Download Normoxia

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Document related concepts
no text concepts found
Transcript
Estrogen receptor-α directly regulates the
hypoxia inducible factor 1 pathway associated
with antiestrogen response in breast cancer
PNAS 2015 112(49) 15172-15177
Speaker:Wu Po-Huei
Adviser : Jung-Yie Kao
Date:2016.11.01
2
Introduction
Estrogen receptor-α (ERα )
Estrogen
Estrogen receptor α
Estrogen receptor α dimer
(Transcription factor)
Estrogen response element
Mol Endocrinol 20(8):1707–1714
3
Tamoxifen (Tam)
CoR = Corepressor
HDAC = Histone deacetylase
4
Mol Endocrinol 16(8) : 1778–1792
Hypoxia inducible factor 1(HIF-1α) 5
Normoxia
Proc Natl Acad Sci USA 100(11):6517–6522
ERα and HIF-1α
6
We have previously shown that HIF-1α and ERα can coordinate
expression of genes, such as lysine-specific demethylase
4B/Jumonji domain-containing 2B (KDM4B/JMJD2B), an
H3K9me3/me2 histone demethylase, which is targeted by both
ERα and HIF-1α and epigenetically regulates cell cycle
progression. The genomic locus of KDM4B bears both HIF-1α
and ERα binding elements.
Cancer Res 70(16):6456–6466
Q : The role of ERα in the regulation of HIF-1 signaling
Q : How HIF-1 signaling is involved in
endocrine drug response
7
Results
ERα signaling regulates hypoxia/HIF-1α pathway
8
We have previously shown that knockdown of ERα
significantly down-regulated histone demethylase KDM4B
expression, a HIF-1α transcriptional target.
J Biol Chem 283(52):36542–36552
ERα signaling regulates hypoxia/HIF-1α pathway
ER antagonist
MCF7
N
C
H
ICI
24hr
C
ICI
(ICI182780 : 1μM)
Extracted RNA
Microarray
Global gene-expression
profile analysis
Dually responsive gene
9
ERα signaling regulates hypoxia/HIF-1α pathway
10
Library of Integrated
Network-based Cellular
Signatures
 Conclusion :
ERα
ERβ
HIF-1
These data
indicate that a
subgroup of
genes that are
targeted by
hypoxia/HIF-1α is
also regulated by
ERα signaling
DNA-protein
Cross-linking
ChIP
Chromatin Immunoprecipitation
11
Cell lysis
Sonication or
enzyme digestion
Protein
Fragmented
chromatin
Immunoprecipitation
with specific antibody
DNA purification
Analysis of
bound DNA
PCR
qPCR
Sequencing
Microarray
ERα signaling regulates hypoxia/HIF-1α pathway
12
Library of Integrated
Network-based Cellular
Signatures
 Conclusion :
ERα
ERβ
HIF-1
These data
indicate that a
subgroup of
genes that are
targeted by
hypoxia/HIF-1α is
also regulated by
ERα signaling
ERα and HIF-1α directly bind their response elements
in a subgroup of genes
13
High-resolution genome-wide mapping of HIF-binding sites by ChIP-seq
Blood 117(23):e207–e217
A CTCF-independent role for cohesin in tissue-specific transcription
Genome Res 20(5):578–588
Kyoto Encyclopedia of
Genes and Genomes
 Conclusion : We found that among the 356 genes bound by
HIF-1α, 202 (57%) of them were identified as the
common genes bound by ERα as well
Estrogen regulates HIF-1α expression
Hypoxia mimetic
Deferoxifine
14
MCF7
N
C
E2
DFO
H
500μm
1%O2
C
E2
C
E2
(Estrogen : 100nm)
6hr
Western blot
 Conclusion : E2 greatly enhanced HIF-1α expression in hypoxia
ERα signaling regulates HIF-1α expression
15
Condition
Hypoxia(1%O2)
1μm ICI182780
24hr
Breast cancer
cell
ERα (+)
Breast cancer
cell
ERα(-)
 Conclusion : ERα signaling regulates HIF-1α expression
ERα signaling regulates HIF-1α expression
Hypoxia mimetic
Dimethyloxalylglycine
siERα
Transfect into MCF7
16
Proteasome inhibitor
200 μm DMOG
10 μm MG132
1%O2 Hypoxia
16hr
Western blot
 Conclusion : ERα signaling pathway regulates
HIF-1α expression
ERα signaling regulates HIF-1α gene expression
17
Cell Culture
(Normoxia)
+E2 / +drug
100nM
1μM
24hr
RNA extraction
RT-PCR
 Conclusion : ERα signaling pathway directly regulates
HIF-1α gene expression
ERα directly binds EREs on the HIF-1α gene to
enhance HIF-1α transcription
Cell Culture
(Normoxia)
C/+E2/+Tam
(100nM)
24hr
18
ChIP
C/+E2/+Tam
ERα ERα ERα
| | |
NB1 ERE NB2
RT-PCR
 Conclusion :
ERα directly binds EREs on the HIF-1α gene
ERα directly binds EREs on the HIF-1α gene to
enhance HIF-1α transcription
Wt → Wild type
Mut → ERE mutant
19
Wild type ERE
Mutant type ERE
clone into
pGL3-Luciferase reporter
Transfect
Luciferase assay
 Conclusion : The luciferase activity was significantly
high, but the ERE mutant abrogated the
activity in MCF7 cells
ERα directly binds EREs on the HIF-1α gene to
enhance HIF-1α transcription in hypoxia
Cell Culture
ChIP
N / H
N / H
48hr
IgG ERα
| |
ERE ERE
RT-PCR
 Conclusion : The results showed that ERα still bound at
the ERE of HIF-1α under hypoxia
20
Machanisms of Tamoxifen and ICI182780
21
Histone deacetylase
Cell Culture
(Normoxia)
C/+Tam/+ICI
(1000nM)
48hr
ChIP
C/+Tam/+ICI
IgG HDAC ERα
| |
|
ERE ERE ERE
RT-PCR
 Conclusion : These data indicate that the two compounds
inhibit ERα function through different
mechanisms
Tamoxifen-bound ERα inhibits HIF-1α expression
Resistant to Tamoxifen
22
Condition
Tam-MCF7
BT474
100 nM Tamoxifen
 Result : When ERα was depleted in tamoxifen-resistant
cell, HIF-1α expression was up-regulated
Tamoxifen-bound ERα inhibits HIF-1α expression
Short Exposure
ERα expression
plasmid
Transfection
(Normoxia)
Long Exposure
48hr
Western Blot
 Result : Longer exposure of the film showed that
overexpression of ERα enhanced HIF-1α in parental cells
 Result : Overexpression of ERα in TamR-MCF7 cells
significantly reduced HIF-1α expression
23
The working model between E2 ERα and HIF-1α
The working model between Tamoxifen and HIF-1α24
 Conclusion : E2-bound ERα induces—but tamoxifen-bound
ERα suppresses—HIF-1α expression
HIF-1α confers tamoxifen resistance to
ER+ breast cancer cells
Colony Formation assay
HIF-1α cDNA
Retroviral vector
Transfection
25
18 d
4 wk
western blot
 Result : HIF-1α–expressing cells were at least twofold more
resistant in normoxia and long-term treatment
showed more remarkable effect
HIF-1α confers tamoxifen resistance to
ER+ breast cancer cells
26
Tumorsphere Formation Assay
Inhibits HIF-1α expression
Induces apoptosis of MCF7
 Result :
HIF-1α conferred significant
resistance to tamoxifen and ICI182780
compared with the parental control
High HIF-1α gene expression show a poor response to
27
Tamoxifen treatment in ERα+ breast cancer
Relapse free survival
Tamoxifen treatment
 Result : Patients with high level of HIF-1α gene expression had a
poorer relapse-free survival to endocrine therapy or
tamoxifen treatment alone
High HIF-1α gene expression show a poor response to
Tamoxifen treatment in ERα+ breast cancer
28
Overall survival
Tamoxifen treatment
with chemotherapy
 Result : When chemotherapy was included for those patients who
received tamoxifen, HIF-1α is also associated with poor
overall survival
HIF-1α Overexpression Confers Advantage of Tumor
Growth and Resistance to Tamoxifen Treatment 29
NSG mice
C
C
Tam
HIF-1
C
Tam
(Tamoxifen : 5mg)
 Result : Tamoxifen
treatment only modestly
delayed tumor growth with
HIF-1α overexpression ,
similar to the in vitro data
 Conclusion : HIF-1α is able to confer tamoxifen resistance
30
Discussion
The working model between ERα and HIF-1 pathway
31
HIF-1 might not be required for ERα activity
but synergizes with ERα
32
Previous studies also show that some genes, such as KDM4B,
STC2, and VEGFA, bear both a hypoxia response element and
ERE.
Cancer Res 62(5):1289–1295
Exp Cell Res 316(3):466–476
We previously showed that depletion of HIF-1α only partially
affected KDM4B expression in hypoxia, whereas depletion of ERα
nearly abrogated KDM4B expression.
Cancer Res 70(16):6456–6466
HIF-1 might not be required for ERα
activity but synergizes with ERα.
33
THE END
Related documents