Search

NFIB (Nuclear Factor I B) haploinsufficiency has recently been identified as a cause of intellectual disability and macrocephaly. Here we describe two patients with pathogenic variants in NFIB. The first is a 6-year-old Latino male with developmental delays, mild hypotonia, facial anomalies, and brain magnetic resonance imaging findings comprising mild thinning of the corpus callosum, with more marked thinning of the splenium and blunting of the rostrum and cavum septum pellucidum. Exome sequencing identified a previously described de novo variant in NFIB, c.265C>T, predicting p.Arg89Ter. The second is a 5-year-old Latino male with developmental delays, hypotonia, dysmorphic features, a preauricular tag and pit, a small ventricular septal defect, and brain magnetic resonance imaging findings including a dysmorphic corpus callosum and a small posterior fossa. A single nucleotide polymorphism microarray identified a 92 kb interstitial deletion at 9p23 including several exons of NFIB and no other known genes. Our two patients add to the knowledge of this rare condition through our addition of new brain MRI findings and dysmorphic features. Additionally, these are the first known Latino patients to be described with NFIB haploinsufficiency, expanding our understanding of the associated facial features in diverse populations. Further data are needed to determine genotype-phenotype relationships for NFIB.

Next-generation sequencing strategies have resulted in mutation detection rates of 21-61% in small cohorts of patients with microphthalmia, anophthalmia and coloboma (MAC), but despite progress in identifying novel causative genes, many patients remain without a genetic diagnosis. We studied a cohort of 19 patients with MAC who were ascertained from a population with high rates of consanguinity. Using single nucleotide polymorphism (SNP) arrays and whole exome sequencing (WES), we identified one pathogenic variant in TENM3 in a patient with cataracts in addition to MAC. We also detected novel variants of unknown significance in genes that have previously been associated with MAC, including KIF26B, MICU1 and CDON, and identified variants in candidate genes for MAC from the Wnt signaling pathway, comprising LRP6, WNT2B and IQGAP1, but our findings do not prove causality. Plausible variants were not found for many of the cases, indicating that our current understanding of the pathogenesis of MAC, a highly heterogeneous group of ocular defects, remains incomplete. This article is protected by copyright. All rights reserved.

Baraitser-Winter cerebrofrontofacial syndrome (BWCS) is a rare, autosomal dominant condition that is characterized by intellectual disability, distinctive craniofacial features, structural brain abnormalities, seizures, microcephaly, hearing loss, and ocular colobomas. The first three cases were described in 1988 by Baraitser and Winter and included two siblings and an unrelated third patient. Subsequently, causative missense variants in the ACTB and ACTG1 genes were identified, with de novo occurrence in patients with the condition. Herein, we describe two adult siblings who were born to unaffected parents and who were diagnosed with BWCS in their fourth and sixth decade of life following exome sequencing performed for intellectual disability. We review the literature reports of adult patients with BWCS to document the clinical features and phenotypic variability that can occur later in life. This is the first molecularly confirmed report of germline mosaicism in BWCS and one of only a few reports to describe two BWCS patients belonging to the same family.

We describe an 11-year old boy with severe global developmental delays, failure to thrive and growth retardation, refractory seizures with recurrent status epilepticus, hypogammaglobulinemia, hypergonadotropic hypogonadism, and duodenal strictures. He had facial and skin findings compatible with trichothiodystrophy, including sparse and brittle hair, thin eyebrows, and dry skin. Exome sequencing showed a hemizygous, truncating variant in RNF113A, c.903_910delGCAGACCA, predicting p.(Gln302fs*12), that was inherited from his mother. Although his clinical features overlap closely with features described in the two previously reported male first cousins with RNF113A loss of function mutations, the duodenal strictures seen in this patient have not been reported. Interestingly, the patient's mother had short stature and 100% skewed X-inactivation as seen in other obligate female carriers. A second male with developmental delays, microcephaly, seizures, ambiguous genitalia, and facial anomalies that included sparse and brittle hair, thin eyebrows and dry skin was recently reported to have c.897_898delTG, predicting p.(Cys299*) in RNF113A and we provide additional clinical details for this patient. This report further supports deleterious variants in RNF113A as a cause of a novel trichothiodystrophy syndrome.

Contains fulltext : 218289.pdf (Publisher’s version ) (Closed access) MN1 encodes a transcriptional co-regulator without homology to other proteins, previously implicated in acute myeloid leukaemia and development of the palate. Large deletions encompassing MN1 have been reported in individuals with variable neurodevelopmental anomalies and non-specific facial features. We identified a cluster of de novo truncating mutations in MN1 in a cohort of 23 individuals with strikingly similar dysmorphic facial features, especially midface hypoplasia, and intellectual disability with severe expressive language delay. Imaging revealed an atypical form of rhombencephalosynapsis, a distinctive brain malformation characterized by partial or complete loss of the cerebellar vermis with fusion of the cerebellar hemispheres, in 8/10 individuals. Rhombencephalosynapsis has no previously known definitive genetic or environmental causes. Other frequent features included perisylvian polymicrogyria, abnormal posterior clinoid processes and persistent trigeminal artery. MN1 is encoded by only two exons. All mutations, including the recurrent variant p.Arg1295* observed in 8/21 probands, fall in the terminal exon or the extreme 3' region of exon 1, and are therefore predicted to result in escape from nonsense-mediated mRNA decay. This was confirmed in fibroblasts from three individuals. We propose that the condition described here, MN1 C-terminal truncation (MCTT) syndrome, is not due to MN1 haploinsufficiency but rather is the result of dominantly acting C-terminally truncated MN1 protein. Our data show that MN1 plays a critical role in human craniofacial and brain development, and opens the door to understanding the biological mechanisms underlying rhombencephalosynapsis.

Developmental and epileptic encephalopathies are genetic disorders in which both the developmental disability and the frequent epileptic activity are the effect of a specific gene variant. While heterozygous variants in SCN1B have been described in families with generalized epilepsy with febrile seizures plus, Type 1, only three cases of homozygous, missense variants in SCN1B have been reported in association with autosomal recessive inheritance of a severe developmental and epileptic encephalopathy. We present two siblings who are homozygous for a novel, missense variant in SCN1B, c.265C>T, predicting p.Arg89Cys. The proband is an 11-year-old female with infantile-onset, fever-induced, intractable generalized tonic-clonic seizures, myoclonic seizures, and developmental slowing and autism spectrum disorder occurring later in the course of the disease. Her 4-year-old brother had a similar epilepsy phenotype, but still displays normal development. This variant has not been previously reported in the homozygous state in control databases. The variant was predicted to be damaging and occurred in the vicinity of other epileptic encephalopathy-associated missense variants that are biallelic and located in the extracellular immunoglobulin loop domain of the protein, which mediates interaction of the beta-1 subunit with cellular adhesion molecules. Our report is the first set of siblings with homozygosity for the p.Arg89Cys variant in SCN1B and further implicates biallelic mutations in this gene as a cause of epileptic encephalopathy mimicking Dravet syndrome. Interestingly, the phenotype we observed was milder compared to that previously described in patients with recessive SCN1B mutations.

CONTEXT AND AIMS:Breast cancer prognosis and predictive biomarkers development would allow sparing some patients from chemotherapy or identifying patients for whom chemotherapy would be indicated. In this context, in 2009, the French National Cancer Institute, a National Health and Science Agency dedicated to cancer, in collaboration with the French society of senology and breast pathology (SFSPM) published a report on the assessment of the prognostic and the predictive clinical validity of tissular biomarkers, uPA/PAI-1, Oncotype DX™ and MammaPrint(®), in breast cancer management. They concluded that only the uPA/PAI-1 prognosis value reached the highest level of evidence (LOE I according to Hayes 1998 classification). In 2012, it was decided to update this report since new data have emerged and because information disparities among clinicians have been identified. This article aims to present the main conclusions together with the levels of evidence associated with those conclusions.METHODS:The updating process was based on literature published since 2009 appraisal and on multidisciplinary and independent experts' opinion. The levels of evidence (LOE) used are those of the classification defined by Simon in 2009 (updated Hayes 1998 classification): LOE IA and LOE IB: high level of evidence; LOE IIB and LOE IIC: intermediate level of evidence; LOE IIIC and LOE IV-VD: low level of evidence.CONCLUSIONS:Among patients without lymph-node involvement, uPA/PAI-1, invasion process biomarkers, reach the highest level of evidence for 10 years recurrence free survival prognosis (LOE IA according to Simon). The predictive value to anthracyclins chemotherapy remains to be confirmed. Oncotype DX™ and MammaPrint(®) prognosis and predictive value do not reach the LOE I level. This updating' process confirms the 2009 levels of evidence for all the three biomarkers prognosis value. Besides, concerning Oncotype DX™ and MammaPrint(®), new data do not allow to conclude neither to their complementary clinical information to other clinicopathological existing biomarkers nor to a favorable cost-efficiency ratio in therapeutic decision making and this because of the methodological weakness and uncertainty that are identified in the selected studies. Practically, beyond the prognosis and predictive biomarkers validity, the clinical utility of a new biomarker for chemotherapy indication depends on its clinical added information with regard to validated biomarkers (HR, HER2 and Ki67) and to clinicopathological parameters. Since they are the sole validated biomarkers of the invasion process, uPA/PAI-1 could complete clinical information of other clinicopathological factors and consequently could confer an added clinical value. However, data concerning the impact of this information on chemotherapy clinical indication are lacking.Copyright © 2014. Published by Elsevier Masson SAS., IntroductionDans le cancer du sein, le développement des marqueurs biologiques pronostiques ou prédictifs a pour objectif de mieux identifier les patientes pour lesquelles un traitement par chimiothérapie pourrait être évité ou a contrario indiqué. Dans ce contexte, en 2009, l’Institut national du cancer (INCa), agence sanitaire et scientifique de l’État chargée de coordonner les actions de lutte contre le cancer, avait publié en partenariat avec la Société française de sénologie et de pathologie mammaire un rapport sur l’état des connaissances relatives aux biomarqueurs uPA/PAI-1, Oncotype DX™ et MammaPrint® dans la prise en charge du cancer du sein. Ce rapport avait montré que seule la valeur pronostique d’uPA/PAI-1 atteignait le plus haut niveau de preuve (LOE I selon la grille de Hayes 1998). En 2012, devant la parution de nouvelles publications et la divergence des messages diffusés sur les signatures moléculaires, il a été décidé d’actualiser le rapport de 2009. Cet article présente les principales conclusions accompagnées de leurs niveaux de preuve.MéthodeLe processus de mise à jour s’est appuyé sur l’analyse des données publiées depuis la recherche bibliographique de 2009, complétée par l’avis d’un groupe de travail multidisciplinaire indépendant. Les niveaux de preuve employés sont ceux de la classification définie par Simon en 2009 (grille de Hayes 1998 après mise à jour) : LOE IA et LOE IB : niveau de preuve élevé ; LOE IIB and LOE IIC : niveau de preuve intermédiaire ; LOE IIIC and LOE IV-VD : niveau de preuve faible.ConclusionsChez les patientes sans envahissement ganglionnaire (pN0), uPA/PAI-1, marqueurs d’invasion, ont un niveau de preuve élevé (LOE IA selon Simon) pour la valeur pronostique de la survie sans récidive à 10 ans. Il reste à confirmer leur valeur prédictive de réponse aux anthracyclines. Pour Oncotype DX™ et MammaPrint®, les valeurs pronostique et prédictive n’ont pas atteint à ce jour le niveau de preuve LOE I. Ce travail confirme les niveaux de preuve précédemment établis dans le rapport de 2009. Par ailleurs, les données ne permettent pas de conclure à une valeur ajoutée de Oncotype DX™ et MammaPrint® par rapport aux outils existants. Les données médico-économiques ne permettent pas de statuer sur le rapport coût/efficacité des stratégies utilisant ces tests dans la décision thérapeutique compte tenu d’un niveau de qualité insuffisant pour la plupart des études et d’une forte incertitude mise en évidence par les quelques études bien menées. En pratique, au-delà des niveaux de preuve attribuables à la valeur pronostique et prédictive d’un biomarqueur, l’utilité clinique d’un nouveau marqueur dans l’aide à la prescription d’une chimiothérapie repose sur sa valeur ajoutée par rapport aux marqueurs validés (RH, HER2 et les marqueurs de prolifération comme Ki67) et aux critères anatomo-cliniques. Puisqu’ils sont les seuls marqueurs validés à témoigner du processus d’invasion, uPA/PAI-1 peuvent apporter une information complémentaire et donc avoir une valeur ajoutée par rapport aux marqueurs existants. Les données de la littérature manquent pour apprécier le poids de cette valeur ajoutée dans la décision de prescrire ou non une chimiothérapie.

Mice lacking DIX domain containing-1 (DIXDC1), an intracellular Wnt/β-catenin signal pathway protein, have abnormal measures of anxiety, depression and social behavior. Pyramidal neurons in these animals' brains have reduced dendritic spines and glutamatergic synapses. Treatment with lithium or a glycogen synthase kinase-3 (GSK3) inhibitor corrects behavioral and neurodevelopmental phenotypes in these animals. Analysis of DIXDC1 in over 9000 cases of autism, bipolar disorder and schizophrenia reveals higher rates of rare inherited sequence-disrupting single-nucleotide variants (SNVs) in these individuals compared with psychiatrically unaffected controls. Many of these SNVs alter Wnt/β-catenin signaling activity of the neurally predominant DIXDC1 isoform; a subset that hyperactivate this pathway cause dominant neurodevelopmental effects. We propose that rare missense SNVs in DIXDC1 contribute to psychiatric pathogenesis by reducing spine and glutamatergic synapse density downstream of GSK3 in the Wnt/β-catenin pathway.

The remodeling of calcium homeostasis contributes to the cancer hallmarks and the molecular mechanisms involved in calcium channel regulation in tumors remain to be characterized. Here, we report that SigmaR1, a stress-activated chaperone, is required to increase calcium influx by triggering the coupling between SK3, a Ca2+-activated K+ channel (KCNN3) and the voltage-independent calcium channel Orai1. We show that SigmaR1 physically binds SK3 in BC cells. Inhibition of SigmaR1 activity, either by molecular silencing or by the use of sigma ligand (igmesine), decreased SK3 current and Ca2+ entry in breast cancer (BC) and colorectal cancer (CRC) cells. Interestingly, SigmaR1 inhibition diminished SK3 and/or Orai1 levels in lipid nanodomains isolated from BC cells. Analyses of tissue microarray from CRC patients showed higher SigmaR1 expression levels in cancer samples and a correlation with tumor grade. Moreover, the exploration of a cohort of 4937 BC patients indicated that high expression of SigmaR1 and Orai1 channels was significantly correlated to a lower overall survival. As the SK3/Orai1 tandem drives invasive process in CRC and bone metastasis progression in BC, our results may inaugurate innovative therapeutic approaches targeting SigmaR1 to control the remodeling of Ca2+ homeostasis in epithelial cancers.

Objective To try and identify a molecular signature for pathological staging and/or grading. through microarray analysis. Patients and Methods We performed a prospective multicentre study between September 2007 and May 2008 that included 108 bladder tumours (45 pTa, 35 pT1 and 28>pT1). Microarray analysis was performed using Agilent Technologies Human Whole Genome 4 × 44K oligonucleotide microarrays (Agilent, Santa Clara, CA, USA). A ‘dual colour’ method was used vs a reference pool of tumours. From the lists of genes provided by the Biometric Research Branch class comparison analyses, we validated the microarray results of 38 selected differentially expressed genes using reverse transcriptase quantitative PCR in another bladder tumour cohort (n = 95). Results The cluster ‘superficial vs invasive stage’ correctly classified 92.9% of invasive stages and 66.3% of superficial stages. Among the superficial tumours, the cluster analysis showed that pT1b tumours were closer to invasive stages than pT1a tumours. We also found molecular differences between low and high grade superficial tumours, but these differences were less well defined than the difference observed for staging. Conclusions We confirmed that the histopathological classification into subgroups pTa, pT1a and pT1b can be translated into a molecular signature with a continuous progression of deregulation (overexpression or repression of these genes) from superficial (pTa) to more invasive (pT1a then b) stages.

International audience; The nodes of Ranvier are essential regions for action potential conduction in myelinated fibers. They are enriched in multimolecular complexes composed of voltage-gated Nav and Kv7 channels associated with cell adhesion molecules. Cytoskeletal proteins ankyrin-G (AnkG) and βIV-spectrin control the organization of these complexes and provide mechanical support to the plasma membrane. IQCJ-SCHIP1 is a cytoplasmic protein present in axon initial segments and nodes of Ranvier. It interacts with AnkG and is absent from nodes and axon initial segments of βIV-spectrin and AnkG mutant mice. Here, we show that IQCJ-SCHIP1 also interacts with βIV-spectrin and Kv7.2/3 channels and self-associates, suggesting a scaffolding role in organizing nodal proteins. IQCJ-SCHIP1 binding requires a βIV-spectrin-specific domain and Kv7 channel 1-5-10 calmodulin-binding motifs. We then investigate the role of IQCJ-SCHIP1 in vivo by studying peripheral myelinated fibers in Schip1 knock-out mutant mice. The major nodal proteins are normally enriched at nodes in these mice, indicating that IQCJ-SCHIP1 is not required for their nodal accumulation. However, morphometric and ultrastructural analyses show an altered shape of nodes similar to that observed in βIV-spectrin mutant mice, revealing that IQCJ-SCHIP1 contributes to nodal membrane-associated cytoskeleton organization, likely through its interactions with the AnkG/βIV-spectrin network. Our work reveals that IQCJ-SCHIP1 interacts with several major nodal proteins, and we suggest that it contributes to a higher organizational level of the AnkG/βIV-spectrin network critical for node integrity.

// Zohra Benyahia 1 , Nadege Dussault 1 , Mylene Cayol 1 , Romain Sigaud 1 , Caroline Berenguer-Daize 1 , Christine Delfino 1 , Asma Tounsi 1 , Stephane Garcia 2 , Pierre-Marie Martin 1 , Kamel Mabrouk 3 , L’Houcine Ouafik 1, 4 1 Aix Marseille Univ, The Institut National pour la Recherche Medicale, Centre de Recherche en Oncologie et Oncopharmacologie, UMR 911, 13005, Marseille, France 2 Assistance Publique Hopitaux de Marseille, Laboratoire d’Anatomie Pathologique, 13015, Marseille, France 3 Aix Marseille Univ, CNRS, ICR, UMR 7273 CROPS, 13397, Marseille, France 4 Assistance Publique Hopitaux de Marseille, Service de Transfert d’Oncologie Biologique, 13015, Marseille, France Correspondence to: L’Houcine Ouafik email: lhoucine.ouafik@univ-amu.fr Keywords: adrenomedullin, breast cancer, myofibroblasts, invasion, tumor growth Received: July 20, 2016 Accepted: January 03, 2017 Published: February 02, 2017 ABSTRACT Tumor- or cancer-associated fibroblasts (TAFs or CAFs) are active players in tumorigenesis and exhibit distinct angiogenic and tumorigenic properties. Adrenomedullin (AM), a multifunctional peptide plays an important role in angiogenesis and tumor growth through its receptors calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). We show that AM and AM receptors mRNAs are highly expressed in CAFs prepared from invasive breast carcinoma when compared to normal fibroblasts. Immunostaining demonstrates the presence of immunoreactive AM and AM receptors in the CAFs (n = 9). The proliferation of CAFs is decreased by anti-AM antibody (αAM) and anti-AM receptors antibody (αAMR) treatment, suggesting that AM may function as a potent autocrine/paracrine growth factor. Systemic administration of αAMR reduced neovascularization of in vivo Matrigel plugs containing CAFs as demonstrated by reduced numbers of the vessel structures, suggesting that AM is one of the CAFs-derived factors responsible for endothelial cell-like and pericytes recruitment to built a neovascularization. We show that MCF-7 admixed with CAFs generated tumors of greater volume significantly different from the MCF-7 xenografts in nude mice due in part to the induced angiogenesis. αAMR and AM 22-52 therapies significantly suppressed the growth of CAFs/MCF-7 tumors. Histological examination of tumors treated with AM 22-52 and aAMR showed evidence of disruption of tumor vasculature with depletion of vascular endothelial cells, induced apoptosis and decrease of tumor cell proliferation. Our findings highlight the importance of CAFs-derived AM pathway in growth of breast carcinoma and in neovascularization by supplying and amplifying signals that are essential for pathologic angiogenesis.

International audience; Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through calcitonin receptor-like receptor/receptor activity-modifying protein-2 and-3 (CLR/RAMP2 and CLR/RAMP3). In this study, real-time quantitative reverse transcription demonstrated a significant expression of AM mRNA in tumor samples from colorectal cancer (CRC) patients in clinical stage II, III, and IV when compared with normal colorectal tissue. AM, CLR, RAMP2, and RAMP3 proteins were immunohistochemically localized in the carcinomatous epithelial compartment of CRC tissue. Tissue microarray analysis revealed a clear increase of AM, CLR, RAMP2, and RAMP3 staining in lymph node and distant metastasis when compared with primary tumors. The human colon carcinoma cells HT-29 expressed and secreted AM into the culture medium with a significant increase under hypoxia. Treatment of HT-29 cells with synthetic AM stimulated cell proliferation and invasion in vitro. Incubation with anti-AM antibody (aAM), anti-AM receptors antibodies (aAMR), or AM antagonist AM 22–52 inhibited significantly basal levels of proliferation of HT-29 cells, suggesting that AM may function as an autocrine growth factor for CRC cells. Treatment with aAM significantly suppressed the growth of HT-29 tumor xenografts in vivo. Histological examination of aAM-treated tumors showed evidence of disruption of tumor vascularity with decreased microvessel density, depletion of endothelial cells and pericytes, and increased tumor cell apoptosis. These findings highlight the potential importance of AM and its receptors in the progression of CRC and support the conclusion that aAM treatment inhibits tumor growth by suppression of angiogenesis and tumor growth, suggesting that AM may be a useful therapeutic target.

To systematically review the evidence for the use of PSA and other biomarkers in the early detection of prostate cancer, we searched PubMed for clinical trials and studies assessing PSA and other biomarkers in the early detection of prostate cancer, published between 2000 and May 2013 that included >200 subjects. The level of evidence (LOE) for clinical utility was evaluated using the tumor marker utility grading system. A total of 84 publications, corresponding to 70 trials and studies were selected for inclusion in this review. We attributed a level of evidence (LoE) of IA to PSA for early PCa detection, but we do not recommend its use in mass screening. Emerging biomarkers were assessed in prospective case-control and cohort studies: PCA3 (n=3); kallikreins (n=3); [-2]proPSA (n=5); fusion oncogenes (n=2). These studies used biopsy results for prostate cancer to determine specificity and sensitivity, but they did not assess the effect on PCa mortality. The LoE attributed was III-C. PSA can be used for early prostate cancer detection but mass screening is not recommended. Studies on other biomarkers suggest that they could be used, individually or in combination, to improve the selection of patients with elevated PSA levels for biopsy, but RCTs assessing their impact on prostate cancer management and mortality are needed. A better use of available tests is possible for men at risk in order to maximize the risk-benefit ratio.

Withdrawn by Author Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-13-02.

Clinicians can use biomarkers to guide therapeutic decisions in estrogen receptor positive (ER+) breast cancer. One such biomarker is cellular proliferation as evaluated by Ki-67. This biomarker has been extensively studied and is easily assayed by histopathologists but it is not currently accepted as a standard. This review focuses on its prognostic and predictive value, and on methodological considerations for its measurement and the cut-points used for treatment decision. Data describing study design, patients’ characteristics, methods used and results were extracted from papers published between January 1990 and July 2010. In addition, the studies were assessed using the REMARK tool. Ki-67 is an independent prognostic factor for disease-free survival (HR 1.05–1.72) in multivariate analyses studies using samples from randomized clinical trials with secondary central analysis of the biomarker. The level of evidence (LOE) was judged to be I-B with the recently revised definition of Simon. However, standardization of the techniques and scoring methods are needed for the integration of this biomarker in everyday practice. Ki-67 was not found to be predictive for long-term follow-up after chemotherapy. Nevertheless, high KI-67 was found to be associated with immediate pathological complete response in the neoadjuvant setting, with an LOE of II-B. The REMARK score improved over time (with a range of 6–13/20 vs. 10–18/20, before and after 2005, respectively). KI-67 could be considered as a prognostic biomarker for therapeutic decision. It is assessed with a simple assay that could be standardized. However, international guidelines are needed for routine clinical use.

Antiangiogenic therapies are used for advanced clear-cell renal carcinomas (cRCC), but without curative possibilities, underlining the need for new therapeutic targets. Adrenomedullin (AM), a multifunctional peptide, is highly expressed in several tumors and plays an important role in angiogenesis and tumor growth through its receptors: calcitonin receptor-like receptor/receptor activity-modifying protein 2 and 3 (CLR/RAMP2 and CLR/RAMP3). In this study, real-time quantitative reverse-transcription-PCR showed AM mRNA levels were higher in cRCC and in chromophobe renal carcinomas (chRCC) than in normal renal tissue. Interestingly, AM mRNA expression in cRCC correlated strongly with VEGF-A mRNA expression. Immunohistochemically, AM, CLR and RAMP2 were localized in the carcinomatous epithelial compartment of cRCC. Interestingly, RAMP3 immunostaining was found only in the inflammatory cells that infiltrated tumors, suggesting a cross talk between tumor cells and the microenvironment. We also observed that cRCC cells BIZ and 786-O expressed and secreted AM into the culture medium. In vitro, exogenous AM treatment stimulated cell proliferation, migration and invasion, indicating the cell can respond to AM. The action of AM was specific and was mediated by the CLR/RAMP2 and CLR/RAMP3 receptors. Clinical data showed the prognostic value of AM. High AM mRNA levels were associated with an increased risk of relapse after curative nephrectomy for cRCC. These findings highlight the implication of the AM pathway in the metastatic process and the prognostic relevance of AM in cRCC and point to a potential new therapeutic target.

Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). Previously, we reported on the development of an anti-AM antibody that potently inhibits tumor cell proliferation in vitro and tumor growth in vivo. Here, we report the effect of anti-AM receptor antibodies (alphaAMRs) on angiogenesis and tumor growth. We demonstrate that alphaAMRs decrease in a dose-dependent manner the growth of U87 glioblastoma cells and HT-29 colorectal cancer cells, but not A549 lung cancer cells, in vitro. In vivo, AM in Matrigel plugs induces angiogenesis by promoting recruitment of endothelial cells, pericytes, myeloid precursor cells, and macrophages and by promoting channel formation. Remarkably, systemic administration of alphaAMRs every 3 d markedly reduced neovascularization of Matrigel plugs in a dose-dependent fashion, as demonstrated by reduced numbers of the recruited cells and vessel structures. Several human tumor xenografts in athymic mice were used to examine the effect of alphaAMR treatment on tumor angiogenesis and growth. AlphaAMR treatment significantly suppressed the growth of glioblastoma, lung, and colon tumors. Histological examination of alphaAMR-treated tumors showed evidence of disruption of tumor vascularity with decreased microvessel density, depletion of endothelial and pericyte cells, and increased tumor cell apoptosis. These findings support the conclusion that alphaAMR treatment inhibits tumor growth by suppression of angiogenesis and tumor growth and suggest that AMRs may be useful therapeutic targets.

Purpose This phase II study evaluated the efficacy and safety of a 7-day on/7-day off regimen of temozolomide before radiotherapy (RT) in patients with inoperable newly diagnosed glioblastoma. Patients and Methods Patients received temozolomide (150 mg/m2/d on days 1 to 7 and days 15 to 21 every 28 days; 7 days on/7 days off) for up to four cycles before conventional RT (2-Gy fractions to a total of 60 Gy) and for four cycles thereafter or until disease progression. The primary end point was tumor response. Tumor tissue from 25 patients was analyzed for O6-methylguanine-DNA methyltransferase (MGMT) expression. Results Twenty-nine patients with a median age of 60 years were treated, and 28 were assessable for response. Seven (24%) of 29 patients had a partial response, nine patients (31%) had stable disease, and 12 patients (41%) had progressive disease. Median progression-free survival (PFS) time was 3.8 months, and median overall survival (OS) time was 6.1 months. Patients with low MGMT expression, compared with patients with high MGMT expression, had a significantly higher response rate (55% v 7%, respectively; P = .004) and improved PFS (median, 5.5 v 1.9 months, respectively; P = .009) and OS (median, 16 v 5 months, respectively; P = .003). The most common grade 3 and 4 toxicities were thrombocytopenia (20%) and neutropenia (17%). Conclusion This dose-dense temozolomide regimen resulted in modest antitumor activity with an acceptable safety profile in the neoadjuvant setting, and expression of MGMT correlated with response to temozolomide. However, this treatment approach seems to be inferior to standard concomitant RT plus temozolomide.

Molecular analyses of large numbers of patient samples are increasingly used for diagnostic applications as well as for understanding cancer biology. Their accuracy depends on the quality and quantity of nucleic acids extracted from human cells or tissues. To optimize these preanalytical steps, we evaluated several automated technologies for nucleic acid purification from clinical samples. Three automated platforms were compared. DNA was extracted from peripheral blood leukocytes from five normal individuals, and its quality was assessed by D-HPLC and sequencing after PCR. Clinical samples from acute leukemia patients were used for automated RNA extractions; results were compared to our standard manual technique. RNA qualification was done using capillary gel electrophoresis and analysis of the Abelson gene transcript by real-time PCR. One robot produced higher total output for both DNA and RNA. While the quality of DNAs obtained from the three workstations allowed implementation of their analysis for det...

Background Triple negative breast cancers (TNBC) are a more aggressive subset of breast cancer. A better understanding of its biology could allow the rational development of targeted therapies. Methods We extensively analyzed the EGFR/PI3K/PTEN axis in a large, homogeneous population of TNBC to help defining the putative role of anti-EGFR and -PI3K targeted therapies in this setting. EGFR gene amplification, EGFR protein expression, PIK3CA and PTEN gene alterations (two members of EGFR downstream pathways) and their clinicopathological and prognostic implications were analyzed in 204 TNBC samples from European patients. Results EGFR amplification was detected in 18 of the 204 TNBC specimens (8.9 %) and was significantly associated with higher EGFR protein levels. Fourteen PIK3CA mutations were identified in exon 9 (6.7 %), and 17 in exon 20 (8.3 %). PIK3CA mutations, especially in exon 9, were significantly associated with grade I-II tumors. PTEN deletions were detected in 43 samples (21.50 %) and were significantly associated with grade III tumors (p

Members of the Dact protein family initially were identified through binding to Dishevelled (Dvl), a cytoplasmic protein central to Wnt signaling. During mouse development, Dact1 is detected in the presomitic mesoderm and somites during segmentation, in the limb bud mesenchyme and other mesoderm-derived tissues, and in the central nervous system (CNS). Dact2 expression is most prominent during organogenesis of the thymus, kidneys, and salivary glands, with much lower levels in the somites and in the developing CNS. Dact3, not previously described in any organism, is expressed in the ventral region of maturing somites, limb bud and branchial arch mesenchyme, and in the embryonic CNS; of the three paralogs, it is the most highly expressed in the adult cerebral cortex. These data are consistent with studies in other vertebrates showing that Dact paralogs have distinct signaling and developmental roles and suggest they may differentially contribute to postnatal brain physiology.

OBJECTIVE: To investigate the respective role of fractionated radiotherapy (FR) and gamma knife stereotactic (GKS) radiosurgery in cavernous sinus meningioma (CSM) treatment. METHODS: The authors report the long-term follow-up of two populations of patients harboring CSMs treated either by FR (Group I, 38 patients) or GKS radiosurgery (Group II, 36 patients). There were 31 females with a mean age of 53 years in Group I and 29 females with a mean age of 51.2 years in Group II. In 20 patients (Group I) and 13 patients (Group II), FR and GKS radiosurgery were performed as an adjuvant treatment. In 18 patients (Group I) and in 23 patients (Group II), FR and GKS radiosurgery were performed as first line treatment. In our early experience with GKS radiosurgery (1992, date of gamma knife availability in the department), patients with tumors greater than 3 cm, showing close relationship with the optic apparatus ( RESULTS: The median follow-up period was 88.6 months (range, 42–168 mo) for Group I and 63.6 months (range, 48–92 mo) for Group II. According to Sekhar's classification, 26 (68.4%) patients were Grade III to IV in Group I and 10 (27.8%) patients in Group II (P < 0.05); 23 (60.5%) patients had extensive lesions in Group I and 7 (19.4%) patients in Group II (P < 0.05). Mean tumor volume was 13.5 cm3 in Group I and 5.2 cm3 in Group II (P < 0.05). Actuarial progression-free survival was 94.7% and 94.4% in Group I and II, respectively. Clinically, improvement was seen for 24 (63.2%) patients in Group I and for 21 (53.8%) patients in Group II (P > 0.05). Radiologically, 11 (29%, Group I) patients and 19 (Group II, 52.7%) patients showed tumor shrinkage (P = 0.04). Transient morbidity was 10.5% in Group I and 2.8% in Group II. Permanent morbidity was 2.6% in Group I and 0% in Group II. CONCLUSION: FR and GKS radiosurgery are safe and efficient techniques in treatment of CSMs, affording comparable satisfactory long-term tumor control. However, GKS radiosurgery provides better radiological response, is far more convenient, and fits into most patients lives much better than FR. Therefore, in the authors' opinion, GKS radiosurgery should be advocated in first intention for patients with CSMs, whereas conventional radiotherapy should be reserved for cases that are not amenable to this technique, thus making these two therapeutic modalities not alternative but complementary tools in CS meningioma treatment strategy.

Amphireguline (AR) is an epidermal growth factor (EGF)-related peptide that seems to play an important role in breast cancer progression. We have demonstrated recently that suppression of AR expression in transformed breast epithelial cells considerably reduced both size and neovascularization of tumors developed in nude mice. We show that the reduction of AR expression allowed to an important decrease of the levels of urokinase-type plasminogen activator (uPA) and transforming growth factor-beta1 (TGFbeta1). According to these data, exogenous AR (10(-10) M-10(-8) M) stimulated the production of uPA and TGFbeta1 in AR antisense-transfected A2-15 and A2-P17F25 cells. The addition of 2 x 10(-10) M TGFbeta1 into culture medium increased the level of uPA produced by AR-expressing parental cells but not by A2-15 and A2-P17F25 cell clones. Whereas AR alone stimulated uPA production to 200% of control, combined AR and TGFbeta1 treatment increased protease level in A2-15 and A2-P17F25 cells to 500-600% of control, demonstrating a synergism between TGFbeta1 and AR. This was accompanied by an important augmentation of the number of tumoral cells that invaded matrigel in vitro. The synergistic induction of uPA protein resulted of an early and transient augmentation of steady state mRNA level and was blocked in the presence of the MAP kinase kinase inhibitor PD098059, strongly suggesting that synergistic effect of AR and TGFbeta1 on uPA expression required MAPK pathway. This data demonstrates concerted action between AR and TGFbeta1 that may have profound effect on protease production and consequently on breast cancer progression.

International audience; PURPOSE: To develop a prognostic nomogram to predict freedom from recurrence for patients treated with adjuvant hormonal therapy (HT) for localised breast cancer (BC).METHODS: We performed a retrospective analysis of 142 patients treated with adjuvant HT between 1996 and 2000. Clinical and pathological parameters were analysed.RESULTS: A nomogram that predicts the probability of remaining free of recurrence for 5 years after surgery with adjuvant HT was developed using a Cox proportional hazards regression model. The progesterone receptor status (p < 0.001), nodal status (p = 0.008) and cathepsin-D (p < 0.001) were retained to construct the nomogram (C-index 0.734).CONCLUSIONS: The nomogram we developed may be useful for estimating the probability of successful treatment 5 years after surgery for localised BC.

International audience; The presence of the Epstein-Barr-virus (EBV) has been reported to be a pathogenic factor in breast cancer (BC). We previously demonstrated the aggressiveness of EBV-positive BC. The purpose of the present study was to evaluate the effect of EBV on the prognosis of BC according to the BC phenotype. A total of 117 patients with primary BC previously tested for the presence of EBV were evaluated. The presence of the virus was evaluated in breast specimens using quantitative PCR (qPCR). Disease-free survival (DFS) and overall survival (OS) were evaluated for 4 molecular subtypes, namely luminal A and B (lumA and lumB, respectively), human epidermal growth factor receptor 2 (HER2) and triple-negative (TN) subtypes and according to the EBV status. EBV positivity was observed in 32.5% of the cases. TN, HER2 and lumB tumours were more frequent among EBV-BC cases (P=0.02). The DFS rates were different between BC subtypes (P=0.002), but the differences were not statistically significant when the cases were stratified according to the EBV status (P=0.08 for EBV-negative and 0.06 for EBV-positive cases). The OS rates were similar for BC subtypes (P= 0.50) and when the cases were stratified according to the EBV status (P=0.16 and P=0.67 for EBV-positive and -negative cases, respectively). EBV was not associated with DFS or OS, in contrast to BC phenotypes, tumour size or nodal status. Therefore, EBV positivity was found to exert no effect on survival, despite its association with aggressive BC phenotypes.

SCHIP1 is a cytoplasmic partner of cortical cytoskeleton ankyrins. The IQCJ-SCHIP1 isoform is a component of axon initial segments and nodes of Ranvier of mature axons in peripheral and central nervous systems, where it associates with membrane complexes comprising cell adhesion molecules. SCHIP1 is also expressed in the mouse developing central nervous system during embryonic stages of active axonogenesis. Here, we identify a new and early role for SCHIP1 during axon development and establishment of the anterior commissure (AC). The AC is composed of axons from the piriform cortex, the anterior olfactory nucleus and the amygdala. Schip1 mutant mice displayed early defects in AC development that might result from impaired axon growth and guidance. In addition, mutant mice presented a reduced thickness of the piriform cortex, which affected projection neurons in layers 2/3 and was likely to result from cell death rather than from impairment of neuron generation or migration. Piriform cortex neurons from E14.5 mutant embryos displayed axon initiation/outgrowth delay and guidance defects in vitro. The sensitivity of growth cones to semaphorin 3F and Eph receptor B2, two repulsive guidance cues crucial for AC development, was increased, providing a possible basis for certain fiber tract alterations. Thus, our results reveal new evidence for the involvement of cortical cytoskeleton-associated proteins in the regulation of axon development and their importance for the formation of neuronal circuits.

PURPOSE: Most primary oligodendrogliomas and mixed gliomas (oligoastrocytoma) respond to treatment with procarbazine, lomustine, and vincristine (PCV), with response rates of approximately 80%. However, limited data on second-line treatments are available in patients with recurrent tumors. A novel second-generation alkylating agent, temozolomide, has recently demonstrated efficacy and safety in patients with recurrent glioblastoma multiforme and anaplastic astrocytoma. This study describes the effects of temozolomide in patients with recurrent anaplastic oligodendroglioma (AO) and anaplastic mixed oligoastrocytoma (AOA). PATIENTS AND METHODS: Forty-eight patients with histologically confirmed AO or AOA who had received previous PCV chemotherapy were treated with temozolomide (150 to 200 mg/m2/d for 5 days per 28-day cycle). The primary end point was objective response. Secondary end points included progression-free survival (PFS), time to progression, overall survival (OS), safety, and tolerability. RESULTS: Eight patients (16.7%) experienced a complete response, 13 patients (27.1%) experienced a partial response (objective response rate, 43.8%), and 19 patients (39.6%) experienced stable disease. For the entire treatment group, median PFS was 6.7 months and median OS was 10 months. For objective responders, median PFS was 13.1 months and median OS was 16 months. For complete responders, PFS was more than 11. 8 months and OS was more than 26 months. Response correlated with improved survival. Temozolomide was safe and well tolerated. Twelve patients developed grade 1/2 thrombocytopenia and three patients developed grade 3/4 thrombocytopenia. CONCLUSION: Temozolomide is safe and effective in the treatment of recurrent AO and AOA.

Amphiregulin (AR) is a heparin-binding epidermal growth factor (EGF)-related peptide that seems to play an important role in mammary epithelial cell growth regulation. We have investigated the regulation of AR-gene expression and -protein secretion by EGF in normal breast epithelial cells (HMECs), as well as in the tumoral breast epithelial cell lines MCF-7 and MDA-MB231. EGF induced a dose-dependent increase of AR mRNA level in both normal and tumoral cells. Thus, 10−8M EGF stimulated AR expression in HMECs to 140–300% of control. A similar EGF concentration increased AR mRNA level to 550% and 980% of control in MCF-7 and MDA-MB231 cells, respectively. This was accompanied by an accumulation of AR into conditioned culture media. However, HMECs secreted in response to EGF, 5–10 fold more AR than tumour cells. Furthermore, the potential participation of AR in the regulation of the plasminogen activator (PA)/plasmin system was investigated. Whereas HMEC-proliferation was stimulated by AR, the levels of secreted urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAi-1) remained unaffected. Conversely, AR failed to regulate the proliferation of tumoral cell lines but induced an accumulation of uPA and PAi-1 into culture media. This was accompanied by an increase of the number of tumoral cells that invaded matrigel in vitro. Moreover, the presence of a neutralizing anti-uPA receptor antibody reversed the increased invasiveness of MDA-MB231 cells induced by AR. These data reveal differential behaviour of normal versus tumoral breast epithelial cells in regard to the action of AR and demonstrate that, in a number of cases, AR might play a significant role in tumour progression through the regulation of the PA/plasmin system. © 2001 Cancer Research Campaign http://www.bjcancer.com

S-phase fraction (SPF) is a reference for cell-kinetic analysis. In this study, the links between SPF and the essential enzymes participating in the pyrimidine synthesis were investigated in breast cancer and their relationships with the natural history of the disease were compared. We measured thymidine kinase (TK) for salvage synthesis, thymidylate synthase (TS) for de novo synthesis and thymidylate kinase (TMK), which is required for both pathways. Our study population consisted of 211 premenopausal women with node-negative tumors. SPF was assessed prospectively by flow cytometry, whereas enzyme activities were measured retrospectively in cytosols using radioenzymatic methods. Among the enzymes analyzed, only TK demonstrated a strong correlation with SPF (rs = 0.59). In univariate analysis, high SPF and high levels of TK were associated with increased risk of developing distant recurrences (p < 0.001). Correlations with other prognostic factors (histological grade, steroid receptors, DNA ploidy status, urokinase plasminogen activator and plasminogen activator inhibitor type 1) confirmed a parallel association of SPF and TK with the most aggressive tumors. In contrast, TS and TMK were not associated with prognosis. After adjustment for SPF, the risk of relapse increased significantly with TK values. Subgroup analysis showed that additional information was provided by TK in the tumors with low SPF. When urokinase plasminogen activator (uPA) was a candidate variable in multivariate analysis, TK remained significant. Combined with SPF and uPA, TK could be useful to define premenopausal node-negative patients with rapidly proliferating tumors at a high risk of metastatic disease. © 2001 Wiley-Liss, Inc.

Recently, we developed a method to quantitatively study tumour cell heterogeneity in terms of both nuclear size and estrogen receptor (ER) content by image cytometry. The method, previously used to analyse the proliferation of the breast cancer cell line MCF‐7, was applied here to analyse the growth of this cell line under estradiol (E2), hydroxytamoxifen (OH‐TAM), and both E2 and OH‐TAM treatments. The method extracts characteristic parameters of single nuclei and features that measure the global and local organisation of the cells in their growing phase. Modifications of the heterogeneity of the cell line are emphasised through phenotypic changes and modifications of the spatial organisation of the cells. The hormone (E2) generates a very fast growth of cells with small nuclei that became ER negative in the long term. The antihormone (OH‐TAM) produces a gradual selection of ER negative or poorly positive cells with large nuclei. These modifications are reversed when E2 and OH‐TAM are simultaneously used. Moreover, estradiol induces a permissive context of proliferation, whereas hydroxytamoxifen acts only on some subpopulations. The combination of cell count, cytomorphology, and cell organisation revealed the magnitude of the potential of structuration of hormones or antihormones onin vitrogrowing cells.

Breast cancer (one of the most common malignancy in Western societies), as well as esophagus, stomach, lung, bladder, and prostate cancer, depend on environmental factors and diet for growth and evolution. Dietary micronutriments have been proposed as effective inhibitory agents for cancer initiation, progression, and incidence. Among them, polyphenols, present in different foods and beverages, have retained attention in recent years. Red wine is a rich source of polyphenols, and their antioxidant and tumor arresting effects have been demonstrated in different in vitro and in vivo systems. In the present study, we have measured the antiproliferative effect of red wine concentrate, its total polyphenolic pool, and purified catechin, epicatechin, quercetin, and resveratrol, which account for more than 70% of the total polyphenols in red wine, on the proliferation of hormone sensitive (MCF7, T47D) and resistant (MDA-MB-231) breast cancer cell lines. Our results indicate that polyphenols, at the picomolar or the nanomolar range, decrease cell proliferation in a dose- and a time-dependant manner. In hormone sensitive cell lines, a specific interaction of each polyphenol with steroid receptors was observed, with IC(50)s lower than previously described. Interaction of polyphenols with steroid receptors cannot fully explain their inhibitory effect on cell proliferation. In addition, discrete antioxidant action on each cell line was detected under the same concentrations, both by modifying the toxic effect of H(2)O(2), and the production of reactive oxygen species (ROS), after phorbol ester stimulation. Our results suggest that low concentrations of polyphenols, and consecutively, consumption of wine, or other polyphenol-rich foods and beverages, could have a beneficial antiproliferative effect on breast cancer cell growth.

High epidermal growth factor (EGF) concentration (10–8 M) induces inhibition of A431 cell proliferation, resulting in part from an apoptotic process. For some cells escaping this process, proliferation was associated with a decrease in apoptosis. Moreover, these surviving cells displayed marked morphological changes consisting of filopodia formation and cell aggregation. Disrupting cell–cell contacts by lowering extracellular calcium concentration reversed the resistance process, suggesting that apoptosis protection by aggregation may involve intercellular adhesion and cell–cell survival signals probably mediated by calcium-requiring molecules such as integrins. From a panel of integrins tested, only α2β1 integrin cell-surface expression was up-regulated after high apoptotic EGF treatment, and this up-regulation was not observed under a growth-stimulatory EGF concentration (10–11 M). Double-labeling analysis (α2β1/DNA) implicated α2β1 integrin in the resistance process since 99% of cells that up-regulated α2β1 integrin survived a high dose of EGF. Moreover, the involvement of α2β1 integrin up-regulation in the survival of A431 cells that escape EGF-induced apoptosis was verified using the blocking anti-α2β1 integrin antibody, which was shown to decrease the survival of EGF-stimulated cells. Furthermore, under our culture conditions, α2β1 integrin–dependent cell–cell adhesion can be inhibited without affecting other cell-adhesive interactions, suggesting that α2β1 integrin is involved more directly in cell–cell interaction than in cell–substrate adhesion. Our results provide evidence that EGF-induced up-regulation of α2β1 integrin contributes to the enhancement of cell–cell adhesion, leading to cell aggregate formation, which permits the escape of A431 cells to EGF-induced death by α2β1 integrin signaling. Int. J. Cancer 87:360–367, 2000. © 2000 Wiley-Liss, Inc.

The presence of epidermal-growth-factor receptors (EGFR) and of its ligands (TGFalpha and amphiregulin) in breast-cancer tissues suggests that they play a paracrine/autocrine role in tumor growth or progression. This hypothesis was tested on 3 cell lines, S2T2, NS2T2A and NS2T2A1. These epithelial cells are derived from a normal human breast-epithelial-cell culture transformed by SV40-T Ag, are of the same clonal origin, have respectively increasing levels of EGFR, TGFalpha, amphiregulin and of thymidine-kinase activity associated with increasing tumorigenic potential in nude mice (tumor intake and tumor volume). The monoclonal antibody MAb 425, which blocks ligands interaction with EGFR, reduced by more than 90% anchorage-independent growth of the most tumorigenic cells, NS2T2A1. Another anti-EGFR MAb, 528, reduced to 25% of controls the mean tumor mass after NS2T2A1 grafting in mice. Anti-sense RNA expression of EGFR in these cells confirmed the importance of this receptor in tumor progression, since it reduced significantly the tumor volume and tumor weight of NS2T2A1 cells to 16% of those in mock-transfected control cells.

In the mammary gland, mesenchymal-epithelial interactions are of paramount importance during normal and tumoral developments. We have studied the paracrine growth regulation of a variety of breast epithelial cells in coculture with normal or pathological breast fibroblasts. Two models of coculture were used in which the two cell types were seeded and grown, either together in microchamber slides or separated by a microporous membrane. Under these two conditions, all fibroblasts were shown to stimulate the proliferation of the hormono-responsive breast carcinoma MCF-7 cell line, suggesting that cell contacts were not indispensable for the paracrine stimulation of MCF-7 cell growth by fibroblasts. Moreover, in the Transwell coculture system, the proliferation of a variety of other breast carcinoma cells (MDA-MB231, T47D, and BT-20) was also stimulated by fibroblasts. However, the amplitude of the proliferative response seemed to be dependent on the carcinoma cell line considered. Moreover, the proliferative response of normal mammary epithelial cells to the presence of fibroblasts was shown to be significantly higher than the tumor cell response. The nature of the tissue of fibroblast origin, normal or pathological, did not influence the growth response of the epithelial cells. In this study, we thus demonstrate that fibroblasts are able to stimulate the proliferation of normal and carcinoma cells through paracrine exchange mechanisms. We also conclude that the target epithelial cell phenotype will essentially determine the extent of the proliferative response.

The aim of this study on testosterone (T) metabolism in benign prostatic hyperplasia (BPH) and prostatic cancer was to compare the formation of metabolites in freshly isolated epithelial cells and in cells of long-term cultures (2 passages) and to identify the 5alpha-reductase (5alpha-R) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms responsible for metabolite formation. Androst-4-enedione (A), dihydrotestosterone (DHT) and 5alpha-androstanedione (5alpha-A) formation were measured by high-performance liquid chromatography coupled to a Flo-one HP radioactivity detector. Enzyme isoforms were studied by Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR). T conversion into A by 17beta-HSD, rather than reduction into DHT by 5alpha-R, was by far the predominant activity in cultured epithelial cells. The metabolic profile did not differ substantially between BPH and cancer cells. Long-term cell culture led to an increase in A formation compared with the level recorded in freshly isolated cells, with no significant incidence on the relative DHT level. According to RT-PCR results, both 5alpha-R isoforms (1 and 2) and 2 17beta-HSD isoforms (2 and 3) are present in epithelial cell cultures and in tissues. According to Northern blot analyses, the mRNAs for 5alpha-R2 and 17beta-HSD4 are expressed in tissue and those for 5alpha-R1 and types 2 and 4 17beta-HSD in isolated cell cultures. Moreover, finasteride, a specific 5alpha-R2 inhibitor, inhibits DHT and 5alpha-A formation in long-term cell culture of adenocarcinoma epithelial cells plated on Matrigel, suggesting a 5alpha-R2 expression. Thus, although 5alpha-R2 is present in freshly isolated epithelial cell cultures and in long-term epithelial cells cultured on Matrigel and predominates in prostate tissue, it is the 5alpha-R1 isoform that is preferentially expressed in epithelial cell cultures.

The relationship between pregnancy and the outcome of breast cancer remains controversial. The purpose of this study was to determine the prognostic value of pregnancy at the time of diagnosis of primary infiltrating breast cancer. In a retrospective multi-center study we compared a group of 154 patients presenting pregnancy-associated (PA) breast cancer with a control group of 308 patients presenting non-pregnancy-associated (non-PA) breast cancer. Classic prognostic factors, treatment modalities, disease-free survival and overall survival were compared in the 2 groups. The relative importance of pregnancy was assessed by Cox multivariate analysis. There was a significantly higher proportion of inflammatory breast cancer, large tumors and negative receptor status in the PA group. Five-year recurrence-free survival, metastasis-free survival and overall survival were lower both in the whole PA group and in the PA sub-group excluding patients with inflammatory breast cancer than in the corresponding non-PA groups. According to clinical stage, histoprognostic grade and microscopic lymph-node involvement, probability of 5-year metastasis-free survival and overall survival was lower in the PA group. Outcome was significantly poorer after chemotherapy for patients in the PA sub-group than in the non-PA sub-group. Multivariate analysis demonstrated that pregnancy was an independent and significant prognostic factor. Pregnancy has an adverse effect on the outcome of breast cancer. Concurrent or recent pregnancy should be taken into account in the development of new systemic therapies. Our findings have important implications for further research into the basic mechanisms of cancer.

Thymidine kinase (TK) and thymidylate synthase (TS) play a key role in, respectively, the salvage and the de novo DNA synthesis pathways. TS is a crucial target for 5-fluoroura-cil(5-FU) and may also influence methotrexate(MTX) efficiency. Tyrosine kinase(TPK) has been associated with the cytoplasmic domain of growth factor receptors as well as oncoproteins. We investigated whether TK, TS and TPK are predictive factors for drug sensitivity evaluated in terms of relapse-free improvement in breast-cancer patients receiving adjuvant chemotherapy. TK, TS and TPK activities were determined in the cytosols of 154 node-positive primary breast cancers. All patients received 5-FU containing adjuvant chemotherapy. Measurements were performed using radioenzymatic methods. The levels of TK were correlated with those of TS and TPK. The levels of TS and TPK were less strongly correlated with each other. High TK levels were more often found in larger tumours, and the levels of both TK and TPK were negatively correlated with those of PgR. Patients whose tumours contained high levels of TK had increased risks of relapse and death. TS was not of prognostic value, while a high level of TPK was associated with early death. In Cox analysis, TK and TPK retained their independent prognostic value. While target enzyme activities on the de novo DNA synthesis pathway could determine response to anti-metabolics mainly inhibiting this pathway, high activities on the alternative salvage pathway could circumvent induced growth inhibition.

Stromal and epithelial components surrounding neoplastic cells are believed to be important in tumor regulation. We have studied the effects of stromal and epithelial cells on the proliferation of a variety of breast-cancer epithelial cell lines. Co-culture experiments were performed in which the 2 cell types were separated by a microporous membrane. Under these conditions, fibroblasts from normal breast tissues inhibited the proliferation of MCF-7 cells, but not that of immortalized normal S2T2 cells. In contrast, fibroblasts from cancerous breast tissues did not influence the proliferation of the 2 cell lines tested. Conditioned media (CM) of breast fibroblasts derived from normal tissues were not able to affect MCF-7 cell growth, suggesting complex paracrine interactions between both cell types. Normal breast epithelial cells (NBEC) have also been tested for their ability to regulate the proliferation of breast-cancer epithelial cell lines. Co-culture experiments demonstrated that NBEC inhibited a variety of breast-cancer cell lines. CM from NBEC induced similar results and the inhibitory effect appeared to be specific for epithelial cells from tumorous breast. Moreover, CM from NBEC and normal fibroblasts were shown to contain more TGF beta 1 and amphiregulin than those of MCF-7 cells. We conclude that both the tissue origin and the target tumor cell's phenotype will determine the extent of proliferative response. More important, the tumor-cell growth inhibition induced by fibroblasts and epithelial cells of normal breast tissue may constitute a tumor-growth-regulatory mechanism.

Purpose: To study the role of the adrenomedullin system [adrenomedullin and its receptors (AMR), CLR, RAMP2, and RAMP3] in prostate cancer androgen-independent growth. Experimental Design: Androgen-dependent and -independent prostate cancer models were used to investigate the role and mechanisms of adrenomedullin in prostate cancer hormone-independent growth and tumor-associated angiogenesis and lymphangiogenesis. Results: Adrenomedullin and AMR were immunohistochemically localized in the carcinomatous epithelial compartment of prostate cancer specimens of high grade (Gleason score >7), suggesting a role of the adrenomedullin system in prostate cancer growth. We used the androgen-independent Du145 cells, for which we demonstrate that adrenomedullin stimulated cell proliferation in vitro through the cAMP/CRAF/MEK/ERK pathway. The proliferation of Du145 and PC3 cells is decreased by anti-adrenomedullin antibody (αAM), supporting the fact that adrenomedullin may function as a potent autocrine/paracrine growth factor for prostate cancer androgen-independent cells. In vivo, αAM therapy inhibits the growth of Du145 androgen-independent xenografts and interestingly of LNCaP androgen-dependent xenografts only in castrated animals, suggesting strongly that adrenomedullin might play an important role in tumor regrowth following androgen ablation. Histologic examination of αAM-treated tumors showed evidence of disruption of tumor vascularity, with depletion of vascular as well as lymphatic endothelial cells and pericytes, and increased lymphatic endothelial cell apoptosis. Importantly, αAM potently blocks tumor-associated lymphangiogenesis, but does not affect established vasculature and lymphatic vessels in normal adult mice. Conclusions: We conclude that expression of adrenomedullin upon androgen ablation in prostate cancer plays an important role in hormone-independent tumor growth and in neovascularization by supplying/amplifying signals essential for pathologic neoangiogenesis and lymphangiogenesis. Clin Cancer Res; 19(22); 6138–50. ©2013 AACR.

Plasminogen activators (PAs) play a key role in malignant transformation. PA secretion by tumoral cells is strongly correlated with their aggressive phenotype. Regulation of invasive potential by growth factors has been also demonstrated. This study was designed to investigate the effects of 5alpha-dihydrotestosterone (DHT), epidermal growth factor (EGF), transforming growth factor beta1 (TGFbeta1), retinoic acid and basic fibroblastic growth factor (bFGF) on cell growth and PA expression and secretion in DU145 and PC3 cells, 2 human prostatic-cancer cell lines. The proliferation of 2 cell lines was significantly increased only by EGF (about 30%), but decreased by TGFbeta1 (40% inhibition). However, EGF-treated cells showed significant enhancement (about 400%) of u-PA secretion. A similar effect was observed when cells were cultured with DHT (200%) and with TGFbeta1 (300%). Nevertheless, u-PA mRNA level in EGF-, TGFbeta1 - or DHT-treated cells was amplified only between 110 and 180% of control, suggesting that growth factors differently controlled the steps of PA expression. Furthermore, our results clearly showed the divergent effect of TGFbeta1, i.e., an inhibition of prostatic-cell-line growth accompanied by an increase in proteolytic activity.

We compare testosterone (T) metabolism in primary cultures of epithelial cells and fibroblasts separated from benign prostate hypertrophy (BPH) and prostate cancer tissues. In all cultures, androstenedione (delta 4) formed by oxidation of T by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) represented 80% of the metabolites recovered. The amounts of 5 alpha-dihydrotestosterone (DHT), formed by reduction of T by 5 alpha-reductase (5 alpha-R), were small: 5 and 2% (BPH) and 8 and 15% (adenocarcinoma) for epithelial cells and fibroblasts, respectively. Northern blot analysis of total RNA from epithelial cells (BPH or adenocarcinoma) attributed the reductive activity to the 5 alpha-reductase type 1 isozyme and oxidative activity to the 17 beta-HSD type 2. In cancer fibroblasts, only little 17 beta-HSD type 2 mRNA was detected. The 5 alpha-reductase inhibitors, 4-MA (17 beta-(N,N-diethyl)carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one) and finasteride, inhibited DHT formation with a preferential action of 4-MA on epithelial cells (BPH or adenocarcinoma) and of finasteride on fibroblasts from adenocarcinoma. Neither inhibitor acted on delta 4 formation. On the other hand, the lipido-sterol extract of Serenoa repens (LSESr, Permixon) inhibited the formation of all the T metabolites studied [IC50 S = 40 and 200 micrograms/ml (BPH) and 90 and 70 micrograms/ml (adenocarcinoma) in epithelial cells and fibroblasts, respectively]. These results have important therapeutic implications when selecting appropriate treatment options for BPH.