Mammalian CAP (Cyclase-associated protein) in the world of cell migration

Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.     

Mammalian CAP (Cyclase-associated protein) in the world of cell migration: Roles in actin filament dynamics and beyond

Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.     

Rho-associated coiled-coil kinase (ROCK) signaling and disease

Abstract

The small Rho GTPase family of proteins, encompassing the three major G-protein classes Rho, Rac and cell division control protein 42, are key mitogenic signaling molecules that regulate multiple cancer-associated cellular phenotypes including cell proliferation and motility. These proteins are known for their role in the regulation of actin cytoskeletal dynamics, which is achieved through modulating the activity of their downstream effector molecules. The Rho-associated coiled-coil kinase 1 and 2 (ROCK1 and ROCK2) proteins were the first discovered Rho effectors that were primarily established as players in RhoA-mediated stress fiber formation and focal adhesion assembly. It has since been discovered that the ROCK kinases actively phosphorylate a large cohort of actin-binding proteins and intermediate filament proteins to modulate their functions. It is well established that global cellular morphology, as modulated by the three cytoskeletal networks: actin filaments, intermediate filaments and microtubules, is regulated by a variety of accessory proteins whose activities are dependent on their phosphorylation by the Rho-kinases. As a consequence, they regulate many key cellular functions associated with malignancy, including cell proliferation, motility and viability. In this current review, we focus on the role of the ROCK-signaling pathways in disease including cancer.     

METCAM/MUC18 augments migration, invasion, and tumorigenicity of human breast cancer SK-BR-3 cells

Previous research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family, as a promoter or a suppressor in the development of human breast cancer by MCF7, MDA-MB-231, and MDA-MB-468. To resolve these conflicting results we have investigated the role of this CAM in the progression of the three aforementioned cell lines plus one additional human breast cancer cell line, SK-BR-3. We transfected the SK-BR-3 cells with human METCAM/MUC18 cDNA to obtain G418-resistant clones, which expressed different levels of the protein and which were used to test the effect of human METCAM/MUC18 expression on in vitro motility, invasiveness, anchorage-independent colony formation in soft agar, disorganized growth in a 3D basement membrane culture assay, and in vivo tumorigenesis in athymic nude mice. Enforced METCAM/MUC18 expression increased in vitro motility, invasiveness, and anchorage-independent colony formation of SK-BR-3 cells and favored disorganized growth of the cells in 3D basement membrane culture. Enforced expression also increased tumorigenicity and final tumor weights of SK-BR-3 clones/cells after subcutaneous injection of the cells under the left third nipple of female athymic nude mice. To understand the mechanisms, we also determined the expression of several downstream key effectors in the tumors. Tumor cells from METCAM/MUC18 expressing clones exhibited elevated expression of an anti-apoptotic and survival index (Bcl2), an aerobic glycolysis index (LDH-A), and pro-angiogenesis indexes (VEGF and VAGFR2). We concluded that human METCAM/MUC18 promotes the development of breast cancer cells by increasing an anti-apoptosis and survival pathway and augmenting aerobic glycolysis and angiogenesis.     

Focal adhesion kinase stabilizes the cytoskeleton

Focal adhesion kinase (FAK) is a central focal adhesion protein that promotes focal adhesion turnover, but the role of FAK for cell mechanical stability is unknown. We measured the mechanical properties of wild-type (FAKwt), FAK-deficient (FAK−/−), FAK-silenced (siFAK), and siControl mouse embryonic fibroblasts by magnetic tweezer, atomic force microscopy, traction microscopy, and nanoscale particle tracking microrheology. FAK-deficient cells showed lower cell stiffness, reduced adhesion strength, and increased cytoskeletal dynamics compared to wild-type cells. These observations imply a reduced stability of the cytoskeleton in FAK-deficient cells. We attribute the reduced cytoskeletal stability to rho-kinase activation in FAK-deficient cells that suppresses the formation of ordered stress fiber bundles, enhances cortical actin distribution, and reduces cell spreading. In agreement with this interpretation is that cell stiffness and cytoskeletal stability in FAK−/− cells is partially restored to wild-type level after rho-kinase inhibition with Y27632.     

Breast Cancer Antiestrogen Resistance 3 (BCAR3) Promotes Cell Motility by Regulating Actin Cytoskeletal and Adhesion Remodeling in Invasive Breast Cancer Cells

Metastatic breast cancer is incurable. In order to improve patient survival, it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility. Here, we focus on the role of the adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) in cellular processes that contribute to cell motility, including protrusion, adhesion remodeling, and contractility. Previous work from our group showed that elevated BCAR3 protein levels enhance cell migration, while depletion of BCAR3 reduces the migratory and invasive capacities of breast cancer cells. In the current study, we show that BCAR3 is necessary for membrane protrusiveness, Rac1 activity, and adhesion disassembly in invasive breast cancer cells. We further demonstrate that, in the absence of BCAR3, RhoA-dependent signaling pathways appear to predominate, as evidenced by an increase in RhoA activity, ROCK-mediated phosphorylation of myosin light chain II, and large ROCK/mDia1-dependent focal adhesions. Taken together, these data establish that BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells through its ability to influence the balance between Rac1 and RhoA signaling. Considering that BCAR3 protein levels are elevated in advanced breast cancer cell lines and enhance breast cancer cell motility, we propose that BCAR3 functions in the transition to advanced disease by triggering intracellular signaling events that are essential to the metastatic process.     

Intracellular Theileria annulata Promote Invasive Cell Motility through Kinase Regulation of the Host Actin Cytoskeleton

The intracellular, protozoan Theileria species parasites are the only eukaryotes known to transform another eukaryotic cell. One consequence of this parasite-dependent transformation is the acquisition of motile and invasive properties of parasitized cells in vitro and their metastatic dissemination in the animal, which causes East Coast Fever (T. parva) or Tropical Theileriosis (T. annulata). These motile and invasive properties of infected host cells are enabled by parasite-dependent, poorly understood F-actin dynamics that control host cell membrane protrusions. Herein, we dissected functional and structural alterations that cause acquired motility and invasiveness of T. annulata-infected cells, to understand the molecular basis driving cell dissemination in Tropical Theileriosis. We found that chronic induction of TNFα by the parasite contributes to motility and invasiveness of parasitized host cells. We show that TNFα does so by specifically targeting expression and function of the host proto-oncogenic ser/thr kinase MAP4K4. Blocking either TNFα secretion or MAP4K4 expression dampens the formation of polar, F-actin-rich invasion structures and impairs cell motility in 3D. We identified the F-actin binding ERM family proteins as MAP4K4 downstream effectors in this process because TNFα-induced ERM activation and cell invasiveness are sensitive to MAP4K4 depletion. MAP4K4 expression in infected cells is induced by TNFα-JNK signalling and maintained by the inhibition of translational repression, whereby both effects are parasite dependent. Thus, parasite-induced TNFα promotes invasive motility of infected cells through the activation of MAP4K4, an evolutionary conserved kinase that controls cytoskeleton dynamics and cell motility. Hence, MAP4K4 couples inflammatory signaling to morphodynamic processes and cell motility, a process exploited by the intracellular Theileria parasite to increase its host cell''s dissemination capabilities.     

MicroRNA-10b Promotes Migration and Invasion through KLF4 in Human Esophageal Cancer Cell Lines

Recently, microRNAs have emerged as regulators of cancer metastasis through acting on multiple signaling pathways involved in metastasis. In this study, we have analyzed the level of miR-10b and cell motility and invasiveness in several human esophageal squamous cell carcinoma cell lines. Our results reveal a significant correlation of miR-10b level with cell motility and invasiveness. Overexpression of miR-10b in KYSE140 cells increased cell motility and invasiveness, whereas inhibition of miR-10b in EC9706 cells reduced cell invasiveness, although it did not alter cell motility. Additionally, we identified KLF4, a known tumor suppressor gene that has been reported to suppress esophageal cancer cell migration and invasion, as a direct target of miR-10b. Furthermore, overexpression of miR-10b in KYSE140 and KYSE450 cells led to a reduction of endogenous KLF4 protein, whereas silencing of miR-10b in EC9706 cells caused up-regulation of KLF4 protein. Coexpression of miR-10b and KLF4 in KYSE140 cells and coexpression of small interfering RNA for KLF4 mRNA and miR-10b-AS in EC9706 cells partially abrogated the effect of miR-10b on cell migration and invasion. Finally, analyses of the miR-10b level in 40 human esophageal cancer samples and their paired normal adjacent tissues revealed an elevated expression of miR-10b in 95% (38 of 40) of cancer tissues, although no significant correlation of the miR-10b level with clinical metastasis status was observed in these samples.     

Potential antitumor activity of digitoxin and user-designed analog administered to human lung cancer cells

BackgroundCardiac glycosides (CGs), such as digitoxin, are traditionally used for treatment of congestive heart failure; recently they also gained attention for their anticancer properties. Previous studies showed that digitoxin and a synthetic L-sugar monosaccharide analog treatment decreases cancer cell proliferation, increases apoptosis, and pro-adhesion abilities; however, no reports are available on their potential to alter lung cancer cell cytoskeleton structure and reduce migratory ability. Herein, we investigated the anticancer effects of digitoxin and its analog, digitoxigenin-α-L-rhamnoside (D6MA), to establish whether cytoskeleton reorganization and reduced motility are drug-induced cellular outcomes.MethodsWe treated non-small cell lung carcinoma cells (NSCLCs) with sub-therapeutic, therapeutic, and toxic concentrations of digitoxin and D6MA respectively, followed by both single point and real-time assays to evaluate changes in cellular gene and protein expression, adhesion, elasticity, and migration.ResultsDigitoxin and D6MA induced a decrease in matrix metalloproteinases expression via altered focal adhesion signaling and a suppression of the phosphoinositide 3-kinases / protein kinase B pathway which lead to enhanced adhesion, altered elasticity, and reduced motility of NSCLCs. Global gene expression analysis identified dose-dependent changes to nuclear factor kappa-light-chain-enhancer, epithelial tumor, and microtubule dynamics signaling.ConclusionsOur study demonstrates that digitoxin and D6MA can target antitumor signaling pathways to alter NSCLC cytoskeleton and migratory ability to thus potentially reduce their tumorigenicity.SignificanceDiscovering signaling pathways that control cancer's cell phenotype and how such pathways are affected by CG treatment will potentially allow for active usage of synthetic CG analogs as therapeutic agents in advanced lung conditions.     

P21-activated kinase 4 overexpression in metastatic gastric cancer patients

INTRODUCTION: P21-activated kinase 4 (PAK), a subfamily of serine/threonine kinases originally known as a regulator of cytoskeletal dynamics and cell motility, has recently been revealed to play a key role in oncogenic signaling pathways. We studied the frequency and clinical features of PAK4-overexpressed metastatic gastric cancer. PATIENTS AND METHODS: PAK4 overexpression was screened by Western blot in 18 human gastric cancer cell lines. Immunohistochemical staining of PAK4 protein was performed in tumor specimens of 49 metastatic gastric cancer patients who received palliative capecitabine/cisplatin as first-line treatment. RESULTS: PAK4 protein overexpression was detected strongly in five gastric cell lines (AGS, MGK-28, MKN-74, SNU-216, SNU-601) and weakly in four cell lines (KATOIII, MKN-1, SNU-620, and SNU-719). PAK4 knockdown by small interfering RNA induced apoptosis in PAK4-overexpressed AGS gastric cancer cells. Immunohistochemical staining revealed PAK4 overexpressions in 4 (8.1%) of 49 metastatic gastric cancer specimens. None of the four patients with PAK4(+) responded to capecitabine/cisplatin chemotherapy, and PAK4(+) gastric cancer patients had a trend of poorer survival compared with PAK(-)(P = .876). CONCLUSIONS: We demonstrated PAK4 overexpression in a subset of gastric cancer patients, implicating a role in gastric cancer tumorigenesis. Its prognostic significance and efficacy as a drug target should be further studied.     

Activated protein C enhances cell motility of endothelial cells and MDA-MB-231 breast cancer cells by intracellular signal transduction

Activated protein C (APC), an anticoagulant serine protease, has been shown to have non-hemostatic functions related to inflammation, cell survival, and cell migration. In this study we investigate the mechanism by which APC promotes angiogenesis and breast cancer invasion using ex vivo and in vitro methods. When proteolytically active, APC promotes cell motility/invasion and tube formation of endothelial cells. Ex vivo aortic ring assays verify the role of APC in promoting angiogenesis, which was determined to be dependent on EGFR and MMP activation. Given the capacity of APC to promote angiogenesis and the importance of this process in cancer pathology, we investigated whether the mechanisms by which APC promotes angiogenesis can also promote motility and invasion in the MDA-MB-231 breast cancer cell line. Our results indicate that, extracellularly, APC engages EPCR, PAR-1, and EGFR in order to increase the invasiveness of MDA-MB-231 cells. APC activation of matrix metalloprotease (MMP) -2 and/or -9 is necessary but not sufficient to increase invasion, and APC does not utilize the endogenous plasminogen activation system to increase invasion. Intracellularly, APC activates ERK, Akt, and NFκB, but not the JNK pathway to promote MDA-MB-231 cell motility. Similar to the hemostatic protease thrombin, APC has the ability to enhance both endothelial cell motility/angiogenesis and breast cancer cell migration.     

Cytoskeletal organization and cell motility correlates with metastatic potential and state of differentiation in prostate cancer.

The actin cytoskeleton is the key cellular machinery responsible for cellular movement. Changes in the organization and distribution of actin and actin binding protein are necessary for several cellular processes such as focal adhesion formation, cell motility and cell invasion. Here we examined differences in cytoskeletal protein distribution, cell morphometry and cell motility of metastatic and non-metastatic cells. Correlations were found between metastatic potential phenotypic properties such as cell motility, cell spreading and cytoskeletal organization in prostate cancer. As a cell progresses from a normal state to a malignant state, it loses its ability to function normally and also become poorly differentiated. Differentiation therapy is concerned with the redirection of malignant cells toward a terminal, non-dividing state using non-cytotoxic agents. Two well acknowledged differentiation agents, retinoic acid (RA) and diflouromethylomithine (DFMO) were examined for their ability to alter cellular phenotypes associated with metastatic potential in rat prostate cancer cell lines. The results of these studies indicate that there are sub-cellular differences between non-metastatic and highly metastatic cells relative to cytoskeletal organization. We also show that treatment of highly metastatic cells with either RA or DFMO significantly alters cell morphology, cell morphometry and motility to states similar to non-metastatic cells.     

miR-204 Targeting of Ankrd13A Controls Both Mesenchymal Neural Crest and Lens Cell Migration

Loss of cell adhesion and enhancement of cell motility contribute to epithelial-to-mesenchymal transition during development. These processes are related to a) rearrangement of cell-cell and cell-substrate adhesion molecules; b) cross talk between extra-cellular matrix and internal cytoskeleton through focal adhesion molecules. Focal adhesions are stringently regulated transient structures implicated in cell adhesion, spreading and motility during tissue development. Importantly, despite the extensive elucidation of the molecular composition of focal adhesions, the complex regulation of their dynamics is largely unclear. Here, we demonstrate, using live-imaging in medaka, that the microRNA miR-204 promotes both mesenchymal neural crest and lens cell migration and elongation. Overexpression of miR-204 results in upregulated cell motility, while morpholino-mediated ablation of miR-204 activity causes abnormal lens morphogenesis and neural crest cell mislocalization. Using a variety of in vivo and in vitro approaches, we demonstrate that these actions are mediated by the direct targeting of the Ankrd13A gene, which in turn controls focal cell adhesion formation and distribution. Significantly, in vivo restoration of abnormally elevated levels of Ankrd13A resulting from miR-204 inactivation rescued the aberrant lens phenotype in medaka fish. These data uncover, for the first time in vivo, the role of a microRNA in developmental control of mesenchymal cell migration and highlight miR-204 as a “master regulator” of the molecular networks that regulate lens morphogenesis in vertebrates.     

Identification of a Dithiol-disulfide Switch in Collapsin Response Mediator Protein 2 (CRMP2) That Is Toggled in a Model of Neuronal Differentiation

Vertebrate-specific glutaredoxin 2 (Grx2) is expressed in at least two isoforms, mitochondrial Grx2a and cytosolic Grx2c. We have previously shown that cytosolic Grx2 is essential for embryonic development of the brain. In particular, we identified collapsin response mediator protein 2 (CRMP2/DPYSL2), a mediator of the semaphorin-plexin signaling pathway, as redox-regulated target of Grx2c and demonstrated that this regulation is required for normal axonal outgrowth. In this study, we demonstrate the molecular mechanism of this regulation, a specific and reversible intermolecular Cys-504-Cys-504 dithiol-disulfide switch in homotetrameric CRMP2. This switch determines two conformations of the quaternary CRMP2 complex that controls axonal outgrowth and thus neuronal development.     

Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.     

单核细胞增多性李斯特菌谷胱甘肽还原酶GR的生物学特性

[目的]本文旨在构建单核细胞增多性李斯特菌谷胱甘肽还原酶(glutathione reductase,GR)基因lmo1433(gr)缺失株,并研究GR在细菌生长和运动过程中发挥的作用及与谷氧还蛋白(glutaredoxin,Grx)系统间的调控关系,探究GR参与细菌抗氧化应激和致病力的生物学功能,为阐明氧化还原蛋白介...     

Breakdown of the endothelial barrier function in tumor cell transmigration

The ability of tumor cells to metastasize is associated with a poor prognosis for cancer. During the process of metastasis, tumor cells circulating in the blood or lymph vessels can adhere to, and potentially transmigrate through, the endothelium and invade the connective tissue. We studied the effectiveness of the endothelium as a barrier against the invasion of 51 tumor cell lines into a three-dimensional collagen matrix. Only nine tumor cell lines showed attenuated invasion in the presence of an endothelial cell monolayer, whereas 17 cell lines became invasive or showed a significantly increased invasion. Endothelial cells cocultured with invasive tumor cells increased chemokine gene expression of IL-8 and Gro-β. Expression of the IL-8 and Gro-β receptor, CXCR2, was upregulated in invasive tumor cells. Addition of IL-8 or Gro-β increased tumor cell invasiveness by more than twofold. Tumor cell variants selected for high CXCR2 expression were fourfold more invasive in the presence of an endothelial cell layer, whereas CXCR2 siRNA knock-down cells were fivefold less invasive. We demonstrate that Gro-β and IL-8 secreted by endothelial cells, together with CXCR2 receptor expression on invasive tumor cells, contribute to the breakdown of the endothelial barrier by enhancing tumor cell force generation and cytoskeletal remodeling dynamics.