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. 2009 Jun;11(6):583-93.
doi: 10.1593/neo.09266.

"VSports注册入口" Cross talk initiated by endothelial cells enhances migration and inhibits anoikis of squamous cell carcinoma cells through STAT3/Akt/ERK signaling

Kathleen G Neiva  1 , Zhaocheng Zhang, Marta Miyazawa, Kristy A Warner, Elisabeta Karl, Jacques E Nör
Affiliations

Cross talk initiated by endothelial cells enhances migration and inhibits anoikis of squamous cell carcinoma cells through STAT3/Akt/ERK signaling

Kathleen G Neiva et al. Neoplasia. 2009 Jun.

Abstract

It is well known that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effect of endothelial cell-secreted factors on the phenotype and behavior of tumor cells. The hypothesis underlying this study is that endothelial cells initiate signaling pathways that enhance tumor cell survival and migration. Here, we observed that soluble mediators from primary human dermal microvascular endothelial cells induce phosphorylation of signal transducer and activator of transcription 3 (STAT3), Akt, and extracellular signal-regulated kinase (ERK) in a panel of head and neck squamous cell carcinoma (HNSCC) cells (OSCC-3, UM-SCC-1, UM-SCC-17B, UM-SCC-74A). Gene expression analysis demonstrated that interleukin-6 (IL- 6), interleukin-8 (CXCL8), and epidermal growth factor (EGF) are upregulated in endothelial cells cocultured with HNSCC. Blockade of endothelial cell-derived IL-6, CXCL8, or EGF by gene silencing or neutralizing antibodies inhibited phosphorylation of STAT3, Akt, and ERK in tumor cells, respectively. Notably, activation of STAT3, Akt, and ERK by endothelial cells enhanced migration and inhibited anoikis of tumor cells. We have previously demonstrated that Bcl-2 is upregulated in tumor microvessels in patients with HNSCC. Here, we observed that Bcl-2 signaling induces expression of IL-6, CXCL8, and EGF, providing a mechanism for the upregulation of these cytokines in tumor-associated endothelial cells. This study expands the contribution of endothelial cells to the pathobiology of tumor cells. It unveils a new mechanism in which endothelial cells function as initiators of molecular crosstalks that enhance survival and migration of tumor cells. VSports手机版.

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Figures

Figure 1
Figure 1
Endothelial cell-derived soluble factors activate STAT3, Akt, and ERK pathways in tumor cells. (A) OSCC-3, (B) UM-SCC-17B, (C) UM-SCC-1, or (D) UM-SCC-74A were serum-starved overnight and exposed to HDMEC CM or control unconditioned medium (EBM) for the indicated time points. Phosphorylated and total STAT3, Akt, and ERK were detected by Western blot.
Figure 2
Figure 2
Endothelial cells showed upregulated IL-6, CXCL8, and EGF expression when cocultured with tumor cells. (A) Genome-wide messenger RNA expression analysis was performed in HDMEC cocultured with OSCC-3 using HDMEC cultured alone (single culture) as control. Black bars represent the fold increase of IL-6, CXCL8, and EGF expression in HDMEC cocultured with OSCC-3 compared with HDMEC cultured alone (gray bars). (B) Western blot for IL-6R, gp130, CXCR1, CXCR2, EGFR, and VEGFR2 expression in HDMEC, OSCC-3, UM-SCC-17B, UM-SCC-1A, and UM-SCC-74A. (C) ELISA for IL-6, CXCL8, and EGF expression in HDMEC, OSCC-3, or HDMEC exposed to 50 ng/ml VEGF165. Asterisk depicts significant difference (P < .001) compared with HDMEC. (D) Western blot for phosphorylated and total STAT3, Akt, and ERK in OSCC-3 serum-starved overnight and exposed to 1 to 50 ng/ml rhIL-6, 1 to 100 ng/ml rhCXCL8, or 1 to 100 ng/ml rhEGF for 30 minutes.
Figure 3
Figure 3
Effect of endothelial cell-secreted IL-6, CXCL8, and EGF on the phosphorylation of STAT3, Akt, and ERK in HNSCC cells. (A) Western blot for phosphorylated and total STAT3, Akt, and ERK in OSCC-3 serum-starved overnight and exposed to EBM; HDMEC CM containing 0 to 2 µg/ml anti-IL-6, anti-EGF, anti-CXCL8 neutralizing antibodies; or IgG control for 30 minutes. (B) ELISA for IL-6, CXCL8, or EGF expression in HDMEC transfected with shRNA-control (shRNA-C), shRNA-IL-6, shRNA-CXCL8, or shRNA-EGF. Asterisk depicts significant difference (P < .001) compared with shRNA-C. (C) Western blot for phosphorylated and total STAT3, Akt, and ERK in OSCC-3 serum-starved overnight and exposed to serum-free EBM or to CM from HDMEC-shRNA-C, HDMEC-shRNA-IL-6, HDMEC-shRNA-CXCL8, or HDMEC-shRNA-EGF for 30 minutes.
Figure 4
Figure 4
Bcl-2 induces IL-6, CXCL8, and EGF expression in endothelial cells. (A) Pools of endothelial cells overexpressing Bcl-2 (HDMEC-Bcl-2) were generated using retroviral vectors. Transgene expression was examined by Western Blot from HDMEC-LXSN (empty vector control) and HDMEC-Bcl-2. (B) ELISA for IL-6, CXCL8, or EGF expression in HDMEC-LXSN or HDMEC-Bcl-2. Asterisk depicts significant difference (P < .001). (C) OSCC-3 were serum-starved overnight and exposed to EBM or to CM from HDMEC-LXSN or from HDMEC-Bcl-2 at the indicated time points. Western blots were used to determine phosphorylated and total STAT3, Akt, and ERK in OSCC-3.
Figure 5
Figure 5
Endothelial cell-secreted IL-6, CXCL8, and EGF enhance tumor cell survival and migration through STAT3, Akt, and ERK pathways. (A) To optimize the dose of STAT3, Akt, and ERK inhibitors, OSCC-3 were serum-starved overnight, preincubated for 1 to 2 hours with (a) 0 to 20 µM Stattic, (b) 0 to 100 µM LY294002, or (c) 0 to 100 µM U0126, and then exposed to EBM or HDMEC CM for 30 minutes. DMSO added to HDMEC CM was used as vehicle control. Inhibition of STAT3, Akt, and ERK phosphorylation was determined by Western blot. (B) To evaluate tumor cell survival, OSCC-3 were maintained in low-attachment plates (LAP) for 24 hours in EBM: (a) CM HDMEC containing 1 µg/ml anti-IL-6, anti-CXCL8, anti-EGF-neutralizing antibodies, or IgG control; (b) CM from HDMEC-shRNA-IL-6, HDMEC-shRNA-CXCL8, HDMEC-shRNA-EGF, or HDMEC-shRNA-C; or (c) CM HDMEC containing 5 µM Stattic, 10 µM LY294002, 10 µM U0126, or DMSO vehicle control. OSCC-3 cells cultured in regular plates (NP) with EBM were used as controls. (C) To evaluate tumor cell migration, OSCC-3 were serum-starved overnight and allowed to migrate for 24 hours toward EBM: (a) HDMEC CM containing 1 µg/ml anti-IL-6, anti-CXCL8, anti-EGF, or IgG control; (b) CM from HDMEC-shRNA-IL-6, HDMEC-shRNA-CXCL8, HDMEC-shRNA-EGF, or HDMEC-shRNA-C; or (c) HDMEC CM containing 5 µM Stattic, 10 µM LY294002, 10 µM U0126, or DMSO vehicle control. Data presented here were normalized by control groups. Asterisk depicts significant difference (P < .001) compared with controls.
Figure 6
Figure 6
Diagram proposing a model for the endothelial cell-initiated cross talk with tumor cells that is described in this study. Endothelial cells secrete IL-6, CXCL8, and EGF that induce phosphorylation of STAT3, Akt, and ERK in tumor cells. These phosphorylation events enhance tumor cell survival andmigration. We hypothesize that a positive feedback loop can be generated by STAT3 phosphorylation in tumor cells. It is known that STAT3 can signal up-regulation of VEGF expression in squamous cell carcinomas. It is also known that tumor cell-secreted VEGF signals through VEGFR1 and VEGFR2 to induce Bcl-2 expression in endothelial cells. Here, we showed that Bcl-2 signaling is sufficient to induce IL-6, CXCL8, and EGF secretion by endothelial cells, which would in turn maintain this feedback loop.
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