Modulation of the rat angiotensin type 1a receptor by an upstream short open reading frame.

The rat angiotensin type 1a receptor (AT1aR) is a peptide hormone G protein-coupled receptor (GPCR) that plays a key role in electrolyte homeostasis and blood pressure control. There is a highly conserved short open reading frame (sORF) in exon 2 (E2) that is downstream from exon 1 (E1) and upstream of the AT1aR coding region located in exon 3 (E3). To determine the role of this E2 sORF in AT1aR signaling, human embryonic kidney-293 (HEK293) cells were transfected with plasmids containing AT1aR cDNA with either an intact or disrupted E2 sORF. The intact sORF attenuated the efficacy of angiotensin (Ang) II (p < 0.001) and sarcosine1,Ile4,Ile8-Ang II (SII), (p < 0.01) to activate AT1aR signaling through extracellular signal-related kinases 1/2 (ERK1/2). A time-course showed agonist-induced AT1aR-mediated ERK1/2 activation was slower in the presence of the intact compared to the disrupted sORF [Ang II: p < 0.01 and SII: p < 0.05]. Ang II-induced ERK1/2 activation was completely inhibited by the protein kinase C (PKC) inhibitor Ro 31-8220 regardless of whether the sORF was intact or disrupted. Flow cytometric analyses suggested the intact sORF improved cell survival; the percentage of live cells increased (p < 0.05) while the percentage of early apoptotic cells decreased (p < 0.01) in cells transfected with the AT1aR plasmid containing the intact sORF. These findings have implications for the regulation of AT1Rs in physiological and pathological conditions and warrant investigation of sORFs in the 5' leader sequence (5'LS) of other GPCRs.

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy.

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Antimitotic activity of DY131 and the estrogen-related receptor beta 2 (ERRß2) splice variant in breast cancer.

Breast cancer remains a leading cause of cancer-related death in women, and triple negative breast cancer (TNBC) lacks clinically actionable therapeutic targets. Death in mitosis is a tumor suppressive mechanism that occurs in cancer cells experiencing a defective M phase. The orphan estrogen-related receptor beta (ERRß) is a key reprogramming factor in murine embryonic and induced pluripotent stem cells. In primates, ERRß is alternatively spliced to produce several receptor isoforms. In cellular models of glioblastoma, short form (ERRßsf) and beta2 (ERRß2) splice variants differentially regulate cell cycle progression in response to the synthetic agonist DY131, with ERRß2 driving arrest in G2/M.The goals of the present study are to determine the cellular function(s) of ligand-activated ERRß splice variants in breast cancer and evaluate the potential of DY131 to serve as an antimitotic agent, particularly in TNBC. DY131 inhibits growth in a diverse panel of breast cancer cell lines, causing cell death that involves the p38 stress kinase pathway and a bimodal cell cycle arrest. ERRß2 facilitates the block in G2/M, and DY131 delays progression from prophase to anaphase. Finally, ERRß2 localizes to centrosomes and DY131 causes mitotic spindle defects. Targeting ERRß2 may therefore be a promising therapeutic strategy in breast cancer.

Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network.

Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM.

SYK allelic loss and the role of Syk-regulated genes in breast cancer survival.

Heterozygotic loss of SYK, a non-receptor tyrosine kinase, gives rise to mouse mammary tumor formation where Syk protein levels are reduced by about half; loss of SYK mRNA is correlated with invasive cell behavior in in vitro models; and SYK loss has been correlated with distant metastases in patients. Here, allelic loss of the SYK gene was explored in breast ductal carcinoma in situ (DCIS) using fluorescence in situ hybridization and pyrosequencing, respectively, and in infiltrating ductal carcinoma (IDC) using genomic data from The Cancer Genome Atlas (TCGA). Allelic loss was present in a subset of DCIS cases where adjacent IDC was present. SYK copy number loss was found in about 26% of 1002 total breast cancer cases and 30% of IDC cases. Quantitative immunofluorescence revealed Syk protein to be six-fold higher in infiltrating immune cells compared with epithelial cells. This difference distorted tumor cell mRNA and protein levels in extracts. 20% of 1002 IDC cases contained elevated immune cell infiltration as estimated by elevated immune-specific mRNAs. In cases without immune cell infiltration, loss of SYK copy number was associated with a significant reduction of SYK mRNA. Here we define a 55 Gene Set consisting of Syk interacting, motility- and invasion-related genes. We found that overall survival was significantly reduced in IDC and Luminal A+B cases where copy number and mutations of these 55 genes were affected (Kaplan-Meier, Logrank test p-value 0.007141 and Logrank test p-value 0.001198, respectively). We conclude that reduction in Syk expression and contributions of genomic instability to copy number and mutations in the 55 Syk interacting genes significantly contribute to poorer overall patient survival. A closer examination of the role of Syk interacting motility and invasion genes and their prognostic and/or causative association with metastatic disease and patient outcome is warranted.

Three dimensional multiphoton imaging of fresh and whole mount developing mouse mammary glands.

BACKGROUND: The applications of multiphoton microscopy for deep tissue imaging in basic and clinical research are ever increasing, supplementing confocal imaging of the surface layers of cells in tissue. However, imaging living tissue is made difficult by the light scattering properties of the tissue, and this is extraordinarily apparent in the mouse mammary gland which contains a stroma filled with fat cells surrounding the ductal epithelium. Whole mount mammary glands stained with Carmine Alum are easily archived for later reference and readily viewed using bright field microscopy to observe branching architecture of the ductal network. Here, we report on the advantages of multiphoton imaging of whole mount mammary glands. Chief among them is that optical sectioning of the terminal end bud (TEB) and ductal epithelium allows the appreciation of abnormalities in structure that are very difficult to ascertain using either bright field imaging of the stained gland or the conventional approach of hematoxylin and eosin staining of fixed and paraffin-embedded sections. A second advantage is the detail afforded by second harmonic generation (SHG) in which collagen fiber orientation and abundance can be observed. METHODS: GFP-mouse mammary glands were imaged live or after whole mount preparation using a Zeiss LSM510/META/NLO multiphoton microscope with the purpose of obtaining high resolution images with 3D content, and evaluating any structural alterations induced by whole mount preparation. We describe a simple means for using a commercial confocal/ multiphoton microscope equipped with a Ti-Sapphire laser to simultaneously image Carmine Alum fluorescence and collagen fiber networks by SHG with laser excitation set to 860 nm. Identical terminal end buds (TEBs) were compared before and after fixation, staining, and whole mount preparation and structure of collagen networks and TEB morphologies were determined. Flexibility in excitation and emission filters was explored using the META detector for spectral emission scanning. Backward scattered or reflected SHG (SHG-B) was detected using a conventional confocal detector with maximum aperture and forward scattered or transmitted SHG (SHG-F) detected using a non-descanned detector. RESULTS: We show here that the developing mammary gland is encased in a thin but dense layer of collagen fibers. Sparse collagen layers are also interspersed between stromal layers of fat cells surrounding TEBs. At the margins, TEBs approach the outer collagen layer but do not penetrate it. Abnormal mammary glands from an HAI-1 transgenic FVB mouse model were found to contain TEBs with abnormal pockets of cells forming extra lumens and zones of continuous lateral bud formation interspersed with sparse collagen fibers. CONCLUSIONS: Collagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen. In addition to providing a bright signal, Carmine Alum stained whole mount slides can be imaged retrospectively such as performed for the HAI-1 mouse gland revealing new aspects of abnormal TEB morphology. These studies demonstrated the intimate contact, but relatively sparse abundance of collagen fibrils adjacent to normal and abnormal TEBS in the developing mammary gland and the ability to obtain these high resolution details subject to the discussed limitations. Our studies demonstrated that the TEB architecture is essentially unchanged after processing.

A novel 3D fibril force assay implicates src in tumor cell force generation in collagen networks.

New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM) environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction and motility in 3D fibrillar environments. The assay consists of a controlled-density fibrillar collagen gel atop a controlled-stiffness polyacrylamide (PAA) surface. Forces generated by living cells and their migration in the 3D collagen gel were measured with the 3D motion of tracer beads within the PAA layer. Here, this 3D fibril force assay is used to study the role of the invasion-associated protein kinase Src in mechanotransduction and motility. Src expression and activation are linked with proliferation, invasion, and metastasis, and have been shown to be required in 2D for invadopodia membranes to direct and mediate invasion. Breast cancer cell line MDA-MD-231 was stably transfected with GFP-tagged constitutively active Src or wild-type Src. In 3D fibrillar collagen matrices we found that, relative to wild-type Src, constitutively active Src: 1) increased the strength of cell-induced forces on the ECM, 2) did not significantly change migration speed, and 3) increased both the duration and the length, but not the number, of long membrane protrusions. Taken together, these results support the hypothesis that Src controls invasion by controlling the ability of the cell to form long lasting cellular protrusions to enable penetration through tissue barriers, in addition to its role in promoting invadopodia matrix-degrading activity.

Dynamic membrane remodeling at invadopodia differentiates invadopodia from podosomes.

Invadopodia are specialized actin-rich protrusions of metastatic tumor and transformed cells with crucial functions in ECM degradation and invasion. Although early electron microscopy studies described invadopodia as long filament-like protrusions of the cell membrane adherent to the matrix, fluorescence microscopy studies have focused on invadopodia as actin-cortactin aggregates localized to areas of ECM degradation. The absence of a clear conceptual integration of these two descriptions of invadopodial structure has impeded understanding of the regulatory mechanisms that govern invadopodia. To determine the relationship between the membrane filaments identified by electron microscopy and the actin-cortactin aggregates of invadopodia, we applied rapid live-cell high-resolution TIRF microscopy to examine cell membrane dynamics at the cortactin core of the invadopodia of human carcinoma cells. We found that cortactin docking to the cell membrane adherent to 2D fibronectin matrix initiates invadopodium assembly associated with the formation of an invadopodial membrane process that extends from a ventral cell membrane lacuna toward the ECM. The tip of the invadopodial process flattens as it interacts with the 2D matrix, and it undergoes constant rapid ruffling and dynamic formation of filament-like protrusions as the invadopodium matures. To describe this newly discovered dynamic relationship between the actin-cortactin core and invadopodial membranes, we propose a model of the invadopodial complex. Using TIRF microscopy, we also established that - in striking contrast to the invadopodium - membrane at the podosome of a macrophage fails to form any process- or filament-like membrane protrusions. Thus, the undulation and ruffling of the invadopodial membrane together with the formation of dynamic filament-like extensions from the invadopodial cortactin core defines invadopodia as invasive superstructures that are distinct from the podosomes.

Tumor suppressor function of Syk in human MCF10A in vitro and normal mouse mammary epithelium in vivo.

The normal function of Syk in epithelium of the developing or adult breast is not known, however, Syk suppresses tumor growth, invasion, and metastasis in breast cancer cells. Here, we demonstrate that in the mouse mammary gland, loss of one Syk allele profoundly increases proliferation and ductal branching and invasion of epithelial cells through the mammary fat pad during puberty. Mammary carcinomas develop by one year. Syk also suppresses proliferation and invasion in vitro. siRNA or shRNA knockdown of Syk in MCF10A breast epithelial cells dramatically increased proliferation, anchorage independent growth, cellular motility, and invasion, with formation of functional, extracellular matrix-degrading invadopodia. Morphological and gene microarray analysis following Syk knockdown revealed a loss of luminal and differentiated epithelial features with epithelial to mesenchymal transition and a gain in invadopodial cell surface markers CD44, CD49F, and MMP14. These results support the role of Syk in limiting proliferation and invasion of epithelial cells during normal morphogenesis, and emphasize the critical role of Syk as a tumor suppressor for breast cancer. The question of breast cancer risk following systemic anti-Syk therapy is raised since only partial loss of Syk was sufficient to induce mammary carcinomas.

Breast cancer cell movement: imaging invadopodia by TIRF and IRM microscopy.

Invadopodia are hair-like membrane protrusions projecting from the ventral side of the plasma membrane of tumor cells invading into an extracellular matrix (ECM). Formation of invadopodia and phagocytosis of partially degraded ECM is correlated with invasiveness of cancer cells. Many proteins associated with actin-rich punctae associated with invadopodia have been identified. However, the dynamic temporal and spatial relationship of invadopodia-related proteins and the mechanisms required for invadopodia formation remain largely unknown. Total Internal Reflection Fluorescence (TIRF) microscopy provides a powerful tool to directly visualize the dynamic membrane transportation of invadopodia-related, GFP-tagged proteins and to simultaneously monitor invadopodia formation by observation of localized degradation and phagocytosis of fluorescently labeled gelatin. Cell-substratum contacts can be visualized using a related technique, Interference Reflection Microscopy (IRM). In this chapter, we provide detailed methodologies to monitor the dynamic localizations of c-Src-eGFP using two-color TIRF microscopy along with IRM to simultaneously visualize translocation of c-Src-eGFP and invadopodia formation by degradation of AlexaFluor 568-labeled gelatin.

The role of the exocyst in matrix metalloproteinase secretion and actin dynamics during tumor cell invadopodia formation.

Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Invadopodia formation involves membrane protrusions driven by Arp2/3-mediated actin polymerization and secretion of matrix metalloproteinases (MMPs) at the focal degrading sites. The exocyst mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for exocytosis and has recently been implicated in regulating actin dynamics during cell migration. Here, we report that the exocyst plays a pivotal role in invadopodial activity. With RNAi knockdown of the exocyst component Exo70 or Sec8, MDA-MB-231 cells expressing constitutively active c-Src failed to form invadopodia. On the other hand, overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by siEXO70 or siSEC8 treatment or by expression of a dominant negative fragment of Exo70 inhibited the secretion of MMPs. We have also found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Together, our results suggest that the exocyst plays important roles in cell invasion by mediating the secretion of MMPs at focal degrading sites and regulating Arp2/3-mediated actin dynamics.

ECM degradation assays for analyzing local cell invasion.

Proteolytic degradation of extracellular matrix (ECM) is a critical step during cell invasion and tissue transmigration that is required for many physiological and pathological processes. Cellular structures that mediate cell adhesion to, degradation of, and invasion into ECM are invadopodia of transformed and tumor cells and podosomes of normal monocytic, endothelial, and smooth muscle cells. Detecting the ability of the cell to form invadopodia and podosomes and to degrade ECM is required for studying the invasive capability of the cell. We have developed approximately 50 nm thick fluorescent gelatin matrices that provide a rapid, sensitive, and reliable in vitro system for detection of invadopodia and podosomes, and measurements of the extent of ECM degradation. In this chapter, we provide a detailed protocol for preparation of thin fluorescent gelatin matrices and for evaluation of the results from this degradation assay.

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells.

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

Progenitor/stem cells give rise to liver cancer due to aberrant TGF-beta and IL-6 signaling.

Cancer stem cells (CSCs) are critical for the initiation, propagation, and treatment resistance of multiple cancers. Yet functional interactions between specific signaling pathways in solid organ "cancer stem cells," such as those of the liver, remain elusive. We report that in regenerating human liver, two to four cells per 30,000-50,000 cells express stem cell proteins Stat3, Oct4, and Nanog, along with the prodifferentiation proteins TGF-beta-receptor type II (TBRII) and embryonic liver fodrin (ELF). Examination of human hepatocellular cancer (HCC) reveals cells that label with stem cell markers that have unexpectedly lost TBRII and ELF. elf(+/-) mice spontaneously develop HCC; expression analysis of these tumors highlighted the marked activation of the genes involved in the IL-6 signaling pathway, including IL-6 and Stat3, suggesting that HCC could arise from an IL-6-driven transformed stem cell with inactivated TGF-beta signaling. Similarly, suppression of IL-6 signaling, through the generation of mouse knockouts involving a positive regulator of IL-6, Inter-alpha-trypsin inhibitor-heavy chain-4 (ITIH4), resulted in reduction in HCC in elf(+/-) mice. This study reveals an unexpected functional link between IL-6, a major stem cell signaling pathway, and the TGF-beta signaling pathway in the modulation of mammalian HCC, a lethal cancer of the foregut. These experiments suggest an important therapeutic role for targeting IL-6 in HCCs lacking a functional TGF-beta pathway.

Disruption of c-Jun reduces cellular migration and invasion through inhibition of c-Src and hyperactivation of ROCK II kinase.

The spread of metastatic tumors to different organs is associated with poor prognosis. The metastatic process requires migration and cellular invasion. The protooncogene c-jun encodes the founding member of the activator protein-1 family and is required for cellular proliferation and DNA synthesis in response to oncogenic signals and plays an essential role in chemical carcinogenesis. The role of c-Jun in cellular invasion remains to be defined. Genetic deletion of c-Jun in transgenic mice is embryonic lethal; therefore, transgenic mice encoding a c-Jun gene flanked by LoxP sites (c-jun(f/f)) were used. c-jun gene deletion reduced c-Src expression, hyperactivated ROCK II signaling, and reduced cellular polarity, migration, and invasiveness. c-Jun increased c-Src mRNA abundance and c-Src promoter activity involving an AP-1 site in the c-Src promoter. Transduction of c-jun(-/-) cells with either c-Jun or c-Src retroviral expression systems restored the defective cellular migration of c-jun(-/-) cells. As c-Src is a critical component of pathways regulating proliferation, survival, and metastasis, the induction of c-Src abundance, by c-Jun, provides a novel mechanism of cooperative signaling in cellular invasion.

Src-dependent phosphorylation of ASAP1 regulates podosomes.

Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.

Akt1 governs breast cancer progression in vivo.

The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Here, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1, in the mouse demonstrates a requirement for Akt1 in ErbB2-induced mammary tumorigenesis. Akt1 deficiency delayed tumor growth and reduced lung metastases, correlating with a reduction in phosphorylation of the Akt1 target, tuberous sclerosis 2 (TSC2) at Ser-939. Akt1-deficient mammary epithelial tumor cells (MEC) were reduced in size and proliferative capacity, with reduced cyclin D1 and p27(KIP1) abundance. Akt1 deficiency abrogated the oncogene-induced changes in polarization of MEC in three-dimensional culture and reverted oncogene-induced relocalization of the phosphorylated ezrin-radixin-moesin proteins. Akt1 increased MEC migration across an endothelial cell barrier, enhancing the persistence of migratory directionality. An unbiased proteomic analysis demonstrated Akt1 mediated MEC migration through paracrine signaling via induction of expression and secretion of CXCL16 and MIP1gamma. Akt1 governs MEC polarity, migratory directionality and breast cancer onset induced by ErbB2 in vivo.

Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function.

Metastatic tumor cells that actively migrate and invade surrounding tissues rely on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are membrane protrusions that localize enzymes required for ECM degradation. Little is known about the formation, function, and regulation of invadopodia. Here, we show that invadopodia have two distinct aspects: (a) structural for organizing the cellular actin cytoskeleton to form membrane protrusions and (b) functional for using proteolytic enzyme(s) for ECM degradation. Small interfering RNA (siRNA) inhibition established that organization of invadopodia structure requires cortactin, whereas protease inhibitor studies identified membrane type 1 matrix metalloproteinase (MT1-MMP) as the key invadopodial enzyme responsible for gelatin matrix degradation in the breast carcinoma cell line MDA-MB-231. The inhibition of invadopodial structure assembly by cortactin depletion resulted in a block of matrix degradation due to failure of invadopodia formation. Either protease inhibition or MT1-MMP siRNA depletion moderately decreased the formation of invadopodial structures that were identified as actin-cortactin accumulations at the ventral cell membrane adherent to matrix. The invadopodia that were able to form upon MT1-MMP inhibition or depletion retained actin-cortactin accumulations but were unable to degrade matrix. Examination of cells at different time points as well as live-cell imaging revealed four distinct invadopodial stages: membrane cortactin aggregation at membranes adherent to matrix, MT1-MMP accumulation at the region of cortactin accumulation, matrix degradation at the invadopodia region, and subsequent cortactin dissociation from the area of continued MT1-MMP accumulation associated with foci of degraded matrix. Based on these results, we propose a stepwise model of invadopodia formation and function.

Co-localization of cortactin and phosphotyrosine identifies active invadopodia in human breast cancer cells.

Invadopodia are filopodia-like projections possessing protease activity that participate in tumor cell invasion. We demonstrate that co-localization of cortactin and phosphotyrosine identifies a subset of cortactin puncta termed "invadopodial complexes" that we find to be closely associated with the plasma membrane at active sites of focal degradation of the extracellular matrix in MDA-MB-231 breast cancer cells. Manipulation of c-Src activity in cells by transfection with kinase activated c-Src(527) or kinase inactive c-Src(295) results in a dramatic increase or decrease, respectively, in the number of these structures associated with changes in the number of sites of active matrix degradation. Overexpression of kinase-inactive c-Src(295) does not prevent localization of cortactin at the membrane; however, co-localized phosphotyrosine staining is decreased. Thus, elevated phosphotyrosine at invadopodial complexes is specifically associated with the proteolytic activity of invadopodia. Further, invadopodial complexes are spatially, morphologically and compositionally distinct from focal adhesions as determined by localization of focal adhesion kinase (FAK), which is not present in invadopodial complexes. Expression of kinase-inactive c-Src(295) blocks invadopodia activity, but does not block filopodia formation. Thus, invadopodia, but not filopodia, are highly correlated with matrix invasion, and sites of invadopodial activity can be identified by the formation of invadopodial complexes.

The Syk tyrosine kinase: a new negative regulator in tumor growth and progression.

The spleen tyrosine kinase Syk was long thought to be a hematopoietic cell-specific signaling molecule. Recent evidence demonstrated that it is also expressed by many non-hematopoietic cell types and that it plays a negative role in cancer. A significant drop in its expression was first observed during breast cancer progression, but an anomalous Syk expression has now also been evidenced in many other tumor types. Mechanistic studies using Syk re-expression demonstrated its suppressive function in tumorigenesis and metastasis formation, which is surprising for a tyrosine kinase. Loss of Syk expression is regulated, albeit not exclusively, by its promoter hypermethylation. The molecular mechanism of its tumor-suppressive function remains largely unknown; the identification of its activators and effectors in non-hematopoietic cells will be a challenge for the years to come. An increasing number of clinical studies reveal a correlation between reduced Syk expression and an increased risk for metastasis formation, and assign Syk as a potential new prognostic marker in different tumor types.