world congress on breast cancer 2015 · ther., 351, 510-518 (2014) hdac3 is the leading candidate...

Downregulation of Ca2+-activated Cl-

channel TMEM16A by histone deacetylase

inhibition in breast cancer cells

Susumu Ohya, Ph.D.

Department of Pharmacology

Kyoto Pharmaceutical University

Kyoto, Japan

World Congress on

Breast Cancer 2015

Breast Cancer-2015, Birmingham, UK

August 4, 2015

Overview

1. Role of Ca2+-activated Cl- channel in cancer cells and

epigenetic modification of gene expression

2. Downregulation of Ca2+-activated Cl- channel, TMEM16A by

a histone deacetylase (HDAC) inhibitor, vorinostat in human

breast cancer cells

3. Effects of pharmacological and siRNA-based blockade of

HDAC on TMEM16A transcription

4. Regulation of HER2 expression by TMEM16A

Overview

1. Role of Ca2+-activated Cl- channel in cancer cells and

epigenetic modification of gene expression

2. Downregulation of Ca2+-activated Cl- channel, TMEM16A by

a histone deacetylase (HDAC) inhibitor, vorinostat in human

breast cancer cells

3. Effects of pharmacological and siRNA-based blockade of

HDAC on TMEM16A transcription

4. Regulation of HER2 expression by TMEM16A

Feske et al., Ann. Rev. Immunol., 33, 291-353 (2015)

Principal interplay between the Na+-, K+-, Ca2+- and Cl--

permeable channels

Gene regulation

DNA fragmentation

proliferation

migration

invasion

apoptosis

Ion channels and cancer

1. Ion channels contribute to various cancer

processes by the regulation of the resting

membrane potential and Ca2+ signaling.

2. Pharmacological inhibition of ion channels is

an attractive target to prevent cancer cell

proliferation and metastasis.

TMEM16A (ANO1) overexpression enhances cancer

cell proliferation through signaling pathways

Overexpression of TMEM16A (ANO1)

breast tumor Britschgi et al., Proc. Natl. Acad. Sci. U.S.A. (2013)

breast cancer cell lines Leanza et al., Front. Physiol. (2013)

Qu et al., Cancer Med. 3, 453-461 (2014)

Picollo et al., J. Mol. Biol. 427, 94-105 (2015)

chromosome 11q13

migration

TMEM16A is an attractive therapeutic target and a novel biomarker for cancer.

Epigenetic modification of gene expression

Histone

Deacetylase

Inhibitor

X Cancer

Cell

Death

histone acetylation

histone methylation

Epigenetic modification therapy with

HDAC inhibitors is one of the novel strategies for cancer treatment.

Overview

1. Role of Ca2+-activated Cl- channel in cancer cells and

epigenetic modification of gene expression

2. Downregulation of Ca2+-activated Cl- channel, TMEM16A by

a histone deacetylase (HDAC) inhibitor, vorinostat in human

breast cancer cells

3. Effects of pharmacological and siRNA-based blockade of

HDAC on TMEM16A transcription

4. Regulation of HER2 expression by TMEM16A

Vorinostat is approved for the treatment of T cell lymphoma and is

developed for the other solid tumors with combination drug therapy.

vorinostat pan-HDAC inhibitor

IC50=100-1000 nM for HDAC1-11

0.0

0.5

1.0

rela

tive c

ell v

iab

ilit

y

(3) (3)

**

0.00

0.05

0.10

0.15

rati

o t

o A

CT

B

YM

B-1

MD

A-M

B

-453

MC

F-7

MD

A-M

B

-231

MD

A-M

B

-468

n=3

BT

54

9

Hs

57

8T

A. TMEM16A mRNA

ER

PR

HER2

-

-

+

-

-

+

+

+/-

-

-

-

-

-

-

-

-

-

-

-

-

-

Functional expression of TMEM16A in YMB-1 cells

B. TMEM16A activity

in YMB-1

-50 mV -80 mV

500 ms

1000 pA

C. Cell viability

Matsuba et al., J. Pharmacol. Exp. Ther., 351, 510-518 (2014)

0.0

0.5

1.0

rela

tiv

e c

ell

via

bilit

y

A. Cell viability

**

**

(5) (5) (5) (5)

1 10

vorinostat

(µM)

0.1

B. TMEM16A mRNA

0.00

0.05

0.10

0.15

rela

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RN

A e

xp

res

sio

n

(ra

tio

to

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TB

)

1 10

vorinostat

(µM)

**

#

** (4) (4) (4)

Downregulation of TMEM16A by treatment with

vorinostat in YMB-1 cells (1)

0.0

0.4

0.8

1.2

rela

tive p

rote

in e

xp

ress

ion

**

C. TMEM16A protein

10 μm

vehicle

vorinostat (10 µM)

vorinostat pan-HDAC inhibitor

IC50=100-1000 nM for HDAC1-11

Matsuba et al., J. Pharmacol. Exp. Ther., 351, 510-518 (2014)

-50 mV -80 mV

500 ms

1000 pA

-60 -40 -20 0 20 40 600

1000

2000

3000

4000

voltage (mV)

cu

rren

t (p

A)

■ vehicle (8)

● vorinostat (7)

pCa6.0

**

Downregulation of TMEM16A by treatment with

vorinostat in YMB-1 cells (2)

A. vehicle

B. vorinostat (10 µM)

C. I-V relationship

500 ms

1000 pA

Matsuba et al., J. Pharmacol. Exp. Ther., 351, 510-518 (2014)

Overview

1. Role of Ca2+-activated Cl- channel in cancer cells and

epigenetic modification of gene expression

2. Downregulation of Ca2+-activated Cl- channel, TMEM16A by

a histone deacetylase (HDAC) inhibitor, vorinostat in human

breast cancer cells

3. Effects of pharmacological and siRNA-based blockade of

HDAC on TMEM16A transcription

4. Regulation of HER2 expression by TMEM16A Of 11 HDAC subtypes, HDAC1, 2, 3 and 6 are highly expressed in

YMB-1 cells in consistent with the expression patterns in human

tumor breast tissues.

0.0

0.4

0.8

1.2

rela

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rote

in e

xp

res

sio

n

0.00

0.05

0.10

0.15

rela

tive m

RN

A e

xp

ress

ion

(rati

o t

o A

CT

B)

*

**

A. TMEM16A mRNA AATB

T247

NCT-14b

HDAC1＆HDAC2 inhibitor

IC50= 7 nM for HDAC1

IC50= 49 nM for HDAC2

HDAC6 inhibitor

IC50= 82 nM for HDAC6

HDAC3 inhibitor

IC50= 240 nM for HDAC3

Downregulation of TMEM16A by HDAC2/3 inhibition

in YMB-1 cells (1)

Matsuba et al., J. Pharmacol. Exp. Ther., 351, 510-518 (2014)

*

**

B. TMEM16A protein

n=4 n=4

-50 mV -80 mV

-60 -40 -20 0 20 40 600

1000

2000

3000

4000

voltage (mV)

cu

rren

t (p

A)

■ vehicle (7)

● T247 (4)

pCa6.0

** 500 ms

1000 pA

Downregulation of TMEM16A by HDAC2/3 inhibition

in YMB-1 cells (2)

0.00

0.04

0.08

0.12

rela

tive m

RN

A e

xp

ressio

n

(rati

o t

o A

CT

B)

**

**

B. vehicle

C. T247 (1 µM)

D. I-V relationship

500 ms

1000 pA

A. TMEM16A mRNA

Matsuba et al., J. Pharmacol. Exp. Ther., 351, 510-518 (2014)

n=4

HDAC3 is the leading candidate for the

downregulaion of TMEM16A in YMB-1 cells.

Overview

1. Role of Ca2+-activated Cl- channel in cancer cells and

epigenetic modification of gene expression

2. Downregulation of Ca2+-activated Cl- channel, TMEM16A by

a histone deacetylase (HDAC) inhibitor, vorinostat in human

breast cancer cells

3. Effects of pharmacological and siRNA-based blockade of

HDAC on TMEM16A transcription

4. Regulation of HER2 expression by TMEM16A

Lipid rafts, ion channel complexes and EGFR signaling

1) In the plasma membrane, ion channels and receptors

can functionally communicate by forming clusters of

lipid microdomains.

2) EGF receptor forms functional complexes with Ca2+-

activated Cl- channel and store-operated Ca2+ channels

and contributes to the Ca2+ signal pathway with them.

Gueguinou et al., Biochim. Biophys. Acta., 2736 (2014)

Lipid rafts, ion channel complexes and EGFR signaling

HER signaling pathway and its related cell processes

Rimawi et al., Annu. Rev. Med., 66, 111-128 (2015)

HER2 is

1) an EGF receptor family with

tyrosine kinase activity.

2) overexpressed in about 30 %

of patients with breast cancer.

3) the mainstay of targeted

therapy for the treatment of

invasive breast cancers.

0.0

0.2

0.4

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(ra

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0.0

0.2

0.4

rela

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RN

A e

xp

res

sio

n

(ra

tio

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TB

)(3) (3)

*

A. HER2 mRNA

(4) (4)

**

HER2

HER2

Downregulation of HER2 by TMEM16A inhibition

in YMB-1 cells

B. HER2 mRNA C. HER2 protein

D. HER2 protein

Conclusions

1. Ca2+-activated Cl- channel, TMEM16A is a potential

therapeutic target for breast cancer, and inhibition of

TMEM16A suppresses proliferation and invasion.

2. In malignancies with a frequent gene amplification of

TMEM16A, TMEM16A dysfunction by HDAC3 inhibition

is at least in part responsible for the suppressive

effects on cell viability in breast cancer cells, and it

may be related to the poor invasion capacity observed

in metastatic breast cancer cells.

3. TMEM16A may play an important role in the regulation

of HER2-downstream signaling in breast cancer cells.

Acknowledgements

Kyoto Pharmaceutical University,

Kyoto,Japan

Masanori Fujii, Ph.D.

Hiroaki Kito, Ph.D.

Satomi Niwa, Ph.D.

Sayo Matsuba

Saki Kanatsuka

Yurika Nakazono

Aichi Gakuin University,

Nagoya, Japan

Electrophysiology

Katsuhiko Muraki, Ph.D.

Noriyuki Hatano, Ph.D.

Kyoto Prefectural University of Medicine,

Kyoto Japan

Chemical synthesis of HDAC inhibitors

Takayoshi Suzuki, Ph.D.

Peng Zhan, Ph.D.