Defining the genomic landscape of head and neck cancers through next-generation sequencing.

Next-generation sequencing (NGS) has revolutionized the field of genomics and improved our understanding of cancer biology. Advances have been achieved by sequencing tumor DNA and using matched normal DNA to filter out germ line variants to identify cancer-specific changes. The identification of high incidences of activating mutations in head and neck squamous cell carcinoma (HNSCC) amenable to drug targeting has been made, with clear distinctions between the mutational profile of HPV-positive and HPV-negative tumors. This wealth of new understanding undoubtedly ameliorates our understanding of HNSCC cancer biology and elucidates clear targets for drug targeting which will guide future personalized medicine.

Dissection of the C-terminal region of E1A redefines the roles of CtBP and other cellular targets in oncogenic transformation.

Human adenovirus E1A makes extensive connections with the cellular protein interaction network. By doing so, E1A can manipulate many cellular programs, including cell cycle progression. Through these reprogramming events, E1A functions as a growth-promoting oncogene and has been used extensively to investigate mechanisms contributing to oncogenesis. Nevertheless, it remains unclear how the C-terminal region of E1A contributes to oncogenic transformation. Although this region is required for transformation in cooperation with E1B, it paradoxically suppresses transformation in cooperation with activated Ras. Previous analysis has suggested that the interaction of E1A with CtBP plays a pivotal role in both activities. However, some C-terminal mutants of E1A retain CtBP binding and yet exhibit defects in transformation, suggesting that other targets of this region are also necessary. To explore the roles of these additional factors, we performed an extensive mutational analysis of the C terminus of E1A. We identified key residues that are specifically required for binding all known targets of the C terminus of E1A. We further tested each mutant for the ability to both localize to the nucleus and transform primary rat cells in cooperation with E1B-55K or Ras. Interaction of E1A with importin α3/Qip1, dual-specificity tyrosine-regulated kinase 1A (DYRK1A), HAN11, and CtBP influenced transformation with E1B-55K. Interestingly, the interaction of E1A with DYRK1A and HAN11 appeared to play a role in suppression of transformation by activated Ras whereas interaction with CtBP was not necessary. This unexpected result suggests a need for revision of current models and provides new insight into transformation by the C terminus of E1A.

The epidemic of human papillomavirus and oropharyngeal cancer in a Canadian population.

BACKGROUND: Sexually transmitted infection with the human papillomavirus (hpv) is responsible for a significant burden of human cancers involving the cervix, anogenital tract, and oropharynx. Studies in the United States and Europe have demonstrated an alarming increase in the frequency of hpv-positive oropharyngeal cancer, but the same direct evidence does not exist in Canada. METHODS: Using the London Health Sciences Centre pathology database, we identified tonsillar cancers diagnosed between 1993 and 2011. Real-time polymerase chain reaction was then used on pre-treatment primary-site biopsy samples to test for dna from the high-risk hpv types 16 and 18. The study cohort was divided into three time periods: 1993-1999, 2000-2005, and 2006-2011. RESULTS: Of 160 tumour samples identified, 91 (57%) were positive for hpv 16. The total number of tonsillar cancers significantly increased from 1993-1999 to 2006-2011 (32 vs. 68), and the proportion of cases that were hpv-positive substantially increased (25% vs. 62%, p < 0.002). Those changes were associated with a marked improvement in 5-year overall survival (39% in 1993-1999 vs. 84% in 2006-2011, p < 0.001). When all factors were included in a multivariable model, only hpv status predicted treatment outcome. INTERPRETATION: The present study is the first to provide direct evidence that hpv-related oropharyngeal cancer is increasing in incidence in a Canadian population. Given the long lag time between hpv infection and clinically apparent malignancy, oropharyngeal cancer will be a significant clinical problem for the foreseeable future despite vaccination efforts.

Establishment of four head and neck squamous cell carcinoma cell lines: importance of reference DNA for accurate genomic characterisation.

OBJECTIVE: There is significant interest in developing early passage cell lines with matched normal reference DNA to facilitate a precision medicine approach in assessing drug response. This study aimed to establish early passage cell lines, and perform whole exome sequencing and short tandem repeat profiling on matched normal reference DNA, primary tumour and corresponding cell lines. METHODS: A cell culture based, in vitro study was conducted of patients with primary human papillomavirus positive and human papillomavirus negative tumours. RESULTS: Four early passage cell lines were established. Two cell lines were human papillomavirus positive, confirmed by sequencing and p16 immunoblotting. Short tandem repeat profiling confirmed that all cell lines were established from their index tumours. Whole exome sequencing revealed that the matched normal reference DNA was critical for accurate mutational analysis: a high rate of false positive mutation calls were excluded (87.6 per cent). CONCLUSION: Early passage cell lines were successfully established. Patient-matched reference DNA is important for accurate cell line mutational calls.

pRB-E2F1 complexes are resistant to adenovirus E1A-mediated disruption.

Disruption of pRB-E2F interactions by E1A is a key event in the adenoviral life cycle that drives expression of early viral transcription and induces cell cycle progression. This function of E1A is complicated by E2F1, an E2F family member that controls multiple processes besides proliferation, including apoptosis and DNA repair. Recently, a second interaction site in pRB that only contacts E2F1 has been discovered, allowing pRB to control proliferation separately from other E2F1-dependent activities. Based on this new insight into pRB-E2F1 regulation, we investigated how E1A affects control of E2F1 by pRB. Our data reveal that pRB-E2F1 interactions are resistant to E1A-mediated disruption. Using mutant forms of pRB that selectively force E2F1 to bind through only one of the two binding sites on pRB, we determined that E1A is unable to disrupt E2F1's unique interaction with pRB. Furthermore, analysis of pRB-E2F complexes during adenoviral infection reveals the selective maintenance of pRB-E2F1 interactions despite the presence of E1A. Our experiments also demonstrate that E2F1 functions to maintain cell viability in response to E1A expression. This suggests that adenovirus E1A's seemingly complex mechanism of disrupting pRB-E2F interactions provides selectivity in promoting viral transcription and cell cycle advancement, while maintaining cell viability.

p400 function is required for the adenovirus E1A-mediated suppression of EGFR and tumour cell killing.

We have recently shown that E1A protein of human adenovirus downregulates epidermal growth factor receptor (EGFR) expression and induces apoptosis in head and neck (HNSCC) and lung cancer cells independently of their p53 status. E1A has five isoforms of which the major ones E1A12S and E1A13S regulate transcription of cellular genes by binding to transcriptional modulators such as pRB, CtBP, p300 and p400. In this study, we have identified E1A12S isoform to have the highest effect on EGFR suppression and induction of apoptosis in HNSCC cells. Similar to Ad5, E1A12S from human adenovirus types 2, 3, 9 and 12 suppressed EGFR, whereas E1A12S of adenovirus types 4 and 40 had no effect on EGFR expression. Using deletion mutants of E1A12S we have shown that interaction of E1A with p400, but not p300 or pRB, is required for EGFR suppression and apoptosis. Inhibition of p400 by short hairpin RNA confirmed that HNSCC cells with reduced p400 expression were less sensitive to E1A-induced suppression of EGFR and apoptosis. p300 function was shown to be dispensable, as cells expressing E1A mutants that are unable to bind p300, or p300 knockout cells, remained sensitive to E1A-induced apoptosis. In summary, this study identifies p400 as an important mediator of E1A-induced downregulation of EGFR and apoptosis.

Mouse mammary tumor virus chromatin in human breast cancer cells is constitutively hypersensitive and exhibits steroid hormone-independent loading of transcription factors in vivo.

We have stably introduced a reporter gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) into human T47D breast cancer cells to study the action of the progesterone receptor (PR) on transcription from a chromatin template. Unexpectedly, the chromatin organization of the MMTV LTR in these human breast cancer cells differed markedly from what we have observed previously. The region adjacent to the transcription start site (-221 to -75) was found to be constitutively hypersensitive to restriction enzyme cleavage in the absence of hormone. This region is normally encompassed within the second nucleosome of a phased array of six nucleosomes that is assembled when the MMTV LTR is stably maintained in mouse cells. Characteristically, in these rodent cells, the identical DNA sequences show increased restriction enzyme cleavage only in the presence of glucocorticoid. The increased access of restriction enzymes observed in the human PR+ cells was not observed in adjacent nucleosomes and was unaffected by treatment with the progesterone antagonist RU486. In addition, exonuclease III-dependent stops corresponding to the binding sites for nuclear factor 1 and the PR were observed before and after hormone treatment. These results indicate that MMTV chromatin replicated in these cells is organized into a constitutively open architecture and that this open chromatin state is accompanied by hormone-independent loading of a transcription factor complex that is normally excluded from uninduced chromatin.

Interaction of the E1A oncoprotein with Yak1p, a novel regulator of yeast pseudohyphal differentiation, and related mammalian kinases.

The C-terminal portion of adenovirus E1A suppresses ras-induced metastasis and tumorigenicity in mammalian cells; however, little is known about the mechanisms by which this occurs. In the simple eukaryote Saccharomyces cerevisiae, Ras2p, the homolog of mammalian h-ras, regulates mitogen-activated protein kinase (MAPK) and cyclic AMP-dependent protein kinase A (cAMP/PKA) signaling pathways to control differentiation from the yeast form to the pseudohyphal form. When expressed in yeast, the C-terminal region of E1A induced pseudohyphal differentiation, and this was independent of both the MAPK and cAMP/PKA signaling pathways. Using the yeast two-hybrid system, we identified an interaction between the C-terminal region of E1A and Yak1p, a yeast dual-specificity serine/threonine protein kinase that functions as a negative regulator of growth. E1A also physically interacts with Dyrk1A and Dyrk1B, two mammalian homologs of Yak1p, and stimulates their kinase activity in vitro. We further demonstrate that Yak1p is required in yeast to mediate pseudohyphal differentiation induced by Ras2p-regulated signaling pathways. However, pseudohyphal differentiation induced by the C-terminal region of E1A is largely independent of Yak1p. These data suggest that mammalian Yak1p-related kinases may be targeted by the E1A oncogene to modulate cell growth.

Ability of adenovirus 5 E1A proteins to suppress differentiation of BC3H1 myoblasts correlates with their binding to a 300 kDa cellular protein.

We have used deletion mutants to define the regions in Ad5 E1A proteins necessary to suppress differentiation of mouse BC3H1 myoblasts. We examined the differentiation of cells infected at a low multiplicity with viruses containing the E1A deletions and constructed so as to produce only the smaller of the two major E1A proteins. Only four of the mutant viruses containing deletions within the N-terminal 69 residues failed to suppress differentiation as judged by changes in morphology and in levels of muscle-specific alpha-actin mRNA and creatine kinase activity. The results were confirmed by analyses of lines of cells stably transfected with representative E1A mutants. The mouse cellular proteins to which mutant E1A proteins bound were identified by immunoprecipitating E1A proteins specifically from infected BC3H1 cells and by analyzing the precipitates on denaturing gels. Bands of proteins of 300, 130, 107, 105 (the retinoblastoma product), and 60 kDa (cyclin A) were distinguished. Failure to suppress differentiation correlated with loss of binding to the 300-kDa protein but not to any of the others. The regions of E1A defined in this way have been shown to be required for several other activities, including enhancer repression and transformation. One function of the 300-kDa protein appears to be to facilitate the action of transcriptional enhancers of differentiation-specific genes.

Tumour suppressive properties of the adenovirus 5 E1A oncogene.

The transforming oncogenes of DNA tumour viruses have proven useful as tools to dissect the mechanisms of complex cellular processes. In particular, studies of the multifunctional proteins encoded by the early region 1A (E1A) of human adenovirus types 2 and 5 have provided insight into the regulation of cellular gene expression, growth and differentiation. Despite their well known ability to immortalize primary rodent cells and transform them in cooperation with a second oncogene, the E1A proteins also exhibit significant anti-tumour/tumour suppressive activity. This review focuses on the surprising ability of E1A to function as a tumour suppressor gene.

Induction of apoptosis by adenovirus type 5 E1A in rat cells requires a proliferation block.

Infection with Ad5dl520EIB-, an adenovirus producing only the 243 residue E1A protein and lacking the E1B region, caused apoptosis in normal rat kidney (NRK) cells as judged by the production of nucleosomal DNA fragments. Apoptosis occurred only when the cells were growth-inhibited by cell-cell contacts in confluent cultures or by serum starvation and not when they were actively growing. In uninfected cultures, apoptosis also occurred at confluency, but more slowly than after infection. Studies with E1A deletion mutants of dl520E1B- showed that the regions of the E1A protein essential for induction of apoptosis were those in exon 1 required for binding to the cellular proteins p300 and pRb. Mutants defective at inducing apoptosis were previously found to be defective at inducing baby rat kidney cells to synthesize cellular DNA. In our experiments, cells underwent apoptosis when stimulated by E1A to proliferate under conditions where proliferation was blocked. It is possible that it was the proliferation block opposing the induction of proliferation that led directly to apoptosis. Circumstances leading to induction of apoptosis by c-myc (Evan et al., 1992) are similar and can be interpreted in a similar way.

Adenovirus early region 1A protein binds to mammalian SUG1-a regulatory component of the proteasome.

Adenovirus early region 1A (Ad E1A) is a multifunctional protein which is essential for adenovirus-mediated transformation and oncogenesis. Whilst E1A is generally considered to exert its influence on recipient cells through regulation of transcription it also increases the level of cellular p53 by increasing the protein half-life. With this in view, we have investigated the relationship of Ad E1A to the proteasome, which is normally responsible for degradation of p53. Here we have shown that both Ad5 and Ad12 E1A 12S and 13S proteins can be co-immunoprecipitated with proteasomes and that the larger Ad12 E1A protein binds strongly to at least three components of the 26S but not 20S proteasome. One of these interacting species has been identified as mammalian SUGI, a proteasome regulatory component which also plays a role in the cell as a mediator of transcription. In vitro assays have demonstrated a direct interaction between Ad12 E1A 13S protein and mouse SUGI. Following infection of human cells with Ad5 wt and Ad5 mutants with lesions in the E1A gene it has been shown that human SUG1 can be co-immunoprecipitated with full-length E1A and with E1A carrying a deletion in conserved region 1 which is the region considered to be responsible for increased expression of p53. We have concluded therefore that Ad EIA binds strongly to SUGI but that this interaction is not responsible for inhibition of proteasome activity. This is consistent with the observation that purified Ad12 E1A inhibits the activity of the purified 20S but not 26S proteasomes. We have also demonstrated that SUGI can be co-immunoprecipitated with SV40 T and therefore we suggest that this may represent a common interaction of transforming proteins of DNA tumour viruses.

Influence of hormone antagonists on chromatin remodeling and transcription factor binding to the mouse mammary tumor virus promoter in vivo.

Steroid hormones act via a group of high affinity receptors that regulate transcription by binding to hormone response elements located in the promoters of hormone-inducible genes. Our understanding of these processes has been greatly enhanced by the use of steroid hormone antagonists in both clinical and experimental procedures. However, despite their usefulness in these applications, much about their mechanisms of action remains to be elucidated. Using in vivo analysis techniques, we investigated the influence of type I (ZK98299) and type II (RU486 and ZK112993) steroid hormone antagonists on glucocorticoid-regulated transcription from the mouse mammary tumor virus promoter. Both type I and type II antagonists substantially reduced glucocorticoid-induced expression from the mouse mammary tumor virus promoter stably maintained as chromatin. Concurrent treatment with glucocorticoid and type I or type II antagonists reduced the receptor-dependent chromatin remodeling and loading of transcription factor NF1 that are signature responses of this promoter in the context of chromatin. Treatment with either type I or type II antagonists alone did not induce chromatin remodeling or transcription factor loading. Although type II antagonist-occupied receptor can bind DNA, our results show that this binding is not functionally equivalent to that of agonist-occupied receptor, as it can not interact productively with the cellular apparatus required to open a repressive chromatin structure.

Differential steroid hormone induction of transcription from the mouse mammary tumor virus promoter.

The Mouse Mammary Tumor Virus (MMTV) contains sequences in its proximal promoter region to which both glucocorticoid and progesterone receptors can bind. In transient transfection experiments both hormones are able to stimulate transcription from reporter plasmids containing either native or consensus hormone response elements (glucocorticoid response element/progesterone response element). Previous experiments have demonstrated that the MMTV long terminal repeat is reproducibly assembled into a phased array of nucleosomes when stably introduced into cells. Stimulation by glucocorticoids of endogenous templates led to a rapid but transient increase in transcription initiation and mRNA accumulation that can be correlated with increased sensitivity to restriction enzymes. In contrast, experiments using progesterone or a truncated glucocorticoid receptor failed to elicit a similar increase in mRNA levels as dexamethasone from stable chromatin templates. In an attempt to understand this differential response, we have compared the responsiveness of the MMTV promoter to glucocorticoids and progesterone when it is organized in either stable chromatin or in transiently acquired plasmids. Our results demonstrate that the native chromatin structure prevents activation of this locus by progesterone, but permits stimulation by glucocorticoids.

Multiple pathways for activation of E2A expression in human KB cells by the 243R E1A protein of adenovirus type 5.

Adenovirus type 5 (Ad5) mutant dl520, which produces only the smaller 243 residue (243R) E1A protein, induced efficient production of the viral E2A 72-kDa DNA binding protein (DBP) in human KB cells, but not in human WI38, 143, or HeLa cells. In transient expression assays, the 243R E1A protein induced transcription from the E2 early promoter in KB but not in HeLa cells; there was no transcription from the E3 promoter in either cell line. In KB cells, truncation of the E2 promoter from -285 to -97 basepairs dramatically reduced transactivation by the 243R E1A product but not by wt E1A, suggesting that the 243R protein acts through factors binding in this region. Multiple deletions in both exon 1 and exon 2 of the 243R E1A protein failed to disrupt its ability to induce DBP expression. The possible redundant pathways for this induction are discussed. The multiplicity of these pathways and the fact that they are all inactivated in the WI38 and 143 lines are surprising.

Adenovirus e1a proteins and transformation (review).

The E1A gene region of human adenovirus can cooperate with a second gene such as adenovirus E1B or activated ras to transform rodent cells oncogenically. On its own, E1A induces quiescent cells to divide, represses cellular differentiation, induces programmed cell death and affects gene expression. E1A acts through its products, two similar nuclear proteins of 289 and 243 residues, which bind to cellular proteins, particularly p300 and a group of interrelated proteins, p130, p107 and pRb. This review describes what is known of the ways E1A interferes with growth regulation by these and other cellular proteins, such as cyclins and transcription factors, so as to bring about oncogenic transformation.

Steroid hormone receptor status defines the MMTV promoter chromatin structure in vivo.

The ability to respond to small signalling molecules such as steroid hormones is important for many physiological processes. Steroid hormones act through a group of high affinity receptors that regulate transcription by binding to hormone response elements (HREs) located within the promoters of target genes, which themselves are organized with nuclear proteins to form chromatin. To dissect the mechanisms(s) of steroid hormone action we have used the steroid inducible mouse mammary tumor virus (MMTV) promoter as a model system. The MMTV promoter is assembled into a phased array of nucleosomes that are specifically positioned in rodent cells. Induction of transcription by glucocorticoids is accompanied by the appearance of a hypersensitive region in the proximal promoter which allows the hormone dependent assembly of a preinitiation complex including transcription factors such as nuclear factor 1 (NF1) and the octamer transcription factor (OTF). Surprisingly, when introduced by transient transfection, the progesterone receptor (PR) is unable to activate this promoter in vivo, a finding that may result from its inability to alter MMTV promoter chromatin. In an attempt to investigate the failure of the PR to activate the promoter, we have stably introduced the MMTV promoter into human T47D breast cancer cells that express high levels of the PR. In contrast to what has been observed previously in rodent cells, the MMTV templates resident in human breast cancer cells adopt a novel and constitutively open chromatin structure. The constitutively open chromatin structure is accompanied by the hormone independent loading of transcription factors including the PR and NF1. In T47D cells that stably express the glucocorticoid receptor, the MMTV promoter responds to glucocorticoids, but not progestins, and displays glucocorticoid induced restriction enzyme hypersensitivity and transcription factor loading. These findings suggest that the organization of the MMTV chromatin structure is dependent upon the cell type and receptor status of the recipient cell into which the MMTV promoter is stably introduced.

Adenovirus evasion of interferon-mediated innate immunity by direct antagonism of a cellular histone posttranslational modification.

Overcoming the cellular type I interferon (IFN) host defense response is critical for a virus to ensure successful infection. Investigating the effects of human adenovirus (HAdV) infection on global cellular histone posttranslational modification (hPTM), we discovered that virus infection-induced activation of IFN signaling triggers a global increase in the monoubiquitination of histone 2B (H2B) at lysine 120, which is a mark for transcriptionally active chromatin. This hPTM, catalyzed by the hBre1/RNF20 complex, is necessary for activation of the cellular IFN-stimulated gene (ISG) expression program in response to viruses. To establish effective infection, the HAdV E1A protein binds to and dissociates the hBre1 complex to block IFN-induced H2B monoubiquitination and associated ISG expression. Together, these data uncover a key role for H2B monoubiquitination in the type I IFN response and a viral mechanism of antagonizing this hPTM to evade the IFN response.

LINA: a laboratory inventory system for oligonucleotides, microbial strains, and cell lines.

In this article, we present the Laboratory Inventory Network Application (LINA), a software system that assists research laboratories in keeping track of their collections of biologically relevant materials. This open source application uses relational Microsoft Access database technology as a back end and a Microsoft .NET application as a front end. Preconstructed table templates are provided that contain standardized and customizable data fields. As new samples are added to the inventory, each is provided with a unique laboratory identifier, which is assigned automatically and sequentially, allowing rapid retrieval when a given reagent is required. The LINA contains a number of useful search tools including a general search, which allows database searches using up to four user-defined criteria. The LINA represents an easily implemented and useful organizational tool for biological laboratories with large numbers of strains, clones, or other reagents.

Identification of a molecular recognition feature in the E1A oncoprotein that binds the SUMO conjugase UBC9 and likely interferes with polySUMOylation.

Hub proteins have central roles in regulating cellular processes. By targeting a single cellular hub, a viral oncogene may gain control over an entire module in the cellular interaction network that is potentially comprised of hundreds of proteins. The adenovirus E1A oncoprotein is a viral hub that interacts with many cellular hub proteins by short linear motifs/molecular recognition features (MoRFs). These interactions transform the architecture of the cellular protein interaction network and virtually reprogram the cell. To identify additional MoRFs within E1A, we screened portions of E1A for their ability to activate yeast pseudohyphal growth or differentiation. This identified a novel functional region within E1A conserved region 2 comprised of the sequence EVIDLT. This MoRF is necessary and sufficient to bind the N-terminal region of the SUMO conjugase UBC9, which also interacts with SUMO noncovalently and is involved in polySUMOylation. Our results suggest that E1A interferes with polySUMOylation, but not with monoSUMOylation. These data provide the first insight into the consequences of the interaction of E1A with UBC9, which was initially described in 1996. We further demonstrate that polySUMOylation regulates pseudohyphal growth and promyelocytic leukemia body reorganization by E1A. In conclusion, the interaction of the E1A oncogene with UBC9 mimics the normal binding between SUMO and UBC9 and represents a novel mechanism to modulate polySUMOylation.