Sam68 Mediates the Activation of Insulin and Leptin Signalling in Breast Cancer Cells

Obesity is a well-known risk factor for breast cancer development in postmenopausal women. High insulin and leptin levels seem to have a role modulating the growth of these tumours. Sam68 is an RNA-binding protein with signalling functions that has been found to be overexpressed in breast cancer. Moreover, Sam68 may be recruited to insulin and leptin signalling pathways, mediating its effects on survival, growth and proliferation in different cellular types. We aimed to study the expression of Sam68 and its phosphorylation level upon insulin and leptin stimulation, and the role of Sam68 in the proliferative effect and signalling pathways that are activated by insulin or leptin in human breast adenocarcinoma cells. In the human breast adenocarcinoma cell lines MCF7, MDA-MB-231 and BT-474, Sam68 protein quantity and gene expression were increased upon leptin or insulin stimulation, as it was checked by qPCR and immunoblot. Moreover, both insulin and leptin stimulation promoted an increase in Sam68 tyrosine phosphorylation and negatively regulated its RNA binding capacity. siRNA was used to downregulate Sam68 expression, which resulted in lower proliferative effects of both insulin and leptin, as well as a lower activation of MAPK and PI3K pathways promoted by both hormones. These effects may be partly explained by the decrease in IRS-1 expression by down-regulation of Sam68. These results suggest the participation of Sam68 in both leptin and insulin receptor signaling in human breast cancer cells, mediating the trophic effects of these hormones in proliferation and cellular growth.肥胖是绝经后女性乳腺癌发生的公认危险因素。高胰岛素与瘦素（leptin）水平似乎在调控此类肿瘤的生长过程中发挥作用。Sam68是一种兼具信号转导功能的RNA结合蛋白（RNA-binding protein），现已被发现在乳腺癌组织中呈过表达状态。此外，Sam68可被招募至胰岛素与瘦素信号通路中，介导其在不同细胞类型中对细胞存活、生长及增殖的调控作用。本研究旨在探究人乳腺腺癌细胞中，Sam68的表达及其在胰岛素与瘦素刺激下的磷酸化水平，以及Sam68在胰岛素或瘦素激活的增殖效应与信号通路中所发挥的作用。在人乳腺腺癌细胞系MCF7、MDA-MB-231及BT-474中，经瘦素或胰岛素刺激后，Sam68的蛋白含量与基因表达水平均有所升高，该结果通过实时定量聚合酶链反应（quantitative PCR，qPCR）与免疫印迹（immunoblot）实验得以验证。此外，胰岛素与瘦素的刺激均可促进Sam68的酪氨酸磷酸化水平升高，并负向调控其RNA结合能力。本研究采用小干扰RNA（small interfering RNA，siRNA）下调Sam68的表达，结果发现胰岛素与瘦素介导的增殖效应均有所减弱，同时这两种激素所激活的丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）与磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）通路的活化水平也均降低。上述效应可部分通过下调Sam68表达所导致的胰岛素受体底物1（insulin receptor substrate 1，IRS-1）表达降低来解释。上述研究结果表明，Sam68可参与人乳腺癌细胞中瘦素与胰岛素受体的信号转导过程，介导这两种激素在细胞增殖与生长过程中的营养支持效应。