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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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Hypoxia sensitizes ECs to NPYA. HMVECs were cultured in 0.1% oxygen. Hif-1α protein was detected in soluble protein fractions by Western blot at desired time points, while Y2R and DPPIV proteins were measured in cell membrane preparations upon 24h incubation in normoxia or hypoxia. B. HMVECs were growth arrested for 24h under normoxia or hypoxia and treated for the following 24h with NPY (10−7M) with or without Y2R antagonist (10−6M) at the respective oxygen concentrations. Proliferation was measured by [3H]thymidine uptake. C. HMVECs were pretreated as above, stimulated with conditioned media from ES cells cultured under basal conditions, and incubated in normoxia or hypoxia for the following 24h. Proliferation was measured as above.
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Figure 7: Hypoxia sensitizes ECs to NPYA. HMVECs were cultured in 0.1% oxygen. Hif-1α protein was detected in soluble protein fractions by Western blot at desired time points, while Y2R and DPPIV proteins were measured in cell membrane preparations upon 24h incubation in normoxia or hypoxia. B. HMVECs were growth arrested for 24h under normoxia or hypoxia and treated for the following 24h with NPY (10−7M) with or without Y2R antagonist (10−6M) at the respective oxygen concentrations. Proliferation was measured by [3H]thymidine uptake. C. HMVECs were pretreated as above, stimulated with conditioned media from ES cells cultured under basal conditions, and incubated in normoxia or hypoxia for the following 24h. Proliferation was measured as above.

Mentions: Having established the hypoxia-induced increase in NPY release from ES cells, we sought to determine if its angiogenic activity can be augmented by parallel changes in ECs. HMVECs were cultured in 0.1% oxygen, which up-regulated Hif-1α within 6h (Fig. 7A). After 24h, hypoxia induced Y2R, which was not detectable in normoxic HMVECs (Fig. 7A). Simultaneously, hypoxia increased DPPIV protein levels (Fig. 7A) and augmented its activity up to 140% of the normoxic control (data not shown). This hypoxia-induced activation of the Y2R/DPPIV system sensitized HMVECs to NPY. The mitogenic activity of the peptide was elevated in hypoxic HMVECs, as compared to the normoxic cells (Fig. 7B). In normoxia and hypoxia, NPY effects were blocked by Y2R antagonist. Similarly, NPY-rich SK-ES1 conditioned media exerted enhanced proliferative activity in hypoxic HMVECs, as compared to normoxic cells (Fig. 7C). Y2R antagonist blocked this effect, thereby confirming its dependency on activation of the Y2R/DPPIV system in ECs. The proliferative activity of ES925 conditioned media, which lacks NPY, was also higher in hypoxic HMVECs (Fig. 7C). However, Y2R antagonist did not block this increase.


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)

Hypoxia sensitizes ECs to NPYA. HMVECs were cultured in 0.1% oxygen. Hif-1α protein was detected in soluble protein fractions by Western blot at desired time points, while Y2R and DPPIV proteins were measured in cell membrane preparations upon 24h incubation in normoxia or hypoxia. B. HMVECs were growth arrested for 24h under normoxia or hypoxia and treated for the following 24h with NPY (10−7M) with or without Y2R antagonist (10−6M) at the respective oxygen concentrations. Proliferation was measured by [3H]thymidine uptake. C. HMVECs were pretreated as above, stimulated with conditioned media from ES cells cultured under basal conditions, and incubated in normoxia or hypoxia for the following 24h. Proliferation was measured as above.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926843&req=5

Figure 7: Hypoxia sensitizes ECs to NPYA. HMVECs were cultured in 0.1% oxygen. Hif-1α protein was detected in soluble protein fractions by Western blot at desired time points, while Y2R and DPPIV proteins were measured in cell membrane preparations upon 24h incubation in normoxia or hypoxia. B. HMVECs were growth arrested for 24h under normoxia or hypoxia and treated for the following 24h with NPY (10−7M) with or without Y2R antagonist (10−6M) at the respective oxygen concentrations. Proliferation was measured by [3H]thymidine uptake. C. HMVECs were pretreated as above, stimulated with conditioned media from ES cells cultured under basal conditions, and incubated in normoxia or hypoxia for the following 24h. Proliferation was measured as above.
Mentions: Having established the hypoxia-induced increase in NPY release from ES cells, we sought to determine if its angiogenic activity can be augmented by parallel changes in ECs. HMVECs were cultured in 0.1% oxygen, which up-regulated Hif-1α within 6h (Fig. 7A). After 24h, hypoxia induced Y2R, which was not detectable in normoxic HMVECs (Fig. 7A). Simultaneously, hypoxia increased DPPIV protein levels (Fig. 7A) and augmented its activity up to 140% of the normoxic control (data not shown). This hypoxia-induced activation of the Y2R/DPPIV system sensitized HMVECs to NPY. The mitogenic activity of the peptide was elevated in hypoxic HMVECs, as compared to the normoxic cells (Fig. 7B). In normoxia and hypoxia, NPY effects were blocked by Y2R antagonist. Similarly, NPY-rich SK-ES1 conditioned media exerted enhanced proliferative activity in hypoxic HMVECs, as compared to normoxic cells (Fig. 7C). Y2R antagonist blocked this effect, thereby confirming its dependency on activation of the Y2R/DPPIV system in ECs. The proliferative activity of ES925 conditioned media, which lacks NPY, was also higher in hypoxic HMVECs (Fig. 7C). However, Y2R antagonist did not block this increase.

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