赤尾幸博 岐阜大学大学院連合創薬医療情報研究科

創薬科学

ワーバーグ効果を制御するPKMスプライサーPTB1の癌病態への関与

H28.7.24 JST.東京

SCIENCE 2009

Introduction Warburg effect

Cancer research, 2010

PTB1 associated miRs

エネルギー代謝機構

Table 1

PKM isoforms の組織発現

PKM isoforms のがん細胞における発現

Tissue PKM1/PKM2 ratio Skeletal muscle 28.49507 Brain 4.530955 Heart 3.38437 F-Brain 1.450338 Spinal cord 0.828757 Testis 0.508398 Uterus 0.298704 Lung 0.235336 Trachea 0.133593 Thymus 0.116841 F-Liver 0.112939 Spleen 0.099795 Stomach 0.08402 Bone marrow 0.064059 Small intestine 0.057837 Colon 0.05469 Liver 0.053422 Kidney 0.049505 Placenta 0.041559

**

** p value < 0.001

PTB1 miR-1,133 miR-9,124,137

PKM2 PKM1

High energy demanded organ Brain, Skeletal muscle, Heart

miR-1, miR-124, miR-133b

Eugene, et al. Molecular Cell, 2007 Li-Chun Cheng, et al. Nature neuroscience, 2009

miR-1 miR-133 miR-206

miR-9 miR-124 miR-137

miR-1, miR-133b Muscle-specific microRNAs

0

2

4

6

8

10

12

14

Rel

ativ

e ex

pres

sion

of

miR

-1/R

NU

6B

***

***

miR-1

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Rel

ativ

e ex

pres

sion

of

miR

-133

b/R

NU

6B

***

miR-133b

***

miR-1 and -133b are muscle-specific microRNAs

miR-1 and/or -133b were highly expressed in colon tissue except muscle

0

0.5

1

1.5

2

2.5

3

3.5

4

MiR

-1 re

lativ

e ex

pres

sion

/RN

U6B

***

0

2

4

6

8

10

12

14

16

18

MiR

-133

b re

lativ

e ex

pres

sion

/RN

U6B

**

***

miR-1 miR-133b

0

2

4

6

8

10

12

14

16

MiR

-1 a

nd m

iR-1

33b

rela

tive

ex

pres

sion

/RN

U6B

*** *** *** *** ***

***

***

*

miR-133b miR-1

miR-1 and -133b were frequently down-regulated in clinical colorectal tumor samples and colon cancer cells

The expression of miR-1 and -133b in clinical samples.

Colon cancer cell lines

Colon cancer cell lines

Charactaristic nExpression of

miR-1(↓)case(%)

Expression ofmiR-

133b(↓)case(%)

75 67(89%) 73(97%)

Cancer 51 45(88%) 50(98%)Adenoma 24 22(92%) 23(96%)

Total

Tumor

n Expression of PTB1 (↑) case (%)

Cancer 25 23(92%)

Adenoma 5 5(100%)

N：Normal T：Tumor

case8 N T

case9 N T

case10 N T

case7 N T

β-actin

case1 N T

PTB1

case4 N T

case5 N T

case6 N T

case2 N T

case3 N T

case19 N T

β-actin

case11 N T

PTB1

case12 N T

case14 N T

case15 N T

case16 N T

case17 N T

case20 N T

PTB1

case21 N T

case22 N T

case23 N T

case24 N T

case25 N T

case26 N T

β-actin

case13 N T

case18 N T

case30 N T

case29 N T

case28 N T

case27 N T

PTB1 was highly expressed in colorectal tumor samples

PTB1

β-actin

Cancer samples

Adenoma samples

・・・siR-PTB1-1

3340 3’-UTR 107 1780 ORF

ORF: open reading frame 3’-UTR: Three prime untranslated region

212 GCTTCTGCAGCAAACGGAAATGACA

1857 GAAGTGACCTTAGCAGACCAGAGAT

5’ 3’

・・・siR-PTB1-2

We examined the function of PTB1 by gene silencing PTB1

(48 h)

C C

DLD-1 WiDr

PTB1

β-actin

siR-PTB1 induced growth inhibition in colon cancer cells DLD-1

***

***

0

100

200

300

400

500

600

700

800

24 48 72 96

Viab

le c

ell r

ate

(%) o

f con

trol

h

control

siR-PTB1-1

siR-PTB1-2

*** ***

***

*** ***

***

***

**

**

***

WiDr

Control siR-PTB1-1 siR-PTB1-2

(60h)

Control siR-PTB1-1 siR-PTB1-2

(60h)

0

100

200

300

400

500

600

700

24 48 72 96

Viab

le c

ell r

ate

(%) o

f con

trol

h

control

siR-PTB1-1

siR-PTB1-2

Control siR-PTB1-2 (5 nM)

siR-PTB1 induced a slight apoptotic cell death

00.5

11.5

22.5

33.5

44.5

5Ap

opto

tic c

ell r

ate

(%) o

f con

trol

***

***

***

DLD-1

C (5 nM)

(48 h) WiDr

C (5 nM)

(48 h)

*** ***

**

00.5

11.5

22.5

33.5

44.5

55.5

66.5

Apop

totic

cel

l rat

e

(%) o

f con

trol

Control siR-PTB1-2 (5 nM)

siR-PTB1 induced autophagy in colon cancer cells

DLD-1

siR-PTB1-2

WiDr

siR-PTB1-2

C C

LC3I

LC3II

β-actin

DLD-1 WiDr (48 h)

(48 h)

Autophagy

3MA ULK1

complex

PI3K class Ⅲ

Beclin-1

LC3-1

LC3-2

Lysosome

Fusion

Viab

le c

ell c

ount

Time

Autophasic cell death

Viab

le c

ell c

ount

Time

Autophasic cell survival

Phagopher Autophagosome Autolysosome

Includes cytoplasm or organelle

Breaks down contents

control siR-PTB1-2

DLD-1 (48 h)

0

20

40

60

80

100

120

Varia

ble

cell

rate

(%) o

f con

trol

*** **

LC3I

LC3II

1 0.73 1.31 1.75

β-actin

control siR-PTB1-2

(48 h)

siR-PTB1 induced an autophagic cell survival in colon cancer cells

0

10

20

30

40

50

60

70

80

subＧ1 Ｇ0/Ｇ1 Ｓ Ｇ2/Ｍ

Cell

cycl

e di

strib

utio

n (%

)

Control

siR-PTB1-1

siR-PTB1-2

*

* **

** *

(48 h) WiDr

0

10

20

30

40

50

60

subＧ1 Ｇ0/Ｇ1 Ｓ Ｇ2/Ｍ

Cell

cycl

e di

strib

utio

n (%

)

Control

siR-PTB1-1

siR-PTB1-2

siR-PTB1 induced cell-cycle arrest in colon cancer cells

DLD-1

**

**

**

** **

***

(48 h)

Pre (0 h) Post (24 h)

siR-PTB1 inhibited cell invasion in colon cancer cells

** **

60

65

70

75

80

85

90

95

100

Wou

nd a

rea

rem

aini

ng (%

)

C siR-PTB1-1 siR-PTB1-2 60

65

70

75

80

85

90

95

100

Wou

nd a

rea

rem

aini

ng (%

)

*** ***

DLD-1 WiDr

Control

siR-PTB1-2

Pre (0 h) Post (24 h)

C siR-PTB1-1 siR-PTB1-2

E-cadherin

C C

β-actin

DLD-1 WiDr

Down-regulation of E-cadherin contributed to inhibition of cell migration

E-cadherin

(48 h)

Gl ucose

PEP

Pyruvat e

Lact ate

Gl ucose

PEP

Pyruvat e

Lact ate

Bi osynt hesi s

TCA cycl e

Hi gh act i vi ty

TCA cycl e

Low act i vi ty

Bi osynt hesi s

８ 10 11 ８ ９ 11

PKM１ PKM２

Di f f erent i ated Cel l s Tumor Cel l s

ON OFF

PTB1

ROS

miR-1 miR-133b

siR-PTB1 induced switching PKM isoforms expression from PKM2 to PKM1 and affected the Warburg effect

PKM1

PKM2

Control siR-PTB1-2

Two upper pictures Green：PKM1 , Red：cytoskeleton , Blue：nucleus Two lower pictures Green：PKM2 , Red：cytoskeleton , Blue：nucleus

(48 h)

DLD-1

Western blotting Immunochemistry

(48 h)

C C

DLD-1 WiDr

PTB1

β-actin

PKM1

PKM2

Gl ucose

PEP

Pyruvat e

Lact ate

Gl ucose

PEP

Pyruvat e

Lact ate

Bi osynt hesi s

TCA cycl e

Hi gh act i vi ty

TCA cycl e

Low act i vi ty

Bi osynt hesi s

８ 10 11 ８ ９ 11

PKM１ PKM２

Di f f erent i ated Cel l s Tumor Cel l s

ON OFF

PTB1

ROS

miR-1 miR-133b

-1500

-1000

-500

0

500

1000

1500

miRs-1 and -133b increased oxidative stress estimated by ESR

Control miR-1

ｖ

miR-133b

ｖ

NAC canceled a part of apoptosis and autophagy induced by siR-PTB1

(48 h)

DLD-1

PTB1

ROS

siR-PTB1 significantly decreased Lactate production

(48 h)

C siR-PTB1-2

-50

0

50

100

150

200

250

300

350

400

450

Tum

or v

olum

e (m

m3 )

系列1

系列2

0 10 12 14 17 19 21 24 days

* ** **

Control

siR-PTB1-2

control

siR-PTB1-2 PKM1

PKM2

β-actin

PTB1

siR-PTB1 topical injection exhibited anti-tumor activity and switched the expression of PKM isoforms in tumor samples in vivo

(24days)

Gl ucose

PEP

Pyruvat e

Lact ate

Gl ucose

PEP

Pyruvat e

Lact ate

Bi osynt hesi s

TCA cycl e

Hi gh act i vi ty

TCA cycl e

Low act i vi ty

Bi osynt hesi s

８ 10 11 ８ ９ 11

PKM１ PKM２

Di f f erent i ated Cel l s Tumor Cel l s

ON OFF

PTB1

ROS

miR-1 miR-133b

Colorectal Tumor

ROS

PTB1

Autophagy

miR-1 miR-133b Oncogene

Promotion of TCA cycle

PKM1 PKM2

PKM2 PKM1

Promotion of glycolysis

PTB1

miR-1 miR-133b Oncogene

Introduction of miR-1 and -133b

Conclusion

Inhibition of cell migration

Colorectal Tumor

PTB1 associated miRs (miR-1,124,133b ,…)

Modulate Warburg effects

ROS

PTB1

Apoptosis and/or Autophagy

PTB1

Cell cycle arrest

① PTB1はがん細胞に比較的高発現していることから 標的分子になり得る。これまでの分子標的薬とは異なり、がんのエネルギー代謝を標的にしていることから代償機構を誘導しにくい。 ② PTB1が高発現のがんにおいてはVEGFを標的にした薬物が

効果的である可能性があり、使用適応のバイオマーカーになる可能性がある。

PTB1を標的にした創薬、バイオマーカーの利点・欠点

新技術の特徴・従来技術との比較

本技術に関する知的財産権 •発明の名称 ：抗がん剤 •出願番号 ：特願2014-127560 •出願人 ：岐阜大学、シーシーアイ株式会社 •発明者 ：赤尾幸博、村瀬博宣

今後の研究 現在、バイオインフォマティックスによるPTB1のRNA-binding 領域と特異的に結合する 低分子化合物候補を検索し、その活性を検証中である。---------共同研究を

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