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. 2009 May;7(5):755-64.
doi: 10.1158/1541-7786.MCR-08-0472. Epub 2009 May 12.

A key role for early growth response-1 and nuclear factor-kappaB in mediating and maintaining GRO/CXCR2 proliferative signaling in esophageal cancer

Bo Wang  1 , Levon M Khachigian, Luke Esau, Michael J Birrer, Xiaohang Zhao, M Iqbal Parker, Denver T Hendricks
Affiliations

A key role for early growth response-1 and nuclear factor-kappaB in mediating and maintaining GRO/CXCR2 proliferative signaling in esophageal cancer

Bo Wang et al. Mol Cancer Res. 2009 May.

Abstract

Although early growth response-1 (EGR-1) has been shown as a key transcription factor in controlling cell growth, proliferation, differentiation, and angiogenesis, its role in the development of esophageal cancer is poorly understood despite the high frequency of this disease in many parts of the world. Here, immunohistochemistry showed that EGR-1 is overexpressed in 80% of esophageal tumor tissues examined. Furthermore, EGR-1 is constitutively expressed in all esophageal cancer cell lines analyzed. Esophageal squamous carcinoma WHCO1 cells stably transfected with EGR-1 short hairpin RNA displayed a 55% reduction in EGR-1 protein levels, 50% reduction in cell proliferation, a 50% reduction in cyclin-dependent kinase 4 levels, and a 2-fold induction in p27(Kip1) levels associated with a G(2)-M cell cycle arrest. EGR-1 knockdown also caused a marked induction in IkappaBalpha expression, an effect also observed in GRObeta RNA interference-expressing WHCO1 cells, because EGR-1 lies downstream of GRO/CXCR2 signaling. Furthermore, p65 mRNA levels were also reduced in cells treated with either short hairpin RNA EGR-1 or small interfering RNA EGR-1. Immunohistochemical analysis indicated that p65 is elevated in 78% (n = 61) of esophageal tumor sections analyzed. Moreover, nuclear factor-kappaB inhibition with either sodium salicylate or p65 RNA interference led to a significant reduction in GROalpha and GRObeta expression. These results indicate that EGR-1 and nuclear factor-kappaB mediate GRO/CXCR2 proliferative signaling in esophageal cancer and may represent potential target molecules for therapeutic intervention.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

FIGURE 1.
FIGURE 1.
Expression of EGR-1 in normal and tumor esophageal tissues. A. Formalin-fixed, paraffin-embedded esophageal tumor tissues (top, II and IV) and adjacentnormal tissues (I and III) were subjected to immunohistochemical staining using polyclonal antibodies to human EGR-1 as described in Materials and Methods. EGR-1 was primarily located in the nucleus (see arrows in II and IV). Bar, 10 μm. b, basal cells of esophageal epithelium; m, mature epithelial cells; n, nucleus. An analysis of the stained sections shows that 80% (52 of 65) of the tumor tissues express EGR-1 at higher levels than observed in the corresponding normal tissues (right). B. Total RNA extracted from biopsies (normal and tumor tissue) obtained from patients diagnosed with esophageal squamous cell carcinoma was subjected to real-time RT-PCR, and the PCR products were analyzed by agarose gel electrophoresis; real-time RT-PCR was carried out with EGR-1-specific primers as described in Materials and Methods. Data represent the standardized expression of EGR-1 mRNA in esophageal squamous cell carcinoma tumor samples and adjacent uninvolved normal esophageal samples. Bars, SD for each sample analyzed in triplicate. *, P < 0.05, Student’s t test relative to normal control. Inset, real-time RT-PCR products separated on the agarose gel for EGR-1 and glyceraldehyde-3-phosphate dehydrogenase (lane 2) and the water blank control (lane 1).
FIGURE 2.
FIGURE 2.
Inhibitory effect of EGR-1 RNAi on cellular proliferation of esophageal cancer. A. To explore the potential role of EGR-1 in cellular proliferation of esophageal cancer, WHCO1 cells were stably transfected with either EGR-1 RNAi or EGR-1 sister control. Total cellular RNA was isolated from clones transfected with either EGR-1 RNAi or its sister control and subjected to real-time RT-PCR using primers to EGR-1. Data represent the standardized expression of EGR-1 mRNA in the indicated cell lines. Bars, SD for each sample analyzed in triplicate. *, P < 0.05, Student’s t test relative to normal control. B. Immunofluorescence staining was done using an EGR-1-specific antibody to determine the EGR-1 protein level and its subcellular localization as described in Materials and Methods. C. To detect the effect of silencing EGR-1 on the proliferation of WHCO1 cells, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was done as described in Materials and Methods and the absorbance was measured at 595 nm using a microtiter plate reader. Each point represents four analyses with the experiment repeated. D. To determine the effect of silencing EGR-1 on the progress of cell cycle, cell cycle analysis was carried out using propidium iodide staining as described in Materials and Methods and each sample was done in triplicate. *, P < 0.05.
FIGURE 3.
FIGURE 3.
Interference with EGR-1 signaling affects expression of key cell cycle regulators. Whole-cell lysates prepared from WHCO1 cells stably transfected with either EGR-1 RNAi, EGR-1 sister, GROα RNAi, GROβ RNAi, or vector control were separated on 7% to 15% gradient SDS-PAGE. Western blot analysis was carried out using antibodies specific to either EGR-1 or CDK4 or p27Kip1 as described in Materials and Methods.
FIGURE 4.
FIGURE 4.
EGR-1 contributes to constitutive activation of NF-κB in esophageal cancer. A. Total RNA extracted from WHCO1 and WHCO1 clones containing GROα RNAi, GROβ RNAi, EGR-1 RNAi was subjected to real-time RT-PCR carried out with IκBα-specific primers as described in Materials and Methods, and the PCR products were analyzed by agarose gel electrophoresis (lane 1, water blank; lane 2, IκBα). Data represent the standardized expression of IκBα mRNA in WHCO1 transfected with vector and WHCO1 clones containing GROα RNAi, GROβ RNAi, and EGR-1 RNAi. Bars, SD for each sample measured in triplicate. *, P < 0.05, Student’s t test relative to vector control WHCO1. B. Total RNA extracted from EGR-1 stable clones and KYSE 450 transiently transfected with EGR-1 siRNA was subjected to real-time RT-PCR using p65-specific primers as described in Materials and Methods. Data represent the standardized expression of p65 mRNA in the indicated cell lines. Bars, SD for each sample measured in triplicate. C. Formalin-fixed, paraffin-embedded esophageal tumor tissues (II and IV) and adjacentnormal tissues (I and III) were subjected to immunohistochemical staining using monoclonal antibody to human p65 as described in Materials and Methods; p65 was located in both cytoplasm and nucleus. Bar, 10 μm. An analysis of the stained sections shows that 78% (48 of 61) of the tumor tissues express p65 at higher levels than observed in the corresponding normal tissues (bottom).
FIGURE 5.
FIGURE 5.
Activation of NF-κB is essential for GROα and GROβ expression. A. Nuclear extracts prepared from WHCO1 treated with 5 mmol/L sodium salicylate for indicated times were subjected to 10% SDS-PAGE, and Western blot analysis was done using monoclonal antibody to p65 (top). Western blot analysis of β-tubulin was carried out using nuclear extracts and whole-cell lysate to determine cytoplasmic contamination of nuclear extracts as described in Materials and Methods. WL, whole-cell lysate. B. WHCO1 cells were grown on coverslips in the same dish for preparation of nuclear extracts and exposed to 5 mmol/L sodium salicylate for the indicated times, and immunofluorescence staining was done to detect GROα (top) and GROβ (bottom) protein levels using polyclonal antibodies to either GROα or GROβ. C. Whole-cell lysates prepared from WHCO1 cells stably transfected with either p65 RNAi or control RNAi were separated on 10% gradient SDS-PAGE. Western blot analysis was carried out using monoclonal antibody specific to p65 as described in Materials and Methods. D. Total RNA isolated from p65 RNAi-expressing WHCO1 cells and control RNAi WHCO1 cells was subjected to real-time RT-PCR using GROα- and GROβ-specific primers as described in Materials and Methods. Data represent the standardized expression of GROα and GROβ mRNA in the indicated cells. Bars, SD for each sample measured in triplicate. *, P < 0.05, Student’s t test relative to normal control.
FIGURE 6.
FIGURE 6.
NF-κB contributes significantly to proliferation in esophageal cancer cells. To determine the effect of p65 knockdown on the proliferation of WHCO1 cells, cells were maintained in serum-free DMEM, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was done as described in Materials and Methods, and absorbance was measured at 595 nm using a microtiter plate reader. Each point represents four analyses with the experiment done in duplicate.
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