Publications by authors named "Silvia Bresolin"

48 Publications

Neonatal-Onset Familial Mediterranean Fever in an Infant with Human Parainfluenza Virus-4 Infection.

Neonatology 2021 Mar 31:1-5. Epub 2021 Mar 31.

Maternal and Child Health Department, Padua University, Padua, Italy.

Unusual, severe infections or inflammatory episodes in newborns and infants are largely unexplained and often attributed to immature immune responses. Inborn errors of immunity (IEI) are increasingly recognized as the etiology of life-threatening inflammatory and infectious diseases in infancy. We describe a patient with a unique neonatal-onset Familial Mediterranean Fever (FMF) due to compound heterozygous variants in MEFV, presenting as pleuritis following human parainfluenza virus-4 infection. Diagnostic challenges of FMF in infancy include the interpretation of the attacks as infectious episodes. Newborns and infants with acute, recurrent, or chronic, unusually severe infectious or inflammatory conditions should be screened for IEI, including both disorders with defective immunological responses and autoinflammatory disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000514694DOI Listing
March 2021

CircRNAs Dysregulated in Juvenile Myelomonocytic Leukemia: CircMCTP1 Stands Out.

Front Cell Dev Biol 2020 6;8:613540. Epub 2021 Jan 6.

Department of Molecular Medicine, University of Padova, Padua, Italy.

Juvenile myelomonocytic leukemia (JMML), a rare myelodysplastic/myeloproliferative neoplasm of early childhood, is characterized by clonal growth of RAS signaling addicted stem cells. JMML subtypes are defined by specific RAS pathway mutations and display distinct gene, microRNA (miRNA) and long non-coding RNA expression profiles. Here we zoom in on circular RNAs (circRNAs), molecules that, when abnormally expressed, may participate in malignant deviation of cellular processes. CirComPara software was used to annotate and quantify circRNAs in RNA-seq data of a "discovery cohort" comprising 19 JMML patients and 3 healthy donors (HD). In an independent set of 12 JMML patients and 6 HD, expression of 27 circRNAs was analyzed by qRT-PCR. CircRNA-miRNA-gene networks were reconstructed using circRNA function prediction and gene expression data. We identified 119 circRNAs dysregulated in JMML and 59 genes showing an imbalance of the circular and linear products. Our data indicated also circRNA expression differences among molecular subgroups of JMML. Validation of a set of deregulated circRNAs in an independent cohort of JMML patients confirmed the down-regulation of circOXNAD1 and circATM, and a marked up-regulation of circLYN, circAFF2, and circMCTP1. A new finding in JMML links up-regulated circMCTP1 with known tumor suppressor miRNAs. This and other predicted interactions with miRNAs connect dysregulated circRNAs to regulatory networks. In conclusion, this study provides insight into the circRNAome of JMML and paves the path to elucidate new molecular disease mechanisms putting forward circMCTP1 up-regulation as a robust example.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2020.613540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815690PMC
January 2021

Posttranslational Regulation of the Exon Skipping Machinery Controls Aberrant Splicing in Leukemia.

Cancer Discov 2020 Sep 22;10(9):1388-1409. Epub 2020 May 22.

Department of Biochemistry and Molecular Genetics, Northwestern University, Chicago, Illinois.

Splicing alterations are common in diseases such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T-cell acute lymphoblastic leukemia (T-ALL) that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease, affecting proteasomal subunits, cell-cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination, and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL. SIGNIFICANCE: Our study provides a new proof-of-principle for posttranslational regulation of splicing factors independently of mutations in aggressive T-cell leukemia. It further suggests a new drug combination of splicing and proteasomal inhibitors, a concept that might apply to other diseases with or without mutations affecting the splicing machinery..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-19-1436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483384PMC
September 2020

iWhale: a computational pipeline based on Docker and SCons for detection and annotation of somatic variants in cancer WES data.

Brief Bioinform 2020 May 20. Epub 2020 May 20.

Whole exome sequencing (WES) is a powerful approach for discovering sequence variants in cancer cells but its time effectiveness is limited by the complexity and issues of WES data analysis. Here we present iWhale, a customizable pipeline based on Docker and SCons, reliably detecting somatic variants by three complementary callers (MuTect2, Strelka2 and VarScan2). The results are combined to obtain a single variant call format file for each sample and variants are annotated by integrating a wide range of information extracted from several reference databases, ultimately allowing variant and gene prioritization according to different criteria. iWhale allows users to conduct a complex series of WES analyses with a powerful yet customizable and easy-to-use tool, running on most operating systems (macOs, GNU/Linux and Windows). iWhale code is freely available at https://github.com/alexcoppe/iWhale and the docker image is downloadable from https://hub.docker.com/r/alexcoppe/iwhale.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bib/bbaa065DOI Listing
May 2020

Pro-inflammatory cytokines favor the emergence of ETV6-RUNX1-positive pre-leukemic cells in a model of mesenchymal niche.

Br J Haematol 2020 07 2;190(2):262-273. Epub 2020 Mar 2.

Centro Ricerca Tettamanti, University of Milano-Bicocca, MBBM Foundation, Monza, Italy.

ETV6-RUNX1 (E/R) fusion gene, arising in utero from translocation t(12;21)(p13:q22), is the most frequent alteration in childhood acute lymphoblastic leukemia (ALL). However, E/R is insufficient to cause overt leukemia since it generates a clinically silent pre-leukemic clone which persists in the bone marrow but fails to out-compete normal progenitors. Conversely, pre-leukemic cells show increased susceptibility to transformation following additional genetic insults. Infections/inflammation are the most accredited triggers for mutations accumulation and leukemic transformation in E/R pre-leukemic cells. However, precisely how E/R and inflammation interact in promoting leukemia is still poorly understood. Here we demonstrate that IL6/TNFα/ILβ pro-inflammatory cytokines cooperate with BM-MSC in promoting the emergence of E/R Ba/F3 over their normal counterparts by differentially affecting their proliferation and survival. Moreover, IL6/TNFα/ILβ-stimulated BM-MSC strongly attract E/R Ba/F3 in a CXCR2-dependent manner. Interestingly, E/R-expressing human CD34 IL7R progenitors, a putative population for leukemia initiation during development, were preserved in the presence of BM-MSC and IL6/TNFα/ILβ compared to their normal counterparts. Finally, the extent of DNA damage increases within the inflamed niche in both control and E/R-expressing Ba/F3, potentially leading to transformation in the apoptosis-resistant pre-leukemic clone. Overall, our data provide new mechanistic insights into childhood ALL pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bjh.16523DOI Listing
July 2020

Patient-Derived Scaffolds of Colorectal Cancer Metastases as an Organotypic 3D Model of the Liver Metastatic Microenvironment.

Cancers (Basel) 2020 Feb 5;12(2). Epub 2020 Feb 5.

First Surgical Clinic, Department of Surgery, Oncology and Gastroenterology, University of Padova, 35128 Padova, Italy.

The liver is the most common site for colorectal cancer (CRC) metastasis and there is an urgent need for new tissue culture models to study colorectal cancer liver metastasis (CRLM) as current models do not mimic the biological, biochemical, and structural characteristics of the metastatic microenvironment. Decellularization provides a novel approach for the study of the cancer extracellular matrix (ECM) as decellularized scaffolds retain tissue-specific features and biological properties. In the present study, we created a 3D model of CRC and matched CRLM using patient-derived decellularized ECM scaffolds seeded with the HT-29 CRC cell line. Here, we show an increased HT-29 cell proliferation and migration capability when cultured in cancer-derived scaffolds compared to same-patient healthy colon and liver tissues. HT-29 cells cultured in CRLM scaffolds also displayed an indication of epithelial-mesenchymal transition (EMT), with a loss of E-cadherin and increased Vimentin expression. EMT was confirmed by gene expression profiling, with the most represented biological processes in CRLM-seeded scaffolds involving demethylation, deacetylation, a cellular response to stress metabolic processes, and a response to the oxygen level and starvation. HT-29 cells cultured in cancer-specific 3D microenvironments showed a reduced response to treatment with 5-fluorouracil and 5-fluorouracil combined with Irinotecan when used at a standard IC (as determined in the 2D culture). Our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the metastatic microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of CRLM progression and assessing the response to chemotherapy agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12020364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072130PMC
February 2020

SYK Targeting Represents a Potential Therapeutic Option for Relapsed Resistant Pediatric B-Acute Lymphoblastic Leukemia Patients.

Int J Mol Sci 2019 Dec 7;20(24). Epub 2019 Dec 7.

Istituto di Ricerca Pediatrica Città della Speranza, 35127 Padova, Italy.

The presence of the chromosomal rearrangement t(12;21)() in childhood B-acute lymphoblastic leukemia (B-ALL) is an independent predictor of favorable prognosis, however relapses still occur many years later after stopping therapy, and patients often display resistance to current treatments. Since spleen tyrosine kinase (SYK), a cytosolic nonreceptor tyrosine kinase interacting with immune receptors, has been previously associated with malignant transformation and cancer cell proliferation, we aimed to assess its role in cell survival and prognosis. We evaluated the effects on cell survival of three SYK inhibitors and showed that all of them, in particular entospletinib, are able to induce cell death and enhance the efficacy of conventional chemotherapeutics. By using reverse phase protein arrays we next revealed that activated SYK is upregulated at diagnosis in pediatric patients who will experience relapse, and, importantly, hyperactivation is maintained at a high level also at relapse occurrence. We thus treated primary cells from patients both at diagnosis and relapse with the combination entospletinib + chemotherapeutics and observed that SYK inhibition is able to sensitize resistant primary cells to conventional drugs. Entospletinib could thus represent a new therapeutic option supporting conventional chemotherapy for relapsed patients, and these evidences encourage further studies on SYK for treatment of other relapsed resistant acute lymphoblastic leukemia (ALL) subgroups.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms20246175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940898PMC
December 2019

Serum miRNA Are Promising Biomarkers for the Detection of Early Hepatocellular Carcinoma after Treatment with Direct-Acting Antivirals.

Cancers (Basel) 2019 Nov 11;11(11). Epub 2019 Nov 11.

Department of Internal Medicine-DIMED, University-Hospital of Padova, 35128 Padova, Italy.

Direct antiviral agents (DAAs) have excellent efficacy against chronic hepatitis C virus (HCV) infection. Despite this strength, recent studies raised concerns about an unexpected hepatocellular carcinoma (HCC) occurrence rate after DAA therapy. In this exploratory case-control study, we evaluated the potential use of miRNAs as serum biomarkers for the detection of early HCC in DAA-treated patients. In the discovery phase, the circulating miRNome was assessed in 10 matched patients with (HCC+) or without HCC (HCC-) occurrence. Microarray analysis was performed before (T0) and after one month of the DAA therapy (T1). MiRNAs discriminating HCC+ and HCC- patients were validated in 60 samples by means of RT-qPCR. We estimated the time-averaged difference of a given miRNA between HCC+ and HCC- patients using a bootstrapped random-effect generalized least square regression model (RE-GLS). At T0, miR-1207-5p, miR-1275, miR-3197, miR-4443, miR-3178, miR-483-5p, miR-4706, miR-4793-3p and miR-1246 discriminated HCC+ from HCC- patients ( < 0.05). At T1, only miR-1180-3p, miR-1228-3p, miR-4329 and miR-4484 ( < 0.05) discriminated HCC+ from HCC- patients. The subsequent validation phase identified miR-3197 as changing with both disease and time. Our results suggest that patients might be already committed to HCC occurrence before DAA therapy. MiR-3197 shows some potential for the identification of patients at risk of HCC during DAA treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers11111773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895878PMC
November 2019

Circular RNA differential expression in blood cell populations and exploration of circRNA deregulation in pediatric acute lymphoblastic leukemia.

Sci Rep 2019 10 11;9(1):14670. Epub 2019 Oct 11.

CRIBI Biotechnology Center, University of Padova, Padova, Italy.

Circular RNAs (circRNAs) are abundantly expressed in the haematopoietic compartment, but knowledge on their diversity among blood cell types is still limited. Nevertheless, emerging data indicate an array of circRNA functions exerted through interactions with other RNAs and proteins, by translation into peptides, and circRNA involvement as regulatory molecules in many biological processes and cancer mechanisms. Interestingly, the role of specific circRNAs in leukemogenesis has been disclosed by a few studies, mostly in acute myeloid leukemia. In this study, circRNA expression in B-cells, T-cells and monocytes of healthy subjects is described, including putative new circRNA genes. Expression comparison considered 6,228 circRNAs and highlighted cell population-specific expression and exon usage patterns. Differential expression has been confirmed by qRT-PCR for circRNAs specific of B-cells (circPAX5, circAFF3, circIL4R, and circSETBP1) or T-cells (circIKZF1, circTNIK, circTXK, and circFBXW7), and for circRNAs from intronic (circBCL2) and intergenic regions that were overexpressed in lymphocytes. Starting from this resource of circRNA expression in mature blood cell populations, targeted examination identified striking and generalized upregulated expression of circPAX5, circPVT1 and circHIPK3 in pediatric B-precursor acute lymphoblastic leukemia, and disclosed circRNAs with variable expression across cytogenetic subtypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-50864-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789028PMC
October 2019

HIF-1α/Wnt signaling-dependent control of gene transcription regulates neuronal differentiation of glioblastoma stem cells.

Theranostics 2019 9;9(17):4860-4877. Epub 2019 Jul 9.

Department of Woman and Children's Health, University of Padova, Padova, Italy.

HIF-1α has been suggested to interplay with Wnt signaling components in order to activate a neuronal differentiation process in both normal brain and glioblastoma (GBM). Based on these data, we explored the molecular mechanisms underlying the observed capability of GBM cells to acquire a neuronal phenotype upon Wnt signaling stimulation and how the microenvironment, particularly hypoxia, modulates this process. : here, the employment of ChIP-seq techniques together with co-immunoprecipitation approaches allowed to reconstruct the molecular interactions responsible for activating specific pro-differentiating transcriptional programs in GBM cells. Moreover, gene silencing/over-expression approaches coupled with the functional analysis of cell phenotype were applied to confirm ChIP-driven hypotheses. Finally, we combined the use of publicly available gene expression datasets with protein expression data by immunohistochemistry to test the clinical relevance of obtained results. : our data clearly suggest that HIF-1α is recruited by the β-catenin/TCF1 complex to foster neuronal differentiation gene transcription in hypoxic GBM cells. Conversely, at higher oxygen levels, the increased expression of TCF4 exerts a transcriptional inhibitory function on the same genomic regions, thus counteracting differentiation. Moreover, we demonstrate the existence of a positive correlation between the expression levels of HIF-1α, TCF1 and neuronal phenotype in GBM tumors, accompanied by the over-expression of several Wnt signaling components, finally affecting patient prognosis. : we unveiled a peculiar mechanism by which TCF1 and HIF-1α can induce a reminiscent neuronal differentiation of hypoxic GBM cells, which is hampered, in normoxia, by high levels of TCF4, thus not only controlling the balance between differentiation and stemness, but also impacting on intra-tumoral heterogeneity and eventually patient outcome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7150/thno.35882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691379PMC
August 2020

HSP70/HSF1 axis, regulated via a PI3K/AKT pathway, is a druggable target in chronic lymphocytic leukemia.

Int J Cancer 2019 12 15;145(11):3089-3100. Epub 2019 May 15.

Department of Medicine, Hematology and Clinical Immunology Branch, Padua University School of Medicine, Padua, Italy.

Considering the role played by the heat shock protein of 70 kDa (HSP70) in cancer, we characterized this protein and its major regulator, the heat shock factor 1 (HSF1), in chronic lymphocytic leukemia (CLL). We found both HSP70 and HSF1 overexpressed in CLL patients, correlated to poor prognosis and abnormally localized in the nucleus of leukemic B cells. The two proteins were strictly correlated each other and their levels decreased consensually in those patients responding to in vivo therapeutic regimens. HSP70 and HSF1 inhibition was proved to be effective in inducing a dose-dependent in vitro apoptosis of CLL B cells. Considering that HSF1 is finely regulated by kinases belonging to pathways triggered by rat sarcoma (RAS), we benefited from a previous proteomic study performed in CLL patients aiming to assess the activation/expression of key signaling proteins. We found that patients showing high levels of HSP70 also expressed high Akt-Ser473, thus activating HSF1. Inhibition of PI3K, which activates AKT, reduced the expression of HSF1 and HSP70. By contrast, HSP70-low patients displayed high activation of MEK1/2 and ERK1/2, known to negatively regulate HSF1. These data demonstrate that the HSP70 expression is regulated by the modulation of HSF1 activity through the activation of RAS-regulated pathways and suggest the HSP70/HSF1 interplay as an interesting target for antileukemic therapies. Finally, inhibition of PI3K, that activates AKT, reduced the expression of HSF1 and HSP70.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ijc.32383DOI Listing
December 2019

CircRNAs Are Here to Stay: A Perspective on the Recombinome.

Front Genet 2019 13;10:88. Epub 2019 Feb 13.

Department of Molecular Medicine, University of Padua, Padua, Italy.

Chromosomal translocations harbored by cancer genomes are important oncogenic drivers. In rearranged acute leukemia (MLLre) fuses with over 90 partner genes. Mechanistic studies provided clues of MLL fusion protein leukemogenic potential, but models failed to fully recapitulate the disease. Recently, expression of oncogenic fusion circular RNAs (f-circ) by fusion was proven. This discovery, together with emerging data on the importance and diversity of circRNAs formed the incentive to study the circRNAs of the recombinome. Through interactions with other RNAs, such as microRNAs, and with proteins, circRNAs regulate cellular processes also related to cancer development. CircRNAs can translate into functional peptides too. and most of the 90 translocation partners do express circRNAs and exploration of our RNA-seq dataset of sorted blood cell populations provided new data on alternative circular isoform generation and expression variability of circRNAs of the recombinome. Further, we provided evidence that rearrangements of and three of the main translocation partner genes can impact circRNA expression, supported also by preliminary observations in leukemic cells. The emerging picture underpins the view that circRNAs are worthwhile to be considered when studying MLLre leukemias and provides a new perspective on the impact of chromosomal translocations in cancer cells at large.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fgene.2019.00088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382020PMC
February 2019

Differences in circulating microRNA signature in Prader-Willi syndrome and non-syndromic obesity.

Endocr Connect 2018 Dec;7(12):1262-1274

Istituto Auxologico Italiano, IRCCS, Experimental Laboratory for Auxo-endocrinological Research, Milan and Piancavallo (VB), Italy.

Prader-Willi syndrome (PWS) represents the most common genetic-derived obesity disorder caused by the loss of expression of genes located on the paternal chromosome 15q11.2-q13. The PWS phenotype shows peculiar physical, endocrine and metabolic characteristics compared to those observed in non-syndromic essential obesity. Since miRNAs have now a well-established role in many molecular pathways, including regulatory networks related to obesity, this pilot study was aimed to characterize the expression of circulating miRNAs in PWS compared to essential obesity. The circulating miRNome of 10 PWS and 10 obese subjects, adequately matched for age, BMI and sex, was profiled throughout Genechip miRNA 4.0 microarray analysis. We identified 362 out of 2578 mature miRNAs to be expressed in serum of the studied population. The circulating miRNA signature significantly characterising the two populations include 34 differently expressed RNAs. Among them, miR-24-3p, miR-122 and miR-23a-3p highly differ between the two groups with a FC >10 in obese compared to PWS. In the obese subjects, miR-7107-5p, miR-6880-3p, miR-6793-3p and miR-4258 were associated to the presence of steatosis. A different signature of miRNAs significantly distinguished PWS with steatosis from PWS without steatosis, involving miR-619-5p, miR-4507, miR-4656, miR-7847-3p and miR-6782-5p. The miRNA target GO enrichment analysis showed the different pathway involved in these two different forms of obesity. Although the rarity of PWS actually represents a limitation to the availability of large series, the present study provides novel hints on the molecular pathogenesis of syndromic and non-syndromic obesity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1530/EC-18-0329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240145PMC
December 2018

ActivinA: a new leukemia-promoting factor conferring migratory advantage to B-cell precursor-acute lymphoblastic leukemic cells.

Haematologica 2019 03 27;104(3):533-545. Epub 2018 Sep 27.

Centro Ricerca Tettamanti, Department of Pediatrics, University of Milano-Bicocca, Fondazione MBBM, Monza

B-cell precursor-acute lymphoblastic leukemia modulates the bone marrow (BM) niche to become leukemia-supporting and chemo-protective by reprogramming the stromal microenvironment. New therapies targeting the interplay between leukemia and stroma can help improve disease outcome. We identified ActivinA, a TGF-β family member with a well-described role in promoting several solid malignancies, as a factor favoring leukemia that could represent a new potential target for therapy. ActivinA resulted over-expressed in the leukemic BM and its production was strongly induced in mesenchymal stromal cells after culture with leukemic cells. Moreover, MSCs isolated from BM of leukemic patients showed an intrinsic ability to secrete higher amounts of ActivinA compared to their normal counterparts. The pro-inflammatory leukemic BM microenvironment synergized with leukemic cells to induce stromal-derived ActivinA. Gene expression analysis of ActivinA-treated leukemic cells showed that this protein was able to significantly influence motility-associated pathways. Interestingly, ActivinA promoted random motility and CXCL12-driven migration of leukemic cells, even at suboptimal chemokine concentrations, characterizing the leukemic niche. Conversely, ActivinA severely impaired CXCL12-induced migration of healthy CD34 cells. This opposite effect can be explained by the ability of ActivinA to increase intracellular calcium only in leukemic cells, boosting cytoskeleton dynamics through a higher rate of actin polymerization. Moreover, by stimulating the invasiveness of the leukemic cells, ActivinA was found to be a leukemia-promoting factor. Importantly, the ability of ActivinA to enhance BM engraftment and the metastatic potential of leukemic cells was confirmed in a xenograft mouse model of the disease. Overall, ActivinA was seen to be a key factor in conferring a migratory advantage to leukemic cells over healthy hematopoiesis within the leukemic niche.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2018.188664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395324PMC
March 2019

USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia.

Clin Cancer Res 2019 01 17;25(1):222-239. Epub 2018 Sep 17.

Department of Biochemistry and Molecular Genetics, Northwestern University, Chicago, Illinois.

Purpose: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes.

Experimental Design: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models.

Results: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth and .

Conclusions: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-18-1740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320313PMC
January 2019

Simultaneous B and T cell acute lymphoblastic leukemias in zebrafish driven by transgenic MYC: implications for oncogenesis and lymphopoiesis.

Leukemia 2019 02 15;33(2):333-347. Epub 2018 Aug 15.

Section of Pediatric Hematology-Oncology, Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.

Precursor-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common pediatric cancer, but there are no useful zebrafish pre-B ALL models. We describe the first highly- penetrant zebrafish pre-B ALL, driven by human MYC. Leukemias express B lymphoblast-specific genes and are distinct from T cell ALL (T-ALL)-which these fish also develop. Zebrafish pre-B ALL shares in vivo features and expression profiles with human pre-B ALL, and these profiles differ from zebrafish T-ALL or normal B and T cells. These animals also exhibit aberrant lymphocyte development. As the only robust zebrafish pre-B ALL model and only example where T-ALL also develops, this model can reveal differences between MYC-driven pre-B vs. T-ALL and be exploited to discover novel pre-B ALL therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41375-018-0226-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365377PMC
February 2019

SRC/ABL inhibition disrupts CRLF2-driven signaling to induce cell death in B-cell acute lymphoblastic leukemia.

Oncotarget 2018 May 1;9(33):22872-22885. Epub 2018 May 1.

M. Tettamanti Research Center, Pediatric Clinic, University of Milano Bicocca, Monza, Italy.

Children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) overexpressing the gene () have poor prognosis. CRLF2 protein overexpression leads to activated JAK/STAT signaling and trials are underway using JAK inhibitors to overcome treatment failure. Pre-clinical studies indicated limited efficacy of single JAK inhibitors, thus additional pathways must be targeted in cells. To identify additional activated networks, we used single-cell mass cytometry to examine 15 BCP-ALL primary patient samples. We uncovered a coordinated signaling network downstream of CRLF2 characterized by co-activation of JAK/STAT, PI3K, and CREB pathways. This CRLF2-driven network could be more effectively disrupted by SRC/ABL inhibition than single-agent JAK or PI3K inhibition, and this could be demonstrated even in primary minimal residual disease (MRD) cells. Our study suggests SCR/ABL inhibition as effective in disrupting the cooperative functional networks present in 2 BCP-ALL patients, supporting further investigation of this strategy in pre-clinical studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.25089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955419PMC
May 2018

AKR1C enzymes sustain therapy resistance in paediatric T-ALL.

Br J Cancer 2018 04 8;118(7):985-994. Epub 2018 Mar 8.

Istituto di Ricerca paediatrica Città della Speranza-IRP, corso Stati Uniti 4, Padova, 35127, Italy.

Background: Despite chemotherapy intensification, a subgroup of high-risk paediatric T-cell acute lymphoblastic leukemia (T-ALL) patients still experience treatment failure. In this context, we hypothesised that therapy resistance in T-ALL might involve aldo-keto reductase 1C (AKR1C) enzymes as previously reported for solid tumors.

Methods: Expression of NRF2-AKR1C signaling components has been analysed in paediatric T-ALL samples endowed with different treatment outcomes as well as in patient-derived xenografts of T-ALL. The effects of AKR1C enzyme modulation has been investigated in T-ALL cell lines and primary cultures by combining AKR1C inhibition, overexpression, and gene silencing approaches.

Results: We show that T-ALL cells overexpress AKR1C1-3 enzymes in therapy-resistant patients. We report that AKR1C1-3 enzymes play a role in the response to vincristine (VCR) treatment, also ex vivo in patient-derived xenografts. Moreover, we demonstrate that the modulation of AKR1C1-3 levels is sufficient to sensitise T-ALL cells to VCR. Finally, we show that T-ALL chemotherapeutics induce overactivation of AKR1C enzymes independent of therapy resistance, thus establishing a potential resistance loop during T-ALL combination treatment.

Conclusions: Here, we demonstrate that expression and activity of AKR1C enzymes correlate with response to chemotherapeutics in T-ALL, posing AKR1C1-3 as potential targets for combination treatments during T-ALL therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41416-018-0014-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931104PMC
April 2018

Somatic mutations activating Wiskott-Aldrich syndrome protein concomitant with RAS pathway mutations in juvenile myelomonocytic leukemia patients.

Hum Mutat 2018 04 19;39(4):579-587. Epub 2018 Jan 19.

Laboratory of Onco-Hematology, Department of Women's and Children's Health, University of Padova, Padova, Italy.

The WAS gene product is expressed exclusively in the cytoplasm of hematopoietic cells and constitutional genetic abrogation of WASP leads to Wiskott-Aldrich syndrome (WAS). Moreover, mutational activation of WASP has been associated with X-linked neutropenia. Although studies reported that patients with constitutional WAS mutations affecting functional WASP expression may present juvenile myelomonocytic leukemia (JMML)-like features, confounding differential diagnosis above all in the copresence of mutated RAS, an activating somatic mutation of WASP has not been previously described in JMML patients. In our ongoing studies on JMML genomics, we at first detected a somatic WAS mutation in a major clone found at two consecutive relapses in one of two twins with JMML. Both twins were treated with hematopoietic stem cell transplantation after diagnosis of JMML. The somatic WAS mutation detected here displayed an activating WASP phenotype. Screening of 46 sporadic JMML patients at disease onset for mutations in the same PBD domain of WAS revealed two additional singleton patients carrying minor mutated clones. This is the first study to associate somatically acquired WASP mutations with a hematopoietic malignancy and increases insight in the complexity of the genomic landscape of JMML that shows low recurrent mutations concomitant with general hyperactivation of RAS pathway signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.23399DOI Listing
April 2018

Glucocorticoid resistance is reverted by LCK inhibition in pediatric T-cell acute lymphoblastic leukemia.

Blood 2017 12 3;130(25):2750-2761. Epub 2017 Nov 3.

Department of Woman's and Child's Health and.

Pediatric T-acute lymphoblastic leukemia (T-ALL) patients often display resistance to glucocorticoid (GC) treatment. These patients, classified as prednisone poor responders (PPR), have poorer outcome than do the other pediatric T-ALL patients receiving a high-risk adapted therapy. Because glucocorticoids are administered to ALL patients during all the different phases of therapy, GC resistance represents an important challenge to improving the outcome for these patients. Mechanisms underlying resistance are not yet fully unraveled; thus our research focused on the identification of deregulated signaling pathways to point out new targeted approaches. We first identified, by reverse-phase protein arrays, the lymphocyte cell-specific protein-tyrosine kinase (LCK) as aberrantly activated in PPR patients. We showed that LCK inhibitors, such as dasatinib, bosutinib, nintedanib, and WH-4-023, are able to induce cell death in GC-resistant T-ALL cells, and remarkably, cotreatment with dexamethasone is able to reverse GC resistance, even at therapeutic drug concentrations. This was confirmed by specific gene silencing and ex vivo combined treatment of cells from PPR patient-derived xenografts. Moreover, we observed that LCK hyperactivation in PPR patients upregulates the calcineurin/nuclear factor of activated T cells signaling triggering to interleukin-4 () overexpression. GC-sensitive cells cultured with IL-4 display an increased resistance to dexamethasone, whereas the inhibition of IL-4 signaling could increase GC-induced apoptosis in resistant cells. Treatment with dexamethasone and dasatinib also impaired engraftment of leukemia cells in vivo. Our results suggest a quickly actionable approach to supporting conventional therapies and overcoming GC resistance in pediatric T-ALL patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2017-05-784603DOI Listing
December 2017

ZNF521 sustains the differentiation block in MLL-rearranged acute myeloid leukemia.

Oncotarget 2017 Apr;8(16):26129-26141

Department of Woman and Child Health, University of Padova, Italy.

Zinc finger protein 521 (ZNF521) is a multiple zinc finger transcription factor and a strong candidate as regulator of hematopoietic stem cell homeostasis. Recently, independent gene expression profile studies have evidenced a positive correlation between ZNF521 mRNA overexpression and MLL-rearranged acute myeloid leukemia (AML), leaving open the question on the role of ZNF521 in this subtype of leukemia. In this study, we sought to analyze the effect of ZNF521 depletion on MLL-rearranged AML cell lines and MLL-AF9 xenograft primary cells. Knockdown of ZNF521 with short-hairpin RNA (shRNA) led to decreased leukemia proliferation, reduced colony formation and caused cell cycle arrest in MLL-rearranged AML cell lines. Importantly, we showed that loss of ZNF521 substantially caused differentiation of both MLL-rearranged cell lines and primary cells. Moreover, gene profile analysis in ZNF521-silenced THP-1 cells revealed a loss of MLL-AF9-directed leukemic signature and an increase of the differentiation program. Finally, we determined that both MLL-AF9 and MLL-ENL fusion proteins directly interacted with ZNF521 promoter activating its transcription. In conclusion, our findings identify ZNF521 as a critical effector of MLL fusion proteins in blocking myeloid differentiation and highlight ZNF521 as a potential therapeutic target for this subtype of leukemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.15387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432245PMC
April 2017

ETV6/RUNX1-like acute lymphoblastic leukemia: A novel B-cell precursor leukemia subtype associated with the CD27/CD44 immunophenotype.

Genes Chromosomes Cancer 2017 08 5;56(8):608-616. Epub 2017 May 5.

CLIP-Childhood Leukaemia Investigation Prague, Czech Republic.

We have shown previously that ETV6/RUNX1-positive acute lymphoblastic leukemia (ALL) is distinguishable from other ALL subtypes by CD27 /CD44 immunophenotype. During diagnostic immunophenotyping of 573 childhood B-cell precursor ALL (BCP-ALL), we identified eight cases with this immunophenotype among "B-other ALL" (BCP-ALL cases negative for routinely tested chromosomal/genetic aberrations). We aimed to elucidate whether these cases belong to the recently described ETV6/RUNX1-like ALL defined by the ETV6/RUNX1-specific gene expression profile (GEP), harboring concurrent ETV6 and IKZF1 lesions. We performed comprehensive genomic analysis using single nucleotide polymorphism arrays, whole exome and transcriptome sequencing and GEP on microarrays. In unsupervised hierarchical clustering based on GEP, five out of seven analyzed CD27 /CD44 B-other cases clustered with ETV6/RUNX1-positive ALL and were thus classified as ETV6/RUNX1-like ALL. The two cases clustering outside ETV6/RUNX1-positive ALL harbored a P2RY8/CRLF2 fusion with activating JAK2 mutations and a TCF3/ZNF384 fusion, respectively, assigning them to other ALL subtypes. All five ETV6/RUNX1-like cases harbored ETV6 deletions; uniform intragenic ARPP21 deletions and various IKZF1 lesions were each found in three ETV6/RUNX1-like cases. The frequency of ETV6 and ARPP21 deletions was significantly higher in ETV6/RUNX1-like ALL compared with a reference cohort of 42 B-other ALL. In conclusion, we show that ETV6/RUNX1-like ALL is associated with CD27 /CD44 immunophenotype and identify ARPP21 deletions to contribute to its specific genomic profile enriched for ETV6 and IKZF1 lesions. In conjunction with previously published data, our study identifies the ETV6 lesion as the only common genetic aberration and thus the most likely key driver of ETV6/RUNX1-like ALL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/gcc.22464DOI Listing
August 2017

Expression Profiling of Circulating Microvesicles Reveals Intercellular Transmission of Oncogenic Pathways.

Mol Cancer Res 2017 06 15;15(6):683-695. Epub 2017 Feb 15.

Department of Women's and Children's Health, University of Padova, Padova, Italy.

Circulating microvesicles have been described as important players in cell-to-cell communication carrying biological information under normal or pathologic condition. Microvesicles released by cancer cells may incorporate diverse biomolecules (e.g., active lipids, proteins, and RNA), which can be delivered and internalized by recipient cells, potentially altering the gene expression of recipient cells and eventually impacting disease progression. Leukemia model systems were used to investigate microvesicles as vehicles of protein-coding messages. Several leukemic cells (K562, LAMA-87, TOM-1, REH, and SHI-1), each carrying a specific chromosomal translocation, were analyzed. In the leukemic cells, these chromosomal translocations are transcribed into oncogenic fusion transcripts and the transfer of these transcripts was monitored from leukemic cells to microvesicles for each of the cell lines. Microarray gene expression profiling was performed to compare transcriptomes of K562-derived microvesicles and parental K562 cells. The data show that oncogenic transcripts and mRNAs related to basic functions of leukemic cells were included in microvesicles. Further analysis of microvesicles cargo revealed a remarkable enrichment of transcripts related to cell membrane activity, cell surface receptors, and extracellular communication when compared with parental K562 cells. Finally, coculturing of healthy mesenchymal stem cells (MSC) with K562-derived microvesicles displayed the transfer of the oncogenic message, and confirmed the increase of target cell proliferation as a function of microvesicle dosage. This study provides novel insight into tumor-derived microvesicles as carriers of oncogenic protein-coding messages that can potentially jeopardize cell-directed therapy, and spread to other compartments of the body. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1541-7786.MCR-16-0307DOI Listing
June 2017

Profiling B cell chronic lymphocytic leukemia by reverse phase protein array: Focus on apoptotic proteins.

J Leukoc Biol 2016 11 16;100(5):1061-1070. Epub 2016 Jun 16.

Department of Medicine, Hematology and Clinical Immunology Branch, Padua University School of Medicine, Padova, Italy;

B cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B lymphocytes from proliferative activity and apoptosis resistance. The increased awareness of the importance of B cell receptor signaling in CLL has raised new opportunities for targeted intervention. Herein, we describe a study performed with the high-throughput RPPA (reverse phase protein array) technique, which allowed us to simultaneously study different molecules in a large series of patients. We analyzed B lymphocytes from 57 patients with CLL and 11 healthy subjects. Different pathways were assessed for activation/expression of key signaling proteins. Data obtained were validated by Western blotting and confocal microscopy. The RPPA investigation and its validation, identified 3 series of proteins: 1) molecules whose expression levels reached statistically significant differences in CLL vs. healthy controls (HSP70, Smac/DIABLO, cleaved PARP, and cleaved caspase-6); 2) proteins with a positive trend of difference in CLL vs. healthy controls (HS1, γ-tubulin, PKC α/β-II Thr-638/641, p38 MAPK Thr-180/Tyr-182, NF-κB Ser-536, Bcl2 Ser-70 and Src Tyr-527); and 3) molecules differentially expressed in patients with IGHV mutations vs. those without mutations (ZAP70, PKC-ζλ, Thr-410/403, and CD45). This study identified some molecules, particularly those involved in apoptosis control, which could be considered for further studies to design new therapeutic strategies in CLL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1189/jlb.2AB0715-301RDOI Listing
November 2016

Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis.

Sci Rep 2016 10 4;6:34449. Epub 2016 Oct 4.

Department of Woman and Child Health, University of Padua, Via Giustiniani 3, 35128 Padua, Italy.

To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep34449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5048141PMC
October 2016

Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ.

Cell Stem Cell 2016 12 15;19(6):725-737. Epub 2016 Sep 15.

Department of Molecular Medicine, University of Padua School of Medicine, viale Colombo 3, 35126 Padua, Italy. Electronic address:

The ability to induce autologous tissue-specific stem cells in culture could have a variety of applications in regenerative medicine and disease modeling. Here we show that transient expression of exogenous YAP or its closely related paralogue TAZ in primary differentiated mouse cells can induce conversion to a tissue-specific stem/progenitor cell state. Differentiated mammary gland, neuronal, and pancreatic exocrine cells, identified using a combination of cell sorting and lineage tracing approaches, efficiently convert to proliferating cells with properties of stem/progenitor cells of their respective tissues after YAP induction. YAP-induced mammary stem/progenitor cells show molecular and functional properties similar to endogenous MaSCs, including organoid formation and mammary gland reconstitution after transplantation. Because YAP/TAZ function is also important for self-renewal of endogenous stem cells in culture, our findings have implications for understanding the molecular determinants of the somatic stem cell state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2016.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5145813PMC
December 2016

Proteomic Alterations in Response to Hypoxia Inducible Factor 2α in Normoxic Neuroblastoma Cells.

J Proteome Res 2016 10 28;15(10):3643-3655. Epub 2016 Sep 28.

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli "Federico II" , via Pansini, 5, 80131 Naples, Italy.

Hypoxia inducible factor (HIF)-2α protein expression in solid tumors promotes stem-like phenotype in cancer stem cells and increases tumorigenic potential in nonstem cancer cells. Recently, we have shown that HIF-1/2α gene expression is correlated to neuroblastoma (NB) poor survival and to undifferentiated tumor state; HIF-2α protein was demonstrated to enhance aggressive features of the disease. In this study, we used proteomic experiments on NB cells to investigate HIF-2α downstream-regulated proteins or pathways with the aim of providing novel therapeutic targets or bad prognosis markers. We verified that pathways mostly altered by HIF-2α perturbation are involved in tumor progression. In particular, HIF-2α induces alteration of central metabolism and splicing control pathways. Simultaneously, WNT, RAS/MAPK, and PI3K/AKT activity or expression are affected and may impact the sensitivity and the intensity of HIF-2α-regulated pathways. Furthermore, genes coding the identified HIF-2α-related markers built a signature able to stratify NB patients with unfavorable outcome. Taken together, our findings underline the relevance of dissecting the downstream effects of a poor survival marker in developing targeted therapy and improving patient stratification. Future prospective studies are needed to translate the use of these data into the clinical practice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.6b00457DOI Listing
October 2016

MicroRNA fingerprints in juvenile myelomonocytic leukemia (JMML) identified miR-150-5p as a tumor suppressor and potential target for treatment.

Oncotarget 2016 Aug;7(34):55395-55408

Division of Hematology, Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.

Juvenile myelomonocytic leukemia (JMML) is an aggressive leukemia of early childhood characterized by aberrant proliferation of myelomonocytic cells and hypersensitivity to GM-CSF stimulation. Mutually exclusive mutations in the RAS/ERK pathway genes such as PTPN11, NRAS, KRAS, CBL, or NF1 are found in ~90% of the cases. These mutations give rise to disease at least in part by activating STAT5 through phosphorylation and by promoting cell growth. MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression, which are often deregulated in leukemia. However, little is known about their role in JMML. Here, we report distinctive miR expression signatures associated with the molecular subgroups of JMML. Among the downregulated miRs in JMML, miR-150-5p was found to target STAT5b, a gene which is often over-activated in JMML, and contributes to the characteristic aberrant signaling of this disorder. Moreover, loss of miR-150-5p and upregulation of STAT5b expression were also identified in a murine model of JMML. Ectopic overexpression of miR-150-5p in mononuclear cells from three JMML patients significantly decreased cell proliferation. Altogether, our data indicate that miR expression is deregulated in JMML and may play a role in the pathogenesis of this disorder by modulating key effectors of cytokine receptor pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.10577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342425PMC
August 2016