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 Cortactin is an F-actin binding protein involved in a variety of cellular processes, including endocytosis, vesicle trafficking, and the formation of cellular protrusions such as lamellipodia (14). In order to mediate these functions, cortactin interacts with a variety of proteins depending on the cell type and subcellular compartment, and this is achieved via distinct binding domains located within the cortactin molecule. The N terminus of cortactin harbors an acidic region that represents the interaction site for the actin-polymerizing Arp2/Arp3 complex, and this is followed by a repeat region containing the F-actin binding site. A Src homology 3 (SH3) domain located at the C terminus of cortactin recruits diverse proteins, including components of the endocytosis machinery (e.g., CD2AP and dynamin) and regulators of Rho family GTPases (e.g., BPGAP1 and Fgd1) and actin polymerization (e.g., N-WASP), while an adjacent proline-rich region contains phosphorylation sites for Src family kinases (14).The cortactin gene (CTTN) is located at chromosome band 11q13, a region that is commonly amplified in many human malignancies, particularly breast, ovarian, and bladder cancers and head and neck squamous cell carcinoma (HNSCC) (43).Several genes lie within this chromosomal region and are overexpressed upon its amplification. However, of these genes, the amplification of cyclin D1 (CCND1) and CTTN is most frequently associated with poor clinical outcomes such as decreased patient survival and increased metastasis (34,43). Chromosomal mapping of the 11q13 locus has revealed four distinct regions that can be individually or coordinately amplified (12,20,34). Within this locus, CCND1 and CTTN are located on different amplicons, and independent amplification of these genes has been demonstrated (34). In HNSCC, a tumor type in which 11q13 amplification occurs at the relatively high frequency of ϳ30% (43), CTTN amplification has been identified as an independent predictor of reduced disease-specific survival while CCND1 amplification is not prognostic in this tumor type (16,18,38,39). This strongly suggests that cortactin overexpression can act independently to promote tumor progression in cases of HNSCC.Due to the ability of cortactin to promote actin polymerization, many previous studies on cancer cells have focused on the role of cortactin in promoting cell motility and invasion (35,40,54), effects mediated by increased lamellipodial persistence (5), invadopodia formation (4), and protease secretion (10, 11). In agreement with this, cortactin overexpression has b...

Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

“…The pSIREN-RetroQ-ZsGreen (Clontech) constructs containing short hairpin RNA (shRNA) targeting SgK269 and GFP (negative control) were constructed by the ligation of synthesized oligonucleotides into the BamHI and EcoRI sites of pSIREN. The negative control sequence targeting GFP has been previously described (13). The pLKO.1 (Thermo Fisher) constructs containing shRNA targeting SgK269 and a nontargeting control were generated by the ligation of synthesized oligonucleotides into the AgeI and EcoRI sites of pLKO.1.…”

“…Cell lines were authenticated by short tandem repeat polymorphism, single-nucleotide polymorphism, and fingerprint analyses, passaged for less than 6 months, and cultured as previously described (15). The packaging cell line PlatE was used for retrovirus production, and MCF-10A and breast cancer cells were infected with retrovirus as previously described (13). Stable knockdown of SgK269 expression in MDA-MB-231 cells was achieved by lentiviral-mediated transduction of the corresponding pLKO.1 construct, packaged with VSVG pseudotype in HEK-293 cells, followed by puromycin (0.7 mg/mL) selection.…”

“…Stable knockdown of SgK269 expression in MDA-MB-231 cells was achieved by lentiviral-mediated transduction of the corresponding pLKO.1 construct, packaged with VSVG pseudotype in HEK-293 cells, followed by puromycin (0.7 mg/mL) selection. Cell proliferation assays were conducted using MTS reagent (13). Live cell microscopy, tracking analysis, and invasion assays were as previously described (2,16).…”

“…Negative control siRNA was ON-TARGET plus nontargeting pool (Dharmacon). siRNA was applied to cells as previously described (13).…”

Cortactin in cell migration and cancer at a glance

“…Although this is, indeed, the case, overexpression of cortactin also promotes cell cycle progression through a RhoAdependent pathway in cancer cells (Croucher et al, 2010). Cortactin overexpression also inhibits the degradation of epidermal growth factor (EGF) receptor, leading to increased pro-mitotic receptor signaling (Patel et al, 1998;Timpson et al, 2005).…”