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SUMMARYLoss of homologous recombination repair (HRR) via germline and somaticBRCA1orBRCA2gene mutations and viaBRCA1promoter methylation has been associated with better response to platinum agents and PARP inhibitors, in both triple negative breast cancer (TNBC) and ovarian carcinoma (OvCa). A major conundrum arising from recent clinical studies is why cancers withBRCA1promoter methylation (BRCA1meth) respond more poorly as compared to those bearing mutations inBRCA1andBRCA2(BRCAmut), given the biologically equivalent HRR deficiency in both states. We dissected this problem through detailed genomic analyses of primary TNBC and OvCa cohorts, as well as experimentation with patient-derived xenograft (PDX) models and genetically engineered cell lines. Using the precise genomic scar of the tandem duplicator phenotype as a precise genomic indicator of BRCA1 deficiency, we found that, in all cohorts,BRCA1mut andBRCA1meth cancers share an equivalent degree of BRCA1-linked genomic rearrangements. Nonetheless, we consistently found that patients withBRCAmut cancers, but not those withBRCA1meth cancers, had significantly better response outcomes when compared to those withBRCAproficient cancers. When fully promoter methylatedBRCA1PDX TNBCs were exposed to a single short course of platinum chemotherapy an unmethylatedBRCA1promoter allele emerged in resultant tumors associated with an increase inBRCA1expression. A separate analysis of PDXs derived from treatment naïve TNBCs featured complete methylation of theBRCA1promoter, whereas those derived from post-chemotherapy TNBCs invariably had only partial methylation. PDXs with partial methylation were significantly associated with lower response rates toin vivoplatinum-based therapy compared to those with complete promoter methylation. Using single cell clonal expansions from a partiallyBRCA1meth PDX, we confirmed that the reduced level of methylation was due to the demethylation of one of theBRCA1promoter alleles and not to the outgrowth of a non-methylated clone. Clinically, analysis of primary OvCas confirmed that high levels ofBRCA1methylation were significantly associated with reducedBRCA1gene expression whereas cancers with lower levels ofBRCA1methylation had expression levels approaching those found inBRCA1proficient cancers. These data suggest that unlikeBRCAmut cancers, where HRR deficiency is achieved via mutations that are genetically ‘fixed’,BRCA1meth cancers are highly adaptive to genotoxin exposure and more likely to recoverBRCA1expression, which may explain their poorer therapeutic response. We further found that an increased immune transcriptional signal, especially an elevated M1 macrophage signature, is associated with enhanced response to platinum-based chemotherapy only in patients withBRCAproficient cancers, in both TNBC and OvCa cohorts underscoring the importance of characterizing molecular heterogeneity to enhance predictive precision in assigning response probabilities in TNBC and OvCa.

SummaryIn coronaviridae such as SARS-CoV-2, subgenomic RNAs (sgRNA) are replicative intermediates, therefore, their abundance and structures could infer viral replication activity and severity of host infection. Here, we systematically characterized the sgRNA expression and their structural variation in 81 clinical specimens collected from symptomatic and asymptomatic individuals with a goal of assessing viral genomic signatures of disease severity. We demonstrated the highly coordinated and consistent expression of sgRNAs from individuals with robust infections that results in symptoms, and found their expression is significantly repressed in the asymptomatic infections, indicating that the ratio of sgRNAs to genomic RNA (sgRNA/gRNA) is highly correlated with the severity of the disease. Using long read sequencing technologies to characterize full-length sgRNA structures, we also observed widespread deletions in viral RNAs, and identified unique sets of deletions preferentially found primarily in symptomatic individuals, with many likely to confer changes in SARS-CoV-2 virulence and host responses. Furthermore, based on the sgRNA structures, the frequently occurred structural variants in SARS-CoV-2 genomes serves as a mechanism to further induce SARS-CoV-2 proteome complexity. Taken together, our results show that differential sgRNA expression and structural mutational burden both appear to be correlated with the clinical severity of SARS-CoV-2 infection. Longitudinally monitoring sgRNA expression and structural diversity could further guide treatment responses, testing strategies, and vaccine development.

It is estimated that up to 80% of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are asymptomatic and asymptomatic patients can still effectively transmit the virus and cause disease. While much of the effort has been placed on decoding single nucleotide variation in SARS-CoV-2 genomes, considerably less is known about their transcript variation and any correlation with clinical severity in human hosts, as defined here by the presence or absence of symptoms. To assess viral genomic signatures of disease severity, we conducted a systematic characterization of SARS-CoV-2 transcripts and genetic variants in 81 clinical specimens collected from symptomatic and asymptomatic individuals using multi-scale transcriptomic analyses including amplicon-seq, short-read metatranscriptome and long-read Iso-seq. Here we show a highly coordinated and consistent pattern of sgRNA expression from individuals with robust SARS-CoV-2 symptomatic infection and their expression is significantly repressed in the asymptomatic infections. We also observe widespread inter- and intra-patient variants in viral RNAs, known as quasispecies frequently found in many RNA viruses. We identify unique sets of deletions preferentially found primarily in symptomatic individuals, with many likely to confer changes in SARS-CoV-2 virulence and host responses. Moreover, these frequently occurring structural variants in SARS-CoV-2 genomes serve as a mechanism to further induce SARS-CoV-2 proteome complexity. Our results indicate that differential sgRNA expression and structural mutational burden are highly correlated with the clinical severity of SARS-CoV-2 infection. Longitudinally monitoring sgRNA expression and structural diversity could further guide treatment responses, testing strategies, and vaccine development. Wong and Ngan et al. characterize the expression and structural variation of SARS-CoV-2 subgenomic RNAs (sgRNAs) in diagnostic specimens from symptomatic and asymptomatic COVID-19 patients. In a series of genomic and transcriptomic analyses, the authors observe reduced sgRNA expression and a distinct set of structural deletions in asymptomatic infections compared with symptomatic infections. Infection with SARS-CoV-2 can lead to symptoms of different severity, with some individuals remaining entirely asymptomatic but still responsible for much of the viral transmission. Here, we sought to identify markers of the severity of symptoms of SARS-CoV-2 infection, as defined by the presence or absence of symptoms. We used a combination of methods to study SARS-CoV-2 genes and their readouts, known as transcripts, in clinical samples from people with symptomatic and asymptomatic infections. We demonstrate that transcripts responsible for making viral proteins are seen at lower levels in asymptomatic infections. We also identify structural changes in the viral genes and transcripts that potentially influence the host’s response to the virus. Our study defines potential markers of symptom severity that may ultimately guide risk mitigation and testing strategies.

SummaryConservative repair of stalled replication forks is important for the maintenance of a stable genome. However, the mechanisms that regulate repair pathway “choice” at stalled mammalian forks remain poorly understood. The Fanconi anemia complementation group M gene, FANCM, encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here we use a chromosomally integrated reporter of stalled fork repair, in combination with defined mutations engineered within the endogenous Fancm gene in primary mammalian cells, to study how Fancm regulates stalled fork repair. We identify separation-of-function Fancm mutants, which reveal that distinct repair functions of FANCM are enacted by modular, molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, a mutation that inactivates the ATPase function of FANCM disables all FANCM-mediated repair functions and appears to “trap” FANCM at stalled forks. We find that Fancm null cells do not survive genetic inactivation of Brca1. This synthetic lethal interaction is recapitulated in Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising “druggable” target for therapy of BRCA1 mutant cancers.

Acquired genomic structural variants (SVs) are major hallmarks of cancer genomes, but they are challenging to reconstruct from short-read sequencing data. Here we exploited the long reads of the nanopore platform using our customized pipeline, Picky ( https://github.com/TheJacksonLaboratory/Picky ), to reveal SVs of diverse architecture in a breast cancer model. We identified the full spectrum of SVs with superior specificity and sensitivity relative to short-read analyses, and uncovered repetitive DNA as the major source of variation. Examination of genome-wide breakpoints at nucleotide resolution uncovered micro-insertions as the common structural features associated with SVs. Breakpoint density across the genome is associated with the propensity for interchromosomal connectivity and was found to be enriched in promoters and transcribed regions of the genome. Furthermore, we observed an over-representation of reciprocal translocations from chromosomal double-crossovers through phased SVs. We demonstrate that Picky analysis is an effective tool for comprehensive detection of SVs in cancer genomes from long-read data.

Background. We recently discovered a unique chromotype, the Tandem Duplicator Phenotype (TDP), characterized by hundreds of somatic tandem duplications (TDs) scattered throughout the genome of a large percentage of triple negative breast cancers (TNBCs). Importantly, we observed that the TDP associates with a better response to cisplatin therapy in vitro and in vivo, suggesting that it is a tractable and quantitative biomarker of response to platinum-based therapy. Here, we expand on our initial findings by analyzing Whole-Genome (WG) sequences of over 2,700 tumors. Methods. TD coordinates from WG sequences relative to 2717 tumors were assembled from over 30 independent studies representing several cancer types, including 254 TNBCs. WG sequencing of mouse breast tumors was carried out using standard Illumina protocols. The number, distribution and span-size of somatic TDs from a training set of 992 tumors were used to develop a TDP classifier that identifies highly recurrent but clearly distinct TDP profiles. The TDP classifier was then applied to the remaining tumor sequences. WG mutation and copy number datasets were investigated to identify the genetic drivers associated with each TDP profile, and the genomic consequences of different TDPs were evaluated through identification of genomic hotspots for gene duplication and transection. Results. We describe six different TDPs featuring distinct TD span size distributions, with peaks at 10Kb (group 1), 300Kb (group 2) and 3Mb (group 3), or different combinations of these (mix12, mix13 and mix23). More than half of all TNBC display a TDP. Of these, 55% classify as group 1, 14% as group 2 and 15% as group mix12. Whereas all TDP groups show a higher TP53 mutation rate compared to non-TDP tumors, each TDP profile is characterized by specific additional gene perturbations, with loss of BRCA1 occurring in groups 1, mix12 and mix13; CCNE1 amplification in group 2; and CDK12 mutations in group mix23. We show that different TDPs are subject to the perturbation of specific oncogenic networks resulting from the duplication of oncogenes by larger TDs (>300Kb) or the disruption of tumor suppressors via double transections by shorter TDs (10Kb). Indeed, tumor suppressor genes such as PTEN, RB1 and MLL3 are frequently disrupted by TDs in TNBC TDP group 1 tumors, whereas TNBC TDP group 2 tumors commonly feature duplication of oncogenes such as MYC and MALAT1. Finally, through WG analyses of 18 mouse models (GEMMs) of breast cancer, we provide the first mechanistic evidence of the driving role of conjoint loss of TP53 and BRCA1, but not of BRCA2, in inducing the TDP group 1 profile. Conclusions. Our study shows a definitive genetic induction of one specific form of TDP (group 1) characterized by 10kb TD span. Different TDP profiles are characterized by alternative somatic genetic origins but always couple with disruptive TP53 mutations. The consequences of the massive TD formation in TDP TNBCs suggest a systems strategy to tumor induction involving heterogeneous combinations of oncogenes and tumor suppressors. That these TDP forms, accounting for ˜50% of TNBC, are associated with significant sensitivity to cisplatin suggest that this chromotype may identify TNBC patients who would benefit from upfront platinum-based chemotherapy. Citation Format: Liu ET, Menghi F, Barthel F, Yadav V, Tang M, Ji B, Carter G, Jonkers J, Verhaak R. Tandem duplicator phenotypes define 50% of triple negative breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS1-05.

Small, approximately 10-kilobase microhomology-mediated tandem duplications are abundant in the genomes of BRCA1-linked but not BRCA2-linked breast cancer. Here we define the mechanism underlying this rearrangement signature. We show that, in primary mammalian cells, BRCA1, but not BRCA2, suppresses the formation of tandem duplications at a site-specific chromosomal replication fork barrier imposed by the binding of Tus proteins to an array of Ter sites. BRCA1 has no equivalent role at chromosomal double-stranded DNA breaks, indicating that tandem duplications form specifically at stalled forks. Tandem duplications in BRCA1 mutant cells arise by a replication restart-bypass mechanism terminated by end joining or by microhomology-mediated template switching, the latter forming complex tandem duplication breakpoints. Solitary DNA ends form directly at Tus-Ter, implicating misrepair of these lesions in tandem duplication formation. Furthermore, BRCA1 inactivation is strongly associated with ~10 kilobase tandem duplications in ovarian cancer. This tandem duplicator phenotype may be a general signature of BRCA1-deficient cancer.

The usefulness of a specific technology often hits a ceiling based on technical limitations. Then, a single advance, frequently orthogonal to the core methodology, dramatically expands the utility of this technology. The effectiveness of early surgeons wielding a scalpel was severely limited by how much a patient could withstand the pain of an operation. Anesthesia was the core discovery that permitted surgery to become a consistently effective medical intervention. Molecular cloning was a powerful technology to understand the importance of genes in biology, but it had limited utility in population genetics and in medicine, because of the arduous requirements for cloning a single gene. The invention of polymerase chain reaction dramatically expanded the utility of molecular biology into high‐throughput sequencing, epidemiology, forensics, diagnostics, and gene therapy. We believe that the advent of powerful gene editing technologies such as the CRISPR/CAS system will be this transformative technology for murine genetics. The subsequent massive sequencing efforts to define the mutational spectrum of human diseases have uncovered a bewildering number of genetic changes. It is now recognized that most diseases are genetically complex and that their dissection requires the enrollment of many hundreds of thousands of individuals to achieve adequate statistical power to ensure the validity of an association. This purely statistical approach is clearly impracticable, given the many variants and mutations that are being uncovered with each sequence‐based study. Thus, the translation of human genome research into health care heavily relies on appropriate organismal models that accurately reflect the human condition. This is where the laboratory mouse ( Mus musculus ) comes in. For many diseases that require an intact organ system in an intact animal—such as neurological disorders, renal disease, complex cardiopulmonary syndromes, and aging—the mouse has been a primary experimental model for mammalian biology since the turn of the last century. > … …

The processes by which tumors evolve are essential to the efficacy of treatment, but quantitative understanding of intratumoral dynamics has been limited. Although intratumoral heterogeneity is common, quantification of evolution is difficult from clinical samples because treatment replicates cannot be performed and because matched serial samples are infrequently available. To circumvent these problems we derived and assayed large sets of human triple-negative breast cancer xenografts and cell cultures from two patients, including 86 xenografts from cyclophosphamide, doxorubicin, cisplatin, docetaxel, or vehicle treatment cohorts as well as 45 related cell cultures. We assayed these samples via exome-seq and/or high-resolution droplet digital PCR, allowing us to distinguish complex therapy-induced selection and drift processes among endogenous cancer subclones with cellularity uncertainty

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphomas (EBV(+)-DLBLs) tend to occur in immunocompromised patients, such as the elderly or those undergoing solid organ transplantation. The pathogenesis and genomic characteristics of EBV(+)-DLBLs are largely unknown because of the limited availability of human samples and lack of experimental animal models. We observed the development of 25 human EBV(+)-DLBLs during the engraftment of gastric adenocarcinomas into immunodeficient mice. An integrated genomic analysis of the human-derived EBV(+)-DLBLs revealed enrichment of mutations in Rho pathway genes, including RHPN2, and Rho pathway transcriptomic activation. Targeting the Rho pathway using a Rho-associated protein kinase (ROCK) inhibitor, fasudil, markedly decreased tumor growth in EBV(+)-DLBL patient-derived xenograft (PDX) models. Thus, alterations in the Rho pathway appear to contribute to EBV-induced lymphomagenesis in immunosuppressed environments.

The processes by which tumors evolve are essential to the efficacy of treatment, but quantitative understanding of intratumoral dynamics has been limited. Although intratumoral heterogeneity is common, quantification of evolution is difficult from clinical samples because treatment replicates cannot be performed and because matched serial samples are infrequently available. To circumvent these problems we derived and assayed large sets of human triple-negative breast cancer xenografts and cell cultures from two patients, including 86 xenografts from cyclophosphamide, doxorubicin, cisplatin, docetaxel, or vehicle treatment cohorts as well as 45 related cell cultures. We assayed these samples via exome-seq and/or high-resolution droplet digital PCR, allowing us to distinguish complex therapy-induced selection and drift processes among endogenous cancer subclones with cellularity uncertainty AUTHOR SUMMARYAn overarching challenge of cancer is that patients develop resistance to treatment -- an essentially evolutionary process. However, there is currently very little understanding of how tumor evolution can be exploited to improve treatment. One reason for this is that usually only 1-2 samples can be obtained per patient, so cancer evolutionary processes are still poorly understood. To solve this problem, we created many dozens of copies of the tumors from two breast cancer patients using xenografting and cell culture methods. We then compared the evolution in these tumor copies in response to different treatments, including four of the most common breast cancer chemotherapies. These studies present the most exhaustive comparisons of treatment-induced evolution that have yet been performed for individual cancer patients. Unexpectedly, high-resolution sequencing of these samples revealed a special dynamically treatable ecology in one tumor, in which tumor growth during platinum therapy was determined by the ecological balance of two tumor cell populations. Our work shows that ecologies that can be targeted by dynamic treatment strategies arise spontaneously in breast cancers. Population heterogeneity is common within cancers, and our work suggests how tracking of intratumoral evolution can be used to optimize treatment.

SUMMARY The tandem duplicator phenotype (TDP) is a genome-wide instability configuration primarily observed in breast, ovarian, and endometrial carcinomas. Here, we stratify TDP tumors by classifying their tandem duplications (TDs) into three span intervals, with modal values of 11 kb, 231 kb, and 1.7 Mb, respectively. TDPs with ~11 kb TDs feature loss of TP53 and BRCA1. TDPs with ~231 kb and ~1.7 Mb TDs associate with CCNE1 pathway activation and CDK12 disruptions, respectively. We demonstrate that p53 and BRCA1 conjoint abrogation drives TDP induction by generating short-span TDP mammary tumors in genetically modified mice lacking them. Lastly, we show how TDs in TDP tumors disrupt heterogeneous combinations of tumor suppressors and chromatin topologically associating domains while duplicating oncogenes and super-enhancers., Graphical Abstract, In Brief Menghi et al. stratify tandem duplicator phenotype tumors by classifying their tandem duplications (TDs) into three span sizes associated with different pathway alterations and show how TDs disrupt tumor suppressors and chromatin topologically associating domains while duplicating oncogenes and super-enhancers.

Acquired genomic structural variants (SVs) are major hallmarks of the cancer genome. Their complexity has been challenging to reconstruct from short-read sequencing data. Here, we exploit the long-read sequencing capability of the nanopore platform using our customized pipeline,Picky, to reveal SVs of diverse architecture in a breast cancer model. From modest sequencing coverage, we identified the full spectrum of SVs with superior specificity and sensitivity relative to short-read analyses and uncovered repetitive DNA as the major source of variation. Examination of the genome-wide breakpoints at nucleotide-resolution uncovered micro-insertions as the common structural features associated with SVs. Breakpoint density across the genome is associated with propensity for inter-chromosomal connectivity and transcriptional regulation. Furthermore, an over-representation of reciprocal translocations from chromosomal double-crossovers was observed through phased SVs. The comprehensive characterization of SVs using the robust long-read sequencing approach in cancer cohorts will facilitate strategies to monitor genome stability during tumor evolution and improve therapeutic intervention.

Since the emergence of the pandemic H1N1pdm09 virus in Mexico and California, biannual increases in the number of cases have been detected in Mexico. As observed in previous seasons, pandemic A/H1N1 09 virus was detected in severe cases during the 2011-2012 winter season and finally, during the 2013-2014 winter season it became the most prevalent influenza virus. Molecular and phylogenetic analyses of the whole viral genome are necessary to determine the antigenic and pathogenic characteristics of influenza viruses that cause severe outcomes of the disease. In this paper, we analyzed the evolution, antigenic and genetic drift of Mexican isolates from 2009, at the beginning of the pandemic, to 2014. We found a clear variation of the virus in Mexico from the 2011-2014 season due to different markers and in accordance with previous reports. In this study, we identified 13 novel substitutions with important biological effects, including virulence, T cell epitope presented by MHC and host specificity shift and some others substitutions might have more than one biological function. The systematic monitoring of mutations on whole genome of influenza A pH1N1 (2009) virus circulating at INER in Mexico City might provide valuable information to predict the emergence of new pathogenic influenza virus.

A recent publication questions the suitability of mice as a model for the human inflammatory response and has fueled the continuing debate about the suitability of mice as models for human disease. We discuss recent advances in disease modeling using mice, and the genetic factors that need to be considered when trying to recapitulate aspects of human disease. Failure to appreciate the important differences between human and mouse biology and genetics underlying attempts to generate faithful models frequently leads to poor outcomes. Closely coordinated human and model organism studies are essential to provide traction for translational research.

Watkins et al. (1) note that tandem duplications (TDs) are a feature of two distinct cancer phenotypes, distinguished based on TD span size (1 Kb–2 Mb vs. 2–10 Mb) and breast cancer 1 ( BRCA1 ) status (inactivation vs. wild-type), present at different frequencies in triple-negative breast cancer (TNBC). The authors suggest that our recent study (2) only captures the first type of TD phenotype (TDP), and that the second form of TDP may have implications in the assessment of genomic instability-based biomarkers of drug response. We agree that the TDP manifests in at least two distinct genomic configurations. However, we believe that Watkins et al. (1) misidentified the two prevalent TDP configurations by exclusively basing their analysis on SNP array data and focusing on predetermined TD span sizes. Indeed, when we compared the analytical precision of … [↵][1]1To whom correspondence should be addressed. Email: edison.liu{at}jax.org. [1]: #xref-corresp-1-1

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.

Although elongation of telomeres is thought to be the prime function of reactivated telomerase in cancers, this activity alone does not account for all of the properties that telomerase reactivation attributes to human cancer cells. Here, we uncover a link between telomerase and NF-κB, a master regulator of inflammation. We observe that while blocking NF-κB signalling can inhibit effects of telomerase overexpression on processes relevant to transformation, increasing NF-κB activity can functionally substitute for reduced telomerase activity. Telomerase directly regulates NF-κB-dependent gene expression by binding to the NF-κB p65 subunit and recruitment to a subset of NF-κB promoters such as those of IL-6 and TNF-α, cytokines that are critical for inflammation and cancer progression. As NF-κB can transcriptionally upregulate telomerase levels, our findings suggest that a feed-forward regulation between them could be the key mechanistic basis for the coexistence of chronic inflammation and sustained telomerase activity in human cancers.

SUMMARY Chromatin interactions play important roles in transcription regulation. To better understand the underlying evolutionary and functional constraints of these interactions, we implemented a systems approach to examine RNA polymerase-II-associated chromatin interactions in human cells. We found that 40% of the total genomic elements involved in chromatin interactions converged to a giant, scale-free-like, hierarchical network organized into chromatin communities. The communities were enriched in specific functions and were syntenic through evolution. Disease-associated SNPs from genome-wide association studies were enriched among the nodes with fewer interactions, implying their selection against deleterious interactions by limiting the total number of interactions, a model that we further reconciled using somatic and germline cancer mutation data. The hubs lacked disease-associated SNPs, constituted a nonrandomly interconnected core of key cellular functions, and exhibited lethality in mouse mutants, supporting an evolutionary selection that favored the nonrandom spatial clustering of the least-evolving key genomic domains against random genetic or transcriptional errors in the genome. Altogether, our analyses reveal a systems-level evolutionary framework that shapes functionally compartmentalized and error-tolerant transcriptional regulation of human genome in three dimensions.

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 transcription factor Ebf1 is an important determinant of early B lymphopoiesis. To gain insight into the functions of Ebf1 at distinct stages of differentiation, we conditionally inactivated Ebf1. We found that Ebf1 is required for the proliferation, survival, and signaling of pro-B cells and peripheral B-cell subsets, including B1 cells and marginal zone B cells. The proliferation defect of Ebf1-deficient pro-B cells and the impaired expression of multiple cell cycle regulators are overcome by transformation with v-Abl. The survival defect of transformed Ebf1fl/fl pro-B cells can be rescued by the forced expression of the Ebf1 targets c-Myb or Bcl-xL. In mature B cells, Ebf1 deficiency interferes with signaling via the B-cell-activating factor receptor (BAFF-R)- and B-cell receptor (BCR)-dependent Akt pathways. Moreover, Ebf1 is required for germinal center formation and class switch recombination. Genome-wide analyses of Ebf1-mediated gene expression and chromatin binding indicate that Ebf1 regulates both common and distinct sets of genes in early and late stage B cells. By regulating important components of transcription factor and signaling networks, Ebf1 appears to be involved in the coordination of cell proliferation, survival, and differentiation at multiple stages of B lymphopoiesis.

Secretory factors that drive cancer progression are attractive immunotherapeutic targets. We used a whole-genome data-mining approach on multiple cohorts of breast tumours annotated for clinical outcomes to discover such factors. We identified Serine protease inhibitor Kazal-type 1 (SPINK1) to be associated with poor survival in estrogen receptor-positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. In ER- cases, the prognostic effect did not reach statistical significance. Forced expression and/or exposure to recombinant SPINK1 induced invasiveness without affecting cell proliferation. However, down-regulation of SPINK1 resulted in cell death. Further, SPINK1 overexpressing cells were resistant to drug-induced apoptosis due to reduced caspase-3 levels and high expression of Bcl2 and phospho-Bcl2 proteins. Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain. Thus, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.

The JAX® Onco-Hu® platform utilizes humanized mice engrafted with tumors to enable in vivo investigation of the interactions between the human immune system and human cancer. We have recently shown that humanized NOD-scid IL2Rγnull (NSG™) mice bearing patient-derived xenografts (PDX) allow efficacy studies of checkpoint inhibitors. A major avenue of our investigation is to generate murine humanized models containing a more complete human hematopoietic system and robust innate immune cell population. Next-generation NSG strains include triple transgenic NSG mice (NSG-SGM3) expressing myelosupportive human cytokines KITLG, CSF2, and IL-3. When engrafted with CD34+ human hematopoietic progenitor cells (HPCs) from CD3-depleted umbilical cord blood, NSG-SGM3 mice produce higher myeloid and Treg populations in the circulation as compared to NSG mice over 18 weeks post engraftment. We implanted an array of PDX tumors into humanized NSG-SGM3 mice at 2-3 months post engraftment. Tumors were dissociated and single-cell infiltrates were analyzed by multicolor flow cytometry with a focus on examining overall immune cell infiltration and the levels of hCD33+ myeloid cells. In the PS4050 melanoma PDX model, we found that hCD45+ cell infiltration was significantly increased in hu-NSG-SGM3 mice as compared to hu-NSG mice engrafted with the same HPC donor (3.7% vs. 1% of viable cells). The majority of tumor-infiltrating cells in hu-NSG-SGM3 mice expressed hCD33 (55% of hCD45+) and the percentage was significantly higher than that in hu-NSG mice (13%). hCD3+T cell infiltration level was similar between these two strains (~20% of hCD45+). PS4050-bearing hu-NSG-SGM3 mice treated with the anti-PD-1 antibody pembrolizumab (Keytruda) showed a significant reduction in tumor growth and the PD-1 levels in tumor-infiltrating T cells were greatly reduced by flow cytometry analysis. The overall hCD45+ cell infiltration and the frequencies of hCD4+T, hCD8+T, and hCD33+myeloid cells in tumors remained similar after treatment. Lastly, we observed that the effect of Keytruda on tumor growth reduction in hu-NSG-SGM3 mice is PD-L1-dependent using the human lung carcinoma cell line NCI-H460 depleted of PD-L1 expression by CRISPR. Keytruda treatment significantly reduced mock-transfected NCI-H460 cell growth. By comparison, PD-L1 KO NCI-H460 cells grew more slowly than the mock cells and lost the response to Keytruda. Together, these results indicate that PDX tumor-implanted hu-NSG-SGM3 mice serve as an important platform for understanding human immune system and tumor microenvironment interactions and for preclinical immuno-oncology efficacy studies. Citation Format: Li-Chin Yao, Mingshan Cheng, Ken-Edwin Aryee, Pooja Kumar, Nicole Walsh, Dale Greiner, Leonard Shultz, Edison T. Liu, Michael Brehm, James G. Keck. Patient-derived tumor xenografts in humanized NSG-SGM3 mice: An improved immuno-oncology platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5676.

To identify genetic susceptibility loci for nasopharyngeal carcinoma (NPC), a genome-wide association study was performed using 464,328 autosomal SNPs in 1,583 NPC affected individuals (cases) and 1,894 controls of southern Chinese descent. The top 49 SNPs from the genome-wide association study were genotyped in 3,507 cases and 3,063 controls of southern Chinese descent from Guangdong and Guangxi. The seven supportive SNPs were further confirmed by transmission disequilibrium test analysis in 279 trios from Guangdong. We identified three new susceptibility loci, TNFRSF19 on 13q12 (rs9510787, Pcombined=1.53x10(-9), odds ratio (OR)=1.20), MDS1-EVI1 on 3q26 (rs6774494, Pcombined=1.34x10(-8), OR=0.84) and the CDKN2A-CDKN2B gene cluster on 9p21 (rs1412829, Pcombined=4.84x10(-7), OR=0.78). Furthermore, we confirmed the role of HLA by revealing independent associations at rs2860580 (Pcombined=4.88x10(-67), OR=0.58), rs2894207 (Pcombined=3.42x10(-33), OR=0.61) and rs28421666 (Pcombined=2.49x10(-18), OR=0.67). Our findings provide new insights into the pathogenesis of NPC by highlighting the involvement of pathways related to TNFRSF19 and MDS1-EVI1 in addition to HLA molecules.

We have previously identified an oncogenic role of artemin (ARTN), a member of glial cell derived neurotrophic factor family of ligands, in mammary carcinoma. We herein report that ARTN is an estrogen-inducible gene. Meta-analysis of gene expression data sets showed that ARTN expression is positively correlated to estrogen receptor (ER) status in human mammary carcinoma. Furthermore, in patients with ER-positive mammary carcinoma treated with tamoxifen, high ARTN expression is significantly correlated with decreased survival. Forced expression of ARTN in ER-positive human mammary carcinoma cells increased ER transcriptional activity, promoted estrogen-independent growth and produced resistance to tamoxifen and fulvestrant in vitro and to tamoxifen in xenograft models. ARTN-stimulated resistance to tamoxifen and fulvestrant is mediated by increased BCL-2 expression. Conversely, depletion of endogenous ARTN by small-interfering RNA or functional antagonism of ARTN by antibody enhanced the efficacy of antiestrogens. Tamoxifen decreased ARTN expression in tamoxifen-sensitive mammary carcinoma cells whereas ARTN expression was increased in tamoxifen-resistant cells and not affected by tamoxifen treatment. Antibody inhibition of ARTN in tamoxifen-resistant cells improved tamoxifen sensitivity. Functional antagonism of ARTN therefore warrants consideration as an adjuvant therapy to enhance antiestrogen efficacy in ER-positive mammary carcinoma.

Estrogen receptors ERalpha and ERbeta, members of the nuclear receptor superfamily, exert profound effects on the gene expression and biological response programs of their target cells. Herein, we explore the dynamic interplay between these two receptors in their selection of chromatin binding sites when present separately or together in MCF-7 breast cancer cells. Treatment of cells (containing ERalpha only, ERbeta only, or ERalpha and ERbeta) with estradiol or ER subtype-selective ligands was followed by chromatin immunoprecipitation analysis with a custom-designed tiling array for ER binding sites across the genome to examine the effects of ligand-occupied and unoccupied ERalpha and ERbeta on chromatin binding. There was substantial overlap in binding sites for these estradiol-liganded nuclear receptors when present alone, but many fewer sites were shared when both ERs were present. Each ER restricted the binding site occupancy of the other, with ERalpha generally being dominant. Binding sites of both receptors were highly enriched in estrogen response element motifs, but when both ERs were present, ERalpha displaced ERbeta, shifting it into new sites less enriched in estrogen response elements. Binding regions of the two ERs also showed differences in their enrichments for other transcription factor binding motifs. Studies with ER subtype-specific ligands revealed that it was the liganded subtype that principally determined the spectrum of chromatin binding. These findings highlight the dynamic interplay between the two ERs in their selection of chromatin binding sites, with competition, restriction, and site shifting having important implications for the regulation of gene expression by these two nuclear receptors.

Most commonly reported non-commercial protein isolation methods require the use of detergents and/or chaotropes for better yield, and they all need additional Purification or clean-up steps for subsequent mass spectrometry analysis. Moreover, there is no simple procedure available for obtaining both soluble and membrane proteins from the same sample. Here we describe a simple and detergent-free chloroform-assisted protein isolation (ChlAPI) method for mammalian cells and tissues, and demonstrated its suitability to mass spectrometry based proteome analysis. In this single-step method, cultured cells or grounded tissue were mixed in 10% chloroform in ammonium bicarbonate buffer to separate whole cell proteome into biphasic layers. Total number of aqueous phase proteins, as assessed by 2DE, was comparable to the proteins isolated with commonly used detergent containing buffer. Shotgun proteomics analysis of the aqueous and organic phase proteome fractions of MCF7 cells by LC-MS/MS resulted in Identification of a total of 752 and 593 proteins, respectively from IPI human protein database. Among the total of 1134 distinct and nonredundant proteins, 29.5% were predicted to be membrane localized; 78% of them wereidentified from organic phase fraction. Application of this new procedure to estrogen-treated MCF cells lead to the Identification of previously known as well as unknown estrogen-responsive gene products. These fi ndings suggest that the simple and inexpensive ChlAPI method described here is suitable for protein isolation from mammalian samples, and is readily compatible with 2DE and LC-MS/MS analyses.

Genomes are organized into high-level three-dimensional structures, and DNA elements separated by long genomic distances can in principle interact functionally. Many transcription factors bind to regulatory DNA elements distant from gene promoters. Although distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Here we describe the development of a new strategy, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for the de novo detection of global chromatin interactions, with which we have comprehensively mapped the chromatin interaction network bound by oestrogen receptor α (ER-α) in the human genome. We found that most high-confidence remote ER-α-binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ER-α functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes. © 2009 Macmillan Publishers Limited. All rights reserved.

The precision of genome sequences, together with advanced computational approaches, allows complex, clinically relevant biological systems to be examined. Here, I describe the experiences of the Genome Institute of Singapore (GIS) in using genome-to-systems strategies to accelerate biomedical research. To maximize clinically relevant output, we have explored an organizational strategy that encourages coordinated experimentation among investigators with diverse skills and interests through building a culture of integrative science. Our experience suggests that systems biomedicine is a real and potentially fruitful strategy for translational research.

Nuclear signaling by estrogens rapidly induces the global recruitment of estrogen receptors (ERs) to thousands of highly specific locations in the genome. Here, we have examined whether ER binding sites that are located distal from the transcription start sites of estrogen target genes are functionally relevant. Similar to ER binding sites near the proximal promoter region, ER binding sites located at distal locations are occupied by ERs after estrogen stimulation. And, like proximal bound ERs, ERs occupied at distal sites can recruit coactivators and the RNA polymerase transcription machinery and mediate specific structural changes to chromatin. Furthermore, ERs occupied at the distal sites are capable of communicating with ERs bound at the promoter region, possibly via long range chromosome looping. In functional analysis, disruption of the response elements in the distal ER binding sites abrogated ER binding and significantly reduced transcriptional response. Finally, sequence comparison of the response elements at the distal sites suggests a high level of conservation across different species. Together, our data indicate that distal ER binding sites are bona fide transcriptional enhancers that are involved in long range chromosomal interaction, transcription complex formation, and distinct structural modifications of chromatin across large genomic spans.

Cancer genomics has focused on the discovery of genetic mutations and chromosomal structural rearrangements that either increase susceptibility to cancer or support the cancer phenotype. Though each individual mutation may induce specific cancer phenotypes, it is the interaction of the functional changes in transcription and proteins that give the characteristics of cancer. Whereas molecular biology focuses on the impact of individual genes on the cancer state, functional genomics assesses the comprehensive genetic alterations in a cancer cell and seeks to integrate the dynamic changes in these networks so that cancer phenotypes can be explained. Most commonly, the transcriptome is the target of analysis because of the maturity, completeness, and speed of the technologies, but progressively the proteome is being studied in the same comprehensive manner. The focus of this review, however, will be on the functional consequences of cancer genomic alterations with special reference to the transcriptome and in the perturbed gene expression found in cancer states. The developments in the past two years (which is our time horizon) have been heavily driven by the applications of the new ultra high-throughput sequencing approaches assisted by computational discovery strategies. The precision and comprehensiveness of the analyses are astonishing. The collective results, when taken together, suggest that despite the large range of mutational and epigenetic events, there is a convergence onto a finite number of pathways that drive cancer behavior. Moreover, the interconnectivity of regulatory control mechanisms suggest that the earlier concepts distinguishing driver from passenger abnormalities may undervalue the contribution of the numerous aberrations that have small but additive effects on cancer virulence.

The expression of carbonic anhydrase XII (CA12), a gene that encodes a zinc metalloenzyme responsible for acidification of the microenvironment of cancer cells, is highly correlated with estrogen receptor α (ERα) in human breast tumors. Here, we show that CA12 is robustly regulated by estrogen via ERα in breast cancer cells, and that this regulation involves a distal estrogen-responsive enhancer region. Upon the addition of estradiol, ERα binds directly to this distal enhancer in vivo, resulting in the recruitment of RNA polymerase II and steroid receptor coactivators SRC-2 and SRC-3, and changes in histone acetylation. Mutagenesis of an imperfect estrogen-responsive element within this enhancer region abolishes estrogen-dependent activity, and chromosome conformation capture and chromatin immunoprecipitation assays show that this distal enhancer communicates with the transcriptional start site of the CA12 gene via intrachromosomal looping upon hormone treatment. This distal enhancer element is observed in the homologous mouse genomic sequence, and the expression of the mouse homologue, Car12, is rapidly and robustly stimulated by estradiol in the mouse uterus in vivo, suggesting that the ER regulation of CA12 is mechanistically and evolutionarily conserved. Our findings highlight the crucial role of ER in the regulation of the CA12 gene, and provide insight into the transcriptional regulatory mechanism that accounts for the strong association of CA12 and ER in human breast cancers. [Cancer Res 2008;68(9):3505–15]

The nuclear hormone receptors liver X receptor α (LXRα) (NR1H3) and LXRβ (NR1H2) are established regulators of cholesterol, lipid, and glucose metabolism and are attractive drug targets for the treatment of diabetes and cardiovascular disease. Adrenal steroid hormones including glucocorticoids and mineralocorticoids are known to interfere with glucose metabolism, insulin signaling, and blood pressure regulation. Here we present genome-wide expression profiles of LXR-responsive genes in both the adrenal and the pituitary gland. LXR activation in cultured adrenal cells inhibited expression of multiple steroidogenic genes and consequently decreased adrenal steroid hormone production. In addition, LXR agonist treatment elevated ACTH mRNA expression and hormone secretion from pituitary cells both in vitro and in vivo. Reduced expression of the glucocortioid-activating enzyme 11β-hydroxysteroid dehydrogenase 1 in pituitary cells upon LXR activation suggests blunting of the negative feedback of glucocorticoids by LXRs. In conclusion, LXRs independently interfere with the hypothalamic-pituitary-adrenal axis regulation at the level of the pituitary and the adrenal gland.

Previous studies showed that expression of the novel candidate tumor suppressor gene, DEC1 (Deleted in Esophageal Cancer 1), is reduced in esophageal carcinoma and suppresses cancer cell growth in vitro and tumor growth in vivo in nude mice. This study shows that DEC1 gene expression was downregulated in 100% of 16 esophageal squamous cell carcinoma (ESCC) cell lines and 52 and 45%, respectively, of esophageal tumor specimens from Hong Kong and a high-risk ESCC region of Henan, China. Using epitope tagging, the DEC1 protein was localized to both the cytoplasm and nucleus of the cell. In 3D Matrigel culture, no significant difference in colony numbers formed was observed for DEC1 stable transfectants, as compared to vector-alone transfectant controls. However, significantly smaller colony sizes were observed for the DEC1 transfectants. In in vitro cell migration, invasion and soft agar assays of DEC1 transfectants, only the soft agar assay showed statistically significant differences in colony numbers with the vector-alone controls, indicating that DEC1 may be involved in anchorage-independent cell growth. In addition, the global gene expression affected by DEC1 in tumor-suppressive stable transfectants was investigated using cDNA oligonucleotide microarray hybridization. Three candidate genes, TFPI-2, GDF15 and DUSP6, were identified through this approach; they are downregulated in tumor segregants of DEC1 stable transfectants, ESCC cell lines and esophageal tumors and have a potential role in tumor growth and progression. These studies show that DEC1 is involved in esophageal cancer development and help elucidate its functional role in tumor development.

International audience; Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.

Family history of endometrial cancer increases the risk of developing the disease, but it is still largely unknown which germ-line genetic factors are involved in the aetiology of endometrial cancer. In a Swedish population-based case-control study including 705 cases and 1565 controls, we examined common variation in the ATM, CHEK2 and ERBB2 genes in relation to endometrial cancer risk overall, restricted to tumours of certain characteristics or stratified by various endometrial cancer risk factors. We genotyped a large number of single-nucleotide polymorphisms (SNPs) in the genes and selected seven haplotype-tagging SNPs (tagSNPs) in ATM, six tagSNPs in CHEK2 and seven tagSNPs in ERBB2 that could predict common variants and haplotypes (frequency > or =0.03) in each gene with R(2) > or = 0.8. We included the tagSNPs or their haplotypes as explanatory variables in unconditional logistic regression models adjusted for age. Our results indicated an increased risk of developing endometroid endometrial cancer for homozygous carriers of the rare allele (AA) of a tagSNP (rs4987886) in CHEK2 (P = 0.005) when contrasted with GG carriers. We also found a decreased endometrial cancer risk among non-smoking carriers of a haplotype in ATM (P = 0.0007) and among carriers of a haplotype in CHEK2, who had experienced menopause below 49 years of age (P = 0.0009) compared with non-carriers of these haplotypes. We found no effect of genetic variation in ERBB2 on endometrial cancer risk. In conclusion, it is possible that common variants in the ATM and CHEK2 genes, in interaction with oestrogen-related exposures, are involved in endometrial cancer aetiology.

Arsenic is a prominent environmental toxicant and carcinogen; however, its molecular mechanism of toxicity and carcinogenicity remains poorly understood. In this study, we performed microarray-based expression profiling on liver of zebrafish exposed to 15 parts/million (ppm) arsenic [As(V)] for 8–96 h to identify global transcriptional changes and biological networks involved in arsenic-induced adaptive responses in vivo. We found that there was an increase of transcriptional activity associated with metabolism, especially for biosyntheses, membrane transporter activities, cytoplasm, and endoplasmic reticulum in the 96 h of arsenic treatment, while transcriptional programs for proteins in catabolism, energy derivation, and stress response remained active throughout the arsenic treatment. Many differentially expressed genes encoding proteins involved in heat shock proteins, DNA damage/repair, antioxidant activity, hypoxia induction, iron homeostasis, arsenic metabolism, and ubiquitin-dependent protein degradation were identified, suggesting strongly that DNA and protein damage as a result of arsenic metabolism and oxidative stress caused major cellular injury. These findings were comparable with those reported in mammalian systems, suggesting that the zebrafish liver coupled with the available microarray technology present an excellent in vivo toxicogenomic model for investigating arsenic toxicity. We proposed an in vivo, acute arsenic-induced adaptive response model of the zebrafish liver illustrating the relevance of many transcriptional activities that provide both global and specific information of a coordinated adaptive response to arsenic in the liver.

The HER-2 (also known as ERBB2/ErbB2/c-erbB2/HER-2/neu) oncogene is the most frequently amplified oncogene in breast cancer and is also amplified in other forms of cancer. Beside its important role in tumor induction, growth and progression, HER-2 is also a target for new therapeutic approaches such as Herceptin (trastuzumab), a recombinant antibody designed to block signaling through the HER-2 receptor. In addition to Herceptin, which is in a wide clinical use for HER-2 amplified breast cancer, a number of various HER-2 directed immunological and genetic strategies, either targeting the HER-2 receptor, its signaling pathways or both HER-2 and epidermal growth factor receptor (EGFR) simultaneously, have demonstrated promising pre-clinical activity in HER-2 amplified carcinomas. Moreover, the HER-2 amplicon is known to contain more than 30 genes with altered copy numbers that could be therapeutic targets for chemotherapy. The topoisomerase IIalpha gene, TOP2A, is located adjacent to the HER-2 oncogene at the chromosome location 17q12-q21 and is either amplified or deleted (with equal frequency) in a great majority of HER-2 amplified primary breast tumors and also in tumors without HER-2 amplification. Recent experimental as well as numerous, large, multi-center trials suggest that amplification (and/or deletion) of TOP2A may account for both sensitivity or resistance to commonly used cytotoxic drugs, i.e. topoII-inhibitors (anthracyclines etc.), depending on the specific genetic defect at the TOP2A locus. The understanding of HER-2 amplification and its role in the pathogenesis of cancer is expanding, and a number of therapeutic strategies targeting either the HER-2 or its signaling pathways in cancer therapy are being investigated. Combining HER-2 targeting therapies with conventional forms of cytotoxic chemotherapy, where additional diagnostic tests such as those ascertaining TOP2A status, may be helpful for the ideal selection of patients for the combination therapy of an HER-2 targeting drug together with a cytotoxic drug such as topoII-inhibitor especially in the case of TOP2A amplification.

The beneficial effect of the selective estrogen receptor (ER) modulator tamoxifen in the treatment and prevention of breast cancer is assumed to be through its ability to antagonize the stimulatory actions of estrogen, although tamoxifen can also have some estrogen-like agonist effects. Here, we report that, in addition to these mixed agonist/antagonist actions, tamoxifen can also selectively regulate a unique set of >60 genes, which are minimally regulated by estradiol (E2) or raloxifene in ERα-positive MCF-7 human breast cancer cells. This gene regulation by tamoxifen is mediated by ERα and reversed by E2 or ICI 182,780. Introduction of ERβ into MCF-7 cells reverses tamoxifen action on ∼75% of these genes. To examine whether these genes might serve as markers of tamoxifen sensitivity and/or the development of resistance, their expression level was examined in breast cancers of women who had received adjuvant therapy with tamoxifen. High expression of two of the tamoxifen-stimulated genes, YWHAZ/14-3-3z and LOC441453, was found to correlate significantly with disease recurrence following tamoxifen treatment in women with ER-positive cancers and hence seem to be markers of a poor prognosis. Our data indicate a new dimension in tamoxifen action, involving gene expression regulation that is tamoxifen preferential, and identify genes that might serve as markers of tumor responsiveness or resistance to tamoxifen therapy. This may have a potential effect on the choice of tamoxifen versus aromatase inhibitors as adjuvant endocrine therapy. (Cancer Res 2006; 66(14): 7334-40)

Protocadherins (PCDH), localized to synaptic junctions, contribute to the formation of neuronal networks during brain development; thus, it is speculated that protocadherins may play a role in evolution of neuronal complexity. While protocadherin genes are highly conserved in vertebrates, EST evidence from the locus suggests apparently species-specific cis-antisense transcripts. Novel cis-antisense transcripts, which partially overlap the PCDHalpha12 variable exon, PCDHbeta3 single-exon gene, and PCDHpsi5 unprocessed pseudogene in the human 5q31 PCDHalpha/beta/gamma gene cluster and which are coexpressed with sense-strand transcripts in fetal and adult brain, were identified computationally and validated by gene-specific strand-specific reverse transcriptase PCR (SSRTPCR) and sequencing. Absence of antisense transcripts arising from equivalent genomic locations in mouse indicates that the antisense transcripts originated in the primates after the primate-rodent divergence. Furthermore, not all expected orthologues of human sense and antisense PCDH transcripts were detected in rhesus macaque brain, implying that protocadherin expression patterns differ between primate species. RT followed by quantitative real-time PCR (QPCR) analysis of the three genes in the brain of all three species, and of the PCDHbeta15 gene paralogous to PCDHpsi5 in human and rhesus, revealed that the presence of antisense transcripts was significantly associated with lower sense expression levels across all orthologues. This inverse relationship, along with the pattern of sense and antisense coexpression in the brain, is consistent with a regulatory role for the primate-specific PCDH cis-antisense transcripts, which may represent recent evolutionary inventions modulating the activity of this conserved gene cluster.

Progression of hepatocellular carcinoma (HCC) is a stepwise process that proceeds from pre-neoplastic lesions—including low-grade dysplastic nodules (LGDNs) and high-grade dysplastic nodules (HGDNs)—to advanced HCC. The molecular changes associated with this progression are unclear, however, and the morphological cues thought to distinguish pre-neoplastic lesions from well-differentiated HCC are not universally accepted. To understand the multistep process of hepato-carcinogenesis at the molecular level, we used oligo-nucleotide microarrays to investigate the transcription profiles of 50 hepatocellular nodular lesions ranging from LGDNs to primary HCC (Edmondson grades 1-3). We demonstrated that gene expression profiles can discriminate not only between dysplastic nodules and overt carcinoma but also between different histological grades of HCC via unsupervised hierarchical clustering with 10,376 genes. We identified 3,084 grade-associated genes, correlated with tumor progression, using one-way ANOVA and a one-versus-all unpooled t test. Functional assignment of these genes revealed discrete expression clusters representing grade-dependent biological properties of HCC. Using both diagonal linear discriminant analysis and support vector machines, we identified 240 genes that could accurately classify tumors according to histological grade, especially when attempting to discriminate LGDNs, HGDNs, and grade 1 HCC. In conclusion, a clear molecular demarcation between dysplastic nodules and overt HCC exists. The progression from grade 1 through grade 3 HCC is associated with changes in gene expression consistent with plausible functional consequences. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2005;42:809–818.)

Activation of p53 tumor suppressor induces either cell cycle arrest or apoptosis through transcription-dependent and independent pathways; however, their relative roles in apoptosis induction and how these pathways are regulated remains elusive. Here, we report a unique role for glycogen synthesis kinase-3β (GSK-3β) in regulating p53 functions in human colorectal cancer cells. Pharmacologic modulation of GSK-3β markedly impaired p53-dependent transactivation of targets including p21 and Puma but promoted p53-dependent conformational activation of Bax, resulting in cytochrome c release, loss of mitochondrial membrane potential, and caspase-9 processing. Thus, p53-mediated damage response is converted from cell cycle arrest to apoptosis following exposure to a variety of chemotherapeutic agents. We found that this effect is associated with the modulation of inhibitory Ser9 phosphorylation of GSK-3β but not with the activating tyrosine phosphorylation. We further show that the induction of apoptosis is through a direct mitochondrial pathway that requires Bax but not Puma. Our results underscore the importance of transcription-independent mechanism in p53-induced apoptosis and indicate that GSK-3β plays distinct dual roles in regulating p53 pathways: promoting p53 transcriptional activity in the nucleus but suppressing p53-mediated direct apoptotic function at the mitochondria. Importantly, our data suggest that small-molecule inhibition of GSK-3β might represent a novel approach for modulating chemotherapy.

We have exploited a discrepancy in the oncogenic potential of autocrine and exogenous human growth hormone (hGH) in an attempt to identify molecules that could potentially be involved in oncogenic transformation of the human mammary epithelial cell. Microarray analysis of 19,000 human genes identified a subset of 305 genes in a human mammary carcinoma cell line that were remarkably different in their response to autocrine and exogenous hGH. Autocrine and exogenous hGH also regulated 167 common genes. Semiquantitative reverse transcription-PCR confirmed differential regulation of genes by either autocrine or exogenous hGH. Functional analysis of one of the identified autocrine hGH-regulated genes, TFF3, determined that its expression is sufficient to support anchorage-independent growth of human mammary carcinoma cells. Small interfering RNA-mediated knockdown of TFF3 concordantly abrogated anchorage-independent growth of mammary carcinoma cells and abrogated the ability of autocrine hGH to stimulate oncogenic transformation of immortalized human mammary epithelial cells. Further functional characterization of the identified subset of specifically autocrine hGH regulated genes will delineate additional novel oncogenes for the human mammary epithelial cell.

Valproate (VPA) and trichostatin A (TSA), inhibitors of zinc-dependent deacetylase activity, induce reduction in the levels of mRNA encoding oestrogen receptor-alpha (ERalpha), resulting in subsequent clearance of ERalpha protein from breast and ovarian cell lines. Inhibition of oestrogen signalling may account for the endocrine disorders, menstrual abnormalities, osteoporosis and weight gain that occur in a proportion of women treated with VPA for epilepsy or for bipolar mood disorder. Transcriptome profiling revealed that VPA and TSA also modulate the expression of, among others, key regulatory components of the cell cycle. Meta-analysis of genes directly responsive to oestrogen indicates that VPA and TSA have a generally antioestrogenic profile in ERalpha positive cells. Concomitant treatment with cycloheximide prevented most of these changes in gene expression, including downregulation of ERalpha mRNA, indicating that a limited number of genes signal a hyperacetylated state within cells. Three members of the NAD-dependent deacetylases, the sirtuins, are upregulated by VPA and by TSA and sirtuin activity contributes to loss of ERalpha expression. However, prolonged inhibition of the sirtuins by sirtinol also induces loss of ERalpha from cells. Mechanistically, we show that VPA invokes reversible promoter shutoff of the ERalpha, pS2 and cyclin D1 promoters, by inducing recruitment of methyl cytosine binding protein 2 (MeCP2) with concomitant exclusion of the maintenance methylase DNMT1. Furthermore, we demonstrate that, in the presence of VPA, local DNA methylation, deacetylation and demethylation of activated histones and recruitment of inhibitory complexes occurs on the pS2 promoter.

Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found ∼140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC .

Human embryonic stem cells (hESCs) are an important source of stem cells in regenerative medicine, and much remains unknown about their molecular characteristics. To develop a detailed genomic profile of ESC lines in two different species, we compared transcriptomes of one murine and two different hESC lines by massively parallel signature sequencing (MPSS). Over 2 million signature tags from each line and their differentiating embryoid bodies were sequenced. Major differences and conserved similarities between species identified by MPSS were validated by reverse transcription polymerase chain reaction (RT-PCR) and microarray. The two hESC lines were similar overall, with differences that are attributable to alleles and propagation. Human-mouse comparisons, however, identified only a small (core) set of conserved genes that included genes known to be important in ESC biology, as well as additional novel genes. Identified were major differences in leukemia inhibitory factor, transforming growth factor-beta, and Wnt and fibroblast growth factor signaling pathways, as well as the expression of genes encoding metabolic, cytoskeletal, and matrix proteins, many of which were verified by RT-PCR or by comparing them with published databases. The study reported here underscores the importance of cross-species comparisons and the versatility and sensitivity of MPSS as a powerful complement to current array technology.

We have developed a DNA tag sequencing and mapping strategy called gene identification signature (GIS) analysis, in which 5' and 3' signatures of full-length cDNAs are accurately extracted into paired-end ditags (PETs) that are concatenated for efficient sequencing and mapped to genome sequences to demarcate the transcription boundaries of every gene. GIS analysis is potentially 30-fold more efficient than standard cDNA sequencing approaches for transcriptome characterization. We demonstrated this approach with 116,252 PET sequences derived from mouse embryonic stem cells. Initial analysis of this dataset identified hundreds of previously uncharacterized transcripts, including alternative transcripts of known genes. We also uncovered several intergenically spliced and unusual fusion transcripts, one of which was confirmed as a trans-splicing event and was differentially expressed. The concept of paired-end ditagging described here for transcriptome analysis can also be applied to whole-genome analysis of cis-regulatory and other DNA elements and represents an important technological advance for genome annotation.

Functional genomics refers to the study of whole genomes and the function of its constituent parts to explain biological processes. Though these investigations may involve whole proteome analysis, the primary focus is on the transcriptome and how it is regulated. Recent advances in technologies that can interrogate cellular transcripts on a genome-wide scale seek the complete disclosure of the transcriptome over time-intervals and across many different cellular states. This massively complex data when viewed as a whole can provide surprisingly precise assessment of cellular conditions. Moreover, these data can define hierarchies of importance and have shown us new transcriptional elements. Herein, we describe the technologies and the experimental strategies to study the transcriptome that would be pertinent to cancer and ageing research.

Activating mutations of KIT and platelet-derived growth factor receptor alpha (PDGFRA) are known to be alternative and mutually exclusive genetic events in the development of gastrointestinal stromal tumors (GISTs). We examined the effect of the mutations of these two genes on the gene expression profile of 22 GISTs using the oligonucleotide microarray. Mutations of KIT and PDGFRA were found in 17 cases and three cases, respectively. The remaining two cases had no detectable mutations in either gene. The mutation status of KIT and PDGFRA was directly related to the expression levels of activated KIT and PDGFRA, and was also related to the different expression levels of activated proteins that play key roles in the downstream of the receptor tyrosine kinase III family. To evaluate the impact of mutation status and the importance of the type of mutation in gene expression and clinical features, microarray-derived data from 22 GISTs were interpreted using a principal component analysis (PCA). Three relevant principal component representing mutation of KIT, PDGFRA and chromosome 14q deletion were identified from the interpretation of the oligonucleotide microarray data with PCA. After supervised analysis, there was at least a two fold difference in expression between GISTs with KIT and PDGFRA mutation in 70 genes. Our findings demonstrate that mutations of KIT and PDGFRA affect differential activation and expression of some genes, and can be used for the molecular classification of GISTs.

Previous studies transferring an intact chromosome 11 into HONE1 cells demonstrated the functional significance of chromosome regions, 11q13 and 11q22-23, in nasopharyngeal carcinoma (NPC) development. In our study the 11q22-23 region was comprehensively re-investigated by detailed microsatellite and single nucleotide polymorphism genotyping and by fluorescence in situ hybridization to map precisely the regions containing tumor suppressive activity. We observed 3 chromosomal intervals within 11q22-23 that were commonly lost in the tumor segregants derived from HONE1/chromosome 11 hybrids. One critical region of 0.36 Mb was mapped near the marker D11S2000 and a second 0.44 Mb region was located around the markers D11S1300 and D11S1391. In a third region high allelic loss was also observed at marker D11S4484, where a newly cloned tumor suppressor gene, TSLC1 (tumor suppressor in lung cancer 1), is located. The gene expression analysis showed absence or low expression levels of TSLC1 mRNA in 4 highly tumorigenic NPC cell lines. In addition, the methylation study results show that the TSLC1 promoter region was hypermethylated in all 4 NPC cell lines and re-expression of the gene occurs in HONE1 cells after 5-aza-2'-deoxycytidine treatment. Hence, the mode of silencing of this candidate TSG in NPC may be attributed to promoter hypermethylation. We have obtained functional evidence for multiple critical tumor suppressive regions in 11q22-23 by fine deletion mapping and for inactivation of TSLC1 being one of these candidate TSGs in NPC development.