Susette C. Mueller

Activation of Arp2/3 complex-mediated actin polymerization by cortactin

Cortactin, a filamentous actin (F-actin)-associated protein and prominent substrate of Src, is implicated in progression of breast tumours through gene amplification at chromosome 11q13. However, the function of cortactin remains obscure. Here we show that cortactin co-localizes with the Arp2/3 complex, a de novo actin nucleator, at dynamic particulate structures enriched with actin filaments. Cortactin binds directly to the Arp2/3 complex and activates it to promote nucleation of actin filaments. The interaction of cortactin with the Arp2/3 complex occurs at an amino-terminal domain that is rich in acidic amino acids. Mutations in a conserved amino-acid sequence of DDW abolish both the interaction with the Arp2/3 complex and complex activation. The N-terminal domain is not only essential but also sufficient to target cortactin to actin-enriched patches within cells. Interestingly, the ability of cortactin to activate the Arp2/3 complex depends on an activity for F-actin binding, which is almost 20-fold higher than that of the Arp2/3 complex. Our data indicate a new mechanism for activation of actin polymerization involving an enhanced interaction between the Arp2/3 complex and actin filaments.

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.

An invasion-related complex of cortactin, paxillin and PKCμ associates with invadopodia at sites of extracellular matrix degradation

Invasive breast cancer cells have the ability to extend membrane protrusions, invadopodia, into the extracellular matrix (ECM). These structures are associated with sites of active matrix degradation. The amount of matrix degradation associated with the activity of these membrane protrusions has been shown to directly correlate with invasive potential. We demonstrate here that microinjection of polyclonal anti-cortactin antibodies blocks matrix degradation at invadopodia supporting the hypothesis that cortactin has a direct role in invasive behavior. MDA-MB-231, invasive breast cancer cells were sheared from the surface of a gelatin matrix to isolate invadopodia. Cortactin, paxillin and protein kinase C (PKC) μ, a serine kinase, were co-immunoprecipitated as a complex from invadopodia-enriched membranes. We confirmed the subcellular distribution of these proteins by immunolocalization and Western blotting. We also determined that, in contrast to its presence in invasive cells, this complex of proteins was not detected in lysates from non-invasive cells that do not form invadopodia. Taken together, these data suggest that the formation of this cortactin-containing complex correlates with cellular invasiveness. We hypothesize that this complex of molecules has a role in the formation and function of invadopodia during cellular invasion.

The Syk tyrosine kinase suppresses malignant growth of human breast cancercells

Syk is a protein tyrosine kinase that is widely expressed in haematopoietic cells. It is involved in coupling activated immunoreceptors to downstream signalling events that mediate diverse cellular responses including proliferation, differentiation and phagocytosis. Syk expression has been reported in cell lines of epithelial origin, but its function in these cells remains unknown. Here we show that Syk is commonly expressed in normal human breast tissue, benign breast lesions and low-tumorigenic breast cancer cell lines. Syk messenger RNA and protein, however, are low or undetectable in invasive breast carcinoma tissue and cell lines. Transfection of wild-type Syk into a Syk-negative breast cancer cell line markedly inhibited its tumour growth and metastasis formation in athymic mice. Conversely, overexpression of a kinase-deficient Syk in a Syk-positive breast cancer cell line significantly increased its tumour incidence and growth. Suppression of tumour growth by the reintroduction of Syk appeared to be the result of aberrant mitosis and cytokinesis. We propose that Syk is a potent modulator of epithelial cell growth and a potential tumour suppressor in human breast carcinomas.

Progenitor/stem cells give rise to liver cancer due to aberrant TGF-β 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-β-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-β 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-β 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-β pathway.

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 p27KIP1 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 MIP1γ. Akt1 governs MEC polarity, migratory directionality and breast cancer onset induced by ErbB2 in vivo.

Effect of estradiol on estrogen receptor- α gene expression and activity can be modulated by the ErbB2/PI 3-K/Akt pathway

Epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and heregulin-β1 (HRG-β1), can modulate the expression and activity of the estrogen receptor-α (ER-α) via the phosphatidylinositol 3-kinase (PI 3-K)/Akt pathway in the ER-α-positive breast cancer cell line, MCF-7. Estradiol can also rapidly activate PI 3-K/Akt in these cells (nongenomic effect). The recent study examines whether Akt is involved in the ER-α regulation by estradiol (genomic effect). Stable transfection of parental MCF-7 cells with a dominant-negative Akt mutant, as well as the PI 3-K inhibitors wortmannin and LY 294,002, blocked the effect of estradiol on ER-α expression and activity by 70–80 and 55–63%, respectively. Stable transfection of MCF-7 cells with a constitutively active Akt mimicked the effect of estradiol. The changes in ER-α expression and activity were abrogated in response to estradiol by an arginine to cysteine mutation in the pleckstrin homology (PH) domain of Akt (R25C), suggesting the involvement of this amino acid in the interaction between Akt and ER-α. Experiments employing selective ErbB inhibitors demonstrate that the effect of estradiol on ER-α expression and activity is mediated by ErbB2 and not by EGFR. Moreover, anchorage-dependent and -independent growth assays, cell cycle and membrane ruffling analyses showed that Akt exerts estrogen-like activity on cell growth and membrane ruffling and that a selective ErbB2 inhibitor, but not anti-ErbB2 antibodies directed to the extracellular domain, can block these effects. In the presence of constitutively active Akt, tamoxifen only partially inhibits cell growth. In contrast, in cells stably transfected with either a dominant-negative Akt or with R25C-Akt, as well as in parental cells in the presence of a selective ErbB2 inhibitor, the effect of estradiol on anchorage-dependent and -independent cell growth was inhibited by 50–75 and 100%, respectively. Dominant-negative Akt inhibited membrane ruffling by 54%; however, R25C-Akt did not have any effect, suggesting that kinase activity plays an important role in this process. Scatchard analysis demonstrated a 67% reduction in estrogen-binding capacity in cells transfected with constitutively active Akt. No change in binding affinity of estradiol to the receptor was observed upon transfection with either Akt mutant. Taken together, our results suggest that estradiol treatment results in binding to membrane ER-α and interaction with a heterodimer containing ErbB2, leading to tyrosine phosphorylation. This results in the activation of PI 3-K and Akt. Akt, in turn, may interact with nuclear ER-α, altering its expression and activity.

Src-Dependent Phosphorylation of ASAP1 Regulates Podosomes

ABSTRACT 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.

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.

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.