Gemma Macchia

Gene amplification as double minutes or homogeneously staining regions in solid tumors: Origin and structure

Double minutes (dmin) and homogeneously staining regions (hsr) are the cytogenetic hallmarks of genomic amplification in cancer. Different mechanisms have been proposed to explain their genesis. Recently, our group showed that the MYC-containing dmin in leukemia cases arise by excision and amplification (episome model). In the present paper we investigated 10 cell lines from solid tumors showing MYCN amplification as dmin or hsr. Particularly revealing results were provided by the two subclones of the neuroblastoma cell line STA-NB-10, one showing dmin-only and the second hsr-only amplification. Both subclones showed a deletion, at 2p24.3, whose extension matched the amplicon extension. Additionally, the amplicon structure of the dmin and hsr forms was identical. This strongly argues that the episome model, already demonstrated in leukemias, applies to solid tumors as well, and that dmin and hsr are two faces of the same coin. The organization of the duplicated segments varied from very simple (no apparent changes from the normal sequence) to very complex. MYCN was always overexpressed (significantly overexpressed in three cases). The fusion junctions, always mediated by nonhomologous end joining, occasionally juxtaposed truncated genes in the same transcriptional orientation. Fusion transcripts involving NBAS (also known as NAG), FAM49A, BC035112 (also known as NCRNA00276), and SMC6 genes were indeed detected, although their role in the context of the tumor is not clear.

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.

Gene expression and single nucleotide polymorphism array analyses of spindle cell lipomas and conventional lipomas with 13q14 deletion

Spindle cell lipomas (SCL) are circumscribed, usually s.c. tumors that typically occur on the posterior neck, shoulder, and back of middle aged men. Cytogenetically, almost all SCL are characterized by deletions of chromosome arm 13q, often in combination with loss of 16q. Deletions of 13q are seen also in approximately 15% of conventional lipomas. Through single nucleotide polymorphism (SNP) array analyses, we identified two minimal deleted regions (MDR) in 13q14 in SCL. In MDR1, four genes were located, including the tumor suppressor gene RB1. MDR1 in SCL overlapped with the MDR detected in conventional lipomas with 13q14 deletion. In MDR2 in SCL there were 34 genes and the two microRNA (miRNA) genes miR‐15a and miR‐16‐1. Global gene expression analysis was used to study the impact of the deletions on genes mapping to the two SCL‐associated MDR. Five genes (C13orf1, DHRS12, ATP7B, ALG11, and VPS36) in SCL and one gene (C13orf1) in conventional lipomas with 13q‐deletions were found to be significantly underexpressed compared with control tissues. Quantitative real‐time PCR showed that miR‐16‐1 was expressed at lower levels in SCL than in the control samples. No mutations were found at sequencing of RB1, miR‐15a, and miR‐16‐1. Our findings further delineate the target region for the 13q deletion in SCL and conventional lipomas and show that the deletions are associated with down‐regulated expression of several genes, notably C13orf1, which was the only gene to be significantly down‐regulated in both tumor types.

FOSL1 as a candidate target gene for 11q12 rearrangements in desmoplastic fibroblastoma.

Desmoplastic fibroblastoma (DF) is a benign fibroblastic/myofibroblastic tumor. Cytogenetic analyses have revealed consistent rearrangement of chromosome band 11q12, strongly suggesting that this region harbors a gene of pathogenetic importance. To identify the target gene of the 11q12 rearrangements, we analyzed six cases diagnosed as DF using chromosome banding, fluorescence in situ hybridization (FISH), single-nucleotide polymorphism array and gene expression approaches. Different structural rearrangements involving 11q12 were found in five of the six cases. Metaphase FISH analyses in two of them mapped the 11q12 breakpoints to an ∼20-kb region, harboring FOSL1. Global gene expression profiling followed by quantitative real-time PCR showed that FOSL1 was expressed at higher levels in DF with 11q12 rearrangements than in desmoid-type fibromatoses. Furthermore, FOSL1 was not upregulated in the single case of DF that did not show cytogenetic involvement of 11q12; instead this tumor was found to display a hemizygous loss on 5q, including the APC (adenomatous polyposis coli) locus, raising the possibility that it actually was a misdiagnosed Gardner fibroma. 5′RACE-PCR in two 11q12-positive DF did not identify any fusion transcripts. Thus, in agreement with the finding at chromosome banding analysis that varying translocation partners are involved in the 11q12 rearrangement, the molecular data suggest that the functional outcome of the 11q12 rearrangements is deregulated expression of FOSL1.

Integrative Genome and Transcriptome Analyses Reveal Two Distinct Types of Ring Chromosome in Soft Tissue Sarcomas.

Gene amplification is a common phenomenon in malignant neoplasms of all types. One mechanism behind increased gene copy number is the formation of ring chromosomes. Such structures are mitotically unstable and during tumor progression they accumulate material from many different parts of the genome. Hence, their content varies considerably between and within tumors. Partly due to this extensive variation, the genetic content of many ring-containing tumors remains poorly characterized. Ring chromosomes are particularly prevalent in specific subtypes of sarcoma. Here, we have combined fluorescence in situ hybridization (FISH), global genomic copy number and gene expression data on ring-containing soft tissue sarcomas and show that they harbor two fundamentally different types of ring chromosome: MDM2-positive and MDM2-negative rings. While the former are often found in an otherwise normal chromosome complement, the latter seem to arise in the context of general chromosomal instability. In line with this, sarcomas with MDM2-negative rings commonly show complete loss of either CDKN2A or RB1 -both known to be important for genome integrity. Sarcomas with MDM2-positive rings instead show co-amplification of a variety of potential driver oncogenes. More than 100 different genes were found to be involved, many of which are known to induce cell growth, promote proliferation or inhibit apoptosis. Several of the amplified and overexpressed genes constitute potential drug targets.

Molecular genetic characterization of the 11q13 breakpoint in a desmoplastic fibroma of bone

Desmoplastic fibroma (DFB) is a benign primary bone tumor that usually occurs in adolescents and young adults. The genetic information on DFB is very limited. We here present cytogenetic, fluorescence in situ hybridization and single nucleotide polymorphism array findings in a case that had a rearrangement involving chromosomes 11 and 19 at G-banding analysis. The results showed that the breakpoint in 11q was different from that in desmoplastic fibroblastomas, and a segment containing five genes was hemizygously deleted from 11q13.

Analysis of clock gene-miRNA correlation networks reveals candidate drivers in colorectal cancer

Altered functioning of the biological clock is involved in cancer onset and progression. MicroRNAs (miRNAs) interact with the clock genes modulating the function of genetically encoded molecular clockworks. Collaborative interactions may take place within the coding-noncoding RNA regulatory networks. We aimed to evaluate the cross-talk among miRNAs and clock genes in colorectal cancer (CRC). We performed an integrative analysis of miRNA-miRNA and miRNA-mRNA interactions on high-throughput molecular profiling of matched human CRC tissue and non-tumor mucosa, pinpointing core clock genes and their targeting miRNAs. Data obtained in silico were validated in CRC patients and human colon cancer cell lines. In silico we found severe alterations of clock gene–related coding-noncoding RNA regulatory networks in tumor tissues, which were later corroborated by the analysis of human CRC specimens and experiments performed in vitro. In conclusion, specific miRNAs target and regulate the transcription/translation of clock genes and clock gene-related miRNA-miRNA as well as mRNA-miRNA interactions are altered in colorectal cancer. Exploration of the interplay between specific miRNAs and genes, which are critically involved in the functioning of the biological clock, provides a better understanding of the importance of the miRNA-clock genes axis and its derangement in colorectal cancer.

Deregulated expression of cryptochrome genes in human colorectal cancer

BackgroundCircadian disruption and deranged molecular clockworks are involved in carcinogenesis. The cryptochrome genes (CRY1 and CRY2) encode circadian proteins important for the functioning of biological oscillators. Their expression in human colorectal cancer (CRC) and in colon cancer cell lines has not been evaluated so far.MethodsWe investigated CRY1 and CRY2 expression in fifty CRCs and in the CaCo2, HCT116, HT29, SW480 cell lines.ResultsCRY1 (p = 0.01) and CRY2 (p < 0.0001) expression was significantly changed in tumour tissue, as confirmed in a large independent CRC dataset. In addition, lower CRY1 mRNA levels were observed in patients in the age range of 62-74 years (p = 0.018), in female patients (p = 0.003) and in cancers located at the transverse colon (p = 0.008). Lower CRY2 levels were also associated with cancer location at the transverse colon (p = 0.007). CRC patients displaying CRY1 (p = 0.042) and CRY2 (p = 0.043) expression levels over the median were hallmarked by a poorer survival rate. Survey of selected colon cancer cell lines evidenced variable levels of cryptochrome genes expression and time-dependent changes in their mRNA levels. Moreover, they showed reduced apoptosis, increased proliferation and different response to 5-fluorouracil and oxaliplatin upon CRY1 and CRY2 ectopic expression. The relationship with p53 status came out as an additional layer of regulation: higher CRY1 and CRY2 protein levels coincided with a wild type p53 as in HCT116 cells and this condition only marginally affected the apoptotic and cell proliferation characteristics of the cells upon CRY ectopic expression. Conversely, lower CRY and CRY2 levels as in HT29 and SW480 cells coincided with a mutated p53 and a more robust apoptosis and proliferation upon CRY transfection. Besides, an heterogeneous pattern of ARNTL, WEE and c-MYC expression hallmarked the chosen colon cancer cell lines and likely influenced their phenotypic changes.ConclusionCryptochrome gene expression is altered in CRC, particularly in elderly subjects, female patients and cancers located at the transverse colon, affecting overall survival. Altered CRY1 and CRY2 expression patterns and the interplay with the genetic landscape in colon cancer cells may underlie phenotypic divergence that could influence disease behavior as well as CRC patients survival and response to chemotherapy.

Ring chromosomes, breakpoint clusters, and neocentromeres in sarcomas.

Gene amplification is relatively common in tumors. In certain subtypes of sarcoma, it often occurs in the form of ring and/or giant rod‐shaped marker (RGM) chromosomes whose mitotic stability is frequently rescued by ectopic novel centromeres (neocentromeres). Little is known about the origin and structure of these RGM chromosomes, including how they arise, their internal organization, and which sequences underlie the neocentromeres. To address these questions, 42 sarcomas with RGM chromosomes were investigated to detect regions prone to double strand breaks and possible functional or structural constraints driving the amplification process. We found nine breakpoint cluster regions potentially involved in the genesis of RGM chromosomes, which turned out to be significantly enriched in poly‐pyrimidine traits. Some of the clusters were located close to genes already known to be relevant for sarcomas, thus indicating a potential functional constraint, while others mapped to transcriptionally inactive chromatin domains enriched in heterochromatic sites. Of note, five neocentromeres were identified after analyzing 13 of the cases by fluorescent in situ hybridization. ChIP‐on‐chip analysis with antibodies against the centromeric protein CENP‐A showed that they were a patchwork of small genomic segments derived from different chromosomes, likely joint to form a contiguous sequence during the amplification process.

The Hidden Genomic and Transcriptomic Plasticity of Giant Marker Chromosomes in Cancer

Neocentromeres contribute to cancer progression by mitotically stabilizing acentric chromosomes containing amplified oncogenes. Macchia et al. show that... Genome amplification in the form of rings or giant rod-shaped marker chromosomes (RGMs) is a common genetic alteration in soft tissue tumors. The mitotic stability of these structures is often rescued by perfectly functioning analphoid neocentromeres, which therefore significantly contribute to cancer progression. Here, we disentangled the genomic architecture of many neocentromeres stabilizing marker chromosomes in well-differentiated liposarcoma and lung sarcomatoid carcinoma samples. In cells carrying heavily rearranged RGMs, these structures were assembled as patchworks of multiple short amplified sequences, disclosing an extremely high level of complexity and definitely ruling out the existence of regions prone to neocentromere seeding. Moreover, by studying two well-differentiated liposarcoma samples derived from the onset and the recurrence of the same tumor, we documented an expansion of the neocentromeric domain that occurred during tumor progression, which reflects a strong selective pressure acting toward the improvement of the neocentromeric functionality in cancer. In lung sarcomatoid carcinoma cells we documented, extensive “centromere sliding” phenomena giving rise to multiple, closely mapping neocentromeric epialleles on separate coexisting markers occur, likely due to the instability of neocentromeres arising in cancer cells. Finally, by investigating the transcriptional activity of neocentromeres, we came across a burst of chimeric transcripts, both by extremely complex genomic rearrangements, and cis/trans-splicing events. Post-transcriptional editing events have been reported to expand and variegate the genetic repertoire of higher eukaryotes, so they might have a determining role in cancer. The increased incidence of fusion transcripts, might act as a driving force for the genomic amplification process, together with the increased transcription of oncogenes.