The Expression Of SOX4and Its Invasion And Progression Mechanism In Cholangiocarcinoma

Cholangiocarcinoma (CCA) is a highly aggressive neoplasm with an extremely poor prognosis, which is featured of silent clinical course, early regional invasiveness and distant metastasis. The morbidity and mortality of CCA have been steadily increasing worldwide in recent years. Anatomically, CCA is classified into intrahepatic cholangiocarcinomas (IHCCs) and extrahepatic cholangiocarcinomas (EHCCs), the latter being further divided into perihilar CCAs and distal CCAs. So far, complete resection is the only curative therapeutic option for patients with CCA but can be applied only in a limited number of patients with localized or locally advanced disease. Even after the curative resection the recurrence rates are high. Besides the effect of adjuvant therapy(chemotherapy and radiation therapy) are not satisfied, and the relative studies not had been clearly shown they can reduce recurrence risk. So far the ideal tumor biomarkers with high sensitivity and specificity, used for early diagnosis and prognostic evaluation of hilarcholangiocarcinoma, have not been discovered or screened. Therefore, identifying themechanism of its malignant biological behavior may help provide new therapeutic strategies.Cholangiocarcinoma (CCA) is a highly aggressive neoplasm with an extremely poor prognosis, which is featured of silent clinical course, early regional invasiveness and distant metastasis. The morbidity and mortality of CCA have been steadily increasing worldwide in recent years. Anatomically, CCA is classified into intrahepatic cholangiocarcinomas (IHCCs) and extrahepatic cholangiocarcinomas (EHCCs), the latter being further divided into perihilar CCAs and distal CCAs. So far, complete resection is the only curative therapeutic option for patients with CCA but can be applied only in a limited number of patients with localized or locally advanced disease. Therefore, identification of new biomarkers for early detection and/or development of novel therapeutic regiments are urgently needed.Epidermal growth factor receptor (EGFR) and HER2are members of the family of tyrosine kinase growth factor receptors (TKGFRs). They share approximately50%overall homology and play a significant role in cellular growth and proliferation signaling. In CCA, overexpressions of EGFR and HER2are thought to be prognostic factors and it has been proposed that EGFR could be potential promising target for treatment of CCA. To date, although a series of studies have revealed amplification, copy number alterations, and mutations of this gene, the exact molecular mechanisms underlying EGFR overexpression in CCA remains imperfectly understood.The SOX4(sex-determining region Y-box4) gene, a transcription factor and member of the SOX family, has been shown to play important roles in development and cell fate decision. It is overexpressed in a wide variety of malignancies, including leukemia, and cancers of the breast liver, lung and prostate. In these tumors, deregulated expression of SOX4has been associated with increased cancer cell proliferation and cell survival and tumor progression through the induction of an epithelial mesenchymal transition (EMT) and metastasis. However, the clinicopathological significance of SOX4in CCA remains largely unknown.In the current study, we systematically characterized the expression and prognostic significance of SOX4/EGFR/HER2in a cohort of Chinese CCA patients. For the first time, we demonstrated that SOX4expression is an independent prognostic factor in CCA patients and may promotes EMT process in CCA cells. SOX4overexpression is significantly associated with EGFR expression and ERG+/SOX4+defines a subset of CCA patients with poor prognosis. Part1. Characterization of SOX4gene aberrations in cholangiocarcinoma Objective:To explore the expressions of SOX4in cholangiocarcinoma and their associations with clinicopathological characteristicsMethods:1. Establishing database of cholangiocarcinoma patients and construct152cases of cholangiocarcinoma patients from2005-2009which treated at the Qilu Hospital of Shandong University, the Central Hospital of Jinan and General hospital of Liaocheng were adopted in this experiment. Clinical information concerning gender, age, clinical stage, tumor size, lymph node metastasis, surgical methods, calculus, and hepatitis infection were collected. Two tissue microarrays of cholangiocarcinomas was constructed.2. Using immunohistochemical method to detect SOX4expressions in cholangiocarcinomas, Monoclonal antibody of EGFR were used to detect theexpression of EGFR family members’immunohistochemically on TMA. analysis. Each SOX4expression and inner link and its relationship clinicopathological features were analyzed accordingly.3. The prognostic significance of SOX4family members in cholangiocarcinomas in the survival analysis, the starting point for the survival time was the date three months after surgery. Cumulative overall survival rates were calculated by Kaplan-Meier method and statistical significance for survival curves comparison was analyzed by log-rank test. Univariate and multivariate survival analyses were performed using the Cox multiple hazards model to estimate hazard ratio.differences for all the tests were regarded as statistically significant when the P-value from a two-tailed test as P<0.05. RESULTS1. SOX4and EGFR expression in cholangiocarcinomas and the association with clinicopathological variables.The relationship between SOX4expression level and clinicopathologic variables in IHCC and EHCC cases were summarized in Table2and Table3. Overall, SOX4overexpression was identified in17(29.3%) of the58IHCCs, and28(29.8%) of the94EHCCs, respectively. Interestingly, SOX4expression was significantly associated with older age in IHCC (P=0.009). The incidence of SOX4overexpression was more frequently observed in elderly patients (≥60years) than younger ones. Similarly, increased SOX4expression was marginally associated with poorly histological differentiation (P=0.061), gender (P=0.078) and higher UICC stage (P=0.089), but not with tumor size (P=0.758), T stage (P=0.753) or lymph node metastasis (P=0.494). In EHCC, there was no significant association between SOX4expression and any clinicopathological factors.Overexpression of EGFR was demonstrated in13(22.4%) of the58IHCC, and33(35.1%) of the94EHCC cases. Increased expression of EGFR was significantly correlated with older age (P=0.045) in EHCC, and poorly histological differentiation both in IHCC (P=0.031) and EHCC cases (P=0.012). No other clinicopathological parameters were associated with EGFR expression in our cohort. In all, amplification of EGFR was identified in1.8%(1/52) of IHCC cases, and in2%(3/82) in patients with EHCC.2. Prognostic value of SOX4expression in CCATo investigate the possible associations between the protein levels of SOX4and overall survival, we compared overall survival rates between patients with or without SOX4overexpression in univariate and multivariate models. In IHCC, the group of the patients who were with SOX4overexpression had a much greater rate of mortality than patients who are not (P=0.038). On the basis of the Kaplan-Meier survival estimates, SOX4overexpression was significantly linked to cancer-related mortality in our cohort. By contrast, no statistical significance was identified between SOX4overexpression and overall survival in EHCC.In univariate Cox regression analysis, SOX4overexpression was a prognostic factor for cancer mortality (hazard ratio=2.35,95%CI=1.104-5.786, P=0.029) in IHCC. Additionally, lymph node metastasis and EGFR overexpression were also significantly related to overall survival. In a multivariate analysis, lymph node metastasis remained its predictive value, whereas SOX4and EGFR expression lost.In EHCC, SOX4expression failed to be related to overall survival of CCA patients.3factors including EGFR overexpression were identified as prognostic factors by univariate analysis. In multivariate analysis, as shown in Table5, only UICC stage was an independent prognostic factor (HR (95%CI):3.110(1.602-5.123), P<0.01). Collectively, these data suggested that SOX4was an unfavorable prognostic indicator in Chinese patients with IHCC.3. SOX4expression is associated with EGFR overexpression in CCAGenetic aberrations including gene amplification and gene mutation contribute to EGFR overexpression in multiple cancers. In an attempt of characterizing novel mechanisms for EGFR overexpression in CCA, we successfully analyzed a total of135CCA cases for both SOX4and EGFR expression by IHC. Notably, SOX4overexpression was significantly associated with overexpression of EGFR both in IHCC (P<0.001) and EHCC (P=0.034).4. SOX4+/EGFR+defines a subset of CCA patients with poor prognosisIn the current study, we next determined whether combining markers further improved prognostic value. The prognostic effects of EGFR aberration and SOX4overexpression were directly compared in combination. The Kaplan-Meier analyses were therefore conducted using the group with no SOX4overexpression and no EGFR overexpression as the reference. As shown in Figure3, the largest group, which comprised those who had both EGFR and SOX4overexpression, had a greater survival when compared with the three other groups. Notably, the subset of patients with EGFR and SOX4overexpression had the worst cancer-related survival.ConclusionIn summary, our results revealed expression of the SOX4in CCA development and progression. Our study suggested that SOX4overexpression was significantly associated with overexpression of EGFR both in IHCC and EHCC. We also find that the subset of patients with EGFR and SOX4overexpression had the worst cancer-related survival. Part2. In vitro studies of SOX4influence on cell proliferation, invasion, invasion abilities and the mechanism in cholangiocarcinoma cells Objective:The first part of this study found that the expression of SOX4is associated with the expression of EGFR in CCA, and the SOX4+/EGFR+defines a subset of CCA patients with poor prognosis, but the specific mechanism is unknown; this section we intends to study SOX4RBE in bile duct cancer cells, the influence of the invasion and metastasis ability, to explore the role of SOX4in bile duct cancer mechanism. Methods:1. Construction of SOX4overexpression plasmid and siRNA-SOX4interference fragments. The SOX4cDNA fragment was cloned by PCR, the Human SOX4cDNA was subcloned into the pcDNA3.1eukaryotic expression vectors. SOX4and empty control plasmids were independently transfected into RBE cells using Lipofectamine (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.2. Investigation of the biological functions of SOX4in proliferation, invasion and metastasis:CCA cell line RBE were cultured and is divided into two groups: NC (Lipofectamine+Scramble RNA) and siRNA-SOX4(Lipofectamine+siRNA-SOX4) group. After24hours incubation in each group,5x103cells were seeded in96-well plates, proliferation rate of each group were detected at time points of in24h,48h and72h respectively. Wound healing assay was performed in12-well plates in the rwo differently treated group, the span of scratches were observed at time point of0h,24h,48h respectively. The ability of RBE migration was evaluated by transwell assay. RBE cells transfected with siRNA-SOX4were cultured in transwells for6hours, the invade cells were fixed with4%Formaldehyde and stained with0.05%crystal violet.The invaded cells on the lower surface were counted from3random fields.3. After the transfection, the RT-PCR and Western blotting test were used to measure the expression levels of SOX4and EGFR in the RBE cell line, respectively.4.The cell invasion potential was preformed by Transwell invasion and migration assay, and MTT assay was employed to detect proliferation of the cholangiocarcinoma cell.5. To explore whether SOX4was a regulator of EMT in CCA, expression of epithelial markers (E-cadherin, and p-catenin) and mesenchymal markers (Vimentin, and N-cadherin) were analyzed after transiently siRNA knockdown of SOX4by the RT-PCR and Western blotting test.Result:1.Modulation of EGFR expression by SOX4in CCAsiRNA knockdown of SOX4significantly decreased EGFR protein expression level in RBE cells. By contrast, transfection of SOX4vector in RBE cells increased expression level of EGFR levels. Interestingly, protein level of phosphorylated EGFR was not altered in above treatment. These data suggested SOX4modulates EGFR expression in vitro in CCA.2. siRNA knockdown of SOX4inhibits cellular migration in CCA cellsWound healing assay indicated that siRNA-SOX4-transfected RBE cells displayed a significant decrease in cell migration ability compared to control conditions.3. In vitro effect of SOX4on EMTTo explore whether SOX4was a regulator of EMT in CCA, expression of epithelial markers (E-cadherin, and β-catenin) and mesenchymal markers (Vimentin, and N-cadherin) were analyzed after transiently siRNA knockdown of SOX4. Expressions of E-cadherin and β-catenin were significantly downregulated, while Fibronectin and N-cadherin were upregulated by Western blot analysis in RBE cells.Conclusion:1.In vitro, siRNA knockdown of SOX4can inhibits cellular capacity of proliferation, invation and migration.2.SOX4was a regulator of EMT in vitro, siRNA knockdown of SOX4can affect the expression of EMT in cholangiocarcnioma.3.our results also define an important role for SOX4modulation on EGFR expression in CCA.