Diverse mechanisms of somatic structural variations in human cancer genomes.

Identification of somatic rearrangements in cancer genomes has accelerated through analysis of high-throughput sequencing data. However, characterization of complex structural alterations and their underlying mechanisms remains inadequate. Here, applying an algorithm to predict structural variations from short reads, we report a comprehensive catalog of somatic structural variations and the mechanisms generating them, using high-coverage whole-genome sequencing data from 140 patients across ten tumor types. We characterize the relative contributions of different types of rearrangements and their mutational mechanisms, find that ~20% of the somatic deletions are complex deletions formed by replication errors, and describe the differences between the mutational mechanisms in somatic and germline alterations. Importantly, we provide detailed reconstructions of the events responsible for loss of CDKN2A/B and gain of EGFR in glioblastoma, revealing that these alterations can result from multiple mechanisms even in a single genome and that both DNA double-strand breaks and replication errors drive somatic rearrangements.

The somatic genomic landscape of glioblastoma.

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

Analyzing Somatic Genome Rearrangements in Human Cancers by Using Whole-Exome Sequencing.

Although exome sequencing data are generated primarily to detect single-nucleotide variants and indels, they can also be used to identify a subset of genomic rearrangements whose breakpoints are located in or near exons. Using >4,600 tumor and normal pairs across 15 cancer types, we identified over 9,000 high confidence somatic rearrangements, including a large number of gene fusions. We find that the 5' fusion partners of functional fusions are often housekeeping genes, whereas the 3' fusion partners are enriched in tyrosine kinases. We establish the oncogenic potential of ROR1-DNAJC6 and CEP85L-ROS1 fusions by showing that they can promote cell proliferation in vitro and tumor formation in vivo. Furthermore, we found that ∼4% of the samples have massively rearranged chromosomes, many of which are associated with upregulation of oncogenes such as ERBB2 and TERT. Although the sensitivity of detecting structural alterations from exomes is considerably lower than that from whole genomes, this approach will be fruitful for the multitude of exomes that have been and will be generated, both in cancer and in other diseases.

Integrated genomic characterization of endometrial carcinoma.

We performed an integrated genomic, transcriptomic and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies. Uterine serous tumours and ∼25% of high-grade endometrioid tumours had extensive copy number alterations, few DNA methylation changes, low oestrogen receptor/progesterone receptor levels, and frequent TP53 mutations. Most endometrioid tumours had few copy number alterations or TP53 mutations, but frequent mutations in PTEN, CTNNB1, PIK3CA, ARID1A and KRAS and novel mutations in the SWI/SNF chromatin remodelling complex gene ARID5B. A subset of endometrioid tumours that we identified had a markedly increased transversion mutation frequency and newly identified hotspot mutations in POLE. Our results classified endometrial cancers into four categories: POLE ultramutated, microsatellite instability hypermutated, copy-number low, and copy-number high. Uterine serous carcinomas share genomic features with ovarian serous and basal-like breast carcinomas. We demonstrated that the genomic features of endometrial carcinomas permit a reclassification that may affect post-surgical adjuvant treatment for women with aggressive tumours.

MIIP haploinsufficiency induces chromosomal instability and promotes tumour progression in colorectal cancer.

The gene encoding migration and invasion inhibitory protein (MIIP), located on 1p36.22, is a potential tumour suppressor gene in glioma. In this study, we aimed to explore the role and mechanism of action of MIIP in colorectal cancer (CRC). MIIP protein expression gradually decreased along the colorectal adenoma-carcinoma sequence and was negatively correlated with lymph node and distant metastasis in 526 colorectal tissue samples (p < 0.05 for all). Analysis of The Cancer Genome Atlas (TCGA) data showed that decreased MIIP expression was significantly associated with MIIP hemizygous deletion (p = 0.0005), which was detected in 27.7% (52/188) of CRC cases, and associated with lymph node and distant metastasis (p < 0.05 for both). We deleted one copy of the MIIP gene in HCT116 CRC cells using zinc finger nuclease technology and demonstrated that MIIP haploinsufficiency resulted in increased colony formation and cell migration and invasion, which was consistent with the results from siRNA-mediated MIIP knockdown in two CRC cell lines (p < 0.05 for all). Moreover, MIIP haploinsufficiency promoted CRC progression in vivo (p < 0.05). Genomic instability and spectral karyotyping assays demonstrated that MIIP haploinsufficiency induced chromosomal instability (CIN). Besides modulating the downstream proteins of APC/CCdc20 , securin and cyclin B1, MIIP haploinsufficiency inhibited topoisomerase II (Topo II) activity and induced chromosomal missegregation. Therefore, we report that MIIP is a novel potential tumour suppressor gene in CRC. Moreover, we characterized the MIIP gene as a novel CIN suppressor gene, through altering the stability of mitotic checkpoint proteins and disturbing Topo II activity. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Next-generation sequencing identifies rare variants associated with Noonan syndrome.

Noonan syndrome (NS) is a relatively common genetic disorder, characterized by typical facies, short stature, developmental delay, and cardiac abnormalities. Known causative genes account for 70-80% of clinically diagnosed NS patients, but the genetic basis for the remaining 20-30% of cases is unknown. We performed next-generation sequencing on germ-line DNA from 27 NS patients lacking a mutation in the known NS genes. We identified gain-of-function alleles in Ras-like without CAAX 1 (RIT1) and mitogen-activated protein kinase kinase 1 (MAP2K1) and previously unseen loss-of-function variants in RAS p21 protein activator 2 (RASA2) that are likely to cause NS in these patients. Expression of the mutant RASA2, MAP2K1, or RIT1 alleles in heterologous cells increased RAS-ERK pathway activation, supporting a causative role in NS pathogenesis. Two patients had more than one disease-associated variant. Moreover, the diagnosis of an individual initially thought to have NS was revised to neurofibromatosis type 1 based on an NF1 nonsense mutation detected in this patient. Another patient harbored a missense mutation in NF1 that resulted in decreased protein stability and impaired ability to suppress RAS-ERK activation; however, this patient continues to exhibit a NS-like phenotype. In addition, a nonsense mutation in RPS6KA3 was found in one patient initially diagnosed with NS whose diagnosis was later revised to Coffin-Lowry syndrome. Finally, we identified other potential candidates for new NS genes, as well as potential carrier alleles for unrelated syndromes. Taken together, our data suggest that next-generation sequencing can provide a useful adjunct to RASopathy diagnosis and emphasize that the standard clinical categories for RASopathies might not be adequate to describe all patients.

Characterization of HPV and host genome interactions in primary head and neck cancers.

Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twenty-five cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter- and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis.

Germline gain-of-function mutations in SOS1 cause Noonan syndrome.

Noonan syndrome, the most common single-gene cause of congenital heart disease, is characterized by short stature, characteristic facies, learning problems and leukemia predisposition. Gain-of-function mutations in PTPN11, encoding the tyrosine phosphatase SHP2, cause approximately 50% of Noonan syndrome cases. SHP2 is required for RAS-ERK MAP kinase (MAPK) cascade activation, and Noonan syndrome mutants enhance ERK activation ex vivo and in mice. KRAS mutations account for <5% of cases of Noonan syndrome, but the gene(s) responsible for the remainder are unknown. We identified missense mutations in SOS1, which encodes an essential RAS guanine nucleotide-exchange factor (RAS-GEF), in approximately 20% of cases of Noonan syndrome without PTPN11 mutation. The prevalence of specific cardiac defects differs in SOS1 mutation-associated Noonan syndrome. Noonan syndrome-associated SOS1 mutations are hypermorphs encoding products that enhance RAS and ERK activation. Our results identify SOS1 mutants as a major cause of Noonan syndrome, representing the first example of activating GEF mutations associated with human disease and providing new insights into RAS-GEF regulation.

Spectrum of somatic mitochondrial mutations in five cancers.

Somatic mtDNA mutations have been reported in some human tumors, but their spectrum in different malignancies and their role in cancer development remain incompletely understood. Here, we describe the breadth of somatic and inherited mutations across the mitochondrial genome by sequence analyses of paired tumor and normal tissue samples from 226 individuals with five types of cancer using whole-genome data generated by The Cancer Genome Atlas Research Network. The frequencies of deleterious tumor-specific somatic mutations found in mtDNA varied across tumor types, ranging from 13% of glioblastomas to 63% of rectal adenocarcinomas. Compared with inherited mtDNA variants, somatic mtDNA mutations were enriched for nonsynonymous vs. synonymous changes (93 vs. 15; P < 2.2E-16) and were predicted to functionally impact the encoded protein. Somatic missense mutations in tumors were distributed uniformly among the mitochondrial protein genes, but 65% of somatic truncating mutations occurred in NADH dehydrogenase 5. Analysis of staging data in colon and rectal cancers revealed that the frequency of damaging mitochondrial mutations is the same in stages I and IV tumors. In summary, these data suggest that damaging somatic mtDNA mutations occur frequently (13-63%) in these five tumor types and likely confer a selective advantage in oncogenesis.

Apc deficiency alters pulmonary epithelial cell fate and inhibits Nkx2.1 via triggering TGF-beta signaling.

Wnt signaling is critical for cell fate specification and cell differentiation in many organs, but its function in pulmonary neuroendocrine cell (PNEC) differentiation has not been fully addressed. In this study, we examined the role of canonical Wnt signaling by targeting the gene for Adenomatous Polyposis Coli (Apc), which controls Wnt signaling activity via mediating phosphorylation of beta-catenin (Ctnnb). Targeting the Apc gene in lung epithelial progenitors by Nkx2.1-cre stabilized Ctnnb and activated canonical Wnt signaling. Apc deficiency altered lung epithelial cell fate by inhibiting Clara and ciliated cell differentiation and activating Uchl1, a marker of neuroendocrine cells. Similar to PNEC in normal lung, Uchl1(positive) cells were innervated. In mice with targeted inactivation of Ctnnb by Nkx2.1-cre, PNEC differentiation was not interrupted. These indicate that, after lung primordium formation, Wnt signaling is not essential for PNEC differentiation; however, its over-activation promotes PNEC features. Interestingly, Nkx2.1 was extinguished in Apc deficient epithelial progenitors before activation of Uchl1. Examination of Nkx2.1 null lungs suggested that early deletion of Nkx2.1 inhibits PNEC differentiation, while late repression does not. Nkx2.1 was specifically inhibited in Apc deficient lungs but not in Ctnnb gain-of-function lungs indicating a functional difference between Apc deletion and Ctnnb stabilization, both of which activate Wnt signaling. Further analysis revealed that Apc deficiency led to increased TGF-beta signaling, which inhibited Nkx2.1 in cultured lung endodermal explants. In contrast, TGF-beta activity was not increased in Ctnnb gain-of-function lungs. Therefore, our studies revealed an important mechanism involving Apc and TGF-beta signaling in regulating the key transcriptional factor, Nkx2.1, for lung epithelial progenitor cell fate determination.

Impaired lymphatic contraction associated with immunosuppression.

To trigger an effective immune response, antigen and antigen-presenting cells travel to the lymph nodes via collecting lymphatic vessels. However, our understanding of the regulation of collecting lymphatic vessel function and lymph transport is limited. To dissect the molecular control of lymphatic function, we developed a unique mouse model that allows intravital imaging of autonomous lymphatic vessel contraction. Using this method, we demonstrated that endothelial nitric oxide synthase (eNOS) in lymphatic endothelial cells is required for robust lymphatic contractions under physiological conditions. By contrast, under inflammatory conditions, inducible NOS (iNOS)-expressing CD11b(+)Gr-1(+) cells attenuate lymphatic contraction. This inhibition of lymphatic contraction was associated with a reduction in the response to antigen in a model of immune-induced multiple sclerosis. These results suggest the suppression of lymphatic function by the CD11b(+)Gr-1(+) cells as a potential mechanism of self-protection from autoreactive responses during on-going inflammation. The central role for nitric oxide also suggests that other diseases such as cancer and infection may also mediate lymphatic contraction and thus immune response. Our unique method allows the study of lymphatic function and its molecular regulation during inflammation, lymphedema, and lymphatic metastasis.

Copy number variation detection in whole-genome sequencing data using the Bayesian information criterion.

DNA copy number variations (CNVs) play an important role in the pathogenesis and progression of cancer and confer susceptibility to a variety of human disorders. Array comparative genomic hybridization has been used widely to identify CNVs genome wide, but the next-generation sequencing technology provides an opportunity to characterize CNVs genome wide with unprecedented resolution. In this study, we developed an algorithm to detect CNVs from whole-genome sequencing data and applied it to a newly sequenced glioblastoma genome with a matched control. This read-depth algorithm, called BIC-seq, can accurately and efficiently identify CNVs via minimizing the Bayesian information criterion. Using BIC-seq, we identified hundreds of CNVs as small as 40 bp in the cancer genome sequenced at 10× coverage, whereas we could only detect large CNVs (> 15 kb) in the array comparative genomic hybridization profiles for the same genome. Eighty percent (14/16) of the small variants tested (110 bp to 14 kb) were experimentally validated by quantitative PCR, demonstrating high sensitivity and true positive rate of the algorithm. We also extended the algorithm to detect recurrent CNVs in multiple samples as well as deriving error bars for breakpoints using a Gibbs sampling approach. We propose this statistical approach as a principled yet practical and efficient method to estimate CNVs in whole-genome sequencing data.

Mutation in Rpa1 results in defective DNA double-strand break repair, chromosomal instability and cancer in mice.

Most cancers have multiple chromosomal rearrangements; the molecular mechanisms that generate them remain largely unknown. Mice carrying a heterozygous missense change in one of the DNA-binding domains of Rpa1 develop lymphoid tumors, and their homozygous littermates succumb to early embryonic lethality. Array comparative genomic hybridization of the tumors identified large-scale chromosomal changes as well as segmental gains and losses. The Rpa1 mutation resulted in defects in DNA double-strand break repair and precipitated chromosomal breaks as well as aneuploidy in primary heterozygous mutant mouse embryonic fibroblasts. The equivalent mutation in yeast is hypomorphic and semidominant and enhanced the formation of gross chromosomal rearrangements in multiple genetic backgrounds. These results indicate that Rpa1 functions in DNA metabolism are essential for the maintenance of chromosomal stability and tumor suppression.

Tbx1: identification of a 22q11.2 gene as a risk factor for autism spectrum disorder in a mouse model.

Although twin studies indicate clear genetic bases of autism spectrum disorder (ASD), the precise mechanisms through which genetic variations causally result in ASD are poorly understood. Individuals with 3 Mb and nested 1.5 Mb hemizygosity of the chromosome 22q11.2 represent genetically identifiable cases of ASD. However, because more than 30 genes are deleted even in the minimal deletion cases of 22q11.2 deficiency, the individual 22q11.2 gene(s) responsible for ASD remain elusive. Here, we examined the impact of constitutive heterozygosity of Tbx1, a 22q11.2 gene, on the behavioral phenotypes of ASD and characterized the regional and cellular expression of its mRNA and protein in mice. Congenic Tbx1 heterozygous (HT) mice were impaired in social interaction, ultrasonic vocalization, memory-based behavioral alternation, working memory and thigmotaxis, compared with wild-type (WT) mice. These phenotypes were not due to non-specific alterations in olfactory function, exploratory behavior, motor movement or anxiety-related behavior. Tbx1 mRNA and protein were ubiquitously expressed throughout the brains of C57BL/6J mice, but protein expression was enriched in regions that postnatally retain the capacity of neurogenesis, and in fact, postnatally proliferating cells expressed Tbx1. In postnatally derived hippocampal culture cells of C57BL/6J mice, Tbx1 levels were higher during proliferation than during differentiation, and expressed in neural progenitor cells, immature and matured neurons and glial cells. Taken together, our data suggest that Tbx1 is a gene responsible for the phenotypes of 22q11.2 hemizygosity-associated ASD possibly through its role in diverse cell types, including postnatally and prenatally generated neurons.

A novel mouse model of cerebral cavernous malformations based on the two-hit mutation hypothesis recapitulates the human disease.

Cerebral cavernous malformations (CCMs) are vascular lesions of the central nervous system appearing as multicavernous, blood-filled capillaries, leading to headache, seizure and hemorrhagic stroke. CCM occurs either sporadically or as an autosomal dominant disorder caused by germline mutation of one of the three genes: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. Surgically resected human CCM lesions have provided molecular and immunohistochemical evidence for a two-hit (germline plus somatic) mutation mechanism. In contrast to the equivalent human genotype, mice heterozygous for a Ccm1- or Ccm2-null allele do not develop CCM lesions. Based on the two-hit hypothesis, we attempted to improve the penetrance of the model by crossing Ccm1 and Ccm2 heterozygotes into a mismatch repair-deficient Msh2(-/-) background. Ccm1(+/-)Msh2(-/-) mice exhibit CCM lesions with high penetrance as shown by magnetic resonance imaging and histology. Significantly, the CCM lesions range in size from early-stage, isolated caverns to large, multicavernous lesions. A subset of endothelial cells within the CCM lesions revealed somatic loss of CCM protein staining, supporting the two-hit mutation mechanism. The late-stage CCM lesions displayed many of the characteristics of human CCM lesions, including hemosiderin deposits, immune cell infiltration, increased endothelial cell proliferation and increased Rho-kinase activity. Some of these characteristics were also seen, but to a lesser extent, in early-stage lesions. Tight junctions were maintained between CCM lesion endothelial cells, but gaps were evident between endothelial cells and basement membrane was defective. In contrast, the Ccm2(+/-)Msh2(-/-) mice lacked cerebrovascular lesions. The CCM1 mouse model provides an in vivo tool to investigate CCM pathogenesis and new therapies.

In vivo wide-area cellular imaging by side-view endomicroscopy.

In vivo imaging of small animals offers several possibilities for studying normal and disease biology, but visualizing organs with single-cell resolution is challenging. We describe rotational side-view confocal endomicroscopy, which enables cellular imaging of gastrointestinal and respiratory tracts in mice and may be extensible to imaging organ parenchyma such as cerebral cortex. We monitored cell infiltration, vascular changes and tumor progression during inflammation and tumorigenesis in colon over several months.

Cis lethal genetic interactions attenuate and alter p53 tumorigenesis.

Rpa1, an essential gene involved in DNA replication and genome maintenance, is syntenic and linked to Trp53 in mice and humans. To study the genetic interaction between Rpa1 and Trp53 in tumorigenesis, we generated compound Rpa1(L230P/+); Trp53(+/-) mutant mice with the mutant alleles in either trans or cis configuration. We demonstrate that the Rpa1(L230P) missense mutation significantly alters the tumor phenotype and spectrum of Trp53 mutant mice by modifying the genetic mechanisms underlying tumorigenesis. Importantly, when the Rpa1(L230P) and Trp53 mutant alleles are in cis, the tumor phenotype is attenuated and altered and loss of heterozygosity (LOH) at the Trp53 wild-type locus is selected against, whereas in the trans configuration, Rpa1(L230P) enhances the Trp53(+/-) tumor phenotype even though Rpa1(L230P) is ultimately lost by LOH. These studies indicate that polymorphic genetic variants in cell essential genes can genetically affect closely linked tumor suppressor loci via allelic phasing, which can result in profound phenotypic variations in tumorigenesis.

Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment.

Most genetically engineered mouse (GEM) models for colon cancer are based on tissuewide or germline gene modification, resulting in tumors predominantly of the small intestine. Several of these models involve modification of the adenomatous polyposis coli (Apc) gene and are excellent models for familial cancer predisposition syndromes. We have developed a stochastic somatic mutation model for sporadic colon cancer that presents with isolated primary tumors in the distal colon and recapitulates the entire adenoma-carcinoma-metastasis axis seen in human colon cancer. Using this model, we have analyzed tumors that are either solely mutant in the Apc gene or in combination with another colon cancer-associated mutant gene, the Kras G12D allele. Because of the restricted location in the distal colon, the natural history of the tumors can be analyzed by serial colonoscopy. As the mammalian target of rapamycin (mTOR) pathway is a critical component of the complex signaling network in colon cancer, we used this model to assess the efficacy of mTOR blockade through rapamycin treatment of mice with established tumors. After treatment, Apc mutant tumors were more than 80% smaller than control tumors. However, tumors that possessed both Apc and Kras mutations did not respond to rapamycin treatment. These studies suggest that mTOR inhibitors should be further explored as potential colorectal cancer therapies in patients whose tumors do not have activating mutations in KRAS.

In vivo optical molecular imaging of matrix metalloproteinase activity following celecoxib therapy for colorectal cancer.

We present an optical molecular imaging approach to measure the efficacy of the cyclooxygenase-2 (COX-2) inhibitor celecoxib on tumor growth rate through its effect on matrix metalloproteinase (MMP) activity. A xenograft model of colorectal cancer was generated in nude mice, which were then randomized to receive celecoxib versus vehicle. MMP activity was measured by an enzyme-activatable optical molecular probe. A novel genetically engineered mouse (GEM) model of colorectal cancer was also used to assess celecoxib's effect on MMP activity, which was measured by quantitative fluorescence colonoscopy. Subcutaneously implanted xenograft tumors were 84% (SD 20.2%) smaller in volume in the treatment group versus the control group. Moreover, treated animals exhibited only a 7.6% (SEM 9%) increase in MMP activity versus 106% (SEM 8%) for untreated animals. There was an apparent linear relationship (r  =  .91) between measured MMP activity and tumor growth rate. Finally, in the GEM model experiment, treated murine tumors remained relatively unchanged in volume and MMP activity; however, untreated tumors grew significantly and showed an increase in MMP activity. This method may provide for the improved identification of patients for whom COX-2 inhibition therapy is indicated by allowing one to balance the patient's cardiovascular risk with the cancer's responsiveness to celecoxib.