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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.

Supplementary Movie S2 from ERBB1 and ERBB2 Have Distinct Functions in Tumor Cell Invasion and Intravasation

Supplementary Movie S1 from ERBB1 and ERBB2 Have Distinct Functions in Tumor Cell Invasion and Intravasation

Supplementary Figures S1-S2 from ERBB1 and ERBB2 Have Distinct Functions in Tumor Cell Invasion and Intravasation

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

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.

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.

BACKGROUND Superficial parotidectomy has traditionally been completed with a drain and overnight hospital stay. We report perioperative and postoperative outcomes for patients undergoing drainless outpatient parotidectomy vs traditional drained extended stay parotidectomy. METHODS Retrospective chart review from a single surgeon from 2009 to 2017 of patients undergoing parotidectomy, including demographic data, surgical approach, tumor pathology and size, blood loss, drain placement, postoperative pain control, and complications, was done. A comparison was performed between patients undergoing drain placement and those treated with "drainless" technique. RESULTS Ninety-one patients underwent parotidectomy (42 drainless; 49 drained). Intraoperative blood loss was lower in the "drainless" group (16.0 mL vs 34.9 mL, P

Objective The objective of this study was to evaluate the clinical outcomes in a cohort of patients with early-stage oral tongue squamous cell carcinoma (OTSCC). Materials and methods We conducted a retrospective analysis of patients with pT1-T2N0 (American Joint Committee on Cancer [AJCC] seventh edition) OTSCC treated from 2000 to 2018. Two-year actuarial rates of local regional control, cancer-specific survival, and overall survival were calculated for the entire cohort and patients with/without adjuvant radiation. Results Ninety-six patients met the criteria with a median follow-up of 4 years; 14 had adjuvant radiation, while 82 had surgery alone. Two-year local regional control was 82.7% (75.4% to 90.8%) for the entire cohort, 84.9% (77.8% to 93.2%) for surgery only, and 70.7% (50.2% to 99.6%) for patients with adjuvant radiation. Two-year progression-free survival was 82.7% (75.3% to 90.8%). Of the 20 patients with recurrence, 11 (55%) were successfully salvaged. Conclusion Local regional recurrence remains modest in early-stage OTSCC, but salvage is possible with high survival rates. Level of evidence Level III-retrospective cohort study.

To evaluate outcomes of in-office versus operating room (OR) sialendoscopy/sialolithotomy and to recognize the efficiency of outpatient salivary gland surgery with significant time and facility charge reductions.Case series with chart review.State hospital OR and ambulatory clinic.Retrospective review was performed of adult patients treated for inflammatory salivary diseases by a single surgeon from 2011 to 2016. The patients were divided into 2 groups based on procedure setting (office vs OR) and compared by various baseline features, including demographics, symptom onset and duration, stone size, symptomatic improvement, and recurrence. Patient time burden was compared via office procedure records and OR time charting from the electronic health record. Retrospective clinic and hospital charge sheets were tallied and similarly compared.The 2 cohorts (office, n = 111; OR, n = 96) were comparable in all demographics, including sialolith number and size (7.36 vs 6.69 mm, P = .45). Additional subgrouping was statistically similar. Both cohorts had similar postprocedure symptom improvement (97% vs 95.8%, P = .65) and recurrence rates (8.9% vs 14.5%, P = .22) independent of subgroup. Overall time burden for patients was 39 minutes in the office versus 277 minutes in the OR ( P ≤ .0001). Procedure and hospital charge data were tallied and compared (office, $719.21; OR, $13,956.14; P ≤ .0001).Bothcohorts were statistically similar in all features. There was significant reduction in patient time burden and health care charges with office-based procedures while maintaining similar symptom improvement and recurrence rates.

Objectives The 8th edition AJCC staging guidelines for head and neck squamous cell carcinoma (HNSCC) recently introduced pathologic staging criteria for nodal disease among p16-positive patients. In this study we evaluate pathologic staging in p16-negative HNSCC. Materials and Methods We compared pathologic staging to the 7th and 8th edition AJCC staging systems using a statewide population-based cohort. All M0 p16-negative surgical patients were included. The outcome was five-year overall survival. Results Of 304 patients identified, 113 were N0, 157 had 1–4 positive nodes, and 34 had ≥4 nodes. Survival was 71% (95% CI 61–78%) with no nodes, 48% (36%−60%) for 1–4 nodes, and 24% (11 – 39%) for > 4 nodes. When compared to the AJCC systems, the pathologic staging yielded a larger total survival gradient, more montonic survival, better consistency across primary sites, and a slightly lower Bayesian information criterion (1510 vs 1538). After adjusting for disease characteristics, demographics, and tobacco use, hazard ratios for survival were similar using pathologic and AJCC criteria. Conclusion In this cohort, pathological staging was more prognostic than AJCC staging. This is the first study to evaluate pathologic staging in p16-negative cancer; if these findings are verified, a universal nodal staging system could be introduced.

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.

In the metamorphic basement of the Sierra de Comechingones, the southernmost extension of Sierras de Cordoba (Argentina), it is hosted a group of polymetallic Pb-Zn-Cu-Ag-Au veins associated with hydrothermal alteration zones, which are the result of a Late Paleozoic metallogenic period. In this paper, the application of spectral analysis in the SWIR (Short-Wave Infrared) region as well as magnetic techniques, unusual in Argentina for this style of mineralization, proved to be an effective tool in the study of altered areas and also useful to establish targets for mineral prospecting and/or exploration. A sericite-quartz assemblage is the main hydrothermal alteration product of the migmatic host rocks. It is developed into narrow but continuous altered zones which follow the main direction of the mineralized structures and can reach up to 5 meters to each side of the veins. Subordinately and locally, a carbonate siderite-calcite assemblage takes place as a result of altered amphibolites, which are interspersed within the metamorphic sequence. In the reflectance spectra, the wavelength position of the Al-OH absorption feature revealed compositional variations and cation substitutions in the hydrothermal white micas, ranging from potassic (wavelengths near 2200 nm) to micas with a lower octahedral Al content (wavelengths > 2210 nm) towards phengite. These variations and the spatial distribution within the altered areas are primarily controlled by three main factors: 1) hydrothermal fluid temperature, 2) pH and/or 3) Mg-Fe/Al ratio in the fluid-rock system. The crystallinity indexes (CI) calculated from the spectral data, indicate the predominance of high crystallinity illites with a slight increase of this parameter towards the proximal zones to the mineralized veins. The illite/smectite mix-layered clays indicate a fluid temperature decrease towards the distal zones and they can be used as vectors to mineralization. Both sericite-quartz and siderite-calcite hydrothermal assemblages are frequently affected by supergene oxidation processes that led to a conspicuous precipitation of hematite-goethite within the altered areas. The oxidation state of Fe+3 in these minerals is the main cause of the decrease of the magnetic susceptibility parameter in the host rocks. With a regional perspective, these hydrothermal alteration zones characterized by a high abundance of white micas of moderate to high crystallinity (CI=1,21-3,92 and IK=0,21-0,23), compositions ranging from potassic to phengitic (Al-OH wavelengths between 2200-2215 nm) and associated with hematization zones and low magnetic susceptibility values (< 0,2 x 10-3SI), may be considered as potential targets for future prospecting and/or exploration of polymetallic veins hosted in the metamorphic environment of the Sierras Pampeanas. En el basamento metamórfico de la Sierra de Comechingones, extensión más austral de las Sierras de Córdoba (Argentina), se emplaza un conjunto de vetas polimetálicas con Pb, Zn, Cu, Ag, Au asociadas con zonas de alteración hidrotermal, que constituyen el registro de una época metalogenética ocurrida hacia fines del Paleozoico. En este trabajo la aplicación de técnicas espectrales en la región del SWIR (infrarrojo de onda corta) y magnéticas, poco usual en Argentina para este estilo de mineralización, demostró ser una herramienta eficaz en el estudio de las zonas alteradas y de utilidad para establecer targets de prospección y/o exploración. La asociación sericita-cuarzo es el principal producto de alteración hidrotermal de las rocas de caja migmáticas, se desarrolla en franjas estrechas pero continuas que siguen el rumbo principal de las estructuras mineralizadas y pueden alcanzar hasta 5 metros de potencia hacia los laterales. En sectores localizados, una asociación carbonática de siderita-calcita se desarrolla como consecuencia de la alteración de anfibolitas intercaladas dentro de la secuencia metamórfica. En los espectros de reflectancia, la posición del rasgo de absorción Al-OH indicó una variación composicional y procesos de sustitución iónica en las micas blancas de origen hidrotermal, desde micas potásicas (longitudes de onda cercanas a 2200 nm) a micas con menor contenido relativo de Al en el sitio octaédrico (longitudes de onda > a 2210 nm) tendientes a la variedad fengita. Estas variaciones y su distribución espacial están controladas fundamentalmente por tres factores: 1) temperatura del fluido hidrotermal, 2) pH y/o 3) relación Mg-Fe/Al dentro del sistema fluido-roca. Los índices de cristalinidad (IC) calculados a partir de los datos espectrales revelaron la predominancia de illitas de alta cristalinidad con un leve incremento de este parámetro hacia las zonas proximales a las vetas mineralizadas. Las interestratificaciones de illita/esmectita hacia las zonas distales de algunas estructuras, reflejan la disminución de la temperatura del fluido y pueden ser utilizadas como vectores hacia las mineralizaciones. Las asociaciones hidrotermales sericita-cuarzo y siderita-calcita están frecuentemente afectadas por los procesos de oxidación supergénica que condujeron a una conspicua precipitación de hematita-goethita dentro de las zonas alteradas. El estado de oxidación del Fe+3 en estos minerales es la principal causa de la disminución del parámetro de susceptibilidad magnética de las rocas hospedantes. Desde una perspectiva regional, estas zonas de alteración hidrotermal caracterizadas por una alta abundancia de micas blancas de moderada a alta cristalinidad (IC=1,21-3,92 e IK=0,21-0,23), composiciones variables entre potásicas y fengíticas (absorción del enlace Al-OH entre 2200 y 2215 nm) y asociadas con zonas de hematización y bajos valores de susceptibilidad magnética (< 0,2 x 10-3SI), pueden considerase como potenciales targets para futuras etapas de prospección y/o exploración de vetas polimetálicas emplazadas en el ambiente metamórfico de las Sierras Pampeanas.

Glioblastoma Multiforme (GBM) is the most aggressive form of adult brain tumor with a median survival time of twelve months. GBM is highly resistant to conventional therapy which includes surgical resection of the tumor, radiation treatment and chemotherapy. GBM cells are highly motile and invasive resulting in infiltrative tumors with poorly defined borders. GBM tumors are heavily infiltrated with microglia cells which are known to stimulate GBM cell invasion. Our laboratory has previously demonstrated that microglia strongly stimulates GBM invasion both in-vitro and in orthotopic animal models. This interaction was found to be dependent on CSF-1R which is expressed on all tumor infiltrating macrophages/microglia. Blockade of the CSF-1R using compounds such as pexidartinib (PLX3397) can inhibit microglia/macrophage-stimulated GBM invasion in-vitro and in vivo. A variety of chemokines are upregulated in the GBM tumor microenvironment and facilitate “cross-talk” between microglia and GBM cells eliciting a chemotactic response. We have demonstrated that the chemotactic ligand, CCL3, is similarly upregulated in GBM tumors. We postulated that inhibition of CCL3 associated receptors such as C-C receptor 1 (CCR1) might also inhibit GBM invasion, thus, a CCR1 antagonist could prove efficacious for blockade of microglia-induced glioblastoma invasion in vitro. Many potent CCR1 antagonists have been described in the literature. We chose four of these compounds with two distinct structural cores, all with reported IC50’s of less than 200 nM for inhibition of CCR1 binding versus CCL3. We examined the ability of these antagonists to block microglia-stimulated glioblastoma invasion using an in-vitro coculture invasion assay. Using quantitative PCR arrays, we also show that expression of chemokines and chemokine receptor genes is greatly altered in GBM conditioned media-treated microglia. Understanding the pattern of tumor-associated macrophage/microglia chemokine secretion in GBM may present additional targets for chemotherapeutic intervention and enhance immunotherapy. Citation Format: Salvatore J. Coniglio, Poornema Ramasundaram, Neshama Fournier, Danielle S. Hamilton, Gregory Marshall, Keia Smith, Diana Habib, James R. Merritt. The chemokine receptor CCR1 is involved in microglia stimulated glioblastoma invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4548.

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.

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.

Ginsenoside Rg3 is a natural active ingredient that is extracted from Korean red ginseng root. It elevates the therapeutic effect of radiotherapy and chemotherapy, but previous studies found that the application of Rg3 is heavily limited by its low bioavailability and poor absorption via oral administration. To overcome these problems, Rg3-loaded PEG-PLGA-NPs (Rg3-NPs) were prepared by the modified spontaneous emulsification solvent diffusion (SESD) method, and the physicochemical characteristics of Rg3-NPs were investigated. We treated primary glioblastoma with 50 mM Rg3-NPs for 48h. We then used gene expression arrays (Illumina) for genome-wide expression analysis and validated the results for genes of interest by means of real-time PCR. Functional annotations were then performed using the DAVID and KEGG online tools. The results showed that the Rg3-NPs are slick and uniform, the average diameter of the nanoparticles is 75-90 nm, and their entrapment efficiency is 89.7+1.7%. MTT showed that the growth of cells can be significantly inhibited by Rg3-NPs in a dose-dependent manner. FCM testing showed Rg3-NPs can be released from the conjugate nanoparticle and react with the genes in the cell nuclei, causing changes in the gene molecules. We also found that cancer cells treated with Rg3-NPs undergo cell-cycle arrest at different checkpoints. This arrest was associated with a decrease in the mRNA levels of core regulatory genes BUB1, CDC20, TTK, and CENPE, as determined by microarray analysis and verified by real-time PCR. Furthermore, Rg3-NPs induced the expression of the apoptotic and antimigratory protein p53 in cell lines. The results of the present study, together with the results of earlier studies, show that Rg3-NPs target genes involved in theprogressionoftheM-phaseofthecellcycle.Itisassociatedwithseveralimportant pathways, which include apoptosis (p53). Rg3-NPs may be a potent cell-cycle regulation drug targeting the M-phase in glioblastoma cell lines.

Many malignancies show increased expression of the epidermal growth factor (EGF) receptor family member ErbB3 (HER3). ErbB3 binds heregulin β-1 (HRGβ1) and forms a heterodimer with other ErbB family members, such as ErbB2 (HER2) or EGF receptor (EGFR; HER1), enhancing phosphorylation of specific C-terminal tyrosine residues and activation of downstream signaling pathways. ErbB3 contains six YXXM motifs that bind the p85 subunit of phosphoinositide 3 (PI3)-kinase. Previous studies demonstrated that overexpression of ErbB3 in mammary tumor cells can significantly enhance chemotaxis to HRGβ1 and overall metastatic potential. We tested the hypothesis that ErbB3-mediated PI3-kinase signaling is critical for heregulin-induced motility, and therefore crucial for ErbB3-mediated invasion, intravasation and metastasis. The tyrosines in the six YXXM motifs on the ErbB3 C-terminus were replaced with phenylalanine. In contrast to overexpression of the wild-type ErbB3, overexpression of the mutant ErbB3 did not enhance chemotaxis towards HRGβ1 in vitro or in vivo. We also observed reduced tumor cell motility in the primary tumor by multiphoton microscopy, as well as a dramatically reduced ability of these cells to cross the endothelium and intravasate into the circulation. Moreover, whereas mutation of the ErbB3 C-terminus had no effect on tumor growth, it had a dramatic effect on spontaneous metastatic potential. Treatment with the PI3-kinase inhibitor PIK-75 similarly inhibited motility and invasion in vitro and in vivo. Our results indicate that stimulation of the early metastatic steps of motility and invasion by ErbB3 requires activation of the PI3-kinase pathway by the ErbB3 receptor.

Fifteen children with prenatally diagnosed myelomeningocele were followed for at least three years to determine the relations between prenatal anatomic level visualized on high-resolution ultrasound, radiographic level, neuromotor level and functional motor outcome. Prenatal anatomic level accurately predicted neuromotor level and functional motor outcome in 12 of the children and was a better predictor than motor level at birth. Clinicians involved in prenatal counseling may use the anatomic level determined on high-resolution ultrasound to discuss motor prognosis with families.

UMMARY Ten children with cerebral palsy (CP) and growth failure underwent assessment of the growth hormone (GH) axis, including spontaneous GH secretion, GH secretion in response to pharmacological stimulation, and circulating levels of insulin-like growth factor-1 (IGF-1) and IGF binding protein-3 (IGFBP-3). Six of the children had subnormal GH secretion consistent with GH deficiency. Subnormal growth velocity was the best clinical predictor of GH deficiency. The large percentâge of these children with apparent GH deficiency is surprising. Possible mechanisms include anatomic abnormalities of the hypothalamic-pituitary axis, psychosocial deprivation, and an interaction between suboptimal nutritional status and an abnormal central nervous system. RESUME Deficience apparente de I'hormone de croissance chez ľ‘enfant IMC ’ Une appreciation de l'hormone de croissance (GH) a ete effectuee chez dix enfants IMC presentant un defaut dc croissance, incluant la secretion spontanee de GH, la secretion de GH en reponse a une stimulation pharmacologique, et le taux circulant du facteur de croissance 1 (IGF-1) ressemblant aľ insuline et de la proteine qui liea l'IGF-3 (IGFBP-3). Six des enfants presentaient une secretion anormale de GH en faveur d'une deficience de GH. La vitesse de croissance inferieure a la normale etait le meillcur predicteur clinique de deficience de GH. Le pourcentâge eleve de ces enfants avec apparence de deficience en GH est surprenant. Les mecanismes possibles incluent les anomalies anatomiques de l'axe hypothalamo-hypophysaire, la deprivation psychosociale, et une interaction entre un etat nutritionnel sous-optimal et un systeme nerveux central anormal. ZUSAMMENFASSUNG Wachstumshormonmangel bet Kindern mit Cerebralparese Bei 10 Kindern mit Cerebralparese (CP) und Wachstumsstorung wurde die Wachstumshormon (GH) Achsc untersucht, einschlieslich der spontanen GH Sekretion, der GH Sekretion nach pharmakologischer Stimulation und der Serumspiegel des insulin-like growth factor-1 (IGF-1) sowie des IGF binding protein-3 (IGFBP-3). Sechs der Kinder hatten einc subnormale GH Sekretion entsprechend einem Wachstumshormonmangel. Eine subnormale Wachstumsgeschwindigkeit war der beste Parameter fur einen GH Mangel. Der hohe Prozentsatz von Kindern mit Wachstumshormonmangel ist uberraschend. Mogliche Ursachen sind in anatomischen Anomalien der Hypothalamus-Hypophysen-Achse, psychosozialer Deprivation und einer Interaktion zwischen suboptimalem Ernahrungsstatus und einem abnormen Zentralnervensystem zu suchen. RESUMEN Aparente deftciencia de hormona de crechniento en ninos con pardlisis cerebral Diez ninos con pardlisis cerebral (PC) y fallo en el crecimiento fueron evaluados en su hormona de crecimiento (HC) incluyendo la secrecion espontanea de HC, la secrecion de_HC en respuesta a una estimulacion farmacologica y los nivels hematicos del factor de crecimientoNI semejante a la insulina (ifc-1) y del IFC ligado a la proteina N3 (IFCBP-3; en ingles IGFBP-3). Seis de los ninos tenian una secreci6n subnormal de HC con deficiencia de HC. La velocidad de crecimiento subnormal fue el mejor predictor de la deficiencia de HC. Es sorprendente el elevado porcentaje de estos ninos con aparente deficiencia de HC. Los posibles mecanismos consisten en anomalies anatomicas del eje hipotalamico-pituitario, la deprivacion psicosocial y una interaccion entre un estado nutritivo suboptimo y un sistema nervioso central anomalo.

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.

Cell migration involves the localized extension of actin-rich protrusions, a process that requires Class I phosphoinositide 3-kinases (PI 3-kinases). Both Rac and Ras have been shown to regulate actin polymerization and activate PI 3-kinase. However, the coordination of Rac, Ras and PI 3-kinase activation during epidermal growth factor (EGF)-stimulated protrusion has not been analyzed. We examined PI 3-kinase-dependent protrusion in MTLn3 rat adenocarcinoma cells. EGF-stimulated phosphatidyl-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] levels showed a rapid and persistent response, as PI 3-kinase activity remained elevated up to 3 minutes. The activation kinetics of Ras, but not Rac, coincided with those of leading-edge PtdIns(3,4,5)P3 production. Small interfering RNA (siRNA) knockdown of K-Ras but not Rac1 abolished PtdIns(3,4,5)P3 production at the leading edge and inhibited EGF-stimulated protrusion. However, Rac1 knockdown did inhibit cell migration, because of the inhibition of focal adhesion formation in Rac1 siRNA-treated cells. Our data show that in EGF-stimulated MTLn3 carcinoma cells, Ras is required for both PtdIns(3,4,5)P3 production and lamellipod extension, whereas Rac1 is required for formation of adhesive structures. These data suggest an unappreciated role for Ras during protrusion, and a crucial role for Rac in the stabilization of protrusions required for cell motility.

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.

Demetalation of an electron-rich spirolactam-based (cyclohexadienyl)Ru(II) complex is achieved upon treatment with boron trifluoride and a source of chloride ion. The cyclohexadienyl ligand is converted to a cyclohexadienone, cyclohexenone, or cyclohexenone epoxide derivative as a function of the specific reaction conditions. Stable (cyclohexadienyl)ruthenium substrates potentially suitable for demetalation under these conditions are prepared via intramolecular spirocyclization of (η6-arene)Ru precursors; thus, demetalation completes a net Ru-mediated dearomatization sequence. Moreover, the functionalized spirolactam products obtained are structurally distinct from those produced via direct oxidative demetalation.

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.

Electron-rich alkoxy- and chloro-substituted azaspirocyclic cyclohexadienyl Ru(II) complexes have been converted to either azaspiro[4.5]decane derivatives or functionalized tetrahydroisoquinolines by treatment with suitable oxidizing agents. Copper(II) chloride was found to provide demetalated products in high yield relative to the other oxidants examined (FeCl(3), DDQ, CAN, [Cp(2)Fe][PF(6)], phenyliodine diacetate, IBX). In certain instances, the efficiency of oxidative demetalation was enhanced by the inclusion of chloride ion additives in the reaction. Pyridinium dichromate (PDC) was also found to effect the demetalation of a wide range of cyclohexadienyl Ru complexes; however, isolated yields of metal-free products were exceedingly low. The cyclohexadienyl ruthenium complexes used in this study were prepared from (arene)Ru(II) precursors; thus, the isolation of alicyclic cyclohexadienone derivatives upon demetalation constitutes completion of a Ru-mediated dearomatization process.

Cyclohexadienyl ruthenium(II) complexes incorporating an azaspirocyclic ring system have been prepared from (η6-N-benzyl acetoacetamide)CpRu(II) precursors via an intramolecular nucleophilic aromatic addition/enolate trapping reaction sequence. The spirocyclization process was found to be applicable to a variety of alkyl-, alkoxy-, and chloro-substituted N-benzyl acetoacetamide ligands, and isolated yields of the cyclohexadienyl products ranged from 44% to 86%. In contrast, related arene ruthenium complexes prepared from benzyl acetoacetate and phenethyl acetoacetamide failed to undergo spirocyclization, but were found to participate in intramolecular SNAr reactions (leading to formation of tetralone and benzazepinone ring systems, respectively). Thus, the conformational mobility of the side chain linking the nucleophilic center to the coordinated arene ring appears to be important in governing the regioselectivity of aromatic addition in these reactions. Several spirolactam complexes were reduced with Li...

Objective: To determine whether a single injection of intravenous secretin results in measurable improvements in socialization and/or communication skills in children with autism. Study design: Sixty subjects with autism were randomly selected and assigned to either treatment or placebo group. Subjects in the treatment group received 2.0 clinical units of secretin per kilogram of body weight as a single intravenous dose. Subjects in the placebo group received normal saline solution. Neurodevelopmental and behavioral assessments were performed for all subjects before injection and at 3 and 6 weeks after injection. Results: Assessment of language skills and parents’ behavioral assessments revealed no significant differences between the treatment and placebo groups. Raters’ assessments of severity of autistic symptoms did not differ for the 2 groups at 6 weeks after injection. A marginally statistically significant improvement in autistic behaviors was seen in the treatment group at 3 weeks after injection (P =.051). Conclusions: A single dose of intravenous secretin does not appear to have significant effects on either parents’ perception of autistic behaviors or language skills at 6 weeks after injection. Transient, marginally significant improvements in autistic behaviors may occur in some children. (J Pediatr 2001;138:649-55)

The rates of mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1), progression to AIDS following HIV-1 infection, and AIDS-associated mortality are all inversely correlated with serum vitamin A levels (R. D. Semba, W. T. Caiaffa, N. M. H. Graham, S. Cohn, and D. Vlahov, J. Infect. Dis. 171:1196–1202, 1995; R. D. Semba, N. M. H. Graham, W. T. Caiaffa, J. B. Margolik, L. Clement, and D. Vlahov, Arch. Intern. Med. 153:2149–2154, 1993; R. D. Semba, P. G. Miotti, J. D. Chiphangwi, A. J. Saah, J. K. Canner, G. A. Dallabetta, and D. R. Hoover, Lancet 343:1593–1596, 1994). Here we show that physiological concentrations of vitamin A, as retinol or as its metabolite, all- trans retinoic acid, repressed HIV-1 Ba-L replication in monocyte-derived macrophages (MDMs). Repression required retinoid treatment of peripheral monocytes during their in vitro differentiation into MDMs. Retinoids had no repressive effect if they were added after virus infection. Retinol, as well as all- trans retinoic acid and 9- cis retinoic acid, also repressed HIV-1 long terminal repeat (LTR)-directed expression up to 200-fold in transfected THP-1 monocytes. Analysis of HIV-1 LTR deletion mutants demonstrated that retinoids were able to repress activation of HIV-1 expression by both NF-κB and Tat. A cis -acting sequence required for retinoid-mediated repression of HIV-1 transcription was localized between nucleotides −51 and +12 of the HIV-1 LTR within the core promoter. Protein-DNA cross-linking experiments identified four proteins specific to retinoid-treated cells that bound to the core promoter. We conclude that retinoids render macrophages resistant to virus replication by modulating the interaction of cellular transcription factors with the viral core promoter.

The purpose of the current investigation was to determine cognitive developmental outcomes for a cohort of children with prenatally detected myelomeningocele and to determine whether the variables of (1) severity of ventriculomegaly and (2) anatomic level of lesion were predictive of cognitive development.Prenatal ultrasonographic examinations were reviewed by a single perinatologist to determine the degree of ventriculomegaly and the anatomic level of the lesion. Ventriculomegaly was defined as a lateral ventricular atrial width10 mm. Anatomic level of lesion was defined as (1) thoracic, (2) high lumbar, (3) midlumbar, (4) low lumbar-high sacral, or (5) sacral. Cognitive developmental quotients for surviving children were determined by one of two developmental pediatricians with use of a modified version of the Clinical Adaptive Test/Clinical Linguistic Auditory Milestone Scale, a measure of visual-motor and language abilities.The mean cognitive developmental quotient for subjects with absent to mild ventriculomegaly was 90.3 (range 54 to 120, SD 17.4), whereas the mean cognitive developmental quotient for those with moderate to severe ventriculomegaly was 74.0 (range 65 to 100, SD 17.1) (p0.01). There was a negative correlation between the degree of ventriculomegaly and the cognitive developmental quotient (r = -0.43, p0.025) and a positive correlation between the level of the lesion and the cognitive developmental quotient (r = 0.50, p0.01).The degree of ventriculomegaly determined on high-resolution prenatal ultrasonography is predictive of early cognitive development in children with myelomeningocele, with worsening ventriculomegaly being associated with lower cognitive developmental quotients. The anatomic level of the lesion also has predictive value, with lower level lesions being associated with more favorable cognitive outcomes. However, because of the high degree of variance in developmental quotients within the two ventriculomegaly groups, we advise clinicians to use caution in the interpretation and use of our data.

In many sensory systems, perception of stimuli is influenced by previous stimulus exposure such that subsequent stimuli may be perceived as more neutral. This phenomenon is known as an aftereffect and has been studied for vision, audition, and some vestibular stimuli including roll and translation. Previous data on yaw rotation perception has focused on low-frequency stimuli on the order of a minute which may not be directly applicable to frequencies during ambulation. The aim of the current study is to look at the influence of yaw rotation on subsequent perception near 1 Hz, the predominant frequency of yaw rotation during human ambulation. Humans were rotated with 12 ° whole body adapting stimulus over 1 or 1.5 s. After an interstimulus interval (ISI) of 0.5, 1.0, 1.5, or 3 s, a test stimulus the same duration as the adapting stimulus was presented, and subjects pushed a button to identify the direction of the test stimulus as right or left. The direction and magnitude of the test stimulus was adjusted based on prior responses to find the stimulus at which no rotation was perceived. Experiments were conducted both in darkness and with a visual fixation point. The presence of a fixation point did not influence the aftereffect which was largest at 0.5 s with an average size of 0.78 ± 0.18 °/s (mean ± SE). The aftereffect diminished with a time constant of ~1 s. Thresholds were elevated after the adapting stimulus and also decreased with a time constant of ~1 s. These findings demonstrate that short adapting stimuli can induce significant aftereffects in yaw rotation perception and that these aftereffects are independent from the previously described velocity storage.

The signal transducer and activator of transcription (STAT) family of transcription factors is triggered by cytokine and growth factor receptors in a number of cell types, and binds to a consensus sequence defined in part by the IFN-gamma activation site (GAS). It is not known whether these transcription factors respond to other kinds of growth stimuli, and, with particular relevance to lymphocytes, it is not known whether STAT proteins participate in Ag-specific responses. To determine the role of STAT proteins, coupling between Ag-receptor cross-linking and nuclear expression of DNA-binding protein complexes that recognize GAS sequences was evaluated. Ag-receptor triggering in primary B lymphocytes stimulated nuclear expression of a complex that specifically binds the IFN response factor-1 (IRF-1) GAS sequence, and is distinguished by electrophoretic mobility and GAS preference from IRF-1 GAS-binding complexes induced by IFN-gamma. Activation of nuclear IRF-1 GAS-binding activity by sIg was inhibited by the tyrosine kinase inhibitor, herbimycin A, and binding activity was eliminated by tyrosine phosphatase treatment. Activation of IRF-1 GAS-binding activity was blocked by depletion of protein kinase C. The IRF-1 GAS-binding activity induced by sIg engagement in B cells was transcriptionally active, and was found to consist of immunoreactive STAT5 and STAT6 proteins. This work demonstrates that the STAT signaling pathway previously associated with cytokine signaling is triggered in B lymphocytes through Ag-receptor engagement in a protein kinase C-dependent fashion. This heretofore described cytokine signaling pathway may play a role in bringing about Ag-specific proliferative and differentiative responses.

GH-secreting carcinomas of the pituitary are extremely rare. We describe a 37-yr-old woman with refractory acromegaly 15 yr after transphenoidal surgery and radiotherapy, with no evidence of a recurrent pituitary mass. Scanning with 111-indium pentetreotide revealed an area of intense activity in the left neck. A 3.5 x 2.5-cm mass was excised from the neck after demonstrating an arterio-venous GH gradient of 7:1. GH levels (50 ng/mL) dropped to 0.8 ng/mL 3 h after surgery and remained normal. GH gene expression was demonstrated in the metastasis by Northern and Western blot analyses and by positive immunocytochemistry and immunoelectron microscopy. In vitro cultured cells responded to GHRH and TRH by increasing GH levels (P < 0.01). Medium GH was identical to authentic pituitary GH, as demonstrated by high pressure liquid chromatography. RT-PCR of hypothalamic hormone receptor messenger RNA in the mass revealed somatostatin receptor subtypes 2, 3, and 5 and GHRH, TRH, and dopamine receptor expression. No GH gene amplification, rearrangement, or gsp mutation was found. RB gene deletion and H-ras mutations, previously reported in PRL- and ACTH-secreting carcinomas, were not detected. In conclusion, clinical and molecular features of a GH-secreting pituitary carcinoma are presented. This metastatic lesion synthesized GH and expressed functional hypothalamic hormone receptors.

Children treated for acute lymphoblastic leukemia, the most common cancer in childhood, are at risk for learning problems, including decreased IQ scores and deficits in mathematics, attention, and memory, both during their treatment phase and after cure. Psychosocial problems, particularly behavioral problems and social skills deficits, are reported as well. These problems occur across all age ranges but are most frequent in children who are less than 4 years old at the time of diagnosis. Up to two thirds of these children may have educational difficulties. To examine these issues more closely, the literature on developmental and psychosocial outcomes of childhood leukemia is reviewed, with particular attention to studies reporting on preschool-age children. Studies are categorized according to outcome measures used: (a) IQ or scholastic achievement tests, (b) neuropsychological performance tests, (c) memory or attention tests, and (d) psychosocial evaluation measurements. In each of these categories, evidence exists that preschool children treated for leukemia are at high risk for developmental difficulties. Suggestions are offered for developmental surveillance and educational interventions.

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.

International audience; ABSTRACT: 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 tissue. 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 from reduction mammoplasty patients, as well as primary mammary gland adenocarcinomas derived from the Polyoma Virus Middle T antigen mouse model (PyMT). MCF7 clones expressing individual GFP-tagged SEPT9 isoforms were used to determine their respective intracellular distribution and affect on cell migration. RESULTS: An overall increase in gene amplification and altered expression of SEPT9 was observed during breast tumorigenesis. We identified an intragenic alternative promoter whose 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.

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.

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.

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.

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.

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.

The authors describe two children with cerebral palsy and linear growth failure secondary to growth hormone deficiency. One of the children was successfully treated with growth hormone replacement therapy. His linear growth velocity increased from 3cm/year before therapy to 8.3 cm/year during the first two years of therapy. Potential complications such as worsening orthopedic status did not occur. Psychosocial benefits were noted. The authors conclude that growth hormone deficiency may play a role in linear growth failure in some children with cerebral palsy and that some of these children may benefit from growth hormone therapy.

A 46-year-old male sought periodontal care for a swelling on his right mandibular gingiva. An excisional biopsy revealed a well-differentiated squamous cell carcinoma. Surgical treatment consisted of a right segmental mandibulectomy with ipsilateral right neck dissection and fibular free flap reconstruction. Two days after the surgical procedure, a weakened Doppler signal suggested vascular compromise of the graft. The patient was returned to the operating room where complete thrombosis of the internal jugular vein (recipient vessel) was observed. This event prompted a complete hematological evaluation that disclosed low serum levels of protein S. The patient was started on systemic heparin and local medicinal leeches. A week later, systemic warfarin sodium was added and successfully resolved the vascular compromise of the graft. Two years later, the patient is active and lives a full life with occasional adjustments of warfarin sodium. This case represents the first report on the treatment of gingival carcinoma that led to the serendipitous discovery of an unrelated and unusual systemic condition, protein S deficiency.

Rho family members play a critical role in malignant transformation. Anchorage-independent growth and the ability to avoid apoptosis caused by loss of anchorage (anoikis) are important features of transformed cells. Here we show that constitutive activation of Rac1 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. Constitutively active Rac1-V12 decreases DNA fragmentation and caspase activity by 50% in MDCK cells kept in suspension. In addition, expression of Rac1-V12 in MDCK cells in suspension conditions causes an increase in the number of surviving cells. We also investigated the signaling pathways that are activated by Rac1 to stimulate cell survival. We show that expression of Rac1-V12 in MDCK cells in suspension stimulates a number of signaling cascades that have been implicated in the control of cell survival, including the p42/44 ERK, p38, protein kinase B, and nuclear factor kappaB pathways. Using specific chemical or protein inhibitors of these respective pathways, we show that Rac1-mediated cell survival strongly depends on phosphatidylinositol 3-kinase activity and that activation of ERK, p38, and NF-kappaB are largely dispensable for Rac1 survival signaling. In conclusion, these studies demonstrate that Rac1 can suppress apoptosis in epithelial cells in anchorage-independent conditions and suggest a potential role for Rac1-mediated survival signaling in cell transformation.

Arene ruthenium complexes possessing beta-amido phosphonate side chains participate in intramolecular spirocyclization reactions to deliver stable cyclohexadienyl ruthenium adducts. Spirocyclization is accomplished via a tandem two-step sequence that involves stereoselective nucleophilic aromatic addition to the ipso position of the coordinated arene, followed by intermolecular Horner-Wadsworth-Emmons olefination. The resulting eta5-cyclohexadienyl complexes can then be diastereoselectively converted to metal-free azaspiro[4.5]decane derivatives upon oxidative demetalation in the presence of suitable nucleophiles. An asymmetric spirocyclization was demonstrated through application of this procedure to a beta-amido phosphonate prepared from (S)-(-)-alpha-methyl benzylamine. The expected spirolactam product was obtained as a single enantiomer.

The ability of glioma tumor cells to infiltrate the surrounding brain parenchyma makes complete removal of these tumors nearly impossible. We investigated how microglia, the resident macrophages of the brain, stimulate glioma cell invasion. Coculture with microglia stimulates the invasion of GL261 glioma cells in-vitro. Pharmacological inhibition of EGFR using Iressa strongly inhibits microglia-stimulated invasion. Furthermore, we demonstrate using novel CSF-1R (c-fms) inhibitors that CSF-1R signaling is also required for microglial enhancement of glioma invasion. GL261 cells were found to constitutively secrete CSF-1, the levels of which are unaffected by EGF stimulation, EGFR inhibition or coculture with microglia. GL261 tumors orthotopically injected into the brains of C57 mice fed with the CSF-1R inhibitor PLX3397 showed a dramatic reduction in microglia/macrophages infiltration compared with tumors from animals fed control chow. Tumors from the PLX3397-fed animals also showed a 50% reduction in invasion into the parenchyma. We discovered that GL261 cell secretion of CSF-1 is involved in upregulating EGFR ligands in microglia including AREG, EREG and TGFA. Microglia isolated from GL261 tumors express AREG mRNA whereas naïve microglia from control animals do not. Depletion of AREG from microglia using siRNA interfered with their ability to promote GL261 invasion in vitro. These data indicate that during invasion, glioma and microglia cells signal to each other using EGFR Ligands and CSF-1 similar to what has been observed in breast cancer models. The results of this study indicate the possibility of inhibiting glioblastoma invasion by targeting functions of tumor-associated microglia in addition to glioma cells. Citation Format: Salvatore J. Coniglio, Jeffrey E. Segall. Mechanisms of microglia-stimulated glioma invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4928. doi:10.1158/1538-7445.AM2013-4928

The signal transducer and activator of transcription (STAT) family of transcription factors is triggered by cytokine and growth factor receptors in a number of cell types, and binds to a consensus sequence defined in part by the IFN-gamma activation site (GAS). It is not known whether these transcription factors respond to other kinds of growth stimuli, and, with particular relevance to lymphocytes, it is not known whether STAT proteins participate in Ag-specific responses. To determine the role of STAT proteins, coupling between Ag-receptor cross-linking and nuclear expression of DNA-binding protein complexes that recognize GAS sequences was evaluated. Ag-receptor triggering in primary B lymphocytes stimulated nuclear expression of a complex that specifically binds the IFN response factor-1 (IRF-1) GAS sequence, and is distinguished by electrophoretic mobility and GAS preference from IRF-1 GAS-binding complexes induced by IFN-gamma. Activation of nuclear IRF-1 GAS-binding activity by sIg was inhibited by the tyrosine kinase inhibitor, herbimycin A, and binding activity was eliminated by tyrosine phosphatase treatment. Activation of IRF-1 GAS-binding activity was blocked by depletion of protein kinase C. The IRF-1 GAS-binding activity induced by sIg engagement in B cells was transcriptionally active, and was found to consist of immunoreactive STAT5 and STAT6 proteins. This work demonstrates that the STAT signaling pathway previously associated with cytokine signaling is triggered in B lymphocytes through Ag-receptor engagement in a protein kinase C-dependent fashion. This heretofore described cytokine signaling pathway may play a role in bringing about Ag-specific proliferative and differentiative responses.

Glioblastoma multiforme (GBM) is the most malignant and lethal form of astrocytoma. GBM patients have a median survival time of approximately one year and therefore it is essential to identify novel targets and effective therapies. There is mounting evidence that microglia (specialized brain-resident macrophages) play a significant role in the development and progression of GBM tumors. We have established a microglia-glioma cell co-culture system consisting of normal microglia from C57Bl/6J mice and syngeneic GL261 glioma cells. We have demonstrated that microglia strongly stimulate the invasive behavior of GL261 cells in this system. Our preliminary data also indicate that the microglia significantly stimulate GL261 cell proliferation and resistance to ionizing radiation (IR) in these conditions. In a search for small molecule inhibitors that block the stimulatory effects of microglia on glioma cells, we have examined CNI-1493, a drug that is known to selectively interfere with macrophage function. We found that CNI-1493 potently inhibits microglia-stimulated GL261 invasion. We also examined the effect of intracranially administered CNI-1493 on orthotopic GL261 tumors in the presence or absence of fractionated whole brain irradiation (10 Gy total). We found that although CNI-1493 only has a marginal benefit in the absence of IR, it strongly stimulates animal survival in combination with IR: whereas IR treatment increases survival by 10 days, IR in combination with CNI-1493 treatment extends survival beyond 40 days. In conclusion, our observations support the notion that pharmacological targeting of microglia may be beneficial as adjuvant therapy in conjunction with ionizing radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-503. doi:1538-7445.AM2012-LB-503