Carcinogenesis Advance Access originally published online on November 12, 2008
Carcinogenesis 2009 30(1):165-174; doi:10.1093/carcin/bgn252
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Role for EPS8 in squamous carcinogenesis
1 Philips Institute of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University, PO Box 980566, 521 North 11th Street, Richmond, VA 23298-0566, USA
2 Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, Bethesda, MD 20892, USA
3 Department of Biochemistry and Molecular Biology
4 Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA
* To whom correspondence should be addressed. Tel: +1 804 828 6415; Fax: +1 804 828 0150; Email: wayeudall{at}vcu.edu
We have investigated the role of the signaling intermediate, EPS8, in tumor progression using a model system and in vivo. HN4 primary tumor cells express low levels of EPS8, similar to normal keratinocytes, and show minimal invasion in vitro in response to epidermal growth factor, whereas HN12 cells express high levels of EPS8 and are highly motile in vitro and tumorigenic in vivo. Additional independent tumor cell lines also showed elevated EPS8 expression compared with normal keratinocytes. Using retroviral transduction, we generated HN4 cell lines expressing EPS8 (HN4/EPS8) at levels equivalent to those present in HN12 cells. HN4/EPS8 cells showed increased proliferation and migration compared with controls, together with elevated expression and activity of matrix metalloprotease (MMP)-9, which was dependent on protein kinase B (AKT) activity. Introduction of plasmids that direct synthesis of EPS8 short hairpin RNA (shRNA) into HN12 cells resulted in decreased EPS8 expression in these cells, which correlated with a decrease in their capacity to migrate and invade in vitro. In addition, shRNA-mediated knockdown of EPS8 reduced expression and activity of MMP-9 produced by these cells and reduced MMP-9 promoter activity. EPS8 knockdown cells showed decreased tumorigenicity in vivo compared with controls and lower MMP-9 expression. Conversely, overexpression of EPS8 in HN4 cells was sufficient to induce growth of these non-tumorigenic cells in orthotopic transplantation assays. Furthermore, EPS8 expression in clinical samples of squamous cell carcinoma showed variable expression levels and broadly paralleled expression of MMP-9. The data support a role for EPS8 in squamous carcinogenesis.
Abbreviations: AKT, protein kinase B; BSA, bovine serum albumin; cDNA, complementary DNA; DMEM, Dulbecco’s modified Eagle’s medium; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; HNSCC, head and neck squamous cell carcinoma; MMP, matrix metalloprotease; PCR, polymerase chain reaction; PI3K, phosphoinositide 3-OH kinase; SCC, squamous cell carcinoma; shRNA, short hairpin RNA; qRT, quantitative real-time
Received April 10, 2008; revised October 10, 2008; accepted November 1, 2008.