Cortactin Modulates RhoA Activation and Expression of Cip/Kip Cyclin-Dependent Kinase Inhibitors To Promote Cell Cycle Progression in 11q13-Amplified Head and Neck Squamous Cell Carcinoma Cells

Abstract

The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21^WAF1/Cip1, p27^Kip1, and p57^Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21^WAF1/Cip1 and p27^Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21^WAF1/Cip1 and p27^Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21^WAF1/Cip1, p27^Kip1, and p57^Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27^Kip1 and p57^Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.

Supplemental material for this article may be found at http://mcb.asm.org/.

Grants were received from the Cancer Institute of New South Wales (CINSW), the Cancer Council of New South Wales, and the National Health and Medical Research Council of Australia. D.R.C. and E.A.M. hold CINSW Early Career Development and Career Development and Support fellowships, respectively.