Publications by authors named "Michele Signore"

47 Publications

Control of replication stress and mitosis in colorectal cancer stem cells through the interplay of PARP1, MRE11 and RAD51.

Cell Death Differ 2021 Feb 2. Epub 2021 Feb 2.

IIGM - Italian Institute for Genomic Medicine, c/o IRCSS, Candiolo, Italy.

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations.
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http://dx.doi.org/10.1038/s41418-020-00733-4DOI Listing
February 2021

PI3K-driven HER2 expression is a potential therapeutic target in colorectal cancer stem cells.

Gut 2021 Jan 12. Epub 2021 Jan 12.

Department of Surgical, Oncological and Stomatological Sciences, Università degli Studi di Palermo, Palermo, Italy

Objective: Cancer stem cells are responsible for tumour spreading and relapse. Human epidermal growth factor receptor 2 (HER2) expression is a negative prognostic factor in colorectal cancer (CRC) and a potential target in tumours carrying the gene amplification. Our aim was to define the expression of HER2 in colorectal cancer stem cells (CR-CSCs) and its possible role as therapeutic target in CRC resistant to anti- epidermal growth factor receptor (EGFR) therapy.

Design: A collection of primary sphere cell cultures obtained from 60 CRC specimens was used to generate CR-CSC mouse avatars to preclinically validate therapeutic options. We also made use of the ChIP-seq analysis for transcriptional evaluation of HER2 activation and global RNA-seq to identify the mechanisms underlying therapy resistance.

Results: Here we show that in CD44v6-positive CR-CSCs, high HER2 expression levels are associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which promotes the acetylation at the regulatory elements of the Erbb2 gene. HER2 targeting in combination with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) inhibitors induces CR-CSC death and regression of tumour xenografts, including those carrying and mutation. Requirement for the triple targeting is due to the presence of cancer-associated fibroblasts, which release cytokines able to confer CR-CSC resistance to PI3K/AKT inhibitors. In contrast, targeting of PI3K/AKT as monotherapy is sufficient to kill liver-disseminating CR-CSCs in a model of adjuvant therapy.

Conclusions: While PI3K targeting kills liver-colonising CR-CSCs, the concomitant inhibition of PI3K, HER2 and MEK is required to induce regression of tumours resistant to anti-EGFR therapies. These data may provide a rationale for designing clinical trials in the adjuvant and metastatic setting.
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http://dx.doi.org/10.1136/gutjnl-2020-323553DOI Listing
January 2021

Deregulated expression of the imprinted DLK1-DIO3 region in glioblastoma stemlike cells: tumor suppressor role of lncRNA MEG3.

Neuro Oncol 2020 12;22(12):1771-1784

Department of Oncology and Molecular Medicine Rome, Italy.

Background: Glioblastoma (GBM) stemlike cells (GSCs) are thought to be responsible for the maintenance and aggressiveness of GBM, the most common primary brain tumor in adults. This study aims at elucidating the involvement of deregulations within the imprinted delta-like homolog 1 gene‒type III iodothyronine deiodinase gene (DLK-DIO3) region on chromosome 14q32 in GBM pathogenesis.

Methods: Real-time PCR analyses were performed on GSCs and GBM tissues. Methylation analyses, gene expression, and reverse-phase protein array profiles were used to investigate the tumor suppressor function of the maternally expressed 3 gene (MEG3).

Results: Loss of expression of genes and noncoding RNAs within the DLK1-DIO3 region was observed in GSCs and GBM tissues compared with normal brain. This downregulation is mainly mediated by epigenetic silencing. Kaplan-Meier analysis indicated that low expression of MEG3 and MEG8 long noncoding (lnc)RNAs significantly correlated with short survival in GBM patients. MEG3 restoration impairs tumorigenic abilities of GSCs in vitro by inhibiting cell growth, migration, and colony formation and decreases in vivo tumor growth, reducing infiltrative growth. These effects were associated with modulation of genes involved in cell adhesion and epithelial-to-mesenchymal transition (EMT).

Conclusion: In GBM, MEG3 acts as a tumor suppressor mainly regulating cell adhesion, EMT, and cell proliferation, thus providing a potential candidate for novel GBM therapies.
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http://dx.doi.org/10.1093/neuonc/noaa127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746944PMC
December 2020

A pre-existing population of ZEB2 quiescent cells with stemness and mesenchymal features dictate chemoresistance in colorectal cancer.

J Exp Clin Cancer Res 2020 Jan 8;39(1). Epub 2020 Jan 8.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.

Background: Quiescent/slow cycling cells have been identified in several tumors and correlated with therapy resistance. However, the features of chemoresistant populations and the molecular factors linking quiescence to chemoresistance are largely unknown.

Methods: A population of chemoresistant quiescent/slow cycling cells was isolated through PKH26 staining (which allows to separate cells on the basis of their proliferation rate) from colorectal cancer (CRC) xenografts and subjected to global gene expression and pathway activation analyses. Factors expressed by the quiescent/slow cycling population were analyzed through lentiviral overexpression approaches for their ability to induce a dormant chemoresistant state both in vitro and in mouse xenografts. The correlation between quiescence-associated factors, CRC consensus molecular subtype and cancer prognosis was analyzed in large patient datasets.

Results: Untreated colorectal tumors contain a population of quiescent/slow cycling cells with stem cell features (quiescent cancer stem cells, QCSCs) characterized by a predetermined mesenchymal-like chemoresistant phenotype. QCSCs expressed increased levels of ZEB2, a transcription factor involved in stem cell plasticity and epithelial-mesenchymal transition (EMT), and of antiapototic factors pCRAF and pASK1. ZEB2 overexpression upregulated pCRAF/pASK1 levels resulting in increased chemoresistance, enrichment of cells with stemness/EMT traits and proliferative slowdown of tumor xenografts. In parallel, chemotherapy treatment of tumor xenografts induced the prevalence of QCSCs with a stemness/EMT phenotype and activation of the ZEB2/pCRAF/pASK1 axis, resulting in a chemotherapy-unresponsive state. In CRC patients, increased ZEB2 levels correlated with worse relapse-free survival and were strongly associated to the consensus molecular subtype 4 (CMS4) characterized by dismal prognosis, decreased proliferative rates and upregulation of EMT genes.

Conclusions: These results show that chemotherapy-naive tumors contain a cell population characterized by a coordinated program of chemoresistance, quiescence, stemness and EMT. Such population becomes prevalent upon drug treatment and is responsible for chemotherapy resistance, thus representing a key target for more effective therapeutic approaches.
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http://dx.doi.org/10.1186/s13046-019-1505-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947904PMC
January 2020

A new bioavailable fenretinide formulation with antiproliferative, antimetabolic, and cytotoxic effects on solid tumors.

Cell Death Dis 2019 07 23;10(7):529. Epub 2019 Jul 23.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.

Fenretinide is a synthetic retinoid characterized by anticancer activity in preclinical models and favorable toxicological profile, but also by a low bioavailability that hindered its clinical efficacy in former clinical trials. We developed a new formulation of fenretinide complexed with 2-hydroxypropyl-beta-cyclodextrin (nanofenretinide) characterized by an increased bioavailability and therapeutic efficacy. Nanofenretinide was active in cell lines derived from multiple solid tumors, in primary spheroid cultures and in xenografts of lung and colorectal cancer, where it inhibited tumor growth independently from the mutational status of tumor cells. A global profiling of pathways activated by nanofenretinide was performed by reverse-phase proteomic arrays and lipid analysis, revealing widespread repression of the mTOR pathway, activation of apoptotic, autophagic and DNA damage signals and massive production of dihydroceramide, a bioactive lipid with pleiotropic effects on several biological processes. In cells that survived nanofenretinide treatment there was a decrease of factors involved in cell cycle progression and an increase in the levels of p16 and phosphorylated p38 MAPK with consequent block in G0 and early G1. The capacity of nanofenretinide to induce cancer cell death and quiescence, together with its elevated bioavailability and broad antitumor activity indicate its potential use in cancer treatment and chemoprevention.
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http://dx.doi.org/10.1038/s41419-019-1775-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6646369PMC
July 2019

The kinase inhibitor SI113 induces autophagy and synergizes with quinacrine in hindering the growth of human glioblastoma multiforme cells.

J Exp Clin Cancer Res 2019 May 17;38(1):202. Epub 2019 May 17.

Section of Cellular Networks and Molecular Therapeutic Targets, Proteomics Unit, IRCCS - Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

Background: Glioblastoma multiforme (GBM), due to its location, aggressiveness, heterogeneity and infiltrative growth, is characterized by an exceptionally dismal clinical outcome. The small molecule SI113, recently identified as a SGK1 inhibitor, has proven to be effective in restraining GBM growth in vitro and in vivo, showing also encouraging results when employed in combination with other antineoplastic drugs or radiotherapy. Our aim was to explore the pharmacological features of SI113 in GBM cells in order to elucidate the pivotal molecular pathways affected by the drug. Such knowledge would be of invaluable help in conceiving a rational offensive toward GBM.

Methods: We employed GBM cell lines, either established or primary (neurospheres), and used a Reverse-Phase Protein Arrays (RPPA) platform to assess the effect of SI113 upon 114 protein factors whose post-translational modifications are associated with activation or repression of specific signal transduction cascades.

Results: SI113 strongly affected the PI3K/mTOR pathway, evoking a pro-survival autophagic response in neurospheres. These results suggested the use of SI113 coupled, for maximum efficiency, with autophagy inhibitors. Indeed, the association of SI113 with an autophagy inhibitor, the antimalarial drug quinacrine, induced a strong synergistic effect in inhibiting GBM growth properties in all the cells tested, including neurospheres.

Conclusions: RPPA clearly identified the molecular pathways influenced by SI113 in GBM cells, highlighting their vulnerability when the drug was administered in association with autophagy inhibitors, providing a strong molecular rationale for testing SI113 in clinical trials in associative GBM therapy.
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http://dx.doi.org/10.1186/s13046-019-1212-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525441PMC
May 2019

Organoids as a new model for improving regenerative medicine and cancer personalized therapy in renal diseases.

Cell Death Dis 2019 02 27;10(3):201. Epub 2019 Feb 27.

IRCCS, Regina Elena National Cancer Institute, Rome, Italy.

The pressure towards innovation and creation of new model systems in regenerative medicine and cancer research has fostered the development of novel potential therapeutic applications. Kidney injuries provoke a high request of organ transplants making it the most demanding system in the field of regenerative medicine. Furthermore, renal cancer frequently threaten patients' life and aggressive forms still remain difficult to treat. Ethical issues related to the use of embryonic stem cells, has fueled research on adult, patient-specific pluripotent stem cells as a model for discovery and therapeutic development, but to date, normal and cancerous renal experimental models are lacking. Several research groups are focusing on the development of organoid cultures. Since organoids mimic the original tissue architecture in vitro, they represent an excellent model for tissue engineering studies and cancer therapy testing. We established normal and tumor renal cell carcinoma organoids previously maintained in a heterogeneous multi-clone stem cell-like enriching medium. Starting from adult normal kidney specimens, we were able to isolate and propagate organoid 3D-structures composed of both differentiated and undifferentiated cells while expressing nephron specific markers. Furthermore, we were capable to establish organoids derived from cancer tissues although with a success rate inferior to that of their normal counterpart. Cancer cultures displayed epithelial and mesenchymal phenotype while retaining tumor specific markers. Of note, tumor organoids recapitulated neoplastic masses when orthotopically injected into immunocompromised mice. Our data suggest an innovative approach of long-term establishment of normal- and cancer-derived renal organoids obtained from cultures of fleshly dissociated adult tissues. Our results pave the way to organ replacement pioneering strategies as well as to new models for studying drug-induced nephrotoxicity and renal diseases. Along similar lines, deriving organoids from renal cancer patients opens unprecedented opportunities for generation of preclinical models aimed at improving therapeutic treatments.
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http://dx.doi.org/10.1038/s41419-019-1453-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393468PMC
February 2019

Renal cancer: new models and approach for personalizing therapy.

J Exp Clin Cancer Res 2018 Sep 5;37(1):217. Epub 2018 Sep 5.

IRCCS, Regina Elena National Cancer Institute, Rome, Italy.

Background: Clear cell RCC (ccRCC) accounts for approximately 75% of the renal cancer cases. Surgery treatment seems to be the best efficacious approach for the majority of patients. However, a consistent fraction (30%) of cases progress after surgery with curative intent. It is currently largely debated the use of adjuvant therapy for high-risk patients and the clinical and molecular parameters for stratifying beneficiary categories. In addition, the treatment of advanced forms lacks reliable driver biomarkers for the appropriated therapeutic choice. Thus, renal cancer patient management urges predictive molecular indicators and models for therapy-decision making.

Methods: Here, we developed and optimized new models and tools for ameliorating renal cancer patient management. We isolated from fresh tumor specimens heterogeneous multi-clonal populations showing epithelial and mesenchymal characteristics coupled to stem cell phenotype. These cells retained long lasting-tumor-propagating capacity provided a therapy monitoring approach in vitro and in vivo while being able to form parental tumors when orthotopically injected and serially transplanted in immunocompromised murine hosts.

Results: In line with recent evidence of multiclonal cancer composition, we optimized in vitro cultures enriched of multiple tumor-propagating populations. Orthotopic xenograft masses recapitulated morphology, grading and malignancy of parental cancers. High-grade but not the low-grade neoplasias, resulted in efficient serial transplantation in mice. Engraftment capacity paralleled grading and recurrence frequency advocating for a prognostic value of our developed model system. Therefore, in search of novel molecular indicators for therapy decision-making, we used Reverse-Phase Protein Arrays (RPPA) to analyze a panel of total and phosphorylated proteins in the isolated populations. Tumor-propagating cells showed several deregulated kinase cascades associated with grading, including angiogenesis and m-TOR pathways.

Conclusions: In the era of personalized therapy, the analysis of tumor propagating cells may help improve prediction of disease progression and therapy assignment. The possibility to test pharmacological response of ccRCC stem-like cells in vitro and in orthotopic models may help define a pharmacological profiling for future development of more effective therapies. Likewise, RPPA screening on patient-derived populations offers innovative approach for possible prediction of therapy response.
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http://dx.doi.org/10.1186/s13046-018-0874-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126022PMC
September 2018

The Double Face of Exosome-Carried MicroRNAs in Cancer Immunomodulation.

Int J Mol Sci 2018 Apr 13;19(4). Epub 2018 Apr 13.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, 00161 Rome, Italy.

In recent years many articles have underlined the key role of nanovesicles, i.e., exosomes, as information carriers among biological systems including cancer. Tumor-derived exosomes (TEXs) are key players in the dynamic crosstalk between cancer cells and the microenvironment while promote immune system control evasion. In fact, tumors are undoubtedly capable of silencing the immune response through multiple mechanisms, including the release of exosomes. TEXs have been shown to boost tumor growth and promote progression and metastatic spreading via suppression or stimulation of the immune response towards cancer cells. The advantage of immunotherapeutic treatment alone over combining immuno- and conventional therapy is currently debated. Understanding the role of tumor exosome-cargo is of crucial importance for our full comprehension of neoplastic immonosuppression and for the construction of novel therapies and vaccines based on (nano-) vesicles. Furthermore, to devise new anti-cancer approaches, diverse groups investigated the possibility of engineering TEXs by conditioning cancer cells’ own cargo. In this review, we summarize the state of art of TEX-based immunomodulation with a particular focus on the molecular function of non-coding family genes, microRNAs. Finally, we will report on recent efforts in the study of potential applications of engineered exosomes in cancer immunotherapy.
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http://dx.doi.org/10.3390/ijms19041183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979514PMC
April 2018

Effect of miR-204&211 and RUNX2 control on the fate of human mesenchymal stromal cells.

Regen Med Res 2017 5;5. Epub 2017 Dec 5.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy - Sbarro Institute for Cancer Research and Molecular Medicine & Center of Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania.

MiR-204 and 211 enforced expression in murine mesenchymal stromal cells (MSCs) has been shown to induce adipogenesis and impair osteogenesis, through RUNX2 down-modulation. This mechanism has been suggested to play a role in osteoporosis associated with obesity. However, two further fundamental MSC functions, chondrogenesis and hematopoietic supporting activity, have not yet been explored. To this end, we transduced, by a lenti-viral vector, miR-204 and 211 in a model primary human MSC line, opportunely chosen among our MSC collection for displaying all properties of canonical bone marrow MSCs, except adipogenesis. Enforced expression of miR-204&211 in these cells, rescued adipogenesis, and inhibited osteogenesis, as previously reported in murine MSCs, but, surprisingly, also damaged cartilage formation and hematopoietic supporting activity, which were never explored before. RUNX2 has been previously indicated as the target of miR-204&211, whose down modulation is responsible for the switch from osteogenesis to adipogenesis. However, the additional disruption of chondrogenesis and hematopoietic supporting activity, which we report here, might depend on diverse miR-204&211 targets. To investigate this hypothesis, permanent RUNX2 knock-down was performed. Sh-RUNX2 fully reproduced the phenotypes induced by miR-204&211, confirming that RUNX2 down modulation is the major event leading to the reported functional modification on our MSCs. It seems thus apparent that RUNX2, a recognized master gene for osteogenesis, might rule all four MSC commitment and differentiation processes. Hence, the formerly reported role of miR204&211 and RUNX2 in osteoporosis and obesity, coupled with our novel observation showing inhibition of cartilage differentiation and hematopoietic support, strikingly resemble the clinical traits of metabolic syndrome, where osteoarthritis, osteoporosis, anaemia and obesity occur together. Our observations, corroborating and extending previous observations, suggest that miR-204&211-RUNX2 axis in human MSCs is possibly involved in the pathogenesis of this rapidly growing disease in industrialized countries, for possible therapeutic intervention to regenerate former homeostasis.
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http://dx.doi.org/10.1051/rmr/170004DOI Listing
December 2017

Drug repurposing for the treatment of glioblastoma multiforme.

J Exp Clin Cancer Res 2017 Nov 28;36(1):169. Epub 2017 Nov 28.

Department of Research, Advanced Diagnostics and Technological Innovation, Unit of Cellular Networks and Therapeutic Targets, Proteomics Area, Regina Elena National Cancer Institute, IRCCS, Via Elio Chianesi, 53, Rome, Italy.

Background: Glioblastoma Multiforme is the deadliest type of brain tumor and is characterized by very poor prognosis with a limited overall survival. Current optimal therapeutic approach has essentially remained unchanged for more than a decade, consisting in maximal surgical resection followed by radiotherapy plus temozolomide.

Main Body: Such a dismal patient outcome represents a compelling need for innovative and effective therapeutic approaches. Given the development of new drugs is a process presently characterized by an immense increase in costs and development time, drug repositioning, finding new uses for existing approved drugs or drug repurposing, re-use of old drugs when novel molecular findings make them attractive again, are gaining significance in clinical pharmacology, since it allows faster and less expensive delivery of potentially useful drugs from the bench to the bedside. This is quite evident in glioblastoma, where a number of old drugs is now considered for clinical use, often in association with the first-line therapeutic intervention. Interestingly, most of these medications are, or have been, widely employed for decades in non-neoplastic pathologies without relevant side effects. Now, the refinement of their molecular mechanism(s) of action through up-to-date technologies is paving the way for their use in the therapeutic approach of glioblastoma as well as other cancer types.

Short Conclusion: The spiraling costs of new antineoplastic drugs and the long time required for them to reach the market demands a profoundly different approach to keep lifesaving therapies affordable for cancer patients. In this context, repurposing can represent a relatively inexpensive, safe and fast approach to glioblastoma treatment. To this end, pros and cons must be accurately considered.
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http://dx.doi.org/10.1186/s13046-017-0642-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704391PMC
November 2017

Integrin α7 Is a Functional Marker and Potential Therapeutic Target in Glioblastoma.

Cell Stem Cell 2017 07 9;21(1):35-50.e9. Epub 2017 Jun 9.

Institute of General Pathology, Università Cattolica del Sacro Cuore and Fondazione Policlinico Universitario A. Gemelli, 00168 Rome, Italy; National Cancer Institute Regina Elena, 00144 Rome, Italy. Electronic address:

Functionally relevant markers of glioblastoma stem-like cells (GSCs) have potential for therapeutic targeting to treat this aggressive disease. Here we used generation and screening of thousands of monoclonal antibodies to search for receptors and signaling pathways preferentially enriched in GSCs. We identified integrin α7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses showed that ITGA7 plays a key functional role in growth and invasiveness of GSCs. We also found that targeting of ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling, and it led to a significant delay in tumor engraftment plus a strong reduction in tumor size and invasion. Our data, therefore, highlight ITGA7 as a glioblastoma biomarker and candidate therapeutic target.
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http://dx.doi.org/10.1016/j.stem.2017.04.009DOI Listing
July 2017

Proteomic analysis of cerebrospinal fluid from children with central nervous system tumors identifies candidate proteins relating to tumor metastatic spread.

Oncotarget 2017 Jul;8(28):46177-46190

Pediatric Oncology Unit, Department of Hematology and Pediatric Hematology-Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Central nervous system (CNS) tumors are the most common solid tumors in childhood. Since the sensitivity of combined cerebrospinal fluid (CSF) cytology and radiological neuroimaging in detecting meningeal metastases remains relatively low, we sought to characterize the CSF proteome of patients with CSF tumors to identify biomarkers predictive of metastatic spread. CSF samples from 27 children with brain tumors and 13 controls (extra-CNS non-Hodgkin lymphoma) were processed using core-shell hydrogel nanoparticles, and analyzed with reverse-phase liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS). Candidate proteins were identified with Fisher's exact test and/or a univariate logistic regression model. Reverse phase protein array (RPPA), Western blot (WB), and ELISA were used in the training set and in an independent set of CFS samples (60 cases, 14 controls) to validate our discovery findings. Among the 558 non-redundant proteins identified by LC-MS/MS, 147 were missing from the CSF database at http://www.biosino.org. Fourteen of the 26 final top-candidate proteins were chosen for validation with WB, RPPA and ELISA methods. Six proteins (type 1 collagen, insulin-like growth factor binding protein 4, procollagen C-endopeptidase enhancer 1, glial cell-line derived neurotrophic factor receptor α2, inter-alpha-trypsin inhibitor heavy chain 4, neural proliferation and differentiation control protein-1) revealed the ability to discriminate metastatic cases from controls. Combining a unique dataset of CSFs from pediatric CNS tumors with a novel enabling nanotechnology led us to identify CSF proteins potentially related to metastatic status.
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http://dx.doi.org/10.18632/oncotarget.17579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542258PMC
July 2017

Antibody Validation by Western Blotting.

Methods Mol Biol 2017 ;1606:51-70

George Mason University, Center for Applied Proteomics and Molecular Medicine, Manassas, VA, USA.

Validation of antibodies is an integral part of translational research, particularly for biomarker discovery. Assaying the specificity of the reagent (antibody) and confirming the identity of the protein biomarker is of critical importance prior to implementing any biomarker in clinical studies, and the lack of such quality control tests may result in unexpected and/or misleading results.Antibody validation is the procedure in which a single antibody is thoroughly assayed for sensitivity and specificity. Although a plethora of commercial antibodies exist, antibody specificity must be extensively demonstrated using diverse complex biological samples, rather than purified recombinant proteins, prior to use in clinical translational research. In the simplest iteration, antibody specificity is determined by the presence of a single band in a complex biological sample, at the expected molecular weight, on a Western blot.To date, numerous Western blotting procedures are available, based on either manual or automated systems and spanning the spectrum of single blots to multiplex blots. X-ray film is still employed in many research laboratories, but digital imaging has become a gold standard in immunoblotting. The basic principles of Western blotting are (a) separation of protein mixtures by gel electrophoresis, (b) transfer of the proteins to a blot, (c) probing the blot for a protein or proteins of interest, and (d) subsequent detection of the protein by chemiluminescent, fluorescent, or colorimetric methods. This chapter focuses on the chemiluminescent detection of proteins using a manual Western blotting system and a vacuum-enhanced detection system (SNAP i.d.™, Millipore).
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http://dx.doi.org/10.1007/978-1-4939-6990-6_4DOI Listing
February 2018

CHK1-targeted therapy to deplete DNA replication-stressed, p53-deficient, hyperdiploid colorectal cancer stem cells.

Gut 2018 05 7;67(5):903-917. Epub 2017 Apr 7.

Institute of General Pathology, Catholic University and A. Gemelli Polyclinic, Rome, Italy.

Objective: Cancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies.

Design: To discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents.

Results: The drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368.

Conclusions: LY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.
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http://dx.doi.org/10.1136/gutjnl-2016-312623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890648PMC
May 2018

Gut Mesenchymal Stromal Cells in Immunity.

Stem Cells Int 2017 28;2017:8482326. Epub 2017 Feb 28.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy; Sbarro Institute for Cancer Research and Molecular Medicine and Center of Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA.

Mesenchymal stromal cells (MSCs), first found in bone marrow (BM), are the structural architects of all organs, participating in most biological functions. MSCs possess tissue-specific signatures that allow their discrimination according to their origin and location. Among their multiple functions, MSCs closely interact with immune cells, orchestrating their activity to maintain overall homeostasis. The phenotype of tissue MSCs residing in the bowel overlaps with myofibroblasts, lining the bottom walls of intestinal crypts (pericryptal) or interspersed within intestinal submucosa (intercryptal). In Crohn's disease, intestinal MSCs are tightly stacked in a chronic inflammatory milieu, which causes their enforced expression of Class II major histocompatibility complex (MHC). The absence of Class II MHC is a hallmark for immune-modulator and tolerogenic properties of normal MSCs and, vice versa, the expression of HLA-DR is peculiar to antigen presenting cells, that is, immune-activator cells. Interferon (IFN) is responsible for induction of Class II MHC expression on intestinal MSCs. The reversal of myofibroblasts/MSCs from an immune-modulator to an activator phenotype in Crohn's disease results in the formation of a fibrotic tube subverting the intestinal structure. Epithelial metaplastic areas in this context can progress to dysplasia and cancer.
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http://dx.doi.org/10.1155/2017/8482326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350335PMC
February 2017

Expression of Iron-Related Proteins Differentiate Non-Cancerous and Cancerous Breast Tumors.

Int J Mol Sci 2017 Feb 14;18(2). Epub 2017 Feb 14.

Proteomics Laboratory, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy.

We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer.
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http://dx.doi.org/10.3390/ijms18020410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343944PMC
February 2017

The clinical value of patient-derived glioblastoma tumorspheres in predicting treatment response.

Neuro Oncol 2017 Aug;19(8):1097-1108

Institute of Neurosurgery, Università Cattolica del Sacro Cuore, Rome, Italy; Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy; Institute of Pathology, Università Cattolica del Sacro Cuore, Rome, Italy; Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana.

Background: Advances from glioma stemlike cell (GSC) research, though increasing our knowledge of glioblastoma (GBM) biology, do not influence clinical decisions yet. We explored the translational power of GSC-enriched cultures from patient-derived tumorspheres (TS) in predicting treatment response.

Methods: The relationship between TS growth and clinical outcome was investigated in 52 GBMs treated with surgical resection followed by radiotherapy and temozolomide (TMZ). The effect on TS of radiation (6 to 60 Gy) and of TMZ (3.9 μM to 1 mM) was related with patients' survival.

Results: Generation of TS was an independent factor for poor overall survival (OS) and poor progression-free survival (PFS) (P < .0001 and P = .0010, respectively). Growth rate and clonogenicity of TS predicted poor OS. In general, TS were highly resistant to both radiation and TMZ. Resistance to TMZ was stronger in TS with high clonogenicity and fast growth (P < .02). Shorter PFS was associated with radiation LD50 (lethal dose required to kill 50% of TS cells) >12 Gy of matched TS (P = .0484). A direct relationship was found between sensitivity of TS to TMZ and patients' survival (P = .0167 and P = .0436 for OS and PFS, respectively). Importantly, values for TMZ half-maximal inhibitory concentration <50 μM, which are in the range of plasma levels achieved in vivo, identified cases with longer OS and PFS (P = .0020 and P = .0016, respectively).

Conclusions: Analysis of TS holds translational relevance by predicting the response of parent tumors to radiation and, particularly, to TMZ. Dissecting the clonogenic population from proliferating progeny in TS can guide therapeutic strategies to a more effective drug selection and treatment duration.
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http://dx.doi.org/10.1093/neuonc/now304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737323PMC
August 2017

Exosomes from human colorectal cancer induce a tumor-like behavior in colonic mesenchymal stromal cells.

Oncotarget 2016 Aug;7(31):50086-50098

Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanità, Rome, Italy.

Background: Cancer cells, including colorectal cancer ones (CRC), release high amounts of nanovesicles (exosomes), delivering biochemical messages for paracrine or systemic crosstalk. Mesenchymal stromal cells (MSCs) have been shown to play contradicting roles in tumor progression.

Results: CRC exosomes induce in cMSCs: i) atypical morphology, higher proliferation, migration and invasion; ii) formation of spheroids; iii) an acidic extracellular environment associated with iv) a plasma membrane redistribution of vacuolar H+-ATPase and increased expression of CEA. Colon cancer derived MSCs, which were isolated from tumor masses, produce umbilicated spheroids, a future frequently observed in the inner core of rapidly growing tumors and recapitulate the changes observed in normal colonic MSCs exposed to CRC exosomes.

Materials And Methods: Tissue specific colonic (c)MSCs were exposed to primary or metastatic CRC exosomes and analysed by light and electron microscopy, proliferation in 2D and 3D cultures, migration and invasion assays, Western blot and confocal microscopy for vacuolar H+-ATPase expression.

Conclusions: CRC exosomes are able to induce morphological and functional changes in colonic MSCs, which may favour tumor growth and its malignant progression. Our results suggest that exosomes are actively involved in cancer progression and that inhibiting tumor exosome release may represent a way to interfere with cancer.
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http://dx.doi.org/10.18632/oncotarget.10574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226570PMC
August 2016

UCN-01 enhances cytotoxicity of irinotecan in colorectal cancer stem-like cells by impairing DNA damage response.

Oncotarget 2016 Jul;7(28):44113-44128

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Colorectal cancer (CRC) is one of the most common and lethal cancers worldwide. Despite recent progress, the prognosis of advanced stage CRC remains poor, mainly because of cancer recurrence and metastasis. The high morbidity and mortality of CRC has been recently ascribed to a small population of tumor cells that hold the potential of tumor initiation, i.e. cancer stem cells (CSCs), which play a pivotal role in cancer recurrence and metastasis and are not eradicated by current therapy. We screened CRC-SCs in vitro with a library of protein kinase inhibitors and showed that CRC-SCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. Nonetheless, broad-spectrum inhibition by the staurosporin derivative UCN-01 blocks CRC-SC growth and potentiates the activity of irinotecan in vitro and in vivo CRC-SC-derived models. Reverse-Phase Protein Microarrays (RPPA) revealed that, albeit CRC-SCs display individual phospho-proteomic profiles, sensitivity of CRC-SCs to UCN-01 relies on the interference with the DNA damage response mediated by Chk1. Combination of LY2603618, a specific Chk1/2 inhibitor, with irinotecan resulted in a significant reduction of CRC-SC growth in vivo, confirming that irinotecan treatment coupled to inhibition of Chk1 represents a potentially effective therapeutic approach for CRC treatment.
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http://dx.doi.org/10.18632/oncotarget.9859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5190083PMC
July 2016

Cancer Stem Cell-Based Models of Colorectal Cancer Reveal Molecular Determinants of Therapy Resistance.

Stem Cells Transl Med 2016 Apr 8;5(4):511-23. Epub 2016 Mar 8.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

Unlabelled: Colorectal cancer (CRC) therapy mainly relies on the use of conventional chemotherapeutic drugs combined, in a subset of patients, with epidermal growth factor receptor [EGFR]-targeting agents. Although CRC is considered a prototype of a cancer stem cell (CSC)-driven tumor, the effects of both conventional and targeted therapies on the CSC compartment are largely unknown. We have optimized a protocol for colorectal CSC isolation that allowed us to obtain CSC-enriched cultures from primary tumor specimens, with high efficiency. CSC isolation was followed by in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, thus generating panels of CSC lines with defined patterns of genetic mutations and therapy sensitivity. Colorectal CSC lines were polyclonal and maintained intratumor heterogeneity in terms of somatically acquired mutations and differentiation state. Such CSC-enriched cultures were used to investigate the effects of both conventional and targeted therapies on the CSC compartment in vivo and to generate a proteomic picture of signaling pathways implicated in sensitivity/resistance to anti-EGFR agents. We propose CSC lines as a sound preclinical framework to test the effects of therapies in vitro and in vivo and to identify novel determinants of therapy resistance.

Significance: Colorectal cancer stem cells (CSCs) have been shown to be responsible for tumor propagation, metastatic dissemination, and relapse. However, molecular pathways present in CSCs, as well as mechanisms of therapy resistance, are mostly unknown. Taking advantage of genetically characterized CSC lines derived from colorectal tumors, this study provides an extensive analysis of CSC response to EGFR-targeted therapy in vivo and an overview of factors implicated in therapy response or resistance. Furthermore, the implementation of a biobank of molecularly annotated CSC lines provides an innovative resource for future investigations in colorectal cancer.
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http://dx.doi.org/10.5966/sctm.2015-0214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4798739PMC
April 2016

A simple and predictive phenotypic High Content Imaging assay for Plasmodium falciparum mature gametocytes to identify malaria transmission blocking compounds.

Sci Rep 2015 Nov 10;5:16414. Epub 2015 Nov 10.

Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena n. 299, 00161 Roma, Italy.

Plasmodium falciparum gametocytes, specifically the mature stages, are the only malaria parasite stage in humans transmissible to the mosquito vector. Anti-malarial drugs capable of killing these forms are considered essential for the eradication of malaria and tools allowing the screening of large compound libraries with high predictive power are needed to identify new candidates. As gametocytes are not a replicative stage it is difficult to apply the same drug screening methods used for asexual stages. Here we propose an assay, based on high content imaging, combining "classic" gametocyte viability readout based on gametocyte counts with a functional viability readout, based on gametocyte activation and the discrimination of the typical gamete spherical morphology. This simple and rapid assay has been miniaturized to a 384-well format using acridine orange staining of wild type P. falciparum 3D7A sexual forms, and was validated by screening reference antimalarial drugs and the MMV Malaria Box. The assay demonstrated excellent robustness and ability to identify quality hits with high likelihood of confirmation of transmission reducing activity in subsequent mosquito membrane feeding assays.
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http://dx.doi.org/10.1038/srep16414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4639769PMC
November 2015

Chemotherapy-induced antitumor immunity requires formyl peptide receptor 1.

Science 2015 Nov 29;350(6263):972-8. Epub 2015 Oct 29.

Gustave Roussy Cancer Campus, Villejuif, France. INSERM, U1138, Paris, France. Équipe 11 Labellisée par la Ligue Nationale Contre le Cancer, Centre de Recherche des Cordeliers, Paris, France. Université Paris Descartes, Sorbonne Paris Cité, Paris, France. Université Pierre et Marie Curie, Paris, France. Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France. Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France. Karolinska Institute, Department of Women's and Children's Health, Karolinska University Hospital, 17176 Stockholm, Sweden.

Antitumor immunity driven by intratumoral dendritic cells contributes to the efficacy of anthracycline-based chemotherapy in cancer. We identified a loss-of-function allele of the gene coding for formyl peptide receptor 1 (FPR1) that was associated with poor metastasis-free and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy. The therapeutic effects of anthracyclines were abrogated in tumor-bearing Fpr1(-/-) mice due to impaired antitumor immunity. Fpr1-deficient dendritic cells failed to approach dying cancer cells and, as a result, could not elicit antitumor T cell immunity. Experiments performed in a microfluidic device confirmed that FPR1 and its ligand, annexin-1, promoted stable interactions between dying cancer cells and human or murine leukocytes. Altogether, these results highlight the importance of FPR1 in chemotherapy-induced anticancer immune responses.
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http://dx.doi.org/10.1126/science.aad0779DOI Listing
November 2015

Altered Traffic of Cardiolipin during Apoptosis: Exposure on the Cell Surface as a Trigger for "Antiphospholipid Antibodies".

J Immunol Res 2015 29;2015:847985. Epub 2015 Sep 29.

Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy.

Apoptosis has been reported to induce changes in the remodelling of membrane lipids; after death receptor engagement, specific changes of lipid composition occur not only at the plasma membrane, but also in intracellular membranes. This paper focuses on one important aspect of apoptotic changes in cellular lipids, namely, the redistribution of the mitochondria-specific phospholipid, cardiolipin (CL). CL predominantly resides in the inner mitochondrial membrane, even if the rapid remodelling of its acyl chains and the subsequent degradation occur in other membrane organelles. After death receptor stimulation, CL appears to concentrate into mitochondrial "raft-like" microdomains at contact sites between inner and outer mitochondrial membranes, leading to local oligomerization of proapoptotic proteins, including Bid. Clustering of Bid in CL-enriched contacts sites is interconnected with pathways of CL remodelling that intersect membrane traffic routes dependent upon actin. In addition, CL association with cytoskeleton protein vimentin was observed. Such novel association also indicated that CL molecules may be expressed at the cell surface following apoptotic stimuli. This observation adds a novel implication of biomedical relevance. The association of CL with vimentin at the cell surface may represent a "new" target antigen in the context of the apoptotic origin of anti-vimentin/CL autoantibodies in Antiphospholipid Syndrome.
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http://dx.doi.org/10.1155/2015/847985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4603604PMC
May 2016

miR-135b suppresses tumorigenesis in glioblastoma stem-like cells impairing proliferation, migration and self-renewal.

Oncotarget 2015 Nov;6(35):37241-56

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Glioblastoma multiforme (GBM) is the most common and fatal malignant adult primary brain tumor. Currently, the overall prognosis for GBM patients remains poor despite advances in neurosurgery and adjuvant treatments. MicroRNAs (miRNAs) contribute to the pathogenesis of various types of tumor, including GBM. In this study we analyzed the expression of a panel of miRNAs, which are known to be differentially expressed by the brain and GBM tumor, in a collection of patient-derived GBM stem-like cells (GSCs). Notably, the average expression level of miR-135b, was the most downregulated compared to its normal counterpart, suggesting a potential role as anti-oncogene.Restoration of miR-135b in GSCs significantly decreased proliferation, migration and clonogenic abilities. More importantly, miR-135b restoration was able to significantly reduce brain infiltration in mouse models of GBM obtained by intracerebral injection of GSC lines. We identified ADAM12 and confirmed SMAD5 and GSK3β as miR-135b targets and potential mediators of its effects. The whole transcriptome analysis ascertained that the expression of miR-135b downmodulated additional genes driving key pathways in GBM survival and infiltration capabilities.Our results identify a critical role of miR-135b in the regulation of GBM development, suggesting that miR-135b might act as a tumor-suppressor factor and thus providing a potential candidate for the treatment of GBM patients.
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http://dx.doi.org/10.18632/oncotarget.5925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741927PMC
November 2015

Gene expression analysis of PTEN positive glioblastoma stem cells identifies DUB3 and Wee1 modulation in a cell differentiation model.

PLoS One 2013 12;8(12):e81432. Epub 2013 Dec 12.

Department of Biomedical Sciences, University of Catania, Italy.

The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081432PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861258PMC
September 2014

Pharmacological enhancement of mGlu1 metabotropic glutamate receptors causes a prolonged symptomatic benefit in a mouse model of spinocerebellar ataxia type 1.

Mol Brain 2013 Nov 19;6:48. Epub 2013 Nov 19.

I,R,C,C,S, Neuromed, Pozzilli, Italy.

Background: Spinocerebellar ataxia type 1 (SCA1) is a genetic disorder characterized by severe ataxia associated with progressive loss of cerebellar Purkinje cells. The mGlu1 metabotropic glutamate receptor plays a key role in mechanisms of activity-dependent synaptic plasticity in the cerebellum, and its dysfunction is linked to the pathophysiology of motor symptoms associated with SCA1. We used SCA1 heterozygous transgenic mice (Q154/Q2) as a model for testing the hypothesis that drugs that enhance mGlu1 receptor function may be good candidates for the medical treatment of SCA1.

Results: Symptomatic 30-week old SCA1 mice showed reduced mGlu1 receptor mRNA and protein levels in the cerebellum. Interestingly, these mice also showed an intense expression of mGlu5 receptors in cerebellar Purkinje cells, which normally lack these receptors. Systemic treatment of SCA1 mice with the mGlu1 receptor positive allosteric modulator (PAM), Ro0711401 (10 mg/kg, s.c.), caused a prolonged improvement of motor performance on the rotarod and the paw-print tests. A single injection of Ro0711401 improved motor symptoms for several days, and no tolerance developed to the drug. In contrast, the mGlu5 receptor PAM, VU0360172 (10 mg/kg, s.c.), caused only a short-lasting improvement of motor symptoms, whereas the mGlu1 receptor antagonist, JNJ16259685 (2.5 mg/kg, i.p.), further impaired motor performance in SCA1 mice. The prolonged symptomatic benefit caused by Ro0711401 outlasted the time of drug clearance from the cerebellum, and was associated with neuroadaptive changes in the cerebellum, such as a striking reduction of the ectopically expressed mGlu5 receptors in Purkinje cells, increases in levels of total and Ser880-phosphorylated GluA2 subunit of AMPA receptors, and changes in the length of spines in the distal dendrites of Purkinje cells.

Conclusions: These data demonstrate that pharmacological enhancement of mGlu1 receptors causes a robust and sustained motor improvement in SCA1 mice, and lay the groundwork for the development of mGlu1 receptor PAMs as novel "cerebellum-specific", effective, and safe symptomatic drugs for the treatment of SCA1 in humans.
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http://dx.doi.org/10.1186/1756-6606-6-48DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4225515PMC
November 2013

Approaching the increasing complexity of non-small cell lung cancer taxonomy.

Curr Pharm Des 2014 ;20(24):3973-81

"Regina Elena" National Cancer Institute, via E. Chianesi, n. 53, 00144, Rome, Italy.

The advent of molecular targeted agents is changing the treatment of solid tumors. In non-small-cell lung cancer, compounds directed against oncogenic proteins offer novel therapeutic opportunities for a fraction of patients whose tumors harbor specific genetic defects. With the increased level of resolution achieved by high-throughput technologies, the taxonomy of lung cancer is rapidly changing. For instance, by cataloguing genetic abnormalities in squamous cell lung cancer the Cancer Genome Atlas Network revealed the existence of multiple molecular entities, each one characterized by specific molecular abnormalities, and by a different spectrum of activated/ inactivated molecular networks. Although this increased complexity could be perceived as a further drawback in effective anticancer therapy, on the other hand the combined interrogation of genomic and proteomic data is expected to provide the whole molecular map of each tumor, and to determine the information flow in the explored biological system. In particular, novel genetic and proteomic approaches are offering the opportunity for matching specific genetic defects and aberrant protein-protein interactions with active pathway-targeted inhibitors. Moreover, the isolation and characterization of a cellular pool endowed with stem-like traits, and able to recapitulate the parental disease in animals, is enabling investigators to recreate the individual patient tumor in the laboratory. In this article, we discuss how novel technologies and cellular and animal models, applied to lung cancer research, hold the potential to foster a new wave of biomarker-driven clinical trials.
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http://dx.doi.org/10.2174/13816128113196660759DOI Listing
March 2015

Cancer quasispecies and stem-like adaptive aneuploidy.

F1000Res 2013 6;2:268. Epub 2013 Dec 6.

Schmid College of Science and Technology, Chapman University Chapman University, Orange, CA, 92866, USA.

In this paper we develop a theoretical frame to understand self-regulation of aneuploidy rate in cancer and stem cells. This is accomplished building upon quasispecies theory, by leaving its formal mathematical structure intact, but by drastically changing the meaning of its objects. In particular, we propose a novel definition of chromosomal master sequence, as a sequence of physically distinct whole or fragmented chromosomes, whose length is taken to be the sum of the copy numbers of each whole or fragmented chromosome. This fundamental change in the functional objects of quasispecies theory allows us to show that previously measured aneuploidy rates in cancer populations are already close to a formally derived aneuploid error threshold, and that any value of aneuploidy rate larger than the aneuploid error threshold would lead to a loss of fitness of a tumor population. Finally, we make a phenomenological analysis of existing experimental evidence to argue that single clone cancer cells, derived from an aneuploid cancer subpopulation, are capable of self-regulating their aneuploidy rate and of adapting it to distinct environments, namely primary and metastatic microenvironments. We also discuss the potential origin of this self-regulatory ability in the wider context of developmental and comparative biology and we hypothesize the existence of a diversification factor, i.e. a cellular mechanism that regulates adaptation of aneuploidy rates, active in all embryo, adult and cancer stem cells.
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http://dx.doi.org/10.12688/f1000research.2-268.v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3968898PMC
April 2014

EGFR inhibition abrogates leiomyosarcoma cell chemoresistance through inactivation of survival pathways and impairment of CSC potential.

PLoS One 2012 8;7(10):e46891. Epub 2012 Oct 8.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Background: Tumor cells with stem-like phenotype and properties, known as cancer stem cells (CSC), have been identified in most solid tumors and are presumed to be responsible for driving tumor initiation, chemoresistance, relapse, or metastasis. A subpopulation of cells with increased stem-like potential has also been identified within sarcomas. These cells are endowed with increased tumorigenic potential, chemoresistance, expression of embryonic markers, and side population(SP) phenotype. Leiomyosarcomas (LMS) are soft tissue sarcomas presumably arising from undifferentiated cells of mesenchymal origin, the Mesenchymal Stem Cells (MSC). Frequent recurrence of LMS and chemoresistance of relapsed patients may likely result from the failure to target CSC. Therefore, therapeutic cues coming from the cancer stem cell (CSC) field may drastically improve patient outcome.

Methodology/principal Findings: We expanded LMS stem-like cells from patient samples in vitro and examined the possibility to counteract LMS malignancy through a stem-like cell effective approach. LMS stem-like cells were in vitro expanded both as "tumor spheres" and as "monolayers" in Mesenchymal Stem Cell (MSC) conditions. LMS stem-like cells displayed MSC phenotype, higher SP fraction, and increased drug-extrusion, extended proliferation potential, self-renewal, and multiple differentiation ability. They were chemoresistant, highly tumorigenic, and faithfully reproduced the patient tumor in mice. Such cells displayed activation of EGFR/AKT/MAPK pathways, suggesting a possibility in overcoming their chemoresistance through EGFR blockade. IRESSA plus Vincristine treatment determined pathway inactivation, impairment of SP phenotype, high cytotoxicity in vitro and strong antitumor activity in stem-like cell-generated patient-like xenografts, targeting both stem-like and differentiated cells.

Conclusions/significance: EGFR blockade combined with vincristine determines stem-like cell effective antitumor activity in vitro and in vivo against LMS, thus providing a potential therapy for LMS patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046891PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466184PMC
May 2013