The Effects And Underlying Mechanism Of Leptin On PKM2 Expression And Epithelial-mesenchymal Transition In Breast Cancer Cells

Objective To explore the role of PKM2 in Leptin-induced EMT of human breast cancer cells MCF-7 and SK-BR-3.Methods 1. Expression of OB-Rb and OB-Rt protein of human breast cancer MCF-7, SK-BR-3 and DMA-MB-468 cells were detected by WB and immunofluorescence assay. Morphological changes of MCF-7, SK-BR-3 and MDA-MB-468 cells were determined after Leptin treatment. EMT-associated markers were detected by WB after Leptin treatment.2. Effects of Leptin on PKM2 mRNA and protein expression in MCF-7 and SK-BR-3 cells were investigated by QRT-PCR and WB.3. Effects of Leptin on migration and invasion of MCF-7 and SK-BR-3 cells were investigated by wound healing assay and matrigel invasion assay after PKM2-siRNA treatment. EMT-associated markers were detected by WB after PKM2-si RNA treatment.4. Expression of PKM2 was detected by WB after Leptin addition with PI3K/AKT or JAK/STAT or MAPK/ERK1/2 signaling pathway inhibitors treatment, and then the effects of Leptin on expression of PKM2 and PI3K/AKT signaling pathway were investigated by WB after OBR blocking antibodies treatment. Expression of PKM2 and EMT-associated markers were detected by WB after PI3K/AKT signaling pathway inhibitor treatment.5. Nude mice breast cancer xenograft model was established using SKBR3-shPKM2 cell line. Diameters of tumor were measured and survival time of mice was also monitored. Expression of e-cadherin, vimentin and fibronectin were detected by immunohistochemical analysis. Lung and liver metastasis were determined by HE staining.Results 1. WB and immunofluorescence analysis indicated that both OB-Rb and OB-Rt expressed in all of MCF-7, SK-BR-3 and MDA-MB-468 cells. Treated with 200ng/ml Leptin induced EMT-related morphological changes in MCF-7, SK-BR-3 and MDA-MB-468 cells. Leptin up-regulated vimentin and fibronectin, while down-regulated e-cadherin of MCF-7, SK-BR-3 and MDA-MB-468 cells.2. QRT-PCR and WB analysis indicated that Leptin up-regulated PKM2 mRNA and protein expression in MCF-7 and SK-BR-3 cells.3. Leptin promoted migration and invasion of MCF-7 and SK-BR-3 cells, however treatment with PKM2-siRNA obviously abolished the effect. Leptin up-regulated vimentin and fibronectin, while down-regulated e-cadherin, noteworthily, this effect was weakened by PKM2-siRNA.4. PKM2 Up-regulation induced by Leptin was partially abolished by PI3K/AKT signaling pathway inhibitor. Leptin-induced p-AKT and PKM2 expression were obviously abolished by anti-ObRs antibody. Leptin up-regulated vimentin and fibronectin, while down-regulated e-cadherin; however, this effect was reduced by PI3K/AKT signaling pathway inhibitor.5. Tumor volume of SKBR-3-shPKM2 group was obviously smaller than SKBR-3, SKBR-3-shLV groups. SKBR-3-shPKM2 treatment resulted in a distinct decrease in tumor size compared with SKBR-3 and SKBR-3-shLV groups. The survival curve indicated that mice in SKBR-3-shPKM2 group lived longer than SKBR-3 and SKBR-3-shLV groups. HE staining showed SKBR-3-shPKM2 resulted in suppression metastasis in lungs and livers compared with SKBR-3 and SKBR-3-shLV groups. Immunohistochemistry staining showed the expression of e-cadherin was increased in SKBR-3-shPKM2 group, while vimentin and fibronectin were obviously decreased in SKBR-3 and SKBR-3-shLV groups.Conclusion 1. Leptin induced EMT of human breast cancer cells MCF-7, SK-BR-3 and MDA-MB-468.2. Leptin promoted EMT of human breast cancer cells via up-regulation PKM2 expression via activation of PI3K/AKT signaling pathway.3. RNA interference against PKM2 suppressed tumor growth, metastasis and increased the survival of mice in xenografts. PKM2 may be a potential therapeutic target for breast cancer.