School of Medicine and Health Sciences Poster Presentations
The Role of Neuropeptide Y (NPY), its’ Y5/Y2R Receptors in Neuroblastoma cell migration
Poster Number
113
Document Type
Poster
Status
Medical Student
Abstract Category
Basic Biomedical Sciences
Keywords
Neuroblastoma, Neuropeptide Y, Metastasis, Migration
Publication Date
Spring 2018
Abstract
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.
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Open Access
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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.