Studies On Amplified Oncogene Candidates In Esophageal Squamous Cell Carcinoma

Esophageal cancer is the fourth most common malignancy in China and esophageal squamous cell carcinoma (ESCC) is the most prevalent type. Multiple genetic changes have been found in ESCC, but little is known about major oncogenes and tumor suppressor genes involved in this disease. In pursuit of candidate oncogenes in ESCC, we focused on overexpressed genes within previously identified chromosome regions with frequent amplification in ESCC in the present study.30 cancer-related genes on chromosome 1q, 3q, 8q and 11q were examined in 16 primary esophageal tumors and matched adjacent histological normal tissues by RT-PCR. TXNRD3, RAB7, AMOTL2, CCNL1, EIF5A2, p63, MUC4, TMEM16A and CTTN presented mRNA overexpression in over 30% tumors. The genomic DNA copy number of these nine genes was then evaluated by semi-quantitative PCR of intragenic UniSTS in 20 cases of tumors and matched normal tissues. Amplification of TXNRD3, RAB7, AMOTL2, CCNL1, MUC4, TMEM16A and CTTN was found in more than 20% of cancerous tissues. CCNL1 and CTTN amplifications were confirmed in ESCC by fluorescence in situ hybridization (FISH).The cortactin gene (CTTN, also as EMS1), located at 11q13, plays a pivotal role in coupling membrane dynamics to cortical actin assembly. In the present study, immunohistochemical analysis was performed on tissue microarrays and CTTN overexpression was found in 68.8% (86/125) tumors. Statistical analysis indicated that there were significant correlations between CTTN expression and lymph node metastasis and tumor stage in ESCC. CCND1 is another oncogene on 11q13 and its overexpression was detected in 47.0% (55/117) ESCC. Although a positive correlation between CCND1 and CTTN expression was observed, none of the tumor clinicopathologic features was associated with CCND1 overexpression. These results indicate that CTTN is an independent marker at 11q13 for ESCC lymphoid metastasis and tumor stage.Functional analysis by siRNA-mediated silencing revealed that CTTN did not influence cell proliferation, cell cycle progression and adhesion to Matrigel in esophageal cancer cell line EC9706. Whereas CTTN RNAi decreased EC9706 capabilities in wound healing, haptotactic migration, Matrigel chemoinvasion colony formation in soft agar and anoikis resistance. In vivo assay showed that inhibition of CTTN expression also decreased tumor growth and lung metastasis of EC9706 cells.To explore the molecular mechanism by which CTTN protected cells from anoikis, we examined the potential effect of CTTN on the activation of several known pathway associated with anoikis. Both PI3K and MEK inhibitors abrogated the survival protection of CTTN in EC9706, while only phosphorylated Akt was down-regulated in CTTN RNAi cells. No difference in Erk activation or EGFR expression was observed after CTTN suppression, suggesting that PI3K-Akt, not MEK-Erk or EGFR signaling, contributed to CTTN-related survival in detached EC9706. CTTN also protected KYSE410 esophageal cancer cells from anoikis when overexpressed. Akt activation was observed in these cells after CTTN transfection as well. In EC9706 and KYSE410 cells, CTTN and PI3K could be co-immunoprecipitated by either of the antibodies. These data imply that CTTN may interact with PI3K and activate it to serve a protective function in anoikis resistance.In conclusion, analysis of amplified genes may be helpful to the identification of oncogenes associated with malignant tumors. The CTTN gene in the 11q13 amplicon, through DNA copy number increase and protein overexpression, plays an oncogenic role in ESCC metastasis by promoting cell migration and anoikis resistance.