Over-expression of facilitative glucose transporter genes in human cancer

Vol.

170,

No.

July

16,

1990

1, 1990

BIOCHEMICAL

BIOPHYSICAL

AND

RESEARCH

COMMUNICATIONS Pages

OVER-EXPRESSION TRANSPORTER

223-230

OF FACILITATIVE GLUCOSE GENES IN JIUMAN CANCER

Taizo Yamamoto': Yutaka Seinol, Hirofumi Fukumoto2, Gyohan Kohl, Hideki Yano2, Nobuya Inagaki2, Yuichiro Yamada2, .Kazutomo Inoue3, Tadao Manabe3, and Hiroo Imura 2 'Division of Metabolism and Clinical Nutrition, 2 Second Division, Department of Medicine, and 3.First Department of Surgery, Kyoto University School of Medicine, Kyoto 606, Japan Received

M.sy 31, 1990

SUMMARY The expression of five facilitative glucose transporter genes, GLUT1 (erythrocyte type), GLUT2 (liver type), GLUT3 (brain GILUT4 (muscle/fat type), and GLUT5 (small intestine type), type), was examined in human cancer tissues of the digestive system by RNA blotting analysis. The amounts of the GLUTl, GLUT2, and GLUT3 transcripts were elevated in most cancer tissues studied, although the expression of the GLUT2 gene is primarily restricted to the liver. On the other hand, mRNA levels of GLUT4 and GLUT5 were below sensitivity in all cancer tissues examined. These results ,suggest that over-expression of GLUT1 and GLUT3 might be closely related with tissue development and that the acceleration of glucose uptake by transformed cells may result, at least in part,

from

the

transporters.

It is

is well

for

Pre**, Inc.

the

that

the

expression

2).

However,

glucose

of

the

uptake

of

proliferation

by an acceleration

(1,

increased

in

known

accompanied

metabolism

increase

0 1990Academic

these

of

glucose

molecular

two

transformed

uptake

cells

and its

mechanism

by transformed

glucose

responsible

cells

remains

unclear. Recent

transport

studies

across

structually-related glucose

transporters

have

the

demonstrated

plasma proteins have

that

membrane (3-7).

facilitative

is mediated

glucose

by a family

of

These human facilitative

a distinct

tissue

distribution

and

XTo whomcorrespondence should be addressed. 0006.291x/90$1.50 223

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

BIOCHEMICAL

170, No. 1. 1990

contribute

to

the

mRNAs encoding most

adult

in

and in

observed It

transporter

insulin

responsive

upper

in various

human

(5,8).

GLUT2

in

liver

such

highest

levels is

the

(4).

GLUT4

as skeletal

levels

in

is

muscle

of

GLUT5 mRNA

of

these

(7). how the

regulated the

therefore,

conditions:

at various

placenta

and the

varying

present

tissues

jejunum

is

under

expressed

to study

genes

examined,

(6),

important

transporter

genes

fat in

is

glucose

term

glucose

and subcutaneous are

of

GLUT1 and GLUT3 are

tissues

predominant abundant

disposal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

expression

transformed

cells.

We have

of

glucose

transporter

mRNA levels cancer

glucose

five

tissues.

Materials

and Methods

Tissues

and RNA extraction RNA was isolated from human cancerous and normal tissues by the guanidium thiocyanatelcesium chloride method (9). These tissues were surgically removed from human esophagus, stomach, colon, liver, and pancreas. RNA blotting analysis Twenty pg of total RNA was denatured with glyoxal and, after electrophoresis through a 1.0% agarose gel, transferred to a nylon filter. The filter was hybridized with cDNA probes labeled by nick translation. The hybridization conditions have been described previously (5). The intensity of the autoradiographic spots was quantified by densitometric scanning. Probes The probes used were: GLUT1 cDNA, the 1396-bp EcoRI-Hind111 insert from phGT2-1 (10); GLUT.2 cDNA, the 1395-bp EcoRI insert from AhHTL-14 (4); GLUT3 cDNA, the 2190-bp EcoRI insert from phMGT-31 (5); GLUT4 cDNA, the 1730-bp EcoRI insert from phJHT-3 (6); GLUT5 cDNA, the 1890-bp EcoRI insert from XhJHT-5 (7); and TGF-f3 cDNA, the 1007-bp PstI-BamHI insert from phTGF-B-2. Total

Results

2.8

RNA blotting

analysis

kb in normal

esophagus,

tissues our

examined,

assay

expressed cancer elevated

while liver

at higher

levels

in

examined. l/l

revealed

a single

GLUT1

trancript

colon,

and all

cancer

stomach,

GLUT1 mRNA was

in normal

(5/5)

and Discussion

and pancreas. in

all

The levels

esophageal

224

l/2

the

sensitivity

of

The GLUT1 gene was

hepatoma of

cancer,

below

of

(212)

and pancreatic

GLUT1 mRNA also gastric

cancer,

were and 2/3

Vol.

170, No. 1, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

.a; 28Su9 zas-

18S-

* 18S-

01

02 Figure system. Liver

1. Expression of Positions of 285 metastasis indicates

Figure 2. Positions

Expression of 28s and

of 185

GLUT1 mRNA in human cancer of digestive and 18s ribosomal RNA are indicated. liver metastasis of colon cancer. GLUT1 mRNA in human pancreatic ribosomal RNA are indicated.

cancer.

28s-

18s-

Expression of Figure 3. system. Positions of 28s Liver metastasis indicates

GLUT2 mRNA in human cancer of digestive and 18s ribosomal RNA are indicated. liver metastasis of colon cancer. 225

Vol.

170,

No.

1, 1990

colon

cancer

(Figure

three

transcripts

liver

metastasis

of of

GLUT2 mRNA in (Figure

3).

were

The

higher

esophagus, GLUTl,

present

1 and 2).

The

GLUT2 cDNA probe

5.4,

and 2.8

than

were

the

levels of

show

that

elevated of

the

almost

in

normal amounts

most

GLUT2 gene

cancer is

hybridized normal

in

normal

4.1

the

restricted

4).

GLUT2, studied,

5).

Our

and

GLUT3

although

primarily

to

285

18s

18S-

05 4. Expression of GLUT3 mRNA in human cancer of digestive system. Positions of 28s and 18s ribosomal RNA are indicated.

Figure Liver

metastasis

Figure 5. Positions

indicates

Expression of 285 and

liver

metastasis

of

colon

of GLUT3 mRNA in human pancreatic 18.5 ribosomal RNA are indicated.

226

Like

all

(Figure

GLUTl,

tissues except

(Figure in

pancreas

kb

normal

tissues

observed

tissues

2.7

cancer

same

of liver

and

all

normal

to

liver,

levels

except in

of

COMMUNICATIONS

The

to

respective

the in

in

those

transcripts

were

than

only

tissues

GLUT3 mRNA were

(5/5)

findings

than

cancer

the

kb

hybridized

GLUT3

in

RESEARCH

and hepatoma.

higher

and

of

those

cancer

expression

cancer,

normal

levels

are

3.4,

colon

amounts

higher

transcripts the

all

where

pancreatic

BIOPHYSICAL

GLUT3 cDNA probe

in

pancreas.

AND

hepatoma

The

transcripts

BIOCHEMICAL

cancer. cancer.

the

Vol.

170,

No.

liver. rat

Werner heart

that the

1, 1990

et

and liver

they

are

cDNA libr,ary,

to

and GLUT5 do not because

and were

in not

Recent factor-$ the

Since

genes

of

the

play

specific

detectable

in

study

(TGF-8)

epidermal

has

growth

in

factor

the

cancer

any

It

these in

cancer

tissues that

(EGF)

study

role

uptake receptor

the

human

been

seems in

the

might

result

likely

transporters

respective

tissues

examined. transforming

growth

by co-activation kinase

system

of --in

18S-

Figure 6. Expression of TGF-6 mRNA in human cancer of digestive system. Positions of 28s and 18s ribosomal RNA are indicated. Liver metastasis indicates liver metastasis of colon cancer. 227

that

cell

glucose

the

in

development,

present

manner

glucose

muscle

present

tissue

tissues.

encoding

Although

skeletal

and GLUT3 have

with

but

stage.

fetal

in

stage,

postnatal

GLUT1

demonstrated

stimulates

the

an important

mRNAs

a tissue

embryonic

GLUT3 mRNA are

related

these

growth

proliferation,

(5).

COMMUNICATIONS

GLUT1 mRNA levels

the

a human of

RESEARCH

that

at

from levels

BIOPHYSICAL

at

decreased

be closely of

rapid

expressed

increased

low

muscle

over-expression

GLUT4

shown

have

isolated

very

demonstrated

the

(11) are

is

skeletal

from

AND

markedly

GLUT3 cDNA

adult

al.

BIOCHEMICAL

vitro ---

Vol.

BIOCHEMICAL

170, No. 1, 1990

ESOPHAGUS

STOMACH

AND BIOPHYSICAL

T

COLON

RESEARCH COMMUNICATIONS

LIVER

-1 PANCREAS

GLUT 1 l-l

+t +i n-LM

GLUT 2

-

i -i 1

GLUT 3

nm TGF-P

M nLm 00

n Number of tissue

Figure 7. Comparison of gene expression and TGF-6 in human cancer of digestive each mRNA is expressed relative to the having the highest level of RNA. Open column: normal region Closed column: cancerous region M: liver metastasis of colon cancer f-: no expression detected

r

PANCREAS

GLUT

of glucose transporters system. The abundance of amount present in tissue

PANCREATIC CANCER

1

+ + + + + GLUT

2 r

+

i

+

I

Comparison of gene expression Figure 8. in human pancreatic cancer. The abundance expressed relative to the amount present level of RNA. Open column: normal region Closed column: cancerous region t: no expression detected 228

of glucose transporters of each mRNA is in tissue having the

highest

BIOCHEMICAL

Vol. 170, No. 1, 1990

(12).

Therefore,

TGF-B

gene

cancer

we have

in

cancer

tissues

suggesting GLUT3 genes product.

but

determined, glucose

found

transporter closely

increased

precise

present gene related glucose

of

of TGF-8

the

that

of GLUT1

TGF-B

the

in

and/or gene

glucose

in Figure

of

the

and GLUT3,

of GLUT1

of

oncogenes

of

mRNA levels

activated

expression

several

with

expression

expression

summarized

of

RESEARCH COMMUNICATIONS

to that

the

gene

no significant

the the

are

in

the

is

and src)

Although

the

of

expression

N-ras,

which

increase

and TGF-B the

Ki-ras, genes,

6) was similar

A comparison

compared

the

The change

may be a consequence

transporters

to

the

examined

tissues.

(Figure

that

also

AND BIOPHYSICAL

7 and 8. We also (myc,

glucose

Ha-ras, transporter

correlation.

mechanism results

suggest

expression, with uptake

still

remains that

such

tissue

the

to be induction

of

as GLUT1 and GLUT3,

development,

by transformed

may contribute cells.

Acknowledgments

The assistance of Hiroko Tachikawa in the preparation of this manuscript is gratefully appreciated. We would like to thank Dr. G.I.:Bell for providing the cDNA clones of GLUTl-5 and TGF-B, and Dr. S.Seino for his helpful advice during the course of this study as well as his comments on this manuscript. This work was supported by Grand-in-Aid for Special Project Research on Cancer Bio-Science and Scientific Research 02671093 from the Ministry of Education, Science, and Culture of Japan and by Takeda Medical Research Foundation.

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