Unravelling similarities and differences in the role of circular and linear PVT1 in cancer and human disease

The plasmacytoma variant translocation 1 (PVT1) is a long non-coding RNA gene involved in human disease, mainly in cancer onset/ progression. Although widely analysed, its biological roles need to be further clarified. Notably, functional studies on PVT1 are complicated by the occurrence of multiple transcript variants, linear and circular, which generate technical issues in the experimental procedures used to evaluate its impact on human disease. Among the many PVT1 transcripts, the linear PVT1 (lncPVT1) and the circular hsa_circ_0001821 (circPVT1) are frequently reported to perform similar pathologic and pro-tumorigenic functions when overexpressed. The stimulation of cell proliferation, invasion and drug resistance, cell metabolism regulation, and apoptosis inhibition is controlled through multiple targets, including MYC, p21, STAT3, vimentin, cadherins, the PI3K/AKT, HK2, BCL2, and CASP3. However, some of this evidence may originate from an incorrect evaluation of these transcripts as two separate molecules, as they share the lncPVT1 exon-2 sequence. We here summarise lncPVT1/circPVT1 functions by mainly focusing on shared pathways, pointing out the potential bias that may exist when the biological role of each transcript is analysed. These considerations may improve the knowledge about lncPVT1/circPVT1 and their specific targets, which deserve further studies due to their diagnostic, prognostic, and therapeutic potential

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 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

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

HMGA2 and MDM2 expression in lipomatous tumors with partial, low-level amplification of sequences from the long arm of chromosome 12.

Ordinary lipomas are cytogenetically characterized primarily by simple balanced chromosome aberrations with stable morphologies, most of which affect chromosome segment, 12q13-15, where the HMGA2 gene plays a key pathogenetic role. Atypical lipomatous tumors (ALTs) display supernumerary ring or giant marker chromosomes with amplification of several genes including HMGA2 and MDM2. A study of HMGA2 expression in a variety of adipocytic tumors showed aberrant expression in lipomas with 12q13-15 aberrations and ring chromosomes as well as in ALTs and well-differentiated liposarcomas (WDLSs), and frequent differential expression of HMGA2 exons 1-2 versus that of exons 4-5. A minor subset of adipocytic tumors harbors unbalanced karyotypes with extra copies of 12q sequences in structures that are not giant marker or ring chromosomes. Out of a series of ten such tumors, three lipomas and four ALTs with more than two copies of 12q13-15 and breakpoints in 12q13-15 could be analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) to find out whether HMGA2 and MDM2 expression was more similar to the levels seen in lipomas with cytogenetically balanced aberrations of 12q13-15, or to ALTs with giant ring or marker chromosomes. One of two ALTs with more complex, hyperdiploid karyotypes had expression levels closer to those seen in ALT, whereas the remaining six cases were similar to lipomas with 12q13-15 changes and ring chromosomes. Differential expression was seen in two ALTs and all three lipomas. Two cases showed MDM2 expression levels similar to those found among WDLSs, two cases showed levels similar to those found among lipomas, whereas the remaining three cases displayed intermediate expression levels. The studied cases represent intermediates between lipoma and ALT, insofar as they shared 12q13-15 rearrangements and karyotypic stability with lipomas and gain of 12q sequences with ALTs. Neither of these characteristics can be used to discriminate between lipoma and ALT

Grappa quality from the Chianti and Montepulciano areas (Tuscany, Italy): Monitoring the leaching of copper from distillation columns

The main aim of this work was to evaluate the leaching of copper in grappa from distillation plant that treats Cabernet and Sangiovese marcs (Montepulciano and Chianti, Tuscany). Copper content (determined by atomic absorption spectrometry, AAS) was 1.05 ± 0.03 mg L-1in industrial distillates (Cabernet marc, 82.3% alcohol, v/v), and 3.7 ± 0.2 μg L-1in artificial grappa (62.0%, Cabernet) obtained by distilling marc in a laboratory-scale glassware plant. The artificial grappa distillate was refluxed over copper shavings, and copper content, CCu, was analysed in relation to reflux time and wear of the copper bell. The results showed that CCutrend was almost linear with increasing alcohol concentration and constant reflux time, and vice versa. The ratios of selected volatile components were not influenced by reflux on the shavings

Rearrangements of chromosome bands 15q12-q21 are secondary to HMGA2 deregulation in conventional lipoma.

Rearrangements of chromosome arm 15q are rare but recurrent in conventional lipomas, a tumor type often showing deregulated expression of the HMGA2 gene. In order to assess whether 15q rearrangements could constitute a distinct pathogenetic mechanism, we studied seven cases of conventional lipoma that at G-banding analysis had various rearrangements of 15q12-q21. The breakpoints in 15q were mapped by fluorescence in situ hybridization (FISH) and single nucleotide polymorphism array analyses, and the status of the HMGA2 gene was evaluated by FISH and/or quantitative PCR. We found an overlapping deletion on 15q in two cases, but no recurring breakpoint among the other cases. In addition, all cases displayed rearrangement of HMGA2 at the genomic or the transcriptional level. Although 15q rearrangements sometimes are noted as the sole aberration at cytogenetic analysis of conventional lipomas, they are secondary to HMGA2 deregulation

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. © 2011 Wiley-Liss, Inc

The transcriptome plasticity of genomic amplification in cancer

Genomic amplification, in the form of homogeneously staining regions, double minutes, and ring/giant rod-shaped markers, is a pivotal event in many tumors. It was recently shown that amplifications as extra-chromosomal DNA are present in nearly half of all tumors, representing a driving force towards their accelerated evolution. To achieve a better understanding of the implications of genomic amplifications we focused on their structure and impact upon transcription. Amplified cancer-associated genes are often overexpressed as a direct consequence of the copy number gain. We analyzed the whole genome (WGS) and transcriptome (RNA-seq) sequencing data of nine small lung carcinoma (SCLC), seven neuroblastoma (NB) and three well- differentiated liposarcoma (WDLPS) cell lines, all carrying genomic amplifications. A widespread heterogeneity was detected in the amplicon arrangement of many cell lines, disclosing the progressive evolution of their structure through cell division. By integrating the WGS (structural variation calling) and RNA-seq (chimeras detection) data we detected a burst of chimeric transcripts partially derived from post- transcriptional events (i.e cis- or trans-splicing) in most of the analyzed cell lines. Notably, we found PVT1 and RLF as hotspots for cis- or trans-splicing events in SCLC and NB cell lines with MYC and MYCL1 amplifications, respectively. In WDLPS cell lines we found fusion genes originated by extremely complex genomic rearrangements, such as those involving three partner genes or assembled by multiple interposed non-contiguous, non-collinear genomic fragments (spliced out in the mature transcript). Our results strongly indicate that the “amplification/overexpression” paradigm does not cover all aspects of the genomic amplification impact upon transcription. The extraordinary transcriptome plasticity herein described, enriching the genetic repertoire of cancer cells with genomic amplifications, likely provides a selective advantage and might have a crucial role in cancer establishment and progression