Pergamon ft. Steroid Biochem. Molec. Biol. Vol. 56, Nos 1-6, pp. 93-98, 1996

Copyright © 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain

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P r o g e s t e r o n e Receptor A and B Prote in Express ion in H u m a n Breast Cancer

J. D i n n y G r a h a m , 1. C h r i s t i n e Yeates , 1 R o s e m a r y L. B a l l e i n e , 1 S u z a n n a S. H a r v e y , i t Jane S. M i l l i k e n , 2 A. M i c h a e l B i l o u s 2

a n d C h r i s t i n e L. C l a r k e I 1Department of Medica,I Oncology, University of Sydney Westmead Hospital, Westmead, N S W 2145, Australia and

eDepartment of Anatomical Pathology, Westmead Hospital, IVestmead, N S W 2145, Australia

T h e h u m a n p r o g e s t e r o n e r e c e p t o r ( P R ) is a l i g a n d - a c t i v a t e d n u c l e a r t r a n s c r i p t i o n f a c t o r w h i c h m e d i a t e s p r o g e s t e r o n e a c t i o n in t a r g e t t i s sues . T w o P R p r o t e i n s , P R A (81-83 k D a ) a n d P R B (116-120 k D a ) , h a v e b e e n d e s c r i b e d a n d d i f f e r e n t p h y s i o l o g i c a l a c t i v i t i e s a s c r i b e d to e a c h on the ba s i s of in vi tro s tud i e s , s u g g e s t i n g t h a t t h e i r r a t i o o f e x p r e s s i o n m a y c o n t r o l p r o g e s t e r o n e r e s p o n s i v e n e s s in t a r g e t cells . P r e s e n c e o f P R in b r e a s t t u m o r s is a n i m p o r t a n t i n d i c a t o r o f l ike ly r e s p o n s i v e n e s s to e n d o c r i n e age n t s . H o w e v e r , t he r e l a t i v e e x p r e s s i o n o f P R A a n d B in b r e a s t c a n c e r h a s n o t b e e n d e s c r i b e d a n d i ts c l in i ca l s i g n i f i c a n c e h a s n o t b e e n a d d r e s s e d . We h a v e e x a m i n e d t h e e x p r e s s i o n o f P R A a n d B in P R - p o s i t i v e b r e a s t t u m o r s a n d f o u n d t h a t wh i l e in m o s t t u m o r s P R A a n d B w e r e e x p r e s s e d in s i m i l a r a m o u n t s t h e r e w a s a b r o a d o v e r a l l d i s t r i b u t i o n o f P R A: B r a t i o s w h i c h d e v i a t e d s i g n i f i c a n t l y f r o m a n o r m a l log d i s t r i b u t i o n w i t h t u m o r s c o n t a i n i n g a P R A : B r a t i o g r e a t e r t h a n 4 b e i n g o v e r - r e p r e s e n t e d in t h e g r o u p . L i n e a r r e g r e s s i o n a n a l y s i s r e v e a l e d t h a t h i g h P R A : B r a t i o s , in g e n e r a l , d e r i v e d f i ' o m a low c o n c e n t r a t i o n o f P R B r a t h e r t h a n h i g h e x p r e s s i o n o f P R A. P R A : B p r o t e i n r a t i o s w e r e n o t c o r r e l a t e d w i t h t he age o f t he p a t i e n t o r w i t h t o t a l P R c o n c e n t r a t i o n . A t h i r d P R p r o t e i n b a n d ( P R 78 k D a ) was d e t e c t e d w h i c h c o m p r i s e d g r e a t e r t h a n 20~o o f t o t a l P R p r o t e i n in a q u a r t e r o f t he t u m o r s a m p l e s e x a m i n e d . T h e c h a r a c t e r i s t i c s o f t u m o r s c o n t a i n i n g P R 78 k D a w e r e n o t d i f f e r e n t f r o m the o v e r a l l g r o u p . In s u m m a r y , in P R - p o s i t i v e b r e a s t t u m o r s t he r a t i o o f e x p r e s s i o n o f P R A ..and B p r o t e i n s is c lose to u n i t y as is s een in a n u m b e r o f o t h e r p r o g e s t i n t a r g e t t i s sues . H o w e v e r , a s i g n i f i c a n t p r o p o r t i o n o f t u m o r s e x p r e s s e d v e r y low leve ls o f P R B a n d a c o n s e q u e n t l y h i g h P R A: B r a t i o . A l t h o u g h the c l in ica l c o n s e q u e n c e o f th i s o b s e r v a t i o n is n o t k n o w n , t h e in v i tro finding.,; t h a t P R A m a y ac t as a r e p r e s s o r o f P R B s u g g e s t s t h a t t u m o r s c o n t a i n i n g p r i m a r i l y P R A m a y i d e n t i f y a s u b s e t o f p a t i e n t s w i t h low o r a b e r r a n t r e s p o n s e to e n d o c r i n e ag en t s .

J. Steroid Biochem. Molec. Biol., Vol. 56, Nos 1-6, pp. 93-98, 1996

I N T R O D U C T I O N

T h e progesterone receptor (PR) (Fig. 1) is a specific and effective mediator of progestin action in target tissues such as the breast and endometr ium and is essential in the complex control of proliferation and differentiation at various stages of development [1]. In the chick and human, PR is detected as two distinct proteins of different molecular weights [2, 3] which

Proceedings of the 12th International Symposium of The ffournal of Steroid Biochemistry & Molecular Biology, Berlin, Germany, 21-24 May 1995.

*Correspondence to J. Dinny Graham. tPresent address: Applied BJLosysterns Ltd, Kelvin Close, Birchwood

Science Park North, Warrington, Cheshire WA3 7BP, U.K.

form dimers and function as ligand-activated nuclear transcription factors [4, 5]. In the human the PR A protein has a mobil i ty of 81 to 83 kDa and PR B of 115-120kDa [6-9]. The two proteins, which differ only in that PR A lacks the first 164 amino acids contained in PR B, are translated f rom distinct m R N A subgroups transcribed f rom a single gene under the control of separate A and B promoters [10].

Although both PR A and PR B bind progestins and interact with progestin responsive elements there is increasing evidence that they are functionally different. In transfection studies the two proteins have different abilities to activate progestin responsive promoters and these differences are p romoter - and cell-specific

93

94 J. Dinny Graham et al.

165 567 633 680 933

[ IDNAI [ li~and ] PRA 1

[ [ ] [ [ PRB 1 165 456 546 595 680 933 i i I I AT( ; I 1

AF3 AF1 AF2

Fig. 1. S c h e m a t i c representat ion o f t h e proges terone receptor (PR) A a n d B p r o t e i n s . D o m a i n s i n v o l v e d in D N A - a n d l i g a n d - b i n d i n g are indicated a n d t h e p o s i t i o n s o f previous ly descr ibed a c t i v a t i o n f u n c t i o n (AF) domains 1, 2 a n d 3 [12, 33]. The pos i t ion o f t h e p o t e n t i a l t r a n s l a t i o n s t a r t f o r a P R C p r o t e i n a t ATGs9 s [30]

is a lso s h o w n .

[11-14], suggesting that cellular responsiveness to progestins may be modulated via alterations in the ratio of PR A and B expression. While PR B tends to be a stronger activator of target genes in many settings, PR A has been shown to act as a dominant repressor of PR B [13, 14] suggesting that high PR A expression may result in reduced progestin responsiveness. Fur ther- more, PR A has been shown to diminish the response of other hormone receptors such as the androgen, glucocorticoid, mineralocorticoid and estrogen recep- tors to their cognate ligands [15-17]. It has also been reported that l igand-bound PR A and B have differing antiestrogenic effects on ER [16-19], possibly via quenching of specific transcription factors required for ER and PR action. This effect is reporter and cell-line specific.

Relative expression of PR A and B is dependent on species and cellular context. Approximately equimolar expression of PR A and B is observed in chick oviduct [2] and human uterus [6] and a similar ratio of expression is seen in human breast cancer cells in culture [8]. In contrast, PR in the rabbit uterus is expressed as a single form homologous to human PR B [20]. In the rodent, PR A expression predominates over PR B in a ratio of 3:1 [21, 22]. The biological import- ance of these different ratios of PR expression has not been extensively explored. Li t t le is known of whether relative PR A and B expression is modulated in v ivo

except in the chick oviduct where alterations in the ratio between the two PR forms during late winter or in aged non-laying animals results in a measurable decrease in PR functional activity [23, 24].

The functional differences between PR A and B which have been described in vitro imply that response to progesterone may be modulated by differential ex- pression of the two PR forms. Clearly, if these observations are borne out in vivo, the balance of expression of the two receptor forms may be critical in the control of progesterone responsiveness in normal and malignant tissue. This is particularly relevant since the presence of PR is an important marker of respon- siveness in estrogen receptor (ER) positive breast tumors [25, 26]. Absence of PR is associated with markedly reduced response to endocrine agents [27]. Given that the two PR proteins may be functionally different the ratio of expression of PR A and B may be as important as the absolute PR level in determining

endocrine response. Although PR expression has been well described in a number of breast cancer cell lines, until recently the level of sensitivity required to indi- vidually quantitate PR A and B in tumors has not been available. We have examined the relative expression of PR A and B proteins in a group of PR-positive human breast tumor biopsies and the results of these studies are reviewed in this paper.

PR PROTEIN E X P R E S S I O N IN B R E A S T TUMORS

We have examined PR expression in PR-positive primary tumors by protein immunoblot. Th e age range and ER status of the patient group are compared in Table 1.

Th e PR A and B protein bands were detected, using monoclonal antibodies to human PR [28], with mobil- ities of 81-83kDa and l l 5 -120kDa , respectively, similar to those observed in the T -4 7 D human breast cancer cell line [8]. Th e PR B protein band was visualized as a group of two or three closely migrating bands (Fig. 2). This was consistent with previous observations in other tissues [6, 9, 28, 29]. Most tumors contained both PR proteins in similar proportions. This result agrees with the equal expression of PR A and B observed in chick oviduct [2], human uterus [6] and cultured human breast cancer cells [8], in contrast to the single PR B-like form in rabbit uterus [20] and predominant PR A expression in the rodent [21, 22]. These different tissues have differing functional requirements for PR in terms of progestin responsiveness, and differential PR A and B expression may be one mechanism by which their distinct physio- logical requirements are fulfilled by this relatively well

Table 1. Age distribution in patient population and comparison to ER status

Age

ER group M i n i m u m Mean + SD M a x i m u m n

Total pop. 18 57.6 + 14.8 90 181 E R - 36 52.5 + 13.0 84 17 E R + 18 58.1 + 14.9 90 164

ER status and age were compared in the populat ion of tumors examined. Data were not available for all t umor samples.

Progesterone Receptor in Human Breast Cancer 95

P R B-----~

P R A - - - - ~

P R 7 8 k D a - - - ~

T - 4 7 D T u m o r

Fig. 2. Representative immunoblot of PR. Expression of PR A, B and PR 78 kDa are indicated in a representative tumor cytosol and compared with detection of PR A and B in the

control P R + T-47D breast cancer cell line.

conse rved b u t d i f fe ren t ly exp res sed p ro te in . I n t e r e s t - ingly , whi le c o m p a r a b l e levels o f P R A and B cou ld be d e m o n s t r a t e d in the m a j o r i t y o f t u m o r samples the re la t ive exp re s s ion of the two p ro t e in s some t imes d i f fered qu i t e c o n s i d e r a b l y and in some cases on ly one P R fo rm cou ld be visrLalized.

Table 2. Natural log distribution of PR .4 : B ratios

Number of Percentage In PR A: B tumors total

- 4 to - 3 1 0.5 - 3 to - 2 6 3.0 - 2 to - 1 19 9.5 - 1 to 0 61 30.3

0 to 1 49 24.4 1 to 2 39 19.4 2 to 3 15 7.4 3 to 4 6 3.0 4 to 5 4 2.0 5 to 6 1 0.5

The natural log values were calculated for the ratio between PR A and B proteins in the overall sample population. The number of samples with In PR A:B falling between the indicated inter- vals are shown.

r e l a t ionsh ip o b s e r v e d be tw e e n In P R A : B and In P R B r e m a i n e d l inear for mos t values , th is r e l a t ionsh ip was lost at low levels o f P R B. T h i s sugges ted tha t h igh P R A : B ra t ios were exp la ined b y low levels o f P R B express ion r a the r t han h igh P R A. F u r t h e r m o r e , it was these samples w h i c h gave rise to the very h igh P R A : B rat ios wh ich were o v e r - r e p r e s e n t e d in the t u m o r popu la t ion .

C O M P A R I S O N O F P R A : B R A T I O TO O T H E R T U M O R F E A T U R E S

D I S T R I B U T I O N O F P R A : B R A T I O S

T h e o b s e r v a t i o n tha t P R A and B had an unusua l p a t t e r n o f exp re s s ion in some t u m o r s was b o r n e ou t b y s ta t is t ica l analysis . Re la t ive express ion o f P R A and B p ro t e in s was ca lcu la t ed in all s amples and the f r equency d i s t r i b u t i o n o f na tu ra l log t r a n s f o r m e d P R A : B rat ios was examined . S ta t i s t ica l ana lys is o f the d i s t r i b u t i o n o f these da ta revea led tha t the values d id no t fall w i th in a n o r m a l log d is t r ibu t ic ,n ( P = 0.023 on S h a p i r o - W i l k s tes t for no rma l i t y ) due to an o v e r - r e p r e s e n t a t i o n o f t u m o r s wi th a h igh P R A : B rat io . T h i s is ev iden t in the f r e q u e n c y d i s t r i b u t i o n ( s u m m a r i z e d in T a b l e 2) wh ich is a s y m m e t r i c a l due to the cohor t o f t u m o r s wi th very h igh P R A : B rat ios . I n the samples e x a m i n e d P R A : B ra t ios fell w i t h i n a b r o a d range o f values , w i th a m e d i a n ra t io o f 1.26.

T h e range o f P R A : B ra t ios w i th in quar t i l e g roups is shown in T a b l e 3 to d e m o n s t r a t e the cons ide rab l e var iance in the o u t l y i n g P R A : B ra t ios obse rved . I m m u n o b l o t s revea led tha t t u m o r s wi th P R A : B ra t ios in the u p p e r quar t i l e g roup gene ra l ly con ta ined low levels o f P R B and this was b o r n e out b y s ta t is t ica l analysis . S p e a r m a n ' s rank cor re la t ion revea led a s ignif i - cant nega t ive r e l a t ionsh ip be tween P R B express ion and P R A : B . L i n e a r r eg ress ion analys is o f the na tu ra l log t r a n s f o r m e d da ta showed tha t , whi le the inverse

T h e r e l a t ionsh ip be tw e e n P R A : B ra t io and o t h e r m e a s u r e d p a r a m e t e r s was examined . As w o u l d be expec ted in a g r o u p o f P R - p o s i t i v e t umor s , a h igh p r o p o r t i o n ( 9 0 % ) were also E R - p o s i t i v e b y enzyme i m m u n o - a s s a y . T u m o r s f rom w o m e n over the age o f 50 years had an average E R concen t r a t i on g rea te r t han in those y o u n g e r t han 50 ( P = 0.0034) a l t hough the over - all d i f ference in age be tw e e n E R + and E R - pa t i en t s was no t s ta t i s t ica l ly s ignif icant ( P = 0 . 1 4 1 5 ) . As d e m o n s t r a t e d in T a b l e 4, the re was no s ignif icant co r re la t ion be tw e e n E R s ta tus or age and P R A : B rat io . T h e r e was a t r e n d t o w a r d s h ighe r P R A : B in t u m o r s f rom pa t i en t s aged less than 50 c o m p a r e d to those g rea te r t han 50 (mean P R A : B = 9.25 vs 3.76, respec t ive ly) . H o w e v e r , th is d id no t reach s ta t is t ica l

Table 3. Distribution of PR .4 : B ratios into quartile groups

Quartile PR A: B range Mean _ SD Median

1st 0.04-0.57 0.33 + 0.16 0.36 2nd 0.58-1.26 0.86 + 0.20 0.84 3rd 1.27-3.88 2.22 + 0.76 2.05 4th 3.89-179.28 19.84 + 32.75 7.55

The tumor population was sorted according to PR A:B values and divided into quartile groups. The range of values in each quartile are shown and the mean + SD and median values indicated.

96 J. Dinny Graham et al.

Table 4. Comparison of In P R ~1 : B ratio with E R status and age

ER status ER positive Mean age Age dist. In PR A:B -ve vs +ve (% total) (years) <50 vs >50

< - 1 2:24 92% 55 8:16 - 1 to 0 6:55 90% 60 18:42

0 to 1 5:44 90% 57 12:28 1 to 3 5:49 91% 57 18:30

>3 2:10 83% 51 5:4

ER status and age were compared to the distribution of In PR A: B ratios. Data were not available for all tumor samples.

significance (P = 0.0621) and further studies would be needed to fully explore any relationship between PR expression a n d menopausal status. The re was no correlation between the overall level of PR expression in tumor cytosols and PR A:B ratio.

D E T E C T I O N OF A THIRD IMMUNOREACTIVE P R B A N D

A third immunoreact ive band (PR 78 kDa) migrat ing ahead of PR A at approximately 78 kDa was detected in a proport ion of tumors (Fig. 2). In around one quarter of the tumors PR 78 kDa accounted for greater than 20% of total PR concentration. T h e distribution of PR A: B ratios in samples containing PR 78 kDa did not differ significantly f rom that of the entire tumor cytosol group and tumors containing PR 78 kDa did not represent a unique subgroup of the overall popu- lation in regard to the other parameters examined, since expression of the protein was not correlated differently with total PR concentration, PR A:B ratio (P = 0.76) or age.

T h e source of PR 78 kDa is not yet clear. The 78 kDa band could not be generated by conditions which would allow degradation of the PR A and B proteins in samples which did not initially contain this PR form.

---- 4 0 0 0 -

3 0 0 0 -

• ~ 2 0 0 0 - 0

1 0 0 0 -

0

P r o t e a s e i n h i b i t o r s

60"C I n c u b a t i o n ( m i n u t e s )

- - 4- - 4-

0 15 15 30 30

Fig. 3. P R 78 k D a is n o t f o r m e d in c y t o s o l s b y h e a t i n g . T u m o r c y t o s o l s a m p l e s w e r e h e a t e d to 60°C f o r 15 o r 30 m i n u t e s in t h e p r e s e n c e o r a b s e n c e o f p r o t e a s e i n h i b i t o r s as i n d i c a t e d . P R B ( f i l led bars ) , P R A ( h a t c h e d b a r s ) a n d P R 7 8 k D a ( u n f i l l e d b a r s ) p r o t e i n s w e r e m e a s u r e d o n i m m u n o b l o t s by

d e n s i t o m e t r y .

As shown in Fig. 3, tumor cytosols heated to 60°C for 15 or 30 min in the presence or absence of protease inhibitors showed no evidence of PR 78 kDa formation with respect to untreated controls although significant degradation of PR A and B was seen in the absence of protease inhibitors. Th is suggested that PR 78 kDa was not an artifact formed during cytosol preparation. There are no possible sites of alternative initiation of translation contained in the N- te rmina l region of the PR gene which would predict a protein of this size. A methionine at amino acid position 595 (indicated in Fig. 1) has been suggested as a possible site of alterna- tive initiation, and this would give rise to a 45-50 kDa protein which has been termed PR C [30]. However , no immunoreact ive band of that size was detected in the tumor cytosols examined. I t is possible that the pres- ence of PR 78 kDa reflects expression of a PR variant. There is evidence f rom breast tumor m R N A studies that several such ER variants may exist [27, 31]. In particular these comprise whole exon deletions ascribed to splicing errors. Fur ther characterization of the origin of PR 78 kDa in breast tumors is currently underway.

DISCUSSION

T h e consequences of high PR A : B ratios on progestin responsiveness in human tissues are not known. However , there is increasing evidence that PR A and B are functionally different. In the chick oviduct relative expression of PR A and B is under seasonal and developmental control. Alterations in the ratio between the two PR forms during late winter or in aged non-laying animals results in a measurable decrease in PR functional activity [23, 24]. Therefore , even in a particular tissue type such as the human breast, control over relative PR A and B expression may be important in modulat ing progestin responsiveness as required at different times in the normal ovarian cycle. Although both PR proteins are able to mediate progestin- activated transcription in transfection studies, they differ in their efficacy and specificity for target genes [11, 12, 14]. The observed differences in PR A and B activity have focused attention on the N- te rmina l re- gion of PR which is unique to PR B. PR mediates progestin regulation through specific activation func- tion domains (AF) contained in its amino-terminal (AF1) and l igand-binding (AF2) domains (Fig. 1) [32]. I t has been postulated that the PR B-unique N- te rmi - nal port ion of PR binds an intermediary factor involved in activation by these domains [12] or is, itself, a third activation function domain (AF3) and that this accounts for the functional differences between PR A and B [33].

PR A has been shown to act as a dominant repressor of PR B function [13, 14] and ligand-activated PR A represses transcriptional activation by co-transfected glucocorticoid, mineralocorticoid, androgen and estro- gen receptors [15-17]. In other settings progestin-

Progesterone Receptor in Human Breast Cancer 97

bound PR B and not PR A has been demonstrated to act as a repressor of ER activity [18]. If these findings are true in vivo then relative expression of PR A and B in breast tumors may strongly influence cellular re- sponsiveness to endocrine agents. Clearly a tumor with an unusual ly high PR A : B ratio, containing almost exclusively PR A, will respond in a different way to endocrine agents than one where PR B predominates. If PR B is the more transcriptionally active of the two PR forms, it could be anticipated that tumors with a high PR A : B ratio may be the least likely to respond to endocrine agents. Work is currently underway to address whether any correlation is seen between PR A : B and other known markers of responsiveness.

In summary, while the majority of PR positive breast tumors contain similar levels o f the two PR proteins, a significant proportion demonstrate low PR B and con- sequently highly unbalanced relative expression of PR A and B. Al though the functional consequences o f this unequal expression are', not known, in vitro evidence suggests that an abnormal ratio of PR expression may be indicative of an impaired or aberrant progestin response. T h e implications of altering PR A : B ratio on cellular responsiveness to progestins remain to be determined.

Acknowledgements--This work was supported by grants from the National Health and Medical Research Council (NHMRC) of Aus- tralia and the University of Sydney Cancer Research Fund. J. D. Graham is supported by an NI-IMRC Dora Lush Biomedical Scholarship and by the Westmead Hospital Research Institute. R. L. Balleine is supported by an NHMRC Medical Scholarship. The authors would like to thank Dr Karen Byth (consultant statistician to the Division of Medicine, Westmead Hospital) for statistical analyses and advice and Dr Wilbur Hughes and the Department of Haematology, ICPMR, Westmead Hospital, Westmead, NSW, Australia for their support.

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