Impaired Cell Motility Research Articles

Suppression of mitochondrial respiration with auraptene inhibits the progression of renal cell carcinoma: involvement of HIF-1α degradation.

Renal cell carcinoma (RCC) progression resulting from the uncontrolled migration and enhanced angiogenesis is an obstacle to effective therapeutic intervention. Tumor metabolism has distinctive feature called Warburg effect, which enhances the aerobic glycolysis rapidly supplying the energy for migration of tumor. To manipulate this metabolic change characteristic of aggressive tumors, we utilized the citrus extract, auraptene, known as a mitochondrial inhibitor, testing its anticancer effects against the RCC4 cell line. We found that auraptene impaired RCC4 cell motility through reduction of mitochondrial respiration and glycolytic pathway-related genes. It also strongly disrupted VEGF-induced angiogenesis in vitro and in vivo. Hypoxia-inducible factor 1a (HIF-1a), a key regulator of cancer metabolism, migration and angiogenesis that is stably expressed in RCCs by virtue of a genetic mutation in the von Hippel-Lindau (VHL) tumor-suppressor protein, was impeded by auraptene, which blocked HIF-1a translation initiation without causing cytotoxicity. We suggest that blockade HIF-1a and reforming energy metabolism with auraptene is an effective approach for suspension RCC progression.

Identification and characterization of a HEPN-MNT family type II toxin-antitoxin in Shewanella oneidensis.

Toxin–antitoxin (TA) systems are prevalent in bacteria and archaea. However, related studies in the ecologically and bioelectrochemically important strain Shewanella oneidensis are limited. Here, we show that SO_3166, a member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) superfamily, strongly inhibited cell growth in S. oneidensis and Escherichia coli. SO_3165, a putative minimal nucleotidyltransferase (MNT), neutralized the toxicity of SO_3166. Gene SO_3165 lies upstream of SO_3166, and they are co-transcribed. Moreover, the SO_3165 and SO_3166 proteins interact with each other directly in vivo, and antitoxin SO_3165 bound to the promoter of the TA operon and repressed its activity. Finally, the conserved Rx4-6H domain in HEPN family was identified in SO_3166. Mutating either the R or H abolished SO_3166 toxicity, confirming that Rx4-6H domain is critical for SO_3166 activity. Taken together, these results demonstrate that SO_3166 and SO_3165 in S. oneidensis form a typical type II TA pair. This TA pair plays a critical role in regulating bacterial functions because its disruption led to impaired cell motility in S. oneidensis. Thus, we demonstrated for the first time that HEPN-MNT can function as a TA system, thereby providing important insights into the understanding of the function and regulation of HEPNs and MNTs in prokaryotes.

Arachidonic acid promotes skin wound healing through induction of human MSC migration by MT3-MMP-mediated fibronectin degradation.

Arachidonic acid (AA) is largely released during injury, but it has not been fully studied yet how AA modulates wound repair with stem cells. Therefore, we investigated skin wound-healing effect of AA-stimulated human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in vivo and its molecular mechanism in vitro. We found that transplantation of hUCB-MSCs pre-treated with AA enhanced wound filling, re-epithelization, and angiogenesis in a mouse skin excisional wound model. AA significantly promoted hUCB-MSCs migration after a 24 h incubation, which was inhibited by the knockdown of G-protein-coupled receptor 40 (GPR40). AA activated mammalian target of rapamycin complex 2 (mTORC2) and Aktser473 through the GPR40/phosphoinositide 3-kinase (PI3K) signaling, which was responsible for the stimulation of an atypical protein kinase C (PKC) isoform, PKCζ. Subsequently, AA stimulated phosphorylation of p38 MAPK and transcription factor Sp1, and induced membrane type 3-matrix metalloproteinase (MT3-MMP)-dependent fibronectin degradation in promoting hUCB-MSCs motility. Finally, the silencing of MT3-MMP in AA-stimulated hUCB-MSCs failed to promote the repair of skin wounds owing to impaired cell motility. In conclusion, AA enhances skin wound healing through induction of hUCB-MSCs motility by MT3-MMP-mediated fibronectin degradation, which relies on GPR40-dependent mTORC2 signaling pathways.

CIP4 promotes metastasis in triple-negative breast cancer and is associated with poor patient prognosis.

Signaling via epidermal growth factor receptor (EGFR) and Src kinase pathways promote triple-negative breast cancer (TNBC) cell invasion and tumor metastasis. Here, we address the role of Cdc42-interacting protein-4 (CIP4) in TNBC metastasis in vivo, and profile CIP4 expression in human breast cancer patients. In human TNBC cells, CIP4 knock-down (KD) led to less sustained activation of Erk kinase and impaired cell motility compared to control cells. This correlated with significant defects in 3D invasion of surrounding extracellular matrix by CIP4 KD TNBC cells when grown as spheroid colonies. In mammary orthotopic xenograft assays using both human TNBC cells (MDA-MB-231, HCC 1806) and rat MTLn3 cells, CIP4 silencing had no overt effect on tumor growth, but significantly reduced the incidence of lung metastases in each tumor model. In human invasive breast cancers, high CIP4 levels was significantly associated with high tumor stage, TNBC and HER2 subtypes, and risk of progression to metastatic disease. Together, these results implicate CIP4 in promoting metastasis in TNBCs.

Specific dephosphorylation at tyr-554 of git1 by ptprz promotes its association with paxillin and hic-5.

G protein-coupled receptor kinase-interactor 1 (Git1) is involved in cell motility control by serving as an adaptor that links signaling proteins such as Pix and PAK to focal adhesion proteins. We previously demonstrated that Git1 was a multiply tyrosine-phosphorylated protein, its primary phosphorylation site was Tyr-554 in the vicinity of the focal adhesion targeting-homology (FAH) domain, and this site was selectively dephosphorylated by protein tyrosine phosphatase receptor type Z (Ptprz). In the present study, we showed that Tyr-554 phosphorylation reduced the association of Git1 with the FAH-domain-binding proteins, paxillin and Hic-5, based on immunoprecipitation experiments using the Tyr-554 mutants of Git1. The Tyr-554 phosphorylation of Git1 was higher, and its binding to paxillin was consistently lower in the brains of Ptprz-deficient mice than in those of wild-type mice. We then investigated the role of Tyr-554 phosphorylation in cell motility control using three different methods: random cell motility, wound healing, and Boyden chamber assays. The shRNA-mediated knockdown of endogenous Git1 impaired cell motility in A7r5 smooth muscle cells. The motility defect was rescued by the exogenous expression of wild-type Git1 and a Git1 mutant, which only retained Tyr-554 among the multiple potential tyrosine phosphorylation sites, but not by the Tyr-554 phosphorylation-defective or phosphorylation-state mimic Git1 mutant. Our results suggested that cyclic phosphorylation-dephosphorylation at Tyr-554 of Git1 was crucial for dynamic interactions between Git1 and paxillin/Hic-5 in order to ensure coordinated cell motility.

The prognostic significance of RUNX2 and miR-10a/10b and their inter-relationship in breast cancer.

BackgroundThe major cancer related mortality is caused by metastasis and invasion. It is important to identify genes regulating metastasis and invasion in order to curtail metastatic spread of cancer cells.MethodsThis study investigated the association between RUNX2 and miR-10a/miR-10b and the risk of breast cancer relapse. Expression levels of RUNX2 and miR-10a/b in108 pairs of tumor and non-tumor tissue of breast cancer were assayed by quantitative PCR analysis and evaluated for their prognostic implications.ResultsThe median expression levels of RUNX2 and miR-10b in tumor tissue normalized using adjacent non-tumor tissue were significantly higher in relapsed patients than in relapse-free patients. Higher expression of these three genes were significantly correlated with the hazard ratio for breast cancer recurrence (RUNX2: 3.02, 95% CI = 1.50 ~ 6.07; miR-10a: 2.31, 95% CI = 1.00 ~ 5.32; miR-10b: 3.96, 95% CI = 1.21 ~ 12.98). The joint effect of higher expression of all three genes was associated with a hazard ratio of 12.37 (95% CI = 1.62 ~ 94.55) for relapse. In a breast cancer cell line, RUNX2 silencing reduced the expression of miR-10a/b and also impaired cell motility, while RUNX2 overexpression elicited opposite effects.ConclusionsThese findings indicate that higher expression of RUNX2 and miR-10a/b was associated with adverse outcome of breast cancer. Expression levels of RUNX2 and miR-10a/b individually or jointly are potential prognostic factors for predicting breast cancer recurrence. Data from in vitro studies support the notion that RUNX2 promoted cell motility by upregulating miR-10a/b.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0257-3) contains supplementary material, which is available to authorized users.

Abstract 1145: Cytoplasmic p27 promotes epithelial-mesenchymal transition and tumor progression via Twist1 upregulation

Abstract p27 is a cell cycle regulator that acts largely to restrain normal cell growth. It is rarely mutated or deleted in human cancers, but is often degraded or mislocalized to the cytoplasm in aggressive tumors. Phosphorylation at T157 or T198 by different PI3K effector kinases leads to p27 cytoplasmic accumulation and enhanced cell motility and invasion in part via effects on the actin cytoskeleton, which are independent of its cell cycle role. However, the functional contributions of C-terminally phosphorylated, cytoplasmic p27 to cancer progression remain poorly understood. Targeted inhibition of PI3K/mTOR impaired tumor cell motility and metastasis via modulation of p27 in a bone metastatic model (Wander, S. et al. BCRT 2013). We observed that p27 knockdown in highly metastatic cancer lines with high levels of cytoplasmic p27pT157pT198 reverted EMT and impaired tumor cell invasion in vitro and metastasis in vivo. This led us to investigate whether PI3K-activated, phosphorylated p27 may function as a novel EMT regulator. A cell cycle defective (CK-) and double phosphomimetic p27 mutant (T157D/T198D or DD) was introduced into immortal human mammary epithelial cells with low PI3K activity. This revealed a novel, oncogenic function of p27 in regulating tumor progression, in that p27CK-DD induced epithelial-mesenchymal transition (EMT) and soft agar colony formation. p27CK-DD also increased motility, invasion and formation of metastasis in bladder and breast cancer models. A RT-PCR screen for potential p27-induced EMT mediators revealed a 20-fold increase in expression of the Twist1 transcription factor in p27CK-DD-transduced cells. Knockdown of Twist reversed the p27CK-DD-mediated increase in EMT marker expression. p27 knockdown rapidly attenuated Twist1-promoter activity and reduced Twist1 mRNA levels and also increased E-cadherin expression in metastatic cells, suggesting that Twist1 may play a critical role in p27CK-DD-induced EMT. These data extend our understanding of p27 function in human cancer and suggest that PI3K deregulated p27 may promote EMT and tumor metastasis through transcriptional activation of TWIST1. Citation Format: Dekuang Zhao, Alexandra H. Besser, Wen Zhou, Seth A. Wander, Jun Sun, Michael Durante, Feng Hong, Bin Wang, Tan Ince, Karoline Briegel, Joyce M. Slingerland. Cytoplasmic p27 promotes epithelial-mesenchymal transition and tumor progression via Twist1 upregulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1145. doi:10.1158/1538-7445.AM2014-1145

Wnt5a Suppresses Colon Cancer by Inhibiting Cell Proliferation and Epithelial–Mesenchymal Transition

Colon cancer remains one of the lethal malignancies in the world. Aberrant activation of canonical Wnt/β-catenin signaling pathway has been observed in colon cancer. In contrast, the non-canonical Wnt signaling functions remain obscure. Wnt5a is a representative non-canonical Wnt ligand which has gained extensive attention nowadays. Wnt5a has been shown to play an important role in EMT in prostate cancer and melanoma, but its role in colon cancer is still ambiguous. Here we have evaluated Wnt5a expression in a large cohort of 217 colon cancers by immunohistochemistry and analyzed its correlation with clinicopathologic characteristics. We found that expression of Wnt5a was diminished significantly in majority of primary colon cancers and negatively related with EMT biomarkers. To further enlighten the mechanism which Wnt5a regulates EMT in vitro, we established ectopic Wnt5a expression models. Protein analysis demonstrated that Wnt5a inhibited EMT and antagonized canonical Wnt signaling in colon cancer cells. Overexpression of Wnt5a impaired cell motility and invasion and inhibited cell proliferation by manipulating Bax. Moreover, Wnt5a suppressed the tumor growth in nude mice and impaired tumorigenicity in vivo. Wnt5a also induced intracellular calcium and activated non-canonical Wnt/Ca(2+) signaling in colon cancer. In summary, although Wnt5a was down-regulated in majority of colon cancers, enhanced Wnt5a expression predict preferable outcome in colon cancer patients. Our findings indicate that Wnt5a might act as tumor suppressor by inhibiting cell proliferation and attenuating EMT in colon cancer cells. Wnt5a could be used as a novel prognostic marker and/or therapeutic target for colon cancer in the future.

Binding of the extreme carboxyl-terminus of PAK-interacting exchange factor β (βPIX) to myosin 18A (MYO18A) is required for epithelial cell migration

The PAK2/βPIX/GIT1 (p21-activated kinase 2/PAK-interacting exchange factor-β/G protein-coupled receptor kinase-interactor 1) complex has been shown to distribute to both membrane ruffles and focal adhesions of cells, where it plays an important role in regulating focal adhesion turnover. However, the detailed mechanism underlying this regulation is largely unknown. We previously reported that MYO18Aα interacts via its carboxyl terminus with the PAK2/βPIX/GIT1 complex through direct binding to βPIX, and that knockdown of MYO18Aα in epithelial cells causes accumulation of the complex in focal adhesions and decreased cell migration ability (Hsu et al., 2010). The current study characterized the detailed MYO18Aα–βPIX interaction mechanism and the biological significance of this interaction. We found that deletion of the carboxyl-terminal globular domain of MYO18Aα profoundly altered the cellular localization of βPIX and inhibited cell migration. βPIX interacts through its most carboxyl-terminus, PAWDETNL (639–646), with MYO18Aα and partially colocalized with MYO18Aα in membrane ruffles of cells, whereas βPIX1–638, a mutant with deletion of PAWDETNL, accumulated in focal adhesions. Both focal adhesion numbers and area in βPIX1–638-expressing cells were greater than those in cells expressing wild-type βPIXFL. Further experiments using deletion mutants of MYO18A and βPIX showed that disruption of MYO18A–βPIX interaction not only impaired cell motility but also decreased Rac1 activity. Collectively, our data unravel the interaction regions between MYO18A and βPIX and provide evidence for the critical role of this interaction in regulating cellular localization of βPIX, Rac1 activity, and adhesion and migration in epithelial cells.

Cell motility and biofilm formation in Bacillus subtilis are affected by the ribosomal proteins, S11 and S21.

Bacillus subtilis differentiates into various cellular states in response to environmental changes. It exists in two states during the exponential growth phase: motile cells and connected chains of sessile cells. Here, we identified new regulators of cell motility and chaining, the ribosomal proteins S21 (rpsU) and S11 (rpsK). Their mutants showed impaired cell motility (observed in a laboratory strain) and robust biofilm formation (observed in an undomesticated strain). The two major operons for biofilm formation, tapA-sipW-tasA and epsA-O, were strongly expressed in the rpsU mutant, whereas the flagellin-encoding hag gene and other SigD-dependent motility regulons were not. Genetic analysis revealed that the mutation of remA, the transcriptional activator of the eps operon, is epistatic to that of rpsU, whereas the mutation of antagonistic regulators of SinR is not. Our studies demonstrate that S11 and S21 participate in the regulation of bistability via the RemA/RemB pathway.

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.

1P138 アクチン疎水性ヘリックスに導入した変異は細胞性粘菌の細胞運動と細胞分裂を阻害する(10. 筋肉,ポスター,第52回日本生物物理学会年会(2014年度))

1P138 アクチン疎水性ヘリックスに導入した変異は細胞性粘菌の細胞運動と細胞分裂を阻害する(10. 筋肉,ポスター,第52回日本生物物理学会年会(2014年度))

RSK Isoforms in Cancer Cell Invasion and Metastasis

Metastasis, the spreading of cancer cells from a primary tumor to secondary sites throughout the body, is the primary cause of death for patients with cancer. New therapies that prevent invasion and metastasis in combination with current treatments could therefore significantly reduce cancer recurrence and morbidity. Metastasis is driven by altered signaling pathways that induce changes in cell-cell adhesion, the cytoskeleton, integrin function, protease expression, epithelial-to-mesenchymal transition and cell survival. The ribosomal S6 kinase (RSK) family of kinases is a group of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) effectors that can regulate these steps of metastasis by phosphorylating both nuclear and cytoplasmic targets. However, our understanding of RSK function in metastasis remains incomplete and is complicated by the fact that the four RSK isoforms perform nonredundant, sometimes opposing functions. Although some isoforms promote cell motility and invasion by altering transcription and integrin activity, others impair cell motility and invasion through effects on the actin cytoskeleton. The mechanism of RSK action depends both on the isoform and the cancer type. However, despite the variance in RSK-mediated outcomes, chemical inhibition of this group of kinases has proven effective in blocking invasion and metastasis of several solid tumors in preclinical models. RSKs are therefore a promising drug target for antimetastatic cancer treatments that could supplement and improve current therapeutic approaches. This review highlights contradiction and agreement in the current data on the function of RSK isoforms in metastasis and suggests ways forward in developing RSK inhibitors as new antimetastasis drugs.

CDK6 associates with the centrosome during mitosis and is mutated in a large Pakistani family with primary microcephaly

Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference, reduction in the size of the cerebral cortex with otherwise grossly normal brain structure and variable intellectual disability. MCPH is caused by mutations of 11 different genes which code for proteins implicated in cell division and cell cycle regulation. We studied a consanguineous eight-generation family from Pakistan with ten microcephalic children using homozygosity mapping and found a new MCPH locus at HSA 7q21.11-q21.3. Sanger sequencing of the most relevant candidate genes in this region revealed a homozygous single nucleotide substitution c.589G>A in CDK6, which encodes cyclin-dependent kinase 6. The mutation changes a highly conserved alanine at position 197 into threonine (p.Ala197Thr). Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. CDK6 is an important protein for the control of the cell cycle and differentiation of various cell types. We show here for the first time that CDK6 associates with the centrosome during mitosis; however, this was not observed in patient fibroblasts. Moreover, the mutant primary fibroblasts exhibited supernumerary centrosomes, disorganized microtubules and mitotic spindles, an increased centrosome nucleus distance, reduced cell proliferation and impaired cell motility and polarity. Upon ectopic expression of the mutant protein and knockdown of CDK6 through shRNA, we noted similar effects. We propose that the identified CDK6 mutation leads to reduced cell proliferation and impairs the correct functioning of the centrosome in microtubule organization and its positioning near the nucleus which are key determinants during neurogenesis.

Histone Demethylase RBP2 Promotes Lung Tumorigenesis and Cancer Metastasis

The retinoblastoma binding protein RBP2 (KDM5A) is a histone demethylase that promotes gastric cancer cell growth and is enriched in drug-resistant lung cancer cells. In tumor-prone mice lacking the tumor suppressor gene RB or MEN1, genetic ablation of RBP2 can suppress tumor initiation, but the pathogenic breadth and mechanistic aspects of this effect relative to human tumors have not been defined. Here, we approached this question in the context of lung cancer. RBP2 was overexpressed in human lung cancer tissues where its depletion impaired cell proliferation, motility, migration, invasion, and metastasis. RBP2 oncogenicity relied on its demethylase and DNA-binding activities. RBP2 upregulated expression of cyclins D1 and E1 while suppressing the expression of cyclin-dependent kinase inhibitor p27 (CDKN1B), each contributing to RBP2-mediated cell proliferation. Expression microarray analyses revealed that RBP2 promoted expression of integrin-β1 (ITGB1), which is implicated in lung cancer metastasis. Mechanistic investigations established that RBP2 bound directly to the p27, cyclin D1, and ITGB1 promoters and that exogenous expression of cyclin D1, cyclin E1, or ITGB1 was sufficient to rescue proliferation or migration/invasion, respectively. Taken together, our results establish an oncogenic role for RBP2 in lung tumorigenesis and progression and uncover novel RBP2 targets mediating this role.

MiR-26a inhibits proliferation and motility in bladder cancer by targeting HMGA1

It is increasingly clear that microRNAs play a crucial role in tumorigenesis. Recently, emerging evidence suggested that miR-26a is aberrantly expressed in tumor tissues. In our study, frequent down-regulation of miR-26a was observed in 10 human bladder cancer tissues. Forced expression of miR-26a in the bladder cancer cell line T24 inhibited cell proliferation and impaired cell motility. High mobility group AT-hook 1 (HMGA1), a gene that modulates cell cycle transition and cell motility, was verified as a novel target of miR-26a in bladder cancer. These findings indicate an important role for miR-26a in the molecular etiology of bladder cancer and implicate the potential application of miR-26a in bladder cancer therapy.

In vitro and in vivo effects of short hairpin RNA targeting integrin-linked kinase in prostate cancer cells

Integrin-linked kinase (ILK) localizes at focal adhesion sites, plays an important role in cell-matrix interactions and is involved in the regulation of tumor cell growth and migration. The aim of the present study was to clarify the functional characterization of ILK in prostate cancer (PCa) cells. ILK was shown to be overexpressed in 57.1% (36/63) of PCa samples and 18.2% (2/11) of benign prostatic hyperplasia (BPH) samples using immunohistochemical analysis. DU145 PCa cells knocked down for ILK were examined using western blot analysis, proliferation assay, flow cytometry and wound healing assay. Depletion of ILK significantly impaired cell growth and motility, induced apoptosis in vitro, and delayed xenograft tumor proliferation in nude mice, which were important for oncogenesis and tumor progression. Western blot analysis showed that Akt activity was attenuated in ILK‑depleted cells compared with the control cells. These results indicate that ILK knockdown attenuates the biological behavior of PCa cells by decreasing Akt activity, demonstrating that ILK is involved in the development and progression of PCa. Thus, ILK is suggested to serve as a potential therapeutic target for PCa.

Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y1086, c-Src, Erk1/2 and Akt phosphorylation and smallGTPase activity

The interactions between cancer cells and their microenvironment are crucial for malignant progression, as they modulate invasion-related activities. Tumor-associated macrophages are generally considered allies in the process of tumor progression in several types of cancer, although their role on gastric and colorectal carcinomas is still poorly understood. In this report, we studied the influence of primary human macrophages on gastric and colorectal cancer cells, considering invasion, motility/migration, proteolysis and activated intracellular signaling pathways. We demonstrated that macrophages stimulate cancer cell invasion, motility and migration, and that these effects depend on matrix metalloproteinase (MMP) activity and on the activation of epidermal growth factor receptor (EGFR) (at the residue Y(1086)), PLC-γ (phospholipase C-gamma) and Gab1 (GRB2-associated binding protein-1), as evidenced by siRNA (small interference RNA) experiments. Epidermal growth factor (EGF)-immunodepletion impaired macrophage-mediated cancer cell invasion and motility, suggesting that EGF is the pro-invasive and pro-motile factor produced by macrophages. Macrophages also induced gastric and colorectal cancer cell phosphorylation of Akt, c-Src and ERK1/2, and led to an increase of RhoA and Cdc42 activity. Interestingly, whereas macrophage-mediated cancer cell c-Src and ERK1/2 phosphorylation occurred downstream EGFR activation, Akt phosphorylation seems to be a parallel event, taking place in an EGFR-independent manner. The involvement of EGF, EGFR-downstream signaling partners and MMPs in macrophage-mediated invasion provides novel insights into the molecular crosstalk established between cancer cells and macrophages, opening new perspectives for the design of new and more efficient therapeutic strategies to counteract cancer cell invasion.

A potential role for autocrine VEGF signaling in endothelial cell function

Paracrine‐derived vascular endothelial growth factor (VEGF) is established as an indispensable contributor to the angiogenic cascade. Autocrine VEGF, in contrast, is solely attributed to endothelial cell survival signaling, and its potential regulation of other endothelial cell functions has not been studied. We hypothesized that the deletion of VEGF would disrupt endothelial cell migration and shear stress adaptation. Endothelial cells were isolated from transgenic mice having VEGF exon 3 flanked by loxP sites (VEGFL/L). Cells were transduced with adeno‐Cre recombinase to induce VEGF deletion, or adeno‐β‐galactosidase as a control. When subjected to a scrape migration assay, cells with Cre induced VEGF deletion (VEGFΔ) exhibited impaired cell motility compared VEGFL/L cells (12% compared to 64% wound closure after 48 hours; p<0.05). Shear stress stimulation (15 dynes; 2hr) caused significant increases in phospho‐Akt in VEGFL/L cells, which was repressed in VEGFΔ cells (2.4 vs. 1.5 fold above static, respectively). Shear‐induced increases in phospho‐p38MAPK were unaltered in VEGFΔ cells (3.9 vs 3.3 fold above static, respectively). Shear‐induced endothelial cell alignment (15 dynes; 24hr) also was repressed in VEGFΔ cells. Taken together these results provide novel evidence that autocrine VEGF contributes to multiple aspects of endothelial cell function.

ARHGDIA: a novel gene implicated in nephrotic syndrome

BackgroundCongenital nephrotic syndrome arises from a defect in the glomerular filtration barrier that permits the unrestricted passage of protein across the barrier, resulting in proteinuria, hypoalbuminaemia, and severe oedema. While...