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Endothelial cell-derived interleukin-6 regulates tumor growth.

Neiva KG, Warner KA, Campos MS, Zhang Z, Moren J, Danciu TE, Nör JE - BMC Cancer (2014)

Bottom Line: In search for a possible mechanism for the activation of the STAT3 signaling pathway, we observed that silencing interleukin (IL)-6 in tumor-associated endothelial cells inhibited STAT3 phosphorylation in tumor cells.Notably, tumors vascularized with IL-6-silenced endothelial cells showed lower intratumoral microvessel density, lower tumor cell proliferation, and slower growth than tumors vascularized with control endothelial cells.Collectively, these results demonstrate that IL-6 secreted by endothelial cells enhance tumor growth, and suggest that cancer patients might benefit from targeted approaches that block signaling events initiated by endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Angiogenesis Research Laboratory, Department of Cariology, Restorative Sciences, and Endodontics, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109-1078, USA. jenor@umich.edu.

ABSTRACT

Background: Endothelial cells play a complex role in the pathobiology of cancer. This role is not limited to the making of blood vessels to allow for influx of oxygen and nutrients required for the high metabolic demands of tumor cells. Indeed, it has been recently shown that tumor-associated endothelial cells secrete molecules that enhance tumor cell survival and cancer stem cell self-renewal. The hypothesis underlying this work is that specific disruption of endothelial cell-initiated signaling inhibits tumor growth.

Methods: Conditioned medium from primary human dermal microvascular endothelial cells (HDMEC) stably transduced with silencing RNA for IL-6 (or controls) was used to evaluate the role of endothelial-derived IL-6 on the activation of key signaling pathways in tumor cells. In addition, these endothelial cells were co-transplanted with tumor cells into immunodefficient mice to determine the impact of endothelial cell-derived IL-6 on tumor growth and angiogenesis.

Results: We observed that tumor cells adjacent to blood vessels show strong phosphorylation of STAT3, a key mediator of tumor progression. In search for a possible mechanism for the activation of the STAT3 signaling pathway, we observed that silencing interleukin (IL)-6 in tumor-associated endothelial cells inhibited STAT3 phosphorylation in tumor cells. Notably, tumors vascularized with IL-6-silenced endothelial cells showed lower intratumoral microvessel density, lower tumor cell proliferation, and slower growth than tumors vascularized with control endothelial cells.

Conclusions: Collectively, these results demonstrate that IL-6 secreted by endothelial cells enhance tumor growth, and suggest that cancer patients might benefit from targeted approaches that block signaling events initiated by endothelial cells.

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Endothelial cell-derived factors phosphorylate STAT3, Akt, and ERK in tumor cells in vitro and in vivo. A, western blot for phosphorylated and total STAT3, Akt, and ERK in HeLa serum-starved overnight and exposed to HDMEC conditioned medium (CM), HeLa CM, or control unconditioned medium (EBM) for the indicated time points. B, western blot for phosphorylated and total STAT3, Akt, and ERK in HeLa or HOK-16B serum-starved overnight and exposed to HDMEC CM or EBM for the indicated time points. C, immunohistochemical analysis for phosphorylated STAT3, Akt, and ERK (with nuclear localization) in representative specimens from xenograft human squamous cell carcinomas. Top panels represent 100× and lower panels represent 200× magnification. Red arrows point to blood vessels.
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Figure 1: Endothelial cell-derived factors phosphorylate STAT3, Akt, and ERK in tumor cells in vitro and in vivo. A, western blot for phosphorylated and total STAT3, Akt, and ERK in HeLa serum-starved overnight and exposed to HDMEC conditioned medium (CM), HeLa CM, or control unconditioned medium (EBM) for the indicated time points. B, western blot for phosphorylated and total STAT3, Akt, and ERK in HeLa or HOK-16B serum-starved overnight and exposed to HDMEC CM or EBM for the indicated time points. C, immunohistochemical analysis for phosphorylated STAT3, Akt, and ERK (with nuclear localization) in representative specimens from xenograft human squamous cell carcinomas. Top panels represent 100× and lower panels represent 200× magnification. Red arrows point to blood vessels.

Mentions: We have previously demonstrated that a crosstalk initiated by endothelial cells enhances tumor cell survival and migration in vitro, and that endothelial cell-derived IL-6 induces phosphorylation of STAT3 in tumor cells [26]. The overall hypothesis underlying this study is that the activation of signaling pathways in tumor cells induced by endothelial cell-secreted factors enhances tumor growth. To begin to address this hypothesis, we exposed HeLa cells to serum-free endothelial cell (HDMEC) conditioned medium (CM) or tumor cell (HeLa) CM and analyzed phosphorylation events over time (Figure 1A). We observed that phosphorylation levels of STAT3, Akt, and ERK were higher in tumor cells exposed to HDMEC CM than in tumor cells exposed to HeLa CM, or unconditioned medium (EBM). The induction of phosphorylation was observed primarily at early time points (15 to 30 minutes), decreasing at 1 hour (Figure 1A). Notably, expression levels of IL-6 were higher in HDMEC CM than in HeLa CM, and silencing IL-6 in endothelial cells did not have a measurable impact in endothelial cell proliferation (data not shown). In addition, we analyzed phosphorylation events on HeLa cells and on keratinocytes (HOK-16B) exposed to HDMEC CM or unconditioned medium (EBM) (Figure 1B). We observed that phosphorylation levels of STAT3, Akt, and ERK were higher when both tumor cells and keratinocytes were exposed to HDMEC CM than to EBM. Similarly, phosphorylation was observed mainly at early time points and decreased at 24 hours (Figure 1B).


Endothelial cell-derived interleukin-6 regulates tumor growth.

Neiva KG, Warner KA, Campos MS, Zhang Z, Moren J, Danciu TE, Nör JE - BMC Cancer (2014)

Endothelial cell-derived factors phosphorylate STAT3, Akt, and ERK in tumor cells in vitro and in vivo. A, western blot for phosphorylated and total STAT3, Akt, and ERK in HeLa serum-starved overnight and exposed to HDMEC conditioned medium (CM), HeLa CM, or control unconditioned medium (EBM) for the indicated time points. B, western blot for phosphorylated and total STAT3, Akt, and ERK in HeLa or HOK-16B serum-starved overnight and exposed to HDMEC CM or EBM for the indicated time points. C, immunohistochemical analysis for phosphorylated STAT3, Akt, and ERK (with nuclear localization) in representative specimens from xenograft human squamous cell carcinomas. Top panels represent 100× and lower panels represent 200× magnification. Red arrows point to blood vessels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016552&req=5

Figure 1: Endothelial cell-derived factors phosphorylate STAT3, Akt, and ERK in tumor cells in vitro and in vivo. A, western blot for phosphorylated and total STAT3, Akt, and ERK in HeLa serum-starved overnight and exposed to HDMEC conditioned medium (CM), HeLa CM, or control unconditioned medium (EBM) for the indicated time points. B, western blot for phosphorylated and total STAT3, Akt, and ERK in HeLa or HOK-16B serum-starved overnight and exposed to HDMEC CM or EBM for the indicated time points. C, immunohistochemical analysis for phosphorylated STAT3, Akt, and ERK (with nuclear localization) in representative specimens from xenograft human squamous cell carcinomas. Top panels represent 100× and lower panels represent 200× magnification. Red arrows point to blood vessels.
Mentions: We have previously demonstrated that a crosstalk initiated by endothelial cells enhances tumor cell survival and migration in vitro, and that endothelial cell-derived IL-6 induces phosphorylation of STAT3 in tumor cells [26]. The overall hypothesis underlying this study is that the activation of signaling pathways in tumor cells induced by endothelial cell-secreted factors enhances tumor growth. To begin to address this hypothesis, we exposed HeLa cells to serum-free endothelial cell (HDMEC) conditioned medium (CM) or tumor cell (HeLa) CM and analyzed phosphorylation events over time (Figure 1A). We observed that phosphorylation levels of STAT3, Akt, and ERK were higher in tumor cells exposed to HDMEC CM than in tumor cells exposed to HeLa CM, or unconditioned medium (EBM). The induction of phosphorylation was observed primarily at early time points (15 to 30 minutes), decreasing at 1 hour (Figure 1A). Notably, expression levels of IL-6 were higher in HDMEC CM than in HeLa CM, and silencing IL-6 in endothelial cells did not have a measurable impact in endothelial cell proliferation (data not shown). In addition, we analyzed phosphorylation events on HeLa cells and on keratinocytes (HOK-16B) exposed to HDMEC CM or unconditioned medium (EBM) (Figure 1B). We observed that phosphorylation levels of STAT3, Akt, and ERK were higher when both tumor cells and keratinocytes were exposed to HDMEC CM than to EBM. Similarly, phosphorylation was observed mainly at early time points and decreased at 24 hours (Figure 1B).

Bottom Line: In search for a possible mechanism for the activation of the STAT3 signaling pathway, we observed that silencing interleukin (IL)-6 in tumor-associated endothelial cells inhibited STAT3 phosphorylation in tumor cells.Notably, tumors vascularized with IL-6-silenced endothelial cells showed lower intratumoral microvessel density, lower tumor cell proliferation, and slower growth than tumors vascularized with control endothelial cells.Collectively, these results demonstrate that IL-6 secreted by endothelial cells enhance tumor growth, and suggest that cancer patients might benefit from targeted approaches that block signaling events initiated by endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Angiogenesis Research Laboratory, Department of Cariology, Restorative Sciences, and Endodontics, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109-1078, USA. jenor@umich.edu.

ABSTRACT

Background: Endothelial cells play a complex role in the pathobiology of cancer. This role is not limited to the making of blood vessels to allow for influx of oxygen and nutrients required for the high metabolic demands of tumor cells. Indeed, it has been recently shown that tumor-associated endothelial cells secrete molecules that enhance tumor cell survival and cancer stem cell self-renewal. The hypothesis underlying this work is that specific disruption of endothelial cell-initiated signaling inhibits tumor growth.

Methods: Conditioned medium from primary human dermal microvascular endothelial cells (HDMEC) stably transduced with silencing RNA for IL-6 (or controls) was used to evaluate the role of endothelial-derived IL-6 on the activation of key signaling pathways in tumor cells. In addition, these endothelial cells were co-transplanted with tumor cells into immunodefficient mice to determine the impact of endothelial cell-derived IL-6 on tumor growth and angiogenesis.

Results: We observed that tumor cells adjacent to blood vessels show strong phosphorylation of STAT3, a key mediator of tumor progression. In search for a possible mechanism for the activation of the STAT3 signaling pathway, we observed that silencing interleukin (IL)-6 in tumor-associated endothelial cells inhibited STAT3 phosphorylation in tumor cells. Notably, tumors vascularized with IL-6-silenced endothelial cells showed lower intratumoral microvessel density, lower tumor cell proliferation, and slower growth than tumors vascularized with control endothelial cells.

Conclusions: Collectively, these results demonstrate that IL-6 secreted by endothelial cells enhance tumor growth, and suggest that cancer patients might benefit from targeted approaches that block signaling events initiated by endothelial cells.

Show MeSH
Related in: MedlinePlus