The Role of the Polyomavirus, JC Virus, in the Pathogenesis

of Colorectal CancerII Falk Gastro-Conference

Dresden, GermanyOctober 12, 2007

C. Richard Boland, M.D.Baylor University Medical Center

Dallas, Texas

Normal

5q (APC)alterations*

Adenoma

Colonic epithelium

Benignneoplasia

Advancedadenoma

Ras mutation

17p (p53)alterations*

Carcinoma

Malignantneoplasia

Larger

Fearon and Vogelstein, 1990

(* “alterations” imply both mutations and allelic losses, or CIN)

18q loss

Multistep Colorectal Carcinogenesis (1990)

Multiple Pathways to CRC (2007)

CIN TSG’s lost by LOH: APC, p53, 18q genes

LynchSyndrome

CIMP

MSILose DNA MMR gene

methylate hMLH1

Mutations at target genes:R2, Bax, etc.

TSG’s lost by methylation:APC, PTEN, HIC-1, p16, MGMT, etc. Cancer

Hypothesis (1994)

• Polyomaviruses encode a transforming gene (T-antigen)

• When cells are transformed (in the laboratory) with SV40 (a polyomavirus), they develop CIN and become aneuploid

• Perhaps a polyomavirus is in CRCs

Are Viruses Involved in the Pathogenesis of Any Cancers?

• Rous sarcoma virus (chickens)• Avian leukosis virus, etc (retroviruses)• Murine leukemia viruses, etc.• Oncogenes in NIH 3T3 cells (Bishop and

Varmus)• SV40 caused cancers in rodents (1960’s)

Are viruses involved in any human cancers?

• HPV and genital tract cancers – (possibly esophagus, nasopharyngeal, and other cancers)

• EBV and lymphomas (also gastric cancer?)• Kaposi’s sarcoma and herpes virus (HHV8)• HTLV-1 and lymphomas• Hepatitis B & C and hepatocellular carcinoma• Polyomaviruses

– SV40 and mesothelioma or lymphomas– JCV and CNS tumors

Polyomaviruses

• All encode a potent oncogene (T antigen)• All potentially potent oncogenic viruses

– Polyomavirus (mouse)– SV40 (monkeys)– JCV (human)– BKV (human)

Polyomavirus Structure

JC Virus (Mad1, complete genome)

5 kbp DNA virusClosed circular genomeSupercoiledEncodes 5 genes

T Antigen3 viral capsids

VP1, VP2, VP3Agnoprotein

Why JCV?

• Nearly all humans have antibody titers to JCV– every population studied (including Yanomami)– remains latent in most of us

• causes PML in immunosuppressed patients

• JCV has encodes a potent oncogene– T antigen

• JCV causes tumors when injected into the CNS of rodents or monkeys

• JCV is associated with “rogue” (aneuploid) lymphocytes in humans (James Neel)

T-Antigen Has Multiple FunctionalDomains

J.S. Butel, Baylor College of Medicine

Takes out the RB protein

Takes out p53

JCV DNA in Colorectal Tissues

Initial PCRn = 46 samples12/46 positive (26%)

With topoisomerase (TISPA)n = 54 samples48/54 positive (89%)

JCV in CRC: Confirmatory Studies

• *Laghi et al. PNAS, 1999 (89% CRCs)• *Ricciardiello et al., Gastro, 2000 (81%, nl colon)• *Ricciardiello et al., J. Virology, 2001

(promoters)• Enam et al., Cancer Res, 2002 (83%)• Theodoropoulis et al. Dis Col Rectum, 2005

(quantitated copy number)• Hori et al., Virchows Arch, 2005 (viral proteins)• Weinreb, Virchows Arch, 2006• *Jung et al, Cancer, 2007 (adenomatous polyps)

How Many Copies of JCV in Human Colorectal Neoplasms?

100-250147 (+50)6Normal

9-20 X 10314.0 (+3.1) x 103

15Adenomas

50-450242 (+127)35Adjacent normal

9-20 X 10314.3 (+4.2) X103

49Cancer

RANGEMEAN (SD)NTISSUE

Thoedoropoulos et al, Dis Colon & Rectum 2005

JCV in the GI Tract (I)Upper GI Tract (71%)

• esophagus: 10/15 (67%)• gastric corpus: 9/17 (53%)• gastric antrum: 8/17 (47%)• duodenum: 10/17 (59%)

Lower GI Tract (81%)• cecum: 10/15 (67%)• transverse colon: 11/16 (69%)• sigmoid colon: 8/16 (50%)• rectum: 10/16 (63%)

Interpretation

The GI tract is a reservoir for JCV

Suggests fecal-oral transmissionof the virus

Ricciardiello L at al. JC virus DNA sequences are frequently present in the human upper and lower gastrointestinal tract. Gastroenterology 119:1228-

1235, 2000.

JCV and other GI Cancers

• JCV is also present in cancers of the:

– Esophagus (DelValle et al. Cancer, 2005)– Stomach (Shin et al. Cancer, 2006)– Pancreas (Fuerst et al. Gastro, 2005 (abstr.)– Lung (Zheng et al. J Pathol, 2007)

MODEL 1: JCV T Ag in HCT116 cells

• HCT116 cells– diploid, MSI, no CIN– does not support JCV infection

• T Ag/GFP construct inserted into plasmid

• Plasmid transfected into HCT116

JCV T Ag in HCT116 Cells

• JCV T Ag protein localizes in the nucleus

• Transfected cells develop CIN

• Control constructs (GFP only): – no CIN

JCV TJCV T--Antigen Transfection Causes Antigen Transfection Causes CIN in HCT116 CellsCIN in HCT116 Cells

Dicentrics: A, C, F, G

Breaks: A, B, E

Fused: D, F, I

Rings: H

A B C

D E F

G H I

CIN IN HCT116/T Ag: CIN IN HCT116/T Ag: 90 Population Doublings90 Population Doublings

Clones ChromÕs Breaks Dicentric Rings Others

C1 409 0 0 0 0

C2 401 2 1 0 0

C3 457 1 0 0 FUSION

T Ag-1 332 10 11 17 0

T Ag-2 398 14 7 17 TRICEN-TRIC (2)

T Ag-3 402 17 3 3 FUSION

Model 2:Induction of CIN in RKO cells

A full JCV genome induces CIN in the diploid colon cell line:

RKO

Ricciardiello et al. Cancer Res 63:7256-62, 2003

Transfection of JCV Genome into Diploid CRC cells

• Model: RKO cells– diploid, microsatellite instability (MSI)

• hypermethylated hMLH1– wild type p53, APC, β-catenin– a CIMP model

• JCV cloned into pBR322 plasmid– full length, Mad-1 inserted into plasmid– transfected into RKO

Results: RKO transfected with JCV

• JCV integrates into RKO• T antigen expressed within 7 days

– nuclear localization of T Ag protein• VP1 expressed (late gene; viral capsid)

– low level expression• suggests viral replication

• T Ag and β-catenin interaction• p53 stabilized• CIN induced

Ricciardiello et al, Cancer Res 63:7256-62, 2003

JCV INDUCES CIN

Model 3:Making NMC460 cells tumorigenic

Normal Colonic Cells:NCM460 cells

• Normal, non-transformed colonic epithelial cell lines

• Derived from the normal colon mucosa of a 68-year-old Hispanic male and selected for in vitro growth.

• Cells were not infected or transfected with any exogenous genetic information.

• Expression of colonic epithelial cell-associated antigens, such as cytokeratins and villin.

• Normal colonic physiology (Gastroenterol.1997, Am J Physiol. 1998; 2000, JBC 1997, J Clin Invest 1997)

• Wild type p53 (Cancer Gene Therapy. 2000, Gastroenterol. 2005).

T antigen expression vector:CMV-JCV TAg

5019 of JCVRemoved TAg intronnt 4772/4426

nt 2473of JCV

20 bp AvaII-EcoRIadapter sequence

XbaI site of pVL1392 and pCR3

pCR3 pCR3

Authentic TAg polyA site

ATG5013

TAA2603

m 4274

Stable Transfected NCM460 with T-Ag

T-Ag

p53

- + + T-Ag Transfection

Clon

e_1_

8 wk

sCl

one_

2_8

wks

JCV T-Ag results in tumor formation in nude mice at 3 weeks

JCV Transformed NMC460 Cells in Nude Mouse

(No tumors from cells transfected with control vector)

JCV T Ag and β-Catenin• JCV DNA found in 83% of CRCs

– 22/27 tumors

• T Ag and agnoprotein expressed by IHC in >50% of CRCs (not viral capsid proteins)– β-catenin expression in nucleus of T Ag+ cells– T Ag not expressed in the normal colon

• T Ag and β-catenin co-immunoprecipitated

Enam et al Cancer Res 62:7093, 2002 (Khalili lab)

β-catenin regulates proliferation

nucleus

Tcf-4

β-catTarget genes

β-cat

Wnt-1

Frizzled(Receptor)

Cell membrane

APC

GSK3β

DSH

axin

C-myc, PPARδ, cyclin D, etcExpressed (proliferation program)

β-cat

β-cat

Wnt binds receptor

β-cat

APC inhibited

increasedβ-catenin

Proliferating cells:-nuclear β-catenin-no APC

APC regulates β-catenin

nucleus

Tcf-4 Target genes

cell-cell adhesion inhibitedWnt-1

Frizzled(Receptor)

Cell membrane

β-catAPC

GSK3β

DSH

β-cat

degradedaxin

genes repressed

α-cat

no ligand bound

x

x

Differentiated cells- APC expressed

- β-catenin degraded

0102030405060708090

100

Normal (150) Cancer (100) Adenoma (80) Ad/Ca withLOH of APC

(30)

APC +

beta-catnuc

APC and β-catenin IHC in Normal, Sporadic/FAP Adenomas and Cancers

Nuclear stabilization of β-catenin precedes loss of APC

Normal

5q (APC)alterations*

Adenoma

Colonic epithelium

Benignneoplasia

Advancedadenoma

Ras mutation

17p (p53)alterations*

Carcinoma

Malignantneoplasia

Larger

Fearon and Vogelstein, 1990

(* “alterations” imply both mutations and allelic losses, or CIN)

18q loss

Multistep Colorectal Carcinogenesis

β-catenin chaperoned to the nucleus by

JCV T-Ag

JCV T antigen expression may “chaperone” β-catenin, and initiate the neoplastic phenotype

JCV and Genomic/EpigeneticInstability in CRC

• JCV T Antigen expression occurs in about half of all CRCs with JCV DNA– CIN and CIMP inversely associated with each

other• CRCs develop as a consequence of JCV

– some permit expression of JCV genes, and develop CIN (most tumors)

– some respond to JCV with promoter methylation (CIMP)

Goel et al, Gastroenterology, 2007

CIMP: Summary of Results

• Expression of JCV T antigen in CRC cell lines induces promoter methylation and CIMP (in vitro)

Goel, unpublished data

SUMMARY

• Most people are infected with JCV• JCV is present in most GI cancers

– CRC, gastric, esophageal, pancreatic• T Ag can initiate a neoplastic phenotype

prior to any mutations via β-catenin • T Ag can induce CIN• JCV transfection can cause CIN• T Ag expression may induce CIMP

Conclusions (2007)• JCV is a plausible explanation for:

– CIN– CIMP– MSI (which is caused by either CIMP or CIN)

• This is the best available explanation for how GI cancers begin in humans

• This raises the hypothesis that if one could prevent infection, one might greatly reduce the incidence of GI cancers

Luigi Laghi, MDMilan, Italy

Initially optimizedJCV PCR, quant-itative JCV assays,etc.

KEY RESEARCH COLLABORATORS

Luigi Ricciardiello, MDAjay Goel, PhD

Found JCV throughout gutInduced CIN in RKO cellsRearrangements in promoter

KEY RESEARCH COLLABORATORS

Luigi Ricciardiello, MDAjay Goel, PhD

Association between JCV and CINAssociation between JCV and CIMPJCV in gastric cancerJCV in polypsMechanisms of genetic/epigenetic instability