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. 2011 Jun 15;117(12):2677-89.
doi: 10.1002/cncr.25859. Epub 2011 Jan 10.

Tristetraprolin regulates interleukin-6, which is correlated with tumor progression in patients with head and neck squamous cell carcinoma

Elizabeth Van Tubergen  1 , Robert Vander Broek, Julia Lee, Gregory Wolf, Thomas Carey, Carol Bradford, Mark Prince, Keith L Kirkwood, Nisha J D'Silva
Affiliations

Tristetraprolin regulates interleukin-6, which is correlated with tumor progression in patients with head and neck squamous cell carcinoma

Elizabeth Van Tubergen et al. Cancer. .

Abstract

Background: Tumor-derived cytokines play a significant role in the progression of head and neck squamous cell carcinoma (HNSCC). Targeting proteins, such as tristetraprolin (TTP), that regulate multiple inflammatory cytokines may inhibit the progression of HNSCC. However, TTP's role in cancer is poorly understood. The goal of the current study was to determine whether TTP regulates inflammatory cytokines in patients with HNSCC.

Methods: TTP messenger RNA (mRNA) and protein expression were determined by quantitative real-time-polymerase chain reaction (Q-RT-PCR) and Western blot analysis, respectively. mRNA stability and cytokine secretion were evaluated by quantitative RT-PCR and enzyme-linked immunoadsorbent assay, respectively, after overexpression or knockdown of TTP in HNSCC. HNSCC tissue microarrays were immunostained for interleukin-6 (IL-6) and TTP.

Results: TTP expression in HNSCC cell lines was found to be inversely correlated with the secretion of IL-6, vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE(2) )(.) Knockdown of TTP increased mRNA stability and the secretion of cytokines. Conversely, overexpression of TTP in HNSCC cells led to decreased secretion of IL-6, VEGF, and PGE(2) . Immunohistochemical staining of tissue microarrays for IL-6 demonstrated that staining intensity is prognostic for poor disease-specific survival (P = .023), tumor recurrence and development of second primary tumors (P = .014), and poor overall survival (P = .019).

Conclusions: The results of the current study demonstrated that down-regulation of TTP in HNSCC enhances mRNA stability and promotes secretion of IL-6, VEGF, and PGE(2) . Furthermore, high IL-6 secretion in HNSCC tissue is a biomarker for poor prognosis. In as much as enhanced cytokine secretion is associated with poor prognosis, TTP may be a therapeutic target to reduce multiple cytokines concurrently in patients with HNSCC.

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Conflict of interest statement

The authors report no conflict of interest with this manuscript.

Figures

Figure 1
Figure 1. TTP is downregulated in HNSCC cell lines
A) Total RNA was isolated from normal human keratinocytes (NHK), HOK16B immortalized keratinocytes (IHOK) and HNSCC cell lines. cDNAs were prepared and Q-RT-PCR was performed using SyBr-Green. B) Whole cell lysates from NHK, IHOK and HNSCC cell lines were electrophoresed and blotted with TTP antibody. Actin was used as a loading control. TTP expression was quantified by densitometry, normalized to actin and then to NHK and expressed as percent change. C, D and E) Conditioned media from NHK, IHOK and HNSCC cell lines, was collected at 24 hours. (*p<0.001 compared to NHK). ELISA experiments were performed in triplicate with similar results.
Figure 2
Figure 2. Overexpression of TTP in HNSCC cell lines decreases cytokine secretion
UM-SCC-11A, UM-SCC-17B and OSCC3 cells (A, B and C respectively) were transduced with adenovirus-5 containing β-gal (control) or TTP. Cell lysates and conditioned media were harvested. Cell lysates were electrophoresed and immunoblotted with TTP antibody. Conditioned media was used to quantify IL-6, VEGF, and PGE2 secretion by ELISA. All experiments were performed in triplicate (p<0.001, Standard error bars are present in all graphs).
Figure 3
Figure 3. TTP knockdown induces cytokine secretion in HNSCC cells
UM-SCC-1 (A), UM-SCC-22B (B) and UM-SCC-81B (C) were transfected with siRNA against TTP or non-target siRNA (nt). Whole cell lysates were electrophoresed and blotted with TTP antibody. Actin was used as loading control. Conditioned media was assayed in triplicate for IL-6, VEGF, and PGE2 by ELISA. All experiments were performed in triplicate. Data are representative of the mean and standard deviation of three replicates within an experiment. (*p<0.05).
Figure 4
Figure 4. TTP knockdown increases cytokine mRNA half-life
A) Whole cell lysates from UM-SCC-1 and -22B stably expressing shSVSG or shTTP were immunoblotted. B) and C) RNA was purified, cDNAs were prepared and Q-RT-PCR was performed using SyBr-Green. Ct values for IL-6 and COX-2 were normalized to GAPDH and expressed as a percentage of time zero. Data are representative of the mean and standard deviation of three individual experiments (*p<0.05, **p<0.001).
Figure 5
Figure 5. TTP knockdown increases HNSCC invasion and migration
UM-SCC-1 cells were stably transduced with shTTP or shVSVG. A) Invasion. Transduced cells were plated in serum free media on Matrigel coated inserts or control inserts. Percent invasion was calculated as described in Methods. B) Migration. UM-SCC-1 cells were treated with 25 mmol of Mitomycin C (Sigma). Media was removed and a scratch was made. Immediately following the scratch, complete media DMEM with 25 mmol of Mitomycin C was added and the cells were photographed at 0h and at 24h. Data are representative of two independent experiments with three replicates within an experiment (*p<0.02).
Figure 6
Figure 6. IL-6 intensity is prognostic for disease specific survival and tumor recurrence in HNSCC
A) Immunohistochemistry was performed on tissue sections of normal human epithelium (left panel, bar = 100 µm) and human HNSCC (right panel, bar = 1000 µm) with TTP antibody. Arrows show staining in the basal third of normal epithelium. The inset image is a higher magnification of the outlined box showing invasive epithelium (bar = 60 µm). Arrow shows invasive epithelium. B) Immunohistochemistry was performed on tissue sections of a human HNSCC TMA with the IL-6 and TTP antibodies. The slides were counterstained with hematoxylin. The top and lower panels represent high IL-6 and low TTP staining, respectively, in the same HNSCC tissue (bars = 100 µm). C) Low TTP and high IL-6 are correlated for poor disease specific survival. Patient groups: red line, low TTP, low IL-6 (n = 9); yellow line, low TTP, high IL-6, (n = 3); blue line, high TTP, low IL-6 (n = 11); green line, high TTP, high IL-6 (n = 10). Events (drop in the graph lines) were deaths due to HNSCC. Subjects who did not experience the events, such as deaths from unrelated causes, were censored. D and E) High IL-6 intensity is prognostic for poor disease specific survival (D) and for tumor recurrence and second primary tumor (E). Patient groups, red line, no IL-6 (n=6), yellow line, low IL-6 (n=19), blue line, medium IL-6 (n=14), green line, high IL-6 (n=3). IL-6 intensity is also prognostic for poor overall survival (not shown).
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