Study On Mechanism Of Adrenergic Signal Regulation Of Tumor Angiogenesis

Tumor angiogenesis play a vital role in tumor growth and metastasis. Undernormal physiology, angiogenesis is strictly controlled by the regulatory mechanismsin the body. In contrast, tumor angiogenesis, once initiated, continues indefinitely.Tumor angiogenesis is an intricate process involving numerousproangiogenic orantiangiogenic factors and multiple signaling pathways. In the recent studies, it isindicated that Notch signaling pathway actively participates in regulation of tumorangiogenesis.Stress occurs when an individual perceives that the demands of an externalsituation are beyond his or her perceived ability to cope with them, such as trauma,surgery, blood loss, infection, poisoning, anoxia, starvation, depression, and anxiety.Stress triggers a response of the hypothalamic-pituitary-adrenal axis (HPA), resultingin increased release of stress-related hormones, such as cortisol and catecholamine.Recent studies revealed that stress-related catecholamine plays a key role in tumorangiogenesis. Experimental studies demonstrated that chronic restraint stress, whichresults in high levels of tissue catecholamine [epinephrine (EPI) and norepinephrin(NE)], enhanced vascular density in ovary cancer xenograft tissues in mice. Thestudies by our group and others show that catecholamine upregulated the expressionof VEGF in a variety of tumor cell lines and promote tumor angiogenesis. β-blockercould reverse angiogenensis induced by chronic stress, implicating the potential rolesof catecholamine and β-adrenergic receptor (β-AR)-mediated signaling pathway intumor angiogenesis. However, the precise mechanisms, by which catecholamineregulates tumor angiogenesis, are not fully understood.In the current study, we uncovered a novel mechanism of tumor angiogenesisregulated by catecholamine. We show that catecholamine upregulated the expressionof Jagged-1in breast cancer cells through the β2-AR-PKA-mTOR pathway, activatedNotch signaling pathway in adjacent vascular endothelial cells, and promoted tumorangiogenesis. Major findings are as follows:1. Catecholamine induced tumor angiogenesis in a paracrine independent manner.We treated human breast cancer cell line MDA-453with NE. The cells were collectedat the different time points, total RNA extracted, and the expression of VEGF mRNAdetected by RT-PCR. The results demonstrate that NE significantly stimulated theexpression of VEGF in MDA-453cells. Then we treated HUVEC with thesupernatant from NE-stimulated MDA-453cells and observed the capillary-like network formation of HUVEC. The data show that the supernatant from NE-treatedMDA-453cells remarkably induced the formation ofcapillary-like tubular structuresby HUVEC, implying that NE promoted angiogenesisin a paracrine manner through inducing the VEGF production. Interestingly, an evenmore dramatic pro-angiogenic effect was seen after coculture of HUVEC withMDA-453cells in the presence of NE, as demonstrated by measuring the number ofendothelial cellsprouts and evaluating the integrity of capillary-like tubular structures.Similar results were observed in coculture of HUVEC with MCF-7cells. These datasuggested that tumor cell-endothelial cell contacts may be more important in thedevelopment of neovasculature in tumor induced by catecholamine.2. Catecholamine modulates the Jagged-1/Notch signaling in a breast tumorcell-endothelial cell contact-dependent manner. HUVEC were transfected with Notchluciferase reporter pGA981-6and then cocultured withMCF-7cells in the presence orabsence of NE. Luciferase assays show that NE induction did not evidently affect theNotch reporter activities in HUVEC cultured alone. However, when HUVEC werecocoltured with MCF-7cells, the luciferase activities was dramatically elevated inresponse to NE. Similar results were observed in coculture of HUVEC withMDA-453cells. These data suggest that interactions between vascular endothelialcells and breast cancer cells in response to NE trigger the activation of the Notchsignaling pathway. We speculated that NE may upregulate the expression of the Notchligands in breast cancer, resulting in the activation of Notch pathway in vascularendothelial cells. Thus, we treated breast cancer cells with catecholamine andanalyzed the expression of the Notch ligand Jagged-1. The data show that theJagged-1expression was markedly enhanced in response to catecholamine stimulationin most breast cancer cell lines. Knockdown of the Jagged-1expression in breastcancer cells greatly inhibited NE-induced Notch reporter activities in HUVEC. Theseresults suggested that activationof Notch pathway in vascular endothelial cells istriggered by NE-induced Jagged-1expression in breast cancer cells.3. Expression of Jagged-1in breast cancer cells is upregulated by catecholaminethrough activation of β2-AR-PKA-mTOR pathway. We performed real-time RT-PCRto detect catecholamine-induced the expression of Jagged-1mRNA in variety ofbreast cancer cell lines. The data demonstrate that catecholamine upregulated theJagged-1expression at transcriptional level in breast cancer cells. Animal experimentsshow that isoproterenol (ISO) treatment daily significantly upregulated the Jagged-1 expression in human breast cancer xenograft tissues at both transcriptional and proteinlevels. Additionally, tumor microvessel (CD31positive) density was also greatlyenhanced by ISO treatment. Luciferase assays show that the JAG-1promoter activitieswere markedly enhanced in response to NE stimulation in breast cancer cells. Theinhibitors of β2-AR, PKA, and mTOR could reverse NE-induced the Jagged-1upregulation in breast cancer cells, indicating that the β2-AR-PKA-mTOR pathwayparticipates in this process.4. Catecholamine promotes tumor angiogenesis through regulating theJagged-1/Notch intercellular signaling between breast cancer cells and endothelialcells. The MDA-453cells were stimulated by NE, then harvested using enzyme-freecell dissociation buffer, and cocultured with HUVEC. Compared with vehiclestimulated MDA-453cells, coculture of HUVEC with NE-stimulated MDA-453cellsmore effectively promoted angiogenesis in vitro. In addition, the Notch-dependenttranscription activities were also greatly enhanced in the HUVEC cocultrued with NEtreated MDA-453cells. Similar data were obtained by coculturing HUVEC withNE-treated MDA-231cells. Enforced expression of Jagged-1in breast cancer cellsdramatically promoted the formation of extensive capillary networks by theendothelial cells in coculture systems. Knockdown of the Jagged-1expressionmarkedly abolished NE-induced Jagged-1upregulation in breast cancer cells,impaired the effects of NE on capillary-like structure formationby the HUVEC incoculture systems, and inhibited NE-induced Notch pathway activation.In conclusion, the main findings in the present study include:1. Catecholamineinduces tumor angiogenesis through a tumor cell-endothelial cell contact dependentmanner;2. Catecholamine upregulates the Jagged-1expression at transcriptional andprotein levels in breast cancer cells by triggering β2-AR-PKA-mTOR pathway;3.Catecholamine triggers the angiogenic switch by upregulating the Jagged-1expression in breast cancer cells, resulting in the activation of the Notch signalingpathway in adjacent endothelial cells.