SUMMARY Despite critical roles in development and cancer, the mechanisms that specify invasive cellular behavior are poorly understood. Through a screen of transcription factors in Caenorhabditis elegans, we identified G1 cell-cycle arrest as a precisely regulated requirement of the anchor cell (AC) invasion program. We show that the nuclear receptor nhr-67/tlx directs the AC into G1 arrest in part through regulation of the cyclin-dependent kinase inhibitor cki-1. Loss of nhr-67 resulted in non-invasive, mitotic ACs that failed to express matrix metalloproteinases or actin regulators and lack invadopodia—F-actin rich membrane protrusions that facilitate invasion. We further show that G1 arrest is necessary for the histone deacetylase HDA-1, a key regulator of differentiation, to promote pro-invasive gene expression and invadopodia formation. Together these results suggest that invasive cell fate requires G1 arrest and that strategies targeting both G1 arrested and actively cycling cells may be needed to halt metastatic cancer.

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. (Cancer Res 2006; 66(16): 8017-25)

The basement membrane is a dense, highly cross-linked, sheet-like extracellular matrix that underlies all epithelia and endothelia in multicellular animals. During development, leukocyte trafficking, and metastatic disease, cells cross the basement membrane to disperse and enter new tissues. Based largely on in vitro studies, cells have been thought to use proteases to dissolve and traverse this formidable obstacle. Surprisingly, recent in vivo studies have uncovered a remarkably diverse range of cellular- and tissue-level strategies beyond proteolysis that cells use to navigate through the basement membrane. These fascinating and unexpected mechanisms have increased our understanding of how cells cross this matrix barrier in physiological and disease settings.

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Highlights d In vivo visualization of 29 basement membrane (BM) proteins fused with mNeonGreen d Embryonic BMs share a common template but diversify greatly during development d Stable BM proteins form a scaffold that supports movement of dynamic BM components d Papilin promotes local BM collagen removal by limiting GON-1 protease localization

Summary Invasive cells use small invadopodia to breach basement membrane (BM), a dense matrix that encases tissues. Following the breach, a large protrusion forms to clear a path for tissue entry by poorly understood mechanisms. Using RNAi screening for defects in C. elegans anchor cell (AC) invasion, we found that UNC-6(netrin)/UNC-40(DCC) signaling at the BM breach site directs exocytosis of lysosomes using the exocyst and SNARE SNAP-29 to form a large protrusion that invades vulval tissue. Live-cell imaging revealed that the protrusion is enriched in the matrix metalloprotease ZMP-1 and transiently expands AC volume more than 20%, displacing surrounding BM and vulval epithelium. Photobleaching and genetic perturbations showed that the BM receptor dystroglycan forms a membrane diffusion barrier at the neck of the protrusion that enables protrusion growth. Together these studies define a netrin dependent pathway that builds an invasive protrusion, an isolated lysosome-derived membrane structure specialized to breach tissue barriers.

BackgroundTumor 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 FindingsIn 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/SignificanceCortactin 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.

SummaryThe 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.