Viral T-antigen Research Articles

LB879 Friend or foe? Identification of MCPyV-specific B cells in Merkel cell carcinomas suggests a functional role in tumor immunity

Merkel cell carcinoma (MCC) is a rare and aggressive cancer of the skin with ∼30% mortality. Around 80% of MCC tumors originate from integration of the Merkel cell polyomavirus (MCPyV) DNA into the host genome, leading to expression of the viral T-antigen oncoprotein and ultimately, tumorigenesis. Though PD-1 pathway blockade has significantly improved outcomes for advanced MCC, many patients do not benefit from or develop resistance to this immunotherapy. In recent years, tumor-infiltrating B cells have been generally associated with better outcomes in MCC.

JCPyV miR-J1-5p in Urine of Natalizumab-Treated Multiple Sclerosis Patients.

The use of Natalizumab in Multiple Sclerosis (MS) can cause the reactivation of the polyomavirus JC (JCPyV); this may result in the development of progressive multifocal leukoencephalopathy (PML), a rare and usually lethal disease. JCPyV infection is highly prevalent in worldwide population, but the detection of anti-JCPyV antibodies is not sufficient to identify JCPyV infection, as PML can develop even in patients with negative JCPyV serology. Better comprehension of the JCPyV biology could allow a better understanding of JCPyV infection and reactivation, possibly reducing the risk of developing PML. Here, we investigated whether JCPyV miR-J1-5p—a miRNA that down-regulates the early phase viral protein T-antigen and promotes viral latency—could be detected and quantified by digital droplet PCR (ddPCR) in urine of 25 Natalizumab-treated MS patients. A 24-month study was designed: baseline, before the first dose of Natalizumab, and after 1 (T1), 12 (T12) and 24 months (T24) of therapy. miR-J1-5p was detected in urine of 7/25 MS patients (28%); detection was possible in three cases at T24, in two cases at T12, in one case at T1 and T12, and in the last case at baseline and T1. Two of these patients were seronegative for JCPyV Ab, and viral DNA was never found in either urine or blood. To note, only in one case miR-J1-5p was detected before initiation of Natalizumab. These results suggest that the measurement of miR-J1-5p in urine, could be a biomarker to monitor JCPyV infection and to better identify the possible risk of developing PML in Natalizumab-treated MS patients.

Abstract 286: Regulation of APOBEC3B gene expression through the Rb/E2F pathway

Abstract The propensity of tumors to increase genomic diversity allows for a bottleneck-type adaptation to anti-cancer treatments. The genomic alterations fueling these adaptations in many different tumor types are, in part, brought about by ongoing endogenous activity of the DNA cytosine deaminase APOBEC3B (A3B). The ability of A3B to convert DNA cytosine bases into uracils has been shown to facilitate C-to-T transitions, C-to-G transversions, and DNA breakage. While low in healthy tissues, A3B expression and activity is elevated in tumors and may increase further throughout metastatic disease. However, molecular mechanisms responsible for transcriptional upregulation of A3B in tumor cells are poorly understood. Here, we investigate the possibility that the Rb/E2F pathway, which is often dysregulated in breast cancer, contributes to A3B gene expression. First, we cloned the A3B promoter upstream of a luciferase reporter and used a combination of deletion and site-directed mutagenesis to demonstrate that only one out of five in silico predicted E2F binding sites is functionally relevant, as disrupting this site caused constitutive reporter activation. Moreover, expression of the BK polyomavirus T-antigen, which inactivates Rb, induced the reporter and additional activation was not observed by mutating the functional E2F binding site. Second, SILAC-labelled nuclear protein extracts were subjected to affinity purification coupled to quantitative mass spectrometry to compare transcriptional complexes that bound wild-type promoter sequences, but not sequences with mutant E2F binding site promoter elements. These proteomics experiments showed that proteins belonging to the DREAM and PRC1.6-complexes strongly bind the wild-type but not the mutant E2F binding sites. Third, RNAi-mediated knock-down of candidate E2F family members implicated E2F6, and to a lesser extend E2F4, which are components of the PRC1.6 and DREAM complexes, respectively, in A3B gene repression. Taken together, these studies indicate that A3B expression may be suppressed by the concerted effort of two different E2F binding complexes and that Rb inactivation, by viral T-antigen here and genetic mutation as often seen in breast cancer, is able to upregulate A3B gene expression, promote genomic DNA mutagenesis, and fuel tumor evolution. Citation Format: Pieter A. Roelofs, William L. Brown, Paul N. Span, John W. Martens, Chai Y. Goh, Boon H. Chua, Dennis Kappei, Reuben S. Harris. Regulation of APOBEC3B gene expression through the Rb/E2F pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 286.

Polyomavirus-driven Merkel cell carcinoma: Prospects for therapeutic vaccine development.

Great strides have been made in cancer immunotherapy including the breakthrough successes of anti-PD-(L)1 checkpoint inhibitors. In Merkel cell carcinoma (MCC), a rare and aggressive skin cancer, PD-(L)1 blockade is highly effective. Yet, ~50% of patients either do not respond to therapy or develop PD-(L)1 refractory disease and, thus, do not experience long-term benefit. For these patients, additional or combination therapies are needed to augment immune responses that target and eliminate cancer cells. Therapeutic vaccines targeting tumor-associated antigens, mutated self-antigens, or immunogenic viral oncoproteins are currently being developed to augment T-cell responses. Approximately 80% of MCC cases in the United States are driven by the ongoing expression of viral T-antigen (T-Ag) oncoproteins from genomically integrated Merkel cell polyomavirus (MCPyV). Since T-Ag elicits specific B- and T-cell immune responses in most persons with virus-positive MCC (VP-MCC), and ongoing T-Ag expression is required to drive VP-MCC cell proliferation, therapeutic vaccination with T-Ag is a rational potential component of immunotherapy. Failure of the endogenous T-cell response to clear VP-MCC (allowing clinically evident tumors to arise) implies that therapeutic vaccination will need to be potent anśd synergize with other mechanisms to enhance T-cell activity against tumor cells. Here, we review the relevant underlying biology of VP-MCC, potentially applicable therapeutic vaccine platforms, and antigen delivery formats. We also describe early successes in the field of therapeutic cancer vaccines and address several clinical scenarios in which VP-MCC patients could potentially benefit from a therapeutic vaccine.

Human Polyomavirus JCPyV and Its Role in Progressive Multifocal Leukoencephalopathy and Oncogenesis.

The human neurotropic virus JCPyV, a member of the Polyomaviridiae family, is the opportunistic infectious agent of Progressive Multifocal Leukoencephalopathy (PML), a fatal disease seen in severe immunosuppressive conditions and, during the last decade, in patients undergoing immunotherapy. JCPyV is a ubiquitous pathogen with up to 85% of the adult population word-wide exhibiting antibodies against it. Early experiments demonstrated that direct inoculation of JCPyV into the brain of different species resulted in the development of brain tumors and other neuroectodermal-derived neoplasias. Later, several reports showed the detection of viral sequences in medulloblastomas and glial tumors, as well as expression of the viral protein T-Antigen. Few oncogenic viruses, however, have caused so much controversy regarding their role in the pathogenesis of brain tumors, but the discovery of new Polyomaviruses that cause Merkel cell carcinomas in humans and brain tumors in racoons, in addition to the role of JCPyV in colon cancer and multiple mechanistic studies have shed much needed light on the role of JCPyV in cancer. The pathways affected by the viral protein T-Antigen include cell cycle regulators, like p53 and pRb, and transcription factors that activate pro-proliferative genes, like c-Myc. In addition, infection with JCPyV causes chromosomal damage and T-Antigen inhibits homologous recombination, and activates anti-apoptotic proteins, such as Survivin. Here we review the different aspects of the biology and physiopathology of JCPyV.

John Cunningham Virus T-Antigen Expression on Mild and Severe Dysplasia Adenomatous Polyp, Low and High Grade Adenocarcinoma of The Colon

Background: John Cunningham Virus (JCV) was involved in pre-malignant lessions and carcinogenesis of the colon. The purpose of this study was to detect and analyze JCV T-Ag expression in mild and severe dysplasia adenomatous polyp as well as low and high grade adenocarcinoma of the colon.Materials and Methods: This study used analytic descriptive, cross sectional approach. The samples’ paraffin blocks were taken from colon adenomatous polyp cases (all grades of dysplasia) and cases of colon adenocarcinoma (all degrees) at Anatomical Pathology Laboratory, School of Medicine, Atma Jaya Catholic University of Indonesia from 2010-2014 (5 years period). Samples were reviewed from HE slides to determine histopathologic diagnosis, grades of dysplasia and grading. We performed immunohistochemistry staining with monoclonal antibody anti–SV 40-T-Ag to detect JCV T-Ag expression.Results: We found 7 cases of colon adenomatous polyp, of which 4 (57%) were mild dysplasia and 3 (43%) were severe dysplasia. Positive expression of JCV T-Ag was detected in 1 (14%) mild dysplasia case. Data analysis using Fischer’s Exact Test was p>0.05. We also found 16 cases of colon adenocarcinoma. 14 cases (87.5%) of low grade variant and 2 cases (12.5%) of high grade variant. Positive expression of JCV T-Ag was detected in 2 (12.5%) low grade cases. Data analysis using Fischer’s Exact Test was p>0.05.Conclusion: There was no difference of JCV T-Ag expression in colon adenomatous polyp (mild-severe dysplasia) and colon adenocarcinoma (low-high grade) cases at Anatomical Pathology Laboratory School of Medicine, Atma Jaya Catholic University of Indonesia 2010-2014.Keywords: colon adenomatous polyp, adenocarcinoma, JCV T-Ag

Polyoma virus-associated carcinomas of the urologic tract: a clinicopathologic and molecular study

In recent years, there has been increased interest in carcinomas of the urologic tract, that demonstrate association with the polyoma virus BK arising in immunosuppressed individuals, though the nature of this association is uncertain. To begin to understand this phenomenon, we reviewed the clinical, morphological, and immunohistochemical features of 11 carcinomas of the urologic tract, mainly urothelial (N = 9) and collecting duct carcinomas (N = 2), occurring during immunosuppression, and expressing polyoma virus T-antigen by immunohistochemistry. These were compared to a control group of carcinomas (N = 8), also arising during immunosuppression, but without T-antigen expression. A subset of both groups were also studied by hybrid capture-based DNA sequencing, probing not only for 479 cancer-related human genes, but also for polyoma and other viral sequences. Polyoma T-antigen-expressing tumors arose in 7 males and 4 females, at a median age of 66, and were aggressive, high-grade tumors with more than 1 variant morphologic pattern identified in 81% of cases, and a majority (73%) presenting at high stage category (>pT3). Diffuse polyoma T-antigen staining was seen in 91% of cases, with co-localization of aberrant p53 staining in 89%. Sequencing detected a lower number of deleterious mutations among T-antigen-expressing cases (average 1.62; 1/8 with TP53 mutation) compared to control cases (average 3.5, 2/4 with TP53 mutation). Only BK virus was detected with clonal integration and breakpoints randomly distributed across the human and viral genomes in 5/5 of the polyoma T-antigen-expressing carcinomas, and in none of the controls (0/4). In summary, these findings identify aggressive clinicopathologic features of polyoma T-antigen-expressing carcinomas, document BK as the strain involved, and associate BK viral integration with T-antigen expression and p53 aberrancy. While the apparent randomness of viral insertion sites is functionally unclear, the differing rates of mutations between T-antigen-expressing and control cases is intriguing.

Abstract 4290: Serum biomarkers of polyomavirus infection and risk of lung cancer in never smokers

Abstract Background: Lung cancer in never smokers is a significant contributor of cancer mortality. As the incidence of lung cancer in never smokers is increasing, there is a need to investigate risk factors for lung cancer in this population. There is data to support that polyomaviruses are potentially carcinogenic in the human lung. We explored the role of polyomaviruses in lung cancer development in never smokers using a multiplex assay to detect serum antibodies to viral capsid proteins. Methods: We conducted a nested case-control study of never-smoking cases of lung cancer identified from four established prospective cohorts- NYU Women's Health Study (NYU-WHS), Janus Serum Bank, Shanghai Women's Health Study (SWHS) and Singapore Chinese Health Study (SCHS). Controls were matched to cases on gender, never-smoking status, age and calendar period of entry. Serological analysis was performed using a 100 μL pre-diagnostic serum sample at the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) in Heidelberg, Germany using fluorescent bead-based multiplex serology and included antibodies to viral capsid protein-1 and T-antigens of 9 human polyomaviruses: JC virus, BK virus, KI virus, WU virus, Trichodysplasia Spinulosa-associated Polyoma Virus, Merkel Cell Polyoma Virus, Human Polyoma Virus 6, Human Polyoma Virus 7 and Human Polyoma Virus 10. Results: The final analysis included 511 cases and 508 controls. Mean age of participants was 56.1±11.0 years. SWHS and SCHS included only Asian participants. NYU-WHS and SWHS had only female participants. Nearly 85% of the participants in our pooled analysis were females and 74% were Asians. Seropositivity for each polyomavirus showed significant heterogeneity by study but overall there were no statistical significant differences between cases and controls for any of the polyomaviruses. 72.0% of the cases and 71.5% of the controls were seropositive for JC virus antibody. Seropositivity for BK virus was higher at 89.0% in cases and 89.8% in controls. We did not find any difference in seropositivity between cases and controls in our stratified analysis based on gender, histology or time interval from sample collection to cancer diagnosis. We also performed sensitivity analyses and found the seroprevalence data to be very robust to alterations in the MFI cutpoints. Conclusions: Our study is the largest epidemiological study in never smokers to investigate the role of polyomaviruses in lung cancer development. We did not find a significant difference in serological measurements of antibodies against each of the polyomaviruses between the cases and controls. Future research to evaluate the relationship between polyomaviruses and lung carcinogenesis should focus on evaluating viral replication in tumor in combination with serological markers of infection especially as antibody reactivities can vary considerably across different populations and geographical areas as demonstrated by our study. Citation Format: Jyoti Malhotra, Tim Waterboer, Michael Pawlita, Angelika Michel, Qiuyin Cai, Wei Zheng, Qing Lan, Nathaniel Rothman, Hilde Langseth, Tom K. Grimsrud, Jian-Min Yuan, Woon-Puay Koh, Alan A. Arslan, Anne Zeleniuch-Jacquotte, Paolo Boffetta. Serum biomarkers of polyomavirus infection and risk of lung cancer in never smokers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4290.

Progressive multifocal leukoencephalopathy in an immunocompetent patient.

Progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the brain, is typically diagnosed in immunocompromised persons. Here, we describe the diagnostic challenge of PML in an apparently immunocompetent patient. Thorough analyses, including cytokine release assays and whole exome sequencing, revealed a deficit in the antiviral interferon gamma production capacity of this patient and compound heterozygous mutations in BCL-2-associated athanogene 3. Interestingly, both factors are associated with reduced expression of John Cunningham virus T-antigen, a protein that plays a key role in viral replication in infected cells. After validation in other patients, our findings may contribute to novel insights into the etiology and possibly treatment of PML.

Structural Based Analyses of the JC Virus T-Antigen F258L Mutant Provides Evidence for DNA Dependent Conformational Changes in the C-Termini of Polyomavirus Origin Binding Domains.

The replication of human polyomavirus JCV, which causes Progressive Multifocal Leukoencephalopathy, is initiated by the virally encoded T-antigen (T-ag). The structure of the JC virus T-ag origin-binding domain (OBD) was recently solved by X-ray crystallography. This structure revealed that the OBD contains a C-terminal pocket, and that residues from the multifunctional A1 and B2 motifs situated on a neighboring OBD molecule dock into the pocket. Related studies established that a mutation in a pocket residue (F258L) rendered JCV T-ag unable to support JCV DNA replication. To establish why this mutation inactivated JCV T-ag, we have solved the structure of the F258L JCV T-ag OBD mutant. Based on this structure, it is concluded that the structural consequences of the F258L mutation are limited to the pocket region. Further analyses, utilizing the available polyomavirus OBD structures, indicate that the F258 region is highly dynamic and that the relative positions of F258 are governed by DNA binding. The possible functional consequences of the DNA dependent rearrangements, including promotion of OBD cycling at the replication fork, are discussed.

UV-Associated Mutations Underlie the Etiology of MCV-Negative Merkel Cell Carcinomas.

Merkel cell carcinoma (MCC) is an uncommon, but highly malignant, cutaneous tumor. Merkel cell polyoma virus (MCV) has been implicated in a majority of MCC tumors; however, viral-negative tumors have been reported to be more prevalent in some geographic regions subject to high sun exposure. While the impact of MCV and viral T-antigens on MCC development has been extensively investigated, little is known about the etiology of viral-negative tumors. We performed targeted capture and massively parallel DNA sequencing of 619 cancer genes to compare the gene mutations and copy number alterations in MCV-positive (n = 13) and -negative (n = 21) MCC tumors and cell lines. We found that MCV-positive tumors displayed very low mutation rates, but MCV-negative tumors exhibited a high mutation burden associated with a UV-induced DNA damage signature. All viral-negative tumors harbored mutations in RB1, TP53, and a high frequency of mutations in NOTCH1 and FAT1. Additional mutated or amplified cancer genes of potential clinical importance included PI3K (PIK3CA, AKT1, PIK3CG) and MAPK (HRAS, NF1) pathway members and the receptor tyrosine kinase FGFR2. Furthermore, looking ahead to potential therapeutic strategies encompassing immune checkpoint inhibitors such as anti-PD-L1, we also assessed the status of T-cell-infiltrating lymphocytes (TIL) and PD-L1 in MCC tumors. A subset of viral-negative tumors exhibited high TILs and PD-L1 expression, corresponding with the higher mutation load within these cancers. Taken together, this study provides new insights into the underlying biology of viral-negative MCC and paves the road for further investigation into new treatment opportunities.

Molecular interplay between T-Antigen and splicing factor, arginine/serine-rich 1 (SRSF1) controls JC virus gene expression in glial cells.

BackgroundHuman polyomavirus JCV is the etiologic agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease characterized by lytic infection of glial cells in the central nervous system. PML is seen primarily in immunosuppressed patients and is mainly classified as an AIDS-defining disease. In addition to structural capsid proteins, JCV encodes multiple regulatory proteins, including T-antigen and agnoprotein, which are required for functional lytic infection. Previous studies have suggested that molecular interaction between viral proteins and host factors play an important role in reactivation of JCV and progression of the viral life cycle in glial cells. Recently, serine/arginine rich splicing factor 1 (SRSF1), a cellular alternative splicing factor, was identified as a strong negative regulator of JCV in glial cells. SRSF1 inhibits JCV gene expression and viral replication by directly interacting with viral promoter sequences. Here, we have investigated possible impact of JCV regulatory proteins, T-antigen and agnoprotein, on SRSF1-mediated suppression of JCV gene expression in glial cells.ResultsReporter gene analysis has suggested that T-antigen rescues viral transcriptional suppression mediated by SRSF1. Further analyses have revealed that T-antigen promotes viral gene expression by suppressing SRSF1 gene transcription in glial cells. A subsequent ChIP analysis revealed that T-antigen associates with the promoter region of SRSF1 to induce the transcriptional suppression.ConclusionsThese findings have revealed a molecular interplay between cellular SRSF1 and viral T-antigen in controlling JCV gene expression, and may suggest a novel mechanism of JCV reactivation in patients who are at risk of developing PML.

Karyotype alteration generates the neoplastic phenotypes of SV40-infected human and rodent cells.

BackgroundDespite over 50 years of research, it remains unclear how the DNA tumor viruses SV40 and Polyoma cause cancers. Prevailing theories hold that virus-coded Tumor (T)-antigens cause cancer by inactivating cellular tumor suppressor genes. But these theories don’t explain four characteristics of viral carcinogenesis: (1) less than one in 10,000 infected cells become cancer cells, (2) cancers have complex individual phenotypes and transcriptomes, (3) recurrent tumors without viral DNA and proteins, (4) preneoplastic aneuploidies and immortal neoplastic clones with individual karyotypes.ResultsAs an alternative theory we propose that viral carcinogenesis is a form of speciation, initiated by virus-induced aneuploidy. Since aneuploidy destabilizes the karyotype by unbalancing thousands of genes it catalyzes chain reactions of karyotypic and transcriptomic evolutions. Eventually rare karyotypes evolve that encode cancer-specific autonomy of growth. The low probability of forming new autonomous cancer-species by random karyotypic and transcriptomic variations predicts individual and clonal cancers. Although cancer karyotypes are congenitally aneuploid and thus variable, they are stabilized or immortalized by selections for variants with cancer-specific autonomy. Owing to these inherent variations cancer karyotypes are heterogeneous within clonal margins. To test this theory we analyzed karyotypes and phenotypes of SV40-infected human, rat and mouse cells developing into neoplastic clones. In all three systems we found (1) preneoplastic aneuploidies, (2) neoplastic clones with individual clonal but flexible karyotypes and phenotypes, which arose from less than one in 10,000 infected cells, survived over 200 generations, but were either T-antigen positive or negative, (3) spontaneous and drug-induced variations of neoplastic phenotypes correlating 1-to-1 with karyotypic variations.ConclusionsSince all 14 virus-induced neoplastic clones tested contained individual clonal karyotypes and phenotypes, we conclude that these karyotypes have generated and since maintained these neoplastic clones. Thus SV40 causes cancer indirectly, like carcinogens, by inducing aneuploidy from which new cancer-specific karyotypes evolve automatically at low rates. This theory explains the (1) low probability of carcinogenesis per virus-infected cell, (2) the individuality and clonal flexibility of cancer karyotypes, (3) recurrence of neoplasias without viral T-antigens, and (4) the individual clonal karyotypes, transcriptomes and immortality of virus-induced neoplasias - all unexplained by current viral theories.

IFN-Gamma Inhibits JC Virus Replication in Glial Cells by Suppressing T-Antigen Expression.

ObjectivePatients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), an often lethal disease of the brain characterized by lytic infection of oligodendrocytes in the central nervous system (CNS) with JC virus (JCV). The immune system plays an important regulatory role in controlling JCV reactivation from latent sites by limiting viral gene expression and replication. However, little is known regarding the molecular mechanisms responsible for this regulation.Methods and ResultsHere, we investigated the impact of soluble immune mediators secreted by activated PBMCs on viral replication and gene expression by cell culture models and molecular virology techniques. Our data revealed that viral gene expression and viral replication were suppressed by soluble immune mediators. Further studies demonstrated that soluble immune mediators secreted by activated PBMCs inhibit viral replication induced by T-antigen, the major viral regulatory protein, by suppressing its expression in glial cells. This unexpected suppression of T-antigen was mainly associated with the suppression of translational initiation. Cytokine/chemokine array studies using conditioned media from activated PBMCs revealed several candidate cytokines with possible roles in this regulation. Among them, only IFN-γ showed a robust inhibition of T-antigen expression. While potential roles for IFN-β, and to a lesser extent IFN-α have been described for JCV, IFN-γ has not been previously implicated. Further analysis of IFN-γ signaling pathway revealed a novel role of Jak1 signaling in control of viral T-antigen expression. Furthermore, IFN-γ suppressed JCV replication and viral propagation in primary human fetal glial cells, and showed a strong anti-JCV activity.ConclusionsOur results suggest a novel role for IFN-γ in the regulation of JCV gene expression via downregulation of the major viral regulatory protein, T-antigen, and provide a new avenue of research to understand molecular mechanisms for downregulation of viral reactivation that may lead to development of novel strategies for the treatment of PML.

Prospective study of seroreactivity to JC virus T-antigen and risk of colorectal cancers and adenomas.

John Cunningham virus (JCV) is a common polyomavirus classified as a possible carcinogen by the International Agency for Research on Cancer. JCV may play a role in colorectal carcinogenesis, although we previously reported no association between JCV capsid antibodies and colorectal cancer. No studies have examined the role of seroreactivity to JCV T-antigen (T-Ag) oncoprotein in colorectal cancer. A case-control study nested within a community-based prospective cohort (CLUE II) was conducted. In 1989, 25,080 residents of Washington County, Maryland, were enrolled in CLUE II, completing baseline questionnaires and providing blood samples. At follow-up, 257 incident colorectal cancer cases were identified by linkage to population-based cancer registries through 2006 and matched to controls on age, sex, race, and date of blood draw. One hundred and twenty-three colorectal adenoma cases were identified through self-report during follow-up and matched to controls on age, sex, race, date of blood draw, and colorectal cancer screening. Baseline serum samples were tested for seroreactivity to JCV T-Ag. Associations between JCV T-Ag seroreactivity and colorectal cancer/adenomas were evaluated using conditional logistic regression models. Overall, seroreactivity to JCV T-Ag was not statistically significantly associated with the risk of either colorectal cancer [OR, 1.34; 95% confidence interval (CI), 0.89-2.01] or adenoma (OR, 1.30; 95% CI, 0.70-2.42), while a borderline association with colorectal cancer was observed among women (OR, 1.82; 95% CI, 1.00-3.31). Our past evaluation of JCV capsid seropositivity, combined with current findings, does not support a notable etiologic role for JCV infection in colorectal cancer.

Milestones in the staging, classification, and biology of Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive skin cancer that is causally associated with ultraviolet light exposure and a recently discovered polyomavirus. Before 2010, MCC was staged using any of 5 unique systems in active use. In 2010, a consensus staging system for MCC was adopted worldwide and replaced these systems. This consensus system includes substages that reflect prognostic differences based on whether nodal evaluation was performed by pathologic analysis or clinical assessment alone. MCC-specific disease classification in ICD-9, to be expanded in the upcoming ICD-10, has improved the ability to track and manage this malignancy. Several biomarkers and histopathologic features have been identified that improve understanding of this cancer and may lead to future refinement of the current staging system. In 2008, the Merkel cell polyomavirus was discovered and is now thought to be a critical mechanism of transformation in at least 80% of MCCs. In patients who produce antibodies to the viral T-antigen oncoprotein, the titer increases and decreases with MCC disease burden and can be a clinically useful marker of recurrence. Diverse studies link CD8-positive T-cell function with outcomes in MCC and serve as the rational basis for ongoing trials of therapies to augment cellular immunity. This article reviews basic and translational research insights that will lead to improved staging, prognostic accuracy, and mechanism-based therapy for this often-lethal skin cancer.

Pulmonary tumors associated with the JC virus T-antigen in a transgenic mouse model

Many attempts to demonstrate the oncogenic role of the JC virus (JCV) have been partially successful in producing brain tumors, either by direct inoculation of JCV into the brain or in transgenic models in rodents. We previously reported the presence of JCV DNA with a relatively high incidence in pulmonary and digestive organs. However, we could not prove the oncogenic role of JCV. We prepared a transgene composed of the K19 promoter, specific to bronchial epithelium with the JCV T-antigen and established transgenic (TG) mice. Pulmonary tumors were detected without any metastasis in 2 out of 15 (13.3%) 16-month-old K19/JCV T-antigen TG mice. Using immunohistochemistry (IHC), these tumors showed JCV T-antigen, p53 and CK 19 expression, but not expression of nuclear and cytoplasmic β-catenin and insulin receptor substrate 1 (IRS1). IHC revealed the same expression pattern as in the bronchial epithelium of the TG mice. One tumor, which was examined with laser capture microdissection and molecular biological tools, demonstrated an EGFR mutation but not a K-ras mutation. We propose that the pulmonary tumors were derived from the JCV T-antigen in a TG mouse model. These findings shed light on pulmonary carcinogenesis.

Correction: Neurofibromatosis Type 2 Tumor Suppressor Protein, NF2, Induces Proteasome-Mediated Degradation of JC Virus T-Antigen in Human Glioblastoma

Correction: Neurofibromatosis Type 2 Tumor Suppressor Protein, NF2, Induces Proteasome-Mediated Degradation of JC Virus T-Antigen in Human Glioblastoma

Abstract 4774: JC virus T-antigen-dependent activation of Wnt target genes and cell cycle progression in colon cancer.

Abstract Background: The Wnt signaling pathway is aberrantly activated in many different types of cancer including colon cancer. Transcription of target genes in the Wnt pathway promotes strong proliferation and differentiation signals in the cell. This transcription is mediated by β-catenin, the central signaling protein in the Wnt pathway. β-catenin targets include potent transcription factors and cell cycle regulatory proteins such as c-myc and cyclin D1. We have previously shown that a strong regulator of β-catenin-binding is the polyomavirus JC virus protein large T-antigen (TAg). According to sero-epidemiological studies, JCV establishes a latent infection in 70-90% of the general population by adulthood and TAg has been associated with numerous types of cancer in the CNS and GI tract. We and others have previously shown that TAg is expressed in colon cancer samples and has a significant association with β-catenin in the nuclei of colon cancer cells. Our hypothesis is that the viral protein T-antigen (TAg) interacts with β-catenin in colonic epithelial cells to promote transcription of β-catenin target genes particularly c-myc and cyclin D1, which affect cell cycle progression and lead to unchecked cellular growth. Materials and Methods: To test this hypothesis we transfected colon cancer cell lines with a plasmid containing the TAg gene and measured β-catenin target gene transcription. We used the TOPflash reporter construct to determine general β-catenin / TCF4-mediated promoter activity and reporter constructs for the β-catenin target genes c-myc and cyclin D1. We performed RT-qPCR and Western blot analysis to determine mRNA and protein levels of c-myc and cyclin D1 in TAg- and empty vector-transfected cells. We also utilized β-catenin siRNA or the dominant negative TCF4 plasmid to inhibit Wnt signaling in these groups and measured the same endpoints. Results: TCF-dependent promoter activity with a TOPflash reporter plasmid was increased in cells transfected with the TAg gene. This increased activity resulted in elevated levels of β-catenin target genes c-myc and cyclin D1. Moreover, inhibition of β-catenin, either by β-catenin siRNA or co-transfection with DN-TCF4 ablated this response. Finally, we found that transfection with TAg altered cell cycle distribution in these colon cancer cells with an increased proportion of cells in the G2/M phase. Conclusions: We found that the TAg protein activates the Wnt signaling pathway in our cellular model of colon cancer. This activation was determined to be β-catenin-dependent and mediated by TCF4 binding to promoter regions. Moreover, because Wnt pathway activation in these cells leads to cell cycle progression we suggest an oncogenic role for TAg in colon cancer. Considering the large proportion of the population seropositive for JC virus, it is imperative to elucidate its role in the oncogenesis of colon cancer. Citation Format: Michael J. Ripple, Amanda Struckhoff, Robin McGoey, Luis Del Valle. JC virus T-antigen-dependent activation of Wnt target genes and cell cycle progression in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4774. doi:10.1158/1538-7445.AM2013-4774

Neurofibromatosis Type 2 Tumor Suppressor Protein, NF2, Induces Proteasome-Mediated Degradation of JC Virus T-Antigen in Human Glioblastoma

Neurofibromatosis type 2 protein (NF2) has been shown to act as tumor suppressor primarily through its functions as a cytoskeletal scaffold. However, NF2 can also be found in the nucleus, where its role is less clear. Previously, our group has identified JC virus (JCV) tumor antigen (T-antigen) as a nuclear binding partner for NF2 in tumors derived from JCV T-antigen transgenic mice. The association of NF2 with T-antigen in neuronal origin tumors suggests a potential role for NF2 in regulating the expression of the JCV T-antigen. Here, we report that NF2 suppresses T-antigen protein expression in U-87 MG human glioblastoma cells, which subsequently reduces T-antigen-mediated regulation of the JCV promoter. When T-antigen mRNA was quantified, it was determined that increasing expression of NF2 correlated with an accumulation of T-antigen mRNA; however, a decrease in T-antigen at the protein level was observed. NF2 was found to promote degradation of ubiquitin bound T-antigen protein via a proteasome dependent pathway concomitant with the accumulation of the JCV early mRNA encoding T-antigen. The interaction between T-antigen and NF2 maps to the FERM domain of NF2, which has been shown previously to be responsible for its tumor suppressor activity. Co-immunoprecipitation assays revealed a ternary complex among NF2, T-antigen, and the tumor suppressor protein, p53 within a glioblastoma cell line. Further, these proteins were detected in various degrees in patient tumor tissue, suggesting that these associations may occur in vivo. Collectively, these results demonstrate that NF2 negatively regulates JCV T-antigen expression by proteasome-mediated degradation, and suggest a novel role for NF2 as a suppressor of JCV T-antigen-induced cell cycle regulation.