| Background:Esophageal cancer is one of the most common cancer type worldwide with high mortality rate, which was responsible for an estimated 455,800 new esophageal cancer cases and 400,200 deaths in 2012. Because of the significant difference among various areas, the highest esophageal cancer incidence rates are found in Eastern Asia and in Eastern and Southern Africa. As the highest-risk area, esophageal squamous cell carcinoma (ESCC) accounts for more than 90% of cases in China, compared with about 26% in the United States. Despite major progress in clinical diagnosis and treatment modalities, ESCC still shows a dismal prognosis with 5-year survival rate less than 15%. Traditional methods of characterizing tumors depend on gross visual information (size of the tumor, degree of infiltration, histological features of the tumor) that is coupled to represent the TNM staging system. Although these parameters do provide a capacity to distinguish tumor subtypes with distinct biological characteristics, it becoming clear that these classifications are imprecise to set up heterogeneous groupings of tumors and patients for clinical treatment. Therefore, it is important to explore effective biomarkers to recognize unique biological characteristics of ESCC patients, in order to efficiently predict the prognosis and to improve therapeutic strategies for ESCC.Significant prognostic biomarkers should be chosen based on either the distinguishing features between benign and malignant tumors or the differentiation/pathological staging status of tumors which can influence clinicians’ decisions related to treatment modalities. In recent years, a number of studies focused on identification of new biomarkers of ESCC so as to find additional therapeutic targets besides surgery, radiotherapy and chemotherapy. Although the potential pathogenesis research and targeted therapies of lung cancer, breast cancer and colorectal cancer has making great progress, relatively little is known about esophageal cancer.PAX9 is a member of the paired box gene family, comprised by nine transcription factors in humans (PAX1-PAX9), which plays key roles in several aspects of embryonic development and organogenesis by regulating the expression of the genes involved in cell proliferation, apoptotic resistance, and cell migration. The family is usually described as cell-lineage-specific regulators of tissues where their expression profiles are finely tuned both spatially and temporally, and now recognized as potentially playing important roles in cancer progression. Muratovska Aleksandra’s study showed that PAX2 is frequently expressed in primary human cancers, including breast and ovarian cancers. PAX3 was required for the survival of melanoma cell lines, and was expressed in breast cancer as well. PAX6 was expressed at high levels in brain, breast, and other cancer cell lines. Expression of PAX2 and PAX8 were found in kidney tissues. Chromosomal translocations involving PAX3, PAX5, PAX7 and PAX8 genes in alveolar rhabdomyosarcoma, B-lymphoid malignancies, and thyroid cancer, respectively. PAX5 was considered to be a functional tumor suppressor involved in hepatocellular carcinoma carcinogenesis through direct regulation of the p53 signaling pathway.Human PAX9 is located within 14q12-q13, characterized by a-128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. Due to the G-quadruplex-forming region located near exon 1, which is present in all known sequenced placental mammals, PAX9 intron 1 plays a key role on splicing efficiency. As a transcription factor expressed in the tooth mesenchyme during tooth morphogenesis, heterozygous mutations in PAX9 have been primarily associated with non-syndromic tooth agenesis, predominantly in the molars. In the last decade, the abnormal expression of PAX9 gene was found in a variety of human malignant tumors, which is closely related to tumourigenesis, development, invasion and metastasis of any cancers. However, the expression and value of PAX9 in malignant tumor cells are still inconclusive. An experiment compared genomic abnormalities in a large cohort of esophageal adenocarcinoma (EAC)and ESCC using single-nucleotide polymorphism (SNP) arrays demonstrated that PAX9 displayed much higher amplification frequencies in ESCC than EAC. Therefore, it is possible that PAX9 represents an oncogene specific to ESCC that may be targeted in the future. Until now, the prognostic effect of PAX9 has not been investigated in ESCC.Objectives:The purposes of the research were to investigate the prognostic significance of PAX9 in resectable ESCC by detecting their expression of PAX9 protein and analyzing their overall survival, and to access its value in predicting radiation sensitivity by subgroup analysis.Materials and methods:1. A total of 229 patients who underwent potential radical surgery for ESCC at the department of thoracic surgery in our hospital between January 1st 2008 and December 31th 2009 were included in the study. None of these patients had received chemotherapy or radiotherapy prior to surgery. The histological features of the specimens were independently evaluated by two experienced pathologists according to the classification criteria from the World Health Organization. The pathological stages of esophageal cancer was determined according to the pathological tumor-node-metastasis (pTNM) staging system (defined by the American Joint Committee on Cancer Staging Manual,7th edition,2010). Follow-up visits were performed every 3 months for the first 2 years and every 6 months for the next up to death or the end of the study. Disease-free survival (DFS) and overall survival (OS) was defined as the interval between the surgery date to the recurrence or death date, respectively.2. The surgical tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin. The presence of viable tumor was confirmed on hematoxylin and eosin staining. Sections were incubated with the Rabbit angti-PAX9 antibody as protocol. Histostain-Plus Kits was used as secondary antibody. IHC staining was visualized with substrate solution containing diaminobenzidine (DAB) and hydrogen peroxide. The counter-staining was performed with haematoxylin. Negative controls consisted of tissue sections undergoing similar staining procedures in the absence of the primary antibody.3. At least five horizons were observed in each slice, and no less than 100 cells were counted in each high magnification. The cells in which brown granules appeared in cytoplasm were judged as positive. Using semi-quantitative analysis, the criteria for scoring the stained sections were as follows:Based on the positive cellular rates of expression of PAX9, we scored each tumor sample as (1):10%～25%; (2): 26%～50%; (3):51%～75%; (4):76%-100%. According to the intensity of PAX9 staining, we scored each one as (0):without staining; (1):weak positive (light brown); (2):strong positive(dark brown). On the basis of the final scores which were calculated according to the positive cellular rate integral multiplied by the staining intensity integral, it was divided into PAX9 negative (score 1-3) and PAX9 positive (score 4-8).4. Statistical analyses were performed with the SPSS 19.0 statistical software. Differences between groups were evaluated using the chi-square test for categorical variables. The Kaplan-Meier method was used for analysis of the distribution of 5-year DFS and OS, and log-rank test was performed to compare the difference between the survival curves. Variables that were identified as statistically significant by univariate analysis were included in the multivariate survival analysis using the Cox proportional hazard regression model.Results:1. Patient characteristicsThe median age was 60 years (range,32 to 84 years), and 80.8% of patients were male. Tumor locations were:Cervical in 7 patients, upper thoracic in 16 patients, middle thoracic in 130 patients, and lower thoracic in 76 patients. The mean length of tumors was 4.17 cm (range,0.5-10.0). The histopathological differentiations were well in 55 cases, moderate in 99 cases, and poor in 75 cases.88 patients (38.4%) had T1/T2 tumors and 141 patients (61.6%) had T3/T4 tumors.109 patients (47.6%) had positive lymph nodes.26 patients were in pathological stage I, while 101 in stage II and 102 in stage III.110 patients (48.0%) received surgery alone,53 (23.1%) received postoperative chemotherapy,101 (44.1%) received postoperative radiotherapy and 35 (15.3%) received postoperative chemoradiation.213 patients (93.0%) had recurrence and 182 (79.5%) died during the follow-up.2. Survival analysisThe estimated 1-,3-,5-year DFS and OS rates were 72.2%,35.2%,5.6% and 86.1%,44.4%,23.1%, respectively, in the PAX9 negative group and 76.9%,47.9%, 24.0% and 90.9%,57.9%,38.8%, respectively, in the PAX9 positive group. Median overall survival for patients in the PAX9 negative group was 29.4 months (range,1.7 to 73.1months), compared with 42.0 months (range,3.5 to 83.2months) for patients in the PAX9 positive group. The difference of DFS (P<0.001) and OS (P=0.001) curves between the two groups remained statistically significant.3. Prognostic analysisIn univariate analysis, PAX9(P=0.001), tumor length (P=0.027), differentiation (P<0.001), T stage (P<0.001), lymph node metastasis (P<0.001), and TNM stage (P<0.001) were associated with OS. In multivariate analysis, Besides lymph node metastasis and pTNM stage, PAX9 was an independent predictor for DFS (HR=0.601, 95%CI:0.451-0.801, P=0.001) and OS (HR=0.662,95%CI:0.491-0.893, P=0.007).4. subgroup analysisFor patients who received no adjuvant therapy, no significant difference was observed between the two groups for 5-year DFS (P=0.494) and OS (P=0.663). While for patients who received adjuvant therapy, significant difference was observed between the two groups for 5-year DFS (P<0.001) and OS (P<0.001). For PAX9 positive ESCC patients, those who received radiotherapy after surgery had a significantly higher 5-year DFS (P=0.011) and OS (P=0.009) than those who received surgery only. While in PAX9 negative group, no significant difference was observed for 5-year DFS (P=0.724) and OS (P=0.136).Conclusion:1. This finding suggests that immunohistochemical characteristics of PAX9 and patterns is an independent prognostic factor for ESCC.2. As a possible predictor of radiation sensitivity, PAX9 may assist clinicians in developing a rational approach to individualizing therapeutic options for a given patient with ESCC. It could even represent a new potential therapeutic target for ESCC. Background:Esophageal cancer was respectively, the eighth and the fourth most common cancer in morbidity and mortality worldwide, and it was the fifth most common cancer type and the fourth leading cause of cancer death in China. The incidence of esophageal cancer, characterized by obvious regional differences, is more in less developed and developing countries, of which China is one of the countries with the world’s highest incidence of the disease. Its pathogenic factors include:chemical etiology, biological etiology, lack of trace elements, vitamin deficiency, tobacco, alcohol, hot food, genetic factors, etc.. In our country, more than 90% of the pathological types are squamous cell carcinoma. With the increasing morbidity of esophageal squamous cell carcinoma, radiotherapy will play more important role in the management of this disease. Radiation therapy is one of the most promising therapeutic strategies in unresectable esophageal squamous cell carcinoma. Unfortunately, it was found in the study that the pathological complete response rate is only 29% or so after preoperative radiotherapy or chemoradiotherapy of esophageal squamous cell carcinoma. The overall efficient was 50%-60%, and quite a number of patients’tumor didn’t shrink or even increased, which indicated that the sensitivity to radiation therapy is different in esophageal squamous cell carcinoma. Radiotherapy intensification for tumors could not improve the local control ratio and long-term survival in esophageal squamous cell carcinoma.Discovery of the key factor on radiation sensitivity may lead to prediction on radiosensitivity of cancer so as to formulate reasonable individualized treatment plan, proceed radiotherapy sensitization or reverse radiation resistance, and even open up a new treatment target which is the representative of "precise medicine", and eventually improve the patient’s overall survival. Tumor-related factors that contribute to radiotherapy effects include oxygen status, proliferative potential, and capacity to repair radiation damage, etc.. The involved content are extensive and complex, and it is hard to detect and anticipation in clinical practice. Although radiation oncologists and biologists have studied for decades, they are still unable to accurately and reliably predict individual tumor radiosensitivity.According to previous research, radioresistance of cancer has been identified to be associated with the aberrant expression of many genes, which could be potential therapeutic targets for treatment of cancer. As is well-known, tumorigenesis and progression of cancer are regulated by multiple signaling pathways, and in consequence direct alteration of single gene is usually insufficient to affect cellular radiosensitivity. As a research hotspot in the field of can cancer radioresistance a few years ago, a batch of non-coding microRNAs (miRNAs) were confirmed to be able to influence radiosensitivity because of their potential ability to simultaneously regulate multiple oncogenic pathways. The experiments about radiation sensitivity of esophageal cancer were also been reported from time to time, and a few factors, for example, the aminopeptidase inhibitor CHR-2797, VPA, Ring finger protein (RNF2), Insulin-like growth factor binding protein-3 (IGFBP-3), X-ray repair cross-complementing gene 3 (XRCC3) and Sunitinib and so on, was proved to be relevant to the radiosensitivity of esophageal squamous cancer cells in vitro. In recent years, the rapid growth of knowledge about tumor oncogenes and tumor suppressor genes, as well as the rapid detection technology regarding relevant gene expression and mutation, indeed vastly promote the development of molecular prediction research. But due to significant differences between experiments in vitro and in vivo, the study results of the basic medicine failed to translate into new methods of clinical treatment. It is a pity that no effective predictors have been identified to date to accurately estimate sensitivity to radiation therapy in ESCC patients.PAX9 is a member of the paired box gene family, which plays key roles in several aspects of embryonic development and organogenesis by regulating the expression of the genes involved in cell proliferation, apoptotic resistance, and cell migration, and now is recognized as potentially playing important roles in cancer progression. Human PAX9 is located within 14ql2-q13, characterized by a-128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. the abnormal expression of PAX9 gene was found in a variety of human malignant tumors, which is closely related to tumourigenesis, development, invasion and metastasis of any cancers. However, the expression and value of PAX9 in malignant tumor cells are still inconclusive. An experiment regarding genomic abnormalities in esophageal cancer demonstrated that PAX9 displayed much higher amplification frequencies in ESCC. Therefore, it is possible that PAX9 represents an oncogene specific to ESCC.Our study demonstrated that adjuvant radiotherapy could benefit patients characterized by PAX9 positivity, on the contrary, those who did not receive radiotherapy after surgery had a poor prognosis. It indicates that PAX9 could be a predictor of radiation sensitivity for ESCC patients. And then we speculate that PAX9 expression also have predictive value on radiation sensitivity in patients with unresectable locally advanced esophageal squamous cell carcinoma.The purposes of this research were to detect the PAX9 expression in human esophageal squamous cell carcinoma cell lines including ECA109, EC9706 and TE-1. By means of a recombinant eukaryotic expression plasmid of PAX9 constructed in vitro transfected into TE-1 cells, the important effect of PAX9 in inhibiting proliferation and invasion, promoting apoptosis and improving the radiosensitivity of TE-1 cells was observed.Methods1. Three kinds of human esophageal squamous carcinoma cell lines were selected (ECA109, EC9706 and TE-1) for cell culture and passage. Real-time PCR and Western blotting method were used to detect the expression of PAX9 mRNA and protein. Lower gene expression of PAX9 cell line was screened for subsequent experiments.2. Six overexpressed sequences of PAX9 were designed according the core area of PAX9 gene, which was connected to the vector of the virus vector. After transformed into the E.coli Stbl3, the monoclonal antibody was selected. By sequencing, we got the right virus vectors. After extracted, purified, virus packaged and infected cells, Real-time PCR and Western blotting shows gene PAX9 was highly expressed in RNA and protein level.3. Establishment of a stable TE-1 cell line with PAX9 highly expression. The PAX9 highly expression vector and empty vector lentivirus were infected into TE-1 for 48h, after sieving 5 generation by G418 drug, Real-time PCR and Western blotting were used to detect expression levels of PAX9 gene mRNA and protein in stably transfected cell lines. Real-time PCR and Western blotting transfected cells was detected in RNA and protein level expression to verify whether the transfection was successful by strains of PAX9 gene in 3 successive generations.4. TE-1 cells, Vector cells and PAX9 overexpression cells were irradiated with 6MVX ray source with different doses at room temperature. After 24 hours, MTT assay was performed and data analysis was conducted.5. Cell scratch test was carried out to detecte cells migrate ability in all groups after irradiation (2Gy,4Gy and 8Gy).6. The effect of PAX9 gene on the radiation sensitivity in TE-1 cells was detected by cloning experiment.7. Through apoptosis experiment of all groups, the cell cycle was observed, and the effect of PAX9 gene on the apoptosis in TE-1 cells was analyzed.Results1. Low expression of PAX9 gene was detected in human esophageal squamous carcinoma cell lines including EC9706, Eca109 and TE-1 cells by Western blotting. So we choose TE-1 cell line as the follow-up test cell line.2. After construction of PAX9 high expression vectors in vitro and lentiviral package, Real-time PCR and western blotting showed highly effective expression of PAX9, which proved the successful construction of the vectors.3. The PAX9 high expression vectors and empty vectors lentivirus were infected into TE-1 cell lines, after G418 drug screening, Real-time PCR and Western blotting showed that PAX9 was highly expressed both in mRNA and protein levels in experimental group. There was a significant difference (P<0.05) compared with with the vector group and the control group.4. The results of MTT showed that the cell proliferation ability of PAX9 overexpression cells decreased, and the cell inhibition rate increased after irradiation. Compared with the control group and vector group, the differences were statistically significant (P<0.05).5. Cell scratch experiment results showed that the migrate ability of PAX9 overexpression cells was decreased, and further decreased after irradiation.6. Cloning experiments results show that clone formation rate of PAX9 overexpression group cells was significantly lower than that of TE-1 control group and empty vector group after 6MV X-ray irradiation. The SER of PAX9 for TE-1 cells was 1.4, which indicating that PAX9 gene had better radiation sensitizing effect on TE-1 cells.7. The results of Flow Cytometry Method showed that the cell apoptosis rate in PAX9 overexpression group was significantly higher than that of control group and vector group after 3Gy and 6Gy X-ray irradiation, which indicating that PAX9 gene had a promotion effect on induced apoptosis in TE-1 cells after irradiation.Conclusions1. PAX9 was in a low expression state in human ESCC cell lines EC9706, ECA109 and TE-1.2. Construction of PAX9 vector in vitro based on CDS region as a core region can transfect TE-1 cells effectively with stabley and highly expressed.3. PAX9 can decrease the proliferation and migrate of human ESCC TE-1 cells and increase the radiosensitivity.4. PAX9 gene may have predictive value of radiosensitivity in esophageal squamous cell carcinoma, so that it is helpful to make rational individual treatment plan which is close to Precision Medicine treatment. It provides a theoretical basis for the treatment of enhancement radiosensitivity of esophageal squamous cell carcinoma targeted to PAX9 gene. |
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