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1.
Oncologist ; 29(2): e224-e236, 2024 Feb 02.
Article in English | MEDLINE | ID: mdl-37682776

ABSTRACT

BACKGROUND: FoundationOneCDx is approved in the US and Japan as a companion diagnostic test to identify patients with cancer who may benefit from treatment with 30 drug therapies in the US and 23 in Japan. Tumor profiling with FoundationOneCDx also detects genomic findings with evidence of clinical significance that may inform clinical care decisions beyond companion diagnostic claims. This observational study reports the breadth and impact of clinical decision insights from FoundationOneCDx solid tumor profiles. MATERIALS AND METHODS: Consecutive test result reports for patients with solid tumor diagnoses (n = 109 695) were retrospectively analyzed for clinically significant predictive, prognostic, and diagnostic genomic alterations and signatures, determined in accordance with professional guidelines. Interventional clinical trials with targeted therapies or immune checkpoint inhibitors were matched to tumor profiles based on evidence that the genomic finding may be an actionable, investigational, or hypothetical target in the patient's tumor type. RESULTS: In 14 predefined cancer types (80.7% of analyzed solid tumors), predictive, prognostic, and diagnostic markers were reported in 47.6%, 13.2%, and 4.5% of samples, respectively, accounting for a total of 51.2% of tumor profiles. Pan-cancer predictive markers of tumor mutational burden (TMB) of 10 or more mutations per megabase, high microsatellite instability (MSI), or NTRK1/2/3 fusions were observed in 15.6%, 2.0%, and 0.1% of solid tumors, respectively. Most solid tumor profiles (89.2%) had genomic results that could theoretically inform decisions on the selection of immunotherapy and targeted therapy clinical trials. CONCLUSION: For this real-world population of patients with FoundationOneCDx solid tumor profiles in the routine course of clinical care, clinically significant findings were reported for approximately half of patients with genomic results.


Subject(s)
Clinical Relevance , Neoplasms , Humans , Retrospective Studies , Neoplasms/pathology , Mutation , Biomarkers, Tumor/genetics , Genomics/methods , High-Throughput Nucleotide Sequencing/methods
2.
Viruses ; 9(12)2017 12 19.
Article in English | MEDLINE | ID: mdl-29257050

ABSTRACT

The most highly expressed protein during the productive phase of the human papillomavirus (HPV) life cycle is E1^E4. Its full role during infection remains to be established. HPV E1^E4 is expressed during both the early and late stages of the virus life cycle and contributes to viral genome amplification. In an attempt to further outline the functions of E1^E4, and determine whether it plays a role in viral capsid assembly and viral infectivity, we examined wild-type E1^E4 as well as four E1^E4 truncation mutants. Our study revealed that HPV18 genomes containing the shortest truncated form of E1^E4, the 17/18 mutant, produced viral titers that were similar to wild-type virus and significantly higher compared to virions containing the three longer E1^E4 mutants. Additionally, the infectivity of virus containing the shortest E1^E4 mutation was equivalent to wild-type and significantly higher than the other three mutants. In contrast, infectivity was completely abrogated for virus containing the longer E1^E4 mutants, regardless of virion maturity. Taken together, our results indicate for the first time that HPV18 E1^E4 impacts capsid assembly and viral infectivity as well as virus maturation.


Subject(s)
Capsid/metabolism , Human papillomavirus 18/genetics , Human papillomavirus 18/physiology , Mutation , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Viral/genetics , Virus Assembly , Cells, Cultured , Fibroblasts/virology , Humans , Microbial Viability , Viral Load
3.
PLoS Pathog ; 13(10): e1006661, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28968467

ABSTRACT

Expression of E7 proteins encoded by carcinogenic, high-risk human papillomaviruses (HPVs) triggers increased expression of the histone H3 lysine 27 demethylase KDM6A. KDM6A expression is necessary for survival of high-risk HPV E7 expressing cells, including several cervical cancer lines. Here we show that increased KDM6A in response to high-risk HPV E7 expression causes epigenetic de-repression of the cell cycle and DNA replication inhibitor p21CIP1, and p21CIP1 expression is necessary for survival of high-risk HPV E7 expressing cells. The requirement for KDM6A and p21CIP1 expression for survival of high-risk HPV E7 expressing cells is based on p21CIP1's ability to inhibit DNA replication through PCNA binding. We show that ectopic expression of cellular replication factors can rescue the loss of cell viability in response to p21CIP1 and KDM6A depletion. Moreover, we discovered that nucleoside supplementation will override the loss of cell viability in response to p21CIP1 depletion, suggesting that p21CIP1 depletion causes lethal replication stress. This model is further supported by increased double strand DNA breaks upon KDM6A or p21CIP1 depletion and DNA combing experiments that show aberrant re-replication upon KDM6A or p21CIP1 depletion in high-risk HPV E7 expressing cells. Therefore, KDM6A and p21CIP1 expression are essential to curb E7 induced replication stress to levels that do not markedly interfere with cell viability.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA Replication/genetics , Histone Demethylases/metabolism , Nuclear Proteins/metabolism , Papillomavirus E7 Proteins/genetics , Uterine Cervical Neoplasms/metabolism , Cell Line, Tumor , DNA-Binding Proteins/metabolism , Female , Humans , Keratinocytes/metabolism , Uterine Cervical Neoplasms/genetics
4.
Viruses ; 9(9)2017 09 01.
Article in English | MEDLINE | ID: mdl-28862667

ABSTRACT

Approximately 15-20% of human cancers are caused by viruses, including human papillomaviruses (HPVs). Viruses are obligatory intracellular parasites and encode proteins that reprogram the regulatory networks governing host cellular signaling pathways that control recognition by the immune system, proliferation, differentiation, genomic integrity, and cell death. Given that key proteins in these regulatory networks are also subject to mutation in non-virally associated diseases and cancers, the study of oncogenic viruses has also been instrumental to the discovery and analysis of many fundamental cellular processes, including messenger RNA (mRNA) splicing, transcriptional enhancers, oncogenes and tumor suppressors, signal transduction, immune regulation, and cell cycle control. More recently, tumor viruses, in particular HPV, have proven themselves invaluable in the study of the cancer epigenome. Epigenetic silencing or de-silencing of genes can have cellular consequences that are akin to genetic mutations, i.e., the loss and gain of expression of genes that are not usually expressed in a certain cell type and/or genes that have tumor suppressive or oncogenic activities, respectively. Unlike genetic mutations, the reversible nature of epigenetic modifications affords an opportunity of epigenetic therapy for cancer. This review summarizes the current knowledge on epigenetic regulation in HPV-infected cells with a focus on those elements with relevance to carcinogenesis.


Subject(s)
Cell Transformation, Neoplastic , Cell Transformation, Viral , Epigenesis, Genetic , Epigenomics , Oncogenic Viruses/genetics , Papillomaviridae/genetics , Acetylation , Carcinogenesis , Cell Cycle Checkpoints , Cell Differentiation , Cell Proliferation , DNA Methylation , Genome, Viral , Histones/metabolism , Humans , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/physiology , Oncogenic Viruses/immunology , Papillomavirus Infections/virology , RNA Splicing , Signal Transduction , Tumor Suppressor Proteins
5.
Semin Oncol ; 42(2): 284-90, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25843732

ABSTRACT

Human papillomaviruses (HPVs) infect the squamous epithelium and can induce benign and malignant lesions. To date, more than 200 different HPV types have been identified and classified into five genera, α, ß, γ, µ, and ν. While high-risk α mucosal HPVs have a well-established role in cervical carcinoma and a significant percentage of other anogenital tract and oral carcinomas, the biology of the cutaneous ß HPVs and their contribution to non-melanoma skin cancer (NMSC) has been less studied. Although the association of ß HPV infection with NMSC in patients with a rare, genetically determined condition, epidermodysplasia verruciformis has been well established, the role of ß HPV infection with NMSC in the normal population remains controversial. In stark contrast to α HPV-associated cancers, the presence of the ß HPV genome does not appear to be mandatory for the maintenance of the malignant phenotype. Moreover, the mechanism of action of the ß HPV E6 and E7 oncoproteins differs from the ß HPV oncoproteins.


Subject(s)
Carcinoma, Basal Cell/virology , Carcinoma, Squamous Cell/virology , Papillomavirus Infections/virology , Skin Neoplasms/virology , Humans
6.
Int J Cancer ; 136(2): 322-32, 2015 Jan 15.
Article in English | MEDLINE | ID: mdl-24862444

ABSTRACT

Mitochondrial organization is often altered to accommodate cellular bioenergetic and biosynthetic demands. Changes in metabolism are a hallmark of a number of diseases, including cancer; however, the interdependence between mitochondrial metabolic function and organization is not well understood. Here, we present a noninvasive, automated and quantitative method to assess mitochondrial organization in three-dimensional (3D) tissues using exclusively endogenous two-photon excited fluorescence (TPEF) and show that mitochondrial organization reflects alterations in metabolic activities. Specifically, we examine the organization of mitochondria within live, engineered epithelial tissue equivalents that mimic normal and precancerous human squamous epithelial tissues. We identify unique patterns of mitochondrial organization in the different tissue models we examine, and we attribute these to differences in the metabolic profiles of these tissues. We find that mitochondria are clustered in tissues with high levels of glycolysis and are more highly networked in tissues where oxidative phosphorylation is more dominant. The most highly networked organization is observed within cells with high levels of glutamine consumption. Furthermore, we demonstrate that mitochondrial organization provides complementary information to traditional morphological hallmarks of cancer development, including variations in nuclear size. Finally, we present evidence that this automated quantitative analysis of endogenous TPEF images can identify differences in the mitochondrial organization of freshly excised normal and pre-cancerous human cervical tissue specimens. Thus, this method could be a promising new modality to assess the role of mitochondrial organization in the metabolic activity of 3D tissues and could be further developed to serve as an early cancer clinical diagnostic biomarker.


Subject(s)
Biomarkers/analysis , Carcinoma, Squamous Cell/pathology , Epithelial Cells/pathology , Mitochondria/pathology , Precancerous Conditions/pathology , Uterine Cervical Neoplasms/pathology , Cells, Cultured , Female , Humans , Imaging, Three-Dimensional , Microscopy, Fluorescence, Multiphoton/methods , Prognosis
7.
Cancer Res ; 74(11): 3067-75, 2014 Jun 01.
Article in English | MEDLINE | ID: mdl-24686167

ABSTRACT

Alterations in the balance between different metabolic pathways used to meet cellular bioenergetic and biosynthetic demands are considered hallmarks of cancer. Optical imaging relying on endogenous fluorescence has been used as a noninvasive approach to assess tissue metabolic changes during cancer development. However, quantitative correlations of optical assessments with variations in the concentration of relevant metabolites or in the specific metabolic pathways that are involved have been lacking. In this study, we use high-resolution, depth-resolved imaging, relying entirely on endogenous two-photon excited fluorescence in combination with invasive biochemical assays and mass spectrometry to demonstrate the sensitivity and quantitative nature of optical redox ratio tissue assessments. We identify significant differences in the optical redox ratio of live, engineered normal and precancerous squamous epithelial tissues. We establish that while decreases in the optical redox ratio are associated with enhanced levels of glycolysis relative to oxidative phosphorylation, increases in glutamine consumption to support energy production are associated with increased optical redox ratio values. Such mechanistic insights in the origins of optical metabolic assessments are critical for exploiting fully the potential of such noninvasive approaches to monitor and understand important metabolic changes that occur in live tissues at the onset of cancer or in response to treatment.


Subject(s)
Epithelial Cells/metabolism , Epithelial Cells/pathology , Glutamine/metabolism , Glycolysis/physiology , Precancerous Conditions/metabolism , Biomarkers/metabolism , Cells, Cultured , Diagnostic Imaging/methods , Energy Metabolism , Fluorescence , Humans , Metabolic Networks and Pathways , Microscopy, Fluorescence, Multiphoton/methods , Oxidation-Reduction , Oxidative Phosphorylation , Precancerous Conditions/pathology , Tissue Engineering/methods
9.
Proc Natl Acad Sci U S A ; 110(40): 16175-80, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-24046371

ABSTRACT

The tumor suppressor p16(INK4A) inhibits formation of enzymatically active complexes of cyclin-dependent kinases 4 and 6 (CDK4/6) with D-type cyclins. Oncogenic stress induces p16(INK4A) expression, which in turn triggers cellular senescence through activation of the retinoblastoma tumor suppressor. Subversion of oncogene-induced senescence is a key step during cancer development, and many tumors have lost p16(INK4A) activity by mutation or epigenetic silencing. Human papillomavirus (HPV)-associated tumors express high levels of p16(INK4A) in response to E7 oncoprotein expression. Induction of p16(INK4A) expression is not a consequence of retinoblastoma tumor suppressor inactivation but is triggered by a cellular senescence response and is mediated by epigenetic derepression through the H3K27-specific demethylase (KDM)6B. HPV E7 expression causes an acute dependence on KDM6B expression for cell survival. The p16(INK4A) tumor suppressor is a critical KDM6B downstream transcriptional target and its expression is critical for cell survival. This oncogenic p16(INK4A) activity depends on inhibition of CDK4/CDK6, suggesting that in cervical cancer cells where retinoblastoma tumor suppressor is inactivated, CDK4/CDK6 activity needs to be inhibited in order for cells to survive. Finally, we note that HPV E7 expression creates a unique cellular vulnerability to small-molecule KDM6A/B inhibitors.


Subject(s)
Carcinoma/metabolism , Cell Survival/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Tumor Suppressor Proteins/metabolism , Uterine Cervical Neoplasms/metabolism , Carcinoma/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase Inhibitor p16/genetics , Female , Humans , Jumonji Domain-Containing Histone Demethylases/metabolism , Papillomavirus E7 Proteins/metabolism , RNA Interference , Real-Time Polymerase Chain Reaction , Tumor Suppressor Proteins/genetics , Uterine Cervical Neoplasms/genetics
10.
Viruses ; 5(5): 1231-49, 2013 May 14.
Article in English | MEDLINE | ID: mdl-23673719

ABSTRACT

The role of enzymes involved in polycomb repression of gene transcription has been studied extensively in human cancer. Polycomb repressive complexes mediate oncogene-induced senescence, a principal innate cell-intrinsic tumor suppressor pathway that thwarts expansion of cells that have suffered oncogenic hits. Infections with human cancer viruses including human papillomaviruses (HPVs) and Epstein-Barr virus can trigger oncogene-induced senescence, and the viruses have evolved strategies to abrogate this response in order to establish an infection and reprogram their host cells to establish a long-term persistent infection. As a consequence of inhibiting polycomb repression and evading oncogene induced-senescence, HPV infected cells have an altered epigenetic program as evidenced by aberrant homeobox gene expression. Similar alterations are frequently observed in non-virus associated human cancers and may be harnessed for diagnosis and therapy.


Subject(s)
Host-Pathogen Interactions , Papillomaviridae/physiology , Polycomb-Group Proteins/metabolism , Viral Proteins/metabolism , Cell Transformation, Viral , Epigenesis, Genetic , Gene Expression Regulation , Humans
11.
Curr Opin Virol ; 2(4): 459-66, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22658985

ABSTRACT

A small group of human papillomaviruses (HPVs) cause almost all cervical carcinoma and a significant percentage of other anogenital tract and oral carcinoma. Another group of HPVs causes non-melanoma skin cancers in genetically predisposed or immune suppressed patients upon UV exposure. HPV genome replication requires the host cell's DNA synthesis machinery and HPVs encode proteins that maintain differentiated epithelial cells in a replication competent state. The resulting rewiring of cellular signal transduction circuits triggers several innate cellular tumor suppressor responses that HPVs need to inactivate in order to establish persistent and/or productive infections. This review emphasizes this interplay between virus and the infected host cells and points out biological similarities and differences between different groups of HPVs.


Subject(s)
Alphapapillomavirus/physiology , Papillomavirus Infections/virology , Uterine Cervical Neoplasms/virology , Alphapapillomavirus/genetics , Animals , Female , Gene Expression Regulation , Humans , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus Infections/genetics , Papillomavirus Infections/metabolism
12.
Proc Natl Acad Sci U S A ; 109(26): 10516-21, 2012 Jun 26.
Article in English | MEDLINE | ID: mdl-22689991

ABSTRACT

Infection by carcinogenic human papillomaviruses (HPV) results in precancers [cervical intraepithelial neoplasia (CIN)] and cancers near the ectoendocervical squamocolumnar (SC) junction of the cervix. However, the specific cells targeted by HPV have not been identified and the cellular origin of cervical cancer remains elusive. In this study, we uncovered a discrete population of SC junctional cells with unique morphology and gene-expression profile. We also demonstrated that the selected junctional biomarkers were expressed by a high percentage of high-grade CIN and cervical cancers associated with carcinogenic HPVs but rarely in ectocervical/transformation zone CINs or those associated with noncarcinogenic HPVs. That the original SC junction immunophenotype was not regenerated at new SC junctions following excision, not induced by expression of viral oncoproteins in foreskin keratinocytes, and not seen in HPV-related precursors of the vagina, vulva, and penis further support the notion that junctional cells are the source of cervical cancer. Taken together, our findings suggest that carcinogenic HPV-related CINs and cervical cancers are linked to a small, discrete cell population that localizes to the SC junction of the cervix, expresses a unique gene expression signature, and is not regenerated after excision. The findings in this study uncover a potential target for cervical cancer prevention, provide insight into the risk assessment of cervical lesions, and establish a model for elucidating the pathway to cervical cancer following carcinogenic HPV infection.


Subject(s)
Uterine Cervical Neoplasms/pathology , Adult , Alphapapillomavirus/immunology , Alphapapillomavirus/isolation & purification , Alphapapillomavirus/pathogenicity , Blotting, Western , Female , Humans , Immunohistochemistry , Middle Aged , Transcription, Genetic , Uterine Cervical Neoplasms/immunology , Uterine Cervical Neoplasms/virology
13.
PLoS One ; 6(9): e24765, 2011.
Article in English | MEDLINE | ID: mdl-21931846

ABSTRACT

BACKGROUND: Multi-photon fluorescence microscopy techniques allow for non-invasive interrogation of live samples in their native environment. These methods are particularly appealing for identifying pre-cancers because they are sensitive to the early changes that occur on the microscopic scale and can provide additional information not available using conventional screening techniques. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we developed novel automated approaches, which can be employed for the real-time analysis of two-photon fluorescence images, to non-invasively discriminate between normal and pre-cancerous/HPV-immortalized engineered tissues by concurrently assessing metabolic activity, morphology, organization, and keratin localization. Specifically, we found that the metabolic activity was significantly enhanced and more uniform throughout the depths of the HPV-immortalized epithelia, based on our extraction of the NADH and FAD fluorescence contributions. Furthermore, we were able to separate the keratin contribution from metabolic enzymes to improve the redox estimates and to use the keratin localization as a means to discriminate between tissue types. To assess morphology and organization, Fourier-based, power spectral density (PSD) approaches were employed. The nuclear size distribution throughout the epithelial depths was quantified by evaluating the variance of the corresponding spatial frequencies, which was found to be greater in the normal tissue compared to the HPV-immortalized tissues. The PSD was also used to calculate the Hurst parameter to identify the level of organization in the tissues, assuming a fractal model for the fluorescence intensity fluctuations within a field. We found the range of organization was greater in the normal tissue and closely related to the level of differentiation. CONCLUSIONS/SIGNIFICANCE: A wealth of complementary morphological, biochemical and organizational tissue parameters can be extracted from high resolution images that are acquired based entirely on endogenous sources of contrast. They are promising diagnostic parameters for the non-invasive identification of early cancerous changes and could improve significantly diagnosis and treatment for numerous patients.


Subject(s)
Microscopy, Fluorescence, Multiphoton/methods , Precancerous Conditions/diagnosis , Cell Line , Cells, Cultured , Flavin-Adenine Dinucleotide/metabolism , Humans , Keratinocytes/cytology , Keratins/metabolism , NAD/metabolism , Tissue Engineering
14.
Proc Natl Acad Sci U S A ; 108(5): 2130-5, 2011 Feb 01.
Article in English | MEDLINE | ID: mdl-21245294

ABSTRACT

Despite the availability of vaccines, human papillomavirus (HPV) infections remain a cause of significant cancer morbidity and mortality. We have previously shown that HPV16 E7 associates with and diminishes E2F6-containing polycomb repressive complexes. Here, we show that repressive trimethyl marks on lysine 27 of histone 3, which are necessary for binding of polycomb repressive complexes, are decreased in HPV16 E7-expressing cells and HPV16-positive cervical lesions. This is caused by transcriptional induction of the KDM6A and KDM6B histone 3 lysine 27-specific demethylases. HPV16 E7-mediated KDM6B induction accounts for expression of the cervical cancer biomarker, p16(INK4A). Moreover, KDM6A- and KDM6B-responsive Homeobox genes are expressed at significantly higher levels, suggesting that HPV16 E7 results in reprogramming of host epithelial cells. These effects are independent of the ability of E7 to inhibit the retinoblastoma tumor suppressor protein. Most importantly, these effects are reversed when E7 expression is silenced, indicating that this pathway may have prognostic and/or therapeutic significance.


Subject(s)
Epigenesis, Genetic , Jumonji Domain-Containing Histone Demethylases/biosynthesis , Papillomavirus E7 Proteins/physiology , Cells, Cultured , Enzyme Induction , Female , Genes, Homeobox , Humans , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Uterine Cervical Neoplasms/virology
15.
Virus Res ; 143(2): 195-208, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19540281

ABSTRACT

Infectious etiologies for certain human cancers have long been suggested by epidemiological studies and studies with experimental animals. Important support for this concept came from the discovery by Harald zur Hausen's group that human cervical carcinoma almost universally contains certain "high-risk" human papillomavirus (HPV) types. Over the years, much has been learned about the carcinogenic activities of high-risk HPVs. These studies have revealed that two viral proteins, E6 and E7, that are consistently expressed in HPV-associated carcinomas, are necessary for induction and maintenance of the transformed phenotype. Hence, HPV-associated tumors are unique amongst human solid tumors in that they are universally caused by exposure to the same, molecularly defined oncogenic agents, and the molecular signal transduction pathways subverted by these viral transforming agents are frequently disrupted in other, non-virus-associated human cancers.


Subject(s)
Cell Transformation, Neoplastic , Papillomaviridae/pathogenicity , Papillomavirus Infections/complications , Tumor Virus Infections/complications , Uterine Cervical Neoplasms/virology , Animals , Female , Humans , Oncogene Proteins, Viral/metabolism , Papillomavirus Infections/virology , Tumor Virus Infections/virology
16.
Virology ; 384(2): 335-44, 2009 Feb 20.
Article in English | MEDLINE | ID: mdl-19007963

ABSTRACT

The human papillomavirus (HPV) E7 oncoprotein shares functional similarities with such proteins as adenovirus E1A and SV40 large tumor antigen. As one of only two viral proteins always expressed in HPV-associated cancers, E7 plays a central role in both the viral life cycle and carcinogenic transformation. In the HPV viral life cycle, E7 disrupts the intimate association between cellular differentiation and proliferation in normal epithelium, allowing for viral replication in cells that would no longer be in the dividing population. This function is directly reflected in the transforming activities of E7, including tumor initiation and induction of genomic instability.


Subject(s)
Cell Transformation, Viral , Papillomaviridae/metabolism , Papillomavirus E7 Proteins/metabolism , Cell Death , Cell Differentiation , Cell Proliferation , Chromosomal Instability , Cytokines/metabolism , E2F Transcription Factors/metabolism , Epigenesis, Genetic , Papillomaviridae/physiology , Papillomavirus Infections/virology , Retinoblastoma Protein/metabolism , Tumor Suppressor Protein p53/metabolism , Virus Replication
17.
Cancer Res ; 68(21): 8715-22, 2008 Nov 01.
Article in English | MEDLINE | ID: mdl-18974113

ABSTRACT

Dynein is a minus end-directed microtubule motor that transports numerous cargoes throughout the cell. During mitosis, dynein motor activity is necessary for the positioning of spindle microtubules and has also been implicated in inactivating the spindle assembly checkpoint. Mutations in dynein motor and/or accessory proteins are associated with human disease, including cancer, and the delocalization of dynein from mitotic spindles has been correlated with an increased incidence of multipolar spindle formation in some cancer cells that contain supernumerary centrosomes. The high-risk human papillomavirus type 16 (HPV16) E7 oncoprotein induces centrosome overduplication and has been shown to cause multipolar mitotic spindle formation, a diagnostic hallmark of HPV-associated neoplasias. Here, we show that HPV16 E7 expression leads to an increased population of mitotic cells with dynein delocalized from the mitotic spindle. This function maps to sequences of HPV16 E7 that are distinct from the region necessary for centrosome overduplication. However, contrary to previous reports, we provide evidence that dynein delocalization by HPV16 E7 is neither necessary nor sufficient to cause the formation of multipolar mitoses.


Subject(s)
Dyneins/metabolism , Mitosis/physiology , Oncogene Proteins, Viral/physiology , Spindle Apparatus/metabolism , Animals , Blotting, Western , Cell Line, Tumor , Dyneins/physiology , Female , Fluorescent Antibody Technique , Humans , Metaphase , Mice , NIH 3T3 Cells , Papillomavirus E7 Proteins , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virology
18.
J Invest Dermatol ; 128(9): 2142-4, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18695686

ABSTRACT

Infections with beta-genus human papillomaviruses (HPVs) have been associated with nonmelanoma skin cancers, particularly in immunocompromised patients and individuals with a rare genetic disease, epidermodysplasia verruciformis (EV). Using a transgenic mouse model, Herbert Pfister's group determined that expression of the HPV8 E2 gene results in skin cancer development and that this process is greatly accelerated by UV irradiation (Pfefferle et al., 2008, this issue).


Subject(s)
Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Viral/physiology , Genes, Viral/physiology , Oncogene Proteins, Viral/physiology , Skin Neoplasms/physiopathology , Skin Neoplasms/virology , Trans-Activators/physiology , Animals , Cell Transformation, Neoplastic/radiation effects , Disease Models, Animal , Humans , Mice , Mice, Transgenic , Neoplasms, Radiation-Induced/genetics , Oncogene Proteins, Viral/genetics , Papillomavirus Infections/complications , Skin Neoplasms/genetics , Trans-Activators/genetics , Ultraviolet Rays/adverse effects
19.
J Virol ; 82(17): 8695-705, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18579589

ABSTRACT

The papillomavirus life cycle is intimately coupled to the differentiation state of the infected epithelium. Since papillomaviruses lack most of the rate-limiting enzymes required for genome synthesis, they need to uncouple keratinocyte differentiation from cell cycle arrest and maintain or reestablish a replication-competent state within terminally differentiated keratinocytes. The human papillomavirus (HPV) E7 protein appears to be a major determinant for this activity and induces aberrant S-phase entry through the inactivation of the retinoblastoma tumor suppressor and related pocket proteins. In addition, E7 can abrogate p21 and p27. Together, this leads to the activation of E2F1 to E2F5, enhanced expression of E2F-responsive genes, and increased cdk2 activity. E2F6 is a pRB-independent, noncanonical member of the E2F transcription factor family that acts as a transcriptional repressor. E2F6 expression is activated in S phase through an E2F-dependent mechanism and thus may provide a negative-feedback mechanism that slows down S-phase progression and/or exit in response to the activation of the other E2F transcription factors. Here, we show that low- and high-risk HPV E7 proteins, as well as simian virus 40 T antigen and adenovirus E1A, can associate with and inactivate the transcriptional repression activity of E2F6, thereby subverting a critical cellular defense mechanism. This may result in the extended S-phase competence of HPV-infected cells. E2F6 is a component of polycomb group complexes, which bind to silenced chromatin and are critical for the maintenance of cell fate. We show that E7-expressing cells show decreased staining for E2F6/polycomb complexes and that this is at least in part dependent on the association with E2F6.


Subject(s)
E2F Transcription Factors/metabolism , Oncogene Proteins, Viral/metabolism , Papillomaviridae/metabolism , Adenovirus E1A Proteins/genetics , Adenovirus E1A Proteins/metabolism , Antigens, Viral, Tumor/metabolism , Cell Culture Techniques , Cell Line , Genes, Reporter , HeLa Cells , Humans , Kidney/cytology , Luciferases/metabolism , Models, Biological , Mutation , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/genetics , Papillomavirus E7 Proteins , Plasmids , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Simian virus 40/metabolism , Time Factors , Transfection
20.
Biochim Biophys Acta ; 1782(3): 127-50, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18201576

ABSTRACT

It is estimated that viral infections contribute to 15-20% of all human cancers. As obligatory intracellular parasites, viruses encode proteins that reprogram host cellular signaling pathways that control proliferation, differentiation, cell death, genomic integrity, and recognition by the immune system. These cellular processes are governed by complex and redundant regulatory networks and are surveyed by sentinel mechanisms that ensure that aberrant cells are removed from the proliferative pool. Given that the genome size of a virus is highly restricted to ensure packaging within an infectious structure, viruses must target cellular regulatory nodes with limited redundancy and need to inactivate surveillance mechanisms that would normally recognize and extinguish such abnormal cells. In many cases, key proteins in these same regulatory networks are subject to mutation in non-virally associated diseases and cancers. Oncogenic viruses have thus served as important experimental models to identify and molecularly investigate such cellular networks. These include the discovery of oncogenes and tumor suppressors, identification of regulatory networks that are critical for maintenance of genomic integrity, and processes that govern immune surveillance.


Subject(s)
Cell Transformation, Neoplastic , Cell Transformation, Viral , Gene Expression Regulation, Neoplastic , Neoplasms/virology , Oncogenic Viruses/physiology , Genes, Tumor Suppressor , Genomic Instability , Humans , Immunologic Surveillance , Models, Biological , Neoplasms/genetics , Oncogenic Viruses/genetics , Signal Transduction/genetics , Tumor Virus Infections/genetics
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