Emily S. Clark

Cortactin Is an Essential Regulator of Matrix Metalloproteinase Secretion and Extracellular Matrix Degradation in Invadopodia

Invadopodia are branched actin-rich structures associated with extracellular matrix (ECM) degradation that collectively form the invasive machinery of aggressive cancer cells. Cortactin is a prominent component and a specific marker of invadopodia. Amplification of cortactin is associated with poor prognosis in head and neck squamous cell carcinomas (HNSCC), possibly because of its activity in invadopodia. Although the role of cortactin in invadopodia has been attributed to signaling and actin assembly, it is incompletely understood. We made HNSCC cells deficient in cortactin by RNA interference knockdown methods. In these cortactin knockdown cells, invadopodia were reduced in number and lost their ability to degrade ECM. In the reverse experiment, overexpression of cortactin dramatically increased ECM degradation, far above and beyond the effect on formation of actin/Arp3-positive invadopodia puncta. Secretion of matrix metalloproteinases (MMP) MMP-2 and MMP-9, as well as plasma membrane delivery of MT1-MMP correlated closely with cortactin expression levels. MMP inhibitor treatment of control cells mimicked the cortactin knockdown phenotype, with abolished ECM degradation and fewer invadopodia, suggesting a positive feedback loop in which degradation products from MMP activity promote new invadopodia formation. Collectively, these data suggest that a major role of cortactin in invadopodia is to regulate the secretion of MMPs and point to a novel mechanism coupling dynamic actin assembly to the secretory machinery, producing enhanced ECM degradation and invasiveness. Furthermore, these data provide a possible explanation for the observed association between cortactin overexpression and enhanced invasiveness and poor prognosis in HNSCC patients.

Extracellular Matrix Rigidity Promotes Invadopodia Activity

Invadopodia are actin-rich subcellular protrusions with associated proteases used by cancer cells to degrade extracellular matrix (ECM) [1]. Molecular components of invadopodia include branched actin-assembly proteins, membrane trafficking proteins, signaling proteins, and transmembrane proteinases [1]. Similar structures exist in nontransformed cells, such as osteoclasts and dendritic cells, but are generally called podosomes and are thought to be more involved in cell-matrix adhesion than invadopodia [2-4]. Despite intimate contact with their ECM substrates, it is unknown whether physical or chemical ECM signals regulate invadopodia function. Here, we report that ECM rigidity directly increases both the number and activity of invadopodia. Transduction of ECM-rigidity signals depends on the cellular contractile apparatus [5-7], given that inhibition of nonmuscle myosin II, myosin light chain kinase, and Rho kinase all abrogate invadopodia-associated ECM degradation. Whereas myosin IIA, IIB, and phosphorylated myosin light chain do not localize to invadopodia puncta, active phosphorylated forms of the mechanosensing proteins p130Cas (Cas) and focal adhesion kinase (FAK) are present in actively degrading invadopodia, and the levels of phospho-Cas and phospho-FAK in invadopodia are sensitive to myosin inhibitors. Overexpression of Cas or FAK further enhances invadopodia activity in cells plated on rigid polyacrylamide substrates. Thus, in invasive cells, ECM-rigidity signals lead to increased matrix-degrading activity at invadopodia, via a myosin II-FAK/Cas pathway. These data suggest a potential mechanism, via invadopodia, for the reported correlation of tissue density with cancer aggressiveness.

Cortactin Promotes Cell Motility by Enhancing Lamellipodial Persistence

BACKGROUND Lamellipodial protrusion, which is the first step in cell movement, is driven by actin assembly and requires activity of the Arp2/3 actin-nucleating complex. However, it is unclear how actin assembly is dynamically regulated to support effective cell migration. RESULTS Cells deficient in cortactin have impaired cell migration and invasion. Kymography analyses of live-cell imaging studies demonstrate that cortactin-knockdown cells have a selective defect in the persistence of lamellipodial protrusions. The motility and protrusion defects are fully rescued by cortactin molecules, provided both the Arp2/3 complex and F-actin binding sites are intact. Consistent with this requirement for simultaneous contacts with Arp2/3 and F-actin, cortactin is recruited by Arp2/3 complex to lamellipodia and binds with a higher affinity to ATP/ADP-Pi-F-actin than to ADP-F-actin. In situ labeling of lamellipodia revealed that the relative levels of free barbed ends of actin filaments are reduced by over 30% in the cortactin-knockdown cells; however, there is no change in Arp2/3-complex localization to lamellipodia. Cortactin-knockdown cells also have a selective defect in the assembly of new adhesions in protrusions, as assessed by analysis of GFP-paxillin dynamics in living cells. CONCLUSIONS Cortactin enhances lamellipodial persistence, at least in part through regulation of Arp2/3 complex. The presence of cortactin also enhances the rate of new adhesion formation in lamellipodia. In vivo, these functions may be important during directed cell motility.

A new role for cortactin in invadopodia: Regulation of protease secretion

Invadopodia are actin-dependent organelles that function in the invasion and remodeling of the extracellular matrix (ECM) by tumor cells. Cortactin, a regulator of the Arp2/3 complex, is of particular importance in invadopodia function. While most of the focus has been on the possible role of cortactin in actin assembly for direct formation of actin-rich invadopodia puncta, our recent data suggest that the primary role of cortactin in invadopodia is to promote protease secretion. In this manuscript, we review our previous work and present new data showing that cortactin is essential for both the localization of key invadopodia matrix metalloproteinases (MMPs) to actin-positive puncta at the cell-ECM interface and for ECM degradation induced by overexpression of MT1-MMP-GFP. Based on these data and results from the literature, we propose potential mechanisms by which cortactin may link vesicular trafficking and dynamic branched actin assembly to regulate protease secretion for invadopodia-associated ECM degradation.

Aggressiveness of HNSCC tumors depends on expression levels of cortactin, a gene in the 11q13 amplicon

11q13 amplification is a late-stage event in several cancers that is often associated with poor prognosis. Among 11q13-amplified genes, the actin assembly protein cortactin/CTTN is considered a likely candidate for direct involvement in tumor progression because of its cell motility-enhancing functions. We modulated cortactin expression in head and neck squamous cell carcinoma (HNSCC) cell lines. Cortactin expression levels directly correlated with tumor size, vascularization and cell proliferation in an orthotopic HNSCC in vivo model. In contrast, under normal in vitro culture conditions, cortactin expression levels had no effect on cell proliferation. However, cell lines in which cortactin expression was reduced by knockdown (KD) grew poorly in vitro under harsh conditions of growth factor deprivation, anchorage independence and space constraint. In contrast, overexpression of cortactin enhanced in vitro growth under the same harsh conditions. Surprisingly, defects in growth factor-independent proliferation of cortactin-KD cells were rescued by coculture with cortactin-expressing cells. As the cocultured cells are separated by permeable filters, cortactin-expressing cells must secrete growth-supporting autocrine factors to rescue the cortactin-KD cells. Overall, cortactin expression modulates multiple cellular traits that may allow survival in a tumor environment, suggesting that the frequent overexpression of cortactin in tumors is not an epiphenomenon but rather promotes tumor aggressiveness.

Dependence of Invadopodia Function on Collagen Fiber Spacing and Cross-Linking: Computational Modeling and Experimental Evidence

Invadopodia are subcellular organelles thought to be critical for extracellular matrix (ECM) degradation and the movement of cells through tissues. Here we examine invadopodia generation, turnover, and function in relation to two structural aspects of the ECM substrates they degrade: cross-linking and fiber density. We set up a cellular automaton computational model that simulates ECM penetration and degradation by invadopodia. Experiments with denatured collagen (gelatin) were used to calibrate the model and demonstrate the inhibitory effect of ECM cross-linking on invadopodia degradation and penetration. Incorporation of dynamic invadopodia behavior into the model amplified the effect of cross-linking on ECM degradation, and was used to model feedback from the ECM. When the model was parameterized with spatial fibrillar dimensions that closely matched the organization, in real life, of native ECM collagen into triple-helical monomers, microfibrils, and macrofibrils, little or no inhibition of invadopodia penetration was observed in simulations of sparse collagen gels, no matter how high the degree of cross-linking. Experimental validation, using live-cell imaging of invadopodia in cells plated on cross-linked gelatin, was consistent with simulations in which ECM cross-linking led to higher rates of both invadopodia retraction and formation. Analyses of invadopodia function from cells plated on cross-linked gelatin and collagen gels under standard concentrations were consistent with simulation results in which sparse collagen gels provided a weak barrier to invadopodia. These results suggest that the organization of collagen, as it may occur in stroma or in vitro collagen gels, forms gaps large enough so as to have little impact on invadopodia penetration/degradation. By contrast, dense ECM, such as gelatin or possibly basement membranes, is an effective obstacle to invadopodia penetration and degradation, particularly when cross-linked. These results provide a novel framework for further studies on ECM structure and modifications that affect invadopodia and tissue invasion by cells.

Mammary-tumor-educated B cells acquire LAP/TGF-β and PD-L1 expression and suppress anti-tumor immune responses

B lymphocytes play a role in inhibiting the immune response against certain tumors, but the underlying mechanisms are poorly understood. EMT-6 mammary tumors grow well in wild-type (WT) mice but show reduced growth in B-cell-deficient μ(-/-) BALB/c mice (BCDM). WT mice demonstrate extensive B-cell infiltration into the tumor bed, reduced CD8(+) T cell and CD49(+) NK cell infiltration, and markedly reduced cytolytic T-cell response relative to BCDM. Expression of LAP/TGF-β1, CD80, CD86 and PD-L1 is significantly increased in tumor-infiltrating B cells (TIL-B) relative to splenic B cells. LAP/TGF-β1 expression on TIL-B progressively increased from 5.4±1.7% on day 8 to 43.1±6.1% by day 21 post tumor implantation. Co-culture of EMT-6 tumor cells with Naive-B cells ex vivo generated B cells (EMT6-B) with a similar immunophenotype to TIL-B. Purified TIL-B, or in-vitro-generated EMT6-B suppressed CD4(+), CD8(+) and CD4(+)CD25(-) T-cell proliferation, and Th1 cytokine secretion, and also suppressed purified NK-cell proliferation in response to IL-15, compared to naive splenic B cells. Acquired B regulatory function required direct tumor cell: B-cell contact, and was partially reversed by antibody to TGF-β or PD-L1, leading to tumor rejection in vivo B-cell acquisition of a suppressive phenotype following tumor infiltration may result in profound inhibition of T-cell anti-tumor responses.

Regulation of late endosomal/lysosomal maturation and trafficking by cortactin affects Golgi morphology

Cortactin is a branched actin regulator and tumor‐overexpressed protein that promotes vesicular trafficking at a variety of cellular sites, including endosomes and the trans‐Golgi network. To better understand its role in secretory trafficking, we investigated its function in Golgi homeostasis. Here, we report that knockdown (KD) of cortactin leads to a dramatic change in Golgi morphology by light microscopy, dependent on binding the Arp2/3 actin‐nucleating complex. Surprisingly, there was little effect of cortactin‐KD on anterograde trafficking of the constitutive cargo vesicular stomatitis virus glycoprotein (VSVG), Golgi assembly from endoplasmic reticulum membranes upon Brefeldin A washout, or Golgi ultrastructure. Instead, electron microscopy studies revealed that cortactin‐KD cells contained a large number of immature‐appearing late endosomal/lysosomal (LE/Lys) hybrid organelles, similar to those found in lysosomal storage diseases. Consistent with a defect in LE/Lys trafficking, cortactin‐KD cells also exhibited accumulation of free cholesterol and retention of the retrograde Golgi cargo mannose‐6‐phosphate receptor in LE. Inhibition of LE maturation by treatment of control cells with Rab7 siRNA or chloroquine led to a compact Golgi morphology similar to that observed in cortactin‐KD cells. Furthermore, the Golgi morphology defects of cortactin‐KD cells could be rescued by removal of cholesterol‐containing lipids from the media, suggesting that buildup of cholesterol‐rich membranes in immature LE/Lys induced disturbances in retrograde trafficking. Taken together, these data reveal that LE/Lys maturation and trafficking are highly sensitive to cortactin‐regulated branched actin assembly and suggests that cytoskeletal‐induced Golgi morphology changes can be a consequence of altered trafficking at late endosomes.

Absence of mature peripheral B cell populations in mice with concomitant defects in B cell receptor and BAFF-R signaling.

Generation of mature B lymphocytes from early (T1) and late transitional (T2) precursors requires cooperative signaling through BCR and B cell-activating factor receptor 3 (BR3). Recent studies have shown that BCR signaling positively regulates NF-κB2, suggesting BCR regulation of BR3 signaling. To investigate the significance of signal integration from BCR and BR3 in B cell development and function, we crossed Btk-deficient mice (btk−/−), which are developmentally blocked between the T2 and the mature follicular B cell stage as a result of a partial defect in BCR signaling, and A/WySnJ mice, which possess a mutant BR3 defective in propagating intracellular signals that results in a severely reduced peripheral B cell compartment, although all B cell subsets are present in relatively normal ratios. A/WySnJ × btk−/− mice display a B cell-autonomous defect, resulting in a developmental block at an earlier stage (T1) than either mutation alone, leading to the loss of mature splenic follicular and marginal zone B cells, as well as the loss of peritoneal B1 and B2 cell populations. The competence of the double mutant T1 B cells to respond to TLR4 and CD40 survival and activation signals is further attenuated compared with single mutations as evidenced by severely reduced humoral immune responses in vivo and proliferation in response to anti-IgM, LPS, and anti-CD40 stimulation in vitro. Thus, BCR and BR3 independently and in concert regulate the survival, differentiation, and function of all B cell populations at and beyond T1, earliest transitional stage.

DNA vaccine molecular adjuvants SP-D-BAFF and SP-D-APRIL enhance anti-gp120 immune response and increase HIV-1 neutralizing antibody titers

ABSTRACT Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating the production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating the molecular adjuvants BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand) with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multitrimers of BAFF and APRIL using surfactant protein D as a scaffold, and we vaccinated mice with these molecular adjuvants using DNA and DNA-protein vaccination strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multitrimers, together with interleukin 12 (IL-12) and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against tier 1 and tier 2 (vaccine strain) viruses. The APRIL-containing vaccine was particularly effective at generating tier 2 neutralizing antibodies following a protein boost. These BAFF and APRIL effects coincided with an enhanced germinal center (GC) reaction, increased anti-gp120 antibody-secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multitrimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1. IMPORTANCE Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, the multiple immune evasion properties of HIV have hampered these efforts. These include the instability of the gp120 trimer, the inaccessibility of the conserved sequences, highly variable protein sequences, and the loss of HIV-1-specific antibody-producing cells during development. We have shown previously that tumor necrosis factor (TNF) superfamily ligands, including BAFF and APRIL, can be multitrimerized using the lung protein SP-D (surfactant protein D), enhancing immune responses. Here we show that DNA or DNA-protein vaccines encoding BAFF or APRIL multitrimers, IL-12p70, and membrane-bound HIV-1 Env gp140 induced tier 1 and tier 2 neutralizing antibodies in a mouse model. BAFF and APRIL enhanced the immune reaction, improved antibody binding, and increased the numbers of anti-HIV-1 antibody-secreting cells. Adaptation of this vaccine design may prove useful in designing preventive HIV-1 vaccines for humans.