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In JoVE (2)
- Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma
- Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts
Other Publications (5)
Articles by Karen E. Hayes in JoVE
JoVE Clinical and Translational Medicine
Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma
1Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, 2Sensory Neuroscience Research Center, West Virginia University, 3Departments of Otolaryngology and Physiology, Center for Neuroscience, West Virginia University
A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.
JoVE General
Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts
We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.
Other articles by Karen E. Hayes on PubMed
Saracatinib Impairs Head and Neck Squamous Cell Carcinoma Invasion by Disrupting Invadopodia Function
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.
Oncogenic Src Requires a Wild-type Counterpart to Regulate Invadopodia Maturation
The proto-oncogene Src tyrosine kinase (Src) is overexpressed in human cancers and is currently a target of anti-invasive therapies. Activation of Src is an essential catalyst of invadopodia production. Invadopodia are cellular structures that mediate extracellular matrix (ECM) proteolysis, allowing invasive cell types to breach confining tissue barriers. Invadopodia assembly and maturation is a multistep process, first requiring the targeting of actin-associated proteins to form pre-invadopodia, which subsequently mature by recruitment and activation of matrix metalloproteases (MMPs) that facilitate ECM degradation. We demonstrate that active, oncogenic Src alleles require the presence of a wild-type counterpart to induce ECM degradation at invadopodia sites. In addition, we identify the phosphorylation of the invadopodia regulatory protein cortactin as an important mediator of invadopodia maturation downstream of wild-type Src. Distinct phosphotyrosine-based protein-binding profiles in cells forming pre-invadopodia and mature invadopodia were identified by SH2-domain array analysis. These results indicate that although elevated Src kinase activity is required to target actin-associated proteins to pre-invadopodia, regulated Src activity is required for invadopodia maturation and matrix degradation activity. Our findings describe a previously unappreciated role for proto-oncogenic Src in enabling the invasive activity of constitutively active Src alleles.
Cortactin Phosphorylated by ERK1/2 Localizes to Sites of Dynamic Actin Regulation and is Required for Carcinoma Lamellipodia Persistence
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.
Revisiting the ERK/Src Cortactin Switch
The filamentous (F)-actin regulatory protein cortactin plays an important role in tumor cell movement and invasion by promoting and stabilizing actin related protein (Arp)2/3-mediated actin networks necessary for plasma membrane protrusion. Cortactin is a substrate for ERK1/2 and Src family kinases, with previous in vitro findings demonstrating ERK1/2 phosphorylation of cortactin as a positive and Src phosphorylation as a negative regulatory event in promoting Arp2/3 activation through neuronal Wiskott Aldrich Syndrome protein (N-WASp). Evidence for this regulatory cortactin "switch" in cells has been hampered due to the lack of phosphorylation-specific antibodies that recognize ERK1/2-phosphorylated cortactin. Our findings with phosphorylation-specific antibodies against these ERK1/2 sites (pS405 and pS418) indicate that cortactin can be co-phosphorylated at 405/418 and tyrosine residues targeted by Src family tyrosine kinases. These results indicate that the ERK/Src cortactin switch is not the sole mechanism by which ERK1/2 and tyrosine phosphorylation events regulate cortactin function in cell systems.
Further Insights into Cortactin Conformational Regulation
The actin regulatory protein cortactin is involved in multiple signaling pathways impinging on the cortical actin cytoskeleton. Cortactin is phosphorylated by ERK1/2 and Src family tyrosine kinases, resulting in neuronal Wiskott Aldrich Syndrome protein (N-WASp) activation and enhanced actin related protein (Arp)2/3-mediated actin nucleation. Cortactin migrates as an 80/85 kDa doublet when analyzed by SDS-PAGE. Phosphorylation by ERK1/2 is associated with conversion of the 80 kDa to the 85 kDa form, postulated to occur by inducing a conformational alteration that releases the carboxyl-terminal SH3 domain from autoinhibition. Our recent analysis of the 80-85 kDa cortactin "shift" in tumor cells indicates that while ERK1/2 phosphorylation is associated with the 85 kDa shift, this phosphorylation event is not required for the shift to occur, nor does ERK1/2 phosphorylation appreciably alter global cortactin confirmation. These data indicate that additional factors besides ERK1/2 phosphorylation contribute to generating and/or maintaining the activated 85 kDa cortactin form in stimulated cells.
