| Background and ObjectiveEsophageal squamous cell carcinoma (ESCC) is a common malignant cancer with poor prognosis. Despite of rapid progress in surgery and adjuvant therapy, the overall 5-year survival rate remains unsatisfactory with less than 30%. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a gene located at chromosome 10q23.3. It is a negative regulator of PI3K/AKT/mTOR pathway, thus regarded as an important tumor suppressor gene to prevent from the development of tumors. Loss of PTEN expression has been reported to be associated with prognosis in breast cancer, prostate cancer and brain tumor et al. However, whether the expression of PTEN is decreased in ESCC and associated with survival rate is still controversial. In addition, it is not determined whether loss of PTEN expression is correlated with postoperative lymphatic metastatic recurrence in ESCC. Furthermore, growing evidences are showing that PTEN hypermethylation could be regarded as early biomarkers for cancer detection and cancer progression in several tumors. PTEN hypermethylation was recently studied to be associated with gender, differention and lymph node metastasis in ESCC. Nevertheless, there was no study on the function of PTEN methylation in the prognosis and loss of PTEN in ESCC. Thus, we aimed to investigate the association between PTEN expression and prognosis or PTEN hypermethylation in ESCC in the present study.MethodsA total of 74 patients with mid-thoracic ESCC who underwent esophagectomy in Provincial Hospital Affiliated to Shandong University were enrolled in the present study. We assessed the mRNA and protein level of PTEN in ESCC by qRT-PCR and immunohistochemistry. Chi-square analysis was used to evaluate the association between negative expression of PTEN protein and clinicopathological factors. The association between negative expression of PTEN protein and 5-year survival rate/3-year lymphatic metastatic recurrence were evaluated using log-rank test. Whether negative expression of PTEN protein was an independent risk factor of 5-year survival rate/3-year lymphatic metastatic recurrence was assessed using Cox regression analysis. After PTEN expression was stably silenced by lentiviral-vectored shRNA (Lenti-shRNA), cell proliferation, migration and invation assay in vitro and growth rate of xenograft tumors in vivo were evaluated. The expression of FAK and p-FAK was detected using Western Blot to study the mechanism of PTEN in ESCC. In addition, the methylation status of PTEN gene was evaluated in 74 ESCC specimens and four esophageal cancer cell lines. Its association with clinicopathological factors or the prognosis was investigated by statistical analysis. The role of PTEN hypermethylation in loss of PTEN expression was analyzed in detail in clinical samples. Next, dementylation of PTEN gene with 5-azaC in EC9706 was performed to confirm the clinical findings of the association between PTEN hypermethylation and loss of PTEN expression in ESCC.ResultsPTEN mRNA level in tumors was significantly lower than that in corresponding non-tumor esophageal epitheliums (p<0.001).41 patients (55.4%) and 38 patients (51.4%) showed reduced mRNA level of PTEN and negative expression of PTEN protein in ESCC. Negative expression of PTEN protein was only correlated with T stage and N stage instead of other clinicopathological factors (p=0.005 and p=0.021). The five-year survival rate (15.8%) in PTEN-negative expression group was lower than that (47.2%) in PTEN-positive expression group (p=0.001). The lymphatic recurrence rate (60.5%) in PTEN-negative expression group was higher than that (36.1%) in PTEN-positive expression group (p=0.019). Multivariate Cox regression analysis showed negative expression of PTEN expression was not an independent risk factor of 5-year survival rate or 3-year lymphatic recurrence (p>0.05). After stable silencing of PTEN expression in Eca109, cell proliferation, cell motility and cell invasion were increased in vitro, and growth rate of xenograft tumors was higher in vivo. The expression of FAK and p-FAK did not show obvious differences in various groups, indicating that FAK/p-FAK was not involved in the mechanism of PTEN in ESCC. PTEN methylation was only found in 14 (18.9%) of 74 ESCC tumor samples and one (EC9706) of four esophageal cancer cell lines. PTEN methylation was not statistically associated with clinicopathological factors and the prognosis (p>0.05). Detailed clinical analysis indicated that PTEN methylation was a possible mechanism of loss of PTEN expression in ESCC. In addition,5-azaC demethylation revealed inversed methylation status and increased mRNA or protein level of PTEN in EC9706. However, due to low frequency of PTEN hypermethylation and common loss of PTEN expression in ESCC, we believed that PTEN hypermethylation was not the leading cause of loss of PTEN expression in ESCC. Maybe some other mechanisms, including transcriptional regulation by transcriptional factors, posttranscriptional regulations by microRNAs and posttranslational modifications, et al., were the main mechanisms leading to loss of PTEN in ESCC.ConclusionNegative expression of PTEN could be a useful biomarker to predict higer lymphatic recurrence and lower survival rate in ESCC. PTEN methylation is a rare event and did not play an important role in the prognosis and loss of PTEN expression in ESCC. |
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