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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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STAT3 phosphorylation in xenograft human oral squamous cell carcinomas correlates with tumor cell proliferation and presence of blood vessels. A, HeLa cells were serum-starved overnight and exposed to 20 ng/ml rhIL-6 for the indicated time points. Phosphorylated and total levels of STAT3, Akt, and ERK were determined by Western blots. B-D, xenograft human tumors were generated in SCID mice by co-implanting HeLa and HDMEC. Tumors were retrieved after 28 days, and tissues were analyzed by immunohistochemistry: B, total STAT3 with cytoplasmic localization, diffused through the tissue; C, phosphorylated STAT3 with nuclear localization, concentrated in the proximity of blood vessels; D, Ki67 with nuclear translocation, localized primarily around blood vessels. Photomicrographs at 200×.
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Figure 3: STAT3 phosphorylation in xenograft human oral squamous cell carcinomas correlates with tumor cell proliferation and presence of blood vessels. A, HeLa cells were serum-starved overnight and exposed to 20 ng/ml rhIL-6 for the indicated time points. Phosphorylated and total levels of STAT3, Akt, and ERK were determined by Western blots. B-D, xenograft human tumors were generated in SCID mice by co-implanting HeLa and HDMEC. Tumors were retrieved after 28 days, and tissues were analyzed by immunohistochemistry: B, total STAT3 with cytoplasmic localization, diffused through the tissue; C, phosphorylated STAT3 with nuclear localization, concentrated in the proximity of blood vessels; D, Ki67 with nuclear translocation, localized primarily around blood vessels. Photomicrographs at 200×.

Mentions: Considering the clinical relevance of the STAT3 signaling pathway in cervical carcinoma [21,22] we focused the remaining studies of this work on the effect of endothelial cell-secreted IL-6 in the biology of adenocarcinoma cells. To understand the Cervical Adenocarcinoma response to IL-6 stimulation, we performed a detailed time course analyzing the phosphorylation events in HeLa cells (Figure 3A). We observed that when tumor cells were exposed to rhIL-6, the phosphorylation of STAT3, Akt, and ERK followed similar patterns as when tumor cells were exposed to HDMEC CM (Figure 1A; Additional file 1: Figure 1A). We then exposed tumor cells to IL-6 in the presence of chemical inhibitors of STAT3, Akt, or ERK pathways and analyzed the phosphorylation responses (Additional file 1: Figure S2). IL-6 strongly activated STAT3 pathway in HeLa, and slightly activated Akt or ERK (Additional file 1: Figure S2A). Blockade of STAT3 phosphorylation had no major effect on Akt but increased ERK phosphorylation (Additional file 1: Figure S2B). Inhibition of Akt had no effect on STAT3, while increased ERK phosphorylation (Additional file 1: Figure S2C). Lastly, inhibition of ERK phosphorylation had no significant effect on STAT3 or Akt phosphorylation (Additional file 1: Figure S2D). Collectively, these results showed that IL-6 is a potent inducer STAT3 signaling, while it has a weaker effect on the phosphorylation of Akt and ERK in Cervical Adenocarcinoma.


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)

STAT3 phosphorylation in xenograft human oral squamous cell carcinomas correlates with tumor cell proliferation and presence of blood vessels. A, HeLa cells were serum-starved overnight and exposed to 20 ng/ml rhIL-6 for the indicated time points. Phosphorylated and total levels of STAT3, Akt, and ERK were determined by Western blots. B-D, xenograft human tumors were generated in SCID mice by co-implanting HeLa and HDMEC. Tumors were retrieved after 28 days, and tissues were analyzed by immunohistochemistry: B, total STAT3 with cytoplasmic localization, diffused through the tissue; C, phosphorylated STAT3 with nuclear localization, concentrated in the proximity of blood vessels; D, Ki67 with nuclear translocation, localized primarily around blood vessels. Photomicrographs at 200×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016552&req=5

Figure 3: STAT3 phosphorylation in xenograft human oral squamous cell carcinomas correlates with tumor cell proliferation and presence of blood vessels. A, HeLa cells were serum-starved overnight and exposed to 20 ng/ml rhIL-6 for the indicated time points. Phosphorylated and total levels of STAT3, Akt, and ERK were determined by Western blots. B-D, xenograft human tumors were generated in SCID mice by co-implanting HeLa and HDMEC. Tumors were retrieved after 28 days, and tissues were analyzed by immunohistochemistry: B, total STAT3 with cytoplasmic localization, diffused through the tissue; C, phosphorylated STAT3 with nuclear localization, concentrated in the proximity of blood vessels; D, Ki67 with nuclear translocation, localized primarily around blood vessels. Photomicrographs at 200×.
Mentions: Considering the clinical relevance of the STAT3 signaling pathway in cervical carcinoma [21,22] we focused the remaining studies of this work on the effect of endothelial cell-secreted IL-6 in the biology of adenocarcinoma cells. To understand the Cervical Adenocarcinoma response to IL-6 stimulation, we performed a detailed time course analyzing the phosphorylation events in HeLa cells (Figure 3A). We observed that when tumor cells were exposed to rhIL-6, the phosphorylation of STAT3, Akt, and ERK followed similar patterns as when tumor cells were exposed to HDMEC CM (Figure 1A; Additional file 1: Figure 1A). We then exposed tumor cells to IL-6 in the presence of chemical inhibitors of STAT3, Akt, or ERK pathways and analyzed the phosphorylation responses (Additional file 1: Figure S2). IL-6 strongly activated STAT3 pathway in HeLa, and slightly activated Akt or ERK (Additional file 1: Figure S2A). Blockade of STAT3 phosphorylation had no major effect on Akt but increased ERK phosphorylation (Additional file 1: Figure S2B). Inhibition of Akt had no effect on STAT3, while increased ERK phosphorylation (Additional file 1: Figure S2C). Lastly, inhibition of ERK phosphorylation had no significant effect on STAT3 or Akt phosphorylation (Additional file 1: Figure S2D). Collectively, these results showed that IL-6 is a potent inducer STAT3 signaling, while it has a weaker effect on the phosphorylation of Akt and ERK in Cervical Adenocarcinoma.

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