a novel therapeutic approach for triple- negative breast cancer raj kumar, ph.d. professor of...
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A Novel Therapeutic Approach For Triple-Negative Breast cancer
Raj Kumar, Ph.D.Professor of Biochemistry
The Commonwealth Medical College, Scranton, PA.
Nothing to Disclose
Basal: derived from myoepithelial cellsER-, PR-, HER-
No specific target for therapies
Breast cancer is classified into clinical subtypes based upon receptor expression
These subtypes dictate possible therapeutic options and vary in their prognosis
Luminal: derived from the luminal cellsER+, PR+, HER+
Can use hormonal therapy
There is no specific therapeutic target for TNBC, and therefore must use cytotoxic chemotherapeutics,
surgery, radiation.
TNBC accounts for 10-20% of all breast cancer, but much higher proportion of all breast cancer mortality.
Younger age at diagnosis, high grade, large tumor size, aggressive relapse.
High proliferation, poor differentiation, and aggressive clinical course with early relapse and
decreased survival.
Standard course of treatment is very aggressive: surgery and radiation therapy combined with adjuvant and neo-adjuvant chemotherapy.
Chemotherapy typically includes combinations of taxanes, anthracyclines, and oxazophorines.
Current Options for TNBC
The search is on for specific therapeutic targets!!!
These results suggest that blocking GR activity could be a useful strategy for increasing tumor cell apoptosis in TNBC. Clin. Cancer Res. 19, 6163–72, 2013
It has previously been reported that because of the anti-apoptotic activity of glucocorticoid receptor (GR) in estrogen receptor (ER)-negative breast epithelial and cancer cells, high GR expression/activity in early-stage TNBC significantly correlates with chemotherapy resistance and increased recurrence.
A recent study has shown that pre-treatment with mifepristone/RU486, a GR antagonist, potentiates the efficacy of chemotherapy in TNBC by inhibiting the anti-apoptotic signaling pathways of GR and increasing the cytotoxic efficiency of chemotherapy.
Another recent study showed that a peptide containing LXXLL motif of steroid receptor coactivator-1 (SRC-1) can severely reduce the viability and proliferation of hormone-unresponsive breast cancer MDA-MB-231 cells. (Int. J. Mol. Sci. 2014, 15, 5680-5698)
SRC-1 is known to function as a coactivator for steroid hormone receptors including GR.
These results suggest that blocking GR/SRC-1 interaction may be an effective drug candidate in the treatment of TNBC.
When viewing steroid receptors (e.g., GR) as therapeutic targets, the challenge is how to selectively control cell/tissue and gene specificity in a manner that affects only deleterious actions of receptor in diseased tissues without altering essential normal functions.
One attractive, but limited, approach is the development of selective steroid receptor modulators (SRMs) that regulate a subset of the normal gene repertoire. SRMs have the clinically useful but enigmatic property of evoking anywhere from full agonist to full antagonist activity in a gene/tissue-dependent manner. Thus SRMs can display between 100% and 0% efficacy.
Human Glucocorticoid Receptor (GR)
?Nature 352 (1991) 497-505. Cell 110 (2002) 93-105
DBDAF1 AF2LBD
NH2 COOH
1 77 262 421 481 777
AF1C
The “Holy Grail” for hormone therapies is to target full SR signaling in a receptor- and tissue/gene-selective manner to maximize therapeutic benefits and to minimize effects on other targets. However, due to available LBD/AF2 crystal structures, the current design of small molecule selective receptor modulators (SRMs) for clinical uses is primarily based on their modulation of AF2 activity, which often fail to inactivate AF1, and thereby missing the whole SR spectrum during endocrine-based therapies. This is despite the fact the most of cell/tissue-specific SRM activities are AF1-dpependent.
Experiments showing intrinsically disordered AF1 conformation
Deuterium/hydrogen exchange /mass (HDXM)
HSQC NMR spectrum of 15N labeled AF1
Similar results are also obtained using several other biophysical methods such as CD and FTIR
The AF1/NTD of all the SHRs studied so far have been found to exit in an intrinsically disordered (ID) conformation.
Conformational flexibility in IDs allows it to interact with binding partners with high specificity and low affinity that means on one hand these provide specific interactions at the same time allow them to be easily dispersed when need arises, an important function of steroid receptors to turn on or turn off signals of target genes as required.
ID proteins normally undergo “disorder-to-order” transition upon interactions with their target molecule(s).
Question: Does GR AF1 adopt a functionally active ordered structure when bound to a
binding partner protein such that its interaction with SRC-1 is facilitated?
Like C-terminal AF-2, AF1 interacts with several specific cofactor proteins including several coactivators
Interaction of TBP to agonist-bound PR alters LBD conformation
Cell-Structure 22, 961–973, July 8, 2014
Color bar (% of deuterium uptake difference)
Interaction of TBP to antagonist-bound PR alters NTD/AF1 conformation
Cell- Structure 22, 961–973, July 8, 2014
A therapeutic Model for Endocrine-related cancers
Structure 22, 961–973, July 8, 2014
GR AF1
TBP
GR AF1 and TBP
Disordered
Ordered
SRC-1
Multi-protein assembly involved in GR-mediated gene regulation
Other binding partner protein
AF1c GR500
1 77 262 421 481 777AF1c
DBD LBDAF1
1 500
GR
AF1 GR
GR500
AF1 GR500
Constructs of GR
CFP-GR500 + YFP-TBP+pTALBefore PB After PB
CFP-GR500 + YFP-TBP
Before PB After PB
Before PB
CFP-AF1_GR500 YFP-TBP
Before PB After PB
FRET analysis showing the GR AF1:TBP interaction in CV-1 cells
Copik et al., Mol. Endocrinol. 2006
-50 0 50 100 150 200 250 300
130
110
90
70
50
30
10
-10
Time (s)
Re
sp
on
se
(RU
)
Fc2
Fc1
Kinetics of binding between GR AF1 and TBP by Surface Plasmon resonance (SPR) method using BIACORE
[AF1] (μM)
0.0 0.2
0.40.8
1.6
3.2
6.4
290 330 370 410 450 490 530 570 610 650
295
245
195
145
95
45
-5Re
sp
on
se
(RU
)
Time (s)
KD = 0.46 µM
Wavelength [nm]
200 220 240 260
Ell
ipti
cit
y [
md
eg
ree
]]
-30
-20
-10
0
10
AF1 AF1-TBP 1:0.25 AF1-TBP 1:0.5 AF1-TBP 1:0.75 AF1-TBP 1:1 AF1-TBP 1:1.25 AF1-TBP 1:1.5 AF1-TBP 1:1.75 AF1-TBP 1:2
AF1:TBPc Ratio0.0 0.5 1.0 1.5 2.0E
llip
tic
ity
@ 2
20
nm
[m
de
g]
0
5
10
15
20
25
AF1:TBPc (Experimental)
AF1+TBPc (Theoretical)
Wavelength [nm]
200 220 240 260
Elli
pti
city
[m
deg
]
-30
-20
-10
0
10
AF1+TBP (1+0) AF1+TBP (1:0.25) AF1+TBP (1+0.50) AF1+TBP (1+0.75) AF1+TBP (1+1) AF1+TBP (1+1.25) AF1+TBP (1+1.50) AF1+TBP (1+1.75) AF1+TBP (1+2)
AF1-TBPc Ratio
0.5 1.0 1.5 2.0 E
llip
tic
ity @
22
0 n
m [
md
eg
]
0.0
0.5
1.0
1.5
2.0
2.5
3.0
D (Exp - Theo) @ 220 nm
Far-UV CD spectra of AF1:TBP complex shows that AF1 adopts ordered structure when bound to TBP
Wavelength [nm]
200 220 240 260
Elli
pti
city
[m
deg
ree]
-15
-10
-5
0
5
10
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00
Concentration Ratio
0.5 1.0 1.5 2.0Elli
pti
city
@ 2
20 n
m [
md
eg]
0
5
10
15
20
TBPc
Far-UV CD spectra of TBP at various concentrations showing no structural changing happening in TBP
AF1 AF1+TBP1 2
Fo
ld In
crea
se
0
2
4
6
8
10
12
*
1 2 3 4 5
AF1+NE AF1+TBP+NE NE
IP = SRC-1 andblot = AF1 antibody
TBP-binding/folding induced conformation in AF1 facilitates AF1’s interaction with SRC-1
Synergistic effects of TBP and SRC-1 on GR AF1 activity. GRE-SEAP
Western blots showing higher levels of TBP and SRC-1 expression in MDA-MB-231 cells compared to MCF-7 cells.
MCF-7 MDA-MB-231
Nuclear GR C Dex C Dex
TBP
SRC-1
MCF-7 MDA-MB-231
TBP-binding to GR AF1 enhances its interaction with SRC-1 in NDA MB 231 cell nuclear extracts (NE)
Fo
ld I
ncr
ease
AF1 + NE
AF1 + TBPc + NE
TBPc +NE NE
MDA-MB-231 NE
IP: SRC-1
IB: GR
Dex treatment results into higher cell viability of MDA MB-231 cells
WS
T1
-Ab
so
rba
nc
e
ControlDex treated
AF1/NTD
TBP
SRC-1
Other binding partner protein
Drug
Tissue specific gene regulation and a drug target for TNBC
Acknowledgements
S. Stoney Simons, Jr., NIDDK, National Institutes of Health, Bethesda, MD.
Patrick R. Griffin, The Scripps Research Institute, Jupiter, FL.
Dean P. Edwards, Baylor College of Medicine, Houston, TX.
Iain J. McEwan, University of Aberdeen, Aberdeen, Scotland, UK.
E. Brad Thompson, University of Houston, Houston, TX
Ravi Jasuja, Harvard Medical School, Boston, MA
Anna S. GarzaAlicja J. CopikShagufta KhanJun LingChristian CarbeSantina Possanza