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