Aims To investigate the expression of epithelial cell transforming sequence 2 (ECT2) in invasive breast cancer and its prognostic significance.
Methods ECT2 immunohistochemical detection was performed in 165 breast cancer specimens and 100 normal control tissues. Univariable and multivariable Cox proportional hazards regression model analysis was used to confirm independent prognostic factors. The PHREG procedure linear hypotheses testing method was used to analyse survival data.
Results Expression of ECT2 in breast cancer was significantly higher than that of the normal control group (p<0.001), and it was related to tumour grade, the status of lymph node metastasis, TNM staging, recurrence status, menopausal status, and the Ki-67 proliferation index (p<0.05), and not related to age, tumour size, tumour type, expression of estrogen receptor, progesterone receptor and human epidermal growth factor 2, and triple-negative disease (p>0.05). Univariable analysis showed that expression of ECT2, the status of lymph node metastasis, triple-negative disease and Ki-67 proliferation index were related to the overall survival of patients with breast cancer (p<0.001, p=0.006, p=0.001, p=0.041, respectively). PHREG procedure linear hypotheses testing results for overall survival revealed that high expression of ECT2, lymph node metastasis, triple-negative disease and high Ki-67 proliferation index predicted lower overall survival rates. Multivariable Cox regression indicated that high expression of ECT2 and triple-negative disease were independent prognostic factors for patients with breast cancer (p<0.001, p=0.004, respectively).
Conclusions Expression of ECT2 may be one of the main causes of the occurrence and development of breast cancer, and high expression of ECT2 as an independent prognostic factor predicts a poor prognosis. ECT2 could also be a potential molecular target for designing therapeutic strategies for breast cancer.
- breast cancer
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Invasive ductal carcinoma is the most common type of breast cancer, followed by invasive lobular carcinoma, and other types are relatively rare. These two types of breast cancer can be definitely diagnosed by pathological findings. The underlying cause of the occurrence and development of this tumour, however, remains a mystery. The occurrence of cancer is directly associated with the malignant transformation of epithelial cells and epithelial cell transforming sequence 2 (ECT2) is considered a major oncogene involved in the onset or progression of human cancers. ECT2 induces the malignant transformation of both epithelial cells and fibroblasts, indicating its vital role in the process of malignant transformation of cells.1 The present study was designed to investigate the expression profile of ECT2 in breast cancer and its relationship with clinical pathological characteristics via detection of ECT2 expression in 165 paraffin-embedded breast cancer tissues and to explore the relationship between ECT2 and the prognosis of patients with breast cancer by survival analysis.
Material and methods
One hundred and sixty-five breast cancer specimens and 100 corresponding non-cancerous tissues(normal tissues) from patients admitted to the Department of Pathology of the First Hospital of Shanxi Medical University from 2006 to 2011 were selected. None of the patients were treated with radiotherapy or chemotherapy before surgery and those with incomplete data were excluded. Patients enrolled were approved by the ethics committee of Shanxi Medical University. All patients signed a written informed consent form . Fluorescence in situ hybridisation (FISH) results were obtained from patients scoring human epidermal growth factor 2 (HER-2) 2+ by immunohistochemistry. All pathological data were reviewed and joint diagnoses were made by two senior pathologists. Follow-up data were available for all patients up to January 2017, with a follow-up time ranging from 13 to 132 months. Of the patients, 107 survived and 58 died.
Main reagents and methods
Anti-ECT2 antibody (catalogue number: AV52381), anti-Ki-67 (catalogue number: PLA0228), estrogen receptor (ER) (catalogue number: E1396), progesterone receptor (PR) (catalogue number: SAB4502184) and HER-2 (catalogue number: SAB4300350) monoclonal antibodies, immunohistochemistry kit, and DAB chromogenic reagent kit (catalogue number: D5637) were purchased from Sigma Company. Immunohistochemistry was performed by the EnVision two-step method according to the kit instructions. The dilution ratio of the ECT2 antibody was 1:100 and phosphate-buffered saline was used as a negative control.
Interpretation of immunohistochemical results
Microscopic results revealed that ECT2 protein was expressed in the nucleus of tumour cells, with slight expression in the cytoplasm of some cells. A count of positive-stained cells was performed and staining intensity was observed, and the percentage of positive cells was calculated (negative=0, 1–10% of positive cells=1, 11–50%=2, 51–80%=3, 81–100%=4) and the staining intensity of positive cells was determined (negative=0, weak positive=1, positive=2, strong positive=3). The product of the percentage and the intensity was used to determine the level of expression: ≤4 was an indicator of low expression or no expression and >4 as high expression. The Ki-67 index was re-evaluated.
Statistical analyses were performed using SPSS for Windows, version 15.0 (SPSS, Chicago, Illinois, USA). χ2 test was used to examine the relationship between ECT2 expression and clinicopathological parameters. We used the SAS (version 9.2) Cox regression procedure PHREG to determine the breast cancer overall survival rate between the variables and the lifetime; if p>0.05, it is shown to be statistically significant.2 The Cox proportional hazards regression model was used to examine univariable and multivariable hazard ratios and identify independent prognostic factors in this study. The univariable Cox proportional hazards regression model was used to screen clinical pathological factors of breast cancer. The multivariable Cox proportional hazards regression model was used to screen the meaningful clinical factors that were screened by univariable Cox proportional hazards regression analysis. Differences were considered statistically significant when p<0.05.
Expression of ECT2 protein in 165 cancer tissues and 100 normal breast tissues
ECT2 was expressed in 95 (57.6%) of the 165 breast cancer tissues to a certain degree (figure 1), and in 13 (13%) of the 100 normal tissues with weakly positive expression. The difference between the two groups was statistically significant (χ2 = 51.239, p<0.001).
Relationship between ECT2 expression and clinical pathological features of breast cancer
HER-2-positive cases were statistically analysed. HER-2-positive cases included those scoring HER-2 2+ with amplification of HER-2 by FISH and those scoring HER-2 3+. HER-2-negative cases consisted of those scoring HER-2 2+without amplification of HER-2 by FISH and those scoring HER-2 1+ (see table 1). It was found from table 1 that the expression of ECT2 protein was related to breast cancer grading, lymph node metastasis, TNM staging, recurrence status, menopausal status, and the Ki-67 proliferation index (p<0.05), and was not related to age, tumour size, tumour type, the expression of ER, PR and HER-2, and triple-negative breast cancer (p>0.05).
Table 2 shows that the survival of patients with breast cancer correlates with the expression of ECT2, lymph node metastasis, triple-negative breast cancer and Ki-67 proliferation index (p<0.05) by univariable analysis. The PHREG procedure linear hypotheses testing results showed that only ECT2 high expression, lymph node metastasis, triple-negative breast cancer and high Ki-67 proliferation index were statistically significant (table 3). The results in table 4 from the multivariable Cox proportional regression model show that the expression of ECT2 and triple-negative breast cancer were independent prognostic factors for patients with breast cancer (p<0.05).
As a guanine nucleotide exchange factor (GEF) of the Rho GTPase family, ECT2 regulates cell division and cell cycle.3 4 Rho GTPases are closely associated with the malignant transformation of human cancers due to the fact that the Rho GTPases are involved in the abnormal signal transduction in tumour cells.5 6 GEF, which promotes the exchange of GDP to GTP and consequently the activation of Rho GTP signal transduction,7 is required for the activation of the Rho GTPase signalling pathway. Therefore, ECT2, as a GEF, plays an important role in the occurrence and development of cancer.
ECT2 is expressed in various cancer tissues and it can upregulate the growth of lung adenocarcinoma cells, including 650, ekvx, hcc4006, HCC827, hcc2935, hop62 and A549. When ECT2 siRNA was used to downregulate the expression of ECT2, the growth, migration and invasion abilities of A549 cells were significantly inhibited, while the overexpression of ECT2 induced significantly enhanced proliferation, migration and invasion of these cells. Therefore, ECT2 plays an important role in the carcinogenesis of lung adenocarcinoma and may serve as a potential therapeutic target for this disease.8 ECT2 was highly expressed in prostate cancer, but it was slightly or not expressed in normal prostate tissue, and the difference was statistically significant. Overexpression of ECT2 was closely correlated with the clinical stage, tumour invasion and lymph node metastasis of prostate cancer, and survival analysis showed that it was an independent prognostic index in patients with prostate cancer.9 In gastrointestinal tumours, such as gastric cancer, colorectal cancer and esophageal cancer, ECT2 was highly expressed and the high expression of ECT2 was an effective marker as well as an important prognostic factor for poor prognosis.3 7 10 In addition to cancer tissues, ECT2 is also expressed in brain tumours such as gliomas. In these gliomas, including patients with grade II–IV glioma and grade II and III oligodendroglioma. Statistical analysis of these patients showed that ECT2 mRNA expression in high-grade gliomas was significantly lower than that of low-grade tumours, and survival analysis demonstrated that overexpression of ECT2 was a marker of poor prognosis in high-grade tumours, suggesting that ECT2 may be a potential therapeutic target for glioma.11 12 In breast cancer cells, ECT2 has been found to be a key factor in the progression of breast cancer.13
Our experimental results showed that the positive rate of ECT2 expression in breast cancer tissues was significantly higher than that in normal tissues, indicating that ECT2 plays an important role in carcinogenesis of breast cancer. ECT2 protein was not only highly expressed in breast cancer, but also related to the clinical pathological parameters. Statistical analysis revealed that the expression of ECT2 protein was associated with tumour grade, the status of lymph node metastasis, TNM staging, recurrence status, menopausal status, and Ki-67 index, but not with the age, tumour size, tumour type, expression of ER, PR and HER-2, and triple-negative breast cancer. Our research shows that high expression of ECT2 occurred in patients with high tumour grade, lymph node metastasis, high TNM staging, relapsed disease and high Ki-67 index, which indicated that the progression of breast cancer may be related to the expression of ECT2, and also indirectly suggested that the expression of ECT2 is related to the prognosis of patients. For this reason, we analysed the relationship between the expression of ECT2 and clinical pathological parameters and survival of patients. The univariable COX proportional hazards regression model showed that the expression of ECT2, the status of lymph node metastasis, triple-negative breast cancer, and Ki-67 proliferation index were risk factors for the prognosis of breast cancer, and the PHREG procedure linear hypotheses testing results for overall survival showed that the prognosis of patients with high expression of ECT2, lymph node metastasis, Ki-67 proliferation index and triple-negative breast cancer was poor. Multivariable analysis of these four risk factors related to breast cancer prognosis showed that ECT2 expression and triple-negative breast cancer were independent prognostic factors of breast cancer. As we all know, triple-negative breast cancer has been reported to be an independent prognostic factor for patients with breast cancer in most reports. Our results are consistent with the reports.14–16 In summary, ECT2 plays an important role in the development of breast cancer, and its high expression is one of the important markers for the poor prognosis of patients with breast cancer.
The mechanism of ECT2 in cancer tissues remains unknown and studies have shown that the expression of ECT2 may be related to the methylation of DNA in pancreatic cancer. Zhang et al examined the methylation level of the promoter region of the ECT2 gene by RT-PCR in pancreatic cancer and the results showed that methylation occurred in the upstream of the ECT2 gene, but not in other regions. Other factors, however, may be related.17 Mansour et al reported E6AP (E3 ligase E6-associated protein) serves as a suppressor of metastasis and was a negative regulator of ECT2 in breast cancers, and may be a therapeutic target for patients with metastatic breast cancer.13 In a follow-up study, we will investigate the expression of ECT2 in breast cancer in vitro and in vivo, hopefully to find out the mechanism of ECT2 in the progression of breast cancer. In conclusion, ECT2 may become a new therapeutic target for cancer treatment due to its role in the occurrence and development of breast cancer as well as its independent prognostic significance.
Take home messages
Epithelial cell transforming sequence 2 (ECT2) is one of another newly discovered important factors in the carcinogenesis and development of breast cancer.
The expression of ECT2 is related to the survival of patients with breast cancer, and Its high expression is significantly associated with unfavourable survival rates.
Immunohistochemistry of ECT2 may be the easiest and most efficient way to assess the prognosis of patients with breast cancer.
Handling editor Cheok Soon Lee.
Contributors All authors participated in discussions. HW wrote the paper.
Competing interests None declared.
Patient consent Obtained.
Ethics approval The ethics committee of Shanxi Medical University.
Provenance and peer review Not commissioned; externally peer reviewed.
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