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. 2010 Nov 29;5(11):e14082.
doi: 10.1371/journal.pone.0014082.

Epithelial cell transforming sequence 2 in human oral cancer

Manabu Iyoda  1 , Atsushi Kasamatsu, Takashi Ishigami, Dai Nakashima, Yosuke Endo-Sakamoto, Katsunori Ogawara, Masashi Shiiba, Hideki Tanzawa, Katsuhiro Uzawa
Affiliations

Epithelial cell transforming sequence 2 in human oral cancer

Manabu Iyoda et al. PLoS One. .

Abstract

Background: Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor for Rho family GTPase, which has been implicated in the malignant phenotype of human cancers. Little is known about the effect of a high level of ECT2 in regulating oral cancer cell behavior. In this study, we investigated the involvement of ECT2 in oral squamous cell carcinoma (OSCC).

Methodology/principal findings: We analyzed ECT2 expression in OSCC-derived cell lines and primary OSCCs compared with matched normal tissue (n = 96) by quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry. We then evaluated the correlation between the ECT2 expression status in primary OSCCs and the clinicopathological features. ECT2 expression was significantly up-regulated in OSCCs in vitro and in vivo (p<0.05). Among the clinical variables analyzed, higher ECT2 expression also was associated with the TNM stage grading (p<0.05). When we performed functional analyses of ECT2 in OSCC-derived cells using the shRNA system, the cellular proliferation of the ECT2 knockdown cells decreased significantly compared with the control cells (p<0.05). Cell cycle analysis by flow cytometry showed arrest of cell cycle progression at the G1 phase in the ECT2 knockdown cells. We also found up-regulation of the Cip/Kip family of the cyclin-dependent kinase inhibitors, p21(cip1) and p27(kip1), and down-regulation of cyclin D1, cyclin E, and CDK4. These data suggested that the elevated Cip/Kip family induced inhibition of the cyclin D1-CDK complex activity leading to cell cycle arrest at the G1 phase.

Conclusions/significance: Our results proposed for the first time that ECT2 is an indicator of cellular proliferation in OSCCs and that ECT2 might be a potential therapeutic target for the development of new treatments for OSCCs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ECT2 exprssion in OSCC-derived cell lines.
(A) Quantification of ECT2 mRNA levels in OSCC-derived cell lines by qRT-PCR analysis. To determine mRNA expression of ECT2 in Oral cancer, we performed qRT-PCR analysis using six OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, H1, Ca9-22, and Sa3) and HNOKs. Significant up-regulation of ECT2 mRNA is seen in six OSCC-derived cell lines compared with that in the HNOKs. Data are expressed as the means ± SEM of values from three assays (*p<0.05; Mann-Whitney U test). (B) Western blot analysis of ECT2 protein in the OSCC-derived cell lines and HNOKs. To investigate protein expression of ECT2 in Oral cancer, we performed Western blot analysis using six OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, H1, Ca9-22, and Sa3) and HNOKs. ECT2 protein expression is up-regulated in OSCC-derived cell lines compared with HNOKs. Densitometric ECT2 protein data are normalized to α-tubulin protein levels. The values are expressed as a percentage of the HNOKs.
Figure 2
Figure 2. Comparison of ECT2 mRNA expression levels between primary OSCCs and matched normal oral tissues.
To investigate the ECT2 mRNA expression levels in primary OSCCs and paired normal oral tissues from 96 patients, we performed qRT-PCR analysis. The relative mRNA expression levels in primary OSCCs and the matched oral tissues (n = 96) range from 0.005 to 4.39 (median, 0.289) and 0.003 to 1.632 (median, 0.081), respectively. ECT2 mRNA expression was up-regulated in 75 (78%) of 96 primary OSCCs compared with the matched normal oral tissues. Significantly higher ECT2 mRNA expression was observed in primary OSCCs than matched normal oral tissues (P<0.05; Mann-Whitney U test).
Figure 3
Figure 3. Evaluation of ECT2 protein expression in primary OSCCs.
(A, B) Representative IHC results of ECT2 in normal oral tissue and primary OSCC. (A) Normal oral tissue has no ECT2 protein expression. Original magnification, ×100. Scale bars, 50 µm. (B) ECT2-positive cases of OSCC. Positive immunoreaction for ECT2 is detected in the nucleus and cytoplasm. Original magnification, ×400. Scale bars, 10 µm. (C) State of ECT2 protein expression in nomal oral tissue and primary OSCC. To investigate protein expression of ECT2 in primary OSCCs, we carried out IHC. The ECT2 IHC scores are calculated as follows: IHC score = 1×(number of weak stained cells in the field)+2×(number of moderately stained cells in the field)+3×(number of intensely stained cells in the field). The ECT2 IHC scores for OSCCs and normal oral tissues range from 55.67 to 211.33 (median, 163.33) and 8.33 to 85.33 (median, 44.00), respectively. The ECT2 protein expression level in OSCCs is significantly higher than that in normal oral tissues (p<0.001; Mann-Whitney U test).
Figure 4
Figure 4. Expression ECT2 in shECT2-transfected cells.
To obtain stable ECT2 knockdown transfectants, we performed transfection of the ECT2 shRNA (shECT2) and the control shRNA (Mock) in OSCC cell lines (Sa3 and H1). We performed qRT-PCR and Western blot analyses to investigate ECT2 mRNA and protein expression in shECT2-transfected cells. (A) Expression of ECT2 mRNA in shECT2- and Mock-transfected Sa3 cells. (B) Western blot analysis of ECT2 protein in shECT2- and Mock-transfected cells. The ECT2 mRNA and proteins are significantly down-regulated in shECT2-transfected cells.
Figure 5
Figure 5. Proliferation of shECT2-transfected cells.
To determine the effect of shECT2 on cellular proliferation, shECT2- and Mock-transfected cells were seeded in 6-well plates at a density of 1×104 viable cells per well. shECT2- and Mock-transfected cells counted on 7 consecutive days. The growth of shECT2-transfected cells is significantly inhibited compared with the Mock-transfected cells after 7 days. The results are expressed as the means ± SEM of values from three assays. The asterisks indicate significant differences between the Mock- and shECT2-transfected cells (p<0.01; Mann-Whitney U test).
Figure 6
Figure 6. shECT2 promotes G1 arrest.
To investigate cell cycle progression, we analyzed Flow cytometric determination of DNA content by a FACScalibur in the G0–G1, S, and, G2–M phases. We then determined the expression level of cyclin-dependent kinase inhibitors (p16INK4A, p21cip1, and p27kip1), cyclin D1, cyclin E, and CDK4 to identify the mechanism by which ECT2 blocks G1 progression. (A) Flow cytometric analysis was performed to investigate cell cycle in shECT2- and Mock-transfected cells. The number of cells in the G1 has increased markedly in the ECT2 knockdown cells. (B) qRT-PCR was performed to investigate mRNA levels of cell cycle related genes. PCR shows up-regulation of p21cip1 and p27kip1 and down-regulation of cyclin D1, cyclin E, and CDK4. Data are expressed as the means ± SEM of values from three assays (*p<0.05; Mann-Whitney U test).
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