Enhanced antitumor activity of doxorubicin in breast cancer through the use of poly(butylcyanoacrylate) nanoparticles.

The use of doxorubicin (DOX), one of the most effective antitumor molecules in the treatment of metastatic breast cancer, is limited by its low tumor selectivity and its severe side effects. Colloidal carriers based on biodegradable poly(butylcyanoacrylate) nanoparticles (PBCA NPs) may enhance DOX antitumor activity against breast cancer cells, thus allowing a reduction of the effective dose required for antitumor activity and consequently the level of associated toxicity. DOX loading onto PBCA NPs was investigated in this work via both drug entrapment and surface adsorption. Cytotoxicity assays with DOX-loaded NPs were performed in vitro using breast tumor cell lines (MCF-7 human and E0771 mouse cancer cells), and in vivo evaluating antitumor activity in immunocompetent C57BL/6 mice. The entrapment method yielded greater drug loading values and a controlled drug release profile. Neither in vitro nor in vivo cytotoxicity was observed for blank NPs. The 50% inhibitory concentration (IC50) of DOX-loaded PBCA NPs was significantly lower for MCF-7 and E0771 cancer cells (4 and 15 times, respectively) compared with free DOX. Furthermore, DOX-loaded PBCA NPs produced a tumor growth inhibition that was 40% greater than that observed with free DOX, thus reducing DOX toxicity during treatment. These results suggest that DOX-loaded PBCA NPs have great potential for improving the efficacy of DOX therapy against advanced breast cancers.

Proangiogenic Factor PlGF Programs CD11b+ Myelomonocytes in Breast Cancer during Differentiation of Their Hematopoietic Progenitors.

Tumor-mobilized bone marrow-derived CD11b(+) myeloid cells promote tumor angiogenesis, but how and when these cells acquire proangiogenic properties is not fully elucidated. Here, we show that CD11b(+) myelomonocytic cells develop proangiogenic properties during their differentiation from CD34(+) hematopoietic progenitors and that placenta growth factor (PlGF) is critical in promoting this education. Cultures of human CD34(+) progenitors supplemented with conditioned medium from breast cancer cell lines or PlGF, but not from nontumorigenic breast epithelial lines, generate CD11b(+) cells capable of inducing endothelial cell sprouting in vitro and angiogenesis in vivo. An anti-Flt-1 mAb or soluble Flt-1 abolished the generation of proangiogenic activity during differentiation from progenitor cells. Moreover, inhibition of metalloproteinase activity, but not VEGF, during the endothelial sprouting assay blocked sprouting induced by these proangiogenic CD11b(+) myelomonocytes. In a mouse model of breast cancer, circulating CD11b(+) cells were proangiogenic in the sprouting assays. Silencing of PlGF in tumor cells prevented the generation of proangiogenic activity in circulating CD11b(+) cells, inhibited tumor blood flow, and slowed tumor growth. Peripheral blood of breast cancer patients at diagnosis, but not of healthy individuals, contained elevated levels of PlGF and circulating proangiogenic CD11b(+) myelomonocytes. Taken together, our results show that cancer cells can program proangiogenic activity in CD11b(+) myelomonocytes during differentiation of their progenitor cells in a PlGF-dependent manner. These findings impact breast cancer biology, detection, and treatment. Cancer Res; 71(11); 3781-91. ©2011 AACR.

Identification and modelling of human breast cancer subtypes

Résumé Le cancer du sein est le cancer le plus commun chez les femmes et est responsable de presque 30% de tous les nouveaux cas de cancer en Europe. On estime le nombre de décès liés au cancer du sein en Europe est à plus de 130.000 par an. Ces chiffres expliquent l'impact social considérable de cette maladie. Les objectifs de cette thèse étaient: (1) d'identifier les prédispositions et les mécanismes biologiques responsables de l'établissement des sous-types spécifiques de cancer du sein; (2) les valider dans un modèle ín vivo "humain-dans-souris"; et (3) de développer des traitements spécifiques à chaque sous-type de cancer du sein identifiés. Le premier objectif a été atteint par l'intermédiaire de l'analyse des données d'expression de gènes des tumeurs, produite dans notre laboratoire. Les données obtenues par puces à ADN ont été produites à partir de 49 biopsies des tumeurs du sein provenant des patientes participant dans l'essai clinique EORTC 10994/BIG00-01. Les données étaient très riches en information et m'ont permis de valider des données précédentes des autres études d'expression des gènes dans des tumeurs du sein. De plus, cette analyse m'a permis d'identifier un nouveau sous-type biologique de cancer du sein. Dans la première partie de la thèse, je décris I identification des tumeurs apocrines du sein par l'analyse des puces à ADN et les implications potentielles de cette découverte pour les applications cliniques. Le deuxième objectif a été atteint par l'établissement d'un modèle de cancer du sein humain, basé sur des cellules épithéliales mammaires humaines primaires (HMECs) dérivées de réductions mammaires. J'ai choisi d'adapter un système de culture des cellules en suspension basé sur des mammosphères précédemment décrit et pat décidé d'exprimer des gènes en utilisant des lentivirus. Dans la deuxième partie de ma thèse je décris l'établissement d'un système de culture cellulaire qui permet la transformation quantitative des HMECs. Par la suite, j'ai établi un modèle de xénogreffe dans les souris immunodéficientes NOD/SCID, qui permet de modéliser la maladie humaine chez la souris. Dans la troisième partie de ma thèse je décris et je discute les résultats que j'ai obtenus en établissant un modèle estrogène-dépendant de cancer du sein par transformation quantitative des HMECs avec des gènes définis, identifiés par analyse de données d'expression des gènes dans le cancer du sein. Les cellules transformées dans notre modèle étaient estrogène-dépendantes pour la croissance, diploïdes et génétiquement normales même après la culture cellulaire in vitro prolongée. Les cellules formaient des tumeurs dans notre modèle de xénogreffe et constituaient des métastases péritonéales disséminées et du foie. Afin d'atteindre le troisième objectif de ma thèse, j'ai défini et examiné des stratégies de traitement qui permettent réduire les tumeurs et les métastases. J'ai produit un modèle de cancer du sein génétiquement défini et positif pour le récepteur de l'estrogène qui permet de modéliser le cancer du sein estrogène-dépendant humain chez la souris. Ce modèle permet l'étude des mécanismes impliqués dans la formation des tumeurs et des métastases. Abstract Breast cancer is the most common cancer in women and accounts for nearly 30% of all new cancer cases in Europe. The number of deaths from breast cancer in Europe is estimated to be over 130,000 each year, implying the social impact of the disease. The goals of this thesis were first, to identify biological features and mechanisms --responsible for the establishment of specific breast cancer subtypes, second to validate them in a human-in-mouse in vivo model and third to develop specific treatments for identified breast cancer subtypes. The first objective was achieved via the analysis of tumour gene expression data produced in our lab. The microarray data were generated from 49 breast tumour biopsies that were collected from patients enrolled in the clinical trial EORTC 10994/BIG00-01. The data set was very rich in information and allowed me to validate data of previous breast cancer gene expression studies and to identify biological features of a novel breast cancer subtype. In the first part of the thesis I focus on the identification of molecular apacrine breast tumours by microarray analysis and the potential imptìcation of this finding for the clinics. The second objective was attained by the production of a human breast cancer model system based on primary human mammary epithelial cells {HMECs) derived from reduction mammoplasties. I have chosen to adopt a previously described suspension culture system based on mammospheres and expressed selected target genes using lentiviral expression constructs. In the second part of my thesis I mainly focus on the establishment of a cell culture system allowing for quantitative transformation of HMECs. I then established a xenograft model in immunodeficient NOD/SCID mice, allowing to model human disease in a mouse. In the third part of my thesis I describe and discuss the results that I obtained while establishing an oestrogen-dependent model of breast cancer by quantitative transformation of HMECs with defined genes identified after breast cancer gene expression data analysis. The transformed cells in our model are oestrogen-dependent for growth; remain diploid and genetically normal even after prolonged cell culture in vitro. The cells farm tumours and form disseminated peritoneal and liver metastases in our xenograft model. Along the lines of the third objective of my thesis I defined and tested treatment schemes allowing reducing tumours and metastases. I have generated a genetically defined model of oestrogen receptor alpha positive human breast cancer that allows to model human oestrogen-dependent breast cancer in a mouse and enables the study of mechanisms involved in tumorigenesis and metastasis.

Identification of cancer stem cells in Ewing's sarcoma.

Cancer stem cells that display tumor-initiating properties have recently been identified in several distinct types of malignancies, holding promise for more effective therapeutic strategies. However, evidence of such cells in sarcomas, which include some of the most aggressive and therapy-resistant tumors, has not been shown to date. Here, we identify and characterize cancer stem cells in Ewing's sarcoma family tumors (ESFT), a highly aggressive pediatric malignancy believed to be of mesenchymal stem cell (MSC) origin. Using magnetic bead cell separation of primary ESFT, we have isolated a subpopulation of CD133+ tumor cells that display the capacity to initiate and sustain tumor growth through serial transplantation in nonobese diabetic/severe combined immunodeficiency mice, re-establishing at each in vivo passage the parental tumor phenotype and hierarchical cell organization. Consistent with the plasticity of MSCs, in vitro differentiation assays showed that the CD133+ cell population retained the ability to differentiate along adipogenic, osteogenic, and chondrogenic lineages. Quantitative real-time PCR analysis of genes implicated in stem cell maintenance revealed that CD133+ ESFT cells express significantly higher levels of OCT4 and NANOG than their CD133- counterparts. Taken together, our observations provide the first identification of ESFT cancer stem cells and demonstration of their MSC properties, a critical step towards a better biological understanding and rational therapeutic targeting of these tumors.

Valeur prédictive de la surexpression/ amplification de Her2/neu pour un traitement ciblé du cancer du sein [Predictive value of Her2/neu expression/amplification for the targeted treatment of breast cancer]

Her2/neu is a tyrosine kinase receptor which stimulates cell growth. The receptor is overexpressed in about 20% of breast cancers. Her2/neu expression is an indicator of poor prognosis but also the target of the treatment of breast cancer using humanised anti-Her2/ neu antibodies. Only cancers overexpressing the protein will respond to this therapy, but which has significant (cardiac) side effects and is expensive. It is therefore important to test for the overexpression of the protein on breast cancer cells. This paper discusses how this can be done and ongoing research into new therapeutic options targeting the involved signaling pathways.

Identification and Characterization of Natural Anti-Breast Cancer Compound: Costunolide

Breast cancer is the most common cancer among women, 23% (1.3 million) of the total of new cases and the second leading cause of cancer death in women exceeded only by lung cancer. Natural medicines have been proven to be a central source of narrative agents with a pharmaceutical potential. Costunolide is sesquiterpene lactones consisting of diverse plant chemicals that exhibit anti cancer action through cytotoxic effects on various cancer cells. The objectives of present study were to explore the effects of natural compounds on the proliferation of MCF-7 cells and to determine the role of ROS in natural compounds-induced apoptosis in breast cancer cells with a therapeutic potential. Results showed that costunolide screened, possess potent anticancer properties against breast cancer MCF-7 cells, Costunolide was observed as strong anti-proliferative agent with IC50 = 50µM. The anti-proliferative effect of costunolide on MCF cells was confirmed by live/dead assay using fluorescent probes calcein AV/PI. The results demonstrated that treatment of cells with costunolide decreased the viability of MCF-7 cells in a dose-dependent manner. To determine the costunolide-induced apoptosis, flow cytometric analysis was carried out. The results showed that costunolide induced apoptosis in a dose-dependent manner in breast cancer MCF-7cells. ROS are well known mediators of intracellular signaling of cascades. The excessive generation of ROS can induce oxidative stress, loss of cell functioning, and apoptosis. In the present study, we assumed that costunolide might arouse ROS level, which could be involved in induction of apoptosis. Therefore, the intracellular ROS level was measured using the ROS-detecting fluorescence dye 2, 7-dichlorofluorescein diacetate (DCF-DA). Interestingly these effects were significantly abrogated when the cells were pretreated with N-acetyl- cysteine (NAC), a specific ROS inhibitor. Costunolide induces apoptosis through extrinsic pathway in MCF-7 breast cancer cells, In order to examine whether costunolide suppresses cell growth inducing apoptotic cell death, we analyzed DNA contents and apoptosis-related proteins expression level by flow cytometry and western blot, respectively in MCF-7 breast cancer cells we investigated whether costunolide activates extrinsic apoptotic pathway. We examined the expression levels of death receptor signaling-related proteins, caspase-3, and PARP. The results showed that procaspase-3 was cleaved to yield 17 and 20kDa fragments and activation of PARP in treated cells with 25 and 50μM of costunolide. Costunolide induce apoptosis through intrinsic mitochondria pathway in MCF-7 breast cancer Cells. We examined the expression levels of mitochondrial apoptotic pathway related proteins such as anti-apoptotic protein, B-cell lymphoma protein-2 (Bcl2), and pro-apoptotic protein Bax. Costunolide involved in the down regulation of Bcl-2 and up regulation of Bax. These results suggest that costunolide may have beneficial effects for the reduction of breast cancer growth, and new therapeutic strategy for the treatment of human cancers.

Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma cancer stem cells.

Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSCs), that have the capacity for self-renewal, tumor initiation, and generation of nontumorigenic progeny. Despite their potentially key role in tumor establishment and maintenance, the energy requirements of these cells and the mechanisms that regulate their energy production are unknown. Here, we show that the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2, IGF2BP2) regulates oxidative phosphorylation (OXPHOS) in primary glioblastoma (GBM) sphere cultures (gliomaspheres), an established in vitro model for CSC expansion. We demonstrate that IMP2 binds several mRNAs that encode mitochondrial respiratory chain complex subunits and that it interacts with complex I (NADH:ubiquinone oxidoreductase) proteins. Depletion of IMP2 in gliomaspheres decreases their oxygen consumption rate and both complex I and complex IV activity that results in impaired clonogenicity in vitro and tumorigenicity in vivo. Importantly, inhibition of OXPHOS but not of glycolysis abolishes GBM cell clonogenicity. Our observations suggest that gliomaspheres depend on OXPHOS for their energy production and survival and that IMP2 expression provides a key mechanism to ensure OXPHOS maintenance by delivering respiratory chain subunit-encoding mRNAs to mitochondria and contributing to complex I and complex IV assembly.

p27 and Skp2 immunoreactivity and its clinical significance with endocrine and chemo-endocrine treatments in node-negative early breast cancer.

BACKGROUND: Low p27 and high Skp2 immunoreactivity are associated with a poor prognosis and other poor prognostic features including resistant phenotypes and antiestrogen drug resistance. We investigated these proteins in two International Breast Cancer Study Group trials studying node-negative early breast cancer. PATIENTS AND METHODS: Trial VIII compared chemotherapy followed by goserelin with either modality alone in premenopausal patients. Trial IX compared chemotherapy followed by tamoxifen with tamoxifen alone in postmenopausal patients. Central Pathology Office assessed p27 and Skp2 expression in the primary tumor by immunohistochemistry among 1631 (60%) trial patients. RESULTS: p27 and Skp2 were inversely related; 13% of tumors expressed low p27 and high Skp2. Low p27 and high Skp2 were associated with unfavorable prognostic factors including larger size and higher grade tumors, absence of estrogen receptor and progesterone receptor, human epidermal growth factor receptor 2 overexpression and high Ki-67 (each P < 0.05). Low p27 and high Skp2 were not associated with disease-free survival (P = 0.42 and P = 0.48, respectively). The relative effects of chemo-endocrine versus endocrine therapy were similar regardless of p27 or Skp2. CONCLUSIONS: We confirm the association of low p27 and high Skp2 with other poor prognostic features, but found no predictive or prognostic value, and therefore do not recommend routine determination of p27 and Skp2 for node-negative breast cancer.

Low E2F1 transcript levels are a strong determinant of favorable breast cancer outcome.

INTRODUCTION: We investigated whether mRNA levels of E2F1, a key transcription factor involved in proliferation, differentiation and apoptosis, could be used as a surrogate marker for the determination of breast cancer outcome. METHODS: E2F1 and other proliferation markers were measured by quantitative RT-PCR in 317 primary breast cancer patients from the Stiftung Tumorbank Basel. Correlations to one another as well as to the estrogen receptor and ERBB2 status and clinical outcome were investigated. Results were validated and further compared with expression-based prognostic profiles using The Netherlands Cancer Institute microarray data set reported by Fan and colleagues. RESULTS: E2F1 mRNA expression levels correlated strongly with the expression of other proliferation markers, and low values were mainly found in estrogen receptor-positive and ERBB2-negative phenotypes. Patients with low E2F1-expressing tumors were associated with favorable outcome (hazard ratio = 4.3 (95% confidence interval = 1.8-9.9), P = 0.001). These results were consistent in univariate and multivariate Cox analyses, and were successfully validated in The Netherlands Cancer Institute data set. Furthermore, E2F1 expression levels correlated well with the 70-gene signature displaying the ability of selecting a common subset of patients at good prognosis. Breast cancer patients' outcome was comparably predictable by E2F1 levels, by the 70-gene signature, by the intrinsic subtype gene classification, by the wound response signature and by the recurrence score. CONCLUSION: Assessment of E2F1 at the mRNA level in primary breast cancer is a strong determinant of breast cancer patient outcome. E2F1 expression identified patients at low risk of metastasis irrespective of the estrogen receptor and ERBB2 status, and demonstrated similar prognostic performance to different gene expression-based predictors.

The biology of cancer stem cells : part 1: nuclear translocation of CD44 : part 2: IMP2 in glioblastoma cancer stem cells

SummaryCancer stem cells (CSC) are poorly differentiated, slowly proliferating cells, with high tumorigenic potential. Some of these cells, as it has been shown in leukemia, evade chemo- and radiotherapy and recapitulate the tumor composed of CSC and their highly proliferative progeny. Therefore, understanding the molecular biology of those cells is crucial for improvement of currently used anti-cancer therapies.This work is composed of two CSC-related projects. The first deals with CD44, a frequently used marker of CSC; the second involves Imp2 and its role in CSC bioenergetics. PART 1. CD44 is a multifunctional transmembrane protein involved in migration, homing, adhesion, proliferation and survival. It is overexpressed in many cancers and its levels are correlated with poor prognosis. CD44 is also highly expressed by CSC and in many malignancies it is used for CSC isolation.In the present work full-lenght CD44 nuclear localization was studied, including the mechanism of nuclear translocation and its functional role in the nucleus. Full-length CD44 can be found in nuclei of various cell types, regardless of their tumorigenic potential. For nuclear localization, CD44 needs to be first inserted into the cell membrane, from which it is transported via the endocytic pathway. Upon binding to transportinl it is translocated to the nucleus. The nuclear localization signal recognized by transportinl has been determined as the first 20 amino acids of the membrane proximal intracellular domain. Nuclear export of CD44 is facilitated by exportin Crml. Investigation of the function of nuclear CD44 revealed its implication in de novo RNA synthesis.PART 2. Glioblastoma multiforme is the most aggressive and most frequent brain malignancy. It was one of the first solid tumors from which CSC have been isolated. Based on the similarity between GBM CSC and normal stem cells expression of an oncofetal mRNA binding protein Imp2 has been investigated.Imp2 is absent in normal brain as well as in low grade gliomas, but is expressed in over 75% GBM cases and its expression is higher in CSC compared to their more differentiated counterparts. Analysis of mRNA transcripts bound by Imp2 and its protein interactors revealed that in GBM CSC Imp2 may be implicated in mitochondrial metabolism. Indeed, shRNA mediated silencing of protein expression led to decreased mitochondrial activity, decreased oxygen consumption and decreased activity of respiratory chain protein complex I. Moreover, lack of Imp2 severely affected self-renewal and tumorigenicity of GBM CSC. Experimental evidence suggest that GBM CSC depend on mitochondrial oxidative phosphorylation as an energy producing pathway and that Imp2 is a novel regulator of this pathway.RésuméLes cellules cancéreuses souches sont des cellules peu différentiées, à proliferation lente et hautement tumorigénique. Ces cellules sont radio-chimio résistantes et sont capable reformer la tumeur dans sont intégralité, reproduisant l'hétérogénéité cellulaire présent dans la tumeur d'origine. Pour améliorer les therapies antitumorales actuelles il est crucial de comprendre les mécanismes moléculaires qui caractérisent cette sous-population de cellules hautement malignes.Ce travail de thèse se compose de deux projets s'articulant autour du même axe :Le CD44 est une protéine multifonctionnelle et transmembranaire très souvent utilisée comme marqueur de cellules souches tumorales dans différents cancers. Elle est impliquée dans la migration, l'adhésion, la prolifération et la survie des cellules. Lors de ce travail de recherche, nous nous sommes intéressés à la localisation cellulaire du CD44, ainsi qu'aux mécanismes permettant sa translocation nucléaire. En effet, bien que principalement décrit comme un récepteur de surface transmembranaire, le CD44 sous sa forme entière, non clivée en peptides, peut également être observé à l'intérieur du noyau de diverses cellules, quel que soit leur potentiel tumorigénique. Pour passer ainsi d'un compartiment cellulaire à un autre, le CD44 doit d'abord être inséré dans la membrane plasmique, d'où il est transporté par endocytose jusqu'à l'intérieur du cytoplasme. La transportai permet ensuite la translocation nucléaire du CD44 via une « séquence signal » contenue dans les 20 acides aminés du domaine cytoplasmique qui bordent la membrane. A l'inverse, le CD44 est exporté du noyau grâce à l'exportin Crml. En plus des mécanismes décrits ci-dessus, cette étude a également mis en évidence l'implication du CD44 dans la synthèse des ARN, d'où sa présence dans le noyau.Le glioblastome est la plus maligne et la plus fréquente des tumeurs cérébrales. Dans ce second projet de recherche, le rôle de IMP2 dans les cellules souches tumorales de glioblastomes a été étudié. La présence de cette protéine oncofoetale a d'abord été mise en évidence dans 75% des cas les plus agressifs des gliomes (grade IV, appelés glioblastomes), tandis qu'elle n'est pas exprimée dans les grades I à III de ces tumeurs, ni dans le cerveau sain. De plus, IMP2 est apparue comme étant davantage exprimée dans les cellules souches tumorales que dans les cellules déjà différenciées. La baisse de l'expression de IMP2 au moyen de shRNA a résulté en une diminution de l'activité mitochondriale, en une réduction de la consommation d'oxygène ainsi qu'en une baisse de l'activité du complexe respiratoire I.L'inhibition de IMP2 a également affecté la capacité de renouvellement de la population des cellules souches tumorales ainsi que leur aptitude à former des tumeurs.Lors de ce travail de thèse, une nouvelle fonction d'un marqueur de cellules souches tumorales a été mise en évidence, ainsi qu'un lien important entre la bioénergétique de ces cellules et l'expression d'une protéine oncofoetale.

Meta-analysis of gene expression profiles in breast cancer: toward a unified understanding of breast cancer subtyping and prognosis signatures.

INTRODUCTION: Breast cancer subtyping and prognosis have been studied extensively by gene expression profiling, resulting in disparate signatures with little overlap in their constituent genes. Although a previous study demonstrated a prognostic concordance among gene expression signatures, it was limited to only one dataset and did not fully elucidate how the different genes were related to one another nor did it examine the contribution of well-known biological processes of breast cancer tumorigenesis to their prognostic performance. METHOD: To address the above issues and to further validate these initial findings, we performed the largest meta-analysis of publicly available breast cancer gene expression and clinical data, which are comprised of 2,833 breast tumors. Gene coexpression modules of three key biological processes in breast cancer (namely, proliferation, estrogen receptor [ER], and HER2 signaling) were used to dissect the role of constituent genes of nine prognostic signatures. RESULTS: Using a meta-analytical approach, we consolidated the signatures associated with ER signaling, ERBB2 amplification, and proliferation. Previously published expression-based nomenclature of breast cancer 'intrinsic' subtypes can be mapped to the three modules, namely, the ER-/HER2- (basal-like), the HER2+ (HER2-like), and the low- and high-proliferation ER+/HER2- subtypes (luminal A and B). We showed that all nine prognostic signatures exhibited a similar prognostic performance in the entire dataset. Their prognostic abilities are due mostly to the detection of proliferation activity. Although ER- status (basal-like) and ERBB2+ expression status correspond to bad outcome, they seem to act through elevated expression of proliferation genes and thus contain only indirect information about prognosis. Clinical variables measuring the extent of tumor progression, such as tumor size and nodal status, still add independent prognostic information to proliferation genes. CONCLUSION: This meta-analysis unifies various results of previous gene expression studies in breast cancer. It reveals connections between traditional prognostic factors, expression-based subtyping, and prognostic signatures, highlighting the important role of proliferation in breast cancer prognosis.

Inhibition of the Kit Ligand/c-Kit Axis Attenuates Metastasis in a Mouse Model Mimicking Local Breast Cancer Relapse after Radiotherapy.

PURPOSE: Local breast cancer relapse after breast-saving surgery and radiotherapy is associated with increased risk of distant metastasis formation. The mechanisms involved remain largely elusive. We used the well-characterized 4T1 syngeneic, orthotopic breast cancer model to identify novel mechanisms of postradiation metastasis. EXPERIMENTAL DESIGN: 4T1 cells were injected in 20 Gy preirradiated mammary tissue to mimic postradiation relapses, or in nonirradiated mammary tissue, as control, of immunocompetent BALB/c mice. Molecular, biochemical, cellular, histologic analyses, adoptive cell transfer, genetic, and pharmacologic interventions were carried out. RESULTS: Tumors growing in preirradiated mammary tissue had reduced angiogenesis and were more hypoxic, invasive, and metastatic to lung and lymph nodes compared with control tumors. Increased metastasis involved the mobilization of CD11b(+)c-Kit(+)Ly6G(high)Ly6C(low)(Gr1(+)) myeloid cells through the HIF1-dependent expression of Kit ligand (KitL) by hypoxic tumor cells. KitL-mobilized myeloid cells homed to primary tumors and premetastatic lungs, to give rise to CD11b(+)c-Kit(-) cells. Pharmacologic inhibition of HIF1, silencing of KitL expression in tumor cells, and inhibition of c-Kit with an anti-c-Kit-blocking antibody or with a tyrosine kinase inhibitor prevented the mobilization of CD11b(+)c-Kit(+) cells and attenuated metastasis. C-Kit inhibition was also effective in reducing mobilization of CD11b(+)c-Kit(+) cells and inhibiting lung metastasis after irradiation of established tumors. CONCLUSIONS: Our work defines KitL/c-Kit as a previously unidentified axis critically involved in promoting metastasis of 4T1 tumors growing in preirradiated mammary tissue. Pharmacologic inhibition of this axis represents a potential therapeutic strategy to prevent metastasis in breast cancer patients with local relapses after radiotherapy. Clin Cancer Res; 18(16); 4365-74. ©2012 AACR.

Mechanisms of CYR61 mediated breast cancer metastasis to the lung

CYR61 (Cysteine-rich angiogenic inducer 61) is a matricellular protein that regulates cell proliferation, adhesion, migration and cell survival through interaction with various types of integrin cell adhesion receptors. At tissue level it is implicated in the regulation of embryonic development, wound healing and angiogenesis. CYR61 has also been involved in cancer progression, however its role appears to be diverse and complex depending on the cancer type and stage. Its contribution to metastasis formation is still unclear. Previous findings reported by our laboratory demonstrated that CYR61 cooperates with avßs integrin to promote invasion and metastasis of cancers growing in a pre-irradiated microenvironment. In this work, we used an orthotopic model of breast cancer to show for the first time that silencing of CYR61 in breast cancer cells suppresses lung metastasis formation. Silencing of MDA-MB-231 reduced both local growth and lung metastasis formation of tumor cells implanted in a pre-irradiated mammary fat pad. CYR61 silencing in tumors growing in non-irradiated mammary fat pads did not impact primary tumor growth but decreased lung metastasis formation. The effect of CYR61 on spontaneous lung metastasis formation during natural cancer progression was further examined by using an experimental model of metastasis. Results from these experiments indicate that CYR61 is critically involved in promoting cancer cells entry into lung parenchyma rather than later steps of colonization. In vitro experiments showed that CYR61 promotes tumor cell spreading, migration and transendothelial migration. CYR61 also supported colony formation under anchorage-independent condition and promotes resistance to anoikis through the involvement of ß1 and ß3 integrin. These results indicate that CYR61 promotes lung metastasis of breast cancer by facilitating extravasation into lung parenchyma through enhanced motility, transendothelial migration and resistance to anoikis. - CYR61 (Cysteine-rich angiogenic inducer 61) est une protéine matricellulaire qui régule la prolifération, l'adhérence, la migration et la survie des cellules par son interaction avec différents types de récepteurs d'adhésion cellulaire de la famille des intégrine. Au niveau des tissus, CYR61 est impliquée dans la régulation du développement embryonnaire, de la cicatrisation et de l'angiogenèse. CYR61 a également été impliquée dans le cancer, mais son rôle semble être divers et complexe en fonction du type du cancer et de son stade. Son rôle dans la formation des métastases n'est pas encore clair. Des résultats antérieurs rapportés par notre laboratoire ont montré que CYR61 coopère avec l'intégrine avß5 pour favoriser l'invasion et la métastase de tumeurs se développant dans un micro-environnement pré-irradié. Dans ce travail, nous avons utilisé un modèle orthotopique de cancer du sein pour démontrer pour la première fois que l'extinction (silencing) du gène CYR61 dans le cancer du sein réduit la formation de métastases pulmonaires. L'extinction de CYR61 dans la lignée cellulaire de cancer du sein humain MDA-MB- 231 réduit à la fois la croissance local ainsi que la formation de métastases pulmonaires à partir de cellules implantés dans les coussinets adipeux mammaires pré-irradié. L'extinction de CYR61 dans des tumeurs grandissant dans les coussinets adipeux mammaires non irradiées n'a pas d'incidence sur la croissance tumorale primaire mais réduit la formation des métastases pulmonaires. Par la suite nous avons examiné l'effet de CYR61 sur la formation de métastases pulmonaires en utilisant un modèle expérimental de métastase. Les résultats de ces expériences indiquent que CYR61 est impliquée de manière cruciale dans les étapes précoces de la formation de métastases, plutôt que dans les étapes tardives de colonisation du poumon. Des expériences in vitro ont montré que CYR61 favorise l'étalement, la migration et la transmigration endothéliale des cellules tumorales. CYR61 favorise également la formation de colonies dans des conditions indépendante de l'ancrage et la résistance à l'anoïkis par l'engagement des intégrines ß1 et ß3. Ces résultats indiquent que CYR61 favorise les métastases pulmonaires du cancer du sein en facilitant l'extravasation dans le parenchyme pulmonaire grâce à la stimulation de la motilità, de la migration transmigration endothéliale et de la résistance à l'anoïkis.

An immune-competent model of spontaneous breast cancer metastasis to the brain

In breast cancer, brain metastases are often seen as late complications of recurrent disease and represent a particularly serious condition, since there are limited therapeutic options and patients have an unfavorable prognosis. The frequency of brain metastases in breast cancer is currently on the rise. This might be due to the fact that adjuvant chemotherapeutic and targeted anticancer drugs, while they effectively control disease progression in the periphery, they only poorly cross the blood-brain barrier and do not reach effectively cancer cells disseminated in the brain. It is therefore of fundamental clinical relevance to investigate mechanisms involved in breast cancer metastasis to the brain. To date experimental models of breast cancer metastasis to the brain described in literature are based on the direct intracarotid or intracardiac injection of breast cancer cells. We recently established a brain metastasis breast cancer model in immunocompetent mice based on the orthotopic injection of 4T1 murine breast carcinoma cells in the mammary gland of syngeneic BALB/c mice. 4T1-derived tumors recapitulate the main steps of human breast cancer progression, including epithelial-to-mesenchymal transition, local invasion and metastatic spreading to lung and lymph nodes. 4T1 cells were engineered to stably express firefly Luciferase allowing noninvasive in vivo and ex vivo monitoring of tumor progression and metastatic spreading to target organs. Bioluminescence imaging revealed the appearance of spontaneous lesions to the lung and lymph nodes and, at a much lower frequency, to the brain. Brain metastases were confirmed by macroscopic and microscopic evaluation of the brains at necropsy. We then isolated brain metastatic cells, re-injected them orthotopically in new mice and isolated again lines from brain metastases. After two rounds of selection we obtained lines metastasizing to the brain with 100% penetrance (named 4T1-BM2 for Brain Metastasis, 2nd generation) compared to lines derived after two rounds of in vivo growth from primary tumors (4T1-T2) or from lung metastases (4T1-LM2). We are currently performing experiments to unravel differences in cell proliferation, adhesion, migration, invasion and survival of the 4T1-BM2 line relative to the 4T1-T2 and 4T1-LM2 lines. Initial results indicate that 4T1-BM2 cells are not more invasive or more proliferative in vitro and do not show a more mesenchymal phenotype. Our syngeneic (BALB/c) model of spontaneous breast carcinoma metastasis to the brain is a unique and clinically relevant model to unravel the mechanisms of metastatic breast cancer colonization of the brain. Genes identified in this model represent potentially clinically relevant therapeutic targets for the prevention and the treatment of brain metastases in breast cancer patients.

European guidelines for antifungal management in leukemia and hematopoietic stem cell transplant recipients: summary of the ECIL 3--2009 update.

In 2005, several groups, including the European Group for Blood and Marrow Transplantation, the European Organization for Treatment and Research of Cancer, the European Leukemia Net and the Immunocompromised Host Society created the European Conference on Infections in Leukemia (ECIL). The main goal of ECIL is to elaborate guidelines, or recommendations, for the management of infections in leukemia and stem cell transplant patients. The first sets of ECIL slides about the management of invasive fungal disease were made available on the web in 2006 and the papers were published in 2007. The third meeting of the group (ECIL 3) was held in September 2009 and the group updated its previous recommendations. The goal of this paper is to summarize the new proposals from ECIL 3, based on the results of studies published after the ECIL 2 meeting: (1) the prophylactic recommendations for hematopoietic stem cell transplant recipients were formulated differently, by splitting the neutropenic and the GVHD phases and taking into account recent data on voriconazole; (2) micafungin was introduced as an alternative drug for empirical antifungal therapy; (3) although several studies were published on preemptive antifungal approaches in neutropenic patients, the group decided not to propose any recommendation, as the only randomized study comparing an empirical versus a preemptive approach showed a significant excess of fungal disease in the preemptive group.