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1.
Int J Oncol ; 53(4): 1455-1468, 2018 Oct.
Article in English | MEDLINE | ID: mdl-30066860

ABSTRACT

Von Hippel-Lindau (VHL) disease is a rare autosomal dominant syndrome that is the main cause of inherited clear-cell renal cell carcinoma (ccRCC), which generally occurs in the form of multiple recurrent synchronized tumors. Affected patients are carriers of a germline mutation in the VHL tumor suppressor gene. Somatic mutations of this gene are also found in sporadic ccRCC and numerous pan-genomic studies have reported a dysregulation of microRNA (miRNA) expression in these sporadic tumors. In order to investigate the molecular mechanisms underlying the pathogenesis of VHL-associated ccRCC, particularly in the context of multiple tumors, the present study characterized the mRNA and miRNA transcriptome through an integrative analysis compared with sporadic renal tumors. In the present study, two series of ccRCC samples were used. The first set consisted of several samples from different tumors occurring in the same patient, for two independent patients affected with VHL disease. The second set consisted of 12 VHL-associated tumors and 22 sporadic ccRCC tumors compared with a pool of normal renal tissue. For each sample series, an expression analysis of miRNAs and mRNAs was conducted using microarrays. The results indicated that multiple tumors within the kidney of a patient with VHL disease featured a similar pattern of miRNA and gene expression. In addition, the expression levels of miRNA were able to distinguish VHL-associated tumors from sporadic ccRCC, and it was identified that 103 miRNAs and 2,474 genes were differentially expressed in the ccRCC series compared with in normal renal tissue. The majority of dysregulated genes were implicated in 'immunity' and 'metabolism' pathways. Taken together, these results allow a better understanding of the occurrence of ccRCC in patients with VHL disease, by providing insights into dysregulated miRNA and mRNA. In the set of patients with VHL disease, there were few differences in miRNA and mRNA expression, thus indicating a similar molecular evolution of these synchronous tumors and suggesting that the same molecular mechanisms underlie the pathogenesis of these hereditary tumors.


Subject(s)
Carcinoma, Renal Cell/genetics , Gene Expression Regulation, Neoplastic , Kidney Neoplasms/genetics , Neoplasm Recurrence, Local/genetics , Neoplasms, Multiple Primary/genetics , von Hippel-Lindau Disease/genetics , Adult , Aged , Aged, 80 and over , Carcinoma, Renal Cell/epidemiology , Carcinoma, Renal Cell/pathology , Case-Control Studies , Cell Line, Tumor , Female , Gene Expression Profiling/methods , Humans , Kidney/pathology , Kidney Neoplasms/epidemiology , Kidney Neoplasms/pathology , Male , MicroRNAs/metabolism , Middle Aged , Neoplasm Recurrence, Local/epidemiology , Neoplasm Recurrence, Local/pathology , Neoplasms, Multiple Primary/epidemiology , Neoplasms, Multiple Primary/pathology , Oligonucleotide Array Sequence Analysis/methods , RNA, Messenger/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Young Adult
2.
Pharm Res ; 33(8): 1913-22, 2016 08.
Article in English | MEDLINE | ID: mdl-27091031

ABSTRACT

PURPOSE: Busulfan-melphalan high-dose chemotherapy followed by autologous stem cell transplantation is an essential consolidation treatment of high-risk neuroblastoma in children. Main treatment limitation is hepatic veno-occlusive disease, the most severe and frequent extra-hematological toxicity. This life threatening toxicity has been related to a drug interaction between busulfan and melphalan which might be increased by prior disturbance of iron homeostasis, i.e. an increased plasma ferritin level. METHODS: We performed an experimental study of busulfan and melphalan pharmacodynamic and pharmacokinetics in iron overloaded mice. RESULTS: Iron excess dramatically increased the toxicity of melphalan or busulfan melphalan combination in mice but it did not modify the clearance of either busulfan or melphalan. We show that prior busulfan treatment impairs the clearance of melphalan. This clearance alteration was exacerbated in iron overloaded mice demonstrating a pharmacokinetic interaction. Additionally, iron overload increased melphalan toxicity without altering its pharmacokinetics, suggesting a pharmacodynamic interaction between iron and melphalan. Based on iron homeostasis disturbance, we postulated that prior induction of ferritin, through Nrf2 activation after oxidative stress, may be associated with the alteration of melphalan metabolism. CONCLUSION: Iron overload increases melphalan and busulfan-melphalan toxicity through a pharmacodynamic interaction and reveals a pharmacokinetic drug interaction between busulfan and melphalan.


Subject(s)
Busulfan/metabolism , Busulfan/toxicity , Iron Overload/metabolism , Melphalan/metabolism , Melphalan/toxicity , Animals , Antineoplastic Agents, Alkylating/metabolism , Antineoplastic Agents, Alkylating/toxicity , Drug Interactions/physiology , Iron Overload/pathology , Male , Mice , Mice, Inbred C57BL
3.
Clin Cancer Res ; 20(13): 3411-21, 2014 Jul 01.
Article in English | MEDLINE | ID: mdl-24658158

ABSTRACT

PURPOSE: Papillary renal cell carcinomas (pRCC) are the most common nonclear cell RCC subtype. Germline mutations of the MET oncogene at 7q31 have been detected in patients with hereditary type I pRCC and in 13% of sporadic type I pRCC. Recent report of MET inhibition strengthened the role of c-Met inhibition across pRCC. EXPERIMENTAL DESIGN: We collected 220 frozen samples of sporadic pRCC through the French RCC Network and quality controlled for percentage of malignant cells >70%. Gene expression was assessed on 98 pRCC using human whole-genome Agilent 8 × 60K arrays. Copy number alterations were analyzed using Agilent Human 2 × 400K and 4× 180K array for type II pRCC and comparative genomic microarray analysis method for type I pRCC. MET gene sequencing was performed on type I pRCC. RESULTS: MET expression level was high across all pRCC. We identified copy number alterations (gain) in 46% of type II pRCC and in 81% of type I pRCC. Correlation between DNA copy number alterations and mRNA expression level was highly significant. Eleven somatic mutations of MET gene were identified amongst 51 type I pRCC (21.6%), including 4 new mutations. We validated LRRK2 cokinase as highly correlated to MET expression. CONCLUSION: The present report expands the role of MET activation as a potential target across all pRCC subtypes. These data support investigating MET inhibitors in pRCC in correlation with MET activation status.


Subject(s)
Carcinoma, Papillary/genetics , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Proto-Oncogene Proteins c-met/genetics , Adult , Aged , Aged, 80 and over , Carcinoma, Papillary/pathology , Carrier Proteins/genetics , Cluster Analysis , Comparative Genomic Hybridization , Computational Biology , DNA Copy Number Variations , Exons , Female , Gene Expression , Gene Expression Profiling , Humans , Male , Middle Aged , Mutation , Neoplasm Grading , Neoplasm Staging , Protein Binding , Sequence Analysis, DNA
4.
PLoS One ; 9(1): e84785, 2014.
Article in English | MEDLINE | ID: mdl-24409309

ABSTRACT

Exome sequence capture and massively parallel sequencing can be combined to achieve inexpensive and rapid global analyses of the functional sections of the genome. The difficulties of working with relatively small quantities of genetic material, as may be necessary when sharing tumor biopsies between collaborators for instance, can be overcome using whole genome amplification. However, the potential drawbacks of using a whole genome amplification technology based on random primers in combination with sequence capture followed by massively parallel sequencing have not yet been examined in detail, especially in the context of mutation discovery in tumor material. In this work, we compare mutations detected in sequence data for unamplified DNA, whole genome amplified DNA, and RNA originating from the same tumor tissue samples from 16 patients diagnosed with non-small cell lung cancer. The results obtained provide a comprehensive overview of the merits of these techniques for mutation analysis. We evaluated the identified genetic variants, and found that most (74%) of them were observed in both the amplified and the unamplified sequence data. Eighty-nine percent of the variations found by WGA were shared with unamplified DNA. We demonstrate a strategy for avoiding allelic bias by including RNA-sequencing information.


Subject(s)
Genome, Human , Genomics/methods , High-Throughput Nucleotide Sequencing , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Female , Genetic Variation , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Mutation , Reproducibility of Results , Sequence Analysis, DNA , Sequence Analysis, RNA
5.
BMC Med Genomics ; 6: 53, 2013 Dec 03.
Article in English | MEDLINE | ID: mdl-24299561

ABSTRACT

BACKGROUND: Non-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC. METHODS: Comparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC. RESULTS: At DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1 and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of ~800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944. CONCLUSIONS: Integrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Genomics , Lung Neoplasms/genetics , Adult , Aged , Aged, 80 and over , DNA Copy Number Variations/genetics , Female , Gene Expression Profiling , Humans , Male , MicroRNAs/genetics , Middle Aged , RNA, Messenger/genetics
6.
Cancer Res ; 73(7): 2271-80, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23554447

ABSTRACT

Non-small cell lung carcinoma patients are frequently treated with cisplatin (CDDP), most often yielding temporary clinical responses. Here, we show that PARP1 is highly expressed and constitutively hyperactivated in a majority of human CDDP-resistant cancer cells of distinct histologic origin. Cells manifesting elevated intracellular levels of poly(ADP-ribosyl)ated proteins (PAR(high)) responded to pharmacologic PARP inhibitors as well as to PARP1-targeting siRNAs by initiating a DNA damage response that translated into cell death following the activation of the intrinsic pathway of apoptosis. Moreover, PARP1-overexpressing tumor cells and xenografts displayed elevated levels of PAR, which predicted the response to PARP inhibitors in vitro and in vivo more accurately than PARP1 expression itself. Thus, a majority of CDDP-resistant cancer cells appear to develop a dependency to PARP1, becoming susceptible to PARP inhibitor-induced apoptosis.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/pathology , Cisplatin/pharmacology , Drug Resistance, Neoplasm , Lung Neoplasms/pathology , Poly(ADP-ribose) Polymerase Inhibitors , Animals , Apoptosis/drug effects , Blotting, Western , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Proliferation/drug effects , Female , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Mice , Mice, Nude , Phenanthrenes/pharmacology , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
7.
Blood ; 121(12): 2186-98, 2013 Mar 21.
Article in English | MEDLINE | ID: mdl-23319568

ABSTRACT

Genomic studies in chronic myeloid malignancies, including myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), and MPN/MDS, have identified common mutations in genes encoding signaling, epigenetic, transcription, and splicing factors. In the present study, we interrogated the clonal architecture by mutation-specific discrimination analysis of single-cell-derived colonies in 28 patients with chronic myelomonocytic leukemias (CMML), the most frequent MPN/MDS. This analysis reveals a linear acquisition of the studied mutations with limited branching through loss of heterozygosity. Serial analysis of untreated and treated samples demonstrates a dynamic architecture on which most current therapeutic approaches have limited effects. The main disease characteristics are early clonal dominance, arising at the CD34(+)/CD38(-) stage of hematopoiesis, and granulomonocytic differentiation skewing of multipotent and common myeloid progenitors. Comparison of clonal expansions of TET2 mutations in MDS, MPN, and CMML, together with functional invalidation of TET2 in sorted progenitors, suggests a causative link between early clonal dominance and skewed granulomonocytic differentiation. Altogether, early clonal dominance may distinguish CMML from other chronic myeloid neoplasms with similar gene mutations.


Subject(s)
Clonal Evolution/genetics , Leukemia, Myelomonocytic, Chronic/genetics , Leukemia, Myelomonocytic, Chronic/pathology , Aged , Aged, 80 and over , Case-Control Studies , Cell Differentiation/genetics , Clonal Evolution/immunology , Cohort Studies , Female , Hematopoiesis/genetics , Hematopoiesis/immunology , Humans , Leukemia, Myelomonocytic, Chronic/immunology , Loss of Heterozygosity , Male , Middle Aged , Mutation/physiology , Mutation Rate , Myeloid Cells/metabolism , Myeloid Cells/physiology
8.
FASEB J ; 26(12): 5172-81, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22898924

ABSTRACT

The aspartic protease cathepsin D, a poor prognostic indicator of breast cancer, is abundantly secreted as procathepsin D by human breast cancer cells and self-activates at low pH in vitro, giving rise to catalytically active cathepsin D. Due to a lower extracellular pH in tumor microenvironments compared to normal tissues, cathepsin D may cleave pathophysiological substrates contributing to cancer progression. Here, we show by yeast 2-hybrid and degradomics analyses that cystatin C, the most potent natural secreted inhibitor of cysteine cathepsins, both binds to and is a substrate of extracellular procathepsin D. The amount of cystatin C in the extracellular environment is reduced in the secretome of mouse embryonic fibroblasts stably transfected with human cathepsin D. Cathepsin D extensively cleaved cystatin C in vitro at low pH. Cathepsin D secreted by breast cancer cells also processed cystatin C at the pericellular pH of tumors and so enhancing extracellular proteolytic activity of cysteine cathepsins. Thus, tumor derived cathepsin D assists breast cancer progression by reducing cystatin C activity, which, in turn, enhances cysteine cathepsin proteolytic activity, revealing a new link between protease classes in the protease web.


Subject(s)
Cathepsin D/metabolism , Cystatin C/metabolism , Fibroblasts/metabolism , Tumor Microenvironment , Amino Acid Sequence , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cathepsin D/genetics , Cells, Cultured , Cystatin C/genetics , Embryo, Mammalian/cytology , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , Extracellular Space/metabolism , Female , Fibroblasts/cytology , Humans , Hydrogen-Ion Concentration , Immunoblotting , MCF-7 Cells , Mice , Mice, Knockout , Models, Biological , Molecular Sequence Data , Protein Binding , Proteolysis , RNA Interference , Two-Hybrid System Techniques
9.
Biochem Biophys Res Commun ; 425(2): 379-83, 2012 Aug 24.
Article in English | MEDLINE | ID: mdl-22842464

ABSTRACT

Personalized cancer treatment requires molecular characterization of individual tumor biopsies. These samples are frequently only available in limited quantities hampering genomic analysis. Several whole genome amplification (WGA) protocols have been developed with reported varying representation of genomic regions post amplification. In this study we investigate region dropout using a φ29 polymerase based WGA approach. DNA from 123 lung cancers specimens and corresponding normal tissue were used and evaluated by Sanger sequencing of the p53 exons 5-8. To enable comparative analysis of this scarce material, WGA samples were compared with unamplified material using a pooling strategy of the 123 samples. In addition, a more detailed analysis of exon 7 amplicons were performed followed by extensive cloning and Sanger sequencing. Interestingly, by comparing data from the pooled samples to the individually sequenced exon 7, we demonstrate that mutations are more easily recovered from WGA pools and this was also supported by simulations of different sequencing coverage. Overall this data indicate a limited random loss of genomic regions supporting the use of whole genome amplification for genomic analysis.


Subject(s)
DNA Mutational Analysis/methods , DNA, Neoplasm/analysis , Genomics/methods , Neoplasms/diagnosis , Neoplasms/genetics , Nucleic Acid Amplification Techniques , Tumor Suppressor Protein p53/genetics , DNA, Neoplasm/biosynthesis , DNA-Directed DNA Polymerase/chemistry , Exons , Genome, Human , Humans , Lung Neoplasms/genetics
10.
Blood ; 118(14): 3824-31, 2011 Oct 06.
Article in English | MEDLINE | ID: mdl-21828134

ABSTRACT

Hydroxyurea is the standard therapy of chronic myelomonocytic leukemia (CMML) presenting with advanced myeloproliferative and/or myelodysplastic features. Response to hypomethylating agents has been reported in heterogeneous series of CMML. We conducted a phase 2 trial of decitabine (DAC) in 39 patients with advanced CMML defined according to a previous trial. Median number of DAC cycles was 10 (range, 1-24). Overall response rate was 38% with 4 complete responses (10%), 8 marrow responses (21%), and 3 stable diseases with hematologic improvement (8%). Eighteen patients (46%) demonstrated stable disease without hematologic improvement, and 6 (15%) progressed to acute leukemia. With a median follow-up of 23 months, overall survival was 48% at 2 years. Mutations in ASXL1, TET2, AML1, NRAS, KRAS, CBL, FLT3, and janus kinase 2 (JAK2) genes, and hypermethylation of the promoter of the tumor suppressor gene TIF1γ, did not predict response or survival on DAC therapy. Lower CJUN and CMYB gene expression levels independently predicted improved overall survival. This trial confirmed DAC efficacy in approximately 40% of CMML patients with advanced myeloproliferative or myelodysplastic features and suggested that CJUN and CMYB expression could be potential biomarkers in this setting. This trial is registered at EudraCT (eudract.ema.europa.eu) as #2008-000470-21 and www.clinicaltrials.gov as #NCT01098084.


Subject(s)
Antimetabolites, Antineoplastic/therapeutic use , Azacitidine/analogs & derivatives , Leukemia, Myelomonocytic, Chronic/drug therapy , Leukemia, Myelomonocytic, Chronic/genetics , Aged , Aged, 80 and over , Azacitidine/therapeutic use , Decitabine , Female , Gene Expression Regulation, Leukemic , Humans , Leukemia, Myelomonocytic, Chronic/diagnosis , Male , Middle Aged , Mutation , Prognosis , Survival Analysis
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