The Importance of the Signature of Breast Cancer...
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The Importance of the
Signature of Breast Cancer
Prevention
Prof. Jose Russo, MD, FACP
Professor and Senior Member, Temple- Fox Chase Cancer Center,
Director Breast Research Laboratory Professor of Pathology and Cell Biology
Jefferson Medical School Professor of Biochemistry Temple Medical School
Philadelphia, Pennsylvania
September 2014
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Protective effect of first full term pregnancy
from developing breast cancer after
menopause when:
O Early parity- First child born from early teens
to middle twenties
O Breastfeeding
O Multiparity in early parous women
O Each additional birth confers greater protection
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Question 1
Why Pregnancy
Produces Protection
Against Breast
Cancer?
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Life-time cycle of breast development
Terminal duct
4Alveolar buds
4Lobule type 1 (site of
origin ductal carcinoma)
Lobule type 2 (site of
origin lobular
carcinoma)
Lobule type 3
Russo, J. et al, Breast Cancer Res. and Treat. 23:211-218, 1992.
Russo, J. et al. JNCI. 2000 ,and Microscopy Res. & Tech., 2001
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Life-time cycle of breast development
Nulliparous Parous
involution
by aging
menopause
sexual
maturation
involution
by agingLob.
1
Lob.2
tT£ extra hormonal
stimulation
sexual
maturation
Lob
.2
post lactation
involution Lob.4
Lob.
3lactation
Lob.3
Russo, J. et al, Breast Cancer Res. and Treat. 23:211-218, 1992.
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Working hypothesis
J The breast of parous postmenopausal
women exhibits a specific genomic
signature that has been induced by a full
term pregnancy during the hormonal
protection window (HPW), i.e., 18-24 years
of age.
■ This genomic signature controls cell
differentiation, leading to breast cancer
prevention.
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Study subjectsS Volunteers: Residents of Lulea,
Sweden. SPost-menopausal: age 50-69
^Parous (P)= had a full term pregnancy
SNulliparous (NP)= never pregnant or
uncompleted pregnancy
Interviewed: 389
IIncluded: 251 111 (44%) nulliparous + 140 (56%)
parous
Samples suitable for microarray: 128 (44 nulliparous + 84
parous)
Eligible for the study: 113 (42 nulliparous + 71 parous)
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Methodology
imn̂: l
L
Two-cycle target labeling
for expression arrays
Breast Core BiopsyRNA isolation and
QC assessment
<
Microarray
Analysis
Human Genome U133 plus
2 Microarrays (Affymetrix)
Microarray Hybridization,
washing and scanning
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Genomic Signature of
Pregnancy (GSP)
Up regulated
genes
Down regulated genes
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Question 2
What is the meaning of the
genomic signature of
pregnancy?
How the genomic signature of
pregnancy works?
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Main Pathways of the GSP
J Cell differentiation J Splicing
mechanism J Shifting of Stem or
Progenitor cells. J Chromatin
remodeling:
- Polycomb
- Methylation of Histones
- GpC methylation
- LncRNA
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Up-regulated processes Up-regulated Genes
cell-substrate junction assembly KRT5, LAMA3, LAMC2
ectoderm developmentCOL7A1, KRT5, KRT15, LAMA3,
LAMC2, NTF4, KLK7
epidermis developmentCOL7A1, KRT5, KRT15, LAMA3,
LAMC2, NTF4, KLK7
hemidesmosome assembly KRT5, LAMA3, LAMC2
RNA metabolic process
DDX17, CHD2,CBX3, CIRBP,
ZNF785, EZH2, L3MBTL, GATA3,
RBMX, ZNF789, HNRNPA1,
HNRNPA2B1, HNRNPD, LUC7L3,
PNN, PRPF39, ZNF83, METTL3,
CREBZF, RBM25, RBBP8, RPS24,
CENPK, SFPQ, SFRS1, SFRS5,
SFRS7, ZNF814,
ZNF207, PABPN1, RUNX3, FUBP1,
PRPF4B, HNRPDL
RNA splicing
RBMX, HNRNPA1, HNRNPA2B1,
HNRNPD, LUC7L3, PNN, PRPF39,
RBM25, SFPQ, SFRS1, SFRS5,
SFRS7, PABPN1, PRPF4B
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Nuclear
speckle
Interchrorratin
compartment
Nuclear speckles
Chromosome
territories
Nuclear
pore
Chromosome
territories
METTL3, CCNL1,
CCNL2,
HNRPD, HNRPA2B1, PRPF4B, SFRS7, CLK4,
SFRS5, PABPN1, CSTF3, HNRPU, RBM5,
SNRP70, SFRS14, SNRPA1, CLK2, NXF1,
SFRS8, SFRS2, PTBP2, FUS, SFRS6, SFRS16,
SF3B1, HNRPA3,
SNRPB, PRPF3, SFRS12, U2AF1, PHF5A,
TXNL4A, CUGBP2, MALAT1,
NEAT1,
XIST
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Localization of CCNL2 in the nuclear speckles
CCNL2cyclin L2
Transcriptional regulator which
participates in regulating the pre-mRNA
splicing process. Also modulates the
expression of critical apoptotic factor,
leading to A cell apoptosis
B
D
jit TV .
iS4
1v: •
Nulliparous Parous
Russo J. et al (2011) unpublished data
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Nucleus
- transcribes
DNA to RNA
Transcri
ption
Factors
\
Methylation of mRNA METTL3
Formation of pre-mRNPs HNRPA1,
HNRPA2B1, HNRPD, RBMMX CCNL1,
CCNL2
Formation of Spliceosomal Complexes
SFRS1, SFRS5, SFRS7, CLK4, SFRS8, SFRS14, NXF1, SFPQ
\AA A A A A A
mRNA polyadenylation
PABPN1
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Pathways Affected by Full Term Pregnancy
in the Breast Epithelial Cell
.Differentiation Cell proliferationCIRBP, DSC3, INTU, GIA,
NRXN1, PTN
IKRT15, DSC3, DSG3, KRT5, C0L4A6, KRT17, KRT14, IAMC2 ,C0L4A1, COL4A2 LAMA3, LAMB3,
Cell
Communication! THBS3, LAMAS.LAMCI , GJA4,
LAMA4,VWF,
{METTL3, HNRPD, HNRPA2B1, PRPF4B, SFRS7, CLK4, SFRS5, PABPN1, CSTF3, HNRPU, RBM5, SNRP70, SFRS14, SNRPA1, CLK2, NXF1, SFRS8, FRS2, PTBP2, FUS, SFRS6, SFRS16, SF3B1, HNRPA3, SNRPB, PRPF3, SFRS12, U2AF1, PHF5A,
Estroge
n
Signalin
g
SCGB2A1, TFF1, SCGB2A2, SCGB1D2, STC2, GATA3,SERPINB5, SERPINA3, AZGP1, TP53, DLC1, FHL5 CCNA2, CCNE2, EGFR, PTEN,
■ The differentiation of breast epithelial cells is centered in the mRNA processing reactome.
i The biological importance of the differential expression of genes that control the spliceosome could be an indication of a safeguard mechanism at post-transcriptional level that maintains the fidelity of the transcriptional process.
Russo J. et al (2011) unpublished data
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Nulliparou
s
Parou
s
r'
T-ISSHS9 A HPftJi*usam
><*&{$ - w
3% *.
*■^ v, •
*<
© G£ I?
EUN
HTN
Int. J. of Cancer, 2012
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Chromatin Condensation
■ Chromatin condensation, limits the ability of RNA polymerase II transcription complex to access DNA, resulting in reduced mRNA and protein output.
■ Chromatin condensation can be induced by epigenetic changes that can range from chemical modifications of histone proteins to modifications made to the DNA itself.
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Histone 3 methylated at lysine
9Nulliparous
Breast
Parou
s
Breast
* J * , * . -*
. f
•
1*1? r« ( «, 3
?> a^, ̂ ̂ *
y # f
' > V
.Y _ A ^ ^ rV
ft? *
. V
VA
<* *£' i
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Histone 3 methylated at lysine
27Nulliparous
Breast
Parous
Breast
V nS>
& At*
P* # ^ ■ r
' * ,
f
i A**»■*' Ih -* -
a ^
f ,
A," «f * -* * '
%v
Vf * '• -,.
— . ^*+ ' -i\ "
« > i
%»*'
sm»V'
V •!
"fi . 'A,' *
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Transcriptionally
Active Chromatin
Nulliparous Breast
Parous Breast
H3-Lys 9
Transcriptionally
Inactive
Chromatin
Int. J. of Cancer, 2012
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Table 1: Differentially expressed genes in the parous breast tissue
Gene
SymbolGene Title Fold
changeFDR q-val
SOX6
SRY (sex determining region Y)-box 6-1.913 0.016
SOX17 SRY (sex determining region Y)-box
17-1.956 0.011
CBX3 chromobox homolog 3 (HP1 gamma
homolog, Drosophila) 2.110 0.001
EZH2 enhancer of zeste homolog 2
(Drosophila) 2.001 0.001
CHD2chromodomain helicase DNA binding
protein 21.956 0.005
GAT A3 GATA binding protein 3 2.217 0.002
CREBZF CREB/ATF bZIP transcription factor 1.711 0.005
XIST X (inactive )-specific transcript 2.230 0.001
NEAT1Nuclear Paraspeckle assembly transcript I
1.980 0.015
MALAT1 Metastasis associated lung
adenocarcinomas transcript 11.899 0.001
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" ^ EUN
N 1 w
• ' "
v 4 toiV
A
CpG island
CpG island
iH3Lys27
Gene
Gene
« ,JL̂ -
D
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Pre and post-menopausal womenPremenopausal period
__
Postmenopausal period
OJ
■Q
Immune responseRNA processing
Cell adhesion
Differentiation / Development / Chromatin Remodeling
&s
F
Years after pregnancy
There are not many overlapping genes between both
premenopausal and post-menopausal;
We observed that genes related to development, differentiation and
chromatin remodeling are up-regulated after pregnancy and remain up-
regulated in pre and post-menopausal women.
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Impact of GSP
J An established genomic signature of pregnancy
would open myriad of research possibilities,
including:
- a surrogate end point to evaluate the degree of mammary gland differentiation.
- its association with breast cancer risk.
- for determining in which women parity has been protective.
- development of interventions to induce protection against breast cancer.
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Period
Windows of Susceptibility
'u.
,0Nvgr.<o~
%®+**
Age
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Future Perspectives in
Personalized Interventions in Breast
Transform the breast GSP to a blood Signature of Prevention.
To implement the hormonal treatment with hCG, that mimic pregnancy, to nulliparous women.
To target the preventive strategies in young women during the window of susceptibility.
Cancer Preventionv Developing a
Blood Test
c
Human
tDES
t t _Conception Birth F jberty Sexual maturity
2-16 18-24
t
tMaturity
End reproductive cycle
30-35
Radiation
Smoking
tPregnan
cy
50-55
years
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Acknowledgments
The women of Norrbotter Count, Sweden for their
contribution to the project.
Jose Russo .
FOX CHASEC A N C E R C E N T E R
T EMPLE HEALTH
Irma Russo
Ricardo Lopez
Yanrong Su
Theresa Nguyen
Yubo Zhai
Fathima Sheriff
Kara Snider
Patricia Russo
E
Eric Ross Suraj
Peri Michael
Slifker Luis
Sigal
A V O NF O U N D A T I O N
N E W Y O R K U N I V E R S I T Y c
Sequencing:
Maria Barton Dominic
Strohmeyer Derek
Jacob Colleen
O'Malley Sandy Weng
Cody Watson
v-
aj
\
Umea University