Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, and growth retardation. The NBS gene product, NBS1, is a part of hMre11 complex which plays a central role in the DNA double-strand break repair. We previously demonstrated that c-MYC activates NBS1 expression (1). Our recent results showed that overexpression of NBS1 contributes to transformation through the activation of PI 3-kinase/Akt (2). Increased NBS1 expression was present in 45% of advanced head and neck squamous cell carcinoma (HNSCC) patients and was associated with a worse prognosis (3). We therefore speculate that overexpression of NBS1 may contribute to the late-stage tumor progression and metastasis. Epithelial-to-mesenchymal transition (EMT) has been demonstrated to be the mechanism responsible for mediating the invasive and metastasic behavior of late stage cancers. In this study, we investigate the possible role of NBS1 overexpression in mediating EMT. We established HNSCC cell line FADU with NBS1 overexpression (FADUNBS) and lung cancer cell line H1299 with NBS1 repression by siRNA (H1299NBSi). The results showed that FADUNBS cells exhibited the shift to EMT phenotypes including downregulation of E-cadherin and upregulation of vimentin, markers of EMT presentation, by RT-PCR and Western blot analysis. Loss of cell junction protein E-cadherin and increased cytoplasmic vimentin expression was also confirmed by immunofluorescence staining. FADUNBS cells had increased invasive and migratory ability by Matrigel invasion and migration assay. Increased expression of metalloproteases-2 & 9 were observed in FADUNBS cells, consistent with their increased invasiveness. NBS1 knockdown in H1299 cells (H1299NBSi) reversed the EMT phenotype. One of the EMT inducer, the transcriptional repressor Snail, was upregulated in FADUNBS cells as shown by RT-PCR and immunoprecipitation-Western blot analysis. In order to correlate the in vitro results with clinical outcome, tissue microarray (TMA) was constructed with primary tumors and metastatic lesions (if present) of 125 HNSCC cases using non-cancerous matched tissues as controls. Immunohistochemistry (IHC) analysis was performed to analyze the expression of NBS1 and related molecular markers in TMA. The results showed that NBS1 overexpression was a significant predictor of metastasis (p<0.001), and cytoplasmic co-localization of NBS1 and phosporylated Akt was identified in both the primary and metastatic lesions in the same cases. The TMA-IHC of Snail and in vivo metastatic assay are ongoing in our laboratory. Our results indicate that NBS1 overexpression induces EMT through the upregulation of Snail expression, contributing to tumor progression and metastasis. References: 1. J Biol Chem 2003;278:19286-91. 2. J Biol Chem 2005;280:32505-11. 3. Clin Cancer Res 2005(in press).
[Proc Amer Assoc Cancer Res, Volume 47, 2006]