Download Gene Methylation

Discovery of Novel Methylated Genes in
Cervical Carcinogenesis:
Methylation of GREM1 and NID2 is Detected in the Majority
of Invasive Cervical Cancers and Defines a Subset of HSILs
KS Gustafson, AR Davis, L Van Neste, BM Ronnett, JG Herman
The Johns Hopkins Hospital, Baltimore, MD

No relevant financial relationships
with commercial interests
“Heritable changes in the
pattern of gene expression
that do not involve changes
to the underlying DNA
sequence.”
 Covalent modifications

Nucleosome
(e.g. methylation, acetylation)
that alter chromatin structure
 DNA: CpG sites
 Histone proteins
Luger et al, Nature 1997
Critical for normal development
and differentiation
 Aberrant changes occur during
tumorigenesis

 Diagnostic/prognostic biomarkers
 Therapeutic targets
▪ Demethylating agents
▪ Histone deacetylase inhibitors
Ting et al, Genes & Dev, 2006
Candidate Tumor Suppressor Genes
Hypermethylated in Cervical Cancer
Gene
Frequency (%)
Function
TP73
39
Apoptosis
TNFRSF10C
100
Apoptosis
PTEN
58
WNT-pathway
CDH1
28-80.5
WNT-pathway
MGMT
5-81
DAPK1
45-100
Metastasis/cell death
IGSF4/CADM1
58-65
Cell adhesion
HIC1
18-45
Transcription factor
RARB
33-66
Cell differentiation
DNA repair
Adapted from Duenas-Gonzalez et al, Molecular Cancer (2005)
Gene Methylation
(Percentage of Samples)
70
DAPK1
p=.0027
60
IGSF4
p=.0081
50
SPARC
40
p=.0015
30
TFPI2
p=.0066
20
Any TSG
10
p<.0001
(Fisher exact)
0
LSIL/NILM
HSIL
(n=60)
(n=39)
Kahn et al, Cancer 2008

Discover novel cancer-specific
hypermethylated genes in
cervical cancer

Determine if these novel
hypermethylated genes can
serve as molecular biomarkers
of HSIL

Gene expression microarray analysis
 Agilent 44K human genome arrays
 SiHa and CaSki cervical cancer cell lines
 Pharmacologic treatment
▪ Demethylating agent: 5-aza-2’-deoxycytidine (DAC)
▪ Histone deacetylase inhibitor: Trichostatin A (TSA)
 Selection of CpG island-containing
candidate genes
▪ Increased expression with DAC and no change
with TSA
▪ No basal expression in mock-treated cells

Validation studies
 Gene expression and methylation status
▪ SiHa and CaSki cell lines
▪ Primary human foreskin keratinocytes (HFK)
 Methylation status in cervical tissues
▪ Normal cervix (n=20)
▪ Invasive cervical cancer (n=20)

Biomarker analysis in precursor lesions
 Methylation in HSIL vs LSIL/NILM Pap Tests
▪ HSIL (n=36), LSIL (n=29), NILM (n=28)
Hypermethylome spike
SiHa
CaSki
All genes CGI All genes CGI
Top Tier (≥2-fold change)
236
126
234
122
Next Tier (>1.4- and <2-fold
change)
256
138
292
159
Total candidate genes
492
264
526
281

85 shared CpG islandcontaining genes
CaSki
SiHa
 36 genes (in TT of both)
 31 genes (in TT of one)
 39 genes selected for validation
179
85
196
Fisher exact
P<.0001
P<.0001
P=1.000
P=0.3203
Relative Methylation (%)
60.00
Cervical Tissues:
Normal (n=20)
50.00
40.00
Invasive Cervical
Cancer (ICC; n=20):
30.00
•GREM1
Mean = 22.65%
Median = 21.10%
20.00
10.00
0.00
Normal
Mann-Whitney
ICC
Normal
GREM1
NID2
P<.0001
P<.0001
ICC
•NID2
Mean = 6.03%
Median = 5.24%
Pap Tests: NILM/LSIL (n=57); HSIL (n=36)
Fisher exact
P<.0001
P<.0001
H25b
H63
H57
H79a
H31
H45
H22a
H39a
H67
H55
H32
H48
H29
H2a
H4a
H8
H9
H12
H20
H21
H36
H40
H43a
H44
H51
H58
H60
H61
H62
H64
H73
H74
H76
H77
H78
H80
Cumulative Methylation Score (%)
40.00
35.00
NID2
30.00
GREM1
25.00
20.00
15.00
10.00
5.00
0.00
Positive for Methylation
Negative for Methylation

Relative level of methylation in HSIL
Pap tests does not correlate with
estimated number of lesional cells

Methylation status of HSIL Pap tests is
not associated with higher grade of
cervical intraepithelial neoplasia (CIN)
 CIN 2 versus CIN 3




GREM1 and NID2 are novel cervical cancerspecific methylated genes.
GREM1 and NID2 methylation represents a
molecular signature that defines a subset of
HSILs.
The biologic determinants of gene methylation
and behavior of this subset of HSILs are unknown.
Gene methylation may serve as a prognostic
molecular biomarker of HSIL.

Funding for research:
 Johns Hopkins University
Cervical Cancer SPORE (KSG)
 NIH/NCI CA123612 (KSG)