Relationship between estrogen receptors, 17β-hydroxysteroid dehydrogenase and estrogen content in human breast cancer

217

2471

RELATIONSHIP

BETWEEN ESTROGEN RECEPTORS, 17B-HYOROXYSTEROIO DEHYDROGENASE AND ESTROGEN CONTENT IN HUMAN BREAST CANCER Y.J.

College

Received

Abul-Hajj

of Pharmacy, Minneapolis,

University Minnesota

of Minnesota 5545.5

4-l-79 ABSTRACT

Estrone and estradiol levels in tumor tissue cytosols were determined in 11 premenopausal and 20 postmenopausal women at the same time that 17S-hydroxysteroid dehydrogenase and estrogen receptors (ER) were carried out on their breast cancers. Estrogen receptor positive tumors showed significantly higher levels of estrone and estradiol. However, al 1 ER negative tumors contained measurable amounts of both estradiol and estrone. Higher levels of estrone were observed in ER negative tumors which correlates well with high 17D-hydroxysteroid dehydrogenase activity. These results suggest that false negative receptor assays in the premenopausal women is not 1 ikely to be due to occupancy of receptors by endogenous Furthermore, the higher estrone content in the ER negative estrogens. group is probably due to high 17S-hydroxysteroid dehydrogenase activity inherent to these tumor cells. INTRODUCTION Recent plasmic in

estrogen

have

total

in

occupancy the

(I-5)

women

receptor

are

receptors

premenopausal

patient

several plasma

Even

though

Volume

34,

studies estrogens

Sahadevan

Number 2

that

the

much

lower

.

McGuire

concentrations

of

However , between

shown

receptors

postmenopausal

variations

in

results

and

(7-10) and

et --

by

al

s

high may

(11)

and

TDROXDSJ

in et --

of

available

to

little

to

that

a partial

endogenous negative

or

no correlation

et --

in

breast

al

(12)

or

estrogens

false

receptors Fishman

women than

(6) suggested

al

circulating lead

cyto-

premenopausal

may be related

showed estrogen

levels

results.

cancer.

showed that

August, 1979

6):

218

estrogen

receptor

estrogens proposal for

low

in

tumor

that

high

receptor

Since

of

analyzed

the to

estradiol

dehydrogenase

levels

of

receptor

observed

results

endogenous

higher

levels

do not

support

estrogens

negative

tumors

activity

than

variations

estrone

may be

relation activity

to

determined

estrogen of

the

in

showed estrogen

receptor

dehydrogenase

were

MATERIALS Steroids: estrone Boston, Wilton,

contained

these

17S-hydroxysteroid and

in

cytosols,

dehydrogenase

(13),

be related

tumors

of the responsible

measurements.

estrogen

hydroxysteroid tumors

positive

TDROXDS

in

receptors tumor

higher receptor

positive

concentrations activity.

tumor and

17B-

tissue

The

may levels

cytosols

and

17S-hydroxysteroid

samples.

AND METHODS

[2,4,6,7-3Hlestradiol (100 Ci/mmole) and [2,4,6,7-3H] (100 Ci/mmole) were purchased from New England Nuclear, Non-radioactive steroids were obtained from Steraloids, Mass. N.H. and checked for purity before use.

Specimens of primary breast cancers were obtained at the Tumors : timeof mastectomy, dissected free of fat and weighed. Fresh tissues were used immediately for 17B-hydroxysteroid dehydrogenase activity For estrogen receptor determinaand rad ioimmunoassay of estrogens. tissues were frozen immediately in liquid nitrogen. tion, Estrogen receptor and lj’S-hydroxysteroid dehydrogenase assays: The procedure for measuring 17B-hydroxysteroid dehydrogenase is similar to The cytosol estrogen receptor determinthat desc.ribed earl ier (13). ation was carried out using a sucrose gradient method as described The results are based on 8s type receptors and previously (14). expressed as femtomoles (fmol) of 3H-estradiol specifically bound per Receptor contents of 5 fmol/mg or over milligram of cytosol protein. in premenopausal and 10 fmol/mg or over in postmenopausal were The whereas below these levels as negative. designated as positive, protein concentration was determined by the method of Lowry -et al (15)

*

Determination of estrone and estradiol in tumor tissue cytosols: The measurement of estrone and estradiol was carried out using highly specific antiestradiol and antiestrone antisera supplied to us by Dr. Cross-reaction of estrone S.E. Davis, National Institutes of Health.

S Table

Tumor

Estrogen estrone

1.

TDR.OXDCI

17D-hydroxysteroid dehydrogenase and content in human breast cancers

receptors, and estradiol

l-/S-HSDH” % Convers E2 to El

Menopausa 1 Status & Age

Pi-e-48 Pre-44 Pre-46 Pre-51 Pre-48 Pre-35 Pre-43

a 9

14 15 16 17

20 21 22

23 24 25 26 27 28 29

17f3-HSDH El

Post-a3

52.7

13 27.4 46.2 21.6 16.5

a

(pg/ml

21 37

Post -66

11

Estradiol

(pg/ml

37 27.5 51 0.5

11.4

Pre-50 Pre-53 Pre-37 Pre-49

la 19

Est rone

49 72

95 48.7

Post-55 Post-70 Post-55 Post-54

13

ion

38.8 59.3

62

Post-61 Post-63 Post-50

10 11 12

219

4.3 2.7 7.j

I

22 61

I

63 a8 47 74 28 286 93

78

zz

38 103 99 142 69 70

17

17 21

22

2: la

22

12

62

33

9 0

t:

:z 47 71 34 a

3: 75 13 :; 48 66 47 172 142 161 111

Post -63 Post-66 Post -54 Post-77 Post-57 Post -72 Post -78 Post -59 Post -70

2:

5.7 0 0 0

2

2.7 0

::

Post-a9 Post-59

:83 108 117 97 47 a0 32 126

107 162

77 a.3

:;

6;

=

= estrone;

48

17B-hydroxysteroid E2 = estradiol

dehydrogenase;

with antiestradiol antiserum was less than 6% and that of estradiol with the antiestrone antiserum was less than 5%. Steroids were extracted from tumor cytosols (l/4, W/V) with 3x5 ml diethyl ether and no purification procedures were incorporated in the assay since results, with and without chromatography, showed no significant difference. Free and bound steroid were separated using dextrancoated charcoal. Recoveries of steroids ranged from 86-103% and duplicate samples agreed to within 2 14%. RESULTS Table steroid

1 shows

dehydrogenase

tumor

cytosols

receptors

1 shows the

was

1 and

found

significantly

and

estrogen

levels

breast

of

receptors, estrone

cancers.

17B-hydroxy-

and

The

dehydrogenase

to

60+38

negative

pg/ml mean and

in

be 41+20

(?SD)

estradiol

data

for

in

estrogen

activity

different

2.

(P=O.Ol)

ER+ tumors, for

estradiol

for

the

ER-

between

the

was

tumors,

with

levels

59’31

The

mean

for

the

pg/ml These

reported

premenopausal

and

a mean

estrone

and

values

of

estrone 64t30

Significant showing respectively.

and

were 40flg

of

not

difference

of

112276

respectively. in

the

menopausal

differences values

are

groups,

of

for

premenopausal

level

estradiol

irrespective

in

postmenopausal

(+SD)

postmenopausal

estradiol

level

levels

a significant

for

ER+ tumors,

and

However,

levels

(P-0.01)

estrone

1 groups.

showing

of

respectively.

and

the

pre-and

levels

The

both

respectively. (P=O.l).

positive

Figure

and

was observed, for

of

postmenopausa

groups,

levels

(P=O .005)

The

distribution

different

estradiol

shown

the

31 human

postmenopausal

groups

and

of

17B-hydroxysteroid

premenopausa

estrone

in

results

(13).

Figure

and

in

and

previously

the

the

ER

status,

is

significantly pg/ml

for

were 53+27

ER-

also

and

and

observed

93?47

pg/ml

S

221

TElROLDb

7 :

A

.

:

! F

!-

Estradiol Figure 1. content in preand human breast cancer

The be 48218

data

in

pg/ml

in

postmenopausal 44+20

pg/ml

between

(P=O. 15))

while

premenopausal

97+36

cancer. pg/ml,

group, the

was

found

to

the

distribution

The

(%SD)

mean

estradiol

ER-

the

level

of

levels

There

ER-

pg/ml

in

was

of

group,

and

and

groups and

pg/ml

levels with

found for

in

primary to

be

the

ER+ premenopausal

ER-

ER-

no significant

estradiol

was

There the

the

P~O.025.

patients

74*37

to

group

postmenopausal

estradiol

respectively. both

estrone

premenopausal

means

pg/ml , and

in

of

significant,

and

postmenopausal

group,

I

ER+ premenopausal

and

be very

levels

postmenopausal

147227

&ml,

1

i

:

78Q3

group.

between

pre-and

group,

in

in

(!ISD)

ER+ postmenopausal

difference

ER+ postmenopausal

difference

pg/ml

negative

44t25

premenopausal and

the

and

mean

premenopausal

30+15

the

4 shows

ER positive

the

ER+ postmenopausal

group

Figure

breast

the

3 shows ER-

group, in

difference

the

.I

Figure 2. Estradiol and estrone content in ER+ and ER- human breast cytosol s. (A) Data point not included in calculation

and estrone postmenopausal cytosols.

Figure

2

T

::

ERgroup

was a significant ER+ premenopausal

S

222

T=EIROIDS

. I--*:

,., d

.

:

c /m

.

:

.

7

:

I

,

2.

*

.

*

.

.

t

v is-~

EW

O?E.

postmenopausal

groups

z/*

NC

P,‘P

Estradiol content in Figure 4. ER+ and ER- premenopausal and postmenopausa 1 human breast (0) Data point cancer cytosols. not included in calculation.

Figure 3. Estrone content in ER+ and ER- premenopausal and postmenopausal human breast cancer cytosol s

and

/r.

showing

a P value

of

~0.05

and


respectively. DISCUSS ION Although study is

our

only

results

tumor

content

is

groups. of

et --

compared

to

the

shown

(11).

of

status, for ER-

in

both tumors.

tumor

Figure

However,

data

patients

shows

cytosol

the

1.

receptor

These

and

have

content

a significant

estrone

Higher

the

results

are

in similar

the

content

estrogen

content

been

tumor

(Fig.

this

to

also

pre-

in

compared

and

the

reported

estrone postmenopausal

Z),

difference

estradiol

used

estradiol

endogenous in

been

group

patients

different by

have

premenopausal

premenopausal

significantly

Analysis

content

in

as

in al

not

menopausal

tumor

group

of

show that

higher

cytosols

by Sahadevan

number

clearly

significantly

postmenopausal in

a small

irrespective

in

endogenous

ER+ tumors to

data

obtained

S by

Fishman

et --

different

for

difference

to

on

shown

estrone

to

higher

subjects

are

both

ER+ and

data

compared

Furthermore,

lower to

attributed

the

ER-

estrogen binding estradiol

receptor

levels

the

the

receptors

activity

interesting

to

dehydrogenase fact

that

that

receptor

in note

estradiol

results.

sites

The

of

tumor that

to

at

At31 (162 tumor

and content

Similar

is

#6

found

is

saturated

very

observations

while

very

to

may be

ER+ premenopausal

be

in

protein).

these were

this levels

between in of

measuring of

the

It

is

the

highest

17b-hydroxysteroid

ER negative. in

in

activity

in excess

low

activity

concentration

medium

the

as

competition

the

times

high by

patients

in

fmoles/mg has

1 and in

dehydrogenase

200

content

observed

estradiol

of

(13).

premenopausal

differences

incubation

least

tumors

These

because

the

ER-

postmenopausal

possibility

unlikely,

added

are

ER-

but

been

Table

are

we

has

dehydrogenase

17@-hydroxysteroid very

in

premenopausal

group.

binding

shown

the

results

estradiol

estradiol

ERin

the

tumor

17@-hydroxysteroid

was

activity

of

the

ER+ and in

no

content

which

for

as

these

receptor

activity

are

showed

of

on

endogenous

found

tight

is

pmoles)

content

only

tumors

to

tumors.

and

1 ight

estradiol

levels

are

high

estradiol

dehydrogenase

a higher

compared

to

as

(100

of

but

investigators

different

higher

postmenopausal

to

not

ER+ postmenopausal

partly

as well

based

content

In

tumors.

premenopausal

as

estradiol these

levels in

ER-

223

dehydrogenase

reflected

significantly

negative

ER-

be significantly

ER+ premenopausal

the

the

to

where

circulating

ER+ and 4.

group

regard

content

analyze

earlier

Figure

with

17b-hydroxysteroid

The

in

(12)

between

attempted also

al

TEIECOIDS

tumor, leading

reported

In

view

it

is to

of

the

possible false

by Maass

et --

al

S (7)

on

exceed

the

absence

menopausa higher

though

levels

of

premenopausal

48+18

and

and

estrone

were

higher

to

The

the

observed

activity

in

these

obtained receptor

from

the

be a consequence

levels

of

estrone

of

hydroxysteroid

dehydrogenase

activity

droepiandrosterone produce tumors.

aromatization that

that

these

Whilst

the

physiological

in

the

(+SD)

to

the

ER-

to

ER-

the

levels

preof

which

high

17B-hydroxy-

postmenopausal

could

clearly

account

group for

not

demonstrate

related

the

tumors

with

capable

tumors

were

significance

high

endo-

detect

high

support

observed

androgens

to

that

receptors

by endogenous

Further

tumors

to

fa i 1 ing

of

earl

primarily

able

ERthe

high

17Sier

tumors.

aromatizing were

of

high

our

by mammary

steroids.

17B-

for

from

(3S-hydroxyandrost-5-en-Ii’-one) and

compared

ER+ postmenopausal

the

in

is of

80% of

estrone

slightly

significant

saturation

observed activity.

showed

in

are

dehydrogenase

studies

due

study

hydroxysteroid

These

mean

and

post-

significant

levels

Therefore,

to

on the

ER-

show

as

However,

group,

this

content.

were

study

in

tumors.

likely

(16)

levels

higher

Furthermore,

concentrations

estradiol

this

showing

present

is

studies

estradiol

are

estrone

primarily

tumor

Higher

3.

the

between

genous not

Figure

groups

are

estrone from

respectively.

content

in

in

ER+ postmenopausal

results

variations

plasma

ER+ postmenopausal

between

differences

estrone

the

shown

observed

of

obtained

in

as

dehydrogenase

compared

results

pg/ml,

These

steroid

with

levels

postmenopausal

78f33

differences groups.

the

group were

menopausal

patients

circulating

1 women,

differences

ER in

pg/ml .

i ng 300 Even

of

TB&OXD6

dehyto

postmenopausal levels

of

S 17B-hydroxysteroid it is possible enzymes

may

hormones growth,

dehydrogenase that

play

which

this

enzyme,

a significant

leads

development

in ER negative as well role

to a disturbed and

225

TElROIDb

hormonal

tumors

as other

in the

dependence

steroid

control

hormonal

is not

milieu

metabolizing

of active and

of mammary

clear,

may

steroid affect

the

tumors.

ACKNOWLEDGEMENT This investigation was supported in part by Public Health Cancer Institute. The Service Grant CA 20530 from the National us with the antiauthors wish to thank Dr. S.E. Davis for providing estrogen antisera. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

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