Association of Human Polyomavirus JCV with Colon Cancer ... · JCV is detected in the urine of...

[CANCER RESEARCH 62, 7093–7101, December 1, 2002]

Association of Human Polyomavirus JCV with Colon Cancer: Evidence forInteraction of Viral T-Antigen and �-Catenin1

Sahnila Enam, Luis Del Valle, Cesar Lara, Dai-Di Gan, Carlos Ortiz-Hidalgo, Juan P. Palazzo, and Kamel Khalili2

Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122 [S. E., L. D. V., C. L., D-D. G., K. K.];Departmento de Patologia, The American British Cowdray Medical Center I. A. P., Mexico D.F., Mexico 01120 [C. O-H.]; and Department of Pathology, Thomas JeffersonUniversity Hospital, Philadelphia, Pennsylvania 19107 [J. P. P.]

ABSTRACT

Infection of the gastrointestinal tract by the human polyomavirus, JCV,which has been frequently detected in raw urban sewage, can occur viaintake of contaminated water and food. In light of earlier reports on thetumorigenecity of JCV, we investigated the presence of the JCV genomeand the expression of viral proteins in a collection of 27 well-characterizedepithelial malignant tumors of the large intestine. Results from geneamplification revealed the presence of the viral early genome in 22 of 27samples. Expression of the viral oncogenic early protein, T-antigen, andthe late auxiliary protein, Agnoprotein, was observed in >50% of thesamples. The absence of the viral capsid protein in the tumor cellsexcludes productive replication of the virus in neoplastic cells. Lasercapture microdissection confirmed the presence of the JCV genome andexpression of T-antigen in precancerous villous adenomas and regions ofinvasive adenocarcinoma. The ability of JCV T-antigen to interact with�-catenin and the nuclear detection of �-catenin in T-antigen-positivecells suggests dysregulation of the Wnt pathway in the tumor cells. Thecoproduction of T-antigen and �-catenin in colon cancer cells enhancedtranscription of the c-myc promoter, the downstream target of �-catenin.These observations provide evidence for a possible association of JCVwith colon cancer and suggest a novel regulatory role for T-antigen in thederegulation of the Wnt signaling pathway through �-catenin in tumors ofthe gastrointestinal tract.

INTRODUCTION

Results from serological studies have indicated an asymptomaticinfection of �90% of the adult population by age 15 with the humanpolyomavirus, JCV (1). It is believed that childhood exposure to JCV,most likely through the upper respiratory tract, causes persistentinfection of kidney epithelial cells where the virus remains in a latentstate during the life of the infected individual. In patients with im-paired cell-mediated immunity, JCV enters the brain and its produc-tive replication in glial cells causes the fatal demyelinating disease ofthe central nervous system, Progressive Multifocal Leukoencephalop-athy (for review see Ref. 2). Moreover, recent studies have establishedan association of JCV with a broad range of human brain tumors, mostnotably medulloblastomas (3–5). JCV can transform cells that aremanifested by distinct morphological changes, rapid division, pro-longed life spans, and the ability to form dense foci in culture (6–8).The oncogenic potential of this virus has also been established inseveral experimental animals (for review see Ref. 9). Although theprecise mechanism responsible for JCV-induced cellular transforma-tion and tumor formation is not fully understood, it is believed that theviral early protein, T-antigen, through interaction with several cellregulatory proteins, including tumor suppressors and cell cycle regu-lators, promotes uncontrolled progression of cells through the cell

cycle (10, 11). Furthermore, recent studies have revealed the potentialof JCV T-antigen to modulate several signaling pathways, includinginsulin-like growth factor I and Wnt in cells derived from JCV-induced mouse medulloblastoma (12, 13).

JCV is detected in the urine of 20–80% of adults (14). Results fromgenotyping of JCV excreted by a diverse group of humans hasrevealed that infection with JCV is influenced by the geographicalorigin, ethnic group, and age (14, 15). Earlier surveys of raw sewagefrom urban areas have shown the detection of JC viral particles insewage samples from widely divergent areas (16–18), suggesting apotential reentry of JCV and/or viral DNA into the human populationthrough the intake of virus-contaminated water and food. In support ofthis notion, earlier studies have revealed the presence of JCV DNAsequences in the upper and lower human gastrointestinal tract (19,20). Considering the tumorigenicity of JCV and its potential intakethrough the intestine, we performed a comprehensive study on acollection of well-characterized malignant epithelial tumors of thecolon for the presence of JCV DNA sequence and expression of theviral early and late proteins. Specimens were obtained from ThomasJefferson University Hospital (Philadelphia, PA) and the AmericanBritish Cowdray Medical Center I. A. P. (Mexico D.F., Mexico).

A total of 27 cases of adenocarcinoma in patients between 41 and91 years of age was selected for the initial evaluation of tumorlocation and expression of various marker proteins. Seven tumorswere obtained from the cecum, 2 from the ascendant colon, 3 from thetransverse region, 4 from the descendent colon, and 11 from thesigmoid (Table 1). All tumors were positive for EMA3 and all pro-duced cytokeratin. None of the samples expressed desmin, whereasmost showed various levels of CEA (Table 1). Histological assess-ment of the tumors as compared with a normal area of the colonicmucosa, which is characterized by interdigitated columnar and gobletcells, revealed papillary structures lined with atypical columnar cellscharacteristic of villous adenoma in seven of the samples, as well asisles of in situ and invasive neoplastic epithelial cells. The invasiveglandular structures contained pleomorphic cells with atypical nucleiand frequent mitotic figures. Necrotic areas within the tumors werefrequently observed. Immunohistochemistry against EMA showedpositive reactivity in columnar cells of normal tissue as well as inpreneoplastic and neoplastic cells. Furthermore, tumor cells showedstrong immunoreactivity with antibodies recognizing cytokeratin andCEA (data not shown).

For the detection of the JCV gene sequences, total DNA wasextracted from paraffin-embedded tissues and evaluated by PCRtechniques using three pairs of primers derived from the viral earlygenome, T-antigen, the viral late auxiliary gene encoding Agnopro-tein, and the viral late capsid gene VP1, as detailed in Fig. 1A. Theamplified DNAs were analyzed by Southern blot hybridization usingoligonucleotide DNA probes specific for the amplified JCV se-quences. Results from several experiments revealed that 22 of thesamples (81.5%) contained the early region of the JCV genome,

Received 8/2/02; accepted 10/4/02.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was made possible by grants awarded by NIH (to K. K.).2 To whom requests for reprints should be addressed, at Center for Neurovirology and

Cancer Biology, College of Science and Technology, Temple University, 1900 North 12thStreet, 015-96, Room 203, Philadelphia, PA 19122. Phone: (215) 204-0678; Fax:(215) 204-0679; E-mail: [email protected].

3 The abbreviations used are: EMA, epithelial membrane antigen; CEA, carcino-embryonic antigen; LCM, laser capture microdissection; TCF, T cell factor, LEF, leuke-mia enhancing factor.

7093

Research. on March 6, 2021. © 2002 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

whereas 16 samples (59.2%) contained DNA sequences correspond-ing to Agnogene. The region of the JCV genome spanning VP1 wasdetected in four samples (14.8%). Fig. 1 illustrates a representativeSouthern blot, and Table 2 summarizes the results from PCR ampli-fication/Southern blotting. Detection of JCV DNA sequences pro-vided a rationale to investigate the expression of the viral T-antigen aswell as Agnoprotein and VP1 in these samples. Results from immu-nohistochemistry revealed the expression of T-antigen in 17 (62.9%)samples (Table 2). Although no immunoreactivity with anti-VP1antibody was observed in either tumor or nontumor regions of thesamples, 12 specimens (44.4%) showed positive staining with anti-Agnoprotein antibody (Table 2). Fig. 2 illustrates results from immu-nohistochemical labeling in which T-antigen was detected in an areaof a tumoral emboli located in the submucosal lymphatic vessels andabsent in the suprayacent normal colonic mucosa (Fig. 2A). In thepreneoplastic villous adenoma, the columnar cells show nuclear im-

munoreactivity for T-antigen (Fig. 2B). In the invasive tumor, some ofthe epithelial glandular neoplastic cells exhibited nuclear accumula-tion of JCV T-antigen (Fig. 2C). Cytoplasmic perinuclear accumula-tion of Agnoprotein was evident in a polyp as well as in neoplasticcells (Fig. 2, D and E, respectively). No evidence of VP1 expressioncould be observed in various regions of the samples (Fig. 2, F and G).

Next, the LCM system was used to extract DNA from specificareas of paraffin-embedded sections, which expressed positiveimmunoreactivity with anti-T-antigen antibody. Three regions ofthe specimens were selected representing normal mucosa, villousadenoma, and the invasive adenocarcinoma (Fig. 3A). Results fromgene amplification revealed detection of sequences correspondingto the JCV early genome in DNA obtained from villous adenomasand invasive adenocarcinoma (Fig. 3B). A very weak signal cor-responding to JCV sequences was observed upon amplification ofDNA from a normal appearing region in juxtaposition with a

Fig. 1. Structural organization of the JCV ge-nome and detection of JCV DNA in colon cancer.The numbers within the inner circle show the mapposition with 0.0 being the EcoRI site (33). Thethick solid arrow on the left depicts the viral earlyprotein T-antigen, whereas the shaded arrows onthe right point to the positions of the viral lateproteins, Agnoprotein, and the capsid protein, VP1.The positions of the PCR primers are shown by thinarrows outside of the circle. The size of the ampli-fied DNAs and the location of the DNA probesused for Southern blot hybridization specific for theamplified T-antigen, Agnoprotein, and VP1 se-quences are shown. Numbers over the hybridiza-tion blots indicate the case number on Table 2. �,negative control; �, positive control; M, molecularweight standards. Size of the different JCV PCRproduct is indicated.

Table 1 Clinical and immunohistochemical evaluation of human colon cancera

No. Origin DiagnosisAge (yr)/Gender Location Stage

Immunohistochemistry

EMA Keratin Desmin CEA

1 Mexico Adenocarcinoma 61/Fb Cecum Dukes C1 ��c �� 2 Mexico Adenocarcinoma 55/F Sigmoid colon Dukes B1 �� 3 Mexico Adenocarcinoma 74/F Descendent colon Dukes B1 � � � �4 Mexico Adenocarcinoma 54/F Descendent colon Dukes C1 �� 5 Mexico Mixed Adeno � squamous 62/F Sigmoid colon Dukes B3 �� f6 Mexico Adenocarcinoma � villous polyp 80/F Ascendent colon Dukes B2 �� 7 Mexico Adenocarcinoma � villous polyp 74/M Sigmoid colon Dukes C1 � �� 8 Mexico Adenocarcinoma � villous polyp 69/F Ascendent colon Dukes B2 � � � �9 Mexico Adenocarcinoma � villous polyp 52/F Sigmoid colon Dukes C1 ��

10 Mexico Adenocarcinoma 51/M Sigmoid colon Dukes B2 � � � ��11 Mexico Adenocarcinoma � villous polyp 75/F Cecum Dukes B2 �� 12 Mexico Adenocarcinoma 81/M Cecum Dukes B2 �� 13 Mexico Adenocarcinoma � villous polyp 55/F Sigmoid colon Dukes C2 � �� 14 Mexico Adenocarcinoma (poor dif) 41/M Sigmoid colon Dukes B2 �� 15 Mexico Adenocarcinoma (poor dif) 85/M Descendent colon Dukes B2 �� 16 Mexico Adenocarcinoma 71/M Descendent colon Dukes B2 � � � ��17 Mexico Adenocarcinoma 62/F Sigmoid colon Dukes B3 �� 18 USA Adenocarcinoma � villous polyp 74/M Transv colon Dukes B1 � �� 19 USA Adenocarcinoma 69/F Cecum Dukes B1 �� 20 USA Adenocarcinoma 82/M Sigmoid colon Dukes B1 � �� 21 USA Adenocarcinoma (poor dif) 65/F Hepatic flexure Dukes B2 �� 22 USA Adenocarcinoma 81/M Cecum Dukes C2 �� 23 USA Adenocarcinoma 54/M Sigmoid colon Dukes C2 �� 24 USA Adenocarcinoma 52/F Rectosigmoid Dukes B2 � � � ��25 USA Mucinous adenocarcinoma 71/F Cecum Dukes B2 �� 26 USA Adenocarcinoma 65/F Cecum Dukes B1 �� 27 USA Adenocarcinoma (poor dif) 91/F Transv colon Dukes B2 ��

a Diagnosis of the tumors is based on the WHO Classification of Tumours of the Digestive System, as described in “Materials and Methods.”b F, female; M, male. Age of the patient at the time of surgical resection is shown.c �, indicates negative immunoreactivity; �, 1–30% cell positivity; ��, 31–60% cell positivity; ��, �61% cell positivity; f � indicates focal positivity.

7094

ASSOCIATION OF HUMAN POLYOMAVIRUS JCV WITH COLON CANCER



precancerous areas, suggesting the presence of few JCV-positivecells in this region.

Although the importance of the presence of JCV DNA in thegenesis of human epithelial malignant tumors of the colon remainsunclear, the detection of viral proteins, including T-antigen, in thetumor cells indicate the potential involvement of JCV in pathwaysleading to the development and/or progression of cancer. Examinationof p53, a cell regulatory protein that loses its activity and becomesmore stable upon mutation or association with viral oncoproteins,including JCV T-antigen, in this collection of colon cancers revealedthe detection of p53 in 19 samples (70.3%) of which 13 were alsopositive for T-antigen (Table 2). Fig. 4 represents immunohistochem-istry of a T-antigen-positive sample in which nuclear p53 expressionwas observed in neoplastic glandular cells inside lymph vessels un-derlying a normal epithelium area (Fig. 4A). The columnar cells liningthe villous polyps and the neoplastic cells in the glandular structureswere similar to the pattern of T-antigen expression and showedimmunoreactivity to p53 (Fig. 4, B and C, respectively). Results fromdouble immunofluorescence labeling of sections with anti-p53 andanti-T-antigen showed abundant columnar cells in a villous adenomawith nuclear localization of both p53 and T-antigen (Fig. 4, D–F).Colocalization of T-antigen and p53 suggests a possible interaction ofthese two proteins that can lead to uncontrolled proliferation of thetumor cells.

In addition to p53, T-antigen may affect other regulatory events thatare implicated in cancer development. Earlier studies have demon-strated that �-catenin, a key component of Wnt signaling pathway, isaffected in colon cancer. Mutations in �-catenin that prolong itsstability in the cytoplasm permits the association of �-catenin withTCF/LEF transcription factors (for review see Ref. 21) and facilitatesthe nuclear import of �-catenin where it activates transcription of aseries of cellular genes that are involved in rapid cell proliferation(22). It has been shown that mutations in the phosphorylation residuesof �-catenin within exon 3 stabilize �-catenin by preventing itsproteasome degradation (for review see Ref. 23). Evaluation of the

�-catenin gene in T-antigen-positive colon specimens showed nomutations in the region of the protein-spanning exon 3 (unpublisheddata). However, results from immunohistochemistry revealed nucleardetection of �-catenin in five cases that showed strong positivenuclear immune reaction for T-antigen (Table 2, also see Fig. 5A).Similarly, cytoplasmic and nuclear labeling of TCF-4 was evident inthe neoplastic cells (Table 2 and Fig. 5B), whereas TCF-1 and LEF-1were mostly detected in the cytoplasm (LEF-1 staining is depicted inFig. 5C). These observations suggest that the Wnt signaling pathwaymay be affected by the expression of the JCV genome in these tumorcells. Interestingly, results from double labeling of the tumor cellswith anti-T-antigen and anti-�-catenin antibodies showed colocaliza-tion of T-antigen and �-catenin in the nuclei of neoplastic columnarcells (Fig. 5, compare D and E to F).

To investigate the possible interaction of JCV T-antigen with�-catenin, protein extract from a colon cancer cell line, HCT116,transfected with a plasmid-expressing T-antigen was immunoprecipi-tated with anti-T-antigen antibody, and the immunocomplex wasanalyzed by Western blot with anti-�-catenin antibody. As shown inFig. 6A, a band corresponding to �-catenin was present in the immu-nocomplex pulled down by anti-T-antigen antibody but not withcontrol preimmune sera. Accordingly, the level of c-myc gene expres-sion, the downstream target for �-catenin, was enhanced in the cells(Fig. 6B). No changes in the level of housekeeping protein, Grb-2,was detected. To assess the functional importance of T-antigen and�-catenin’s presence in the cells, transfection of HTC116 cells wascarried out in the presence of plasmids containing the c-myc promoterdriving the reporter luciferase gene. According to the results illus-trated in Fig. 6C, the expression of �-catenin had no stimulatory effectupon the c-myc promoter. However, in the presence of T-antigen,�-catenin significantly enhanced transcription of the c-myc promoter.T-antigen alone caused a modest increase in the activity of c-mycpromoter, suggesting its cooperation with endogenous �-catenin inthese cells. To investigate the subcellular localization of �-catenin inthe absence and presence of JCV T-antigen, HCT116 cells were

Table 2 DNA and protein analysis of human colon cancer

No.

PCR/Southern Immunohistochemistrya

T-antigen VP-1 Agno

Viral proteins Cellular proteins

T-antigen VP-1 Agno p53 APC �-Catenin TCF-4 TCF-1 LEF-1

1 � � � � � � � � � � cy � � cy2 � � � �� 3 � � � � � � � � � � � �4 � � � � � � �� cy � �� cy5 � � � � � � � � � � cy � cy � cy6 � � � � � � �f �� cy � nu � cy � nu � � cy7 � � � � � � � � �� nu � cy � cy �� cy8 � � � � � � � � � � cy � cy �� cy9 � � � � � � � � � � � � cy

10 � � � � � � �� cy � nu � � cy � cy11 � � � �� cy12 � � � �� cy � nu �� cy � cy13 � � � � � � �� 14 � � � � � � � � � � � �15 � � � �� f � � � cy � nu � �16 � � � � � � � � � � cy � nu � � cy17 � � � � � � �� 18 � � � � � � �� cy � nu � cy � nu � �19 � � � � � � �� cy � cy � nu � cy �20 � � � �� cy � nu � cy � nu � �21 � � � � � � �� cy � cy � nu � cy �22 � � � � � � � � � � cy � nu � �23 � � � � � � �f � � � � cy � cy24 � � � � � �� cy � nu � �25 � � � � � � � � � cy � cy � nu � � cy26 � � � � � � �� cy � nu � �27 � � � � � � � � � � � cy �

a Immunohistochemistry: �, indicates negative immunoreactivity; �, 1–30% cell positivity; ��, 31–60% cell positivity; ��, �61% cell positivity; f, indicates focal positivity;cy, cytoplasmic immunoreactivity; nu, nuclear immunoreactivity; APC, adenomatous polyposis coli.

7095




transfected with a plasmid-expressing T-antigen. As shown in Fig.6D, among the group of cells, �-catenin was colocalized in thenucleus of the cells that also expresses T-antigen. In the other cellswhere T-antigen is not present, �-catenin was detected in the cyto-plasm. Taken together, these observations suggest that the associationof T-antigen with �-catenin may translocate this protein to the nu-cleus.

MATERIALS AND METHODS

Clinical Samples. A total of 27 paraffin-embedded tumors of the colonwas collected from the pathology archives of the following institutions: 17samples were obtained from the American British Cowdray Hospital in Mex-

ico City, Mexico, and 10 samples were obtained from Thomas JeffersonUniversity in Philadelphia, Pennsylvania. The tumors were histologicallygraded and immunohistochemically characterized according to the WHO Clas-sification of Tumors of the Gastrointestinal Tract (24).

DNA Extraction and Analysis. A dedicated microtome was used to sec-tion the paraffin blocks. Furthermore, the block and blade holder were peri-odically autoclaved, and a new, disposable blade was used for each specimen.The sections were handled with a disposable, one-time use applicator toprevent contamination. DNA was extracted from �10 sections of 10 �m inthickness from each of the tissue samples by using the QIAamp Tissue Kit,according to the manufacturer’s instructions (Qiagen, Valencia, CA).

PCR amplification was performed by using three individual sets of primers:PEP1 and PEP2 (nucleotides 4255–4272 and 4408–4427, respectively), which

Fig. 2. Expression of JCV proteins in human coloncancer. A, dissection of biopsy with an area of normalmucosa on top and tumoral emboli on the bottom wasimmunostained with anti-T-antigen antibody. Immuno-reactivity was observed in tumor cells but not in normalareas. B, immunostaining of a villous adenoma withT-antigen shows nuclear labeling of columnar cells.C, invasive epithelial glandular neoplastic cells dis-played immunoreactivity with anti-T-antigen antibody.Cytoplasmic perinuclear detection of Agnoprotein in apolyp and in neoplastic cells is shown (D and E, re-spectively). Negative immunoreactivity of both normaland neoplastic areas were observed with anti-VP1 an-tibody (F and G, respectively). Magnification: �200(A, F, and G); �400 (B–E).

7096




amplify sequences in the NH2-terminal region of JCV T-antigen, VP2 and VP3(nucleotides 1828–1848 and 2019–2039, respectively), which amplify a por-tion of the VP1 capsid gene sequence, and AGNO1 and AGNO2 (nucleotides280–298 and 438–458, respectively), which amplify a region within codingregion of JCV Agnoprotein. Amplification was carried out on 500 ng oftemplate DNA with AmpliTaq DNA Polymerase (Perkin-Elmer) in a totalvolume of 50 �l. PCR was performed in the presence of 2.5 mM MgCl2 and0.5 mM of each primer (Oligos, Etc., Guilford, CT). A Perkin-Elmer GeneAmp 9700 PCR System dedicated and used in this study was run using 9600ramping conditions with denaturation at 95°C for 10 min, followed by 45cycles of denaturation at 95°C for 15 s, annealing for 30 s, and extension at72°C for 30 s. The annealing steps were performed at temperatures of 55°C forthe PEP primers, 57°C for the Agno primers, and 54°C for the VP primers. Asa termination step, the extension time of the last cycle was increased to 7 min.Samples amplified in the absence of template DNA served as a negativecontrol, whereas inclusion of serial dilutions of the plasmid, pBJC, containingthe JCV genome as template served as a positive control for each procedure.

Southern blot was performed by resolving 10 �l of each PCR reaction on2% agarose gel electrophoresis. The gels were then treated for 15 min eachwith 0.2 M HCl for depurination, 1.5 M NaCl/0.5 M NaOH for denaturation, and1.5 M NaCl/0.5 M Tris-HCl (pH 7.4) for neutralization, followed by transfer ofthe amplified fragments from the gel to nylon membranes (Hybond-N; Am-ersham). The membranes were then prehybridized for 1 h in Ultrahyb solution(Amersham), followed by hybridization in the same solution containing5 � 106 cpm/ml [�32P]ATP end-labeled oligonucleotide specific for JCV.

Probes used for Southern blotting included JCV probe (nucleotides 4303–4327) to detect fragments amplified with PEP primers, VP probe (nucleotides1872–1891) for those amplified with VP primers, and Agno probe (nucleotides425–445) for sequences amplified with the Agno 1 and 2 primers as shown inFig. 2. Membranes were hybridized overnight, washed, and autoradiographedas described previously (5).

Histological and Immunohistochemical Analysis. Paraffin-embedded tis-sue previously fixed in 10% buffered formalin was sectioned at 4-�m thick-ness and mounted onto charged slides. Sections were placed in an oven at 65°Cto melt the paraffin and then deparaffinized in three changes of xylene for 30min each. Sections were then rehydrated through a graded series of alcohols upto water, and nonenzymatic antigen retrieval was performed in 0.01 M sodiumcitrate (pH 6.0) at 97°C in a vacuum oven for 35 min. After a cooling periodof 25 min, sections were rinsed with PBS, and endogenous peroxidase wasquenched by incubating the slides in methanol/3% H2O2 for 30 min at roomtemperature.

Sections were blocked in 2% horse serum and incubated overnight withprimary antibodies at room temperature in a humidified chamber. Antibodiesto characterize the epithelial nature of the tumors included a mouse mono-clonal anti-EMA, (clone E29, 1:50 dilution; Dako), a rabbit polyclonal anti-body against cytokeratin (wide spectrum, 1:2000 dilution; Dako), a mousemonoclonal anti-desmin to exclude any sarcomas (clone D33, 1:100 dilution;Dako), and a mouse monoclonal antihuman CEA (clone II-7, 1:100 dilution;Dako). Antibodies used to detect viral proteins included a mouse monoclonalantibody against SV40 large T-antigen that cross-reacts with JCV T-antigen

Fig. 3. LCM and gene amplification. A, selectionof the cells for laser capture was guided by immuno-histochemical detection of JCV T-antigen in villousadenoma and invasive adenocarcinoma. Top: threedifferent areas with normal histological features withno visible labeling with anti-T-antigen antibody aswell as precancerous (villous adenoma) and invasiveadenocarcinoma with strong immunoreactivity withanti-T-antigen antibody are shown before laser cap-ture. Middle: after the thermoplastic film is removed,the tissue left behind after laser capture showpunched holes in the remaining section. Bottom: theprecise removal of the target cells is confirmed bymicroscopic visualization before processing for DNAextraction. B, gene amplification followed by South-ern blot hybridization using a pair of primers thatrecognize a sequence of T-antigen followed bySouthern blot hybridization using probes that specif-ically detect the amplified JCV T-antigen gene. Theposition of the 173-bp-amplified JCV DNA fragmentis shown by an arrow. Magnification: �100 (allpanels).

7097




(clone pAb416, 1:100 dilution; Oncogene Science), a rabbit polyclonal anti-body against Agnoprotein (4), and a mouse monoclonal antibody against theJCV capsid protein VP-1 (1:1000 dilution; a kind gift from Dr. Walter Atwood,Brown University, Providence, RI). The antioncogenic gene product p53 wasdetected by using a mouse monoclonal antibody that recognizes both mutantand wild-type p53 (clone DO-7, 1:100 dilution; Dako). Proteins of the Wntpathway were analyzed by using the following antibodies: a mouse mono-clonal antihuman adenomatous polyposis coli (clone F-3, 1:500 dilution;

Dako); a mouse monoclonal anti-�-catenin antibody (clone E-5, 1:100 dilu-tion; Santa Cruz Biotechnology); a goat polyclonal antibody against TCF-1(1:200 dilution; Santa Cruz Biotechnology); a goat polyclonal antibody againstTCF-4 (1:250 dilution; Santa Cruz Biotechnology); and a goat polyclonalantibody for LEF-1 (1:200 dilution; Santa Cruz Biotechnology).

After rinsing the sections in PBS, the slides were incubated for 1 h at roomtemperature with biotinylated antimouse or antirabbit secondary antibodies andthen were rinsed in PBS. The tissue was subsequently incubated with avidin-

Fig. 4. Detection of p53 and its colocalizationwith T-antigen in colon cancer. A, immunohisto-chemistry against p53 shows negative reactivity innormal epithelium (top) and robust labeling in neo-plastic cells inside lymph vessels (bottom). Strongimmunoreactivity with anti-p53 antibody was ob-served in villous polyp (B) and neoplastic cells inglandular structures (C). Double labeling of colum-nar cells with anti-p53 antibody and anti-T-antigenshows nuclear colocalization of these proteins inneoplastic cells. The presence of p53, T-antigen, andcolocalization of both proteins in columnar cells of avillous adenoma are depicted in D–F, respectively.Magnification: �200 (A); �400 (B and C); �1000(D–F).

7098




biotin-peroxidase complexes for 1 h at room temperature according to themanufacturer’s instructions (Vector Laboratories), and finally, the sectionswere developed with a diaminobenzidine substrate (Sigma, St. Louis, MO),counterstained with hematoxylin, and coverslipped with Permount (Fisher,Pittsburgh, PA). To assess the fraction of immunolabeled cells in specimensfrom each patient case, the labeling index defined as the percentage of positivecells of the total number of tumor cells counted was determined.

Double-labeling Immunofluorescence. For immunofluorescence anddouble labeling of paraffin embedded sections, deparaffinization, antigenretrieval, endogenous peroxidase quenching and blocking were performed asdescribed above. For immunofluorescent double labeling of HCT116 cells inculture, the cells were washed with PBS, fixed in cold acetone, and blocked inPBS containing 5% horse serum and 0.1% BSA for 2 h. Sections were thenincubated with mouse anti-T-antigen antibody (Oncogene Science, clonepAb416, 1:100 dilution) for 16 h followed by washing in PBS and incubationin antimouse rhodamine antibody (1:200 dilution; Vector Laboratories). Next,sections were incubated with mouse monoclonal antibody, which recognizeseither �-catenin (clone E-5, 1:100 dilution; Santa Cruz Biotechnology) or p53(Dako, clone DO-7, 1:100 dilution) for 16 h followed by washing in PBS andincubation in antimouse fluorescein antibody (1:200 dilution; Vector Labora-tories). Finally, sections were washed in PBS and mounted in aqueous mount-ing media (Vector Laboratories).

LCM. Representative sections of colon cancer tissue were selected andformalin-fixed, paraffin-embedded sections were cut at 5 �m and mounted onglass slides. Immunohistochemistry against the JCV T-antigen protein wasperformed to identify and selectively dissect T-antigen-positive cell popula-tions. Sections were subsequently dehydrated in graded ethanol solutions (95%ethanol, 2 � 5 min, 100% 3 � 5 min) and cleared in xylene (3 � 5 min). Afterair-drying for 30 min, laser capture was performed under direct microscopicvisualization of the T-antigen-positive immunolabeled areas by laser activationof thermoplastic film mounted on optically transparent LCM caps (ArcturusEngineering, Mountain View, CA). The PixCell II LCM System (ArcturusEngineering) was set to the following parameters: 15-�m laser spot size;40-mW power; and 3.0-ms duration. Cancer cells were captured by focalmelting of the membrane through a carbon dioxide laser pulse activation.

Normal epithelial crypts, villous adenomas, and invasive adenocarcinomacomponents were individually dissected for all cases.

Transient Transfection 15, Protein Extraction, and Analysis. HCT116cells were transfected with 5 �g of pCMV-T-antigen by the calcium phosphateprecipitation method. Total protein extracts were prepared after 48 h accordingto procedures described previously (12). Protein extract (250 �g) was incu-bated with anti-T-antigen antibody or preimmune sera, and the immunocom-plexes were separated by SDS-PAGE, transferred to nitrocellulose, and ana-lyzed by Western blot using anti-�-catenin antibody. For direct Western blot,50 �g of protein extract were analyzed by SDS-PAGE and transferred tonitrocellulose filter incubated with anti-c-myc or anti-Grb-2 antibodies. Forluciferase assay, protein extracts were prepared from HCT116 cells transfectedwith 3 �g pDel-1 (kindly provided by Dr. B. Vogelstein, Johns HopkinsHospital, Baltimore, MD) reporter plasmid alone or together with pC52MT-WT�-cat (kindly provided by Dr. F. Fagotto, Max Placnk Institute, Tubingen,Germany) and pCMV-T-antigen by standard methods (25) and assayed forluciferase activity as described previously (12).

DISCUSSION

Once believed to be a highly neurotropic virus whose genes arepreferentially expressed in astrocytes and oligodendrocytes of humanbrain, recent studies have provided evidence for the presence of theJCV genome in a broad range of human cell types and tissues. Forexample, the JCV genome has been detected in tonsillar stromal cells(26), B lymphoid cells (27), kidney epithelial cells (28), and upper andlower parts of the gastrointestinal tract, including the mucosa of thecolon (19, 20, 29). Moreover, a segment of JCV DNA has beendetected in a broad range of tumors of glial and nonglial origin,including gliomas, ependymomas, and medulloblastomas (3). Thusfar, expression of the viral proteins, including T-antigen and Agno-protein, has been shown in only tumors originating from the centralnervous system in the absence of productive viral infection.

Fig. 5. Expression of Wnt pathway proteins and colocalization of �-catenin and T-antigen. A, immunohistochemical labeling of neoplastic cells with anti-�-catenin shows both,nuclear and cytoplasmic staining. B, immunostaining of the tumor cells with anti-TCF-4 antibody demonstrates strong nuclear staining. C, treatment of tumor cells with anti-LEF-1antibody shows predominant cytoplasmic with scattered nuclear staining (arrows). D, columnar cells show nuclear labeling of tumor cells with anti-T-antigen antibody (shown by anarrow; E) The same section shows nuclear (arrow) and cytoplasmic (arrowhead) accumulation of �-catenin in the tumor areas. F, superimposition of the double-labeled tumor cellswith anti-T-antigen and anti-�-catenin show frequent nuclear colocalization of the two proteins (arrow). Magnification: �400 (A–C); �1000 (D–F).

7099




Results from the studies presented here reveal the expression ofJCV proteins in a series of well-defined nonneural origin clinicalspecimens of the lower gastrointestinal tract and provide some cluesas to the molecular events that are affected by JCV T-antigen in tumorcells. According to the proposed genetic model for colorectal tumor-igenesis (30), accumulation of a series of genetic variations resultingin the activation of an oncoprotein and inactivation of tumor suppres-sors proteins orchestrate the development of tumors at various stagesin the colon. Whereas some of these alterations may be inherited suchas the mutation in chromosome 5, which is seen in familial adenom-atous polyposis (31), others such as viral infection with the ability toalter many regulatory events can be environmentally acquired. TheT-antigen of polyomaviruses, including JCV, has an established on-cogenic capability likely because of its ability to interact with andinactivate several tumor suppressor proteins such as p53 and pRb.These events can lead to suppression of p21WAF-1, the downstreamregulator of p53 and the liberation of the E2F family of transcriptionfactors from pRb. Consequently, p21WAF-1 and E2F stimulate severalproteins, the function of which is essential for cell cycle progressionand rapid cell proliferation. Detection of p53 and its colocalizationwith T-antigen in the tumor cells suggests that p53 is functionallyinactive. This notion is supported by results from immunohistochem-istry where p21WAF-1 was not detected in the tumors cells (data notshown).

�-catenin is an integral component of the Wnt signaling pathwaywhose nuclear import contributes to colorectal carcinogenesis (21).Our results demonstrate that in T-antigen-positive tumor cells, �-cate-nin is found in the nuclei where T-antigen is present. Furthermore, wefound that T-antigen and wild-type �-catenin can form a complexleading to speculate that the association of these two proteins resultsin the nuclear entry of �-catenin in neoplastic cells. Although thefunctional consequence of this interaction in cancer developmentremains to be investigated, results from cotransfection studies showcooperativity between �-catenin and T-antigen in inducing transcrip-tion from the c-myc promoter, a known downstream target gene of�-catenin. This observation is in agreement with earlier reports show-ing the involvement of the Wnt signaling pathway and up-regulationof c-myc in colon cancer (for review see Refs. 23, 32). Takentogether, the results presented in this manuscript extend earlier reportson the presence of JCV DNA sequences in the human upper and lowergastrointestinal tract and colorectal cancers (19, 20) and, for the firsttime, demonstrate the expression of the viral oncoprotein, T-antigen,and late Agnoprotein in tumor cells containing viral DNA sequences.The presence of the JCV genome and, more importantly, expressionof its proteins in tumor cells suggests a role, perhaps as a cofactor, inthe development of tumors of the gastrointestinal tract. These obser-vations should invite additional investigation of the association of thisoncogenic virus with other human tumors, the development of animalmodels for human cancers using JCV as a tool, and the investigationof pathways that may be differentially deregulated by JCV in varioustumors.

Fig. 6. Interaction of JCV T-antigen with �-catenin in colon cancer cells. A, the humancolon cancer cell line, HCT116, was transfected with the T-antigen expression plasmidpCMV-T-antigen. After 48 h, total protein extracts were prepared and incubated witheither anti-T-antigen antibody or control preimmune serum for 16 h. The immunocom-plexes were analyzed by Western blot using anti-�-catenin antibody. Lane 1 representsdirect Western blot analysis of protein extract from HCT116 cells. The band representing�-catenin is depicted by an arrow. B, Western blot analysis of protein extract fromuntransfected and pCMV-T-antigen-transfected cells using anti-c-myc (top) and Grb-2

(bottom) antibodies. C, HCT116 cells were transfected with 3 �g of a plasmid containingthe c-myc promoter upstream of a luciferase reporter gene either alone or together withpCMV-�-catenin and pCMV-T-antigen plasmids. The level of luciferase activity wastested after 36 h. The experiment was repeated three times, and the results with standarderrors are illustrated. D, HCT116 cells were transfected with 2 �g of pCMV-T-antigenplasmid. Cells were fixed after 48 h, and expression and subcellular localization ofT-antigen and �-catenin were examined by immunocytochemistry using specific antibod-ies (as described in “Materials and Methods”). The arrowhead shows cells that expressT-antigen, whereas the arrows depict representative cells that do not receive expressT-antigen.

7100




ACKNOWLEDGMENTS

We thank past and present members of the Center for Neurovirology andCancer Biology for their insightful discussion and sharing of ideas and re-agents. We also thank Cynthia Schriver for editorial assistance and preparationof the manuscript.

REFERENCES

1. Weber, T., and Major, E. O. Progressive multifocal leukoencephalopathy: molecularbiology, pathogenesis and clinical impact. Intervirology, 40: 98–111, 1997.

2. Berger, J. R., and Concha, M. Progressive multifocal leukoencephalopathy: theevolution of a disease once considered rare. J. Neurovirol., 1: 15–18, 1995.

3. Del Valle, L., Gordon, J., Assimakopolou, M., Enam, S., Geddes, J. F., Varakis, J.,Katsetos, C. D., Croul, S. E., and Khalili, K. Detection of JC virus DNA sequencesand expression of the viral regulatory protein, T-antigen, in tumors of the centralnervous system. Cancer Res., 61: 4287–4293, 2001.

4. Del Valle, L., Gordon, J., Enam, S., Delbue, S., Croul, S., Abraham, S., Radhakrishnan,S., Assimakoupoulou, M., Katsetos, C. D., and Khalili, K. Expression of human neuro-tropic polyomavirus JCV late gene product AGNO protein in human medulloblastoma.J. Natl. Cancer Inst. (Bethesda), 94: 267–273, 2002.

5. Krynska, B., Del Valle, L., Croul, S. E., Gordon, J., Katsetos, C., Carbone, M.,Giordano, A., and Khalili, K. Detection of human neurotropic JC virus DNA se-quence and expression of the viral oncogenic protein in pediatric medulloblastomas.Proc. Natl. Acad. Sci. USA, 96: 11519–11524, 1999.

6. Frisque, R. J., Rifkin, D. B., and Walker, D. L. Transformation of primary hamsterbrain cells with JC virus and its DNA. J. Virol., 35: 265–269, 1980.

7. Howley, P. M., Rentier-Delrue, F., Heilman, C. A., Law, M. F., Chowdhury, K.,Israel, M. A., and Takemoto, K. K. Cloned human polyomavirus JC DNA cantransform human amnion cells. J. Virol., 36: 878–882, 1980.

8. Vacante, D. A., Traub, R., and Major, E. O. Extension of JCV virus host range tomonkey cells by insertion of a simian virus 40 enhancer into the JC virus regulatoryregion. Virology, 170: 353–361, 1989.

9. Del Valle, L., Gordon, J., Ferrante, P., and Khalili, K. JV virus in experimental andclinical brain tumorigenesis. In: Human Polyomaviruses: Molecular and ClinicalPerspectives, K. Khalili and G. Stoner (eds.) pp. 409–430. New York: Wiley-Liss,2001.

10. Dyson, N., Bernards, R., Friend, S. H., Gooding, L. R., Hassell, J. A., Major, E. O.,Pipas, J. M., van Dyke, T., and Harlow, E. Large T antigens of many polyomavirusesare able to form complexes with the retinoblastoma protein. J. Virol., 64: 1353–1356,1990.

11. Krynska, B., Otte, J., Franks, R., Khalili, K., and Croul, S. Human ubiquitous JCV�CYT-antigen gene induces brain tumors in experimental animals. Oncogene, 18: 39–46,1998.

12. Gan, D. D., Reiss, K., Carrill, T., Del Valle, L., Croul, S., Giordano, A., Fishman, P.,and Khalili, K. Involvement of Wnt signaling pathway in murine medulloblastomainduced by human neurotropic JC virus. Oncogene, 20: 4864–4870, 2001.

13. Lassak, A., Del Valle, L., Peruzzi, F., Wang, J. Y., Croul, S., Khalili, K., and Reiss,K. Insulin receptor substrate 1 translocation to the nucleus by the human JC virusT-antigen. J. Biol. Chem., 277: 17231–17238, 2002.

14. Agostini, H. T., Yanagihara, R., Davis, V., Ryschkewitsch, C. F., and Stoner, G. L.Asian genotypes of JC virus in Native Americans and in a Pacific Island population:

markers of viral evolution and human migration. Proc. Natl. Acad. Sci. USA, 94:14542–14546, 1997.

15. Kitamura, T., Kunitake, T., Guo, J., Timinaga, T., Kawabe, K., and Yogo, Y.Transmission of the human polyomavirus JC virus occurs both within the family andoutside the family. J. Clin. Microbiol., 32: 2359–2363, 1994.

16. Bofill-Mas, S., Pina, S., and Girones, R. Documenting the epidemiologic patterns ofpolyomaviruses in human populations by studying their presence in urban sewage.Appl. Environ. Microbiol., 66: 238–245, 2000.

17. Bofill-Mas, S., Formiga-Cruz, M., Clemente Casares, P., Calafell, F., and Girones, R.Potential transmission of human polyomaviruses through the gastrointestinal tractafter exposure to virions or viral DNA. J. Virol., 75: 10290–10299, 2001.

18. Bofill-Mas, S., and Girones, R. Excretion and transmission of JCV in human popu-lation. J. Neurovirol., 7: 345–349, 2001.

19. Ricciardiello, L., Laghi, L., Ramamirtham, P., Chang, C. L., Chang, D. K., Randolph,A. E., and Boland, C. R. JC virus DNA sequences are frequently present in the humanupper and lower gastrointestinal tract. Gastroenterology, 119: 1228–1235, 2000.

20. Ricciardiello, L., Chang, D. K., Laghi, L., Goel, A., Chang, C. L., and Boland, R.Mad-1 is the exclusive JC virus strain present in the human colon, and its transcrip-tional control region has a deleted 98-base-pair sequence in colon cancer tissues.J. Virol., 75: 1996–2001, 2001.

21. Alexander, N., Wong, C. S., and Pignatelli, M. �-Catenin: a linchpin in colorectalcarcinogenesis? Am. J. Pathol., 160: 389–401, 2002.

22. Peifer, M. �-Catenin as oncogene: the smoking gun. Science (Wash. DC), 275:1752–1753, 1997.

23. Morin, P. J. �-Catenin signaling and cancer. Bioessays, 21: 1021–1030, 1999.24. Hamilton, S. R., and Aaltonen, L. A. (eds.). World Health Organization classification

of tumours: pathology and genetics of tumours of the digestive system. Lyon, France:International Agency for Research on Cancer, 2001.

25. Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., and Struhl, K.Current protocols in molecular biology. New York: John Wiley and Sons, Inc., 1996.

26. Monaco, M. C., Atwood, W. J., Gravell, M., Tornatore, C. S., and Major, E. O. JCvirus infection of hematopoietic progenitor cells, primary B lymphocytes, and ton-sillar stromal cells: implications for viral latency. J. Virol., 70: 7004–7012, 1996.

27. Tornatore, C., Berger, J. R., Houff, S. A., Curfman, B., Meyers, K., Winfield, D., andMajor, E. O. Detection of JC virus DNA in peripheral lymphocytes from patients withand without progressive multifocal leukoencephalopathy. Ann. Neurol., 31: 454–462, 1992.

28. Dorries, K., and ter Meulen, V. Progressive multifocal leukoencephalopathy: detec-tion of papovavirus JC in kidney tissue. J. Med. Virol., 11: 307–317, 1983.

29. Laghi, L., Randolph, A. E., Chauhan, D. P., Marra, G., Major, E. O., Neel, J. V., andBoland, C. R. JC virus DNA is present in the mucosa of the human colon and incolorectal cancers. Proc. Natl. Acad. Sci. USA, 96: 7484–7489, 1999.

30. Fearon, E. R., and Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell,61: 759–767, 1990.

31. Solomon, E., Voss, R., Hall, V., Bodmer, W. F., Jass, J. R., Jeffreys, A. J., Lucibello,F. C., Patel, I., and Rider, S. H. Chromosome 5 allele loss in human colo-rectalcarcinomas. Nature (Lond.), 328: 616–619, 1987.

32. Dobbie, Z., Muller, P. Y., Heinimann, K., Albrecht, C., D’Orazio, D., Bendik, I.,Muller, H., and Bauerfeind, P. Expression of COX-2 and Wnt pathway gene inadenomas of familial adenomatous polyposis patients treated with meloxicam. Anti-cancer Res., 22: 2215–2220, 2002.

33. Frisque, R. J., Bream, G. L., and Cannella, M. T. Human polyomavirus JC virusgenome. J. Virol., 51: 458–469, 1984.

7101




2002;62:7093-7101. Cancer Res Sahnila Enam, Luis Del Valle, César Lara, et al.

-CateninβEvidence for Interaction of Viral T-Antigen and Association of Human Polyomavirus JCV with Colon Cancer:

Updated version

http://cancerres.aacrjournals.org/content/62/23/7093

Access the most recent version of this article at:

Cited articles

http://cancerres.aacrjournals.org/content/62/23/7093.full#ref-list-1

This article cites 29 articles, 15 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/62/23/7093.full#related-urls

This article has been cited by 16 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/62/23/7093To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/content/62/23/7093.full#ref-list-1

http://cancerres.aacrjournals.org/content/62/23/7093.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Association of Human Polyomavirus JCV with Colon Cancer ... · JCV is detected in the urine of...

Documents

Transcript of Association of Human Polyomavirus JCV with Colon Cancer ... · JCV is detected in the urine of...