factor 21FGF21 or a coding gene thereof, SENP2 protein or a coding gene thereof, 123 th serine of SENP2 protein and/or fatty acid transport receptor CD36 protein or a coding gene thereof as a marker for diagnosing breast cancer and migration or metastasis capacity thereof, predicting migration or metastasis capacity of the breast cancer and survival rate of breast cancer patients. The concentration of FGF21 in the serum of a luminal breast cancer patient and a HER2 breast cancer patient is in positive correlation with breast cancer metastasis, and the FGF21 activates SENP2 through ERK1/2 phosphorylation to promote the lipid metabolic process of breast cancer cells, so that the invasion and metastasis capacity of the breast cancer cells are enhanced. The invention utilizes the intervention measures of reducing the concentration of FGF21 or inhibiting phosphorylation activation of SENP2 in breast cancer cells and the like by using a neutralizing antibody, and can effectively inhibit invasion and metastasis of the breast cancer cells. The invention can obviously inhibit the invasion, migration and metastasis of breast cancer cells by utilizing the inhibitor of PPAR gamma.)

1. The application of fibroblast growth factor 21FGF21 or a coding gene thereof, SENP2 protein or a coding gene thereof, phosphorylation modification of 123 th serine of SENP2 protein, fatty acid transport receptor CD36 protein or a coding gene thereof, or SENP2 protein, CD36 protein or coding genes thereof as markers for diagnosing luminal and HER2 breast cancers and migration or metastasis capacities thereof, predicting the migration or metastasis capacities of luminal and HER2 breast cancers and survival rates of luminal and HER2 breast cancer patients.

2. Application of a detection reagent or inhibitor of fibroblast growth factor FGF21 or a coding gene thereof in preparation of luminal and HER2 breast cancer related products.

Application of a detection reagent or inhibitor of SENP2 protein or coding gene thereof in preparation of luminal and HER2 breast cancer related products.

Application of a detection reagent or dephosphorylation modification reagent for phosphorylation modification of 123 th serine of SENP2 protein in preparation of luminal and HER2 breast cancer related products.

5. Application of a detection reagent or inhibitor of a fatty acid transport receptor CD36 protein or a coding gene thereof in preparation of luminal and HER2 breast cancer related products.

Application of a combined detection reagent or a combined inhibitor of SENP2 protein and CD36 protein or coding genes thereof in preparation of luminal and HER2 breast cancer related products.

7. The use according to any one of claims 2 to 6, wherein the detection reagent is for diagnosing luminal and HER2 breast cancer and its migratory or metastatic potential; the inhibitor is used for preventing or inhibiting migration and metastasis of luminal and HER2 breast cancers and improving survival rates of luminal and HER2 breast cancer patients; the dephosphorylation modification reagent is used for preventing or inhibiting migration and transfer capacity of luminal and HER2 breast cancers, or improving survival rate of luminal and HER2 breast cancer patients.

8. Application of an inhibitor of protein kinase ERK1/2 in preparing a medicament for preventing and/or treating luminal and HER2 breast cancers.

9. Application of an inhibitor capable of inhibiting expression level of fatty acid metabolism-related genes or proteins in preparation of a medicament for preventing and/or treating luminal and HER2 breast cancer.

10. A composition comprising a detection reagent or inhibitor according to any one of claims 2, 3, 5, 6; or comprises a detection reagent or dephosphorylation modification reagent according to claim 4, or comprises an inhibitor according to claim 8 or 9.

11. A method for diagnosing and predicting the migration or transfer capacity of luminal and HER2 breast cancers, predicting the migration or transfer capacity of luminal and HER2 breast cancers and predicting the survival rate of luminal and HER2 breast cancer patients is characterized in that a fibroblast growth factor 21FGF21 or a coding gene thereof, SENP2 protein or a coding gene thereof, phosphorylation modification of serine at position 123 of SENP2 protein, fatty acid transport receptor CD36 protein or a coding gene thereof, or SENP2 protein and CD36 protein or a coding gene thereof are taken as markers to carry out related diagnosis and prediction.

12. A method for screening a compound for inhibiting SENP2S123 phosphorylation modification is characterized in that an FGF21 neutralizing antibody and an antagonistic antibody of a receptor KLB thereof are prepared by taking FGF21 and SENP2S123 phosphorylation modification as markers, and the compound for inhibiting SENP2S123 phosphorylation modification is screened.

13. Use of the composition of claim 10 for predicting the risk of metastasis in luminal and HER2 breast cancer or for predicting survival in luminal and HER2 breast cancer patients.

14. Application of any one or more of GW9662 shown in formula A, T0070907 shown in formula B and GinsenosideRh1 shown in formula C as transcription factor PPAR gamma inhibitor in preparation of medicine for treating luminal and/or HER2 type breast cancer.

Technical Field

The invention belongs to the field of biological medicines, relates to a marker and a composition for diagnosis, treatment and prognosis of luminal and HER2 breast cancers, and particularly relates to application of FGF21 and SENP2 proteins as luminal and HER2 breast cancer markers.

Background

It is well known that breast cancer is the leading cancer that afflicts the health of western women, with a slightly lower incidence in china than in western developed countries. But since the last 90 s, the rate of breast cancer incidence in china has increased more than the average level worldwide. According to incomplete data statistics, more than 17 ten thousand new cases of breast cancer and more than 5 ten thousand cases of death caused by breast cancer in China each year account for 12.2 percent of the number of the breast cancer and 9.6 percent of the number of deaths (Fan et al, 2014) in the world at present. The clinical manifestations of breast cancer are not single, and there are significant differences in biological and clinical features between different subtypes. Traditional indicators for clinical and pathological assessment of breast cancer grade include patient age, tumor size, histological features, and number of metastases to axillary lymph nodes. Currently, the most important identification indexes for breast cancer classification are detection of expression of three marker proteins, namely, Estrogen Receptor (ER), Progesterone Receptor (PR) and human epidermal growth factor receptor 2 (HER 2) by an immunohistochemical method. Breast cancers are generally classified into luminal a, luminal B, HER2, and basal-like (triphobic) types, depending on the amount of marker protein expressed by the breast cancer cells. The clinical treatment measures for different types of breast cancer are also different. Luminal A and B are mainly endocrine therapy, and part of patients need chemotherapy; HER2 type breast cancer patients need to be treated with anti-HER 2 drugs in addition to chemotherapy; basal-like breast cancer is the most malignant, and the current primary treatment is chemotherapy. Although the disease of most breast cancer patients can be greatly improved or even completely eradicated by surgery or drug therapy, treatment for metastatic breast cancer (metastic breast cancer) still lacks effective targeting means, and the survival status of the patients is still extremely severe, with an overall average survival time of only 2 to 3 years. The process of metastasis of tumor cells from the primary to other organs is very elaborate and involves invasion of the primary tumor cells into the surrounding tissue, infiltration into the circulatory system for transport and survival, penetration of the vessel wall into new tissues to form micrometastases, and proliferation of the cells to form new tumor masses (Lambert et al, 2017). The molecular basis for tumor metastasis is not yet elucidated. The most widely accepted theory for analyzing tumor metastasis is the epithelial-mesenchymal transition (EMT) model, i.e., the epithelial-like tumor cells can acquire characteristics of mesenchymal cells, such as extracellular matrix decomposition, invasion and metastasis, and thus the tumor cells can be diffused and metastasized. Numerous experiments have demonstrated that the molecular basis for regulating EMT is primarily related to several important transcription factors, including Snail, Slug, Twist, and Zeb1, among others (lamouillet et al, 2014). Although the EMT model is critical to the metastasis and spread of most tumors, there are still some fundamental problems that have not been solved so far, such as: how such multiple heterogeneous signal molecules can simultaneously converge together to activate the otherwise silent EMT pathway under the physiopathological conditions; whether the extent to which EMT is activated during tumor development is sufficient to trigger tumor metastasis; and what extracellular and intracellular signals maintain sustained activation of EMT after it is initiated. On the other hand, more and more studies have found that not only Cancer Stem Cells (CSCs) but also a group of metastasis initiating cells (metastasis-initiating cells) play a key role in initiating tumor metastasis in primary tumor tissues with diverse heterogeneity. The researchers thought that if the source pathway and action mechanism of the group of cells were analyzed thoroughly, effective intervention could be performed to achieve the purpose of controlling tumor metastasis, and therefore, research on tumor metastasis initiating cells is of great interest.

In 2017, the Benitah research group in spain demonstrated for the first time an important role of lipid metabolism in tumor metastasis (Pascual et al, 2017). When the oral squamous cancer cells marked with fluorescence are injected into mice in situ, the fluorescence signal of single cells gradually weakens along with the proliferation of the cancer cells, and the slowly proliferating cells retain the fluorescence intensity. Sorting CD44 by flow⁺The authors compared the gene expression profiles of two groups of cells with strong and weak fluorescence signals, and found that the group of cells with weak fluorescence signals highly express genes related to cell proliferation and division, and the group of cells with strong fluorescence signals highly express genes related to tumor metastasis and lipid metabolism. Among these varied genes, the authors emphasize that CD36 is a key marker protein for metastasis-initiating cells, and demonstrate that CD36 can significantly promote metastasis of cancer cells through experimental demonstration of RNA interference and neutralizing antibodies, respectively, and further demonstrate that CD36 promotes metastasis of cancer cells by uptake of fatty acids to enhance fatty acid β oxidation. In addition, the authors also analyzed lung, bladder and breast cancer and similarly obtained that CD36 has an important role in promoting metastasis of these tumor cells. Therefore, the mechanism research aiming at regulating CD36 and lipid metabolism will help us to find the action target point for inhibiting tumor metastasis.

The mammary tissue itself is an organ rich in fat cells, and the amount of fat cells in the mammary tissue of women in non-nursing period can account for 56% of all cells, and even in nursing period, the amount of fat cells is 35% of the total amount, so that the fat cells with the largest composition are considered to be closely related to the occurrence and development of breast cancer. The fat cells in the breast cancer not only provide energy substances such as fatty acid for adjacent cancer cells, but also can actively regulate the processes of invasion, metastasis, drug resistance and the like of the breast cancer cells. Adipokines released by adipocytes such as leptin, adiponectin, TNF α, IL-6, etc., all play an important regulatory role in the vital activity of breast cancer cells (Choi et al, 2017). In recent years, the adipokine FGF21 has received increased attention due to its specific efficacy in obesity and insulin resistance. FGF21 belongs to the family of fibroblast growth factors, and FGF21 is expressed in all tissues of the liver, fat, pancreas and cartilage, and its receptor of action consists of FGF receptors (FGFRs) with tyrosine kinase activity and β -Klotho. FGF21 plays an important regulatory role in glucose metabolism and lipid metabolism. FGF21 significantly reduced blood glucose levels in obese or insulin resistant mice, promoted browning of white fat (browning of white adipocytes), reduced body weight, increased body thermogenesis and energy expenditure, and increased insulin sensitivity (khanitonnkov et al, 2005). In addition, FGF21 also decreased serum levels of triglycerides and free fatty acids, in part, due to increased uptake of fatty acids by adipose tissue (Schlein et al, 2016). In molecular mechanism, FGF21 can be absorbed by adipocytes in an autocrine or paracrine form, so that SUMO modification of intracellular PPAR γ is reduced, transcription activity of PPAR γ is activated, expression of downstream genes related to fatty acid uptake and synthesis and the like downstream of PPAR γ is promoted, free fatty acid level in blood is reduced, absorption and utilization of glucose by organs such as muscles are enhanced, and insulin sensitivity is improved (Dutchak et al, 2012). It is to be noted that, although a previous study of the present inventors has demonstrated that FGF21 inhibits SUMO modification of PPAR γ by SENP2 in adipocytes, it is unknown whether this pathway also functions in other cells.

SUMO modification is an important post-translational modification of proteins in eukaryotic cells, and SENP2 belongs to one of the specific protease family members that mediate the maturation of SUMO proteins and the de-SUMO modification of proteins. The present inventors' topic group previously conducted a series of studies on the physiological functions of SENP2, such as cardiac development and muscle formation. Recent studies published by the subject groups of the present inventors have clarified an important regulatory function of SENP2 in adipocytes in lipid metabolism (Zheng et al, 2018). In the process of obesity, SENP2 mainly promotes the expression of transcription factors PPARg and C/EBP alpha, reduces the SUMO modification level of PPAR gamma, and enhances processes such as fatty acid uptake, triglyceride synthesis and storage; in mice with a specific loss of the SENP2 in the adipose tissues, although the weight of the mice fed with a high-fat diet is lighter than that of the mice fed with the high-fat diet compared with that of the wild type, the SENP 2-deficient mice have the phenomena of hyperlipidemia, fatty liver, insulin resistance and the like due to the fact that fat cells cannot effectively store lipid substances, and the SENP2 has a key role in positively regulating lipid metabolism.

Disclosure of Invention

The invention innovatively provides application of fibroblast growth factor 21(FGF21) or a coding gene thereof in serving as a marker for diagnosing luminal and HER2 breast cancers and migration or metastasis capacities thereof, predicting the migration or metastasis capacities of luminal and HER2 breast cancers and survival rates of luminal and HER2 breast cancer patients for the first time.

The invention also provides application of the SENP2 protein or the coding gene thereof as a marker for diagnosing the luminal and HER2 breast cancers and the migration or metastasis capability thereof, predicting the migration or metastasis capability of the luminal and HER2 breast cancers and the survival rate of luminal and HER2 breast cancer patients.

The invention also provides application of phosphorylation modification of 123 th serine of SENP2 protein in diagnosis of luminal and HER2 breast cancers and migration or metastasis capability thereof, prediction of migration or metastasis capability of luminal and HER2 breast cancers and survival rate of luminal and HER2 breast cancer patients.

The invention also provides application of the fatty acid transport receptor CD36 protein or the coding gene thereof as a marker for diagnosing luminal and HER2 breast cancers and migration or metastasis capacities thereof, predicting the migration or metastasis capacities of luminal and HER2 breast cancers and survival rates of luminal and HER2 breast cancer patients.

The invention also provides application of combination of the SENP2 protein and the CD36 protein or coding genes thereof as markers for diagnosing the luminal and HER2 breast cancers and the migration or metastasis capacities thereof, predicting the migration or metastasis capacities of the luminal and HER2 breast cancers and the survival rates of luminal and HER2 breast cancer patients.

The invention also provides application of a detection reagent or an inhibitor of fibroblast growth factor 21(FGF21) or a coding gene thereof in preparation of a luminal and HER2 breast cancer related product; wherein the inhibitor comprises FGF21 neutralizing antibody and antagonist antibody of receptor KLB thereof, and compounds inhibiting SENP2S123 phosphorylation modification are screened.

The invention also provides application of a detection reagent or an inhibitor of SENP2 protein or a coding gene thereof in preparation of a luminal and HER2 breast cancer related product; wherein the inhibitor comprises a compound that inhibits the phosphorylation modification of SENP2S 123.

The invention also provides an application of a detection reagent or a dephosphorylation modification reagent for phosphorylation of serine at position 123 of SENP2 protein in preparation of a luminal and HER2 breast cancer related product.

The invention also provides application of a detection reagent or inhibitor of the fatty acid transport receptor CD36 protein or the coding gene thereof in preparation of a luminal and HER2 breast cancer related product.

The invention also provides application of a combined detection reagent or a combined inhibitor of the SENP2 protein and the CD36 protein or encoding genes thereof in preparation of luminal and HER2 breast cancer related products.

The invention also provides application of the protein kinase ERK1/2 inhibitor in preparing a medicament for preventing and/or treating luminal and HER2 breast cancer. Wherein the inhibitor is used for preventing or inhibiting migration and metastasis of luminal and HER2 breast cancers and improving survival rates of luminal and HER2 breast cancer patients.

The invention also provides application of the inhibitor capable of inhibiting the expression level of the fatty acid metabolism related gene or protein in preparing a medicament for preventing and/or treating the luminal and HER2 breast cancers. Wherein the inhibitor comprises a compound that inhibits fatty acid uptake, a compound that inhibits fatty acid synthesis, a compound that inhibits fatty acid oxidation; the fatty acid metabolism related gene or protein is involved in fatty acid uptake, absorption and oxidative phosphorylation.

The invention also provides application of three inhibitors (one or more of GW9662, T0070907 and Ginsenoside Rh1) of transcription factor PPAR gamma in preparing a medicament for treating luminal and/or HER2 type breast cancer (anti-luminal and/or HER2 type breast cancer). Wherein the inhibitor is used for inhibiting invasion, migration and metastasis abilities of luminal and HER2 breast cancers and improving survival rates of luminal and HER2 breast cancer patients.

In the present invention, the metastasis includes bone metastasis, lung metastasis, lymph metastasis and the like, preferably bone metastasis.

In the invention, the detection reagent is used for diagnosing the breast cancer of the luminal type and the HER2 type and the migration or metastasis capacity thereof; in the invention, the detection reagent is oligonucleotide for detecting mRNA and DNA of related genes.

In the invention, the inhibitor is used for preventing or inhibiting the migration and the transfer capacity of the luminal and HER2 breast cancers and improving the survival rate of luminal and HER2 breast cancer patients.

In the invention, the dephosphorylation modification reagent is used for preventing or inhibiting the migration and transfer capacity of the luminal and HER2 breast cancers and improving the survival rate of luminal and HER2 breast cancer patients.

The invention also provides a composition comprising a detection reagent or inhibitor or dephosphorylation modification reagent as described above. The composition is used for predicting the risk of metastasis of the luminal and HER2 breast cancers; or for predicting survival in both luminal and HER2 breast cancer patients.

The invention provides SENP2 which can improve the fatty acid uptake and oxidation functions of a luminal and HER2 breast cancer cell and promote the transfer capacity of the luminal and HER2 breast cancer cell. The post-operation survival rate of the SENP2 high-expression luminal and HER2 breast cancer patients is lower. Fibroblast growth factor 21 (cytokine or adipokine) FGF21 is able to promote lipid metabolism and the ability to transfer in both luminal and HER2 breast cancer cells, and this effect is SENP2 dependent. Fibroblast growth factor 21(FGF21) phosphorylates serine at position 123 of SENP2 protein through protein kinase ERK1/2, and mutation of this site can inhibit the effect of fibroblast growth factor 21(FGF21) on luminal and HER2 type breast cancer cells.

The invention detects the expression of SENP2 in the specimen section of a luminal breast cancer patient and a HER2 breast cancer patient, and finds that SENP2 is specifically expressed only in tumor cells with invasion and metastasis tendencies. The fatty acid transport receptor CD36 is further used as a marker of a transfer initiating cell, and the SENP2 and CD36 are found to have consistent positioning expression in a breast cancer specimen; after analyzing clinical cases of luminal and HER2 breast cancers, it was also found that the survival rates of the breast cancer patients with high expression of SENP2 and CD36 were significantly lower than those of the breast cancer patients with low expression of SENP2 and CD36, respectively.

According to the invention, the knocking-down of SENP2 in the luminal breast cancer cell line MCF-7 can obviously inhibit CD36 and gene expression related to fatty acid oxidation, and simultaneously weaken the oxygen consumption level and migration capacity of MCF-7 cells. The fibroblast growth factor 21(FGF21) is used for stimulating MCF-7 cells, and the fibroblast growth factor 21(FGF21) is found to be capable of remarkably improving the expression of CD36 and genes related to lipid metabolism, promoting fatty acid uptake and oxidation and enhancing the migration capacity of the MCF-7 cells, but the phenomena are obviously retarded in the MCF-7 cells with the knockdown SENP2, and the fact that the fibroblast growth factor 21(FGF21) plays a role depending on SENP2 is proved.

Further research shows that the fibroblast growth factor 21(FGF21) phosphorylates and modifies serine at the 123 th site of SENP2 protein through protein kinase ERK1/2, so that the uptake and oxidation of fatty acid are promoted, and the migration capacity of MCF-7 cells is enhanced; mutation of the site (serine at position 123 of SENP2 protein) can inhibit the function of fibroblast growth factor 21(FGF21) in promoting MCF-7 cell transfer. Therefore, the target designed for preventing and treating the luminal and HER2 breast cancer metastasis by aiming at the FGF21-SENP2 pathway has a certain application prospect.

The invention further provides a method for diagnosing the luminal and HER2 breast cancers and the migration or transfer capability thereof, predicting the migration or transfer capability of the luminal and HER2 breast cancers and predicting the survival rate of patients with luminal and HER2 breast cancers, which takes fibroblast growth factor 21FGF21 or a coding gene thereof, SENP2 protein or a coding gene thereof, SENP2 protein 123 th serine, fatty acid transport receptor CD36 protein or a coding gene thereof, or SENP2 protein and CD36 protein or a coding gene thereof as markers to carry out related diagnosis and prediction.

The invention further provides a method for screening a compound for inhibiting SENP2S123 phosphorylation modification, which takes FGF21 and SENP2S123 phosphorylation modification as markers to prepare an FGF21 neutralizing antibody and an antagonistic antibody of a receptor KLB thereof and screen the compound for inhibiting SENP2S123 phosphorylation modification.

Compared with the prior art, the invention has the following remarkable beneficial effects:

the invention discovers for the first time that: the phosphorylation modification site of the dessumoylation protease SENP2 is identified, the signal regulation and control pathway of FGF21-SENP2 axis is found, and the significance of the pathway in fatty acid metabolism and breast cancer metastasis is researched. The present invention will further promote the recognition of fatty acid metabolism and tumor metastasis. The invention analyzes the action and mechanism of FGF21-SENP2 axis on luminal and HER2 breast cancer metastasis, and can inspire a new strategy for inhibiting tumor metastasis. The invention proves that three inhibitors (GW9662, T0070907 and Ginsenoside Rh1) of PPAR gamma can obviously inhibit the invasion, migration and metastasis of breast cancer of luminal type and HER2 type.

The invention provides that the phosphorylation of 123 th serine (S123) of SENP2 is necessary for FGF21 to influence the metabolism and metastasis of the lipid of the breast cancer of the luminal and HER2 types. The invention provides a brand new mechanism for regulating and controlling the metastasis of luminal and HER2 breast cancers: FGF21 and SENP2S123 phosphorylation modification are used as marks to prepare FGF21 neutralizing antibody and antagonist antibody of receptor KLB, SENP2S123 phosphorylation modification inhibition compounds are screened, and the antibodies and the compounds can regulate and control luminal and HER2 breast cancer lipid metabolism and metastasis through an FGF21-SENP2 signal channel, so that the application prospect is achieved.

In conclusion, the FGF21-SENP2 axis signal regulation and control pathway disclosed by the invention has an important significance on the influence of lipid metabolism of luminal and HER2 breast cancer cells on the prevention and treatment of luminal and HER2 breast cancer metastasis.

In the present invention, the terms are explained as follows:

fibroblast growth factor 21(FGF21) is a Fibroblast growth factor 21, a protein encoded by the FGF21 gene present in mammals and a member of the endocrinological subfamily of the family of Fibroblast growth factors.

The SENP2 protein is Sentrin-specific protease 2, and is a specific SUMO protease encoded by the human SENP2 gene.

The fatty acid transport receptor CD36 is a protein encoded by the human CD36 gene, and is a member of the B class scavenger receptor family of cell surface proteins. SMA protein is a smoothened muscle actin, which is encoded by the human ACTA2 gene.

The ACSL protein is Acyl-CoA synthetic Long Chain Family Member 1, is a ligase coded by a human ACSL1 gene and is responsible for catalyzing free fatty acid into fatty Acyl-CoA ester.

The FA2H protein is FattyAcid 2-Hydroxylase, is fatty acid Hydroxylase encoded by human FA2H gene and is responsible for catalyzing and synthesizing 2-hydroxy sphingomyelin.

The ERK protein is extracellular signal-regulated kinase, and is mitogen-activated protein kinase coded by human MAPK gene.

GAPDH protein refers to Glyceraldehyde-3-Phosphate Dehydrogenase encoded by human GAPDH gene, i.e., glycoaldehyde-3-Phosphate Dehydrogenase.

The TUBULIN protein is Tubulin Beta Class I, and is a cytoskeletal TUBULIN encoded by the human TUBB gene.

PPAR protein refers to Peroxisome Proliferator Activated Receptor, which is encoded by human PPAR gene.

SCH772984(SCH) refers to a compound that specifically inhibits the activity of ERK1/2 phosphokinase.

GW9662 is a compound specifically inhibiting PPAR γ transcriptional activity and has a molecular formula of C₁₃H₉ClN₂O₃Molecular weight of 276.68, and chemical formula is shown as formula A:

t0070907 is a compound with specific PPAR γ transcriptional activity inhibiting effect and its molecular formula is C₁₂H₈ClN₃O₃Molecular weight of 277.66, and chemical formula B:

ginsenoside Rh1 refers to a compound which specifically inhibits PPAR gamma transcriptional activity and has a molecular formula of C₃₆H₆₂O₉Molecular weight of 638.87, and chemical structural formula as shown in formula C:

drawings

FIG. 1 is a schematic representation of the localization of SENP2 expression in HER2 type breast cancer specimens; panel a shows that SENP2 is expressed at a low level in non-invasive breast cancer nests and has no cell specificity; panel b shows that SENP2 is specific in aggressive cancer nests and expresses cells at the leading edge of the challenge at high levels; panel c shows that SMA expressing myoepithelial cells encapsulate breast cancer cells intact; panel d shows that the invading cancer cells break through the muscle epithelial cell envelope.

FIG. 2 is a schematic representation of SENP2 affecting the ability of breast cancer cells to migrate; wherein panel a is a crystal violet staining pattern of knockdown negative control, knockdown SENP2, and nominal-type breast cancer cells MCF-7 overexpressing SENP2 migrating through a transwell chamber; panel b is a statistical plot of cell counts in panel a. Knocking down SENP2 inhibits the migration ability of MCF-7 cells, and conversely, over-expressing SENP2 can promote the migration of MCF-7 cells.

FIG. 3 is a schematic diagram showing that SENP2 affects the transfer of MCF-7 cells in nude mice; after picture a is knocked down and negative control group luminal breast cancer cell MCF-7 is injected into a nude mouse by tail vein for 6 weeks, living body imaging shows that the lung and spine of the nude mouse have the formation of metastatic cancer nests; b, in-vivo imaging shows that no metastatic cancer nests appear after knocking down SENP2 group breast cancer cells MCF-7 and injecting the breast cancer cells into nude mice by tail vein for 6 weeks; and c, the picture shows that after the breast cancer cells MCF-7 of the SENP2 overexpression group are injected into nude mice by tail vein for 6 weeks, the living body imaging shows that the lung and the spine have the formation of metastatic cancer nests. Knocking down SENP2 in breast cancer cells completely inhibits the transfer of MCF-7 cells in nude mice, and conversely, over-expressing SENP2 can promote the transfer of MCF-7 cells in nude mice.

FIG. 4 is a graph showing the correlation between SENP2 expression levels and survival of both luminal and HER2 breast cancer patients. The overall survival rate of the breast cancer patients with SENP2 expression and HER2 expression in cancer tissues is significantly lower than that of the patients with SENP2 low expression. Wherein, the expression is low, the expression level of SENP2 is low, and the expression is high, the expression level of SENP2 is high.

FIG. 5 is a schematic diagram of SENP2 regulating the MCF-7 fatty acid metabolic pathway of a luminal breast cancer cell; analyzing genes with different expression amounts in MCF-7 cells with the knockdown negative control and the knockdown SENP2, wherein in the genes with the remarkably reduced expression level of the MCF-7 cells with the knockdown SENP2, a fatty acid metabolism pathway and a PPAR signal pathway are two most remarkably reduced signal pathways.

FIG. 6 is a schematic diagram of SENP2 regulating expression of key genes of fatty acid metabolic pathway. In the luminal breast cancer cell MCF-7 with reduced SENP2, the expression of key genes participating in the uptake and oxidation of fatty acid is obviously reduced; on the contrary, in MCF-7 cells over-expressing SENP2, the expression of key genes involved in fatty acid uptake and oxidation was significantly increased.

FIG. 7 is a schematic representation of the co-localization of SENP2 protein and CD36 protein in a HER2 type breast cancer patient specimen; wherein, a picture shows that SENP2 protein is specifically expressed in invasive cells in breast cancer tissues; panel b shows that CD36 protein is also specifically expressed in the same invasive cells in breast cancer tissues. Both SENP2 and the key fatty acid uptake protein CD36 were expressed in breast cancer cells with a propensity for metastasis.

FIG. 8 shows that SENP2 affects fatty acid uptake and oxidation function of luminal breast cancer cells MCF-7; wherein, a is a graph which is a graph of the capacity of the MCF-7 cells of the knockdown negative control group, the knockdown SENP2 and the over-expression SENP2 to take up exogenous fatty acid; panel b is a graph depicting the oxygen-consuming capacity of MCF-7 cells knockdown negative controls, knockdown SENP2, and overexpress SENP 2. The level of fatty acid uptake and mitochondrial oxidative phosphorylation of MCF-7 cells with knocked-down SENP2 is down regulated, and conversely, the fatty acid uptake and oxidative phosphorylation capacity of MCF-7 cells can be remarkably enhanced after SENP2 is over-expressed.

FIG. 9 is a schematic diagram of FGF21 promoting fatty acid uptake and expression of key genes in the oxidation process. As can be seen from FIG. 9, FGF21 can significantly increase the expression levels of CD36, ACSL1 and FA2H genes in the luminal breast cancer cell MCF-7.

FIG. 10 is a schematic representation of FGF 21-dependent SENP2 promoting expression of CD36 protein. FGF21 can promote protein expression of CD36, but in SENP 2-knockdown breast cancer cells, the effect of FGF21 on CD36 was significantly impaired.

FIG. 11 is a graph showing that the ability of FGF21 to promote uptake and oxidation of fatty acids by luminal breast cancer cells MCF-7 is dependent on SENP 2; wherein, a is a diagram showing the capability of MCF-7 cells to take in exogenous fatty acid after being treated by a control reagent or FGF 21; panel b is a graph showing the rate of oxygen consumption by MCF-7 cells treated with a control agent or FGF 21. FGF21 can promote the ability of luminal breast cancer cells MCF-7 to take up and oxidize fatty acids, but in SENP 2-knockdown MCF-7 cells, this effect of FGF21 is significantly impaired.

FIG. 12 is a schematic diagram showing that FGF21 promotes the migration of luminal breast cancer cells MCF-7 depending on SENP 2; panel a shows (i) a picture of the staining of crystal violet that knockdown negative control MCF-7 cells migrated through the transwell cell after negative control treatment, (ii) a picture of the staining of crystal violet that knockdown negative control MCF-7 cells migrated through the transwell cell after FGF21 treatment, (iii) a picture of the staining of crystal violet that knockdown SENP2 MCF-7 cells migrated through the transwell cell after negative control treatment, and (iv) a picture of the staining of crystal violet that knockdown SENP2 MCF-7 cells migrated through the transwell cell after FGF21 treatment; panel b is a statistical analysis of the cell counts of the four panels in panel a. FGF21 can promote the migration ability of MCF-7 cells, but in SENP2 knockdown MCF-7 cells, this effect of FGF21 is significantly attenuated.

FIG. 13 is a schematic representation of the effects of FGF21 on CD36 expression through the ERK1/2 signaling pathway. FGF21 can promote the expression of CD36 in luminal breast cancer cells MCF-7, and can block the promotion effect of FGF21 on CD36 after ERK1/2 inhibitor SCH is added.

FIG. 14 is a schematic diagram of mass spectrometry to identify serine 123 as the phosphorylation site of SENP 2.

FIG. 15 is a schematic diagram showing that FGF21 affects SENP2S123 phosphorylation modification through ERK1/2 signaling pathway. The inhibitor of ERK1/2 can block the promoting effect of FGF21 on phosphorylation modification of SENP2S 123.

Fig. 16 is a schematic diagram of FGF21 effect on CD36 protein expression through SENP2S123 phosphorylation modification. When SENP2S123 is mutated into S123A which can not be subjected to phosphorylation modification, the function of FGF21 in promoting CD36 protein expression is obviously blocked.

FIG. 17 is a schematic representation of the effects of FGF21 on the migratory capacity of the luminal breast cancer cell MCF-7 by SENP2S123 phosphorylation modification; in panels a, (i) a crystal violet staining pattern for SENP2 knockdown of MCF-7 cells migrating through the transwell cell after treatment with a negative control reagent, (ii) a crystal violet staining pattern for SENP2 knockdown of MCF-7 cells migrating through the transwell cell after treatment with FGF21, (iii) a crystal violet staining pattern for SENP2 knockdown and refilling wild-type SENP2 with MCF-7 cells after treatment with a negative control reagent, (iv) a crystal violet staining pattern for SENP2 knockdown and refilling wild-type SENP2 with MCF-7 cells after treatment with FGF21 and (v) a crystal violet staining pattern for SENP2 knockdown and refilling mutant-type SENP2 with MCF-7 cells after treatment with a negative control reagent, (vi) crystal violet staining pattern of re-complementation of MCF-7 cells with mutant SENP2 to knock down SENP2 migrating through the transwell chamber after FGF21 treatment; panel b is a statistical analysis of the cell counts of the six groups in panel a. When SENP2S123 is mutated into S123A which cannot be subjected to phosphorylation modification, the effect of FGF21 on promoting breast cancer cell metastasis is obviously blocked.

FIG. 18 is a graph showing the correlation of serum FGF21 concentrations and breast cancer metastasis in luminal and HER2 breast cancer patients; wherein panel a shows FGF21 concentration values in serum of patients with non-metastatic and metastatic luminal and HER2 type breast cancer; b is a graph showing the proportion of non-metastatic and metastatic patients when the concentration of FGF21 in serum is statistically analyzed to be less than or greater than 50pg/ml, respectively. Among the luminal and HER2 breast cancer patients, the mean concentration of FGF21 in the serum of non-metastatic breast cancer patients is lower than that of metastatic breast cancer patients, and when the concentration of FGF21 in the serum is less than 50pg/ml, only about 40% of breast cancer patients develop tumor metastasis, while when the concentration of FGF21 in the serum is greater than 50pg/ml, nearly 80% of breast cancer patients develop tumor metastasis.

Figure 19 is a graph of the results of three inhibitors of PPAR γ significantly slowing/inhibiting migration and metastasis of luminal breast cancer cells; wherein, in a panels, (i) is a graph showing the crystal violet staining of MCF-7 cells that migrated through the transwell cell after treatment with a negative control reagent and a blank reagent, (ii) is a graph showing the crystal violet staining of MCF-7 cells that migrated through the transwell cell after treatment with a negative control reagent and GW9662 (1. mu.M), (iii) is a graph showing the crystal violet staining of MCF-7 cells that migrated through the transwell cell after treatment with a negative control reagent and T0900077 (1. mu.M), (iv) is a graph showing the crystal violet staining of MCF-7 cells that migrated through the transwell cell after treatment with a negative control reagent and Ginsenoside Rh1 (100. mu.M), (v) is a graph showing the crystal violet staining of MCF-7 cells that migrated through the transwell cell after treatment with FGF21(200ng/ml) and a blank reagent, (vi) is a graph showing the crystal violet staining of MCF-7 cells that migrated through the transwell cell after treatment with FGF21(200ng/ml) and GW9662 (1. mu.M), (vii) (viii) is a graph of crystal violet staining of MCF-7 cells migrating through the transwell chamber after FGF21(200ng/ml) and T0070907(1 μ M) treatment, (viii) is a graph of crystal violet staining of MCF-7 cells migrating through the transwell chamber after FGF21(200ng/ml) and GinsenosideRh1(100 μ M) treatment; panel b is the result of statistical analysis of the cell counts of the eight groups in panel a; in the figure, (i) the lung metastasis cancer nest of the nude mouse is displayed by living body imaging after MCF-7 is injected into the nude mouse through a tail vein and then negative control reagent is injected into the abdominal cavity for 6 weeks, (ii) the lung metastasis cancer nest of the nude mouse is displayed by living body imaging after MCF-7 is injected into the nude mouse through the tail vein and then FGF21 and blank reagent are injected into the abdominal cavity for 6 weeks, (iii) the lung metastasis cancer nest of the nude mouse is displayed by living body imaging after MCF-7 is injected into the nude mouse through the tail vein and then FGF21 and GW9662 reagent is injected into the abdominal cavity for 6 weeks, (iv) the lung metastasis cancer nest of the nude mouse is displayed by living body imaging after MCF-7 is injected into the nude mouse through the tail vein and then FGF21 and T0070907 reagent are injected into the abdominal cavity, and (v) the lung metastasis cancer nest of the nude mouse is displayed by living body imaging after MCF-7 is injected into the nude mouse through the tail vein and then FGF21 and Ginsenoside 1 reagent are injected into, wherein the numbers below the c chart are 10000, 20000, 30000, 40000 and 50000 from left to right in sequence. The results show that three PPAR gamma inhibitors (GW9662, T0070907 and Ginsenoside Rh1) can effectively inhibit the in vitro migration and in vivo metastasis of breast cancer cells MCF-7.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

In the following specific examples, the breast cancer cells MCF-7 are classified into the following 5 types:

knock-down negative control (shNC), which refers to a stably expressing cell line established using interfering RNA that is not directed against any gene sequence;

knockdown of SENP2(shSENP2), which refers to a stably expressing cell line established using interfering RNAs designed against SENP2 encoding gene sequences;

over-expressing SENP2(OE-SENP2), which refers to a cell line stably expressing SENP2 protein established using a method of lentiviral infection;

knocking down SENP2 to complement SENP2 wild type (shSENP2-WT), which is a cell line stably expressing SENP2 wild type protein established by a method of lentivirus infection on the basis of knocking down SENP2(shSENP2) cells;

knocking down SENP2 to complement SENP2 mutant (shSENP2-S123A), which is a cell line stably expressing SENP2 mutant protein established by a method of lentivirus infection on the basis of knocking down SENP2(shSENP2) cells;

the relationship between "OE-SENP 2" and "shSENP 2-WT" or "shSENP 2-S123A":

OE-SENP2 refers to a cell line which expresses exogenous SENP2 wild-type protein in large quantities by a method of lentivirus infection without removing endogenous SENP2 of the cells; shSENP2-WT refers to a cell line which can largely express exogenous SENP2 wild-type protein through a method of lentivirus infection under the condition of clearing endogenous SENP2 of cells; shSENP2-S123A refers to a cell line which can largely express exogenous SENP2 mutant protein by a method of lentivirus infection under the condition of removing endogenous SENP2 of cells.