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Hypoxia shifts activity of neuropeptide Y in Ewing sarcoma from growth-inhibitory to growth-promoting effects.

Tilan JU, Lu C, Galli S, Izycka-Swieszewska E, Earnest JP, Shabbir A, Everhart LM, Wang S, Martin S, Horton M, Mahajan A, Christian D, O'Neill A, Wang H, Zhuang T, Czarnecka M, Johnson MD, Toretsky JA, Kitlinska J - Oncotarget (2013)

Bottom Line: This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death.Hypoxia-driven actions of the peptide such as these may contribute to ES progression.Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES.

View Article: PubMed Central - PubMed

Affiliation: Department of Nursing, Georgetown University Medical Center, Georgetown University, Washington DC.

ABSTRACT
Ewing sarcoma (ES) is an aggressive malignancy driven by an oncogenic fusion protein, EWS-FLI1. Neuropeptide Y (NPY), and two of its receptors, Y1R and Y5R are up-regulated by EWS-FLI1 and abundantly expressed in ES cells. Paradoxically, NPY acting via Y1R and Y5R stimulates ES cell death. Here, we demonstrate that these growth-inhibitory actions of NPY are counteracted by hypoxia, which converts the peptide to a growth-promoting factor. In ES cells, hypoxia induces another NPY receptor, Y2R, and increases expression of dipeptidyl peptidase IV (DPPIV), an enzyme that cleaves NPY to a shorter form, NPY3-36. This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death. Instead, NPY3-36 acts as a selective Y2R/Y5R agonist. The hypoxia-induced increase in DPPIV activity is most evident in a population of ES cells with high aldehyde dehydrogenase (ALDH) activity, rich in cancer stem cells (CSCs). Consequently, NPY, acting via Y2R/Y5Rs, preferentially stimulates proliferation and migration of hypoxic ALDHhigh cells. Hypoxia also enhances the angiogenic potential of ES by inducing Y2Rs in endothelial cells and increasing the release of its ligand, NPY3-36, from ES cells. In summary, hypoxia acts as a molecular switch shifting NPY activity away from Y1R/Y5R-mediated cell death and activating the Y2R/Y5R/DPPIV/NPY3-36 axis, which stimulates ES CSCs and promotes angiogenesis. Hypoxia-driven actions of the peptide such as these may contribute to ES progression. Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES.

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The Y2R/Y5R/DPPIV system is preferentially activated in ALDHhigh ES CSCsA. SK-ES1 cells were cultured in normoxia or hypoxia for 24h, stained with Aldefluor, and ALDH activity was measured by FACS. The non-stained cells or the cells incubated with Aldefluor in the presence of ALDH inhibitor, DEAB, served as negative controls. B. SK-ES1 cells were stained with Aldefluor and FACS-sorted based on the ALDH activity into ALDHhigh (upper 8% of cells) and ALDHlow (lower 10% of cells) cells. The levels of ALDH-1 protein and stem cell marker, Oct-4, were compared by Western blot in soluble protein fractions of ALDHhigh and ALDHlow cells, to confirm the efficiency of cell sorting. C. ALDHhigh and ALDHlow cells were cultured for 24h in normoxia and hypoxia. Protein levels of Y2Rs and DPPIV were detected in cell membrane fractions isolated from these cells by Western blot. Flotillin-1 served as a loading control. Protein levels from 4 independent experiments were quantified by densitometry. D. DPPIV activity was measured in the above cell membrane fractions via luminescent method.
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Figure 4: The Y2R/Y5R/DPPIV system is preferentially activated in ALDHhigh ES CSCsA. SK-ES1 cells were cultured in normoxia or hypoxia for 24h, stained with Aldefluor, and ALDH activity was measured by FACS. The non-stained cells or the cells incubated with Aldefluor in the presence of ALDH inhibitor, DEAB, served as negative controls. B. SK-ES1 cells were stained with Aldefluor and FACS-sorted based on the ALDH activity into ALDHhigh (upper 8% of cells) and ALDHlow (lower 10% of cells) cells. The levels of ALDH-1 protein and stem cell marker, Oct-4, were compared by Western blot in soluble protein fractions of ALDHhigh and ALDHlow cells, to confirm the efficiency of cell sorting. C. ALDHhigh and ALDHlow cells were cultured for 24h in normoxia and hypoxia. Protein levels of Y2Rs and DPPIV were detected in cell membrane fractions isolated from these cells by Western blot. Flotillin-1 served as a loading control. Protein levels from 4 independent experiments were quantified by densitometry. D. DPPIV activity was measured in the above cell membrane fractions via luminescent method.

Mentions: The results of soft agar assay suggested that hypoxia-induced changes in the NPY system affect CSCs. Thus, in the subsequent experiments, we focused on ES cells with high ALDH activity, which have been shown to be rich in CSCs [2]. Culture under hypoxic conditions increased the number of SK-ES1 cells with elevated ALDH activity (Fig. 4A), suggesting hypoxia-driven enrichment in CSCs. To assess expression of the NPY system in ES CSC, SK-ES1 cells were sorted by flow cytometry into ALDHhigh and ALDHlow cells (Supplementary Fig. 2). This approach allowed for testing two cell populations with maximal difference in ALDH activity. The efficiency of sorting was confirmed by elevated expression of one of the main forms of ALDH, ALDH1, and a stem cell marker, Oct-4, in ALDHhigh cells (Fig. 4B). Western blot analysis of ALDHlow and ALDHhigh cells cultured under normoxic and hypoxic conditions revealed hypoxia-induced up-regulation of Y2R protein in both cell fractions (Fig. 4C). However, in normoxia and hypoxia, expression of DPPIV was higher in ALDHhigh cells, as compared to ALDHlow cells. Consequently, hypoxic CSCs had the highest DPPIV protein and activity levels (Fig. 4C, D).


Hypoxia shifts activity of neuropeptide Y in Ewing sarcoma from growth-inhibitory to growth-promoting effects.

Tilan JU, Lu C, Galli S, Izycka-Swieszewska E, Earnest JP, Shabbir A, Everhart LM, Wang S, Martin S, Horton M, Mahajan A, Christian D, O'Neill A, Wang H, Zhuang T, Czarnecka M, Johnson MD, Toretsky JA, Kitlinska J - Oncotarget (2013)

The Y2R/Y5R/DPPIV system is preferentially activated in ALDHhigh ES CSCsA. SK-ES1 cells were cultured in normoxia or hypoxia for 24h, stained with Aldefluor, and ALDH activity was measured by FACS. The non-stained cells or the cells incubated with Aldefluor in the presence of ALDH inhibitor, DEAB, served as negative controls. B. SK-ES1 cells were stained with Aldefluor and FACS-sorted based on the ALDH activity into ALDHhigh (upper 8% of cells) and ALDHlow (lower 10% of cells) cells. The levels of ALDH-1 protein and stem cell marker, Oct-4, were compared by Western blot in soluble protein fractions of ALDHhigh and ALDHlow cells, to confirm the efficiency of cell sorting. C. ALDHhigh and ALDHlow cells were cultured for 24h in normoxia and hypoxia. Protein levels of Y2Rs and DPPIV were detected in cell membrane fractions isolated from these cells by Western blot. Flotillin-1 served as a loading control. Protein levels from 4 independent experiments were quantified by densitometry. D. DPPIV activity was measured in the above cell membrane fractions via luminescent method.
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Related In: Results  -  Collection

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Figure 4: The Y2R/Y5R/DPPIV system is preferentially activated in ALDHhigh ES CSCsA. SK-ES1 cells were cultured in normoxia or hypoxia for 24h, stained with Aldefluor, and ALDH activity was measured by FACS. The non-stained cells or the cells incubated with Aldefluor in the presence of ALDH inhibitor, DEAB, served as negative controls. B. SK-ES1 cells were stained with Aldefluor and FACS-sorted based on the ALDH activity into ALDHhigh (upper 8% of cells) and ALDHlow (lower 10% of cells) cells. The levels of ALDH-1 protein and stem cell marker, Oct-4, were compared by Western blot in soluble protein fractions of ALDHhigh and ALDHlow cells, to confirm the efficiency of cell sorting. C. ALDHhigh and ALDHlow cells were cultured for 24h in normoxia and hypoxia. Protein levels of Y2Rs and DPPIV were detected in cell membrane fractions isolated from these cells by Western blot. Flotillin-1 served as a loading control. Protein levels from 4 independent experiments were quantified by densitometry. D. DPPIV activity was measured in the above cell membrane fractions via luminescent method.
Mentions: The results of soft agar assay suggested that hypoxia-induced changes in the NPY system affect CSCs. Thus, in the subsequent experiments, we focused on ES cells with high ALDH activity, which have been shown to be rich in CSCs [2]. Culture under hypoxic conditions increased the number of SK-ES1 cells with elevated ALDH activity (Fig. 4A), suggesting hypoxia-driven enrichment in CSCs. To assess expression of the NPY system in ES CSC, SK-ES1 cells were sorted by flow cytometry into ALDHhigh and ALDHlow cells (Supplementary Fig. 2). This approach allowed for testing two cell populations with maximal difference in ALDH activity. The efficiency of sorting was confirmed by elevated expression of one of the main forms of ALDH, ALDH1, and a stem cell marker, Oct-4, in ALDHhigh cells (Fig. 4B). Western blot analysis of ALDHlow and ALDHhigh cells cultured under normoxic and hypoxic conditions revealed hypoxia-induced up-regulation of Y2R protein in both cell fractions (Fig. 4C). However, in normoxia and hypoxia, expression of DPPIV was higher in ALDHhigh cells, as compared to ALDHlow cells. Consequently, hypoxic CSCs had the highest DPPIV protein and activity levels (Fig. 4C, D).

Bottom Line: This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death.Hypoxia-driven actions of the peptide such as these may contribute to ES progression.Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES.

View Article: PubMed Central - PubMed

Affiliation: Department of Nursing, Georgetown University Medical Center, Georgetown University, Washington DC.

ABSTRACT
Ewing sarcoma (ES) is an aggressive malignancy driven by an oncogenic fusion protein, EWS-FLI1. Neuropeptide Y (NPY), and two of its receptors, Y1R and Y5R are up-regulated by EWS-FLI1 and abundantly expressed in ES cells. Paradoxically, NPY acting via Y1R and Y5R stimulates ES cell death. Here, we demonstrate that these growth-inhibitory actions of NPY are counteracted by hypoxia, which converts the peptide to a growth-promoting factor. In ES cells, hypoxia induces another NPY receptor, Y2R, and increases expression of dipeptidyl peptidase IV (DPPIV), an enzyme that cleaves NPY to a shorter form, NPY3-36. This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death. Instead, NPY3-36 acts as a selective Y2R/Y5R agonist. The hypoxia-induced increase in DPPIV activity is most evident in a population of ES cells with high aldehyde dehydrogenase (ALDH) activity, rich in cancer stem cells (CSCs). Consequently, NPY, acting via Y2R/Y5Rs, preferentially stimulates proliferation and migration of hypoxic ALDHhigh cells. Hypoxia also enhances the angiogenic potential of ES by inducing Y2Rs in endothelial cells and increasing the release of its ligand, NPY3-36, from ES cells. In summary, hypoxia acts as a molecular switch shifting NPY activity away from Y1R/Y5R-mediated cell death and activating the Y2R/Y5R/DPPIV/NPY3-36 axis, which stimulates ES CSCs and promotes angiogenesis. Hypoxia-driven actions of the peptide such as these may contribute to ES progression. Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES.

Show MeSH
Related in: MedlinePlus