Proto-oncogenes are frequently activated by genomic amplification. Characterization of genes with increased copy numbers and consequent over-expression in tumor tissues can facilitate the identification of tumor-specific oncogenes.
Esophageal cancer is a common cancer worldwide and esophageal squamous cell carcinoma (ESCC) is the most prevalent type in China. Multiple genetic changes have been found in ESCC, but little is known about major oncogenes and tumor suppressor genes involved in the tumorigenesis of ESCC.
As the chromosome locus 11q13 is frequently amplified in ESCC 1, 2, Luo et al. 3 examined several cancer-related genes in the 11q13 region, including MYEOV, ORAOV1, FGF19, FGF4, FGF3, ORAOV2, FADD1, PPFIA1 and CTTN, in primary ESCC and matched normal tissues by RT-PCR. Only the cortactin gene (CTTN, also as EMS1) presented overexpression in most of the examined tumor tissues. The authors subsequently examined CTTN at the genomic DNA and protein levels. CTTN amplification was found in esophageal cancer tissues by FISH and real-time PCR. Immunohistochemical analysis on the tissue microarray (TMA) confirmed the overexpression of this gene. Moreover, a positive correlation between CTTN amplification/overexpression and lymph node metastasis in ESCC was revealed by statistical analysis.
CCND1 is an important gene that promotes cell cycle progression and is also located at 11q13. CCND1 and CTTN are frequently co-amplified in cancers. It thus needs to be clarified whether both CTTN and CCND1 or only one of these two genes constitute the prediction factor for lymph node metastasis in ESCC. Luo et al. 3 found a positive correlation between CCND1 and CTTN protein expression. Nevertheless, CCND1 amplification/overexpression did not correlate with lymph node metastasis, implicating that CTTN was an independent prediction factor for ESCC lymphoid metastasis.
CTTN is an actin-associated scaffolding protein, which binds and activates the actin related protein complex (Arp2/3), and thus regulates the branched actin networks in the formation of dynamic cortical actin associated structures. The cellular role of CTTN is related to cell migration, morphogenesis, adhesion, receptor mediated endocytosis and pathogens invasion, etc 4. More recently, functional analysis in breast cancer, hepatocellular carcinoma, and head and neck squamous cell carcinoma has revealed that CTTN promotes invasiveness of cancer cells 5, 6, 7, 8.
To explore the role of CTTN in ESCC, CTTN RNAi was carried out in esophageal cancer cells that harbor CTTN amplification. Knockdown of CTTN expression drastically decreased the ESCC cell mobility in wound-healing, haptotactic cell migration and Matrigel chemoinvasion assays, without impairing the adhesive ability of these cells.
Luo et al. 3 also examined the impact of CTTN expression on esophageal tumor growth. SiRNA-mediated silencing of CTTN revealed that this gene had no effect on ESCC cell growth in the MTS cell proliferation assay. However, colony-forming ability of CTTN RNAi cells under anchorage-independent condition was markedly decreased in comparison with control cells in the soft agar assay. In vivo experiments revealed that CTTN RNAi suppressed the subcutaneous tumor growth in nude mice.
In view of the fact that tumor growth is determined by the balance of cell proliferation and programmed cell death, the authors analyzed cell proliferation by Ki-67 immunohistochemistry and apoptosis by the TUNEL assay on subcutaneous tumor sections. The results showed that CTTN RNAi in esophageal cancer cells suppressed in vivo tumor growth by promoting apoptosis. However, in the apoptosis analysis in vitro, CTTN down-regulation in ESCC cells did not affect apoptosis induced by UV exposure or doxorubicin treatment. Considering that CTTN was associated with lymph node metastasis in tumor tissues and with anchorage-independent growth in ESCC cells, the authors performed detachment-induced apoptosis (anoikis) assay. Increased anoikis was observed in CTTN RNAi cells as compared to control cells, indicating that CTTN protected ESCC cells from anoikis. To our knowledge, CTTN was only shown previously to affect the metastatic behavior of cancer cells by enhancing cell motility and endothelial cell adhesion. This is the first report that CTTN promotes anoikis resistance, which extends our understanding of the role that CTTN plays in tumor metastasis.
Molecular mechanisms underlying anoikis have been described to involve several signal pathways in different cell types. PI3K-Akt, MEK-Erk and EGFR are important pathways mediating survival signals in detachment-induced apoptosis 9, 10. Although both PI3K and MEK inhibitors induced anoikis in this study, only phosphorylated Akt was down-regulated in CTTN siRNA cells. Activated Erk and EGFR levels did not alter after CTTN suppression. Thus, PI3K-Akt, not MEK-Erk or EGFR signaling, constitutes the main downstream target responsible for the protective effect of CTTN against anoikis.
As increase of cell migration and suppression of anoikis both contribute to metastatic potential of cancer cells, Luo et al. 3 investigated whether inhibition of CTTN in highly metastatic ESCC cells would affect their metastasis ability in vivo. The results demonstrated that inhibition of CTTN expression prevented lung metastasis of ESCC cells and prolonged the survival of tumor-bearing mice.
Capabilities of invasion and metastasis are the hallmarks of cancer cells. Given the complexity of the metastasis process, cancer cells must acquire a series of traits that enable them to overcome multiple barriers erected by normal tissues. Enhancement in migration ability is advantageous to tumor invasion, which is an important mechanism accounting for the role of CTTN in cancer cell metastasis. Loss of adhesion is a stress that metastatic cancer cells encounter en route. Resistance to anoikis may promote survival of cancer cells during systemic circulation and facilitate secondary tumor formation in distant organs. Accumulating evidence has implicated that cytoskeletal alterations are potentially involved in anoikis 9, but the mechanisms are still not completely understood. Since CTTN is a scaffold protein regulating cortical actin assembly, the contribution of CTTN to anoikis resistance described in this study not only reveals a novel and significant biological role for CTTN, but also provides new insights into the influence of cytoskeletal organization on the survival pathway in anoikis.
Taken together, the study by Luo et al. 3 demonstrates that CTTN is an oncogene in the 11q13 amplicon which contributes to the metastasis of ESCC by promoting cell migration and anoikis resistance. At the molecular level, the protective role of CTTN in anoikis resistance was correlated with the activation of the PI3K-Akt pathway. Given these results, this work raises interesting questions which needs further investigations. First, when does CTTN amplification and overexpression occur? Is the alteration of CTTN an early event of tumorigenesis? Moreover, can CTTN be used as a biomarker to predict tumor metastasis or prognosis? Finally, it will be attractive to elucidate the molecular basis for the regulation of PI3K-Akt by CTTN. Addressing these issues will enlarge our view of the function that CTTN serves in tumor initiation and progression as well as the connection between cytoskeletal proteins and survival signaling.
This work was supported by National Natural Science Fundation of China (30470969), National Basic Research Program of China (2004CB518705, 2002CB513101), Specialized Research Fund for the Doctoral Program of Higher Education of China (20060023003, 20050023046) and Program for Cheung Kong Scholars and Innovative Research Team in University (IRT0416).