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Nuclear Receptors and Cancer Cancer Biology 101 February 19

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Title: Nuclear Receptors and Cancer Cancer Biology 101 February 19


1
Nuclear Receptors and Cancer
  • Cancer Biology 101
  • February 19, 2013
  • Joe Lipsick

2
Nuclear Receptors and Cancer
  • Glucocorticoids
  • Stress or therapy?
  • Lipophilic Hormones
  • Nuclear Receptors
  • Estrogens (and Androgens)
  • Retinoids
  • Orphan Receptors

3
(No Transcript)
4
Glucocorticoid Therapy for CLL
white blood cells
CLL chronic lymphocytic leukemia
Damashek, 1967
5
Glucocorticoids and Apoptosis
steroid
steroid
steroid
-steroid
-steroid
-steroid
Thymic T Cells
Wyllie, 1980
6
Glucocorticoids and Apoptosis
MN micrococcal nuclease control for
nucleosomal digestion
nuclease
DNA Extracted from Low Molecular Weight Chromatin
of Thymocytes At Different Times During Treatment
of Cells with Glucocorticoid (3 hours and 5 hours)
Wyllie, 1980
7
Bcl2 (and Friends) vs Glucocorticoids
Murine T Cell Line Dexamethasone (a synthetic
glucocorticoid)
Huang, et al, 1997
8
Nuclear Receptors and Cancer
  • Glucocorticoids
  • Stress or therapy?
  • Lipophilic Hormones
  • Nuclear Receptors
  • Estrogens (and Androgens)
  • Retinoids
  • Orphan Receptors

9
Lipophilic Hormones and Vitamins
from Cooper
10
Lipophilic Hormones and Vitamins
  • Small organic molecules
  • Cross cell membranes by diffusion
  • Easily modified by medicinal chemistry
  • Potent regulators of gene expression

11
Lipophilic Hormones and Cancer
  • Glucocorticoids
  • Cause lymphoid apoptosis
  • Chemotherapy for leukemias and lymphomas
  • Estrogens
  • Promote growth of many breast and uterine cancers
  • Androgens
  • Promote growth of many prostate cancers
  • Retinoids
  • Promote differentiation of specific types of
    leukemia
  • Useful for differentiation therapy of carcinomas?

12
Nuclear Receptors and Cancer
  • Glucocorticoids
  • Stress or therapy?
  • Lipophilic Hormones
  • Nuclear Receptors
  • Estrogens (and Androgens)
  • Retinoids
  • Orphan Receptors

13
Evidence for an Estrogen Receptor
Elwood Jensen
Uptake of radioactive estrogen by different
tissues in the presence or absence of an
anti-estrogen (PD).
Jensen and DeSombre, 1973
14
Sucrose Gradient Sedimentation
Jensen and DeSombre, 1973
15
Estrogen Receptor Transformation
cytosolic (untreated)
nuclear (E2 treated)
5S
8S
salt
E2
4S
E2
5S
Jensen and DeSombre, 1973
16
Glucocorticoid Receptor (GR) Activation
Immuno- fluorescence with anti-GR antibody
- Hormone Hormone
5S 90 KDa
9S 330 KDa
Biochemistry
17
Molecular Cloning of GR
western blot
cDNA expression library screen with anti-GR
antibody reconstruct full-length cDNA
clones in vitro transcribe RNA from cDNA in
vitro translate (IVT) RNA with RRL western blot
with anti-GR antibody

1 Rabbit Reticulocyte Lysate (RRL) 2 RRL
GR-alpha RNA 3 RRL GR-beta RNA 4 9
extracts of cell lines 7 variant
glucocorticoid-resistant T cell line
Hollenberg, et al, 1985
18
Molecularly Cloned GR Binds Hormone
in vitro transcribe RNA from cDNA in vitro
translation (IVT) of RNA radioactive hormone
binding assays -/ unlabeled cold competitor
IVT GR protein T cell extract
Radioactive hormone binding assays in 100x excess
of cold competitor.
Hollenberg, et al, 1985
19
Function of Molecularly Cloned GR
products
substrate
Ron Evans, 1982
radioactive chloramphenicol acetyl
transferase(CAT) assay by thin-layer
chromatography
anti-GR western blot
Giguere, et al, 1986
20
Inducible Gene Expression
Transcriptional Activation
Mut-Reporter WT-Reporter -GC GC -GC GC no no
no yes
Proteins Tested
GR N-term
GR C-term
Keith Yamamoto
GR glucocorticoid receptor GC
glucocorticoid GRE glucocorticoid response
element
Picard, et al, 1988
21
Inducible Gene Expression
Transcriptional Activation
Mut-Reporter WT-Reporter -GC GC -GC GC no no
no yes no no yes yes
Proteins Tested
GR N-term
GR C-term
GR N-term
Keith Yamamoto
GR glucocorticoid receptor GC
glucocorticoid GRE glucocorticoid response
element
Picard, et al, 1988
22
General Structure of Nuclear Receptor
from Berg
23
Glucocorticoid Receptor Activation
AD transcriptional activation domain DBD
DNA-binding domain LBD ligand-binding domain
from Lodish
24
A Modular Response Element
Transcriptional Activation
Proteins Tested
GRE-Reporter E1A-Reporter -GC GC -GC GC no
yes no no yes yes no no no no yes yes no no
no yes
GR
GR N-term
GR C-term
GR N-term
E1A
E1A
E1A
GR C-term
GC glucocorticoid
Picard, et al, 1988
25
Nuclear Receptors and Cancer
  • Glucocorticoids
  • Stress or therapy?
  • Lipophilic Hormones
  • Nuclear Receptors
  • Estrogens (and Androgens)
  • Retinoids
  • Orphan Receptors

26
Nuclear Receptor Family
Evans lab Chambon lab Yamamoto lab (figure from
Lodish)
Pierre Chambon
27
ER-GFP Re-localization within the Nucleus
Hager, et al, 2000
28
Different Modes of NR Regulation
29
Childbearing and Breast Cancer
Alberts, MBoC
30
Estrogen Receptors and Breast Cancer
ER and ER- indicate presence or absence of
estrogen receptor in cancer cells.
Knight, et al, 1977
31
E2-Dependent Breast Cancer Cells
E2
E2
-E2
-E2
Medium FCS Medium
Charcoal-Treated FCS
E2 estradiol FCS fetal calf serum
Darbre, et al, 1983
32
Estrogen Biosynthesis
Observation ER-positive breast cancer cells are
E2-dependent for growth Question Potential
therapeutic interventions?
33
Changes in ER Ligand-Binding Domain
from Berg
34

Estrogen versus Tamoxifen
ER-LBD plus estradiol (hidden)
helix 12
ER-LBD plus tamoxifen (red)
dimeric protein structures
NCBI
35
Tamoxifen as an Adjuvant (Post-Surgical) Therapy
for Breast Cancer Patients
P phenylalanine mustard F fluorouracil
T Tamoxifen
Fisher, et al, 1986
36
Chemoprevention of Cancer in Rats
DMBA-induced mammary carcinoma in rat model
Jensen and Jordan, 2003
37
Prevention of Breast CA by Tamoxifen
13,388 women at increased risk for breast cancer.
Fisher, et al, 1998
38
National Cancer Institute
39
Tamoxifen Causes Endometrial Cancer
13,388 women at increased risk for breast cancer.
Fisher, et al, 1998
40
Tamoxifen No Free Lunch?
0.4
1998
2005
3.3
Fisher, et al, 2005
41
Tamoxifen No Free Lunch?
42/1000
1998
16/1000
2005
3/1000
0.7/1000
1.2/1000
0.3/1000
0.8/1000
Tamoxifen Trial 13,388 women with increased risk
for breast cancer (rates indicated are relative
to those without tamoxifen for the seven year
trial) Lifetime risk of breast CA for all women
is 1/8 risk of death from breast CA is 1/35.
Fisher, et al, 2005
42
National Cancer Institute
43
Another Estrogen Receptor!
Pearce and Jordan, 2004
44
Selective Estrogen Receptor Modulators (SERMs)
Teratogen and carcinogen.
Displaces Helix 12
Note Tamoxifen is actually a partial agonist of
the ER in the uterus Raloxifene may be an
antagonist in breast and uterus, but an agonist
in bone.
45
LCIS lobular carcinoma in situ DCIS ductal
carinoma in situ.
National Cancer Institute
46
Raloxifene vs Tamoxifen in Prevention
19,747 postmenopausal women at increased risk of
breast cancer.
Vogel, et al, 2006
47
Estrogen Biosynthesis
Observation ER-positive breast cancer cells are
E2-dependent for growth Question Other
potential therapeutic interventions?
48
Aromatase Inhibitor Prevents Recurrencein Breast
Cancer Patients
(aromatase inhibitor) (anti-estrogen)
Coombes, et al, 2007
49
Nuclear Receptors and Cancer
  • Glucocorticoids
  • Stress or therapy?
  • Lipophilic Hormones
  • Nuclear Receptors
  • Estrogens (and Androgens)
  • Retinoids
  • Orphan Receptors

50
v-erbA Oncogene Blocks Differentiation
  • v-erbA
  • Retroviral oncogene
  • Blocks erythroid differentiation

blast
RNA splicing
gag-v-erbA v-erbB
RBC
Blocks Differentiation
Maintains Proliferation
Avian Erythroblastosis Virus (AEV)
51
c-erbA Looks Like a Nuclear Receptor
c-erbA
Evans lab Chambon lab Vennstrom lab Yamamoto
lab (figure from Lodish)
52
v-erbA versus c-erbA
Sap, et al, 1986 1989
53
c-erbA Protein Binds Thyroid Hormone
mutations in v-erbA
Open bars no cold competitor Shaded bars
1000-fold excess of cold competitor
125I-T3 bound
Bjorn Vennstrom
Chicken embryo fibroblasts Infected with
indicated virus
Positive Control cells
Sap, et al, 1986
54
v-erbA Dominant Negative?
Transient transfections of cells with endogenous
receptors for indicated hormones, receptor-respons
ive promoters, v-erbA (- or ), and then
hormone-treated (- or ). Triac gt thyroid
hormone receptor E2 gt estrogen receptor RA gt
retinoic acid receptor Dex gt glucocorticoid
receptor
Sharif and Privalsky, 1991
55
v-erbA Off Target Dominant Negative
Mutants of v-erbA DNA-Binding Domain
Sharif and Privalsky, 1991
56
Nuclear Receptor Family
HOMODIMERIC? YES YES YES NO NO
Q How do TR and RAR bind to DNA?
figure from Lodish
57
RXR, a Partner of RAR and TR
deLera, et al, 2007
58
Heterodimeric Receptor DNA Sites
v-erbA mutations
AGGTCA(N)xAGGTCA
Spacing between tandem DNA motifs determines the
heterodimeric protein recognition sites.
Harbers, et al, 1998
59
Summary of v-erbA
  • v-erbA
  • Retroviral oncogene
  • Blocks erythroid differentiation
  • Altered form of thyroid hormone receptor
  • Inhibits function of retinoic acid receptor
  • Off target dominant negative (neomorph)

RNA splicing
AEV
gag-v-erbA v-erbB
Thyroid Hormone Receptor
EGF Receptor
60
APL Responds to Retinoic Acid
Wang Zhenyi and Chen Zhu photo from Shanghai
Daily
Retinoic Acid
from Alberts
61
Differentiation Therapy for Human APL
APL acute promyelocytic leukemia BM bone
marrow PB peripheral blood
from Wang and Chen, 2008
62
Chromosomal Translocation in APL
Wang and Chen, 2008
63
Different Modes of NR Regulation
transcriptional repressor in the absence of
ligand
64
RA Relieves Repression by PML-RAR
histone acetylation
histone deacetylation
Lin, 2001
65
Nuclear Receptors and The Cell Cycle
S phase genes
Cdk2/Cyclin A
Altucci, 2001
66
Summary
  • Nuclear hormone receptors
  • Bind to specific sequences in DNA
  • Alter their conformation upon hormone binding
  • Directly regulate gene expression
  • Nuclear hormones can promote cancer
  • Estrogens and androgens
  • Nuclear receptor inhibitors can treat cancer
  • Tamoxifen, true anti-estrogens, anti-androgens
  • Nuclear hormones as differentiation therapy
  • Retinoic acid analogs, vitamin D, ???
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