Koichiro Inaki

Comprehensive long-span paired-end-tag mapping reveals characteristic patterns of structural variations in epithelial cancer genomes

Somatic genome rearrangements are thought to play important roles in cancer development. We optimized a long-span paired-end-tag (PET) sequencing approach using 10-Kb genomic DNA inserts to study human genome structural variations (SVs). The use of a 10-Kb insert size allows the identification of breakpoints within repetitive or homology-containing regions of a few kilobases in size and results in a higher physical coverage compared with small insert libraries with the same sequencing effort. We have applied this approach to comprehensively characterize the SVs of 15 cancer and two noncancer genomes and used a filtering approach to strongly enrich for somatic SVs in the cancer genomes. Our analyses revealed that most inversions, deletions, and insertions are germ-line SVs, whereas tandem duplications, unpaired inversions, interchromosomal translocations, and complex rearrangements are over-represented among somatic rearrangements in cancer genomes. We demonstrate that the quantitative and connective nature of DNA-PET data is precise in delineating the genealogy of complex rearrangement events, we observe signatures that are compatible with breakage-fusion-bridge cycles, and we discover that large duplications are among the initial rearrangements that trigger genome instability for extensive amplification in epithelial cancers.

Transcriptional consequences of genomic structural aberrations in breast cancer

Using a long-span, paired-end deep sequencing strategy, we have comprehensively identified cancer genome rearrangements in eight breast cancer genomes. Herein, we show that 40%-54% of these structural genomic rearrangements result in different forms of fusion transcripts and that 44% are potentially translated. We find that single segmental tandem duplication spanning several genes is a major source of the fusion gene transcripts in both cell lines and primary tumors involving adjacent genes placed in the reverse-order position by the duplication event. Certain other structural mutations, however, tend to attenuate gene expression. From these candidate gene fusions, we have found a fusion transcript (RPS6KB1-VMP1) recurrently expressed in ∼30% of breast cancers associated with potential clinical consequences. This gene fusion is caused by tandem duplication on 17q23 and appears to be an indicator of local genomic instability altering the expression of oncogenic components such as MIR21 and RPS6KB1.

Molecular‐feature domains with posterodorsal–anteroventral polarity in the symmetrical sensory maps of the mouse olfactory bulb: mapping of odourant‐induced Zif268 expression

Individual glomeruli in the mammalian olfactory bulb presumably represent a single type of odourant receptor. Thus, the glomerular sheet provides odourant receptor maps at the surface of the olfactory bulb. To understand the basic spatial organization of the olfactory sensory maps, we first compared the spatial distribution of odourant‐induced responses measured by the optical imaging of intrinsic signals with that detected immunohistochemically by expressions of Zif268, one of the immediate early gene products in juxtaglomerular cells. In the dorsal surface of the bulb, we detected a clear correlation in the spatial pattern between these responses. In addition, the molecular‐feature domains and their polarities (spatial shifts of responses with an increase in carbon chain length) that were defined by the optical imaging method could be also detected by the Zif268 mapping method. We then mapped the Zif268 signals over the entire olfactory bulb using a homologous series of fatty acids and aliphatic alcohols as stimulus odourants. We superimposed the Zif268 signals onto the standard unrolled map with the help of cell adhesion molecule compartments. Each odourant typically elicited two pairs of clusters of dense Zif268 signals. The results showed that molecular‐feature domains and their polarities were arranged symmetrically at stereotypical positions in a mirror‐image fashion between the lateral and the medial sensory maps. The polarity of each domain was roughly in parallel with the posterodorsal–anteroventral axis that was defined by the cell adhesion molecule compartments. These results suggest that the molecular‐feature domain with its fixed polarity is one of the basic structural units in the spatial organization of the odourant receptor maps in the olfactory bulb.

Structural mutations in cancer: mechanistic and functional insights

Next-generation sequencing (NGS) has enabled the comprehensive and precise identification of many somatic structural mutations in cancer. Analyses integrating point mutation information with data on rearrangements and copy number variation have revealed a higher-order organization of the seemingly random genetic events that lead to cancer. These meta-analyses provide a more refined view of the mutational mechanisms, genomic evolution, and combinations of mutations that contribute to tumorigenesis. Structural mutations, or genome-scale rearrangements of segments of DNA, may play a hitherto unappreciated role in cancer through their ability to move blocks of adjacent genes simultaneously, leading to concurrent oncogenic events. Moreover, whole-genome sequencing (WGS) data from tumors have revealed global rearrangements, such as those seen in the tandem duplicator phenotype and in chromothripsis, suggesting that massive rearrangements are a specific cancer phenotype. Taken together, the emerging data suggest that the chromosome structure itself functions as a systems oncogenic organizer.

The tandem duplicator phenotype as a distinct genomic configuration in cancer

Significance In this study, we provide the first detailed molecular characterization, to our knowledge, of a distinct cancer genomic configuration, the tandem duplicator phenotype (TDP), that is significantly enriched in the molecularly related triple-negative breast, serous ovarian, and endometrial carcinomas. We show here that TDP represents an oncogenic configuration featuring (i) genome-wide disruption of cancer genes, (ii) loss of cell cycle control and DNA damage repair, and (iii) increased sensitivity to cisplatin chemotherapy both in vitro and in vivo. Therefore, the TDP is a systems strategy to achieve a protumorigenic genomic configuration by altering a large number of oncogenes and tumor suppressors. The TDP arises in a molecular context of joint genomic instability and replicative drive, and is consequently associated with enhanced sensitivity to cisplatin. Next-generation sequencing studies have revealed genome-wide structural variation patterns in cancer, such as chromothripsis and chromoplexy, that do not engage a single discernable driver mutation, and whose clinical relevance is unclear. We devised a robust genomic metric able to identify cancers with a chromotype called tandem duplicator phenotype (TDP) characterized by frequent and distributed tandem duplications (TDs). Enriched only in triple-negative breast cancer (TNBC) and in ovarian, endometrial, and liver cancers, TDP tumors conjointly exhibit tumor protein p53 (TP53) mutations, disruption of breast cancer 1 (BRCA1), and increased expression of DNA replication genes pointing at rereplication in a defective checkpoint environment as a plausible causal mechanism. The resultant TDs in TDP augment global oncogene expression and disrupt tumor suppressor genes. Importantly, the TDP strongly correlates with cisplatin sensitivity in both TNBC cell lines and primary patient-derived xenografts. We conclude that the TDP is a common cancer chromotype that coordinately alters oncogene/tumor suppressor expression with potential as a marker for chemotherapeutic response.

Systems consequences of amplicon formation in human breast cancer

Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer.

Abstract 3414: Genomic signatures of melanoma maintenance

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Melanoma is the malignant tumor of melanocytes and considered as one of the most aggressive human cancers. While the worldwide incidence rate of melanoma has increased during the last decade, current therapies for melanoma do not provide long-term effect. The highly heterogeneous expression signature of melanoma underlies the inefficient therapy and accentuates the inevitability of profound molecular understanding of melanoma, its maintenance and progression. In this study, we aimed to reveal the complex patterns of distinct molecular mechanisms underlying the progression and maintenance of melanoma using high-throughput sequencing technologies. Series of melanoma tumor tissues representing discrete stages of the malignant progression were deeply sequenced. Integrative analysis revealed hard-wired genomic aberrations and associated transcriptomic consequences. These genomic aberrations included clusters of oncogenes brought together by structural variations, which cooperated in their amplifications and subsequent up-regulated expressions. We believe that these hard-wired genetic changes are biologically significant; they sustain the survival of the tumor and offer the advantage of the distant metastasis. We reconstituted the architecture of melanoma progression and maintenance and we identified gene cassettes with a putative role in sustaining tumor growth. The correlation of these gene cassettes with the cancer phenotype and the feasibility of these putative oncogenes as candidates for targeted therapy were addressed using cell-based assays on a high content screening platform. Analysis of the matched cell lines derived from the same patient offered a valuable resource for in vitro manipulation, testing of drugs and functional validation. A detailed molecular characterization of the functionally validated genes and their contribution in the cancer signaling pathways will provide a better understanding of the molecular basis of tumor evolution. Citation Format: Faranak Ghazi Sherbaf, Koichiro Inaki, Denis Bertrand, Xing Yi Woo, Dave Hoon, Axel Hillmer, Edison Liu. Genomic signatures of melanoma maintenance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3414. doi:10.1158/1538-7445.AM2014-3414

Abstract 3180: In vitro tool for discovering oncogenes and tumor suppressor genes in a system's manner.

The molecular complexity of cancer still remains as the limiting factor for developing efficient therapies with minimal side effects. However, scientists succeeded in determining common alterations in most human cancer types. Each of these alterations results from disruption of an anti-cancer regulation mechanism and/or activation of cancer-promoting networks by genetic and epigenetic changes. As a consequence, cancer cells become capable of mimicking stem cells’ ability to proliferate indefinitely while maintaining their cellular identity, or to self-renew, which is crucial for any type of stem cell. Indeed, some of the networks which are deregulated in cancer normally regulate self-renewal in stem cells. However, self-renewal is tightly regulated in stem cells, while cancer cells manage to escape the internal and external regulation mechanisms. Hence, cancer cells differ from stem cells by displaying disruptions in feedback mechanism controlling the rate of cell division, aberrant differentiation programs and error-prone replication, so they become invasive and even metastatic. We hypothesize that the stemness regulatory “cassette” that serves limitless replicative potential and undifferentiated state is a measure of cancer virulence and progression. We believe that this gene cassette is established through different mechanisms in each cell type, so it can be separated from invasion and instability in a precise and defined manner. To test our hypotheses, we set up a system reconstruction model consisting of induced pluripotent cells and transformed cells as stem and cancer cell models respectively, and their source of primary mouse embryonic fibroblast cells. From this model system, we created a system9s map for transformation using differential expression patterns from whole genome expression arrays. We discovered gene networks that are common between stem and cancer cells, but different from their primary cells of origin. We also found deregulated/activated cassettes which are unique for cancer cells and which are hardwired by genomic rearrangements (copy number and structural variations) identified by DNA paired end tag (DNA-PET) sequencing and single nucleotide variations using RNA sequencing. We are now investigating the role of candidate genes selected from this analysis in cancer and stem cell maintenance through a high content siRNA screening. Citation Format: Gaye Saginc, Leena Ukil, Xingyi Woo, Francesca Menghi, Denis Bertrand, Charlie Wah Heng Lee, Koichiro Inaki, Edison Tak Bun Liu. In vitro tool for discovering oncogenes and tumor suppressor genes in a system9s manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3180. doi:10.1158/1538-7445.AM2013-3180

Abstract 2001: Genomic signatures of melanoma progression and maintenance.

Melanoma is one of the most aggressive human cancers. The worldwide incidence rate of melanoma has increased during the last decade but with few FDA approved therapeutic options. Patients with lymph node metastasis can show highly variable clinical outcomes, from several years disease free survival after excision of the primary lesion to extremely aggressive metastatic disease; while patients with distant metastasis show poor clinical outcome. Outstanding outcomes using target therapies such as B-RAF V600E inhibitor (Vemurafenib) bring hope for possibility of melanoma cure. Alternatively, since melanoma acquires resistance to Vemurafenib, the necessity of searching for other candidates and simultaneously targeting several pathways for long-term survival is inevitable. Better understanding of the genomic signatures of melanoma progression and maintenance provides new opportunities for developing novel therapeutic targets for successful management of this fatal disease. In this study, we aim to reveal the complex patterns of distinct molecular aberrations and mechanisms underlying the progression and maintenance of melanoma using high-throughput sequencing approaches. Our hypothesis is that there are genetic changes in melanoma which suggest the survival of the distant metastasis; these genetic changes include activated oncogenes and suppressed tumor suppressor genes correlated with melanoma progression and maintenance. As a proof of principal we initiated this study with a poor prognosis melanoma patient; a 46 year old man diagnosed with lymph node metastasis and treated with T-cell vaccination (TCV), who showed the tumor distant metastasis to lung, 14.8 months after complete lymph node dissection. The availability of primary cell lines established from both lymph node and lung metastatic tissues represents a major advantage for the functional validation of individual candidate genes which may serve as novel targets for cancer therapy. Integration of copy number with structural variation data showed a great selection for cell lines generation (derived from lymph node and lung tumors) and we observed that this selection was for a similar clone embedded in lymph node and lung metastatic tumors. With this data, we hypothesized that common sets of events which cut across all samples in this case identify the core sets of putative oncogenes and tumor suppressor genes which drive this patient9s cancer cells. The correlation of these genes with the cancer phenotype will be addressed. Citation Format: Faranak Ghazi Sherbaf, Charlie LEE Wah Heng, Xing Yi Woo, Denis Bertrand, Koichiro Inaki, Kelly Chong Chong, Donald Morton, Sharon Huang, Dave Hoon, Edison Liu. Genomic signatures of melanoma progression and maintenance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2001. doi:10.1158/1538-7445.AM2013-2001

Abstract 4947: Analysis of structural mutations in cancer genomes

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL In our group, we have extensively sequenced various cancer genomes (breast cancer, ovarian cancer and melanoma) using a range of genome-wide sequencing approaches, including DNA-PET, RNA-SEQ and exome-capture sequencing. Taking each tumor as a system of its own, we aim to integrate the different outputs of these technologies to explore the composite of hard-wired changes that drive tumorgenesis in different cancers. One type of hard-wired changes is copy number alterations, where the cancer genome tends to gain oncogenes and lose tumor suppressors by DNA amplifications and deletions respectively. While copy number variation data are now a standard output of the DNA-PET pipeline, the accurate prediction of copy number gains and losses is further complicated by several issues, including normal tissue contamination, tumor heterogeneity and aneuploidy. I will present my current work to deconvolute the contribution of these factors to the copy number profile. I will also discuss the classification of our sequenced cancer genomes into various chromotypes based on their unique copy number signatures. I will show how different chromotypes are enriched in specific types of structural variations, which may underlay alternative mechanisms of genomic instability promoting cancer evolution. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4947. doi:1538-7445.AM2012-4947