Promoter hypermethylation of candidate tumor suppressor genes in urinary bladder and prostate cancer

Identification of the full spectrum of gene promoters methylated in cancer, or the cancer methylome, would greatly advance our understanding of gene regulatory networks involved in tumorigenesis. A robust approach was developed that couples genome-wide probabilistic search algorithms with an established pharmacologic unmasking strategy for unbiased and precise global localization of tumor-specific methylated genes. Through this approach, a set of 175 novel candidate genes was identified that cluster throughout the genome and may harbor cancer-specific promoter methylation. In this study, over sixty genes were tested in bladder and prostate cancer by candidate gene approach to evaluate new promising tumor suppressor genes in genitourinary cancer. An initial screen of the methylation status of the promoter region was conducted by bisulfite genomic sequencing in a panel of over sixty genes in seven bladder cancer and four prostate cancer cell lines.Based on the bisulfite sequencing data, we selected nine genes (camk4, fkbp4, hoxB5, krt14, LPAR2, mal, rgs4, vgf and ZMYM2) where aberrant methylation was detected in both bladder and prostate cancer cell lines. The expression of eight genes in carcinoma cell lines was analyzed by semiquantitative reverse transcription-PCR (RT-PCR) after 5-aza-2\u27-deoxycytidine treatment, alone or in combination with Trichostatin A. Methylation was detected by bisulfite sequencing in these genes at the following frequencies in bladder cancer cell lines: camk4 (16.6%), fkbp4 (71.4%), hoxB5 (42.9%), krt14 (86%), LPAR2 (28.6%), mal (71.4%), rgs4 (33.3%), vgf (57.1%), and ZMYM2 (57.1%). Methylation frequencies in prostate cancer cell lines were: camk4 (50%), fkbp4 (50%), hoxB5 (25%), krt14 (75%), LPAR2 (50%), mal (75%), rgs4 (50%), vgf (75%), and ZMYM2 (75%).RT-PCR analysis revealed re-expression of camk4, hoxB5, LPAR2, mal, rgs4, vgf, and ZMYM2 after 5-aza-2\u27-deoxycytidine treatment at least in one bladder cancer cell line and camk4, hoxB5, LPAR2, mal, rgs4, vgf, and ZMYM2 at least in one prostate cancer cell line. Previously unreported epigenetically altered genes in genitourinary cancer cell lines were identified. While further analysis is warranted in primary tissues, these results identify novel candidate tumor suppressor genes in bladder and prostate cancer

Multiple myeloma primary cells show a highly rearranged unbalanced genome with amplifications and homozygous deletions irrespective of the presence of immunoglobulin-related chromosome translocations

Background and Objectives Multiple myeloma (MM) is a malignant plasma cell neoplasia in which genetic studies have shown that genomic changes may affect almost all chromosomes, as shown by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). Our objective was the genomic characterization of CD 138 positive primary MM samples by means of a high resolution array CGH platform. Design and Methods For the first time, a high resolution array CGH with more than 40,000 probes, has been used to analyze 26 primary MM samples after the enrichment of CD138-positive plasma cells. Results This approach identified copy number imbalances in all cases. Bioinformatics strategies were optimized to perform data analysis allowing the segregation of hyperdiploid and non-hyperdiploid cases by array CGH. Additional analysis showed that structural chromosome rearrangements were more frequently seen in hyperdiploid cases. We also identified the same Xq21 duplication in nearly 20% of the cases, which originated through unbalanced chromosome translocations. High level amplifications and homozygous deletions were recurrently observed in our series and involved genes with meaningful function in cancer biology. Interpretation and Conclusions High resolution array CGH allowed us to identify copy number changes in 100% of the primary MM samples. We segregated different MM subgroups based on their genomic profiles which made it possible to identify homozygous deletions and amplifications of great genetic relevance in MM

Functions of Fibroblast Growth Factor Receptors in cancer defined by novel translocations and mutations

AbstractThe four receptor tyrosine kinases (RTKs) within the family of Fibroblast Growth Factor Receptors (FGFRs) are critical for normal development but also play an enormous role in oncogenesis. Mutations and/or abnormal expression often lead to constitutive dimerization and kinase activation of FGFRs, and represent the primary mechanism for aberrant signaling. Sequencing of human tumors has revealed a plethora of somatic mutations in FGFRs that are frequently identical to germline mutations in developmental syndromes, and has also identified novel FGFR fusion proteins arising from chromosomal rearrangements that contribute to malignancy. This review details approximately 200 specific point mutations in FGFRs and 40 different fusion proteins created by translocations involving FGFRs that have been identified in human cancer. This review discusses the effects of these genetic alterations on downstream signaling cascades, and the challenge of drug resistance in cancer treatment with antagonists of FGFRs

Drosophila HP1c Is Regulated by an Auto-Regulatory Feedback Loop through Its Binding Partner Woc

HP1 is a major component of chromatin and regulates gene expression through its binding to methylated histone H3. Most eukaryotes express at least three isoforms of HP1 with similar domain architecture. However, despite the common specificity for methylated histone H3, the three HP1 isoforms bind to different regions of the genome. Most of the studies so far focused on the HP1a isoform and its role in transcriptional regulation. As HP1a requires additional factors to bind methylated chromatin in vitro, we wondered whether another isoform might also require additional targeting factors. Indeed, we found that HP1c interacts with the DNA binding factors Woc and Row and requires Woc to become targeted to chromatin in vivo. Moreover, we show that the interaction between HP1c and Woc constitutes a transcriptional feedback loop that operates to balance the concentration of HP1c within the cell. This regulation may prevent HP1c from binding to methylated heterochromatin

Pumilio Proteins Regulate Translation in Embryonic Stem Cells and are Essential for Early Embryonic Development

Embryonic stem (ES) cells are defined by their dual abilities to self-renew and to differentiate into any cell type in the body. This vast potential is precisely controlled by spatial and temporal gene regulation at transcriptional, post-transcriptional, and epigenetic levels. Recent studies have revealed several transcription factors that are essential for stem cell self-renewal and pluripotency, but the role of translational control in ES cells is poorly understood. Translational control is a fundamental mechanism of gene regulation during early development, and likely explains the discrepancies between the transcriptome and proteome profiles of stem cells and their differentiated progeny. Pumilio proteins are well-characterized RNA-binding translational repressors that are required for germline stem cell maintenance in Drosophila. However, relatively little is known about the two mammalian Pumilio proteins, Pumilio 1 (Pum1) and Pumilio 2 (Pum2). In this dissertation I characterize the mRNA targets, protein partners, and in vitro and in vivo function of Pum1 and Pum2.Pum1- and Pum2-deficient mouse embryonic stem cell (mES) lines and conditional knockout mice were generated as a means to unravel the function of Pumilio proteins in ES cells and during early development. Pum1-/- and Pum2-/- ES cells grow more slowly than wild type ES cells but remain self-renewing and pluripotent. Pum1-/- and Pum2-/- mice are fertile and viable. Pum1-/- mice are smaller than their littermates, have a hunched appearance that becomes more prominent with age, frequently develop ulcerative dermatitis, and have disorganized, blunted intestinal villi compared to wild type mice. Pum1+/-; Pnm2-/- mice are viable, Pum1-/-; Pum2+/- mice are born alive but have no oral intake and die within 24 hours, and Pum1-/-; Pum2-/- double knockout animals are embryonic lethal by e8.5.Pum1 and Pum2 are highly expressed in the cytoplasm of mES cells. RNA Immunoprecipitation-Microarray (RIP-Chip) analysis of mES lysate reveals that Pum1 binds to 1947 mRNAs and Pum2 binds to 437 mRNAs that comprise almost a complete subset of Pum1 targets. Transcription factors, genes involved in cell cycle control, and genes involved in embryonic patterning are significantly enriched among the mRNA targets of both Pum1 and Pum2. Several targets including Cyclin E, Cyclin B1, and Pum2 are translationally repressed by Pum1, as indicated by changes in protein level without corresponding changes in mRNA level. In mES cells, Pum1 is part of a ∼450 kDa protein complex and Pum2 is part of a ∼350 kDa complex as shown by size exclusion chromatography. Co-immunoprecipitation and mass spectrometry were used to identify three novel binding partners of Pum1: Anaphase-promoting complex subunit 1 (APC1), Regulator of nonsense transcripts 1 (RENT1), and Zinc Finger Protein 198 (ZNF198). Overall, this study reveals an essential function of mammalian Pumilio proteins during early embryogenesis, identifies mRNA targets that are translationally controlled by Pum1 and Pum2 in mES cells, and suggests novel protein-protein interactions that lend insight into the mechanism of action of Pumilio-mediated translational repression

Acute Progression of BCR-FGFR1 Induced Murine B-Lympho/Myeloproliferative Disorder Suggests Involvement of Lineages at the Pro-B Cell Stage

Constitutive activation of FGFR1, through rearrangement with various dimerization domains, leads to atypical myeloproliferative disorders where, although T cell lymphoma are common, the BCR-FGFR1 chimeric kinase results in CML-like leukemia. As with the human disease, mouse bone marrow transduction/transplantation with BCR-FGFR1 leads to CML-like myeloproliferation as well as B-cell leukemia/lymphoma. The murine disease described in this report is virtually identical to the human disease in that both showed bi-lineage involvement of myeloid and B-cells, splenomegaly, leukocytosis and bone marrow hypercellularity. A CD19+ IgM− CD43+ immunophenotype was seen both in primary tumors and two cell lines derived from these tumors. In all primary tumors, subpopulations of these CD19+ IgM− CD43+ were also either B220+ or B220−, suggesting a block in differentiation at the pro-B cell stage. The B220− phenotype was retained in one of the cell lines while the other was B220+. When the two cell lines were transplanted into syngeneic mice, all animals developed the same B-lymphoblastic leukemia within 2-weeks. Thus, the murine model described here closely mimics the human disease with bilineage myeloid and B-cell leukemia/lymphoma which provides a representative model to investigate therapeutic intervention and a better understanding of the etiology of the disease

Stat5 as a diagnostic marker for leukemia

The Jak-Stat-Socs pathway is an important component of cytokine receptor signaling. Not surprisingly, perturbation of this pathway is implicated in diseases of hematopoietic and immune origin, including leukemia, lymphoma and immune deficiencies. This review examines the role of a key component of this pathway, Stat5. This has been shown to be activated in a variety of leukemias and myeloproliferative disorders, including downstream of a range of key oncogenes where it has been shown to play an important role in mediating their effects. Therefore, Stat5 represents a useful pan-leukemia/myeloproliferative disorder diagnostic marker and key therapeutic end point, as well as representing an attractive therapeutic target for these disorders.<br /

ZNF198 (zinc finger protein 198)

Review on ZNF198 (zinc finger protein 198), with data on DNA, on the protein encoded, and where the gene is implicated

Molecular and functional studies of ABL1 and FGFR1 fusion oncogenes in myeloproliferative neoplasms

The tyrosine kinase encoding genes ABL1 and FGFR1 are involved in fusion genes underlying the myeloproliferative neoplasms chronic myeloid leukemia (CML) and the 8p11-myeloproliferative syndrome (EMS). CML and EMS are both myeloproliferative disorders with an initiating, relatively indolent, chronic phase that after some time progresses into acute myeloid or lymphoid leukemia. The mechanisms underlying disease progression are currently unknown, but additional genetic aberrations are commonly found in the progressed phase. The general aim of this thesis was to study BCR/ABL1 and FGFR1 fusion oncogenes in a relevant cellular context, that of primary human hematopoietic cells, in order to increase our understanding of disease mechanisms underlying the development of CML and EMS. In Article I, a secondary translocation between chromosomes 9 and 21, identified in the leukemic cells from a patient in the progressed phase of EMS, was investigated. The translocation was found to result in a truncated RUNX1 gene, suggesting that haploinsufficiency for RUNX1 could be a mechanism behind disease progression in EMS. It was also found that trisomy 21 is a common secondary change in EMS. In Article II, two variants of BCR/ABL1, P190 and P210, were retrovirally expressed in cord-blood derived human CD34-positive cells. Both variants induced erythroid expansion, increased proliferation, and similar gene expression profiles, indicating that P190 and P210 BCR/ABL1 have a similar mode of action. These results indirectly support the theory that the difference in disease manifestation between P190 and P210 BCR/ABL1 depends on separate cellular origins rather than intrinsic differences of the two fusion proteins. In Article III, retroviral expression of BCR/FGFR1 or ZMYM2/FGFR1 in human CD34-positive cells resulted in increased cellular proliferation and erythroid expansion, in similar to the effects caused by BCR/ABL1. Transplantation of BCR/FGFR1- and ZMYM2/FGFR1-expressing cells into immunodeficient mice resulted in engraftment of human cells in the mouse bone marrow. The human cells differentiated into a myeloid and erythroid direction. Both fusion genes induced similar EMS-like disorders in transplanted mice, with eosinophilia, splenomegaly, and accumulation of blasts. The established in-vivo model of EMS should constitute a valuable tool for obtaining further insights into FGFR1 fusion gene mediated leukemogenesis and for the development and evaluation of new treatment strategies in EMS

The Mutation without childrenrgl Causes Ecdysteroid Deficiency in Third-Instar Larvae of Drosophila melanogaster

Larvae homozygous for the recessive lethal allele gene, located in polytene chromosomal region 97F, consists of 11 exons. A 6.8-kb transcript is expressed throughout development but is absent in the mutant gene encodes a protein of 187 kDa. Eight zinc fingers of the C2–C2 type point to a possible function as a transcription factor. The protein shows considerable homology to human proteins which have been implicated in both mental retardation and a leukemia/lymphoma syndrome