JC Virus T-antigen Research Articles

Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells.

ObjectivePML is a rare and fatal demyelinating disease of the CNS caused by the human polyomavirus, JC virus (JCV), which occurs in AIDS patients and those on immunosuppressive monoclonal antibody therapies (mAbs). We sought to identify mechanisms that could stimulate reactivation of JCV in a cell culture model system and targeted pathways which could affect early gene transcription and JCV T-antigen production, which are key steps of the viral life cycle for blocking reactivation of JCV. Two important regulatory partners we have previously identified for T-antigen include Pur-alpha and SRSF1 (SF2/ASF). SRSF1, an alternative splicing factor, is a potential regulator of JCV whose overexpression in glial cells strongly suppresses viral gene expression and replication. Pur-alpha has been most extensively characterized as a sequence-specific DNA- and RNA-binding protein which directs both viral gene transcription and mRNA translation, and is a potent inducer of the JCV early promoter through binding to T-antigen.Methods and ResultsPur-alpha and SRSF1 both act directly as transcriptional regulators of the JCV promoter and here we have observed that Pur-alpha is capable of ameliorating SRSF1-mediated suppression of JCV gene expression and viral replication. Interestingly, Pur-alpha exerted its effect by suppressing SRSF1 at both the protein and mRNA levels in glial cells suggesting this effect can occur independent of T-antigen. Pur-alpha and SRSF1 were both localized to oligodendrocyte inclusion bodies by immunohistochemistry in brain sections from patients with HIV-1 associated PML. Interestingly, inclusion bodies were typically positive for either Pur-alpha or SRSF1, though some cells appeared to be positive for both proteins.ConclusionsTaken together, these results indicate the presence of an antagonistic interaction between these two proteins in regulating of JCV gene expression and viral replication and suggests that they play an important role during viral reactivation leading to development 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.

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.

Insights into the initiation of JC virus DNA replication derived from the crystal structure of the T-antigen origin binding domain.

JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML). PML is a disease that occurs primarily in people who are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV) DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD) of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel “pocket”; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable.

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

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen

JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies.

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.

Relationship between Methyl CpG Binding Protein 2 and JC Viral Proteins

JC virus (JCV) is a causative agent of progressive multifocal leukoencephalopathy (PML). Methyl CpG binding protein 2 (MeCP2) is a transcriptional control nuclear protein that is abundantly expressed in neurons. We previously observed that the MeCP2 protein is expressed in JCV large T antigen (TAg)-expressing glial cells in PML brains. To investigate the relationship between MeCP2 and JCV TAg, we examined the promoter activity and mRNA and protein expression levels of MeCP2 in JCV TAg-expressing cells. We found that JCV TAg enhances the promoter activity of MeCP2, but does not enhance the mRNA and protein levels of MeCP2. These results suggest that post-transcriptional mechanisms may play a role in MeCP2 expression.

Abstract 5490: Prospective study of seroreactivity to JC virus T-Antigen and risk of colorectal cancers and adenomas

Abstract JC virus (JCV) has been hypothesized to play a role in the development of colorectal cancer (CRC), although findings are inconsistent across studies. Case series have reported the presence of JCV DNA in tumors, but two serological studies observed no statistically significant association between circulating antibodies to JCV capsid protein and risk of CRC. No epidemiologic study has examined the association between antibodies to the JCV oncoprotein, T-antigen, and cancer. To investigate the association between circulating levels of JCV T-Ag antibodies and the risk of CRC and adenomas, we conducted a case-control study nested within a community-based cohort established in Washington County, MD, in 1989. Cancer registries were used to identify incident cases of CRC occurring within this cohort through 2006 (n=251), and 123 colonoscopy-detected adenoma cases were initially self-reported through a follow-up questionnaire and subsequently verified through pathology report review. Controls were selected from cohort participants who did not develop CRC or adenomas and matched to cases on age, sex, race, date of blood draw, and for adenoma cases, date of endoscopy. Recombinant protein-based enzyme linked immunosorbent assays (ELISA) were used to measure JCV T-Ag antibodies in baseline pre-diagnostic plasma samples. High versus low seroreactivity was defined using the median value among the controls. Conditional logistic regression was used to calculate odds ratios (OR's) and 95% confidence intervals (CI). Overall, high JCV T-Ag seroreactivity was not associated with development of CRC (OR=1.24, 95% CI=0.84-1.84) or adenoma (OR=1.18, 95% CI=0.67-2.09). Among females, high JCV T-Ag seroreactivity was associated with an increased risk of CRC (OR=1.78, 95% CI=1.00-3.17), whereas no association was observed among males (OR=0.89, 95% CI=0.51-1.54). High JCV T-Ag seroreactivity was associated with non-statistically significant risks of rectal cancer (OR=1.55, 95% CI=0.72-3.30) and CRC diagnosed at distant stage (OR=2.25, 95% CI=0.69-7.31), but not with distal or proximal colon cancer or CRC diagnosed at local/regional stages. High JCV T-Ag seroreactivity was not associated with increased risk of adenomas overall or by gender. However, a statistically significant association was observed between high JCV T-Ag seroreactivity and adenomas of the proximal colon (OR=2.80, 95% CI=1.01-7.77), but not the distal colon. No clear differences in risk were observed when results were stratified by adenoma size or histology. These results suggest that associations between JCV infection and CRC/adenomas may differ by gender and anatomic site. Further studies incorporating multiple serologic and DNA-based markers are needed to better understand the relationship between JCV infection and CRC/adenomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5490. doi:1538-7445.AM2012-5490

JC Virus T-Antigen Regulates Glucose Metabolic Pathways in Brain Tumor Cells

Recent studies have reported the detection of the human neurotropic virus, JCV, in a significant population of brain tumors, including medulloblastomas. Accordingly, expression of the JCV early protein, T-antigen, which has transforming activity in cell culture and in transgenic mice, results in the development of a broad range of tumors of neural crest and glial origin. Evidently, the association of T-antigen with a range of tumor-suppressor proteins, including p53 and pRb, and signaling molecules, such as β-catenin and IRS-1, plays a role in the oncogenic function of JCV T-antigen. We demonstrate that T-antigen expression is suppressed by glucose deprivation in medulloblastoma cells and in glioblastoma xenografts that both endogenously express T-antigen. Mechanistic studies indicate that glucose deprivation-mediated suppression of T-antigen is partly influenced by 5′-activated AMP kinase (AMPK), an important sensor of the AMP/ATP ratio in cells. In addition, glucose deprivation-induced cell cycle arrest in the G1 phase is blocked with AMPK inhibition, which also prevents T-antigen downregulation. Furthermore, T-antigen prevents G1 arrest and sustains cells in the G2 phase during glucose deprivation. On a functional level, T-antigen downregulation is partially dependent on reactive oxygen species (ROS) production during glucose deprivation, and T-antigen prevents ROS induction, loss of ATP production, and cytotoxicity induced by glucose deprivation. Additionally, we have found that T-antigen is downregulated by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the pentose phosphate inhibitors, 6-aminonicotinamide and oxythiamine, and that T-antigen modulates expression of the glycolytic enzyme, hexokinase 2 (HK2), and the pentose phosphate enzyme, transaldolase-1 (TALDO1), indicating a potential link between T-antigen and metabolic regulation. These studies point to the possible involvement of JCV T-antigen in medulloblastoma proliferation and the metabolic phenotype and may enhance our understanding of the role of viral proteins in glycolytic tumor metabolism, thus providing useful targets for the treatment of virus-induced tumors.

Abstract 2711: JC virus T-Antigen (SV40) is an independent prognostic marker for poor disease free survival in epithelial ovarian carcinoma

Abstract Epithelial ovarian cancer is the leading cause of death from gynecologic cancers and because of the pelvic location of the ovaries, the patients present at an advanced stage that is generally incurable. SV40 is a type of polyoma virus has been shown to induce tumors in rodents, transform cells in tissue culture and promote tumor transformation in transgenic mice. Many studies have reported the presence of simian virus 40 (SV40) deoxyribonucleic acid (DNA) or protein in human brain tumors, bone cancers, non-Hodgkin's lymphomas, malignant mesothelioma and other solid epithelial tumors including epithelial ovarian carcinomas. We studied the expression of SV40 in a tissue microarray cohort of 156 epithelial ovarian cancers by immunohistochemistry. The incidence of SV40 expression was 44.3% (58/131) and poorly differentiated carcinomas showed a trend towards higher expression of SV40 as compared to moderately and well differentiated tumors (p=0.0898). Validation of SV40 IHC expression was done by polymerase chain reaction (PCR) assay in selected clinical samples and ovarian cancer cell lines. A good concordance was observed between immunohistochemical expression of SV40 and amplification by PCR. Earlier studies have reported various molecular targets of SV40: up-regulation of c-MET oncogene and IGF-1; inhibition of tumor suppressor genes like p53, RASS1FA and protein phosphotase PP2A. To elucidate the link between these targets and SV40 we assessed the immunohistochemical expression of p53, RASS1FA, PP2A, c-MET and IGF-1 the cohort of epithelial ovarian carcinomas. In addition we also analyzed the association of SV40 with known oncogenic mutations in p53, KRAS and PIK3CA genes. SV40 expression showed a significant association with presence of p53 mutation (p<0.0001.) as well as p53 overexpression (p<0.0001). In addition, SV40 expression was associated with overexpression of c-MET (p=0.0111) and IGF-1(p=0.0334). EOC patients with SV40 expression had a poor progression free survival (PFS) of 13.2 months as compared to 20 months with absent SV40 expression (p=0.0980). In the multivariate analysis using Cox Proportional Hazard model for multiple factors like age, FIGO stage, grade, the relative risk was 1.70 for high SV40 expression (95% CI 1.10 – 2.69; p=0.0227) and 2.32 for high stage group III-IV (95% CI 1.33 – 3.90; p=0.0038). Thus, SV40 over expression was an independent prognostic marker in EOC. Altogether, our data highlights the prognostic utility of SV40 as a prognostic biomarker and confirms the well-known effect of SV40 on down-regulation or inactivation of p53, and activation of c-MET/IGF-1 in the epithelial ovarian carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2711. doi:10.1158/1538-7445.AM2011-2711

STAT3 Expression, Molecular Features, Inflammation Patterns, and Prognosis in a Database of 724 Colorectal Cancers

STAT3 is a transcription factor that is constitutively activated in some cancers. It seems to play crucial roles in cell proliferation and survival, angiogenesis, tumor-promoting inflammation, and suppression of antitumor host immune response in the tumor microenvironment. Although the STAT3 signaling pathway is a potential drug target, clinical, pathologic, molecular, or prognostic features of STAT3-activated colorectal cancer remain uncertain. Utilizing a database of 724 colon and rectal cancer cases, we evaluated phosphorylated STAT3 (p-STAT3) expression by immunohistochemistry. The Cox proportional hazards model was used to compute mortality HR, adjusting for clinical, pathologic, and molecular features, including microsatellite instability (MSI), the CpG island methylator phenotype (CIMP), LINE-1 methylation, 18q LOH, TP53 (p53), CTNNB1 (β-catenin), JC virus T-antigen, and KRAS, BRAF, and PIK3CA mutations. Among the 724 tumors, 131 (18%) showed high-level p-STAT3 expression (p-STAT3-high), 244 (34%) showed low-level expression (p-STAT3-low), and the remaining 349 (48%) were negative for p-STAT3. p-STAT3 overexpression was associated with significantly higher colorectal cancer-specific mortality [log-rank P = 0.0020; univariate HR (p-STAT3-high vs. p-STAT3-negative): 1.85, 95% CI: 1.30-2.63, P(trend) = 0.0005; multivariate HR: 1.61, 95% CI: 1.11-2.34, P(trend) = 0.015]. p-STAT3 expression was positively associated with peritumoral lymphocytic reaction (multivariate OR: 3.23; 95% CI: 1.89-5.53, P < 0.0001). p-STAT3 expression was not associated with MSI, CIMP, or LINE-1 hypomethylation. STAT3 activation in colorectal cancer is associated with adverse clinical outcome, supporting its potential roles as a prognostic biomarker and a chemoprevention and/or therapeutic target.

Interaction and co-localization of JC virus large T antigen and the F-box protein β-transducin-repeat containing protein

Lytic infection and transformation of cultured cells by JC virus (JCV) require five tumor proteins, which interact with factors regulating critical cellular processes. We demonstrate that JCV large T antigen (TAg) binds the F-box proteins β-transducin-repeat containing protein-1 and 2 (βTrCP1/2). These interactions involve a phosphodegron (DpSGX 2–4pS) found in βTrCP substrates. TAg stability is unaltered, suggesting TAg is a pseudo-substrate. βTrCP and TAg co-localize in the cytoplasm, and a functional SCF complex is required. We examined whether TAg influences the levels of β-catenin, a βTrCP substrate. We were unable to demonstrate that TAg elevates β-catenin as previously reported, and a mutant TAg unable to bind βTrCP also had no detectable effect on β-catenin stability. Results presented in this study link JCV TAg to the cellular degradation complex, SCF βTrCP1/2. Proteasomal degradation is essential for proper regulation of cellular functions, and interference with proteasomal pathways highlights possible JCV pathogenic and oncogenic mechanisms.

Patients With Sporadic Colorectal Cancer or Advanced Adenomatous Polyp Have Elevated Anti-JC Virus Antibody Titer in Comparison With Healthy Controls

JC virus (JCV) is thought to infect approximately 80% of the human population. Antibodies against JCV can be found in the sera of many people with and without colorectal carcinoma (CRC). We hypothesized that JCV antibody titer will be higher in CRC patients than in healthy controls. To evaluate this hypothesis in a cohort of patients undergoing colonoscopy. We compared JCV antibody titers in patients with simple adenoma, advanced adenomatous polyp (AAP), CRC, and healthy controls, and evaluated JCV DNA in the tissue. Ninety-seven patients undergoing colonoscopy offered to participate in the study. Normal colonoscopy, simple adenoma, AAP, and CRC were found in 41, 19, 12, and 25 cases, respectively. A blood sample was taken for JCV DNA isolation and serology. In 18 patients with CRC or AAP tissue samples were taken for JCV DNA isolation and T-antigen (T-Ag) detection. A positive correlation was found between a JCV antibody titer and advanced colonic pathology. The average titer for normal controls, simple polyp, AAP, and CRC was 2.61+/-0.72, 2.95+/-0.77, 3.33+/-0.76, and 3.30+/-0.50 log, respectively (P<0.001). Viral DNA could not be shown in the serum. The presence of neoplastic tissue T-Ag (in 33.3% of the patients) was not associated with a difference in the log titer of serum antibody. In this study we showed that patients with advanced neoplasia, compared with patients with normal colonoscopy, harbor a higher JCV antibody titer in the serum. If confirmed, our finding may serve as a marker for CRC or for an earlier stage of AAP.

1033 Genetic and Epigenetic Features in Synchronous Colorectal Cancer Cases Versus Solitary Cancers, Utilizing 1243 Colorectal Cancer Patients From Two Large Prospective Cohort Studies

Background & Aims: Synchronous colorectal cancers refer to two or more primary colorectal carcinomas detected in a single individual at the time of the first diagnosis of colorectal cancer. Synchronous neoplasias, which arise in a background of common etiologic (genetic or environmental) factors, can provide a unique model to examine molecular aberrations. Previous studies have reported several molecular features in synchronous colorectal cancers. However, in all of these studies, control solitary cases were retrospectively selected, thereby subject to potential selection bias. In this study, we analyzed data collected from 32 patients with synchronous colorectal cancers and 1211 solitary cancers (controls) in 2 well-characterized, large prospective cohort studies. Methods: Tumor tissue specimens were analyzed for methylation in LINE-1 and 16 CpG islands (CACNA1G, CDKN2A [p16], CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1, CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14 [CDKN2A/ARF], and WRN); microsatellite instability (MSI) status; CpG island methylator phenotype (CIMP) status; KRAS, BRAF, and PIK3CA mutations; and expressions of DNA methyltransferase-3B (DNMT3B) and JC virus T-antigen (JCVT). Results: BRAF mutations were significantly more common in synchronous cancers (30%=9/30; p=0.0080) than in solitary cancers (13%=158/1198). Likewise, synchronous cancers were more commonly CIMP-high (p=0.047) and MSI-high (p=0.030), compared to solitary controls, whereas no association was found between tumor synchronicity and other molecular markers. Considering the confounding effect by age, we constructed a logistic regression model including age, BRAF mutation, CIMP-high, MSI-high and synchronicity status as an outcome variable. As a result, the association between BRAF mutation (p=0.010), CIMP-high (p=0.038), MSIhigh (p=0.033) and tumor synchronicity persisted, while age was no longer associated with tumor synchronicity. Among the 32 synchronous cancer cases, 12 synchronous cases could provide cancer tissues from both of two cancers in synchronous pairs. We found that methylation levels of LINE-1 (Spearman r=0.76; p=0.0062) and levels of CpG island methylation (p<0.0001) correlated between synchronous pairs from the same individuals. Conclusions: Compared with solitary cancers, synchronous colorectal cancers more commonly show BRAF mutations, CIMP-high, and MSI-high. In addition, synchronous colorectal cancer pairs exhibit a significant correlation of LINE-1 methylation levels and concordance in CpG island methylation. Our study may be an important one step forward to elucidate clinical and molecular characteristics of synchronous colorectal cancers.

M1953 JC Virus T-Antigen Expression in Gastrointestinal Mucosa of Immunosuppressed Patients – A Prospective, Controlled Study

M1953 JC Virus T-Antigen Expression in Gastrointestinal Mucosa of Immunosuppressed Patients – A Prospective, Controlled Study

The presence of JC virus in gastric carcinomas correlates with patient's age, intestinal histological type and aberrant methylation of tumor suppressor genes

JC virus (JCV) is a neurotropic polyomavirus and the causative agent of progressive multifocal leukoencephalopathy. A role for JCV in gastrointestinal malignancies has been recently suggested. This study was carried out to determine the prevalence of polyomaviruses including JCV, BKV and SV40 in gastric cancers in Tunisia and to determine the clinicopathological characteristics of virus-associated gastric carcinomas. The presence of polyomaviruses DNA sequences was surveyed in 61 cases of primary gastric carcinomas and in 53 paired non-tumor gastric mucosa by PCR. Findings were correlated to clinicopathological parameters, p53 expression and methylation status of 11 tumor-related genes. Using PCR assays, JCV T-antigen sequence was more frequently detected in gastric carcinomas than in non-tumor gastric mucosa (26 vs 6%, P=0.03), while those of SV40 and BKV were not detected in any cases. Correlation analysis showed that JCV had higher frequency in patients older than 55 years (P=0.034) and in the intestinal histological type (P=0.04). With regard to methylation status, P16 and P14 showed significantly higher methylation frequencies in JCV-positive gastric carcinomas than in JCV-negative cases (P=0.007 and P=0.003, respectively). Moreover, the mean of the methylation index was significantly higher in JCV-positive than in JCV-negative cases (P=0.024). In multivariate logistic regression analysis, age of patients and the methylation index are only the two independent factors associated with JCV infection. Kaplan–Meier survival analysis showed a trend toward better survival for JCV-associated gastric carcinomas patients (log-rank, P=0.11). Our study suggests a role of JCV as cofactor in the pathogenesis of the intestinal type of gastric carcinomas in older persons.

Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion-induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.

Binding and Internalization of Iron Oxide Nanoparticles Targeted To Nuclear Oncoprotein

A targeted nanoconjugate is being developed for non-invasive detection of gene expression in cells expressing the JC virus oncoprotein, T-antigen, which has been associated with medulloblastoma and other cancers. JC virus T-antigen localizes predominantly to the nucleus via a classical monopartite nuclear localization signal (NLS). An antibody fragment which recognizes JC virus T-antigen was attached to cross-linked dextran coated iron oxide nanoparticles. Radiolabeled conjugates were added to mouse medulloblastoma cells expressing the target T-antigen to test their ability to bind to tumor cells and be internalized by the cells. All conjugates containing targeting antibody bound to cells and were internalized, with increasing levels over time. There was no difference in cell binding or internalization among conjugates containing 2, 4, 6 or 8 antibody fragments per nanoparticle. Conjugates with only nonspecific antibody on nanoparticles, or unconjugated nonspecific antibody, had significantly lower total binding and internalization than conjugates with targeting antibody. Unconjugated targeting antibody had equivalent or lower cell uptake compared with targeted nanoparticle conjugates. Specificity of uptake was demonstrated by >80% reduction of nanoconjugate uptake in the presence of 100 fold excess of unconjugated antibody. The presence of a membrane translocation peptide (Tat) on the nanoparticles in addition to targeting antibody did not improve nanoconjugate internalization over the internalization caused by the antibody alone. This antibody nanoconjugate demonstrates feasibility of targeting a nuclear protein and suggests that a minimum number of antibody fragments per nanoparticle are sufficient for achieving binding specificity and efficient uptake into living cells.