Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines

The recently emerged concept of cancer stem cell (CSC) has led to a new hypothesis on the basis for tumor progression. Basically, the CSC theory hypothesizes the presence of a hierarchically organized and relatively rare cell population, which is responsible for tumor initiation, self-renewal, and maintenance, in addition to accumulation of mutation and resistance to chemotherapy. CSCs have recently been described in breast cancer. Different genetic markers have been used to isolate breast CSCs, none of which have been correlated with the tumorigenicity or metastatic potential of the cells, limiting their precise characterization and clinical application in the development of therapeutic protocols. Here, we sought for subpopulations of CSCs by analyzing 10 judiciously chosen stem cell markers in a normal breast cell line (MCF10-A) and in four human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T) displaying different degrees of metastatic and invasiveness potential. We were able to identify two markers, which are differentially expressed in nontumorigenic versus tumor cells. The CD90 marker was highly expressed in the malignant cell lines. Interestingly, the CD14 molecule displayed higher expression levels in the nontumorigenic cell line. Therefore, we demonstrated that these two markers, which are more commonly used to isolate and characterize stem cells, are differentially expressed in breast tumor cells, when compared with nontumorigenic breast cells. (C) 2012 International Society for Advancement of Cytometry

EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells

Introduction: Cancer stem cells (CSC) display plasticity and self renewal properties reminiscent of normal tissue stem cells but the events responsible for their emergence remain obscure. We have recently identified CSC in Ewing sarcoma family tumors (ESFT) and shown that they arise from mesenchymal stem cells from the bone marrow. Objective of the study: To analyze the mechanisms underlying cancer stem cell development in ESFT. Methods: Primary human mesenchymal stem cells (MSC) isolation from adult and pediatric bone marrow. Retroviral delivery of fusion protein (EWS-FLI1) to primary MSC, and transcriptional and phenotypical analysis. Results: We show that the EWS-FLI-1 fusion gene, associated wit 85-90% of ESFT and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2 and NANOG in human pediatric MSC (hpMSC) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSC expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWSFLI- 1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Conclusion: Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a cancer stem cell phenotype.

Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells.

Cancer stem cells are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. To date, the molecular mechanisms that generate and regulate cancer stem cells are not well defined. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA-binding protein that is expressed in normal tissues only in germ cells and is re-activated in tumors. Recent evidences have highlighted the correlation of BORIS/CTCFL expression with poor overall survival of different cancer patients. We have previously shown an association of BORIS-expressing cells with stemness gene expression in embryonic cancer cells. Here, we studied the role of BORIS in epithelial tumor cells. Using BORIS-molecular beacon that was already validated, we were able to show the presence of BORIS mRNA in cancer stem cell-enriched populations (side population and spheres) of cervical, colon and breast tumor cells. BORIS silencing studies showed a decrease of sphere formation capacity in breast and colon tumor cells. Importantly, BORIS-silencing led to down-regulation of hTERT, stem cell (NANOG, OCT4, SOX2 and BMI1) and cancer stem cell markers (ABCG2, CD44 and ALDH1) genes. Conversely, BORIS-induction led to up-regulation of the same genes. These phenotypes were observed in cervical, colon and invasive breast tumor cells. However, a completely different behavior was observed in the non-invasive breast tumor cells (MCF7). Indeed, these cells acquired an epithelial mesenchymal transition phenotype after BORIS silencing. Our results demonstrate that BORIS is associated with cancer stem cell-enriched populations of several epithelial tumor cells and the different phenotypes depend on the origin of tumor cells.

Metformin and soybean-derived bioactive molecules attenuate the expansion of stem cell-like epithelial subpopulation and confer apoptotic sensitivity in human colon cancer cells

Colorectal cancer (CRC) is a disease whose genesis may include metabolic dysregulation. Cancer stem cells are attractive targets for therapeutic interventions since their aberrant expansion may underlie tumor initiation, progression, and recurrence. To investigate the actions of metabolic regulators on cancer stem cell-like cells (CSC) in CRC, we determined the effects of soybean-derived bioactive molecules and the anti-diabetes drug metformin (MET), alone and together, on the growth, survival, and frequency of CSC in human HCT116 cells. Effects of MET (60 μM) and soybean components genistein (Gen, 2 μM), lunasin (Lun, 2 μM), β-conglycinin (β-con, 3 μM), and glycinin (Gly, 3 μM) on HCT116 cell proliferation, apoptosis, and mRNA/protein expression and on the frequency of the CSC CD133(+)CD44(+) subpopulation by colonosphere assay and fluorescence-activated cell sorting/flow cytometry were evaluated. MET, Gen, and Lun, individually and together, inhibited HCT116 viability and colonosphere formation and, conversely, enhanced HCT116 apoptosis. Reductions in frequency of the CSC CD133(+)CD44(+) subpopulation with MET, Gen, and Lun were found to be associated with increased PTEN and reduced FASN expression. In cells under a hyperinsulinemic state mimicking metabolic dysregulation and without and with added PTEN-specific inhibitor SF1670, colonosphere formation and frequency of the CD133(+)CD44(+) subpopulation were decreased by MET, Lun and Gen, alone and when combined. Moreover, MET + Lun + Gen co-treatment increased the pro-apoptotic and CD133(+)CD44(+)-inhibitory efficacy of 5-fluorouracil under hyperinsulinemic conditions. Results identify molecular networks shared by MET and bioavailable soy food components, which potentially may be harnessed to increase drug efficacy in diabetic and non-diabetic patients with CRC.

Expression profiling of stem cell-related genes in neoadjuvant-treated gastric cancer: a NOTCH2, GSK3B and β-catenin gene signature predicts survival

Cancer stem cell (CSC) based gene expression signatures are associated with prognosis in various tumour types and CSCs are suggested to be particularly drug resistant. The aim of our study was first, to determine the prognostic significance of CSC-related gene expression in residual tumour cells of neoadjuvant-treated gastric cancer (GC) patients. Second, we wished to examine, whether expression alterations between pre- and post-therapeutic tumour samples exist, consistent with an enrichment of drug resistant tumour cells. The expression of 44 genes was analysed in 63 formalin-fixed, paraffin embedded tumour specimens with partial tumour regression (10-50% residual tumour) after neoadjuvant chemotherapy by quantitative real time PCR low-density arrays. A signature of combined GSK3B(high), β-catenin (CTNNB1)(high) and NOTCH2(low) expression was strongly correlated with better patient survival (p<0.001). A prognostic relevance of these genes was also found analysing publically available gene expression data. The expression of 9 genes was compared between pre-therapeutic biopsies and post-therapeutic resected specimens. A significant post-therapeutic increase in NOTCH2, LGR5 and POU5F1 expression was found in tumours with different tumour regression grades. No significant alterations were observed for GSK3B and CTNNB1. Immunohistochemical analysis demonstrated a chemotherapy-associated increase in the intensity of NOTCH2 staining, but not in the percentage of NOTCH2. Taken together, the GSK3B, CTNNB1 and NOTCH2 expression signature is a novel, promising prognostic parameter for GC. The results of the differential expression analysis indicate a prominent role for NOTCH2 and chemotherapy resistance in GC, which seems to be related to an effect of the drugs on NOTCH2 expression rather than to an enrichment of NOTCH2 expressing tumour cells.

Analysis of outcome and cancer stem cells status in patients with resectable pancreatic adenocarcinoma

RESUMO: Actualmente, a única possibilidade de cura para doentes com adenocarcinoma do pâncreas (PDAC) é a ressecção cirúrgica, no início deste estudo, perguntamo-nos se os predictores clínico-patológicos clássicos de prognostico poderiam ser validados em uma grande cohort de doentes com cancro do pâncreas ressecável e se outros predictores clínicos poderiam ter um papel na decisão de que doentes beneficiariam de ressecção cirúrgica. No capítulo 2, observamos que até 30% dos doentes morrem no primeiro ano após a ressecção cirúrgica, pelo que o nosso objectivo foi determinar factores pré-operatórios que se correlacionam com mortalidade precoce após ressecação cirúrgica com recurso a um instrumento estatisticamente validado, o Charlson-Age Comorbidity Index (CACI), determinamos que um CACI score superior a 4 foi preditivo de internamentos prolongados (p <0,001), complicações pós-operatórias (p = 0,042), e mortalidade em 1 ano pós- ressecção cirúrgica (p <0,001). Um CACI superior a 6 triplicou a mortalidade no primeiro ano pós-cirurgia e estes doentes têm menos de 50% de probabilidade de estarem vivos um ano após a cirurgia. No capítulo 3, o nosso objectivo foi identificar uma proteína de superfície que se correlacionasse estatisticamente com o prognostico de doentes com adenocarcinoma do pâncreas e permitisse a distinção de subgrupos de doentes de acordo com as suas diferenças moleculares, perguntamo-nos ainda se essa proteína poderia ser um marcador de células-estaminais. No nosso trabalho anterior observamos que as células tumorais na circulação sanguínea apresentavam genes com características bifenotípica epitelial e mesenquimal, enriquecimento para genes de células estaminais (ALDH1A1 / ALDH1A2 e KLF4), e uma super-expressão de genes da matriz extracelular (colagénios, SPARC, e DCN) normalmente identificados no estroma de PDAC. Após a avaliação dos tumores primários com RNA-ISH, muitos dos genes identificados, foram encontrados co-localizando em uma sub-população de células na região basal dos ductos pancreáticos malignos. Além disso, observamos que estas células expressam o marcador SV2A neuroendócrino, e o marcador de células estaminais ALDH1A1/2. Em comparação com tumores negativos para SV2, os doentes com tumores SV2 positivos apresentaram níveis mais baixos de CA 19-9 (69% vs. 52%, p = 0,012), tumores maiores (> 4 cm, 23% vs. 10%, p = 0,0430), menor invasão de gânglios linfáticos (69% vs. 86%, p = 0,005) e tumores mais diferenciados (69% vs. 57%, p = 0,047). A presença de SV2A foi associada com uma sobrevida livre de doença mais longa (HR: 0,49 p = 0,009) bem como melhor sobrevida global (HR: 0,54 p = 0,018). Em conjunto, esta informação aponta para dois subtipos diferentes de adenocarcinoma do pâncreas, e estes subtipos co-relacionam estatisticamente com o prognostico de doentes, sendo este subgrupo definido pela presença do clone celular SV2A / ALDH1A1/2 positivo com características neuroendócrinas. No Capítulo 4, a expressão de SV2A no cancro do pâncreas foi validado em linhas celulares primárias. Demonstramos a heterogeneidade do adenocarcinoma do pâncreas de acordo com características clonais neuroendócrinas. Ao comparar as linhas celulares expressando SV2 com linhas celulares negativas, verificamos que as linhas celulares SV2+ eram mais diferenciadas, diferindo de linhas celulares SV2 negativas no que respeita a mutação KRAS, proliferação e a resposta à quimioterapia. No capítulo 5, perguntamo-nos se o clone celular SV2 positivo poderia explicar a resistência a quimioterapia observada em doentes. Observamos um aumento absoluto de clones celulares expressando SV2A, em múltiplas linhas de evidência - doentes, linhas de células primárias e xenotransplantes. Embora, tenhamos sido capazes de demonstrar que o adenocarcinoma do pâncreas é uma doença heterogénea, consideramos que a caracterização genética destes clones celulares expressando SV2A é de elevada importância. Pretendemos colmatar esta limitação com as seguintes estratégias: Após o tratamento com quimioterapia neoadjuvante na nossa coorte, realizamos microdissecação a laser das amostras primarias em parafina, de forma a analisar mutações genéticas observadas no adenocarcinoma pancreático; em segundo lugar, pretendemos determinar consequências de knockdown da expressão de SV2A em nossas linhas celulares seguindo-se o tratamento com gemicitabina para determinação do papel funcional de SV2A; finalmente, uma vez que os nossos esforços anteriores com um promotor - repórter e SmartFlare ™ falharam, o próximo passo será realizar RNA-ISH PrimeFlow™ seguido de FACS e RNA-seq para caracterização deste clone celular. Em conjunto, conseguimos provar com várias linhas de evidência, que o adenocarcinoma pancreático é uma doença heterogénea, definido por um clone de células que expressam SV2A, com características neuroendócrinas. A presença deste clone no tecido de doentes correlaciona-se estatisticamente com o prognostico da doença, incluindo sobrevida livre de doença e sobrevida global. Juntamente com padrões de proliferação e co-expressão de ALDH1A1/2, este clone parece apresentar um comportamento de células estaminais e está associado a resistência a quimioterapia, uma vez que a sua expressão aumenta após agressão química, quer em doentes, quer em linhas de células primárias.----------------------------- ABSTRACT: Currently, the only chance of cure for patients with pancreatic adenocarcinoma is surgical resection, at the beginning of my thesis studies, we asked if the classical clinicopathologic predictors of outcome could be validated in a large cohort of patients with early stage pancreatic cancer and if other clinical predictors could have a role on deciding which patients would benefit from surgery. In chapter 2, we found that up to 30% of patients die within the first year after curative intent surgery for pancreatic adenocarcinoma. We aimed at determining pre-operative factors that would correlate with early mortality following resection for pancreatic cancer using a statistically validated tool, the Charlson-Age Comorbidity Index (CACI). We found that a CACI score greater than 4 was predictive of increased length of stay (p<0.001), post-operative complications (p=0.042), and mortality within 1-year of pancreatic resection (p<0.001). A CACI score of 6 or greater increased 3-fold the odds of death within the first year. Patients with a high CACI score have less than 50% likelihood of being alive 1 year after surgery. In chapter 3 we aimed at identifying a surface protein that correlates with patient’s outcome and distinguishes sub-groups of patients according to their molecular differences and if this protein could be a cancer stem cell marker. The most abundant class of circulating tumor cells identified in our previous work was found to have biphenotypic features of epithelial to mesenchymal transition, enrichment for stem-cell associated genes (ALDH1A1/ALDH1A2 and KLF4), and an overexpression of extracellular matrix genes (Collagens, SPARC, and DCN) normally found in the stromal microenvironment of PDAC primary tumors. Upon evaluation of matched primary tumors with RNA-ISH, many of the genes identified were found to co-localize in a sub-population of cells at the basal region of malignant pancreatic ducts. In addition, these cells expressed the neuroendocrine marker SV2A, and the stem cell marker ALDH1A1/2. Compared to SV2 negative tumors, patients with SV2 positive tumors were more likely to present with lower CA 19-9 (69% vs. 52%, p = 0.012), bigger tumors (size > 4 cm, 23% vs. 10%, p= 0.0430), less nodal involvement (69% vs. 86%, p = 0.005) and lower histologic grade (69% vs. 57%, p = 0.047). The presence of SV2A expressing cells was associated with an improved disease free survival (HR: 0.49 p=0.009) and overall survival (HR: 0.54 p=0.018) and correlated linearly with ALDH1A2. Together, this information points to two different sub-types of pancreatic adenocarcinoma, and these sub-types correlated with patients’ outcome and were defined by the presence of a SV2A/ ALDH1A1/2 expressing clone with neuroendocrine features. In Chapter 4, SV2A expression in cancer was validated in primary cell lines. We were able to demonstrate pancreatic adenocarcinoma heterogeneity according to neuroendocrine clonal features. When comparing SV2 expressing cell lines with SV2 negative cell lines, we found that SV2+ cell lines were more differentiated and differ from SV2 negative cell lines regarding KRAS mutation, proliferation and response to chemotherapy. In Chapter 5 we aimed at determining if this SV2 positive clone could explain chemoresistance observed in patients. We found an absolute increase in SV2A expressing cells, with multiple lines of evidence, in patients, primary cell lines and xenografts. Although, we have been able to show evidence that pancreatic adenocarcinoma is a heterogeneous disease, our findings warrant further investigation. To further characterize SV2A expressing clones after treatment with neoadjuvant chemotherapy in our cohort, we have performed laser capture microdissection of the paraffin embedded tissue in this study and will analyze the tissue for known genetic mutations in pancreatic adenocarcinoma; secondly, we want to know what will happen after knocking down SV2A expression in our cell lines followed by treatment with gemcitabine to determine if SV2A is functionally important; finally, since our previous efforts with a promoter – reporter and SmartFlare™ have failed, we will utilize a novel PrimeFlow™ RNA-ISH assay followed by FACS and RNA sequencing to further characterize this cellular clone. Overall our data proves, with multiple lines of evidence, that pancreatic adenocarcinoma is a heterogeneous disease, defined by a clone of SV2A expressing cells, with neuroendocrine features. The presence of this clone in patients’ tissue correlates with patient’s disease free survival and overall survival. Together with patterns of proliferation and ALDH1A1/2 co-expression, this clone seems to present a stem-cell-like behavior and is associated with chemoresistance, since it increases after chemotherapy, both in patients and primary cell lines.

Expression and role of BORIS/CTCFL in human cancer stem cells

Le cancer est défini comme la croissance incontrôlée des cellules dans le corps. Il est responsable de 20 % des décès en Europe. Plusieurs expériences montrent que les tumeurs sont issues et se développent grâce à un petit nombre de cellules, que l'on appelle cellules souches cancéreuses (CSC). Ces CSC sont également responsables de l'apparition de métastases et de la résistance aux médicaments anticancéreux. De ce fait, l'identification des gènes qui contribuent aux propriétés de ces CSC (comme la survie des tumeurs, les métastases et la résistance aux médicaments) est nécessaire pour mieux comprendre la biologie des cancers et d'améliorer la qualité des soins des patients avec un cancer. A ce jour, de nombreux marqueurs ont été proposés ainsi que de nouvelles thérapies ciblées contre les CSC. Toutefois, et malgré les énormes efforts de la recherche dans ce domaine, la quasi-totalité des marqueurs de CSC connus à ce jour sont aussi exprimés dans les cellules saines. Ce projet de recherche visait à trouver un nouveau candidat spécifique des CSC. Le gène BORIS (pour Brother of Regulator of Imprinted Sites), nommé aussi CTCFL (CTCF-like), semble avoir certaines caractéristiques de CSC et pourrait donc devenir une cible prometteuse pour le traitement du cancer. BORIS/CTCFL est une protéine nucléaire qui se lie à l'ADN, qui est exprimée dans les tissus normaux uniquement dans les cellules germinales et qui est réactivée dans un grand nombre de tumeurs. BORIS est impliqué dans la reprogrammation épigénétique au cours du développement et dans la tumorigenèse. En outre, des études récentes ont montré une association entre l'expression de BORIS et un mauvais pronostic chez des patients atteints de différents types de cancers. Nous avons développé une nouvelle technologie basée sur les Molecular Beacon pour cibler l'ARNm de BORIS et cela dans les cellules vivantes. Grâce à ce système expérimental, nous avons montré que seule une toute petite sous-population (0,02 à 5%) de cellules tumorales exprimait fortement BORIS. Les cellules exprimant BORIS ont pu être isolées et elles présentaient les caractéristiques de CSC, telles qu'une forte expression de hTERT et des gènes spécifiques des cellules souches (NANOG, SOX2 et OCT4). En outre, une expression élevée de BORIS a été mise en évidence dans des populations enrichies en CSC ('side population' et sphères). Ces résultats suggèrent que BORIS pourrait devenir un nouveau et important marqueur de CSC. Dans des études fonctionnelles sur des cellules de cancer du côlon et du sein, nous avons montré que le blocage de l'expression de BORIS altère largement la capacité de ces cellules à former des sphères, démontrant ainsi un rôle essentiel de BORIS dans l'auto- renouvellement des tumeurs. Nos expériences montrent aussi que BORIS est un facteur important qui régule l'expression de gènes jouant un rôle clé dans le développement et la progression tumorale, tels le gène hTERT et ceux impliqués dans les cellules souches, les CSC et la transition épithélio-mésenchymateuse (EMT). BORIS pourrait affecter la régulation de la transcription de ces gènes par des modifications épigénétiques et de manière différente en fonction du type cellulaire. En résumé, nos résultats fournissent la preuve que BORIS peut être classé comme un gène marqueur de cellules souches cancéreuse et révèlent un nouveau mécanisme dans lequel BORIS jouerait un rôle important dans la carcinogénèse. Cette étude ouvre de nouvelles voies pour mieux comprendre la biologie de la progression tumorale et offre la possibilité de développement de nouvelles thérapies anti-tumorales et anti-CSC avec BORIS comme molécule cible. - Cancer is defined as the uncontrolled growth of cells in the body. It causes 20% of deaths in the European region. Current evidences suggest that tumors originate and are maintained thanks to a small subset of cells, named cancer stems cells (CSCs). These CSCs are also responsible for the appearance of metastasis and therapeutic resistance. Consequently, the identification of genes that contribute to the CSC properties (tumor survival, metastasis and therapeutic resistance) is necessary to better understand the biology of malignant diseases and to improve care management. To date, numerous markers have been proposed to use as new CSC- targeted therapies. Despite the enormous efforts in research, almost all of the known CSCs markers are also expressed in normal cells. This project aimed to find a new CSC-specific candidate. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA binding protein involves in epigenetic reprogramming in normal development and in tumorigenesis. Recent studies have shown an association of BORIS expression with a poor prognosis in different types of cancer patients. Therefore, BORIS seems to have the same characteristics of CSCs markers and it could be a promising target for cancer therapy. BORIS is normally expressed only in germinal cells and it is re-expressed in a wide variety of tumors. We developed a new molecular beacon-based technology to target BORIS mRNA expressing cells. Using this system, we showed that the BORIS expressing cells are only a small subpopulation (0.02-5%) of tumor cells. The isolated BORIS expressing cells exhibited the characteristics of CSCs, with high expression of hTERT and stem cell genes (NANOG, SOX2 and OCT4). Furthermore, high BORIS expression was observed in the CSC-enriched populations (side population and spheres). These results suggest that BORIS might be a novel and powerful CSCs marker. In functional studies, we observed that BORIS knockdown significantly impairs the capacity to form spheres in colon and breast cancer cells, thus demonstrating a critical role of BORIS in the self-renewal of tumors. The results showed in the functional analysis indicate that BORIS is an important factor that regulates the expression of key-target genes for tumor development and progression, such as hTERT, stem cells, CSCs markers and EMT (epithelial mesenchymal transition)-related marker genes. BORIS could affect the transcriptional regulation of these genes by epigenetic modification and in a cell type dependent manner. In summary, our results support the evidence that BORIS can be classified as a cancer stem cell marker gene and reveal a novel mechanism in which BORIS would play a critical role in tumorigenesis. This study opens new prospective to understand the biology of tumor development and provides opportunities for potential anti-tumor drugs.

Interactions between cancer stem cells and their niche govern metastatic colonization.

Metastatic growth in distant organs is the major cause of cancer mortality. The development of metastasis is a multistage process with several rate-limiting steps. Although dissemination of tumour cells seems to be an early and frequent event, the successful initiation of metastatic growth, a process termed 'metastatic colonization', is inefficient for many cancer types and is accomplished only by a minority of cancer cells that reach distant sites. Prevalent target sites are characteristic of many tumour entities, suggesting that inadequate support by distant tissues contributes to the inefficiency of the metastatic process. Here we show that a small population of cancer stem cells is critical for metastatic colonization, that is, the initial expansion of cancer cells at the secondary site, and that stromal niche signals are crucial to this expansion process. We find that periostin (POSTN), a component of the extracellular matrix, is expressed by fibroblasts in the normal tissue and in the stroma of the primary tumour. Infiltrating tumour cells need to induce stromal POSTN expression in the secondary target organ (in this case lung) to initiate colonization. POSTN is required to allow cancer stem cell maintenance, and blocking its function prevents metastasis. POSTN recruits Wnt ligands and thereby increases Wnt signalling in cancer stem cells. We suggest that the education of stromal cells by infiltrating tumour cells is an important step in metastatic colonization and that preventing de novo niche formation may be a novel strategy for the treatment of metastatic disease.

Stem cell pathways in the basal-like breast carcinoma: the role of beta-catenin and HIF-1alpha

Basal-like tumor is an aggressive breast carcinoma subtype that displays an expression signature similar to that of the basal/myoepithelial cells of the breast tissue. Basal-like carcinoma are characterized by over-expression of the Epidermal Growth Factor receptor (EGFR), high frequency of p53 mutations, cytoplasmic/nuclear localization of beta-catenin, overexpression of the Hypoxia inducible factor (HIF)-1alpha target Carbonic Anhydrase isoenzime 9 (CA9) and a gene expression pattern similar to that of normal and cancer stem cells, including the over-expression of the mammary stem cell markers CD44. In this study we investigated the role of p53, EGFR, beta-catenin and HIF-1alpha in the regulation of stem cell features and genes associated with the basal-like gene expression profile. The findings reported in this investigation indicate that p53 inactivation in ductal breast carcinoma cells leads to increased EGFR mRNA and protein levels. In our experimental model, EGFR overexpression induces beta-catenin cytoplasmatic stabilization and transcriptional activity and, by that, leads to increased aggressive features including mammosphere (MS) forming and growth capacity, invasive potential and overexpression of the mammary stem cell gene CD44. Moreover we found that EGFR/beta-catenin axis promotes hypoxia survival in breast carcinoma cells via increased CA9 expression. Indeed beta-catenin positively regulates CA9 expression upon hypoxia exposure. Interestingly we found that beta-catenin inhibits HIF-1alpha transcriptional activity. Looking for the mechanism, we found that CA9 expression is promoted by HIF-1alpha and cytoplasmatic beta-catenin further increased it post-transcriptionally, via direct mRNA binding and stabilization. These data reveal a functional beta-catenin/HIF-1alpha interplay among hallmarks of basal-like tumors and unveil a new functional role for cytoplasmic beta-catenin in the phenotype of such tumors. Therefore it can be proposed that the interplay here described among EGFR/beta-catenin and HIF-1alpha may play a role in breast cancer stem cell survival and function.

MicroRNA Regulation of Prostate Cancer Stem/Progenitor Cells and Prostate Cancer Development

Most human tumors contain a population of cells with stem cell properties, called cancer stem cells (CSCs), which are believed to be responsible for tumor establishment, metastasis, and resistance to clinical therapy. It’s crucial to understand the regulatory mechanisms unique to CSCs, so that we may design CSC-specific therapeutics. Recent discoveries of microRNA (miRNA) have provided a new avenue in understanding the regulatory mechanisms of cancer. However, how miRNAs may regulate CSCs is still poorly understood. Here, we present miRNA expression profiling in six populations of prostate cancer (PCa) stem/progenitor cells that possess distinct tumorigenic properties. Six miRNAs were identified to be commonly and differentially expressed, namely, four miRNAs (miR-34a, let-7b, miR-106a and miR-141) were under-expressed, and two miRNAs (miR-301 and miR-452) were over-expressed in the tumorigenic subsets compared to the corresponding marker-negative subpopulations. Among them, the expression patterns of miR-34, let-7b, miR-141 and miR-301 were further confirmed in the CD44+ human primary prostate cancer (HPCa) samples. We then showed that miR-34a functioned as a critical negative regulator in prostate CSCs and PCa development and metastasis. Over-expression of miR-34a in either bulk or CD44+ PCa cells significantly suppressed clonal expansion, tumor development and metastasis. Systemic delivery of miR-34a in tumor-bearing mice demonstrated a potent therapeutic effect again tumor progression and metastasis, leading to extended animal survival. Of great interest, we identified CD44 itself as a direct and relevant downstream target of miR-34a in mediating its tumor-inhibitory effects. Like miR-34a, let-7 manifests similar tumor suppressive effects in PCa cells. In addition, we observed differential mechanisms between let-7 and miR-34a on cell cycle, with miR-34a mainly inducing G1 cell-cycle arrest followed by cell senescence and let-7 inducing G2/M arrest. MiR-301, on the other hand, exerted a cell type dependent effect in regulating prostate CSC properties and PCa development. In summary, our work reveals that the prostate CSC populations display unique miRNA expression signatures and different miRNAs distinctively and coordinately regulate various aspects of CSC properties. Altogether, our results lay a scientific foundation for developing miRNA-based anti-cancer therapy.

Decreased Expression Of Stem Cell Markers By Simvastatin In 7,12-dimethylbenz(a)anthracene (dmba)-induced Breast Cancer.

Simvastatin, a competitive inhibitor of HMG-CoA reductase widely used in the treatment and prevention of hyperlipidemia-related diseases, has recently been associated to in vitro anticancer stem cell (CSC) actions. However, these effects have not been confirmed in vivo. To assess in vivo anti-CSC effects of simvastatin, female Sprague-Dawley rats with 7,12-dimethyl-benz(a)anthracene (DMBA)-induced mammary cancer and control animals were treated for 14 days with either simvastatin (20 or 40 mg/kg/day) or soybean oil (N = 60). Tumors and normal breast tissues were removed for pathologic examination and immunodetection of CSC markers. At 40 mg/kg/day, simvastatin significantly reduced tumor growth and the expression of most CSC markers. The reduction in tumor growth (80%) could not be explained solely by the decrease in CSCs, since the latter accounted for less than 10% of the neoplasia (differentiated cancer cells were also affected). Stem cells in normal, nonneoplastic breast tissues were not affected by simvastatin. Simvastatin was also associated with a significant decrease in proliferative activity but no increase in cell death. In conclusion, this is the first study to confirm simvastatin anti-CSC actions in vivo, further demonstrating that this effect is specific for neoplastic cells, but not restricted to CSCs, and most likely due to inhibition of cell proliferation.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches.

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Fourth European Conference on Infections in Leukaemia (ECIL-4): guidelines for diagnosis, prevention, and treatment of invasive fungal diseases in paediatric patients with cancer or allogeneic haemopoietic stem-cell transplantation.

Invasive opportunistic fungal diseases (IFDs) are important causes of morbidity and mortality in paediatric patients with cancer and those who have had an allogeneic haemopoietic stem-cell transplantation (HSCT). Apart from differences in underlying disorders and comorbidities relative to those of adults, IFDs in infants, children, and adolescents are unique with respect to their epidemiology, the usefulness of diagnostic methods, the pharmacology and dosing of antifungal agents, and the absence of interventional phase 3 clinical trials for guidance of evidence-based decisions. To better define the state of knowledge on IFDs in paediatric patients with cancer and allogeneic HSCT and to improve IFD diagnosis, prevention, and management, the Fourth European Conference on Infections in Leukaemia (ECIL-4) in 2011 convened a group that reviewed the scientific literature on IFDs and graded the available quality of evidence according to the Infectious Diseases Society of America grading system. The final considerations and recommendations of the group are summarised in this manuscript.

EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells.

Cancer stem cells (CSCs) display plasticity and self-renewal properties reminiscent of normal tissue stem cells, but the events responsible for their emergence remain obscure. We recently identified CSCs in Ewing sarcoma family tumors (ESFTs) and showed that they retain mesenchymal stem cell (MSC) plasticity. In the present study, we addressed the mechanisms that underlie ESFT CSC development. We show that the EWS-FLI-1 fusion gene, associated with 85%-90% of ESFTs and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2, and NANOG in human pediatric MSCs (hpMSCs) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSCs expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWS-FLI-1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene, SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a CSC phenotype.