Amanda G. Ammer

Since its discovery in the early 1990's, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures.

Carcinoma cell motility and invasion are prerequisites for tumor cell metastasis, which requires regulation of the actin cytoskeleton. Cortactin is an actin-related protein 2/3 (Arp2/3) complex-activating and filamentous (F)-actin-binding protein that is implicated in tumor cell motility and metastasis, partially by its ability to become tyrosine phosphorylated. Cortactin is encoded by the CTTN gene and maps to chromosome 11q13, a region amplified in many carcinomas, including head and neck squamous cell carcinoma (HNSCC). CTTN gene amplification is associated with lymph node metastasis and poor patient outcome, and cortactin overexpression enhances motility in tumor cells lacking 11q13 amplification. However, a direct link between increased motility and invasion has not been reported in tumor cells with chromosome 11q13 amplification and cortactin overexpression. In this study, we have examined the relationship between CTTN amplification and tumor cell motility in HNSCC. In 11 of 39 (28%) HNSCC cases, cortactin overexpression determined by immunohistochemistry correlates with lymph node metastasis and CTTN gene amplification. HNSCC cells containing cortactin gene amplification and protein overexpression display increased binding and activation of Arp2/3 complex, and were more motile and invasive than HNSCC cells lacking CTTN amplification. Down-regulation of cortactin expression in CTTN-amplified HNSCC cells by small interfering RNA impairs HNSCC motility and invasion. Treatment of HNSCC cells with the epidermal growth factor receptor inhibitor gefitinib inhibits HNSCC motility and down-regulates cortactin tyrosine phosphorylation. These data suggest that cortactin may be a valid prognostic and therapeutic marker for invasive and metastatic HNSCC and other carcinomas with 11q13 amplification.

The lack of translatable in vitro blood-tumor barrier (BTB) models creates challenges in the development of drugs to treat tumors of the CNS and our understanding of how the vascular changes at the BBB in the presence of a tumor. In this study, we characterize a novel microfluidic model of the BTB (and BBB model as a reference) that incorporates flow and induces shear stress on endothelial cells. Cell lines utilized include human umbilical vein endothelial cells co-cultured with CTX-TNA2 rat astrocytes (BBB) or Met-1 metastatic murine breast cancer cells (BTB). Cells were capable of communicating across microfluidic compartments via a porous interface. We characterized the device by comparing permeability of three passive permeability markers and one marker subject to efflux. The permeability of Sulforhodamine 101 was significantly (p < 0.05) higher in the BTB model (13.1 ± 1.3 × 10−3, n = 4) than the BBB model (2.5 ± 0.3 × 10−3, n = 6). Similar permeability increases were observed in the BTB model for molecules ranging from 600 Da to 60 kDa. The function of P-gp was intact in both models and consistent with recent published in vivo data. Specifically, the rate of permeability of Rhodamine 123 across the BBB model (0.6 ± 0.1 × 10−3, n = 4), increased 14-fold in the presence of the P-gp inhibitor verapamil (14.7 ± 7.5 × 10−3, n = 3) and eightfold with the addition of Cyclosporine A (8.8 ± 1.8 × 10−3, n = 3). Similar values were noted in the BTB model. The dynamic microfluidic in vitro BTB model is a novel commercially available model that incorporates shear stress, and has permeability and efflux properties that are similar to in vivo data.

BACKGROUND: Tumor cell motility and invasion is governed by dynamic regulation of the cortical actin cytoskeleton. The actin-binding protein cortactin is commonly upregulated in multiple cancer types and is associated with increased cell migration. Cortactin regulates actin nucleation through the actin related protein (Arp)2/3 complex and stabilizes the cortical actin cytoskeleton. Cortactin is regulated by multiple phosphorylation events, including phosphorylation of S405 and S418 by extracellular regulated kinases (ERK)1/2. ERK1/2 phosphorylation of cortactin has emerged as an important positive regulatory modification, enabling cortactin to bind and activate the Arp2/3 regulator neuronal Wiskott-Aldrich syndrome protein (N-WASp), promoting actin polymerization and enhancing tumor cell movement. METHODOLOGY/PRINCIPAL FINDINGS: In this report we have developed phosphorylation-specific antibodies against phosphorylated cortactin S405 and S418 to analyze the subcellular localization of this cortactin form in tumor cells and patient samples by microscopy. We evaluated the interplay between cortactin S405 and S418 phosphorylation with cortactin tyrosine phosphorylation in regulating cortactin conformational forms by Western blotting. Cortactin is simultaneously phosphorylated at S405/418 and Y421 in tumor cells, and through the use of point mutant constructs we determined that serine and tyrosine phosphorylation events lack any co-dependency. Expression of S405/418 phosphorylation-null constructs impaired carcinoma motility and adhesion, and also inhibited lamellipodia persistence monitored by live cell imaging. CONCLUSIONS/SIGNIFICANCE: Cortactin phosphorylated at S405/418 is localized to sites of dynamic actin assembly in tumor cells. Concurrent phosphorylation of cortactin by ERK1/2 and tyrosine kinases enables cells with the ability to regulate actin dynamics through N-WASp and other effector proteins by synchronizing upstream regulatory pathways, confirming cortactin as an important integration point in actin-based signal transduction. Reduced lamellipodia persistence in cells with S405/418A expression identifies an essential motility-based process reliant on ERK1/2 signaling, providing additional understanding as to how this pathway impacts tumor cell migration.

Elevated Src kinase activity is linked to the progression of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Src regulates HNSCC proliferation and tumor invasion, with the Src-targeted small molecule inhibitor saracatinib displaying potent anti-invasive effects in preclinical studies. However, the pro-invasive cellular mechanism(s) perturbed by saracatinib are unclear. The anti-proliferative and anti-invasive effects of saracatinib on HNSCC cell lines were therefore investigated in pre-clinical cell and mouse model systems. Saracatinib treatment inhibited growth, cell cycle progression and transwell Matrigel invasion in HNSCC cell lines. Dose-dependent decreases in Src activation and phosphorylation of the invasion-associated substrates focal adhesion kinase, p130 CAS and cortactin were also observed. While saracatinib did not significantly impact HNSCC tumor growth in a mouse orthotopic model of tongue squamous cell carcinoma, impaired perineural invasion and cervical lymph node metastasis was observed. Accordingly, saracatinib treatment displayed a dose-dependent inhibitory effect on invadopodia formation, extracellular matrix degradation and matrix metalloprotease 9 activation. These results suggest that inhibition of Src kinase by saracatinib impairs the pro-invasive activity of HNSCC by inhibiting Src substrate phosphorylation important for invadopodia formation and associated matrix metalloprotease activity.