Sympathetic Neurotransmitters and Tumor Angiogenesis—Link between Stress and Cancer Progression

Recent evidence supports a longstanding hypothesis that chronic stress can influence tumor growth and progression. It has been shown that sympathetic neurotransmitters, such as catecholamines and neuropeptides, can affect both cancer cell growth and tumor vascularization. Depending on neurotransmitter and type of tumor, these effects can be both stimulatory and inhibitory. Norepinephrine (NE) and epinephrine (E) are potent stimulators of vascularization, acting both by inducing the release of angiogenic factors from tumor cells and directly on endothelial cell (EC) functions. As a result, activation of the adrenergic system increases growth of various types of tumors and has been shown to mediate stress-induced augmentation of tumor progression. Dopamine (DA), on the other hand, interferes with VEGF signaling in endothelial cells, blocks its angiogenic functions and inhibits tumor growth. Another sympathetic neurotransmitter coreleased with NE, neuropeptide Y (NPY), directly stimulates angiogenesis. However, proangiogenic actions of NPY can be altered by its direct effect on tumor cell proliferation and survival. In consequence, NPY can either stimulate or inhibit tumor growth, depending on tumor type. Hence, sympathetic neurotransmitters are powerful modulators of tumor growth and can become new targets in cancer therapy

The Role of Neuropeptide Y (NPY), its’ Y5/Y2R Receptors in Neuroblastoma cell migration

Neuroblastoma is a pediatric tumor that is known for its’ variable clinical presentation, ranging from spontaneous regression to aggressive disease and subsequent metastasis. It is the most common childhood cancer after leukemia and brain tumors. Neuroblastoma tumors originate from sympathetic precursors and are known to express the sympathetic neurotransmitter Neuropeptide Y (NPY). NPY acts through the Y1-Y5 receptors. Tumor cell proliferation, survival, migration and angiogenesis are effects known to be mediated via two specific NPY receptors, Y2R and Y5R. In neuroblastoma, Y2R is constitutively expressed and maintains cell proliferation, while the expression of Y5R is inducible as a survival factor after periods of cellular stress, such as chemotherapy. These findings led us to believe that NPY via its two receptors may play a role in Neuroblastoma dissemination and metastasis. Our goal was to further elucidate the involvement of NPY in Neuroblastoma cell migration. Using Transwell migration assay on SK-N-BE-2 Neuroblastoma cells, we found that NPY stimulated spontaneous migration, while Y5 and combined Y2/Y5R antagonism significantly decreased cell motility. On the other hand, in SK-N-AS Neuroblastoma cells, exogenous NPY did not stimulate migration, yet the basal cell motility was significantly decreased in the presence of the combined Y2R and Y5R antagonists. These data indicate that in SK-N-AS cells, the endogenous NPY released from tumor cells is sufficient to saturate its receptors and stimulate cell migration. Additionally, we used CHO-K1-Y2R-GFP and CHO-K1-Y5R-GFP transfected cells as a model to investigate changes in cell morphology and phenotype induced by individual NPY receptors. Immunohistochemistry showed giant cells and the increased presence of migratory cells in the Y5R transfected group. These cells also expressed multiple cell membrane extensions and filopodia, not present in CHO-K1 cells transfected with other NPY receptors or GFP alone. A strong co-localization of Y5R and F-Actin signal was detected within these Filopodia in addition to the leading edges and trailing ends of migratory cells, further supporting a role for Y5R in migration and cytoskeleton regulation. Further investigation is required to clarify the role of NPY in metastasis. Currently, there is ongoing work to develop NB cell lines with NPY and Y5R knockout using CRISPR/Cas9 technology, which will be used to elucidate the role of the NPY/Y5R pathway in this process. Moreover, additional studies are needed to understand the role of other NPY receptors and its signaling mediators, such RhoA, in cytoskeletal regulation as it relates to metastasis in NB

Stress hormone epinephrine enhances adipogenesis in murine embryonic stem cells by up-regulating the neuropeptide Y system.

Prenatal stress, psychologically and metabolically, increases the risk of obesity and diabetes in the progeny. However, the mechanisms of the pathogenesis remain unknown. In adult mice, stress activates NPY and its Y2R in a glucocorticoid-dependent manner in the abdominal fat. This increased adipogenesis and angiogenesis, leading to abdominal obesity and metabolic syndrome which were inhibited by intra-fat Y2R inactivation. To determine whether stress elevates NPY system and accelerates adipogenic potential of embryo, here we "stressed" murine embryonic stem cells (mESCs) in vitro with epinephrine (EPI) during their adipogenic differentiation. EPI was added during the commitment stage together with insulin, and followed by dexamethasone in the standard adipogenic differentiation medium. Undifferentiated embryonic bodies (EBs) showed no detectable expression of NPY. EPI markedly up-regulated the expression NPY and the Y1R at the commitment stage, followed by increased Y2R mRNA at the late of the commitment stage and the differentiation stage. EPI significantly increased EB cells proliferation and expression of the preadipocyte marker Pref-1 at the commitment stage. EPI also accelerated and amplified adipogenic differentiation detected by increasing the adipocyte markers FABP4 and PPARγ mRNAs and Oil-red O-staining at the end of the differentiation stage. EPI-induced adipogenesis was completely prevented by antagonists of the NPY receptors (Y1R+Y2R+Y5R), indicating that it was mediated by the NPY system in mESC's. Taken together, these data suggest that stress may play an important role in programming ESCs for accelerated adipogenesis by altering the stress induced hormonal regulation of the NPY system

Epinephrine up-regulated the NPY system during mESCs adipogenesis.

<p>(A) Schematic representation of the strategy for differentiation of mESCs into adipocytes. (B–G) Gene expression analysis of NPY system during mESCs adipogenesis. EBs were treated with insulin (standard, STD) or insulin plus 1 µM EPI (EPI) from day 7 to day 17 during commitment stage, mRNA levels of NPY (B), Y1R (C), Y2R (D) and DPPIV (E) were measured by real time PCR. β-actin expression was used as an internal control. Values are from three separate experiments each performed in triplicate. *p<0.05, **p<0.01.</p

Epinephrine augmented and accelerated the differentiation of mESCs into adipocytes at the end of differentiation.

<p>(A) Images of adipocytes developed from EBs treated without insulin at the end of differentiation (day 27) (CTL), with insulin (STD) or insulin plus epinephrine (EPI) added from day 7 to day 17 during commitment stage. Scale bars = 150 µm. (B) Percentage of EB outgrowths with adipocyte colonies and (C) percentage of adipocytes per EB outgrowth were measured and quantified at day 27. (D–E) Gene expression of adipocyte markers was measured by real time PCR at the end of the differentiation (day 27). RNA was isolated from differentiated mESCs and analyzed for expression of adipocyte marker genes FABP4 and PPARγ. β-actin expression was used as an internal control. Values are from three separate experiments each performed in triplicate. *p<0.05, **p<0.01.</p