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. 2009 Jun;11(6):583–593. doi: 10.1593/neo.09266

Cross talk Initiated by Endothelial Cells Enhances Migration and Inhibits Anoikis of Squamous Cell Carcinoma Cells through STAT3/Akt/ERK Signaling1,2

Kathleen G Neiva *, Zhaocheng Zhang *, Marta Miyazawa *,†, Kristy A Warner *, Elisabeta Karl *, Jacques E Nör *,‡,§
PMCID: PMC2685447  PMID: 19484147

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.

Introduction

Tumor angiogenesis requires active interaction between endothelial and tumor cells and plays an important role in cancer progression [1]. However, emphasis has been placed on the assumption that tumor cell-initiated signals are the dominant events in tumor angiogenesis and tumor growth. We have recently reported signaling cross talks between tumor cells and endothelial cells that promote tumor growth. In cell contact-dependent interactions, Jagged1 expressed by squamous cell carcinoma (SCC) cells activates Notch signaling in adjacent endothelial cells and enhances tumor growth [2]. In a cell contact-independent system, we observed that vascular endothelial growth factor (VEGF) secreted by tumor-associated endothelial cells induced Bcl-2, growth-related oncogene(GRO)α (CXCL1), and interleukin 8 (CXCL8) expression in SCC cells [3]. Notably, these endothelial cell-initiated signals significantly enhanced tumor growth in vivo [3]. Collectively, this work indicates that the impact of endothelial cells in the pathobiology of cancer is not limited to making angiogenic blood vessels in response to tumor cell-initiated signals. The identification and characterization of the signaling events initiated by tumor-associated endothelial cells may have important implications in human cancer therapy.

Squamous cell carcinoma is a common cancer in the gastrointestinal track, lung, skin, and head and neck regions. For example, more than 500,000 new patients with head and neck squamous cell carcinomas (HNSCCs) are diagnosed each year worldwide [4,5]. The 5-year survival rate for patients with HNSCC is one of the lowest among major cancer sites and has not improved significantly during the last 30 years despite extensive basic and clinical research [5–7]. Today, we know that several intracellular signaling molecules are key orchestrators of SCC progression. Among them, the signal transducer and activator of transcription 3 (STAT3) has been described to be constitutively active in HNSCC and in several other epithelial malignancies [8,9]. Altered expression or mutation of components of the phosphoinositol 3-kinase (PI3K)/Akt pathway has also been implicated in tumorigenesis [10,11]. In addition, the mitogen-activating protein kinase and extracellular signal-regulated kinase (ERK) pathway has been described as an important regulator of tumor growth and a key target for cancer therapy [12,13]. These three signaling molecules, i.e., STAT3, Akt, and ERK, play critical roles in the control of cell cycle, survival, proliferation, and migration of tumor cells. Notably, deregulation of any of these pathways has been shown to drive oncogenic transformation [14–16]. However, the impact of endothelial cell-initiated signaling on the activation of STAT3, Akt, and ERK signaling in tumor cells is unclear.

It is known that VEGF induces Bcl-2 expression in endothelial cells and that Bcl-2 expression level in tumor-associated endothelial cells is directly correlated with tumor angiogenesis and tumor growth [3,17,18]. Notably, Bcl-2 gene expression is approximately 60,000-fold higher in the endothelial cells lining tumor blood vessels in patients with HNSCC compared with the endothelial cells from normal oral mucosa [3]. It is also known that Bcl-2 induces CXC chemokines' expression by endothelial cells, which results in enhanced HNSCC invasiveness and local recurrence [19,20]. We have shown that Bcl-2 signals through nuclear factor κB (NF-κB) to induce CXCL1 and CXCL8 expression in endothelial cells [19]. However, the effect of Bcl-2 in the expression of interleukin 6 (IL-6; known to activate the STAT3 signaling pathway) and epidermal growth factor (EGF; known to activate the ERK signaling pathway) is unknown. In this study, we unveil a signaling pathway that is initiated by endothelial cells and that results in the activation of STAT3, Akt, and ERK in HNSCC cells. Notably, blockade of endothelial cell-initiated signaling had a direct impact on tumor cell survival and migration.

Materials and Methods

Cell Culture

Oral squamous cell carcinoma (OSCC-3; gift from M. Lingen, University of Chicago), University of Michigan squamous cell carcinoma (UM-SCC-1, UM-SCC-17B, and UM-SCC-74A; gift from T. Carey, University of Michigan) were cultured in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Primary human dermal microvascular endothelial cells (HDMECs; Cambrex, Walkersville, MD) were cultured in endothelial cell growth medium 2 (EGM2-MV; Cambrex). Pools of HDMEC stably transduced with Bcl-2 (HDMEC-Bcl-2) and HDMEC-LXSN (empty vector controls) were generated with retroviruses and cultured in EGM2-MV supplemented with 250 µg/ml G418 (Cellgro; Mediatech, Inc., Herndon, VA), as described [17,18]. Conditioned medium (CM) from HDMEC was prepared in endothelial cell basal medium (EBM) without supplementation with growth factors or serum from 24-hour cultures.

Stable Short Hairpin RNA Transduction

Lentiviruses expressing a short hairpin RNA (shRNA) construct for silencing IL-6, CXCL8, or EGF (Vector Core, University of Michigan) were generated in human embryonic kidney cells (293T) transfected by the calcium phosphate method, as described [3]. A scrambled oligonucleotide sequence (shRNA-C) was used as control. Supernatants were collected 48 hours after transfection and used to infect HDMEC in a 1:1 dilution medium containing 4 µg/ml polybrene (Sigma-Aldrich, St. Louis, MO). Cells were selected and maintained in EGM2-MV supplemented with 1 µg/ml puromycin (InvivoGen, San Diego, CA). Down-regulation of IL-6, CXCL8, or EGF was confirmed by ELISA.

Western Blots

OSCC-3, UM-SCC-1, UM-SCC-17B, or UM-SCC-74A (8 x 105) were plated in 60-mm dishes, starved overnight, and exposed to HDMEC CM or EBM for the indicated time points. Alternatively, tumor cells were exposed to HDMEC CM containing 0.1 to 2 µg/ml anti-IL-6, anti-CXCL8, anti-EGF, or IgG isotype control (R&D Systems, Minneapolis, MN) or CM collected from HDMEC-shRNAIL-6, HDMEC-shRNA-CXCL8, HDMEC-shRNA-EGF, or HDMEC-shRNA-C. Signaling pathways were blocked by preincubating tumor cells for 1 to 2 hours with 0.5 to 20 µM Stattic (STAT3 inhibitor V; Calbiochem, San Diego, CA), 5 to 100 µM LY294002 (PI3 kinase inhibitor; Cell Signaling Technology, Danvers, MA), or 5 to 100 µM U0126 (MEK1/2 inhibitor; Cell Signaling Technology) and exposing to HDMEC CM. Protein (30 µg) was electrophoresed on SDS-polyacrylamide gels and transferred to nitrocellulosemembranes. Primary antibodies were as follows: mouse anti-human phospho-STAT3, rabbit anti-human STAT3, rabbit anti-human phospho-Akt, rabbit anti-human Akt, rabbit anti-human phospho-ERK1/2, and mouse anti-human ERK1/2 (Cell Signaling Technology); rabbit anti-human VEGFR2, rabbit anti-human IL-6R, and rabbit anti-human EGFR (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti- human gp130 (Biosource, Camarillo, CA);mouse anti-human CXCR1 and rabbit anti-human CXCR2 (Abcam, Cambridge, MA); hamster anti-human Bcl-2 (BD Biosciences, San Jose, CA); and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (Chemicon, Millipore, Billerca,MA). Immunoreactive proteins were visualized by SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL).

Affymetrix Microarray

Total RNA was isolated from HDMEC cultured alone or cocultured with OSCC-3 using Trizol (Invitrogen, Carlsbad, CA). The RNA was purified using the RNeasy kit (Qiagen, Valencia, CA), and 10 µg of total RNA was used to perform microarray analysis on Human Genome U133 Plus 2.0 (Affymetrix, Santa Clara, CA). Briefly, cDNA was synthesized from total RNA (One-Cycle cDNA Synthesis Kit; Affymetrix), and in vitro transcription was followed to make biotin-labeled cRNA (IVT Labeling Kit; Affymetrix). Fragmented cRNA was hybridized with microarray. After microarray washing, staining, and scanning, data were collected and further analyzed using Data Mining software and NetAffx Analysis Center (Affymetrix).

Enzyme-Linked Immunosorbent Assay

Supernatants of 24-hour endothelial or tumor cell cultures were collected and centrifuged to eliminate debris. Interleukin 6, CXCL8, and EGF expression was determined using ELISA kits (Quantikine; R&D Systems) according to the manufacturer's instructions. Data were normalized by cell number.

Migration Assays

The HDMEC CM was preincubated with 1 µg/ml anti-IL-6, anti-CXCL8, anti-EGF, or IgG control (R&D Systems) for 1 hour, and 400 µl of CM was added to 24-well companion plates (Fisher Scientific, Pittsburgh, PA). Alternatively, CM collected from HDMEC-shRNA-IL-6, HDMEC-shRNA-CXCL8, HDMEC-shRNA-EGF, or HDMEC-shRNA-C was used. OSCC-3 (2 x 105) were serum-starved overnight, loaded onto 8-µm pore-sized cell culture inserts (Becton Dickinson, Franklin Lakes, NJ), and allowed to migrate for 24 hours toward HDMEC CM. In addition, OSCC-3 were preincubated for 1 hour with 5 µM Stattic (Calbiochem), 10 µM LY294002, or 10 µM U0126 (Cell Signaling Technology). Unconditioned EBM was used as a negative control. Migrated cells were trypsinized, collected, and stained with 2 µM Cell Tracker Green (Invitrogen) for 1 hour. Fluorescence was read at 485/535 nm in a microplate reader (Tecan, Salzburg, Austria).

Survival Assays

OSCC-3 (3 x 105) were seeded in six-well ultra low-attachment plates (Corning Incorporated, Corning, NY) using EBM; HDMEC CM containing 1 µg/ml anti-IL-6, anti-CXCL8, anti-EGF, or IgG control (R&D Systems); CM from HDMEC-shRNA-IL-6, HDMEC-shRNA-CXCL8, HDMEC-shRNA-EGF, or HDMEC-shRNA-C; or HDMEC CM containing 5 µM Stattic (Calbiochem), 10 µM LY294002, or 10 µM U0126 (Cell Signaling Technology). OSCC-3 cells seeded in regular six-well plates (Becton Dickinson) were used as positive control. After 24 hours, propidium iodide staining followed by flow cytometry was used to determine the percentage of dead cells as described [21]. In addition, percentage of dead cells was determined by the Trypan blue (Gibco, Invitrogen Corporation, Grand Island, NY) exclusion method after 24 to 48 hours.

Statistical Analyses

t Tests or one-way analysis of variance was performed using the SigmaStat 2.0 software (SPSS, Chicago, IL). Statistical significance was determined at P < .001.

Results

Endothelial Cell-Secreted Factors Induce Phosphorylation of STAT3, Akt, and ERK in HNSCC Cells

The overall hypothesis underlying this study is that endothelial cell-secreted factors initiate signaling pathways in tumor cells that enhance their survival and migration. To begin to address this hypothesis, we exposed a panel of head and neck tumor cells, i.e., OSCC-3 (Figure 1A), UM-SCC-17B (Figure 1B), UM-SCC-1 (Figure 1C), or UM-SCC-74A (Figure 1D), to serum-free endothelial cell (HDMEC) CM and analyzed the phosphorylation levels of key signaling molecules over time. We observed that STAT3, Akt, and ERK were consistently phosphorylated in the tumor cells on exposure to endothelial cell CM compared with tumor cells treated with unconditioned medium (Figure 1). The induction of phosphorylation by endothelial cell CM was observed primarily between 15 minutes and 1 hour, decreasing after 4 to 24 hours as expected. The four tumor cell lines that we tested showed different phosphorylation levels on exposure to HDMEC CM, but the trends among the cell lines were similar. To verify the results of the CM experiments with a second experimental model, we used a noncontact coculture system with HDMEC and HNSCC cells. Within 1 hour, STAT3, Akt, and ERK phosphorylation was enhanced in OSCC-3 cocultured with HDMEC compared with OSCC-3 in single culture (Figure W1A). Notably, even after 24 hours in coculture, the phosphorylation status of STAT3 was still upregulated in two (of four) HNSCC cell lines tested here (Figure W1B). Collectively, these results showed that endothelial cell-secreted factors induce STAT3, Akt, and ERK phosphorylation in HNSCC cells.

Figure 1.

Figure 1

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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.

IL-6, CXCL8, and EGF Are Upregulated in Endothelial Cells Cocultured with HNSCC Cells

In search for putative factors secreted by endothelial cells that could lead to STAT3, Akt, and ERK phosphorylation, we performed an Affymetrix microarray comparing the gene expression profile of HDMEC cultured alone with HDMEC cocultured with OSCC-3. We observed that IL-6, CXCL8, and EGF were upregulated in HDMEC when cocultured with OSCC-3 (Figure 2A). We then analyzed whether the tumor cells express the receptors for IL-6, CXCL8, and EGF. All tumor cells tested here express IL-6R and gp130 (receptors for IL-6), CXCR1 and CXCR2 (receptors for CXCL8), and EGFR (receptor for EGF; Figure 2B). We selected OSCC-3 cells for most of the remaining experiments presented here and used a second HNSCC cell line to verify reproducibility of results of key experiments. Next, we compared the expression levels of IL-6, CXCL8, and EGF in endothelial and tumor cells. Because it is known that HNSCC cells secret high levels of VEGF [22], we exposed HDMEC to VEGF and evaluated the impact of this treatment on the expression of IL-6, CXCL8, and EGF. Notably, the expression of IL-6 and CXCL8, but not EGF, was upregulated in endothelial cells treated with VEGF (Figure 2C). We observed that IL-6 induced phosphorylation of STAT3, CXCL8 enhanced phosphorylation of Akt and ERK compared with untreated group (first lane), and EGF enhanced phosphorylation of STAT3, Akt, and ERK (Figure 2D). We also exposed OSCC-3 to the combination of IL-6, CXCL8, and EGF and observed similar signaling trends compared with each recombinant protein alone (data not shown).

Figure 2.

Figure 2

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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.

Effect of Endothelial Cell-Secreted IL-6, CXCL8, and EGF on the Phosphorylation of STAT3, Akt, and ERK in HNSCC Cells

To evaluate the role of these cytokines on endothelial cell-initiated signaling pathways in tumor cells, we exposed OSCC-3 to HDMEC CM in the presence of neutralizing antibodies to IL-6, CXCL8, or EGF and analyzed phosphorylation of STAT3, Akt, and ERK. Blockade of IL-6 in HDMEC CM blocked STAT3 phosphorylation in OSCC-3 but had no effect on the phosphorylation levels of Akt and ERK compared with IgG controls (Figure 3A). Blockade of CXCL8 in HDMEC CM slightly decreased phosphorylation levels of STAT3 and Akt but had no significant effect on ERK (Figure 3A). Finally, blockade of EGF in HDMEC CM eliminated ERK phosphorylation in OSCC-3 compared with IgG control, whereas it had no effect on STAT3 and Akt phosphorylation levels (Figure 3A). To evaluate specifically the role of endothelial cell initiated events, we silenced the expression of IL-6, CXCL8, or EGF in HDMEC. The effectiveness of expression knockdown was verified by ELISA (Figure 3B). OSCC-3 were then exposed to CM from HDMEC-shRNA-IL-6, HDMEC-shRNA-CXCL8, HDMEC-shRNA-EGF, or control HDMEC-shRNA-C. Down-regulation of IL-6 and EGF in endothelial cells resulted in the inhibition of phosphorylation of STAT3 and ERK in tumor cells, respectively (Figure 3C). However, down-regulation of CXCL8 in endothelial cells did not result in an observable inhibition of Akt in the tumor cells, presumably because HDMEC-shRNA-CXCL8 cells still secreted substantial amounts of this protein.

Figure 3.

Figure 3

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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.

Bcl-2 Signaling Induces IL-6, CXCL8, and EGF Expression in Endothelial Cells

To begin to understand the mechanisms involved in the up-regulation of IL-6, CXCL8, and EGF in the endothelial cells, we evaluated a potential role for Bcl-2 signaling. Previous work from our laboratory has shown that tumor cell-secreted VEGF induces Bcl-2 expression in endothelial cells, and that up-regulation of Bcl-2 in microvascular endothelial cells is sufficient to enhance tumor progression [17,18]. Notably, Bcl-2 is significantly upregulated in the endothelial cells of head and neck tumor microvessels [3]. We have also shown that Bcl-2 upregulates CXCL1 and CXCL8 expression in endothelial cells through the IKK/I-κB/NF-κB pathway [19], and it is known that NF-κB directly stimulates the transcription of several cytokines and growth factors [23]. These observations led to the hypothesis that Bcl-2 induces the expression of IL-6 and EGF in endothelial cells. To test this hypothesis, we generated pools of endothelial cells overexpressing Bcl-2 (HDMEC-Bcl-2) and empty vector controls (HDMEC-LXSN; Figure 4A). HDMEC-Bcl-2 secreted significantly higher levels of IL-6, CXCL8, and EGF than HDMEC-LXSN (Figure 4B). Next, we analyzed the phosphorylation levels of STAT3, Akt, and ERK in OSCC-3 when exposed to CM collected from HDMEC-LXSN and HDMEC-Bcl-2. At initial time points (i.e., 15 minutes), the phosphorylation levels induced by HDMEC-LXSN and HDMEC-Bcl-2 CM were similar (Figure 4C). However, at later time points, the phosphorylation of STAT3 (4 and 24 hours) and ERK (4 hours) was higher when OSCC-3 cells were exposed to HDMEC-Bcl-2 CM than in OSCC-3 exposed to HDMEC-LXSN CM (Figure 4C). Collectively, these results showed that Bcl-2 signaling induces the expression of IL-6, CXCL8, and EGF in endothelial cells. Moreover, Bcl-2 expression levels in the endothelial cells had a significant impact on the phosphorylation status of STAT3 and ERK, but not Akt, in the tumor cells.

Figure 4.

Figure 4

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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.

Endothelial Cell-Secreted Factors Enhance the Motility and Prevent Anoikis of HNSCC Cells

To understand the biological impact of endothelial cell-induced tumor cell STAT3, Akt, and ERK phosphorylation, we exposed the OSCC-3 to HDMEC CM containing inhibitors of these pathways and evaluated tumor cell proliferation, survival, and migration. We used increasing concentrations of Stattic (STAT3 inhibitor), LY294002 (PI3K/Akt inhibitor), or U0126 (MEK/ERK inhibitor; Figure 5A) and selected the lowest dose able to inhibit activation of STAT3, Akt, or ERK to carry out these experiments. Viability experiments showed that these dosages were not cytotoxic (data not shown). No difference was observed in HNSCC proliferation when cells were exposed to HDMEC CM for up to 72 hours compared with exposure to unconditioned medium. Blockade of IL-6, CXCL8, or EGF in the CM using neutralizing antibodies (Figure W2A) or shRNA in HDMEC (Figure W2B) did not affect HNSCC proliferation. As expected, direct blockade of STAT3, Akt, or ERK pathways with chemical inhibitors decreased tumor cell proliferation (Figure W2C). Notably, HDMEC CM had a significant effect on tumor cell survival. To analyze the effect of endothelial cell-derived factors on tumor cell survival, we cultured OSCC-3 cells in extra low-attachment plates. The percentage of dead cells was significantly higher in low-attachment plates in EBM compared with normal cell culture plates (Figure 5B, a–c). Conditioned medium from HDMEC protected OSCC-3 from anoikis induced by preventing the attachment of the tumor cells to the plate (Figure 5B, a–c). Notably, blockade of IL-6, CXCL8, or EGF in the CM using neutralizing antibodies (Figure 5B, a) or gene silencing in HDMEC (Figure 5B, b) inhibited the protective effect mediated by endothelial cells. Blockade of STAT3, Akt, or ERK activity with Stattic, LY294002, or U0126 showed similar results (Figure 5B, c). These results were confirmed using the Trypan blue exclusion method under the same experimental conditions (Figure W3). To evaluate the effect of endothelial cell-secreted IL-6, CXCL8, or EGF on tumor cell migration, we also used two experimental strategies. Blockade of IL-6, CXCL8, or EGF with neutralizing antibodies (Figure 5C, a) or shRNA in HDMEC (Figure 5C, b) inhibited migration of OSCC-3 compared with controls. In addition, blockade of STAT3, Akt, or ERK using Stattic, LY294002, or U0126 also inhibited OSCC-3 migration (Figure 5C, c). Collectively, these data demonstrate that endothelial cell-initiated signaling has a significant impact on tumor cell survival and migration, two critical elements of the pathobiology of HNSCC.

Figure 5.

Figure 5

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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.

Discussion

This study suggests a new paradigm for endothelial cell and tumor cell interactions in the tumor microenvironment. We demonstrate here that endothelial cells can play an active role in establishing tumor cell phenotypes that are critical for the pathobiology of cancers, namely, tumor cell survival and migration. Several reports suggest a role for inflammation, extracellular matrix, macrophages, and stromal fibroblasts in the initiation and progression of carcinomas [24–26]. However, the role of endothelial cells in activating oncogenic signaling pathways in tumor cells is just starting to be unveiled. Notably, the current paradigm is that tumor cell-initiated signaling is predominant. Yet, this study provides evidence for endothelial cells as key players in the determination of the tumor cell phenotype.

We observed that endothelial cell-initiated signals activate three key intracellular signaling molecules, namely, STAT3, Akt, and ERK in HNSCC. The role of these pathways in cancer has been extensively studied. STAT3 is activated in a wide variety of cancers including HNSCC. Notably, studies have consistently demonstrated an essential role for STAT3 in tumor progression [27–29]. The Akt signaling network is considered a key determinant of the biological aggressiveness of tumors. PI3K/Akt is often activated in HNSCC [30,31]. Finally, ERK is one of the most important signaling molecules in the regulating cell proliferation and is overexpressed in a variety of tumors, including HNSCC [32]. The results of our study suggest that endothelial cells play an active role in the activation of these pathways in HNSCC. Our results demonstrated that STAT3, Akt, and ERK were phosphorylated in tumor cells without requiring cell contact. These results suggested that endothelial cell-secreted factors were able to activate these signaling pathways in the tumor cells. This is in line with observations from other research groups that have characterized soluble factors as key regulators of tumorigenesis [33–35]. One of the challenges of our study was to find out which factors were mediating these effects in the tumor cells. Affymetrix microarrays revealed several genes upregulated in endothelial cells when cocultured with tumor cells. Several validation experiments, together with the existing knowledge of the effects of IL-6, CXCL8, and EGF on STAT3, Akt, and ERK signaling, led us to focus on these three soluble mediators. Although previous work from our laboratory has already shown that endothelial cells overexpressing Bcl-2 secrete high levels CXCL8 [19], the role of endothelial cell-secreted IL-6, CXCL8, and EGF on the activation of signaling pathways in tumor cells has not been described. Interleukin 6 is a cytokine that affects a variety of biological functions including immune response, inflammation, hematopoiesis, and oncogenesis by regulating cell growth, survival, and differentiation [36,37]. Interleukin 6 is one of the major activators of STAT3 signaling [37–39], although it can also stimulate PI3K/Akt and ERK pathways [40]. Recent studies correlate IL-6 levels in HNSCC patients with poor prognosis [41–43]. Interleukin 8 is a member of the CXC chemokine family that contributes to cancer progression through several mechanisms, including the promotion of angiogenesis [44,45]. Many cancer cells constitutively secrete CXCL8 and its receptors CXCR1 and CXCR2, and it has been established that CXCL8 is an autocrine growth factor for a variety of human cancer cells [46]. Interleukin 8 can also activate STAT3, PI3K/Akt, and ERK signaling pathways [47,48]. Overexpression of EGFR has been reported in most HNSCC cases [8,49]. Notably, downstream intracellular targets of EGFR include STAT3, PI3K/Akt, and ERK pathways [41,50,51]. One concludes from these studies that each one of these three cytokines has effects that may converge toward these three pathways in tumor cells. However, under our experimental conditions, we observed that the primary effect of endothelial cell-derived IL-6 was the activation of STAT3, the primary effect of CXCL8 was on the activity of Akt, and the primary effect of EGF was on ERK activity in HNSCC.

Previous work from our laboratory has shown that Bcl-2 expression levels in endothelial cells have a direct effect on tumor growth [3,18]. Here, we demonstrate that IL-6 and EGF are upregulated in endothelial cells overexpressing Bcl-2 and that CM from these cells further enhanced the phosphorylation of STAT3 and ERK, two pathways implicated in the survival of tumor cells. Notably, we have previously demonstrated that the apoptotic index of tumors vascularized with endothelial cells overexpressing Bcl-2 was lower than the index in tumors vascularized with control endothelial cells [18]. At that time, we interpreted these data as simply an increase in endothelial cell survival and tumor microvessel density directly due to the up-regulation of Bcl-2 expression in these cells. However, the data presented here suggest that the activation of major survival pathways for SCC mediated by endothelial cell-secreted factors may also have contributed to those results.

It was critical for us to understand the biological significance of endothelial cell-induced activation of the STAT3/Akt/ERK pathways in SCCs. Surprisingly, we did not observe a significant effect in tumor cell proliferation when we blocked endothelial cell-derived IL-6, CXCL8, or EGF. We attributed these negative results to the exceedingly high proliferation rate of OSCC-3. We reasoned that these cells have such a high basal mitotic activity that blockade of one additional mitogenic pathway derived from the endothelial cells is not sufficient to cause a significant change in OSCC-3 cell numbers. Conversely, we observed that endothelial cell-secreted factors significantly protected the tumor cells from anoikis. Notably, this protective effect was partially inhibited when we downregulated IL-6, CXCL8, or EGF in the endothelial cells. Finally, considering that IL-6, CXCL8, and EGF have been characterized as chemotactic factors, we studied their effect on tumor cell motility using migration assays. These experiments demonstrated that endothelial cells are capable of generating a chemotactic gradient that induces tumor cell motility toward the blood vessels. Such findings may help us to understand the mechanisms underlying the process of field cancerization, which involves the lateral spread of malignant disease and is commonly observed in patients with head and neck cancer [20,52]. We hypothesize that endothelial cells play a key role in field cancerization by providing chemotactic signals that enhance the invasive phenotype of tumor cells, and by activating survival signals that protect the tumor cells against anoikis once these cells are displaced from their original microenvironment. This hypothesis is currently being rigorously tested in our laboratory.

Collectively, these data led to a new model for cross talk between endothelial and SCC cells. It is known that tumor cell-secreted VEGF binds to its cognate receptors in endothelial cells and induce expression of Bcl-2 [3,17]. Bcl-2 enhances IL-6, CXCL8, and EGF synthesis and secretion by endothelial cells. The endothelial cell-secreted factors induce activation of the STAT3, Akt, and ERK signaling pathways in tumor cells. The biological outcome of this cross talk is a significant increase in tumor cell survival and migration (Figure 6). We postulate that better understanding of the complexity of signal transduction processes between tumor cells and other cells from the tumor microenvironment may help to optimize the overall therapeutic benefit of molecularly targeted drugs. The fact that tumor-associated endothelial cells are readily accessible to drugs injected in the circulation makes them a particularly attractive therapeutic target. The work presented here demonstrated that blockade of specific pathways in endothelial cells may have a direct impact on tumor cell survival and migration, two critical components of the pathobiology of SCCs.

Figure 6.

Figure 6

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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.

Supplemental Materials and Methods

Noncontact Coculture Assays

Tumor cells (OSCC-3, UM-SCC-17B, UM-SCC-1, or UM-SCC-74A; 2 x 105) were plated in 1-µm-pore cell culture inserts containing polyethylene terephthalate track-etched membranes (Becton Dickinson) in six-well plates (Multiwell; Becton Dickinson). HDMECs (1 x 105) were cultured in the bottom wells of the Transwell system. Serum-free EBM or EGM2-MV was used for these coculture experiments. Total RNA and protein from both endothelial and tumor cells was collected 24 hours after cells were combined, and microarrays or Western blots were performed.

GEArray RNA Microarrays

Gene expression of tumor cells (OSCC-3, UM-SCC-17B, UM-SCC-1A, or UM-SCC-74A) cocultured with HDMEC was compared with tumor cells cultured alone using GEArray Q series (SuperArray; Bioscience Corporation, Frederick, MD). RNA was purified using the RNeasy kit (Qiagen), and 3 µg of total RNA was analyzed using Human Signaling Transduction in Cancer and Human Angiogenesis gene arrays according to the manufacturer's instructions.

Sulforhodamine B Assays

OSCC-3 (2 x 103) were seeded in 96-well plates (Becton Dickinson) and exposed to HDMEC CM containing 1 µg/ml anti-IL-6, anti-CXCL8, anti-EGF, or IgG isotype control (R&D Systems). Alternatively, OSCC-3 cells were exposed to CM from HDMEC-shRNA-IL-6, HDMEC-shRNA-CXCL8, HDMEC-shRNA-EGF, or HDMEC-shRNA-C. OSCC-3 were also exposed to HDMEC CM containing 5 µM Stattic (Calbiochem), 10 µM LY294002, or 10 µM U0126 (Cell Signaling Technology). After 24 to 72 hours, cells were fixed with 10% trichloroacetic acid and stained with 0.4% sulforhodamine B solution. Plates were read in a microplate reader at 565 nm (Tecan).

Supplementary Material

Supplementary Figures and Tables
neo1106_0583SD1.pdf (146.7KB, pdf)

Acknowledgments

The authors thank Tom Carey (University of Michigan) and Mark Lingen (University of Chicago) for the HNSCC cells used here. The authors also thank Chris Yung for his work with the illustration of the model and Taocong Jin for his technical assistance.

Abbreviations

CM

conditioned medium

CXCL8

interleukin-8

EBM

endothelial cell basal medium

EGF

epidermal growth factor

ERK

extracellular signal-regulated kinase

HDMEC

human dermal microvascular endothelial cell

HNSCC

head and neck squamous cell carcinoma

IL-6

interleukin-6; NF-κB, nuclear factor κB

OSCC-3

oral squamous cell carcinoma

PI3K/Akt

phosphoinositol 3-kinase/Akt

SCC

squamous cell carcinoma

shRNA

short hairpin RNA

STAT3

signal transducer and activator of transcription 3

VEGF

vascular endothelial growth factor

Footnotes

1

This work was supported by grant P50-CA97248 (University of Michigan Head & Neck SPORE) from the National Institutes of Health/National Cancer Institute and grants R01-DE14601, R01-DE15948, R01-DE16586, and R21-DE19279 from the National Institutes of Health/National Institute of Dental and Craniofacial Research (J.E.N.).

2

This article refers to supplementary materials, which are designated by Figures W1 and W2 and are available online at www.neoplasia.com.

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Supplementary Materials

Supplementary Figures and Tables
neo1106_0583SD1.pdf (146.7KB, pdf)

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