|Year : 2016 | Volume
| Issue : 5 | Page : 60-63
The Rho GTPase RhoE exerts tumor-suppressing effects in human esophageal squamous cell carcinoma via negatively regulating epidermal growth factor receptor
Haojie Wang, Yi Wang, Bing Liang, Fei He, Yong Li, Jianbo Che, Xiaohui Li, Hui Zhao, Gongning Shi
Department of Cardiothoracic Surgery, Henan University Huaihe Hospital, Kaifeng, Henan 475000, China
|Date of Web Publication||7-Oct-2016|
Department of Cardiothoracic Surgery, Henan University Huaihe Hospital, Kaifeng, Henan 475000
Department of Cardiothoracic Surgery, Henan University Huaihe Hospital, Kaifeng, Henan 475000
Source of Support: None, Conflict of Interest: None
Objectives: The objective of this study was to investigate the expression pattern, functions, and possible mechanisms of RhoE/Rnd3, a novel member of the Rho GTPases family, in human esophageal squamous cell carcinoma (ESCC) cells by using molecular and cell-based experiments.
Materials and Methods: Quantitative polymerase chain reaction and Western blotting were carried out to determine the mRNA and protein expression of RhoE in ESCC cell lines, respectively. Both 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT) and flow cytometry were applied to evaluate the effects of RhoE overexpression on ESCC cell growth and apoptosis. Furthermore, Western blotting was used to test the expression of epidermal growth factor receptor (EGFR) and phosphorylated-extracellular signal-regulated kinase (p-ERK) in ESCC cells after RhoE was forced and expressed.
Results: RhoE was downregulated in human ESCC tissues. Overexpression of RhoE inhibited cell growth as assessed by MTT assay and induced apoptosis. Importantly, we proved that RhoE could negatively regulate the protein expression of EGFR and p-ERK, suggesting that RhoE might inhibit ESCC progression through the EGFR/ERK pathway.
Conclusion: Our data supported that RhoE could inhibit cell proliferation and promote apoptosis. Moreover, these tumor-suppressing effects might be acted through the negative regulation of EGFR/ERK signaling.
Keywords: Cell growth and apoptosis, epidermal growth factor receptor/extracellular signal-regulated kinase, esophageal squamous cell carcinoma, RhoE
|How to cite this article:|
Wang H, Wang Y, Liang B, He F, Li Y, Che J, Li X, Zhao H, Shi G. The Rho GTPase RhoE exerts tumor-suppressing effects in human esophageal squamous cell carcinoma via negatively regulating epidermal growth factor receptor. J Can Res Ther 2016;12:60-3
|How to cite this URL:|
Wang H, Wang Y, Liang B, He F, Li Y, Che J, Li X, Zhao H, Shi G. The Rho GTPase RhoE exerts tumor-suppressing effects in human esophageal squamous cell carcinoma via negatively regulating epidermal growth factor receptor. J Can Res Ther [serial online] 2016 [cited 2018 Oct 19];12:60-3. Available from: http://www.cancerjournal.net/text.asp?2016/12/5/60/191633
Haojie Wang and Yi Wang contribute equally to this work
| > Introduction|| |
Esophageal cancer represents as the eighth most frequent malignancy and the sixth leading cause of cancer-related deaths worldwide. Esophageal cancer mainly includes two different histological subtypes: Adenocarcinoma and squamous cell carcinoma, in which esophageal squamous cell carcinoma (ESCC) is the predominant type, especially in China and other Asian countries.,, In addition, the 5-year survival rate remains 5–10% for ESCC in Chinese population, and effective options on diagnosis and treatments for this disease are still largely lacking, especially for those at the advanced stage with organ metastasis.,
Rho GTPases, which include RhoA, Rac1, and cell division cycle 42, are a family of proteins participating in cytoskeletal dynamics and other cellular processes important for tumor biology. These proteins are conserved from yeast to mammals, and function by interacting with and stimulating multiple downstream targets. Most Rho proteins function as molecular switches, cycling between an active GTP-bound conformation and an inactive GDP-bound conformation. The Rnd family, as a distinct subgroup of the Rho proteins, contains three proteins in human cells, including Rnd1/Rho6, Rnd2/Rho7, and Rnd3/Rho8/RhoE. Rnd3/RhoE is an atypical member of the Rho family because it is devoid of GDPase activity and predominantly GTP-bound, hence considered as constitutively activated within cells. Increasing evidence had suggested that RhoE exerts important functions in the carcinogenesis and progression of human cancers. For instance, RhoE has been shown to function in different processes of cancer development, including cell proliferation, and apoptosis, cell transformation, cell migration and invasion.,,,,
In ESCC, Zhao et al. had reported that RhoE functions as a tumor suppressor and modulates the PTEN/PI3K/Akt signaling pathway. However, still little is known about whether other mechanisms correspond for its tumor-suppressing roles. The aim of this study was to examine the roles as well as other underlined mechanisms of RhoE in ESCC cells.
| > Materials and Methods|| |
Human esophageal epithelial cells (HEECs) were ordered from ATCC. The ESCC cell lines including KYSE-150, KYSE-410, Eca-109, and Ec-9706 (Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China) were maintained in RPMI1640 supplemented with 10% fetal bovine serum at 37°C in a humidified incubator containing 5% CO2.
Plasmids and transfections
Wild-type human RhoE proteins were amplified from cDNA of HEEC cells by polymerase chain reaction (PCR). And then, it was cloned into pcDNA3.1 vector without myc-tag in C-terminal. Ec-9706 cells were transfected with LipofectAMINE2000 (Invitrogen) according to the manufacturer's instructions.
Total RNA extraction and quantitative polymerase chain reaction
Total RNA was extracted by Trizol reagent (Life Technologies). Quantitative PCR (qPCR) was performed using a SYBR green method on ABI7500 system. β-actin was used as a normalized control.
Immunoblotting and antibodies
Total proteins were extracted by RIPA buffer plus with cocktail protease inhibitor (Roche) and other phosphatase inhibitors. Antibodies were from the following sources: RhoE, phosphorylated-extracellular signal-regulated kinase (p-ERK) and total-extracellular signal-regulated kinase (t-ERK) from Santa Cruz Biotechnology; epidermal growth factor receptor (EGFR) from Cell Signaling Technology; and β-actin from Sigma-Aldrich.
Cell apoptosis assay
106 of Ec-9706 cells after transfections were harvested at 48 h and washed in phosphate-buffered saline (PBS), then fixed in 70% cold ethanol overnight at −20°C. After washing in cold PBS twice, cells were resuspended in 1 ml of PBS with 40 μg of propidium iodide and 100 μg of RNaseA at 37°C for 1 h. Then, the samples were subjected to analysis of their DNA contents by FACScan.
3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT) assay was performed to test cell viability. Ec-9706 cells transfected with pcDNA3.1 RhoE or control vectors were trypsinized and seeded into 96-well plates at a density of 1 × 103 cells in 200 µl of full medium each well. Then, the plate was incubated at 37°C with 5% CO2. MTT assay was performed each day for the next continuous 4 days. Absorbance at 490 nm (OD490) was detected by using a microplate reader.
All experimental results in this study were from three independent experiments. The data were analyzed by two-sided Student's t-test using SPSS software (version 13, http://www-01.ibm.com/software/analytics/spss/). Summary statistics was expressed at means ± standard deviation. In all statistical analyses, P < 0.05 was considered statistically significant.
| > Results|| |
Downregulation of RhoE in esophageal squamous cell carcinoma cells
To examine the roles of RhoE in ESCC, we first detected its mRNA expression in several selected ESCC cell lines. As shown in [Figure 1]a, compared with the immortalized HEEC, the mRNA levels in ESCC cell lines exhibited downregulation. Particularly, RhoE mRNA was most decreased in Ec-9706 cells, approximately 2-fold lower than that in HEEC cells. In addition, the protein expression of RhoE revealed the same obvious downregulation in ESCC cell lines [Figure 1]b.
|Figure 1: Downregulation of RhoE in esophageal squamous cell carcinoma cells. (a) Quantitative polymerase chain reaction analysis of RhoE expression in four human esophageal squamous cell carcinoma cell lines, compared to human esophageal epithelial cells. RhoE mRNA levels were normalized using β-actin, *P < 0.05. (b) Western blotting analysis of RhoE expression in four human esophageal squamous cell carcinoma cell lines, compared to human esophageal epithelial cells. β-actin was used as an internal control|
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Elevation of RhoE expression inhibiting cell growth and induced apoptosis in EC-9706 cells
To investigate the biological function of RhoE in ESCC cells, we took Ec-9706 cell line as the in vitro model, since RhoE was expressed at a low internal level. pcDNA3.1–RhoE or control pcDNA3.1 vector was transiently transfected into Ec-9706 cells, the elevation of RhoE protein was validated by qPCR and Western blotting [Figure 2]a and [Figure 2]b. Twenty-four hours later, cells were seeded into 96-well plates, and MTT assay was applied to analyze cell growth rate. We found that heterogeneous expression of RhoE in Ec-9706 cells led to a slower growth rate compared with that in Ec-9706 cells transfected with empty vector pcDNA3.1 [Figure 2]c. Moreover, flow cytometry experiments demonstrated that RhoE overexpression could induce apoptosis as shown in [Figure 2]d. These results suggest that a high dose of RhoE could inhibit cell growth and induce cell apoptosis in ESCC cells.
|Figure 2: Elevation of RhoE expression inhibiting cell growth and induced cell apoptosis in Ec-9706 cells. (a) Quantitative polymerase chain reaction analysis for RhoE mRNA expression level in RhoE-overexpressing Ec-9706 cells. β-actin was used as an internal control, *P < 0.05. (b) Ec-9706 cells transfected with expressing vector of nontagged RhoE were lysed and analyzed by immunoblotting using RhoE antibody. (c) Ec-9706 cells were transfected with the expression vector of RhoE or pcDNA3.1 control vector. Cell growth was evaluated every day by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide assay as described in the materials and methods section, **P < 0.01. (d) The effect of overexpressing RhoE on Ec-9706 cell apoptosis in vitro by cell flow cytometry, *P < 0.05|
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Negative regulation of epidermal growth factor receptor/extracellular signal-regulated kinase signaling pathway due to RhoE overexpression
Next, we studied the underlined mechanisms that are responsible for the growth-suppressing and apoptosis induction effects of RhoE in ESCC cells. It is well known that the EGFR is a key cellular receptor which has been implicated in many cancers. Upon binded by ligands, the monomeric EGFR leads to dimerization and autophosphorylation, which consequently allows for the docking of adaptor proteins that initiate downstream signaling, including Ras/Raf/MEK/ERK/MAPK signaling and PI3K/PDK1/Akt signaling. We next investigated whether the key factors of EGFR/ERK signaling pathway could be regulated by RhoE. As shown in [Figure 3], we found that overexpression of RhoE could downregulate the protein expression of EGFR as well as p-ERK, whereas the t-ERK level remained constant, which indicated that RhoE might inhibit ESCC progression through the EGFR/ERK pathway.
|Figure 3: Changes of RhoE expression regulate key factors and downstream target proteins of epidermal growth factor receptor/extracellular signal-regulated kinase. Expressions of epidermal growth factor receptor, phosphorylated-extracellular signal-regulated kinase and total-extracellular signal-related kinase proteins were determined by Western blotting analysis in Ec-9706 cells transfected with pcDNA3.1 RhoE or control vectors|
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| > Discussion|| |
Here, we reported that RhoE was downregulated in ESCC cell lines, and we also found that RhoE could inhibit cell proliferation and promote apoptosis. Moreover, these tumor-suppressing effects might be acted through the negative regulation of EGFR/ERK signaling. Previously, RhoE expression was also reported as low in ESCC tissues and cell lines, it was suggested to function as a tumor suppressor by influencing cell proliferation, cell cycle progression, as well as cell invasion in vitro, and inhibited cell apoptosis. Therefore, our results presented in this study seems redundant, but still prove that the tumor-suppressing effects of RhoE in ESCC were true. However, we provided another possibility that EGFR/ERK might be the acting pathway by RhoE in ESCC cells.
There are controversial reports about the functional significance of RhoE in the development and progression of human cancers. Numerous studies indicated that upregulation of RhoE was observed in pancreatic cancer, colon cancer, and melanomas, suggesting RhoE functions as an oncogene. Nevertheless, RhoE expression was markedly decreased in prostate cancer and hepatocellular carcinoma. Complexity and diversity of the functions of RhoE in different tumors impelled us to investigate the role of RhoE in the development and progression of ESCC.
As a member of Rnd subfamily of the Rho GTPases, RhoE is not regulated by the typical GTP/GDP cycle, but was thought to be regulated dominantly at the transcriptional level. In our study, we proved that the mRNA expression of RhoE was indeed reduced in ESCC cell lines. However, the mechanisms for this downregulation of RhoE transcription remain unknown. Notably, Chen et al. previously reported that epigenetic silencing of the RhoE promoter by hypermethylation occurred in gastric cancer. Whether this epigenetic modification exits in ESCC cells stay unclear.
| > Conclusion|| |
Our results suggested that RhoE is a potential tumor suppressor gene in ESCC cells by inhibiting cell growth and promoting apoptosis, and the effects of RhoE on cellular biological responses are tightly associated with the EGFR/ERK signaling pathway. The targeting of this pathway and RhoE may offer a new therapeutic approach for ESCC treatment.
Financial support and sponsorship
This study was financially supported by the Henan Provincial Education Science and technology research key Fund Project (No. 14A320081).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]