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Common fragile sites (cFSs) represent parts of the normal chromosome structure susceptible to breakage under replication stress. Although only a small number of cFSs have been molecularly characterized, genomic damage of cFS genes appears to be critical for the development of various human diseases. In this study, we fine mapped the location of FRA14B and showed that the fragile region spans 765 kb at 14q23.3, containing the large gephyrin (GPHN) gene. The FRA14B sequence is enriched in perfect A/T >24 stretches and R-loop forming sequences (RLFS), and harbors a large palindromic motif in the core region. FRA14B instability is not only limited to lymphocytes, but also occurs in neuroblastoma and breast epithelial cells. Using array comparative genomic hybridization (CGH), we examined copy number alteration patterns within FRA14B in a panel of 180 cancer cell lines and primary tumors. Our CGH data and a survey of 1046 Cancer Cell Line Encyclopedia profiles demonstrate that focal deletions cluster within FRA14B and disrupt the genomic integrity of GPHN in approximately 5% of cancer cells. Moreover, germline CNVs (copy number variants) profiles provided by the Database of Genomic Variants and available literature suggest that germline CNVs and rare pathogenic deletions associated with neurodevelopmental disorders cluster within the core fragile region of GPHN. Overall, our data provide insight into the molecular structure of FRA14B, and identify GPHN, as a large cFS gene in the human genome, whose disruption appears to trigger various neurodevelopmental diseases., (© 2018 Wiley Periodicals, Inc.)

The cytogenetic hypothesis that common fragile sites (cFSs) are hotspots of cancer breakpoints is increasingly supported by recent data from whole-genome profiles of different cancers. cFSs are components of the normal chromosome structure that are particularly prone to breakage under conditions of replication stress. In recent years, cFSs have become of increasing interest in cancer research, as they not only appear to be frequent targets of genomic alterations in progressive tumors, but also already in precancerous lesions. Despite growing evidence of their importance in disease development, most cFSs have not been investigated at the molecular level and most cFS genes have not been identified. In this review, we summarize the current data on molecularly characterized cFSs, their genetic and epigenetic characteristics, and put emphasis on less-studied cFS genes as potential contributors to cancer development.

Specific patterns of genomic aberrations have been associated with different types of malignancies. In colorectal cancer, losses of chromosome arm 8p and gains of chromosome arm 8q are among the most common chromosomal rearrangements, suggesting that the centromeric portion of chromosome 8 is particularly sensitive to breakage. Genomic alterations frequently occur in the early stages of tumorigenesis at specific genomic regions known as common fragile sites (cFSs). CFSs represent parts of the normal chromosome structure that are prone to breakage under replication stress. In this study, we identified the genomic location of FRA8I, spanning 530 kb at 8q11.21 and assessed the composition of the fragile DNA sequence. FRA8I encompasses KIAA0146, a large protein-coding gene with yet unknown function, as well as CEBPD and part of PRKDC, two genes encoding proteins involved in tumorigenesis in a variety of cancers. We show that FRA8I is unstable in lymphocytes and epithelial cells, displaying similar expression rates. We examined copy number alteration patterns within FRA8I in a panel of 25 colorectal cancer cell lines and surveyed publically available profiles of 56 additional colorectal cancer cell lines. Combining these data shows that focal recombination events disrupt the genomic integrity of KIAA0146 and neighboring cFS genes in 12.3% of colorectal cancer cell lines. Moreover, data analysis revealed evidence that KIAA0146 is a translocation partner of the immunoglobulin heavy chain gene in recurrent t(8;14)(q11;q32) translocations in a subset of patients with B-cell precursor acute lymphoblastic leukemia. Our data molecularly describe a region of enhanced chromosomal instability in the human genome and point to a role of the KIAA0146 gene in tumorigenesis., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The TP53 tumor suppressor pathway is abrogated by TP53 mutations in the majority of human cancers. Increased levels of wild-type TP53 in aggressive neuroblastomas appear paradox but are tolerated by tumor cells due to co-activation of the TP53 ubiquitin ligase, MDM2. The role of the MDM2 antagonist, p14(ARF), in controlling the TP53-MDM2 balance in neuroblastoma is unresolved. In the present study, we show that conditional p14(ARF) expression substantially suppresses viability, clonogenicity and anchorage-independent growth in p14(ARF)-deficient or MYCN-amplified neuroblastoma cell lines. Furthermore, ectopic 14(ARF) expression induced accumulation of cells in the G1 phase and apoptosis, which was paralleled by accumulation of TP53 and its targets. Comparative genomic hybridization analysis of 193 primary neuroblastomas detected one homozygous deletion of CDKN2A (encoding both p14(ARF) and p16(INK4A)) and heterozygous loss of CDKN2A in 22% of tumors. Co-expression analysis of p14(ARF) and its transactivator, E2F1, in a set of 68 primary tumors revealed only a weak correlation, suggesting that further regulatory mechanisms govern p14(ARF) expression in neuroblastomas. Intriguingly, analyses utilizing chromatin immunoprecipitation revealed different histone mark-defined epigenetic activity states of p14(ARF) in neuroblastoma cell lines that correlated with endogenous p14(ARF) expression but not with episomal p14(ARF) promoter reporter activity, indicating that the native chromatin context serves to epigenetically repress p14(ARF) in neuroblastoma cells. Collectively, the data pinpoint p14(ARF) as a critical factor for efficient TP53 response in neuroblastoma cells and assign p14(ARF) as a neuroblastoma suppressor candidate that is impaired by genomic loss and epigenetic repression.

Relapse with drug-resistant disease is the main cause of death in MYCN-amplified neuroblastoma patients. MYCN-amplified neuroblastoma cells in vitro are characterized by a failure to arrest at the G(1)-S checkpoint after irradiation- or drug-induced DNA damage. We show that several MYCN-amplified cell lines harbor additional chromosomal aberrations targeting p53 and/or pRB pathway components, including CDK4/CCND1/MDM2 amplifications, p16INK4A/p14ARF deletions or TP53 mutations. Cells with these additional aberrations undergo significantly lower levels of cell death after doxorubicin treatment compared with MYCN-amplified cells, with no additional mutations in these pathways. In MYCN-amplified cells CDK4 expression is elevated, increasing the competition between CDK4 and CDK2 for binding p21. This results in insufficient p21 to inhibit CDK2, leading to high CDK4 and CDK2 kinase activity upon doxorubicin treatment. CDK4 inhibition by siRNAs, selective small compounds or p19(INK4D) overexpression partly restored G(1)-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment. Our results suggest a specific function of p19(INK4D), but not p16(INK4A), in sensitizing MYCN-amplified cells with a functional p53 pathway to doxorubicin-induced cell death. In summary, the CDK4/cyclin D-pRB axis is altered in MYCN-amplified cells to evade a G(1)-S arrest after doxorubicin-induced DNA damage. Additional chromosomal aberrations affecting the p53-p21 and CDK4-pRB axes compound the effects of MYCN on the G(1) checkpoint and reduce sensitivity to cell death after doxorubicin treatment. CDK4 inhibition partly restores G(1)-S arrest and sensitizes cells to doxorubicin-mediated cell death in MYCN-amplified cells with an intact p53 pathway.

Medulloblastomas are the most common malignant brain tumors in childhood. Emerging evidence suggests that medulloblastoma comprises at least four distinct diseases (WNT, SHH, Group 3 and 4) with different biology, clinical presentation, and outcome, with especially poor prognosis in Group 3. The tight connection of biology and clinical behavior in patients emphasizes the need for subgroup-specific preclinical models in order to develop treatments tailored to each subgroup. Herein we report on the novel cell line HD-MB03, isolated from tumor material of a patient with metastasized Group 3 medulloblastoma, and preclinical testing of different histone deacetylase inhibitors (HDACis) in this model. HD-MB03 cells grow long term in vitro and form metastatic tumors in vivo upon orthotopic transplantation. HD-MB03 cells reflect the original Group 3 medulloblastoma at the histological and molecular level, showing large cell morphology, similar expression patterns for markers Ki67, p53, and glial fibrillary acidic protein (GFAP), a gene expression profile most closely matching Group 3 medulloblastomas, and persistence of typical molecular alterations, i.e., isochromosome 17q [i(17q)] and MYC amplification. Protein expression analysis of HDACs 2, 5, 8, and 9 as well as the predictive marker HR23B showed intermediate to strong expression, suggesting sensitivity to HDACis. Indeed, treatment with HDACis Helminthosporium carbonum (HC)-toxin, vorinostat, and panobinostat revealed high sensitivity to this novel drug class, as well as a radiation-sensitizing effect with significantly increased cell death upon concomitant treatment. In summary, our data indicate that HD-MB03 is a suitable preclinical model for Group 3 medulloblastoma, and HDACis could represent a therapeutic option for this subgroup.

Common fragile sites (cFSs) are non-random chromosomal regions that are prone to breakage under conditions of replication stress. DNA damage and chromosomal alterations at cFSs appear to be critical events in the development of various human diseases, especially carcinogenesis. Despite the growing interest in understanding the nature of cFS instability, only a few cFSs have been molecularly characterised. In this study, we fine-mapped the location of FRA2H using six-colour fluorescence in situ hybridisation and showed that it is one of the most active cFSs in the human genome. FRA2H encompasses approximately 530 kb of a gene-poor region containing a novel large intergenic non-coding RNA gene (AC097500.2). Using custom-designed array comparative genomic hybridisation, we detected gross and submicroscopic chromosomal rearrangements involving FRA2H in a panel of 54 neuroblastoma, colon and breast cancer cell lines. The genomic alterations frequently involved different classes of long terminal repeats and long interspersed nuclear elements. An analysis of breakpoint junction sequence motifs predominantly revealed signatures of microhomology-mediated non-homologous recombination events. Our data provide insight into the molecular structure of cFSs and sequence motifs affected by their activation in cancer. Identifying cFS sequences will accelerate the search for DNA biomarkers and targets for individualised therapies.

Common fragile sites (cFS) represent chromosomal regions that are prone to breakage after partial inhibition of DNA synthesis. Activation of cFS is associated with various forms of DNA instability in cancer cells, and is thought to be an initiating event in the generation of DNA damage in early-stage tumorigenesis. Only a few cFS have been fully characterized despite the growing interest in cFS instability in cancer genomes. In this study, six-color fluorescence in situ hybridization revealed that FRA2C consists of two cFS spanning 747 kb FRA2Ctel and 746 kb FRA2Ccen at 2p24.3 and 2p24.2, respectively. Both cFS are separated by a 2.8 Mb non-fragile region containing MYCN. Fine-tiling array comparative genomic hybridization of MYCN amplicons from neuroblastoma (NB) cell lines and primary tumors revealed that 56.5% of the amplicons cluster in FRA2C. MYCN amplicons are either organized as double minutes or as homogeneously stained regions in addition to the single copy of MYCN retained at 2p24. We suggest that MYCN amplicons arise from extra replication rounds of unbroken DNA secondary structures that accumulate at FRA2C. This hypothesis implicates cFS in high-level gene amplification in cancer cells. Complex genomic rearrangements, including deletions, duplications and translocations, which originate from double-strand breaks, were detected at FRA2C in different cancers. These data propose a dual role for cFS in the generation of gross chromosomal rearrangements either after DNA breakage or by inducing extra replication rounds, and provide new insights into the highly recombinogenic nature of cFS in the human cancer genome.