title: Sphingosine kinase 1 contributes to leptin-induced STAT3 phosphorylation through IL-6/gp130 transactivation in oestrogen receptor-negative breast cancer.
abstract: Obesity is a known risk factor for breast cancer. We have recently identified that adipokine leptin regulates the expression of a proto-oncogenic enzyme sphingosine kinase 1 (SK1). Signal transducer and activator of transcription 3 (STAT3) has been linked to breast cancer progression and here we investigate the mechanism of leptin-induced STAT3 activation in ER-negative breast cancer. Gene and protein expression in human primary and secondary breast cancer tissues was analysed using quantitative real-time polymerase chain reaction (qRT-PCR) assay and immunofluorescence. Leptin-induced signalling was analysed in human ER-negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays. Gene expression and receptor signalling was modified using small interfering RNA and neutralising antibodies. In human ER-negative breast tumours and lymph node metastases, the expression of leptin receptor significantly correlated with SK1. In ER-negative breast cancer cells, SK1 knockdown led to a significant reduction in leptin-induced STAT3 phosphorylation. Knockdown of another known activator of STAT3 signalling, gp130 also resulted in a significant decrease in leptin-induced STAT3 phosphorylation. ELISA assay showed that leptin produces a significant amount of IL-6 in an SK1-dependent manner. IL-6 neutralising antibodies significantly reduced p-STAT3. Immunofluorescent staining of human primary and secondary breast tumours showed significant correlation between SK1 and IL-6 (P < 0.001), SK1 and p-STAT3 (P < 0.01) and IL-6 and p-STAT3 (P < 0.01). Our findings demonstrate that leptin-induced STAT3 is partially cross activated through SK1-mediated IL6 secretion and gp130 activation. Positive correlations in human tissues suggest the potential significance of this pathway in ER-negative breast cancer.
authors.affiliation: Department of Surgery and Cancer, Hammersmith Hospital, Imperial College London, 1st Floor ICTEM, Ducane Road, London, W120NN, UK, h.alshaker10@imperial.ac.uk.
authors.forename: Heba
authors.initials: H
authors.lastname: Alshaker
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.forename: Adam E
authors.initials: AE
authors.lastname: Frampton
authors.forename: Jonathan
authors.initials: J
authors.lastname: Krell
authors.forename: Jonathan
authors.initials: J
authors.lastname: Waxman
authors.forename: Mathias
authors.initials: M
authors.lastname: Winkler
authors.forename: Justin
authors.initials: J
authors.lastname: Stebbing
authors.forename: Colin
authors.initials: C
authors.lastname: Cooper
authors.forename: Ernesto
authors.initials: E
authors.lastname: Yagüe
authors.forename: Dmitri
authors.initials: D
authors.lastname: Pchejetski
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D050822:Cytokine Receptor gp130 / Q000096:biosynthesis*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics; D020738:Leptin / Q000235:genetics*; D008207:Lymphatic Metastasis; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics; D054411:Receptors, Leptin / Q000235:genetics; D050796:STAT3 Transcription Factor / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction; D015533:Transcriptional Activation / Q000235:genetics; D000099157:Sphingosine Kinase
chemical_list: D050822:Cytokine Receptor gp130; D015850:Interleukin-6; D020738:Leptin; D017853:Phosphotransferases (Alcohol Group Acceptor); D011960:Receptors, Estrogen; D054411:Receptors, Leptin; D050796:STAT3 Transcription Factor; D000099157:Sphingosine Kinase; C494086:STAT3 protein, human