Phagocytosis Checkpoints in Glioblastoma: CD47 and Beyond.

Glioblastoma multiforme (GBM) is one of the deadliest human cancers with very limited treatment options available. The malignant behavior of GBM is manifested in a tumor which is highly invasive, resistant to standard cytotoxic chemotherapy, and strongly immunosuppressive. Immune checkpoint inhibitors have recently been introduced in the clinic and have yielded promising results in certain cancers. GBM, however, is largely refractory to these treatments. The immune checkpoint CD47 has recently gained attention as a potential target for intervention as it conveys a "don't eat me" signal to tumor-associated macrophages (TAMs) via the inhibitory SIRP alpha protein. In preclinical models, the administration of anti-CD47 monoclonal antibodies has shown impressive results with GBM and other tumor models. Several well-characterized oncogenic pathways have recently been shown to regulate CD47 expression in GBM cells and glioma stem cells (GSCs) including Epidermal Growth Factor Receptor (EGFR) beta catenin. Other macrophage pathways involved in regulating phagocytosis including TREM2 and glycan binding proteins are discussed as well. Finally, chimeric antigen receptor macrophages (CAR-Ms) could be leveraged for greatly enhancing the phagocytosis of GBM and repolarization of the microenvironment in general. Here, we comprehensively review the mechanisms that regulate the macrophage phagocytosis of GBM cells.

Macrophages in the Glioblastoma Tumor Microenvironment.

The prognosis of high-grade glioma remains dismal, with the median survival time being 15 months [...].

The Chemokine Receptor CCR1 Mediates Microglia Stimulated Glioma Invasion.

Glioblastoma multiforme (GBM) is the most aggressive form of adult brain tumor which is highly resistant to conventional treatment and therapy. Glioma cells are highly motile resulting in infiltrative tumors with poorly defined borders. Another hallmark of GBM is a high degree of tumor macrophage/microglia infiltration. The level of these tumor-associated macrophages/microglia (TAMs) correlates with higher malignancy and poorer prognosis. We previously demonstrated that inhibition of TAM infiltration into glioma tumors with the CSF-1R antagonist pexidartinib (PLX3397) can inhibit glioma cell invasion in-vitro and in-vivo. In this study, we demonstrate an important role for the chemokine receptor CCR1 in mediating microglia/TAM stimulated glioma invasion. Using two structurally distinct CCR1 antagonists, including a novel inhibitor "MG-1-5", we were able to block microglial activated GL261 glioma cell invasion in a dose dependent manner. Interestingly, treatment of a murine microglia cell line with glioma conditioned media resulted in a strong induction of CCR1 gene and protein expression. This induction was attenuated by inhibition of CSF-1R. In addition, glioma conditioned media treatment of microglia resulted in a rapid upregulation of gene expression of several CCR1 ligands including CCL3, CCL5, CCL6 and CCL9. These data support the existence of tumor stimulated autocrine loop within TAMs which ultimately mediates tumor cell invasion.

Unsaturated Fatty Acids and Their Immunomodulatory Properties.

Oils are an essential part of the human diet and are primarily derived from plant (or sometimes fish) sources. Several of them exhibit anti-inflammatory properties. Specific diets, such as Mediterranean diet, that are high in ω-3 polyunsaturated fatty acids (PUFAs) and ω-9 monounsaturated fatty acids (MUFAs) have even been shown to exert an overall positive impact on human health. One of the most widely used supplements in the developed world is fish oil, which contains high amounts of PUFAs docosahexaenoic and eicosapentaenoic acid. This review is focused on the natural sources of various polyunsaturated and monounsaturated fatty acids in the human diet, and their role as precursor molecules in immune signaling pathways. Consideration is also given to their role in CNS immunity. Recent findings from clinical trials utilizing various fatty acids or diets high in specific fatty acids are reviewed, along with the mechanisms through which fatty acids exert their anti-inflammatory properties. An overall understanding of diversity of polyunsaturated fatty acids and their role in several molecular signaling pathways is useful in formulating diets that reduce inflammation and increase longevity.

Contribution of CXCL12 secretion to invasion of breast cancer cells.

INTRODUCTION: Neu (HER2/ErbB2) is overexpressed in 25% to 30% of human breast cancer, correlating with a poor prognosis. Researchers in previous studies who used the mouse mammary tumor virus Neu-transgenic mouse model (MMTV-Neu) demonstrated that the Neu-YB line had increased production of CXCL12 and increased metastasis, whereas the Neu-YD line had decreased metastasis. In this study, we examined the role of increased production of CXCL12 in tumor cell invasion and malignancy. METHODS: We studied invasion in the tumor microenvironment using multiphoton intravital imaging, in vivo invasion and intravasation assays. CXCL12 signaling was altered by using the CXCR4 inhibitor AMD3100 or by increasing CXCL12 expression. The role of macrophage signaling in vivo was determined using a colony-stimulating factor 1 receptor (CSF-1R) blocking antibody. RESULTS: The Neu-YD strain was reduced in invasion, intravasation and metastasis compared to the Neu-YB and Neu deletion mutant (activated receptor) strains. Remarkably, in the Neu-YB strain, in vivo invasion to epidermal growth factor was dependent on both CXCL12-CXCR4 and CSF1-CSF-1R signaling. Neu-YB tumors had increased macrophage and microvessel density. Overexpression of CXCL12 in rat mammary adenocarcinoma cells increased in vivo invasion as well as microvessel and macrophage density. CONCLUSIONS: Expression of CXCL12 by tumor cells results in increased macrophage and microvessel density and in vivo invasiveness.

Microglial stimulation of glioblastoma invasion involves epidermal growth factor receptor (EGFR) and colony stimulating factor 1 receptor (CSF-1R) signaling.

Glioblastoma multiforme is a deadly cancer for which current treatment options are limited. The ability of glioblastoma tumor cells to infiltrate the surrounding brain parenchyma critically limits the effectiveness of current treatments. We investigated how microglia, the resident macrophages of the brain, stimulate glioblastoma cell invasion. We first examined the ability of normal microglia from C57Bl/6J mice to stimulate GL261 glioblastoma cell invasion in vitro. We found that microglia stimulate the invasion of GL261 glioblastoma cells by approximately eightfold in an in vitro invasion assay. Pharmacological inhibition of epidermal growth factor receptor (EGFR) strongly inhibited microglia-stimulated invasion. Furthermore, blockade of colony stimulating factor 1 receptor (CSF-1R) signaling using ribonucleic acid (RNA) interference or pharmacological inhibitors completely inhibited microglial enhancement of glioblastoma invasion. GL261 cells were found to constitutively secrete CSF-1, the levels of which were unaffected by epidermal growth factor (EGF) stimulation, EGFR inhibition or coculture with microglia. CSF-1 only stimulated microglia invasion, whereas EGF only stimulated glioblastoma cell migration, demonstrating a synergistic interaction between these two cell types. Finally, using PLX3397 (a CSF-1R inhibitor that can cross the blood-brain barrier) in live animals, we discovered that blockade of CSF-1R signaling in vivo reduced the number of tumor-associated microglia and glioblastoma invasion. These data indicate that glioblastoma and microglia interactions mediated by EGF and CSF-1 can enhance glioblastoma invasion and demonstrate the possibility of inhibiting glioblastoma invasion by targeting glioblastoma-associated microglia via inhibition of the CSF-1R.

Opposing roles of CXCR4 and CXCR7 in breast cancer metastasis.

INTRODUCTION: CXCL12-CXCR4 signaling has been shown to play a role in breast cancer progression by enhancing tumor growth, angiogenesis, triggering cancer cell invasion in vitro, and guiding cancer cells to their sites of metastasis. However, CXCR7 also binds to CXCL12 and has been recently found to enhance lung and breast primary tumor growth, as well as metastasis formation. Our goal was to dissect the contributions of CXCR4 and CXCR7 to the different steps of metastasis - in vivo invasion, intravasation and metastasis formation. METHODS: We overexpressed CXCR4, CXCR7 or both in the rat mammary adenocarcinoma cell line MTLn3. Stable expressors were used to form tumors in severe combined immunodeficiency (SCID) mice, and in vivo invasiveness, intravital motility, intravasation, and metastasis were measured. RESULTS: We found that CXCR4 overexpression increased the chemotactic and invasive behavior of MTLn3 cells to CXCL12, both in vitro and in vivo, as well as in vivo motility and intravasation. CXCR7 overexpression enhanced primary tumor growth and angiogenesis (as indicated by microvessel density and VEGFA expression), but decreased in vivo invasion, intravasation, and metastasis formation. In vitro, expression of CXCR7 alone had no effect in chemotaxis or invasion to CXCL12. However, in the context of increased CXCR4 expression, CXCR7 enhanced chemotaxis to CXCL12 but decreased invasion in response to CXCL12 in vitro and in vivo and impaired CXCL12 stimulated matrix degradation. The changes in matrix degradation correlated with expression of matrix metalloproteinase 12 (MMP12). CONCLUSIONS: We find that CXCR4 and CXCR7 play different roles in metastasis, with CXCR4 mediating breast cancer invasion and CXCR7 impairing invasion but enhancing primary tumor growth through angiogenesis.

Septin 9 isoform expression, localization and epigenetic changes during human and mouse breast cancer progression.

INTRODUCTION: Altered expression of Septin 9 (SEPT9), a septin coding for multiple isoform variants, has been observed in several carcinomas, including colorectal, head and neck, ovarian and breast, compared to normal tissues. The mechanisms regulating its expression during tumor initiation and progression in vivo and the oncogenic function of its different isoforms remain elusive. METHODS: Using an integrative approach, we investigated SEPT9 at the genetic, epigenetic, mRNA and protein levels in breast cancer. We analyzed a panel of breast cancer cell lines, human primary tumors and corresponding tumor-free areas, normal breast tissues from reduction mammoplasty patients, as well as primary mammary gland adenocarcinomas derived from the polyoma virus middle T antigen, or PyMT, mouse model. MCF7 clones expressing individual GFP-tagged SEPT9 isoforms were used to determine their respective intracellular distributions and effects on cell migration. RESULTS: An overall increase in gene amplification and altered expression of SEPT9 were observed during breast tumorigenesis. We identified an intragenic alternative promoter at which methylation regulates SEPT9_v3 expression. Transfection of specific GFP-SEPT9 isoforms in MCF7 cells indicates that these isoforms exhibit differential localization and affect migration rates. Additionally, the loss of an uncharacterized SEPT9 nucleolar localization is observed during tumorigenesis. CONCLUSIONS: In this study, we found conserved in vivo changes of SEPT9 gene amplification and overexpression during human and mouse breast tumorigenesis. We show that DNA methylation is a prominent mechanism responsible for regulating differential SEPT9 isoform expression and that breast tumor samples exhibit distinctive SEPT9 intracellular localization. Together, these findings support the significance of SEPT9 as a promising tool in breast cancer detection and further emphasize the importance of analyzing and targeting SEPT9 isoform-specific expression and function.

Microglial-stimulation of glioma invasion involves the EGFR ligand amphiregulin.

High grade glioma is one of the deadliest human cancers with a median survival rate of only one year following diagnosis. The highly motile and invasive nature of high grade glioma makes it difficult to completely remove surgically. Therefore, increasing our knowledge of the mechanisms glioma cells use to invade normal brain is of critical importance in designing novel therapies. It was previously shown by our laboratory that tumor-associated microglia (TAMs) stimulate glioma cell invasion and this process is dependent on CSF-1R signaling. In this study, we seek to identify pro-invasive factors that are upregulated in microglia in a CSF-1R-dependent manner. We assayed cDNA and protein from microglia treated with conditioned media from the murine glioma cell line GL261, and discovered that several EGFR ligands including amphiregulin (AREG) are strongly upregulated. This upregulation is blocked by addition of a pharmacological CSF-1R inhibitor. Using RNA interference, we show that AREG-depleted microglia are less effective at promoting invasion of GL261 cells into Matrigel-coated invasion chambers. In addition, an AREG blocking antibody strongly attenuates the ability of THP-1 macrophages to activate human glioma cell line U87 invasion. Furthermore, we have identified a signaling pathway which involves CSF-1 signaling through ERK to upregulate AREG expression in microglia. Interfering with ERK using pharmacological inhibitors prevents AREG upregulation in microglia and microglia-stimulated GL261 invasion. These data highlight AREG as a key factor in produced by tumor associated microglia in promoting glioma invasion.

Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin.

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP-Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.

ERBB1 and ERBB2 have distinct functions in tumor cell invasion and intravasation.

PURPOSE: The epidermal growth factor receptor (ERBB1) and related family member HER-2/neu (ERBB2) are often overexpressed in aggressive breast cancers and their overexpression is correlated with poor prognosis. Clinical studies using ERBB inhibitors have focused on tumor growth effects, but ERBBs can contribute to malignancy independent of their effects on tumor growth. Our studies were designed to evaluate the effect of ERBB inhibition on tumor cell motility and intravasation in vivo using clinically relevant small-molecule inhibitors. EXPERIMENTAL DESIGN: Using in vivo mouse models of breast cancer, we test the effects of ERBB1 and ERBB2 inhibitors AC480 and lapatinib, ERBB1 inhibitor gefitinib, and ERBB2 inhibitor AG825 on in vivo tumor cell invasive properties in mammary fat pad tumors. RESULTS: ERBB1 and ERBB2 inhibition rapidly (within 3 h) inhibits both tumor cell motility and intravasation. Using gefitinib, ERBB1 inhibition rapidly inhibits tumor cell motility and invasion but not intravasation, whereas ERBB2 inhibition by AG825 rapidly blocks intravasation. CONCLUSIONS: ERBB1 and ERBB2 inhibition can rapidly block tumor cell invasive properties. In addition, we differentiate for the first time the contributions of ERBB1 and ERBB2 to the key metastatic properties of in vivo tumor cell invasion and intravasation. These experiments temporally and molecularly separate two key stages in tumor cell entry into blood vessels: invasion and intravasation. These results indicate that ERBB inhibition should be considered for blocking other tumor cell malignant properties besides growth.

Successful Extracorporeal Membrane Oxygenation Treatment in an Acquired Immune Deficiency Syndrome (AIDS) Patient with Acute Respiratory Distress Syndrome (ARDS) Complicating Pneumocystis jirovecii Pneumonia: A Challenging Case.

BACKGROUND Patients with HIV infection tend to have poor intensive care unit (ICU) outcomes; however, survival in the modern combination antiretroviral therapy (cART) era has markedly improved, but Pneumocystis jirovecii pneumonia (PJP) still remains a preeminent cause of respiratory failure in AIDS patients. Extracorporeal membrane oxygenation (ECMO) is an adapted cardiopulmonary bypass circuit for temporary life support for patients not responding to conventional treatment. CASE REPORT A 43-year-old male HIV "late presenter" was admitted to our hospital for fever and dyspnea. A chest CT scan revealed bilateral ground-glass opacities. Empiric antibiotic treatment and cART were started. The emergence of ARDS due to PJP dictated urgent veno-venous (VV) ECMO placement. One week later, radiologic findings and respiratory function had improved and the patient was started on a weaning trial from ECMO and removed 12 days after placement. CONCLUSIONS Acute respiratory distress syndrome (ARDS) is a potentially reversible clinical syndrome with a high mortality rate. ECMO is a rescue therapy allowing lung recovery during acute processes and should be considered an adequate treatment option in HIV+ patients with respiratory failure. ECMO should be considered a useful and adequate treatment option in AIDS patients who have a high risk of dying from respiratory failure.

Role of Tumor-Derived Chemokines in Osteolytic Bone Metastasis.

Metastasis is the primary cause of mortality and morbidity in cancer patients. The bone marrow is a common destination for many malignant cancers, including breast carcinoma (BC), prostate carcinoma, multiple myeloma, lung carcinoma, uterine cancer, thyroid cancer, bladder cancer, and neuroblastoma. The molecular mechanism by which metastatic cancer are able to recognize, infiltrate, and colonize bone are still unclear. Chemokines are small soluble proteins which under normal physiological conditions mediate chemotactic trafficking of leukocytes to specific tissues in the body. In the context of metastasis, the best characterized role for the chemokine system is in the regulation of primary tumor growth, survival, invasion, and homing to specific secondary sites. However, there is ample evidence that metastatic tumors exploit chemokines to modulate the metastatic niche within bone which ultimately results in osteolytic bone disease. In this review, we examine the role of chemokines in metastatic tumor growth within bone. In particular, the chemokines CCL2, CCL3, IL-8/CXCL8, and CXCL12 are consistently involved in promoting osteoclastogenesis and tumor growth. We will also evaluate the suitability of chemokines as targets for chemotherapy with the use of neutralizing antibodies and chemokine receptor-specific antagonists.

Roles of the Rac1 and Rac3 GTPases in human tumor cell invasion.

Members of the Rho family of small GTPases have been shown to be involved in tumorigenesis and metastasis. Currently, most of the available information on the function of Rho proteins in malignant transformation is based on the use of dominant-negative mutants of these GTPases. The specificity of these dominant-negative mutants is limited however. In this study, we used small interfering RNA directed against either Rac1 or Rac3 to reduce their expression specifically. In line with observations using dominant-negative Rac1 in other cell types, we show that RNA interference-mediated depletion of Rac1 strongly inhibits lamellipodia formation, cell migration and invasion in SNB19 glioblastoma cells. Surprisingly however, Rac1 depletion has a much smaller inhibitory effect on SNB19 cell proliferation and survival. Interestingly, whereas depletion of Rac3 strongly inhibits SNB19 cell invasion, it does not affect lamellipodia formation and has only minor effects on cell migration and proliferation. Similar results were obtained in BT549 breast carcinoma cells. Thus, functional analysis of Rac1 and Rac3 using RNA interference reveals a critical role for these GTPases in the invasive behavior of glioma and breast carcinoma cells.

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Review: molecular mechanism of microglia stimulated glioblastoma invasion.

Glioblastoma multiforme is one of the deadliest human cancers and is characterized by a high degree of microglia and macrophage infiltration. The role of these glioma infiltrating macrophages (GIMs) in disease progression has been the subject of recent investigation. While initially thought to reflect an immune response to the tumor, the balance of evidence clearly suggests GIMs can have potent tumor-tropic functions and assist in glioma cell growth and infiltration into normal brain. In this review, we focus on the evidence for GIMs aiding mediating glioblastoma motility and invasion. We survey the literature for molecular pathways that are involved in paracrine interaction between glioma cells and GIMs and assess which of these might serve as attractive targets for therapeutic intervention.

Role of Rac1-regulated signaling in medulloblastoma invasion. Laboratory investigation.

OBJECT: Medulloblastomas are the most common malignant brain tumors in children. These tumors are highly invasive, and patients harboring these lesions are frequently diagnosed with distant spread. In this study, the authors investigated the role of Rac1, a member of the Rho family of small guanosine triphosphatases, in medulloblastoma invasion. METHODS: Three established medulloblastoma cell lines were used: DAOY, UW-228, and ONS-76. Specific depletion of Rac1 protein was accomplished by transient transfection of small interfering RNA. Cell invasion through extracellular matrix (Matrigel) was quantified using a transwell migration assay. Mitogen activated protein kinase activation was determined using phospho-MAP kinase-specific antibodies, and inhibition of MAP kinase pathways was achieved by specific small molecule inhibitors. Localization of Rac1 and its expression levels were determined by immunohistochemical analysis using a Rac1-specific antibody, and Rac1 activation was qualitatively assessed by Rac1 plasma membrane association. RESULTS: Small interfering RNA-mediated depletion of Rac1 strongly inhibited medulloblastoma cell invasion. Although depletion of Rac1 inhibited the proliferation of UW-228 cells, and of ONS-76 cells to a lesser extent, it stimulated the proliferation of DAOY cells. Depletion of Rac1 also inhibited the activation of the ERK and JNK MAP kinase pathways, and inhibition of either pathway diminished invasion and proliferation. Immunohistochemical analysis demonstrated that the Rac1 protein was overexpressed in all medulloblastoma tumors examined, and indicated that Rac1 was hyperactive in 6 of 25 tumors. CONCLUSIONS: The authors' data show that Rac1 is necessary for the invasive behavior of medulloblastoma cells in vitro, and plays a variable role in medulloblastoma cell proliferation. In addition, these results indicate that Rac1 stimulates medulloblastoma invasion by activating the ERK and JNK pathways. The authors suggest that Rac1 and signaling elements controlled by this guanosine triphosphatase may serve as novel targets for therapeutic intervention in malignant medulloblastomas.

Pak1 and Pak2 mediate tumor cell invasion through distinct signaling mechanisms.

Pak kinases are thought to play critical roles in cell migration and invasion. Here, we analyze the roles of Pak1 and Pak2 in breast carcinoma cell invasion using the transient transfection of small interfering RNA. We find that although both Pak1 and Pak2 contribute to breast carcinoma invasion stimulated by heregulin, these roles are mediated by distinct signaling mechanisms. Thus, whereas the depletion of Pak1 interferes with the heregulin-mediated dephosphorylation of cofilin, the depletion of Pak2 does not. The depletion of Pak1 also has a stronger inhibitory effect on lamellipodial protrusion than does the depletion of Pak2. Interestingly, Pak1 and Pak2 play opposite roles in regulating the phosphorylation of the myosin light chain (MLC). Whereas the depletion of Pak1 decreases phospho-MLC levels in heregulin-stimulated cells, the depletion of Pak2 enhances MLC phosphorylation. Consistent with their opposite effects on MLC phosphorylation, Pak1 and Pak2 differentially modulate focal adhesions. Pak2-depleted cells display an increase in focal adhesion size, whereas in Pak1-depleted cells, focal adhesions fail to mature. We also found that the depletion of Pak2, but not Pak1, enhances RhoA activity and that the inhibition of RhoA signaling in Pak2-depleted cells decreases MLC phosphorylation and restores cell invasion. In summary, this work presents the first comprehensive analysis of functional differences between the Pak1 and Pak2 isoforms.

The atypical Rho family GTPase Wrch-1 regulates focal adhesion formation and cell migration.

Wrch-1 (Wnt-regulated Cdc42 homolog) is a new member of the Rho family that was identified as a gene transcriptionally upregulated by Wnt-1. Wrch-1 has no detectable GTPase activity and displays very high intrinsic guanine nucleotide exchange, implying that it is constitutively GTP-bound. The biological functions of Wrch-1 largely remain to be characterized. Here, we report that Wrch-1 prominently localizes to focal adhesions. Depletion of Wrch-1 by small interfering RNA increases focal adhesion formation, whereas Wrch-1 overexpression disassembles focal adhesions. Wrch-1 depletion inhibits myosin-light-chain phosphorylation, which in turn leads to an increase in the number of focal adhesions and inhibits cell migration in response to wound healing. Depletion of Wrch-1 also inhibits Akt and JNK activation. Although pharmacological inhibitors of Akt and JNK inhibit cell migration, they do not affect focal adhesions. Thus, our data suggest that Wrch-1 regulates cell migration by multiple mechanisms: on the one hand Wrch-1 controls focal adhesions by regulating myosin light chain and on the other hand Wrch-1 stimulates the activation of Akt and JNK.

Up-regulation of Rac1 by epidermal growth factor mediates COX-2 expression in recurrent respiratory papillomas.

Recurrent respiratory papillomas are epithelial tumors of the airway caused by human papillomaviruses. We previously reported that the epidermal growth factor receptor (EGFR) is overexpressed in papilloma cells, that cyclooxygenase-2 (COX-2) is induced, and that COX-2 expression in primary papilloma cells requires activation of the EGFR but not Erk. Rac1, a member of the Rho family of GTPases, is a key signaling element that is known to control multiple pathways downstream of the EGFR. Here we report that Rac1 is overexpressed in papilloma cells compared with normal laryngeal epithelial cells and that the increased levels of Rac1 are mediated by EGFR activation. Transfecting cells with Rac1-specific siRNA suppressed COX-2 expression. Surprisingly, Rac1 mediated phosphorylation of p38 mitogen-activated kinase in papilloma cells but not normal cells, and inhibition of p38 with the specific inhibitor SB202190 suppressed COX-2 expression in papilloma cells but had no effect on low-level COX-2 expression in normal cells. Thus, the signaling cascades that regulate COX-2 expression are different in HPV-infected papilloma cells, with a significant contribution by the EGFR-- Rac1-->p38 pathway.