C 1
BALANCE BETWEEN EPITHELIAL AND MESENCHYMAL FEATURES REGULATES PLASTICITY AND RESPONSE TO
MICROENVIRONMENTAL CUES IN LUNG CANCER CELLS
Francesca Andriani (1) - Giulia Bertolini (1) - Tiziana Caputo (1) - Federica
Facchinetti (1) - Erika Baldoli (1) - Massimo Moro (1) - Roberto Caserini (1) -
Gabriella Sozzi (1) - Luca Roz (1)
Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy (1)
Introduction: solid cancers can be seen as abnormal organs
with their own hierarchical organization where only a subpopu- lation of cells, defined as “cancer stem cells” (CSCs) has poten- tial to initiate a new tumor. Signals from the microenvironment are involved in modulation of stemness properties and recent evidence related to the plasticity of cancer cells suggests that,
CSCs can be generated de novo by a dynamic phenotypic switch between non-CSC and CSC populations.
Materials and methods: to investigate how signals from mi-
croenvironment could influence stemness properties, six lung cancer cell lines were treated with TGFb. FACS analysis showed an heterogeneous increase of CD133 (1 to 10 fold) and stemness-related genes, associated in general to an up regulation of mesenchymal EMT-related genes and downregulation of epithelial marker CDH1 assessed by Real Time PCR.
Results: extent of response was found to be strictly dependent
on the ratio between mesenchymal (M) and epithelial (E) featu- res estimated by the expression of SNAI2 and E-cadherin (CDH1) markers. Cells displaying both, mesenchymal and epithelial features, responded to microenvironmental stimuli more efficiently than cells with a fully mesenchymal phenotype suggesting that a hybrid state could be more conductive to pla- sticity. Overexpression of miR200c was used to establish whether modulation of epithelial-mesenchymal balance could affect the acquisition of stemness phenotype under microenvi- ronmental stimuli. MiR200c induced upregulation of CDH1 wi- thout any alteration of mesenchymal markers and resulted in a shift, driving cells toward a more epithelial phenotype. Only cell lines that reached a critical threshold in the balance between E and M markers were able to generate stem-like cells in re- sponse to external signals. Moreover, to verify the importance of microenvironment in providing the right cues also for tumor progression in vivo, cell lines were co-injected with fibroblasts in mice. Fibroblasts promoted tumor growth and dissemination in vivo in particular in cells with hybrid features (E/M), while cells with mesenchymal traits resulted more cell-autonomous and not responsive to signals derived from fibroblasts.
Conclusions: together these data provide evidence that external
stimuli can generate cells with stem-like features and that phe- notypic state, mesenchymal (M), epithelial (E) or hybrid (M/E), is critical for the regulation of plasticity induced by the mi- croenvironment.
C 2
IMPAIRMENT OF V-ATPASE AS A THERAPEUTIC MODALITY TARGETING CANCER STEM CELLS OF RHABDOMYOSARCOMA
Sofia Avnet (1) - Manuela Salerno (2) - Shigekuni Hosogi (3) - Nicola Baldini (2)
Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna, Italy (1) - Department of Biomedical and Neu-
romotor Sciences, University of Bologna, Bologna, Italy (2) - Department
of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan, Bologna, Italy (3)
Introduction: Rhabdomyosarcoma (RMS) is a very aggressive
tumor with a high incidence of relapse and metastasis. Since treatment options for RMS patients are limited, the identifica- tion of novel targets is strongly needed. According to the cancer stem cell (CSC) theory, the development of new agents that se- lectively block key pathways in the CSC subpopulation would be predictably successful. We have previously isolated and characterized CSC from several sarcomas, including RMS.1 In
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this work, we investigate the vacuolar proton pump (V-ATPase) activity as a candidate therapeutic target.
Materials and methods : RMS-CSC were obtained by the 3D
sphere system assay, under low attachment conditions plus bFGF and EGF exposure. Stemness, chemoresistance, invasive- ness, lysosome and cytosolic acidity, and V-ATPAse V0c ex- pression of CSC were evaluated and compared to those of pa- rental cells. Finally, we analyzed the effects of the V-ATPase V0c blocker, omeprazole, on survival, invasiveness, and drug resistance of CSC.
Results: isolated RMS-CSC showed stem-like features (high
expression of NANOG and OCT3/4 genes, self-renewal and multiple differentiation ability) and higher invasive ability and chemoresistance to doxorubicin (DXR) in comparison to native cells. Interestingly, RMS-CSC were also resistant to DXR through a mechanism unrelated to the classical multidrug re- sistance process but dependent on a high level of lysosome acidity mediated by an overexpression of vacuolar ATPase (V- ATPase). Inhibition of lysosomal acidification by the V-ATPase inhibitor omeprazole (OME) significantly modified chemo- resistance of RMS-CSC. Unexpectedly, lysosomal targeting al- so blocked cell growth and reduced the invasive potential of RMS CSC, even at a very low dosage (OME 10 and 50 µM, re- spectively).
Conclusions: Based on these observations, we propose lyso-
some acidity as a valuable target to overcome chemoresistance of RMS-CSC, and suggest the use of anti-V-ATPase agents in combination with standard regimens as a promising tool for the eradication of minimal residual disease or the prevention of metastatic disease.
Acknowledgements: This project was founded by the Italian
Association of Cancer Research (AIRC) and Italian Ministry of Education (FIRB).
Reference:
Salerno M, Avnet S, Bonuccelli G, Eramo A, De Maria R, Gambarotti M, Gamberi G, Baldini N. Sphere-forming cell sub- sets with cancer stem cell properties in human musculoskeletal sarcomas. Int J Oncol 2013;43:95-102.
C 3
GENE EXPRESSION AND MICRORNA PROFILES OF GBM STEM- LIKE CELLS DISCRIMINATE DIFFERENTIALLY AGGRESSIVE CELL CLUSTERS
Mariachiara Buccarelli (1) - Michele Signore (2) - Liliana Morgante (2) -
Daniele Runci (1) - Ramona Ilari (2) - Roberto Pallini (3) - Lucia Ricci-vitiani (2)
Department of Hematology, Oncology and Molecular Medicine,Istituto Superiore di Sanità, Rome, Italy, Università di Roma 'La Sapienza', Ro- ma, Italy (1) - Department of Hematology, Oncology and Molecular Medi-
cine,Istituto Superiore di Sanità, Rome, Italy, Department of Hematology, Oncology and Molecular Medicine,Istituto Superiore di Sanità, Rome, Italy, Roma, Italy (2) - Institute of Neurosurgery,Università Cattolica del
Sacro Cuore, Roma, Italy, Institute of Neurosurgery,Università Cattolica del Sacro Cuore, Roma, Italy, Roma, Italy (3)
Glioblastoma multiforme (GBM) is the most common and ag- gressive adult brain tumor. Despite aggressive surgery, radiation and chemotherapies, the life expectancy of patients with GBM is about 14 months after diagnosis. The extremely aggressive behaviour of GBM has been ascribed to a rare fraction of self- renewing, multipotent tumor-initiating cells, called GBM stem- like cells (GSCs), responsible for tumor progression, mainte- nance, and recurrence. GSCs can be maintained in vitro using specific serum-free conditions. During the last years, we have collected GSCs from more than 50 GBM patients. These cells have been expanded and validated for their stem cell properties. The tumorigenic potential of GSCs has been assayed in vivo by orthotopic and heterotopic xenograft in immunodeficient mice. Gene expression profiling (Affymetrix assay) has been per- formed in a large panel of GSC lines. Clustering of the 40 GSC samples using only the top 1000 most variable genes/transcript revealed two distinct clusters. A clustering of GBM SC samples into two distinct clusters has been reported in the literature. Ac- cording to this previous works we also classified the two clus- ters as a “GSf” full stem-like phenotype (more closely related to GBM samples) and a “GSr” restricted stem-like phenotype (more closely related to GBM stem-like cell lines).
MicroRNA (miRNAs) gene profiling (Agilent assay) has also been performed on GSC lines identifying 9 miRNAs as differ- entially expressed between GSf and GSr groups. Some of them were previously reported to be de-regulated in malignancies, including GBM. These miRNAs are mainly involved in cell growth and survival as regulators of the EGFR signaling path- way.
C 4
RESVERATROL EFFECTS ON MIGRATION AND WNT SIGNALING PATHWAY IN CANCER STEM CELL LINES FROM GLIOBLASTOMA MULTIFORME
Chiara Cilibrasi (1) - Gabriele Riva (2) - Valentina Butta (1) - Leda Dalpra' (3)
- Angela Bentivegna (2)
Phd in neuroscience, department of surgery and translational medicine, university of milan-bicocca, monza (mb), italy (1) - department of surgery
and translational medicine, university of milan-bicocca, monza (mb), italy
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(2) - department of surgery and translational medicine / department of
clinical pathology, medical genetics laboratory, university of milan- bicocca / san gerardo hospital, monza (mb), italy (3)
Introduction Glioblastoma multiforme (GBM) is a grade IV
astrocytoma and the least successfully treated solid tumors: cur- rent therapies provide a median survival of 12-15 months after diagnosis, due to the high recurrence rate. GBM is characterized by an highly infitrative nature and a remarkable intratumoral heterogeneity, mirrored by the presence of distinct sub- populations of cells with different tumorigenic capabilities: Gli- oma stem cells (GSCs) are believed to be the real driving force of tumor initiation, progression and relapse. Better therapeutic strategies GSC-targeted are needed.
Resveratrol (RSV) is a polyphenolic phytoalexin with plei- otropic health benefits. Many studies highlighted its antiprolif- erative and proapoptotic effects and its ability to reduce tumor invasion and cell migration in several types of cancers, inclu- ding GBM.
RSV might represent an attractive agent for the treatment of GBM because of its minimal toxicity and blood brain barrier permeability.
Material and Methods In this study, we analyzed the effects of
RSV exposure (48-72-96h) on cell motility, through Wound Healing assay, in six GSC lines isolated from GBM (GBM2, GBM7, G179, GliNS2, G144, GBM04). Moreover we evaluat- ed the effect of RSV administration on WNT signaling pathway using RT-PCR technology on Applied Biosystems platform. The WNT signaling expression profile was performed using PCR custom arrays (Qiagen) on untreated and treated (100mM 96h RSV) cells in order to explore expression variations of 7 WNT-related genes (WNT1, FZD4, CTNNB1, EP300, CREBBP, TCF7, MYC) after RSV exposure.
Results RSV strongly reduces cellular motility in all GSC cell
lines and differently modulates WNT signaling pathway in the GSC cell lines taken into account. WNT1 is upregulated in GBM04 and G144 cell lines, while FZD4 is upregulated in GBM2, GBM04 and G144 cell lines after treatment. CTNNB1 and EP300 show an unchanged transcriptional activity in most of the cell lines. CREBBP and TCF7 are upregulated in some cell lines and downregulated in others. MYC is upregulated in most of the cell lines.
The validation of these data in order to verify, at protein level, the RSV modulation of MMPs and WNT signaling pathway is in progress.
Conclusions RSV treatment could represent, in combination
with other therapeutic strategies, a new approach in order to in- hibit the infiltrative nature of GSCs. Moreover the study of the WNT signaling pathway could suggest new insights on GBM oncogenesis.
Keywords: GBM, Glioma stem cells, Resveratrol, WNT signal- ing pathway
C 5
EFFECTS OF CHEMOPREVENTIVE AGENTS ON MELANOMA STEM CELLS
Katiuscia Dallaglio (1) - Tiziana Petrachi (1) - Martina Chiappelli (1) - Adria-
na Albini (2)
Tranlastional Research Laboratory, IRCCS 'Tecnologie Avanzate e Mod- elli Assistenziali in Oncologia', Arcispedale Santa Maria Nuova, REGGIO EMILIA, Italy (1) - Department of Research and Statistics Infrastructure,
IRCCS 'Tecnologie Avanzate e Modelli Assistenziali in Oncologia', Ar- cispedale Santa Maria Nuova, REGGIO EMILIA, Italy (2)
Introduction: Melanoma is the deadliest form of skin cancer,
displaying resistance to conventional therapies. Recently dis- covered drugs targeting melanoma induce some degree of tumor regression, however relapse often occurs within few months. A possible mechanism explaining this phenomenon is the persis- tence of stem-like tumor cells, namely cancer stem cells (CSC), in treated patients. Therapies targeting the CSC compartment are warranted, however to date no currently available drug kills exclusively or preferentially melanoma stem cells. Chemopre- vention is the use of natural or synthetic chemical agents sup- pressing or preventing the carcinogenic progression. Several chemopreventive agents have shown to be able to kill melano- ma cells, however, little is known on the cellular targets of chemoprevention in melanoma.
Materials and methods: We evaluated the effect of the chem-
opreventive biguanides metformin and phenformin on melano- ma cells using both monolayer and 3D spheroid cultures. Given that in melanoma, cells expressing high levels of aldehyde de- hydrogenase (ALDH) are CSC, we isolated ALDH+ and + mel- anoma cells by flow cytometric cell sorting and analyzed these cells by Real Time PCR. We also tested the sensitivity of ALDH+ and - cells to 10mM metformin and 1mM phenformin by blu trypan exclusion dye.
Results: Metformin and phenformin abrogate melanoma cell
viability in monolayer cell cultures and reduce sphere size and the number of viable cells/sphere at day10. Once sorted, ALDH+ melanoma cells express stem cell markers including SOX2 and CD271 and generate slightly bigger and less necrotic spheres as compared to ALDH- cells. Cell viability is higher in ALDH+ derived spheres as compared to ALDH- derived ones. To test if AMP-Activated Protein Kinase (AMPK), a molecular hub activated by metfomin, is involved in this mechanism we analysed AMPK expression in ALDH+ and – cells. AMPK lev- els are similar in both cell types, consistently with a similar de- crease in size of spheres derived from ALDH+ and – cells after treatment with metformin and phenformin. However, the num- ber of viable cells/sphere is markedly and significantly de- creased in ALDH+ derived spheres treated with metfor- min/phenformin, but not in ALDH- ones.
Conclusions: Preliminary results suggest that both metformin
and phenformin decrease melanoma cell viability, preferentially targeting ALDH+ melanoma cells. Further studies analysing the molecular mechanisms (including AMPK involvement) under- lying metformin/phenformin- effects on melanoma CSC are necessary. Overall these data indicate a possible approach to target melanoma stem cells with cancer chemopreventive thera- pies.
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C 6
INHIBITION OF GSK 3BETA ACTIVITY RESULTS IN
MESENCHYMAL TO EPITHELIAL REVERTING TRANSITION IN PRIMARY COLON CANCER CELL CULTURES
Marina De Rosa (1) - Valeria Costabile (1) - Raffaella Accetta (1) - Paolo
Delrio (2) - Daniela Rega (2) - Ugo Pace (2) - Francesca Duraturo (1) - Raffa-
ella Liccardo (1) - Giovanni Battista Rossi (3) - Rita Genesio (4) - Lucio
Nitsch (4) - Paola Izzo (1)
Department of Molecular Medicine and Medical Biotechnology and CEINGE Biotecnologie Avanzate, University of Naples “Federico II”, Na- ples, Italy (1) - Colorectal Surgical Oncology - Abdominal Oncology De-
partment, Istituto Nazionale per lo studio e la cura dei tumori, “Fonda- zione Giovanni Pascale”-IRCCS, Naples, Italy (2) - Endoscopy Unit, Istituto
Nazionale per lo studio e la cura dei tumori, “Fondazione Giovanni Pascale" -IRCCS, Naples, Italy (3) - Department of Molecular Medicine
and Medical Biotechnology, University of Naples “Federico II”, Naples, Italy (4)
Introduction. Epithelial-to mesenchymal transition (EMT) con-
fers stem cell-like phenotype and more motile properties to car- cinoma cells. During EMT, the expression of E-cadherin de- creases, resulting in loss of cell–cell adhesion and increased mi- gration. Expression of Twist1 and other pleiotropic transcription factors, such as Snail, is known to activate EMT.
We aimed to realize a tissue bio-bank from patients affected by colorectal cancer and to establish primary cell cultures with the main purpose to study EMT.
Materials and methods. We sampled pairs of normal colorec-
tal mucosa and its matched cancer tissues from 110 patients with sporadic and hereditary colorectal cancer, established pri- mary cell cultures from some of these patients and validated coltures with cytogenetic and molecular biology approaches. Western blot assay, quantitative Real-Time PCR and immuno- fluorescence were performed to investigate the expression of several markers, such as: E-cadherin, N-cadherin, vimentin, be- ta-catenin, cytokeratin 20 and cytokeratin 18, twist1, Snail, CD44, ki67, sox-2, oct-4 and nanog. Moreover, cell differentia- tion was induced by incubation with 5% FBS and 30mM LiCl containing medium for 10 days.
Results. Our prymary colorectal cancer cells lose the expression
of E-cadherin epithelial marker, which is instead expressed in cancer and normal colon mucosa of the same patient, while over-express vimentin (mesenchymal marker) Twist1, Snail (EMT markers) and Ki67. Cytokeratin 18 was expressed both in tissues and cell cultures. Expression of stem cell markers, such as CD44, Sox-2, oct-4 and nanog were also observed. Follow- ing differentiation with the GSK3b inhibitor LiCl, the cells be- gan to express E-cadherin and, at once, twist1 expression was strongly down-regulated, suggesting a mesenchymal to epitheli- al reverting transition process.
Discussion. We have established primary colorectal cancer cell
cultures, isolated from CRC patients, that express mesenchymal and epithelial bio-markers together with high level of EMT transcription factors. Moreover, inhibition of GSK3 beta by LiCl causes mesenchymal to epithelial reverting transition. In our opinion this approach could represent an interesting way to further investigate the clinical relevance of EMT in human colo-
rectal cancer and the molecular basis of pharmacological re- sistance and metastasis.
C 7
EFFECTS OF CD133 MUTANTS ON COLON CANCER CELLS PHENOTYPE
Caterina Fanali (1) - Maddalena Corbi (2) - Maria Svelto (3) - Marisa Farina (3) - Donatella Lucchetti (3) - Valerio Cufino (3) - Alma Boninsegna (3) - Fed-
erica Calapà (3) - Achille Cittadini (3) - Alessandro Sgambato (3)
pathology institute, università cattolica del sacro cuore, roma, Italy (1) -
Università Cattolica del Sacro Cuore - Istituto di Patologia Generale (2)
Background: Colorectal cancer (CRC) is the third most com-
mon cause of cancer-related death. It has been reported that a subpopulation of cells within CRC possesses the potential to initiate and sustain tumor growth. These cells are called cancer stem cells (CSCs) and present special surface markers.
The CD133 has proved a useful marker to identify and isolate the colon CSCs and although many study have demonstrated that CD133 expression has prognostic significance, the exact roles of the molecule in human tumorigenesis remain unknown. CD133 contains a short C-terminal cytoplasmatic domain with five tyrosine residues, including a consensus tyrosine phosphor- ylation site. To analyze the role of C-terminal domain of CD133, the HCT116 human colon cancer cells were engineered to express an exogenous CD133 without C-terminal domain and 5 mutants of CD133 in which the tyrosine residues of C- terminal domain have been mutated in phenylalanines.
Materials and Methods: The cDNA encoding the CD133 de-
leted of C-terminal domain was transfected into HCT116 cells; these cells was compared with cells overexpressing the full- length CD133. Mutagenesis experiments were conducted to mu- tate the tyrosine residues in phenylalanine and subsequent ex- periments were performed to test the potential function of these aminoacid residues within the molecule.
Results: Cells overexpressing the CD133 deleted of C-terminal
domain, have shown a reduction in proliferation, migration, in- vasiveness and tumorigenicity compared to cells overexpressing the full size CD133. Moreover cells overexpressing the CD133 wild type have shown an increased phosphorylation of Akt, which was not observed in the cells overexpressing the truncat- ed protein.
To test if CD133 C-terminal domain could be involved in the activation of AKT pathway through the tyrosine phosphoryla- tion, the HCT116 have been engineered to express mutants of CD133 in which the tyrosine residues have been mutated in phenylalanines.
Real time PCR have shown that some of these mutants dis- played a genic expression profile more similar to cells overex- pressing the CD133 deleted of C-terminal domain that to cells overexpressing the CD133 wild type .
Conclusions: Our studies show that the C-terminal domain ti-
rosynes are involved in the activation of intracellular signaling CD133-dependent and that the activation of AKT pathway
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might be involved in this signaling. Further studies are needed to assess which tyrosine are more involved in this activation.
C 8
BONE MORPHOGENETIC PROTEINS SIGNALING PROMOTES CANCER INITIATING CELLS PHENOTYPE
Chiara Garulli (1) - Cristina Kalogris (1) - Lucia Pietrella (1) - Caterina Bar-
tolacci (1) - Cristina Andreani (1) - Valentina Gambini (1) - Martina Tilio (1) -
Maria Elexpuru Zabaleta (1) - Cristina Marchini (1) - Augusto Amici (1)
Department of Bioscience and Biotechnology, University of Camerino, Camerino, Italy. (1)
INTRODUCTION Increasing evidence supports the theory
that tumor growth, homeostasis, and recurrence are dependent on a small subset of cells with stem cell properties, redefined cancer initiating cells (CICs) or cancer stem cells. Bone mor- phogenetic proteins (BMPs) are involved in cell-fate specifica- tion during embryogenesis, in the maintenance of developmen- tal potency in adult stem cells and may contribute to sustain CIC populations in breast carcinoma. Using the mouse A17 cell model previously related to mesenchymal cancer stem cells and displaying properties of CICs, we investigated the role of BMPs in the control of breast cancer cell plasticity.
MATERIALS AND METHODS A17 cells were treated with