ANTI-APOPTOTIC AND PRO-

ANGIOGENIC PROPERTIES OF

ENDOGENOUS THYROID

HORMONE AND ITS ACTION ON

P-GLYCOPROTEIN MAY BLUNT

RESPONSE TO CONVENTIONAL

CHEMOTHERAPY

Paul J. Davis, MD

Albany Medical College and

Pharmaceutical Research

Institute, Albany College of

Pharmacy and Health

Sciences, Albany, NY USA

MECHANISMS OF THYROID

HORMONE ACTION

• Thyroid hormone (L-thyroxine, T4;

3,5,3’-triiodo-L-thyronine, T3) acts

via genomic and nongenomic

mechanisms. The genomic

pathway depends primarily upon

formation of intranuclear complexes

of T3 with the nuclear thyroid

hormone receptor proteins (TRs).

• The nongenomic pathways include

actions initiated at a cell surface

receptor for T4 and T3 on the

extracellular domain of integrin

avb3.

O CH2-CH-COOH

NH2

I - -

I - - I

- -

I - -

3’

5’ 5

3

Thyroxine (T4)

O CH2-CH-COOH

NH2

I - -

I - -

I - -

3’

5’ 5

3

3,5,3’-Triiodothyronine (T3)

HO

Integrin

STAT1a

TRb1, ER

PLC PKC

TRb1 ERa

T4

TRb1 p300 NCoR

mRNA

Trip230

MAPK (ERK1/ERK2)

Cytoplasm

T3

TRa

STAT1a, p53

T4

TR∆a1

rT3

TRb1

PI 3-K-

Akt/PKB

aV β3

T3

mRNA

Extracellular matrix

proteins, e.g.,

laminin, vitronectin

p53

SIGNAL TRANSDUCTION

TRb1

ER

STAT1a

a

a

p53

T3

T3

T3

MAPK (ERK1/ERK2)

Na+/H+

antiporter

Amino acid

transporter

Protein trafficking

Serine phosphorylation of nuclear proteins

Specific gene transcription

Activity of ion transporters

Nongenomic Actions of Thyroid Hormone

Initiated at the Cell Surface

Integrin avb3 is primarily expressed

by dividing cells and thus

concentrated and activated in

cancer cells and rapidly-dividing

endothelial cells that serve cancers.

Usually viewed as a sensing

mechanism for extracellular matrix

(ECM) proteins and critical to cell-

matrix and cell-cell interactions that

underlie tissue organization, avb3

has recently been appreciated to

bind small molecules and to contain

the cell surface receptor for thyroid

hormone.

As a prototypic small molecule

ligand of integrin avb3, thyroid

hormone importantly alters

transcription of differentially-

regulable genes important to

cancer cell function and

angiogenesis—and a) regulates

integrin vascular growth factor

receptor function, b) alters the state

of the actin cytoskeleton, c)

changes intracellular protein

trafficking and d) controls the

function of plasma membrane

NHE1 and Na,K-ATPase.

Thyroid hormone and cancer cell

proliferation

Time (h)

_

2 4 6 24 24 1

_ _ _ _ + ICI (3 nM)

MCF-7 cells: 3H-thymidine

uptake

- F

old

In

cre

ase

in

3H

-th

ym

idin

e In

co

rpo

ratio

n

0

10

20

30

40

10-10

M E2

10-7

M T4

Control

* *

* *

37 kDa-

MCF-7 cells: PCNA

PD + + _ _

+

T4 + + _ _ _ _

T4-A + + _ _ _ _

_

- F

old

In

cre

ase

in

I.O

.D.

0

1

2

3

Thyroid hormone and breast cancer

cell proliferation

ICI = ICI 182,780,

fulvestrant

T4 (10-7 M) _ + _ + _ +

- NCoR

IP: anti-integrin αv

Blot: anti-NCoR

Blot: anti-pSTAT1

- pSTAT1

Blot: anti-SMRT

Blot: anti-p300

Blot: anti-pERK1/2

C siRNA scRNA | | | |

- SMRT

- pERK1

- pERK2

- p300

- Lamin B

T4 (10-7 M) _ + _ + _ +

C siRNA scRNA | | | |

Fig. 3

61 kDa -

37 kDa -

82 kDa -

180 kDa -

180 kDa -

- R

ela

tive I

.O.D

.

*

* *

*

* *

* *

* *

*

* * *

0

2

4

6

8

10

12

14

16

NCoR

SMRT

P300

pSTAT1

pERK1/2

* *

OVCAR-3 cells

A

NCI-H522 cells

Blot: anti-PCNA

- PCNA 37 kDa -

T3 (M) _

10-9 10-8 10-7 10-6

B

Thymidine Incorporation

C

T4 (M)

T3 (M)

_ _

_ _ _ _ 10-8 10-7 10-6

10-9 10-8 10-7

CP

M

x 1

04

_ _

0

5

10

15

20

25

30NCI-H522 cells

NCI-H510A cells

Blot: anti-PCNA

- PCNA

- Lamin-B

37 kDa -

61 kDa -

T4 (M) _

10-9 10-8 10-7 10-6

NCI-H522 cells

- Lamin-B 61 kDa -

*

*

*

*

*

*

*

*

*

*

* *

*

- GAPDH

- Integrin α

- Integrin β

D

H510A, small cell lung carcinoma cells

H522, NSCLC cells

RT-PCR

NCI-H510A cells

ICI (nM)

T3 (10-7 M)

_

_

T4 (10-7 M) _

_ _

_ _ _ _

_ + +

_ _ _ + + + +

20 2 20 20 2

Blot: anti-PCNA - PCNA

37 kDa -

37 kDa -

- pERK1 - pERK2

Blot: anti-pERK1/2

Blot: anti-phosphoER-a (118)

62 kDa -

IP: anti-ER-α

Blot: anti-ER-α

- pER-a

62 kDa -

Thymidine Incorporation

- ER-a

0

5

10

15

20

ICI (nM)

T3 (10-7 M)

_

_

T4 (10-7 M) _

_ _

_ _ _

_ +

_ _ + + +

20 20 20

B

A

CP

M X

10

4

-a

0

1

2

3

4

5

6

pER

pERK1/2

PCNA

-Rela

tive

I.

O.D

.

61 kDa -

- Lamin-B

*

* *

+ +

* *

+

* *

* *

*

+ + +

+

* *

* *

ICI = ICI 182,780,

0

1

2

3

4

5pERK1/2

PCNA

pTyr-p85-PI 3-K

p85-PI 3-K

0

1

2

3

4

5

6

pERK1/2

PCNA

U87MG (GBM) cells

Blot: anti-pERK1/2

Nucleus

Blot: anti-PCNA

Blot: anti-pTyrp85-PI 3-K

Cytosol

T4 (M) _ 10-9 10-8 10-7 10-6

- pERK1 - pERK2

- PCNA

- pTyr-p85

48 kDa -

37 kDa -

37 kDa -

84 kDa -

T3 (10-7 M)

Rela

tive

I.O

.D.

-PI 3-K

T3 (M) _ 10-9 10-8 10-7 10-6

Blot: anti-pTyrp85-PI 3-K

Cytosol

10-10

Blot: anti-p85-PI 3-K

- p85-PI 3-K

Blot: anti-pERK1/2

Nucleus

Blot: anti-PCNA

- pERK1 - pERK2

- PCNA

84 kDa -

84 kDa -

48 kDa -

37 kDa -

37 kDa -

Rela

tive

I.

O.D

.

- pTyr-p85

-PI 3-K

T3 T4

Fig. 1

In vitro stimulation of cell proliferation

(PCNA), activation of ERKs, PI3K by

thyroid hormone analogues

Hypothyroid

Median Survival: 10.1 mos

Non-hypothyroid

Median Survival: 3.1 mos

Hercbergs AA et al, Anticancer Res, 2003

Spontaneous or medically-induced

hypothyroidism has been shown to

favorably affect the clinical courses

of GBM (Cleveland Clinic), breast

cancer (MD Anderson Cancer

Center), renal cell carcinoma (TKI

therapy at multiple centers) an

head-and neck cancers (Cleveland

Clinic).

Thyroid hormone has anti-apoptotic

activity in cancer cells

RV = resveratrol

O CH2-CH-COOH

NH2

I - -

I - - I

- -

I - -

3’

5’ 5

3

Thyroxine (T4)

O CH2-CH-COOH

NH2

I - -

I - -

I - -

3’

5’ 5

3

3,5,3’-Triiodothyronine (T3)

HO

O CH2--COOH

I - -

I - - I

- -

I - -

3’

5’ 5

3

Tetrac

HO

Low-grade thyromimetic within cells

TH antagonist at integrin avb3 TH

receptor

Resveratrol

pERK1/2 activation

PD98059

Cytosol

Nucleus

pSer15-p53 NS-398

Apoptosis

Thyroid Hormone

pERK1/2 activation

PD98059

X

Cell Proliferation

[COX-2]-pERK1/2

complexing

Tetrac

Thyroid hormone inhibits induction by

RV of Ser-15 phosphorylation of p53

Thyroid hormone is pro-angiogenic

by a variety of mechanisms. This is

relevant to support by the hormone

of cancer-related vascularization

and to vascularity of nonmalignant

conditions, such as skin diseases.

PBS

A

T4 T4 + Tetrac

B

Summary of effects of T4, T4-agarose

and tetrac on angiogenesis

Treatment Angiogenesis Index

PBS 67 9

T4 (0.1 nM) 156 16**

Tetrac (0.1 M) 76 9

T4 + tetrac 66 6

T4-agarose (total, 0.1 M) 194 28**

T4-agarose + tetrac 74 7

C PBS T4-ag T4-ag + Tetrac

Angiogenesis in the CAM

Data represent mean ± SD, n=8; *p<0.01, indicating significant

stimulation of angiogenesis. PBS, phosphate-buffered saline.

A

B

b-FGF (1.0 µg/ml)

PBS (Control)

DITPA (0.01 µM) VEGF (2.0 µg/ml)

T4 (0.1 µM) T4-agarose (0.1 µM)

Mean Vessel Branch

Treatment Points ± SD

PBS (Control) 87 ± 9

T4 (0.1 μM) 148 ± 7*

T4-agarose (0.1 μM) 167 ± 8*

DITPA (0.01 μM) 134 ± 11*

DITPA (0.1 μM) 170 ± 9*

VEGF (2.0 μg/ml) 168 ± 10*

b-FGF (1.0 μg/ml) 174 ± 8*

PBS T4 (total, 0.1M)

T4+ LM609(10g)

Inhibitory Effect of avb3 MAB (LM609) on T4-

stimulated Angiogenesis in the CAM Model

CAM Treatment # of branch pts ± SEM % Inhibition ± SEM

PBS 73 ± 8

T4(0.1uM) 170 ± 16 0

T4+LM609(10ug) 109 ± 9 64 ± 9

By a variety of mechanisms, thyroid

hormone, specifically T4, has been

shown to influence the activity and

abundance of P-glycoprotein (P-gp;

MDR1; ABCB1), a plasma

membrane efflux pump that serves

to shorten intracellular retention

time of traditional chemotherapeutic

agents that are ligands for the

protein (doxorubicin, etoposide,

paclitaxel, etc.). Thus, thyroid

hormone may support

chemoresistance.

MDR1

EGFR

transcription

transcription

Plasma

Membrane

Cytoplasm

Na+, K+ -

ATPase

Na+/H+

antiporter P-glycoprotein EGFR

Chemotherapeutic

agent efflux EGF

β3 β3 β3

K+

Na+

H+

Na+

Results of

T4 action Increased EGF

input to P-gp

[Na+]i

Ca2+

pHi

pHe

+ Calmodulin

T4

Bold blue arrows indicate stimulatory

action of tetrac/Nanotetrac on Na+,

K+ - ATPase, Na+/H+ antiporter and

EGF receptor

T4

SUMMARY

There are multiple implications of

all of these functions of thyroid

hormone recently recognized to

occur on cancer cells and on

angiogenesis.

• Normal thyroid function may

support tumor cell proliferation and

limit effectiveness of

chemotherapy.

• Induced or spontaneous

hypothyroidism may impede cancer

cell function.

SUMMARY 2

• It is desirable to have a specific

pharmacologic antagonist of thyroid

hormone actions at integrin avb3 (=

tetrac, Nanotetrac).

• The affinity of the thyroid hormone

receptor on avb3 is higher for T4

than T3; at physiological concen-

trations, free T4 supports tumor cell

proliferation and cancer cell sur-

vival pathway gene transcription.

• Nonmalignant, hypervascular skin

disorders, such rosacea, may be

thyroid hormone-supported.

COLLABORATORS

Shaker A. Mousa, PhD Albany

Hung-Yun Lin, PhD Albany

Heng-Yuan Tang, MA Albany

Thangirala Sudha, PhD Albany

Faith B. Davis, MD Albany

Murat Yalcin, DVM, PhD Turkey

Sandra Incerpi, PhD Italy

Osnat Ashur-Fabian, PhD Israel

VEGF

Tetrac _ 2.5 1.0

+ + +

A

B

Effect of tetrac on tube formation by human

dermal microvascular endothelial cells (HDMEC).

In this 3-dimensional human microvascular endothelial cell sprouting assay,

cells were mixed with gelatin-coated Cytodex-3 beads and the mixture suspended in

endothelial basal medium (EBM) with 15% normal human serum, mixed and cultured

overnight in a CO2 incubator. The “EC-beads” were then placed in a fibrinogen solution and

thrombin added. After polymerization of the fibrin, EBM and 20% human serum were added

and samples incubated for 24-48 h.

Int Angiol, 2006

Green: phosphoER-a

Red: Nucleoprotein

Control

NCI-H522 cells

Tetrac

Tetrac

+

T4

L-T4

A

B

- ER-a

- GAPDH

ER-a gene expression ER-a gene expression

Re

lative

I.

O.D

.

-0.1

0.4

0.9

1.4

1.9

2.4

Con ICI T₄ ICI + T₄

Rela

tive B

ound D

NA

NCI-H-522 cells

C

0

2

4

6

NCI-H510A NCI-H522

ER-α

IP: anti-integrin-αv

T₄

IgG

- ER-α Promoter

_ +

- Input Control

* *

+ +

Nucleosome ELISA

0

1

2

3

4

5

6

7

BHP 18-21 cells

FTC 236 cells

RV (𝜇M) _ _

_ _ _ 1 10 1 10

+ + +

-Fo

ld I

ncre

ase

in

nucle

osom

e c

onte

nt

A

0

5

10

15

BHP 2-7 cells

BHP 18-21 cells

RV (10 𝜇M)

T4 (10-7 M)

Tetrac (10-7 M) _ _ _ _ + + + +

_

_

_ _ _ + + + +

_ _ _ + + + +

-Fo

ld I

ncre

ase in

nucle

osom

e c

onte

nt

B Nucleosome ELISA

T4 (10-7 M)

5

10

15

20

25

EC

Mig

ration (

RF

U)

x 1

03

Effects of L-T4 or T4 Nanoparticles (NP) on

Endothelial Cell Migration toward Vitronectin (VN)

Cha

mb

er

Upper

Lower

0

0

0 T4 T4-NP

VN VN VN

Boyden apparatus