Research On Role Of PPARδ In Tumor Genesis And Development And Its Related Mechanisms

BackgroundPeroxisome proliferators-activated receptors(PPARs)are ligand-activated nuclear transcription factor superfamily,including three subtypes of PPARα,PPARβ/δ and PPARγ,of which PPARδ is the most studied.PPARδ plays an important role in the regulation of inflammation,arthrosclerosis,insulin resistance,glucose metabolism,tumor and obesity.Recent studies have shown that PPAR antagonists may have new applications in the clinical treatment of cancer.According to the 2022 annual report released by A Cancer journal for Clinicians(CA),there are 2.26 million new cases of breast cancer(BRCA)in the world,surpassing lung cancer for the first time and becoming “No.1 Cancer” in the world.As the most common malignant tumor in women,BRCA is also one of the leading causes of cancerrelated deaths in women.Nearly 680,000 women were reported to have died from breast cancer in 2021.At the same time,young women diagnosed with breast cancer(age of onset< 40 years)were more likely to have a poor clinical prognosis and early metastasis.In recent years,immunotherapy,especially immune checkpoint blockade therapy,has provided new treatment ideas and methods for lung,skin,bowel and some breast cancers.In 2019,the U.S.Food and Drug Administration(FDA)approved the use of the immune checkpoint inhibitor atezolizumab,an anti-PD-L1 monoclonal antibody,for the treatment of metastatic triple-negative breast cancer;The combination of atezolizumab and paclitaxel significantly prolonged survival in these breast cancer patients.However,since anti-PD-L1 monoclonal antibody drugs have only been shown to be effective in some patients with triple-negative breast cancer(approximately 18%),other tumor therapeutic targets need to be explored to improve patient outcomes Although PPARδ and its associated signaling pathways have been shown to play important regulatory roles in a variety of malignancies,their role in the development of breast cancer,potential impact on immune invasion,and its predictive ability as a prognostic marker remain unclear.In the field of non-solid tumore,studies have also shown that the PPARδexpression level in chronic lymphocytic leukemia(CLL)is higher than that in normal lymphocytes and other hematological tumors,and that it is directly related to clinical scores.CLL is a malignant tumor mainly caused by clonal proliferation of B lymphocytes,which proliferate and accumulate malignantly in tissues and organs such as peripheral blood,bone marrow,liver,spleen and lymph nodes.Advanced tumor progression can cause bone marrow failure,leading to severe anemia,bleeding,infection,and even death.Combined chemotherapy containing purine analogues or alkylating agents is currently the main clinical treatment for CLL patients,which may achieve good efficacy in early stage,but regardless of initial efficacy,CLL might progress to an active phase and become refractory or relapsing.Therefore,new treatment ideas and treatments are urgently needed to deal with relapsed or refractory CLL.Although previous studies speculated that targeted inhibition of PPARδ may be effective in CLL,the effect of PPARδ on the development of CLL tumor cells and its mechanism are not clear.ObjectiveTo explore the possible influence of PPARδ downregulation on tumorigenesis and its related molecular mechanism through analyzing the association between PPARδexpression level and clinical outcome in patients with breast cancer,a solid tumor,and CLL,a non-solid tumor,and the role of PPARδ inhibitor in vivo and in vitro experiments,so as to evaluate the therapeutic effect of PPARδ inhibitors on tumors and the predictive ability of PPARδ expression level on tumor prognosis.Methods1.Research methods for breast cancer1.1 Bioinformatics analysis was used to compare the difference in expression levels of PPARδ in tumor samples obtained from the TCGA database and paracancerous samples and its possible association with tumorigenesis,development and prognosis.At the same time,the expression level of PPARδ in breast cancer cell lines and normal breast cell lines was detected by cell culture,immunohistochemical staining,and Western blot.1.2 A mouse model of allogeneic tumors was established to verify whether PPARδinhibitor(GSK3787)had an effect on breast cancer progression.Subsequently,cell culture,Western blot,and flow cytometry were used to verify whether GSK3787 could directly affect breast cancer cell lines in vitro experiments.Finally,a mouse model of allogeneic tumor was established again using NOG mice to verify whether the PPARδinhibitor GSK3787 had an effect on breast cancer progression.1.3 Cell culture,Western blot,protein chip technology,ELISA detection and multiple sets of animal experiments were used to explore and verify the secretion of JAK/STAT pathway and its downstream immune-related cytokines.1.4 Bioinformatics methods were employed to predict the association between PPARδ and breast cancer clinical data,so as to provide a theoretical basis for using PPARδ as a new prognostic evaluation index and a new immunotherapy target.2.Research methods for CLL2.1 Cell culture and flow cytometry were used to detect the difference in the expression of PPARδ between mouse CLL cell lines and normal mouse B cells,and between B cells in the peripheral blood of CLL patients and B cells in the peripheral blood of healthy adults.2.2 Cell culture,Western blot,flow cytometry and other experimental methods were used to verify the effects of GSK3787 on proliferation and apoptosis of CLL cell lines.2.3 Cell co-culture and flow cytometry were employed to analyze the relationship between PPARδ expression level and T cell proliferation in CLL cell lines.2.4 Cell culture and Transwell experimental methods were adopted to explore the effect of GSK3787 on the migration ability of CLL cell lines in order to evaluate its control effect on CLL distant metastasis.Results1.Results of breast cancer1.1 Breast cancer tissues had higher expression of PPARδ compared with paracancerous tissues,and breast cancer cell lines had higher expression of PPARδcompared with normal breast cell lines.The high expression of PPARδ in breast cancer may be closely related to clinical score and prognosis.1.2 In animal experiments,GSK3787 inhibited PPARδ expression and breast cancer progression.However,in vitro experiments,although GSK3787 inhibited PPARδ expression,it did not affect the survival status of breast cancer cell lines.1.3 Repeated animal experiments using NOG mice revealed that GSK3787 could not inhibit breast cancer progression,so it was speculated that the anti-tumor effect of GSK3787 might rely on the immune system.Bioinformatics analysis predicted that its anti-tumor effect might be related to the infiltration of immune cells such as Treg cells,NK cells,and B cells and the status of JAK/STAT pathway.1.4 Multiple sets of related experiments verified the association between the antitumor effect of GSK3787 in breast cancer models and the inhibition of JAK1/STAT1 and JAK1/STAT2 pathways,which could affect the cytokines IL2,IL12,IL15,IFNγ,IL4,IL10,TGFβ,CXCR4,CXCL13 and C-AMP,thereby regulating the infiltration of NK cells,B cells and Treg cells in tumor tissues.2.Results of CLL2.1 CLL cell lines had higher expression of PPARδ compared with normal mouse B cells,and B cells in the peripheral blood of CLL patients had higher expression of PPARδ compared with B cells in the peripheral blood of healthy adults.2.2 In vitro experiments proved that GSK3787 inhibited the proliferation and migration ability of CLL cell lines while promoting apoptosis.2.3 There might be an immune synergistic mechanism in the effect of GSK3787 on CLL cell lines,in which high levels of PPARδ in CLL cell lines inhibited T cell proliferation,which could be partially relieved by GSK3787.Conclusion1.Inhibition of PPARδ expression in tumor cells can inhibit tumor growth in breast cancer mouse models with normal immune systems,which rely on the participation of the immune system.2.In breast cancer tissues,the downregulation of PPARδ can inhibit the activation of JAK1/STAT1 and JAK1/STAT2 pathways,and then affect the secretion of their downstream related cytokines(IL2,IL4,CXCR4,etc.),thereby upregulating NK cells and downregulating the infiltration of B cells and Treg cells in tumor tissues.Therefore,in the treatment of breast cancer,PPARδ may become a new prognostic evaluation index and immunotherapy target.3.Inhibition of PPARδ expression may be a new idea for the clinical treatment of CLL,and its mechanism may be directly inducing apoptosis of tumor cells and indirectly promoting the proliferation of T cells.4.Although inhibiting PPARδ expression plays the role of inhibiting the cancer growth through different mechanisms in breast cancer and chronic lymphocytic leukemia,its therapeutic effect on tumors has been preliminarily confirmed.