Criticai Reviews in Oncology]Hematology , 1992; 12:9 1~I49 0 1992 Elsevier Science Publishers B.V. A11 rights reserved 1040-8428/92/$15.00

ONCHEM

91

00019

Hormone receptors in malignancy David N. Danforth Jr. Surger.v Branch, National Cancer Institute, (Accepted

National Institutes 23 October

of Health. Bethesda, MD, USA

1991)

Contents I. Introduction......................................... II. Structure

and function

of hormone

91

......................

B. Estrogen

......................

receptor

E. F.

G.

A. Breast cancer.

of hormone

J.

Tumors

D. E. F. ci. H.

94

receptors

97 98

...

.....

100 101 102

in malignancy

103 103

I.

C. Ovarian

93

.............

..... ........ Cervical carcinoma. ........ Leukemia. ................ Lymphoma ............... Prostate carcinoma. ........ Rena1 cell carcinoma ....... Melanoma ................

B. Endometrial

.

.,........_

receptor

III. Clinical characteristics

.. ..

93

.................. Glucocorticoid receptor. ................ Androgenreceptor..................... Epidermal growth factor receptor ........ Prolactin receptor, .....................

C. Progesterone D.

.

..

receptors.

A. Steroid receptors.

carcinoma

115

carcinoma.

120

.. ..... ..

124 125

.

.... .. .

126 127 128 129 130

of the head and neck

............... L. Soft tissue sarcomas. ....... M Gastric carcinoma ......... N. Colon carcinoma .......... K. Lung cancer

0.

Hepatocellular

P.

Esophageal

carcinoma. carcinoma.

131 131 132

....... .. .

.. .....

133 133

,.........

134

References.............................................................................

Introduction

Hormone receptors are cellular proteins which mediate the effects of plasma hormones on cell growth and metabolism. These receptors may be located intracelluDavid N. Danforth

Jr. received

of New Mexico, Albuquerque, Northwestern ior Investigator, Bethesda,

MD.

University, Surgery

his M.S. degree

from the University

New Mexico and his M.D. degree from

Chicago, Branch,

IL. Dr. Danforth National

Cancer

is presently Institute,

134

larly, as in the case for steroid hormones, or on the cell membrane as for prolactin, epidermal growth factor, and others. Hormone receptors are present in cells of many malignant tumors as well as normal tissues. The structure and function of receptors in malignant tumors appears to be comparable to that in normal cells. The presence of hormone receptors in malignant tumors, therefore, suggests these tumors might also respond to

SenNIH,

Correspondence:

D.N.

Danforth

Bldg IO. Rm 2838, Bethesda,

Jr.,

M.D..

MD 20892, USA.

Surgery

Branch,

NCI,

92

the addition or withdrawal of the appropriate hormone. In 195 1 Huggins and Bergenstal [I] reported that adrenalectomy inhibited human mammary and prostatic cancers. This indicated that not only were these malignancies sensitive to the hormonal environment, but that manipulation of the hormonal environment could represent effective treatment for these tumors. Since that time, receptors for other steroidal as well as nonsteroida1 hormones have been identified in malignant tissues. It is not uncommon for a given tumor to contain receptors for several, or even many, hormones. Human breast cancer, for example, may contain receptors for estrogen, progestin, glucocorticoids, androgens, prolactin, as well as many of the growth factors and even cytokines. Each receptor, when bound with the appropriate hormone, exerts its own set of effects on cell metabolism. While the behavior of the tumor might be analyzed in terms of one or two receptors, the net state of growth and metabolism is probably the sum total of a variety of different hormonal effects. Our understanding of the structure and function of hormone receptors has advanced tremendously in the last l&l5 years. The cDNAs for many of the hormone receptors have been cloned, the amino acid sequence determined, and the physicochemical properties further characterized. For each steroid receptor, binding of ligand activates the receptor and allows it to bind to respective specific DNA sequences (steroid response elements (SRE)), where it modulates gene transcription and DNA synthesis. For cell surface receptors, the effects of ligand-binding on gene expression are mediated by other transcriptional mechanisms. Much of our information about the mechanism of action and specific effects of hormone receptors necessarily comes from in vitro and in vivo studies. These findings, however, have accurately predicted hormone responses in humans and allowed a much better understanding of the changes in target tissues. Hormone receptors in malignant tissues, in addition to providing information about the biology of tumors, play two important roles in the clinical management of tumors. First, the presence of receptors may indicate that the tumor is hormone dependent, and provide for the use of hormonal or antihormonal therapy as treatment for the tumor. Many tumors (e.g., breast, prostate, leukemia) are clearly hormone responsive and hormonal manipulation is an important part of the treatment strategy. And second, the presence or absence of certain receptors in the cell may reflect the aggressiveness of the tumor. For this reason information about receptors may be used prognostically to predict the likelihood of future recurrence of a tumor. Many tumors have been studied for the presence of

hormone receptors and whether their presence may vary according to stage of disease, histology, tumor grade, etc. A word of caution, however, is needed. To simply demonstrate (such as by competitive-binding studies or sucrose density gradients) that specific binding for a hormone is present does not by itself mean the tumor is hormone responsive. Many events in addition to receptor binding are needed in order for a hormone to elicit a response (such as release of heat shock proteins, dimerization, DNA-binding, transcription, translation). In the absence of these events, a response will not occur. It is probably for this reason that some tumors (e.g., renal cell carcinoma, melanoma) do not respond to hormonal manipulation even though specific receptor binding is present. The relevance of hormone receptors in tumors must be confirmed by established clinical and laboratory studies which demonstrate the ability of hormones to alter the behavior/growth of the cell. While many hormone receptors have been, and are being, identified in malignant tissues, those for the steroid hormones (estrogen, progesterone, glucocorticoid, androgen) have been the most studied. The present review will, therefore, focus on these receptors. The prolactin receptor has also received attention in breast cancer and will be included. Finally, an additional receptor, that for epidermal growth factor (EGF), will also be discussed. While EGF does not appear to meet the classic definition of a hormone (secretion by an endocrine organ into the bloodstream to act on a distant target organ), its receptor is present on many tumors and is an important determinant of behavior and useful prognostically for many tumors. It is certainly acknowledged that many other substances, especially the growth factors (transforming growth factor B, insulin-like growth factor- 1, platelet-derived growth factor, fibroblast growth factor) have been shown in vitro to regulate tumor growth and metabolism. Their clinical significance, however, is largely untested, and for this reason will not be discussed in the present review. The structure and function of the steroid receptor, prolactin, and EGF receptors will be discussed. Major characteristics and important new findings will be presented as well as an extensive bibliography for those wishing additional information. Such a review is important for our understanding of receptor regulation of cell growth and metabolism, and may also provide the basis for new hormonal treatment strategies for malignancies. Several excellent recent reviews have discussed assay methods for hormone receptors, and accordingly this subject will not be considered here [2-51. All major tumors which have been analyzed for the presence and role of the above receptors will be considered. This will include breast, endometrial, ovarian and cervical carci-

91

noma, leukemia and lymphoma, prostate and renal cell carcinoma, melanoma, head and neck tumors, lung carcinoma, soft tissue sarcoma, gastric, colon, hepatocellular, and esophageal carcinoma. Hormone receptors are clearly important determinants of the behavior of many malignancies. It is hoped the present review will promote our understanding of these proteins, and encourage further efforts into their use in the management of malignancy. II. Structure and function of hormone receptors II-A. Steroid receptors

The family of steroid receptors includes receptor proteins for the following hormones: estrogen, progesterone, glucocorticoid, androgen, mineralocorticoid, thyroid, retinoic acid, and vitamin Ds. These receptors are all structurally homologous in at least the DNA binding region. and are, therefore, referred to as a ‘superfamily’ of receptors (Fig. 1) [6]. There are, in addition, other proteins whose structures are homologous to the known Maximum activity

Fig. 1. Steroid

hormone

aligned according the percentage

amino

tion to the human sequence

receptor

to regions

acid identity

GR (hGR).

are noted.

superfamily.

of maximum

indicated

Homologies

The NH?-terminal

Receptors

amino

have been

acid similarity,

for each region

according

with

in rela-

to structure

end is to the left and

and the

steroid receptors, but for which a specific ligand has not been identified. This latter group is referred to as ‘orphan receptors’ [7-91. The steroid receptors which have the most important role in malignancy and have been the most actively studied are those for estrogen (ER), progesterone (PR), androgen (AR) and glucocorticoid (GR). These four steroid hormone receptors will be the focus of the present review. The steroid receptors are proteins characterized by low capacity (usually < 50,000 sites per cell), high affinity (&< 10 nM), and specificity for a given class of steroids (e.g., estrogens) [IO]. In the unbound state the steroid receptors were formerly thought to be located in the cytoplasm, however, recent immunohistochemical studies indicate that PR [ 111, ER [ 121 and AR [ 131 are, in the unbound state, located in the nucleus, whereas the GR is located predominantly in the cytoplasm [14]. Once occupied by ligand, all steroid receptors are present in the nucleus where they bind to DNA acceptor sites called ‘steroid response elements’ (SRE); this binding to DNA allows the receptor to initiate transcription and modify the rate of cell proliferation. O’Malley [7] DNA

-COOH corticoid; receptor

Hormone

terminal

end to the right. CR, glucocorticoid;

PR. progesterone; related

ER, estrogen;

1 or 2; VDR, vitamin

MR. mineralo-

ERR1 and ERRZ. estrogen

D,; T,Rs and T,R., thyroid;

V

r’hA,erb-A oncogene product; RAR, retinoic acid. Two ‘orphan‘ receptom. HAP and E75, are included. Reproduced with permission, Evans, R. [6].

94

has summarized four main reactions by which steroid hormone receptors regulate gene expression: (i) ligand induced allosteric activation of the receptor; (ii) specific binding to SREs; (iii) stable complex formation at these DNA enhancer sites; and (iv) recruitment of transcription factors and RNA polymerase to initiate transcription of target genes. These events are summarized in a model, described by Tsai et al. [15] for steroid hormone action. This is illustrated in Fig. 2. In the nonactivated state (nonligand-bound) the steroid receptor is associated with multiple nonsteroid-binding proteins. Ligandbinding prompts the release of these proteins and dimerization of the receptor. The activated receptor subsequently binds to its response element, recruits transcription factors, and initiates gene transcription. These events are discussed in more detail below for each of the steroid receptors. While ligand binding represents the principle means by which steroid receptors are activated, it appears other mechanisms may also exist. Power et al. [ 161found that physiologic concentrations of the neurotransmitter dopamine activated one of the orphan receptors (chicken ovalbumin upstream promoter transcription factor (COUP-TF)) with resultant gene expression. This raised the possibility that classic steroid hormone receptors can be activated by compounds other than their cognant ligands. Fuqua et al. [17] reported that an estrogen receptor negative variant breast cancer cell line constitutively activates transcription of a normally estrogen-dependent gene construct in yeast cells. Alternate nonligand related pathways for activation therefore exist, although activation by ligand-receptor complexes are by far the most common mechanism. The transcription products resulting from receptor induced gene expression, and their subsequent effect on growth and metabolism, may vary both with the recep-

1

H90

Fig. 2. Model

for steroid

the nonligand

bound

hormone

receptor

state is associated

action.

Receptor

with nonsteroid

(R,) in

binding

pro-

teins, including heat shock proteins (H70, H90). Binding of hormone (H) prompts release of associated proteins, receptor dimerization, and to steroid

response

ing of transcription

binding

factors

(F), and polymerase produced

element (SRE). This in turn initiates TFIIB

II (POLII),

(B), TFIID

(D), TFIIE

and subsequent

with permission,

transcription.

Tsai et al. [15].

bind-

(E), TFIIF Re-

tor and the cell type. These products can include intracellular functional or structural proteins as well as secretory proteins. Some of the secretory proteins may in turn act on the same cell (autocrine action) or on other cells (paracrine or endocrine action). The initial binding of steroid to receptor can thus have many and varied consequences. II-B. Estrogen receptor

The human estrogen receptor (ER) gene has been localized to chromosome 6q24-q27 [ 181.The cDNA of the human ER has been cloned, sequenced and expressed [ 19,201. An open reading frame of 1785 nucleotides corresponds to a polypeptide of 595 amino acids with a molecular mass of 66,200 daltons [20]. The isoelectric point is 5.7, and the half-life measured in MCF-7 breast cancer cells and uterine cells is 334 h [21,22]. The ER has the following comparative binding affinity for estrogens: diethylstilbesterol > 17a, ethynyl estradiol > 17b estradial> estrone > estriol > mestranol [23]. The apparent dissociation constant (&) of the ER is in the range of 0.06-2.5 nM [24]. The binding capacity is in the range of 60-150 fmol/mg cytosolic protein [24,25]. The unbound ER is located primarily in the nucleus [12]. Under low salt conditions the ER sediments on sucrose density gradients as an 8s species, and in high salt as a 4s species [26]. Combination with estradiol results in activation of the receptor to a 5S sedimenting species [26]. Whereas the unbound 4S sedimenting form has little or no DNA-binding capacity, the transformed 5S form readily binds to DNA. The 5S form has recently been shown to be a homodimer of two 66,000 hormone-binding subunits which may be released as such from the nontransformed 8-9s ER [27]. Steroid receptors in the untransformed (nonligand bound) state may be associated with one or more nonsteroidal binding components. The most prominant of these are the heat shock proteins, of which at least three have been identified: hsp56, hsp70, hsp90. Other proteins (e.g., ~59) may in turn interact with these heat shock proteins [28]. Inano et al [29]. have reconstituted the 9S ER from the purified ER subunit and hsp90. This reconstituted 9S form was dissociated to the 4.6s form by high salt buffers. Ligand binding also causes dissociation of heat shock proteins, allowing receptor binding to hormone response elements of target genes [7,28,30]. Hsp90 may facilitate the subsequent response of the unliganded receptor to the hormonal signal [3 11. The steroid hormone receptor contains six different domains, designated A-F (Fig. 3) [32-341. These are based on degrees of homology between receptors: regions A, C, E are highly conserved between receptors,

95

whereas B, D, F are less so [33]. The domains have different functions: at the N-terminal end is a hypervariable region (domain A/B) which is a transcriptional modulation domain [7]. This contains information which enhances transcriptional stimulation potency and also contains sequences which allow preferential activation of certain genes. An internal domain C is responsible for DNA binding. This domain contains two zinc binding Cys.-Cysz sequence motifs (zinc fingers) which fold to form a single structural domain (Fig. 4) [35,35]. The structure consists of two helices which are perpendicular to each other. A zinc ion, coordinated by four conserved cysteins, holds the base of a loop at the N terminus of each helix [30,54]. The first zinc finger contains primary information for sequence specific binding while the second finger stabilizes binding of the receptor to its DNA response element [7]. Discrimination among specific binding sites is determined by three amino acids at the first finger. For the ER, these are Glu, Gly, and Ala [36]. Domain D separates domains C and E and is thought to act as a hinge between the DNA-binding and the hormone-binding domains [33]. Region E, near the carboxy terminal end, is the ligand-binding domain. Deletions throughout region E between residues 265-588 of the ER abolish estrogen binding [32]. Region E may, therefore, form a hydrophobic pocket in which only a small number of discrete residues contribute directly to ligand binding [32]. Within the C-terminal domain are

two regions which may participate in either ligand-binding, protein-protein structural interactions, or transcriptional activation. [7] Within the steroid-binding domain is located a region required for both receptor dimerization and high affinity DNA binding [37,38]. Two transactivation domains have been identified within the receptor [39,40]. The major domain is contained within the C-terminal portion of the protein and depends upon estrogen binding for its activity. The second transactivation domain lies within the N-terminal region and is active in the absence of estradiol [40]. The formation of steroid receptor complexes with heat shock protein-90 involves several receptor regions [41]. The activated ligand-bound steroid receptor, as noted, binds to specific DNA sequences where it initiates transcription. These regulatory elements (SREs) are generally 5’, or upstream to, the structural gene, however, they may also occur in the coding region of the gene [7,42]. Kon and Spelsberg [43] have estimated there are 200&3000 acceptor sites per cell. These elements consist of inverted repeats of the sequence TGTTCT for glucocorticoids, progestagens and androgens, and TGACC for estrogens (Fig. 5) [32,34]. Berg [44] has indicated the following consensus sequence for the estrogen response elements: GGTCAnnnTGACC [44]. Peale Jr. et al. [45] derived a consensus sequence for the ER which was 38 nucleotides long and contained an inverted repeat (5’ CAGGTCAGAGTGACCTG 3’) and an A/T rich region. Maximum specificity for ER binding occurred at lOCL-150nM ionic strength and pH 7.58.0. The dissociation constant for the ER bound to the sequence was 0.5 nM. The A/T rich region alone was ineffective in binding ER. The TGGGTCA element has been shown to mediate the transactivation of the ovalalbumin promoter by C-SOS,c-&n, and the ER [46]. Specific binding in vitro of nuclear factors to the TGGGTCA element was not affected by the presence of the ER in cultured cells. Steroid receptors bind to their respective SREs as

Fig. 4. The DNA-binding

finger

discrimination

of the

dimer is illustrated

--

Nuclear Translocation

-

m

Transactivation Dimerization 90 K hsp

Fig. 3. Structural six different

and functional

domains

ed. The designated

(A-F) amino

domains

of the steroid receptor.

and loci for specific functions acid numbers

The

are indicat-

are those for the ER. Modi-

fied from Beato, M. [34]. and Sluyser, M. [33].

motif and regulatory steroid

receptor

of the steroid

folds into two zinc finger binding

to a half site hormone acids (asterisk*)

domain

receptor:zinc

zipper model. The DNA-binding response

domain motifs,

and binds

element (HRE; panel A). Three amino

at the base of the first (N-terminal)

zinc finger allow

main shown.

(DD)

among and

Reproduced

specific

binding

sites. Binding

in panel B. The regulatory transcriptional

of a receptor

zipper dimerization

inactivation

with permission, Forman, H.H. [35].

domain

do-

(T,) are also

B.M. and Samuels,

96

tion of an initial stable preinitiation

CONSENSUS RESPONSIVE ELEMENTS FOR NUCLEAR RECEPTORS 7,

13

template polymerase

15

123456

1. 2. 3. 4.

GRE (+) PRE ARE MRE

8. GRE

3 45678910

12 II

Fig. 5. Consensus

response

sensus

sequence

genes,

GR - = for

elements

for glucocorticoid repressed

(AR), mineralocorticoid

(MR),

tinoic acid (RR) are indicated.

for nuclear receptor

genes), estrogen

13

15

content

receptors. (PR),

(ER), thyroid

Reproduced

of the receptor,

in receptor

the amount

decreases. is thought

properties

and RNA with a tem-

[52]. This

of the steevent,

called

to involve kinetic

and loss of binding

of estradiol-filled

ca-

ER, but rather is due to degra-

dation of the estrogen-receptor complex down-regulates ER mRNA transcription

14

The con-

(GR + = for inducible

progesterone

‘processing’

tability

ATYACNnnnTGATCW

12

to DNA,

receptor

conferring

pacity [25,53]. Recently, it has been demonstrated that ‘processing’ is not the result of a decrease in the extrac-

TCAGGTCA-TGACCTGA I. (-)

binding

TFIIE,

stabily associated conditions

roid-occupied changes

AGGTCAnnnTGACCT AGGGlTnnnTGCACT

6. TRE 7. RRE

do not remain

Following

complex,

while TFIIB,

plate under transcription

GGTACAnnn:G;T:T .I II II

5. ERE 6. EcRE

commitment,

androgen

(TR), and re-

with permission,

Beato,

M. [34].

dimers. The SRE is composed of two half sites, with each half site binding one monomer of the receptor. Only the dimeric form of the receptor binds with an affinity (& = 10w9M) sufficient to influence transcription [7]. Binding of the ER to the SRE appears to require the presence of DNA-binding stimulatory factor [47]. Once bound to an SRE, the receptor dimer can couple with another dimer (or other transcription factor) at an adjacent SRE to create a more stable complex with much higher affinity (& approx. lo-“M) [7]. There are indications that binding of the ligand to the receptor, however, may not be required for binding of the ER to the response element. Using gel shift assays, Murdoch et al. [48] found a dissociation constant of 390+40 pM for the E2 occupied, heated ER to the vitellogenin AR gene, and a dissociation constant of 450 f 170 pM for the unoccupied, heated ER, indicating that estrogen was not necessary for specific binding to DNA. Curtis and Korach [49] also found that the ligand-binding domain of the ER does not exert its regulatory effects at the level of sequence specific DNA binding since its occupancy does not alter binding to the ER. The activated hormone receptor initiates gene transcription by first forming stable preinitiation complexes with transcription factors. One such class, the general transcription factors, is required for transcription of all class II genes and includes seven different protein factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIG. and TFIIH [50]. The transcription complex is initiated by the binding of TFIID to the TATA sequence, followed sequentially by TFIIA, TFIIB, polymerase II, and TFIIE/F [7,50,51]. TFIID alone is sufficient for forma-

[55,56]. It appears

levels through

that estrogen

a decrease in its mRNA

[54]. Estradiol as well as ER regulates

expression

ER [54].

The effects of estradiol have been studied in many cell types, both normal and malignant. Among the most extensively studied are human breast cancer cells. While a multitude of responses to estradiol have been noted, it remains unclear to what degree these also occur in situ in patients with breast cancer. Nevertheless the in vitro and in vivo systems have provided an important opportunity to study these effects under controllled conditions. Estradiol effects which have been noted include increased cell growth and thymidine incorporation in vitro and in vivo [57,58], increased progesterone receptor synthesis [59], antagonism of interleukin-1 growth inhibition [60], increased secretion of 24K and 52K proteins [61,62], increased secretion of growth factors IGF1 [63], PDGF [58], TGFa [64], decreased secretion of TGFB [65], progression to S phase of cell cycle [66], enhanced expression of protooncogenes c-ras, c-fos, c-myc [67], and increased EGF receptor mRNA [59]. Many of the secretory products may influence other cellular events in an autocrine or paracrine manner. Estradiol regulation of cell growth and metabolism thus represents the sum total of its effects on many cell processes. There are a number of substances which have been shown to alter the cellular ER content and/or the effects of estradiol on cell processes. These include the antiestrogens tamoxifen and hydroxytamoxifen [57,59], the pineal gland hormone melatonin [68,69], interferon [70], insulin [71], progestins [72], interleukin-1 [60], and tumor necrosis factor [73]. The most important of these from a clinical standpoint are probably the antiestrogens; within this group there has been the most experience with tamoxifen. The mechanism of action of tamoxifen is thought to be by either (i) binding to a separate nonestrogen receptor which then competes with and antagonizes ER binding to DNA [74]. Or (ii) binding directly to the ER and inhibiting by positive cooperativity the subsequent binding of estradiol, thereby reducing the estrogenic effects [75,76]. Tamoxifen by

97

itself (in the absence of estrogens), however, does not inhibit cell growth. At low doses, hydroxytamoxifen may even stimulate growth [57]. II-C. Progesterone receptor

The progesterone receptor gene maps to human chromosome band 11q 13 [77]. The cDNA of the human PR has been cloned, sequenced, and expressed [78]. The human PR is present in two subunit forms, A and B, with molecular masses of 93 kDa for A and 119 kDa for B as measured on SDS gels [79]. The two forms are present in equimolar amounts [87]. Greene et al. [79] have examined purified forms of the two subunits using monoclonal antibodies. Each of the antibodies recognized cytosolic and nuclear forms of occupied as well unoccupied nuclear receptor. They demonstrated that an amino terminal region of B was not present in A, and that a significant portion of A and B are either identical or very similar in amino acid sequence. Using a cDNA for human PR, Wei et al. [80] and Read et al. [55] have identified 5-6 PR mRNAs ranging in size from 2.5 to 11.4 kb. All six species were present in normal human endometrium and in PR-positive MCF-7 cells breast cancer cells but not in PR-negative cells [80]. MCF-7 contained approx. 16 message molecules per/cell, this level being increased to 45 by estradiol treatment. Another breast cancer cell line T47D, which constitutively synthesizes PR, contained 90 message molecules per cell. The relationship of multiple mRNAs to the two subunit forms is not clear. It is possible that the heterogeneity is due to polyadenylation at different sites [80]. Greene et al. [79] have indicated that only one transcript is translated to produce the two PR forms. Kastner et al. [81], however, recently presented data arguing against the hypothesis that similar amounts of the two PR isoforms are generated by alternative initiation of translation on a single PR transcript. They concluded from their studies that for human PR and chicken PR, isoforms A and B are in fact translated from different mRNAs.

Monomer

Unactwated ComDlex (a- 1OS)

Fig. 6. Proposed

multistep

Synthesis of the PR is stimulated by estrogens. In the absence of estrogens, cells contain low levels of PR (2000 sites/cell) [59]. In the presence of estradiol, PR content is increased substantially (E?, 1 nM =30,000 sites/cell) [55,59]. In MCF-7 breast cancer cells, the major effect of E? on PR content is to increase the rate or PR synthesis, while leaving the degradation rate unaltered. The Ez-stimulated increase in PR protein is associated with increased levels of PR mRNA [82]. In the chick oviduct, estrogen stimulation of PR levels is a post-transcriptional process [83]. Progestational agonists (progesterone, R5020) appear to autoregulate the levels of PR by inhibiting transcription of the PR gene [55,84]. The Kd of the PR is estimated to be 0.80 to 10 nM [85,86]. The presence in the literature of a range of values for the Kd of the PR as well as other receptors reflects a range of cell and tissue types, differences in ligands used for Scatchard analysis, methods and purity of tissue preparations and types of assays. The PR sediments as an 8s species on low salt (0.01 M KCl), and as a 3.5s species on high salt [79,87], sucrose density gradients. Each of the two forms (A and B) sediments as an 8s holoreceptor; that is, they are bound together as dimers [87]. Dimers can be any combination of A and B. Rodriguez et al. [88] found that the PRs assemble in vitro into dimers in the absence of DNA, and that dimerization does not require hormone. They felt dimerization presumably occurs through an undefined dimerization domain. The PR (as well as the ER and GR) in the nonligandbound state are associated with heat-shock proteins which must be stripped from the receptor before the receptor can bind DNA, at least in vitro. Ligand binding is thought to cause dissociation of these heat-shock proteins so that DNA interaction can occur (Fig. 6) [7,89,90]. Smith et al. [91] have identified five nonreceptor proteins which copurified with the unactivated avian progesterone receptor; these proteins had molecular masses of approx. 90,70, 54,50 and 23 kDa. Except for ~70, each could be dissociated from the receptor by salt.

+ hsD

model for the process

70

of progesterone

receptor

activation.

Reproduced

with permission,

Demarzo

A.A., et al, [89].

98

Association of the PR with hsp70 and hsp90 requires the ligand-binding domain [90]. Deletion of 290 amino acids from the C-terminus results in loss of ability to form an 8s complex. The site of interaction between PRA and hsp90 responsible for 8S complex formation appears to be in the region of amino acids 3699506. There may, however, may be more than one site of interaction in this region [92]. Reconstitution of PR with hsp70 and hsp90 does not occur in the presence of progesterone [91]. The ability to form stable PR-A-PR-B oligomers in the absence of DNA correlated with release of 90 kDa heat shock protein (hsp 90) from the unactivated PR complex [89]. The hsp90 may function to suppress DNA-binding activity indirectly by blocking receptor dimerization [89]. When bound by ligand, both A and B forms become ‘transformed’ and acquire tight nuclear-binding capacity within 5 min. Transformation leaves the primary structure of each of the receptor proteins unchanged [87]. In vivo progesterone treatment results in increased receptor phosphorylation, altered interaction with heat shock protein-90, and increased DNA binding [93]. The topographical structure of the PR is thought to be comparable to that for other steroid receptors and consists of six domains [6,7,34] (Fig. 3; see also Estrogen receptor above). The degree to which the A and B subunits differ in their structural/ functional capacity is being actively studied. Multiple epitopes recognized by monoclonal antibodies are present on both A and B forms [79]. Monoclonal antibodes appear to recognize four different immunogenic domains, all of which are located on the amino-terminal half of the protein [94]. Gronemeyer et al. [95] have presented evidence that the cPR from A corresponds to an N-terminally truncated form of B (see also discussion of binding to SRE below). The DNA-binding domain of the PR, like other steroid receptors, is a highly conserved 68 amino acid domain which contains 20 invariant amino acids which fold into two zinc-finger DNA-binding motifs [36]. The three amino acids of the first finger provinding specificity of the PR for recognition of the response element are Gly, Ser, and Val [36]. The N-terminal domain of the PR has been implicated in both the efficiency and specificity of transcriptional transactivation [65]. Deletion of this domain has been shown to reduce gene activation potential in the PR [7]. Studies on mechanisms of nuclear localization of the PR have also indicated at least two regions involved in this process. Guichon-Mantel et al. [l l] defined a region of five amino acids, located around position 638-642, which is the main nuclear signal and whose deletion is sufficient to prevent the nuclear localization of the receptor. A second localization signal, whose activity is

very limited, lies in the steroid-binding domain and necessitates the binding of the hormone to be effective. The SRE for the progesterone receptor is identical to that for the glucocorticoid receptor and consists of the 15 base pair DNA element TGTACAGGATGTTCT [96], located within the 5’ flanking region of hormone responsive genes. The progesterone receptor interacts with its cognate enhancer as a dimer [97]. Exposure of receptor to either progesterone, R5020, or the antiprogestin RU38 486 in vivo or in vitro leads to the formation of two protein-DNA complexes (1 and 2) which are specific for the progesterone responsive element [98]. The complexes 1 and 2 are formed by progesterone receptor forms B and A, respectively. The purine contact site for the subunit A and subunit B form of the progesterone receptor are identical [97]. Tora et al. [65] noted differential activation of the SRE between forms A and B of the receptor. Kastner et al. [99] demonstrated that the two hPR forms similarly activated transcription from reporter genes containing a single palindromic progestin responsive element (PRE), while form B was more efficient at activating the PRE of the mouse mammary tumor virus long terminal repeat. Transcription from the ovalbumin promoter, however, was induced by hPR form A but not by form B. Binding of ligand-bound PR to its SRE results in activation of one or more of a variety of genes depending upon the specific tissue and species. The subsequent effects of the PR at the cellular and tissue level have been discussed in an excellent review by Walters [loo]. This should be consulted for further information on morphological changes. II-D. Glucocorticoid receptor

The gene for the human glucocorticoid receptor (GR) is located on chromosome 5 [loll, locus 5q31 [102]. The cDNA for the GR has been cloned, sequenced, and expressed [ 103,104]. These cDNAs predict two protein forms, a and B, 777 and 742 amino acids long, respectively [ 1041. The glucocorticoid receptor in the unbound state is located predominantly in the cytoplasm [15]. Upon the addition of hormone the GR is translocated to the nucleus, returning rapidly to the cytoplasm upon hormone withdraw1 [105]. The concentration of GR varies with the cell type but is generally in the range of 100 to 800 fm/mg protein in GR positive cells [ 106108J The dissociation constant (&) is 1 to 10 nM [109,1 lo]. The transformed (4s) GR has a reduced [3H]triamcinolone acetate-binding affinity as compared to the nontransformed (9s) GR [ 1111.The half-life of GR mRNA in hepatoma tissue culture cells is approx. 4.5 h and is unaffected by dexamethasone [I 121. In the absence of dexamethasone, GR protein half-life is approx. 25 h, de-

99

creasing

to approx.

Autoregulation and occurs

11 h in the presence

of GR by its cognate at both

transcriptional

tional levels [ 1121. The molybdate stabilized complex

of hormone.

ligand

nonactivated

receptor

with a stokes radius (Rs) of 7-8nM, coefficient

mentation

of

is complex

and posttranscrip-

9-10s

is a

and a sedi(calculated

ly the heat shock

proteins

(hsp; Fig. 2) [15]. Hsp90

is

thought to bind to two sites on the GR [114]. The critical contact site occurs in region of amino acids 632-659. A second

hsp90 contact

632, which acid sequence

contains

site is predicted

the only highly

in the receptor

in region

conserved

superfamily

outside

Fig. 7. Model for activation pied state, binding

the DNA-binding

domain

al change

of glucocorticoid region

receptor.

In the unoccu-

(B) is concealed

by the steroid

(A). Binding of glucocorticoid

in the receptor

with exposure

permission,

574

A

B M

C

M,= 270,00~330,000) [113]. The GR in the nontransformed state is complexed with other proteins, especial-

Gustafsson

causes a conformation-

of region B. Reproduced J., et al.

with

[I 131.

amino of the

DNA-binding domain. The receptor becomes associated with hsp90 late during translation or immediately at the termination of translation [I 161. The hsp90 may play an important role in stabilizing the GR in a high affinity state for steroids [ 1111, and may play a critical role in maintaining the receptor in a nonfunctional state [117]. Recent studies have also indicated the presence of an additional protein of 59 kDa which is bound directly to hsp90 [ 1151. Binding of ligand to the receptor is followed by release of heat shock proteins and activation of the receptor (Fig. 2) [15]. The activated GR has an S~O.~ of 4s (calculated M,=90,000) and a Stokes radius (R,) of 6 nM [91]. The activated GR exists as a homodimer when unbound as well as when bound to DNA [ 1181. The GR, like other steroid receptors, consists of six functional domains. The topography of these domains is illustrated in Fig. 3 [115]. The ligand-binding domain is located at the C-terminal end. There may, however, be two or more steroid-binding sites. Svec et al. [ 1191reported a second binding site which resides in the steroidbinding domain topographically close to the agonistbinding site. Studies using the high potency glucocorticoid cortivazol indicate that this compound recognizes two glucocorticoid binding sites on the human GR or a protein very similar to it [120]. Ligand binding and activation of the GR appear to involve a conformational change resulting in exposure of the DNA binding site (Fig. 7). Gustaffson and collegues [113] proposed that the DNA-binding domain (B) is protected by the steroid-binding domain (A) prior to activation, and steroid binding leads to the opening up of the protein at a hinge region. The most likely localization for such a hinge region would be at the border between the steroid-binding domain and the DNA-binding domain. The DNA binding domain has a globular fold which contains two zincnucleated substructures of distinct conformation and function. When it binds to DNA the domain dimerizes, placing the subunits in adjacent major grooves. One

subunit half-site

interacts

specifically

and the other

with the consensus

nonspecifically

target

with a noncog-

nant element [121]. A short segment has been identified in the proximity of one of the bound zinc ions that is required for cooperative binding of the GR to the GRE. This segment is involved in dimer formation of the native GR and is important for correct positioning of the dimer on the double helix of the DNA [122]. The three amino acids of the first zinc finger which provide specificity of the GR are the same as for the PR and are Gly. Ser, and Val [35]. The immunogenic domain is distinct from the steroid and DNA-binding regions [ 1131. The glucocorticoid receptor, like other steroid receptors, binds to specific DNA sequences referred to as steroid response elements (SRE). The consensus sequence for the glucocorticoid receptor consists of a 15 base pair DNA element TGTACAGGATETTCT located within the 5’ flanking region of hormone responsive genes [96]. The GRE is present as two hexamer half-sites, each halfsite recognized by a single subunit of a receptor dimer, probably in a cooperative fashion [123]. Binding to the low affinity half-site is dependent on previous occupancy of the high affinity half-site [124]. The nonconserved DNA sequences flanking the GRE contribute significantly to the free energy of receptor binding to DNA [125]. The DNA-binding domain of the GR is thought to be composed of distinct subdomains, which interact with the subelements of the recognition sequence [ 1231. Glucocorticoid receptors have high selectivity and affinity only for DNA which contains specific partially symmetric GREs; this high affinity for such DNA sites may be sufficient to account for the selective regulation of gene expression observed in glucocorticoid responsive cells [126]. The GCR and PR, as noted, bind to identical DNA elements. When present together in a cell, the specific cellular effects of these two hormones appear to be determined by differential expression of their respective receptors [7]. In an elegant experiment by Greene and Chambon [ 1271, in which the DNA bind-

100

ing domain of the ER was replaced with that of the GR, the chimeric ER, when exposed to estradiol, behaved as a glucocorticoid receptor [ 1281. The GR, like other steroid receptors, recruits general transcription factors (TFII A-H) [50,51]. In addition, other transcription factors may also be involved. Schule et al. [129] found cooperativity of the progesterone and glucocorticoid receptors and the binding site for transcription factors CPl, Spl, OTF, NFl, the CACCCbox, or a second steroid receptor-binding site. The degree of synergism was inversely related to the strength of the GRE. A steroid responsive unit can thus be composed of several modules that, if positioned correctively, can act synergistically. The transcriptional events following ligand binding of the receptor are being actively studied by many laboratories. A system which has also been extensively examined is that of the mouse mammary tumor virus (MMTV). The MMTV is known to cause mammary adenocarcinoma in mice, and also to be responsive to regulation by glucocorticoids at the transcriptional level [ 128,130]. Glucocorticoid regulation has been well characterized, including the primary sequence structure of the MMTV promoter, promoter regulatory elements, chromatin structure of the MMTV long terminal repeat (LTR), formation of an active transcription initiation complex, and the mechanism of steroid action [ 1281.The GR is also known to suppress many genes, including propriomelanocortin [13 11,the prolactin gene [ 1321, the proliferin gene [133], the chorionic a subunit gene [ 134,135], and the collagenase gene [ 136,137]. The activity of the GR is related to expression of the oncoprotein c-&n. Overexpression of c-&n prevents the glucocorticoid-induced activation of genes carrying a functional glucocorticoid response element. Conversely, GR is able to repress AP- 1-mediated transcriptional activity [ 1381. Finally, other factors may also play a role in regulating the reponse to glucocorticoids. The S115 mammary tumor cells, which contain glucocorticoid and androgen receptors but do not respond to their steroids, are converted to responsive cells when transfected with constructs of long terminal repeat - C3( 1) gene for prostatic steroid binding protein [ 1391. II-E. Androgen receptor

The gene for the androgen receptor is located on the X-chromosome between the centromere and q13 [140,141]. The cDNA for the human and rat AR have been cloned, sequenced, and expressed [140,142-1441. The human AR is encoded by one single gene [ 1451.The cDNA sequence reveals an open reading frame of 2751 nucleotides encoding a protein of 917 amino acid resi-

dues with a calculated molecular mass of 98,845 daltons. One analysis also indicated an additional 43 kDa protein with binding specificity only for R188 1 and which possessed a higher affinity for nuclei than the 110 kDa form. The function of this smaller protein is unknown [ 1461. Brinkman et al. [ 1451 found in the LNCaP prostate carcinoma cell line two major (11 and 8 kb) and one minor (4.7 kb) AR mRNA species which could be down-regulated by androgens. The unbound AR is located predominantly in the nucleus [14]. The cellular concentration of the AR is quite variable. In MCF-7 human breast cancer cells there are approx. 180 fmol/mg DNA [147]. Many studies have identified AR in prostatic carcinoma cells (see discussion under Clinical considerations, below). The dissociation constant (&) of the human AR is 15.0 nM [148,149]. Competition with different unlabeled steroids for the binding of 3 nM [3H]dihydrotestosterone to cytosolic AR revealed the following order of competitor effectiveness: methyltrienolone > dihydrotesterone > testosterone > estradiol > = progesterone > triacinolone acetonide [ 1491.The human AR made from cDNA sediments as a 4s species in high salt [143,150]. Omission of salt, however, does not alter the sedimentation pattern, suggesting the 7-10s or larger forms of AR may be formed only after a modification of the newly made hAR and/or associated with a specific macromolecule [ 1431.Tilley et al. [ 1491examined cytosolic extracts from cells transfected with cDNA for hAR and noted a sedimentation rate of 8.3s in low salt sucrose density gradients. Davies and Rushmore [ 1501analyzed rat ventral prostate AR and reported a sedimentation coefficient of 4.24.5S, a Stokes radius of 4.65 nM and a calculated M, of 78,OOO-82,000. The deduced amino acid sequence of the human AR has been determined [ 1511.The hAR has the same topographical structure, with six domains, as the other steroid receptors. The steroid-binding domain at the C-terminal end (amino acid 669-9 17) of the human androgen receptor exhibits 54%, 50% and 51% amino acid identities with the corresponding domains of the PR, GCR and mineralocorticoid receptor [ 140,15 1,152]. For the DNA-binding domain (amino acid 557-622) the identies are 85%, 79%, and 80%, respectively. The three amino acids of the first zinc finger which provide specificity of the AR are the same as for the PR and GR and are Gly, Ser, and Val [36]. A subtle difference in structure can abolish steroid-dependent transactivation [ 1531. Govindan et al. [152] recently demonstrated that a change in amino acids from Lys+Leu (816) Asp+Phe (819) Glu+Phe (820) with an insertion mutation of TTG (codon for Leu at 821) at the hormone-binding domain of hARB, disrupted high affinity hormone binding.

101

Others (Lubahn et al. [153]) have shown that a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development. The AR also contains a number of distinct regulatory regions important for normal activity [154]. Deletion mapping studies showed that carboxy terminal deletions of approx. 250 amino acids convert AR into a constitutive activator of transcription [154]. Simental et al. [155] also demonstrated that within the amino terminal region is a domain required for full transcriptional activity, and within the steroid binding domains an inhibitory function, deletion of which yields a constitutively active receptor. Ham et al. [ 1561has identified a 15 bp sequence which can function as a steroid response element for androgens. The ARE consists of inverted repeats of the sequence TGTTCT [32]. The androgen receptor acts as a transcription factor, with the potential for inducing multiple genes. Transfection studies, in which mouse mammary tumor virus (MMTV) reporter genes were inserted into AR-containing T47 human breast cancer cells, demonstrated the ability of androgens to induce MMTV directed transcription [157-1601. This was further supported by Rundlett, using cotransfection assays in human HeLa cells, in which it was demonstrated that AR induced chloramphenicol acetyltransferase activity more than 20-fold using the MMTV-LTR as a source of androgen response elements [ 1541. AR has been shown to bind to high affinity sites in or near the promoter region of the prostate binding protein gene C3(1) [161]. The rat AR gene promoter lacks TATA and CCAAT box elements, but it contains one SPl site and several other possible binding sites for transcription factors. Part of the promoter can function in an orientation dependent manner, but the full promoter shows a higher and unidirectional activity [162]. Several in vivo studies have suggested that androgens primarily control mRNA stability [ 163,164]. Rundlett et al. [ 1541,however, has indicated that androgens modulate rates of transcriptional initiation, suggesting that posttranscriptional effects of androgens are secondary responses. Quarmby et al. [165] demonstrated that the amount of AR mRNA increased 2-IO-fold with androgen withdraw1 and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney and brain, and in a human prostate cancer cell line, LNCaP. The AR mRNA thus appears to be downregulated in an autologous manner. The androgen receptors in MCF-7 human breast cancer cells are also down-regulated by estrogens [148]. S 115 mouse mammary tumor cells contain MMTV-related sequences which are transcribed into RNA only in the

long-term presence of androgen [166]. Walters [loo] has recently reviewed other effects of androgens on target tissue function as well as AR interaction with the genome and alteration of gene expression. The reader is referred to this excellent review for additional references. II-F.

Epidermal growth factor

receptor

The epidermal growth factor receptor (EGFR) is located on the plasma membrane. The gene for EGFR is located in the pl4-pl2 region of chromosome 7 [167,168]. The cDNA for the receptor has been cloned and the amino acid sequence determined [ 1691. The mature receptor consists of a single polypeptide chain of 1186 amino acids and a molecular mass of 131,600 [ 170,17 13. A precursor molecule has a signal peptide at the N-terminal end which is cleaved in the endoplasmic reticulum prior to subsequent insertion in the plasma membrane [ 1701. The EGFR is composed of three domains: (i) an extracellular binding domain; (ii) a transmembrane region; and (iii) a cytoplasmic region (Fig. 8) [I 701. The N-terminal end is extracellular and the C-terminal end intracellular. The cytoplasmic portion of the receptor contains a protein kinase domain and three sites of autophosphorylation. The protein kinase of the EGFR phosphorylates tyrosine residues, both on the receptor itself as well as on intracellular proteins [ 1701. The dominant regulatory effect of protein kinase C on the EGF receptor is mediated through phosphorylation at Thr654 which effectively inactivates the receptor [ 1721. The submembrane region of the EGF receptor appears to regulate transmission of conformational information from the extracellular ligand-binding site to the cytoplasmic kinase and regulatory domains. Phosphorylation is thought to play an important role in signal transduction, although the mechanism is not understood [173]. The average receptor concentration ranges from 20,000 to 200,000 sites per cell. Some cell lines (A431

EGF bmdinq domatn

plasma

membrane (23 aa)

cytoplasmlc (542aa)

protean-k~nase domoln

‘a/ . .’ .

Fig. 8. Structure

of the epidermal with permission,

Tyr 1066 Tyr II48 Tyr 1173

growth factor receptor. Schleisinger J. [170].

Reproduced

102

and some epithelial carcinoma cell lines) have much higher receptor numbers, approaching 2,000,OOO for A431 [174]. The latter characteristic has made these cell lines especially valuable for receptor characterization. At least three ligands will bind with high affinity to the extracellular domain: epidermal growth factor (EGF), transforming growth factor-a (TGFa), and vaccinia virus growth factor (VGF) [174]. Secretion of TGFa by breast cancer and other tumors is well known [175]. Whereas many tumors are known to contain EGF receptors (see below), no tumors have been found which secrete EGF [ 1761. Scatchard analysis indicates a dissociation constant (Kd) of lop9 to lo-r0 M for binding of EGF [174]. Binding occurs rapidly, with the surface receptor being saturated in 5-10 min at 37°C [174]. Binding of the ligand in virtually all cells is followed by internalization and then degradation in lysosomes [177]. Kinetic analysis of EGF binding to HeLa cells revealed the presence of three types of receptors, one with high affinity and two with low affinity [ 1781.Activation of the EGFR signal transduction cascade can occur completely through exclusive binding of EGF to the subclass of high affinity EGFR [179]. Binding of ligand (EGF, TGFa, VGF) to the EGF receptor can elicit a spectrum of responses, depending to some degree on the cell type. The effects of EGF on normal tissues include effects on fetal and neonatal development, gastric acid secretion, cornea1 epithelium, and endothelial and epithelial regeneration and wound healing [ 1801. EGF has been shown to promote normal and malignant mammary growth in rodents [181], stimulate proliferation of mouse mammary epithelial cells [1821851 and is necessary for lobular alveolar development of mouse mammary gland in organ culture [183]. EGF has also been shown to stimulate proliferation of human breast cancer cells in vitro [ 186-1881. In A43 1 carcinoma cells, EGF stimulates inositol phosphate production, release of calcium from intracellular stores, rise in intracellular pH, phosphorylation of EGF on threonine residue 654, induction of c-fos gene expression, and alteration in cell morphology [179]. An important relationship has been identified between the EGFR and the v-e&B oncogene. The v-erbB oncogene of the avian erythroblastosis virus (AEV) encodes a truncated EGF receptor [176]. Its product, the GP74V-“h protein, lacks most of the extracellular binding domain, as well as the 32 amino acid residues at the C-terminal end of the receptor (containing the main autophosphorylation site of the receptor) [ 170,189]. Closely related is a second oncogene neu (erbB2). This oncogene, which is located on chromosome 17~1 l-q21, encodes a protein ~185 which is also related to the EGF receptor. More than 50% of the amino acids for neu are

shared with EGFR, and there is 80% homology in the tyrosine kinase domain [190]. ~185 appears to be a substrate for the EGF receptor tyrosine kinase in a tyrosine kinase cascade [ 19 11.Lupu et al. [ 1921has also presented evidence that a 30-kilodalton factor (gp30) secreted from MDA-MB-231 human breast cancer cells is a li. It has been shown that v-erb expresgand for p185 erbB2 sion in breast cancer correlates inversely with both estrogen receptor content and with relapse-free survival (see Clinical considerations below). EGFR cellular content and mRNA expression may be modified by a variety of factors, including progestins [193], estradiol [194,195], EGF [196], and glucocorticoids [197]. II-G. Prolactin receptor

The prolactin receptor (PRLR) is located on the membrane, both the plasma membrane as well as golgi and lysosomes [ 198,199]. The gene for the prolactin receptor is located on chromosome 5 in the region pl3~14. This is also the same locus as the gene for the growth hormone receptor [200]. The cDNA for the rabbit mammary gland PRLR has been cloned and the PRLR sequenced [201]. A strong localized sequence identity was noted between prolactin receptor and growth hormone receptor in both the extracellular and cytoplasmic domains, suggesting that the two receptors originated from a common ancestor [202,203]. The PRLR cDNA encodes a mature prolactin-binding protein of 592 amino acids that contains three domains: (i) the extracellular amino terminal prolactin-binding region of 210 residues; (ii) the transmembrane region of 24 residues; and (iii) the intracellular carboxy terminal domain (57 residues). There was 77% overall amino acid identity and 86% overall homology compared to the rat PRLR [201]. Although the extended cytoplasmic region has additional segments of localized sequence identity with the human GH receptor, there is no identity with any consensus sequences known to be involved in hormonal signal transduction [204]. The cytoplasmic domain of the rabbit mammary gland PRLR is longer than that of rat liver, which may relate to regulation of gene expression for the former as opposed to transport function for the latter [201]. There also appears to be structural differences in the PRLR between species. Recent studies on mouse liver PRLR indicate three PRLR proteins which appear to be encoded by at least two genes [205]. Using monoclonal antibodies, Murakami et al. [206] also identified in the rabbit mammary gland three PRL-binding subunits of M, 77,000,41,000, and 25,000. The molecular weights of these forms vary slightly with tissue and species. Other studies suggest the 42,000 molecular weight form may be the dominant subunit form

103

and the 70-80K form a holoreceptor 12071. Scatchard analysis of purified rat liver prolactin receptor indicates two classes of binding sites. One class is high affinity (&= 3.46 x lo-lo M), and medium capacity (1.26 nmol/mg protein), and the second class lower affinity (& = 1.93 x 10e8 M) and higher capacity (86.4 nmol/mg) [208]. In rat mammary tissue only a single class of receptor with moderate capacity has been noted [208]. The tumor for which prolactin has received the most attention is breast cancer. Binding of prolactin has been demonstrated immunocytochemically and by autoradiography in human breast cancer cells [209] and in DMBA-induced tumors in rats [210,211]. Prolactin receptor has been demonstrated in human breast cancer cells [212,213] and in DMBA-induced tumors [214]. Prolactin induces specific proteins in T47D breast cancer cells [215], promotes induction and growth of DMBAinduced mammary tumor in rats [210], promotes mammary tumor growth in mice [216], stimulates growth of human breast cancer cells in vitro [217], and regulates casein gene expression in rat mammary cells [218,219]. Estradiol has been shown to regulate transcription and translation of prolactin mRNA and protein [220]. III. Clinical characteristics

of hormone receptors in

malignancy III-A. Breast cancer

Among the earliest, if not the first, indication that malignant tumors may be hormone responsive was the report of Beatson in 1896 [221] that metastatic breast cancer regressed following oophorectomy. In the 1960% Jensen and others demonstrated that hormone responsive breast cancer concentrated estrogenic substances [222]. It is now known that breast cancer contains a variety of hormone receptors. These include receptors for estrogens, progestins, glucocorticoids, androgens, and prolactin, as well as epidermal growth factor. Some of these receptors play major roles in defining both the prognosis and treatment of breast cancer. The clinical characteristics of these receptors in breast cancer are reviewed in the following section. Overall distribution of steroid receptors in breast cancer

Among the steroid receptors, those for estrogen (ER) and progesterone (PR) have made the most significant contribution to the management of breast cancer. The overall incidence of ER positivity and PR positivity in breast cancer is summarized in Table 1 for 20 series totaling over 19,000 patients. An attempt has been made

TABLE

1

Overall incidence

of ER and PR in breast cancer Patients

Series

ER+

PR+

Fekete et al. [223]

500

81.6%

68.0%

Brentani

154

54.5

39.0

3 735

77.0

70.0

Kute et al. [226]

113

56.0

48.0

McCarty

500

48.4

23.6

145

70.0

64.0

2 977

75.6

54.2

117

61.0

42.0

Thorpe

Horsfall

et al. 12241 et al. [225] et al. [227] et al. [228]

Clark et al. [229] Smyth et al. [230] Vihko et al. 1755

78.0

60.5

825

65.6

54.9

1262

71.0

54.0

Alexieva et al. [234

506

72.1

58.1

Cooper

380

66.1

Fisher et al. [232] Spyratos

et al. [233] et al. [235]

46.4

252

75.8

Pare et al. [237]

116

77.0

Silfversward

264

76.9

316

73.4

175

66.0

Ferno et al. [241]

4 323

66.0

49.0

Wilking

2 329

75.0

44.0

69.4% & 2.0

52.3% + 3.0

Hawkins

Stewart

et al. [236]

Raemakers

Total

et al. [238]

et al. [239] et al. [240]

et al. [242]

19744

61.6

to include in this summary all recent large series evaluating ER and PR content. The cutoff value for positivity for both ER and PR in these series was usually 10 fmol/ mg cytosolic protein. The average incidence of positivity for the ER was 69.4% and for the PR, 52.3%. This is for all breast cancer patients irrespective of age, grade, lymph node status, menopausal status, etc. The vast majority of these determinations were performed on the primary tumor (although there is a high degree of concordance between primary tumor and metastatic lesions; see below). The distribution of these two receptors alone or in combination has also been recently been reviewed by Hahnel et al. [243]. Their experience totaled 9000 patients from 35 series. They found the following distribution of ER and PR: ER+/PR+, 41.6% (range 1678%); ER+/PR-, 25.7% (range 12-67%); ER-/ PR+, 5.6% (O-15%), and ER-/PR-, 28.4% (656%). Progesterone receptor, when present, is therefore usually accompanied by ER. While laboratory studies have indicated the synthesis of PR is stimulated by estrogens, basal PR expression is not dependent upon estrogen [571. Menopausal status and age

The distribution of ER and PR according to menopausal status is summarized in Table 2. It can be seen that the incidence of ER positivity of postmenopausal patients (71.9%) is slightly higher than that of premenopausal patients (56.7%). No significant differences are

104

TABLE

2

ER and PR according Series

to menopausal

status in breast cancer Postmenopausal

Premenopausal ER+

PR+

ER+

PR+ 42.5%

Ruder et al. [246

32.8%

56.6%

68.2%

Clark et al. [229]

64.0

58.0

79.0

53.0

Thorpe

et al. [225]

68.0

73.0

82.0

68.0

Kohail

et al. [245]

59.1

54.5

67.2

65.5

61.1

73.2

57.6

61.1%+3.3

71.9;9&2.8

57.6k4.6

Skinner

et al. [244] 50.0

Vihko et al. [231]

71.1

Cooper

52.1

Mean

et al. [235

56.7sk4.9

noted for progesterone receptor, however, between premenopausal and postmenopausal women. Among 12 series examining the relationship of receptor content to for ER age, all showed a positive correlation [225,229,231,232,234,235,241,242,244-2471. That is, the tumors of older women have a higher ER content than those of younger women. The relationship of PR content to age, however, appears to be more variable. Five series found no correlation [229,232,234,241,242]. Of note within these five is the fact that four totaled 3802 patients [229,232,241,242]. Clark et al. [229] found that older women were more likely to be ER positive than younger women. When patient age and menopausal status were analyzed together, age was found to be the primary determinant for increased ER concentrations. There appeared to be no relationship for PR for either age or menopausal status when these variables were analyzed separately. Kohail et al. [245] found the combination of ER+/PR+ greater in older than in younger women. Alghanem et al. [247] and Ruder et al. [246] both found ER and PR content to be greater in older patients. Vihko et al. [23 l] found that ER concentration correlated significantly with age in both pre- and postmenopausal patients, while PR concentration correlated with age only in postmenopausal patients. Wilking et al. [242] found only a minor increase in PR with age when menstrual status was ignored. These series, therefore, indicate that the ER content of breast cancer is increased in older patients and in those who are postmenopausal. It is not clear whether age and menopausal status are independent variables, although it appears that age is the major determinant. The relationship of PR to menopausal status and age is equivocal at best. Weimer and Donegan [248] examined ER and PR content of breast cancer in premenopausal women throughout the menstrual cycle. They found the incidence of ER but not PR positivity became significantly

higher after the early follicular phase. An increase in mean ER and PR concentration was found in the late luteal phase. They concluded that the ER and PR values change during the menstrual cycle, probably in response to hormonal fluctuations. Holdaway et al. [249] found a significant circannual variation in the mean monthly PR receptor concentration, with peak PR levels in April and nadir values in August and September. There was no significant cyclic variation in estrogen receptor values. Parity

The relationship of ER and PR status to parity has been examined in a small number of series. These studies suggest, but do not establish, a higher incidence of receptor positivity in nonparous women. Thorpe and Rose [225] noted, among premenopausal women, a higher incidence of ER and PR receptor positivity among nonparous than among parous women, although this was of borderline significance (p=O.O65). The median concentration of PR was also higher among nonparous patients. Among postmenopausal patients there was no difference in the incidence of ER or PR receptor positivity, however, the median ER concentration was 1.5 x higher in nonparous vs. parous patients. They concluded that tumors from nonparous patients appear to be more hormone-dependent than those from parous counterparts. Ruder et al. [246] found that ER positivity was more common in nulliparous women, however PR positivity was more common in parous women (in contrast to the findings of Thorpe and Rose [225]). Cooper et al. [235] found no correlation between ER content and parity. Race

Race may be a determinant of ER and PR positivity in breast cancer. Beverly et al. [250] found a higher incidence of ER and PR positivity in white vs. black women with breast cancer. These differences persisted after standardization for age, menopausal status, tumor size, nodal or distant metastases. These findings were consistent with previous reports indicating a lower incidence of ER positive tumors among black patients [251-2531. Pegarano et al. [254] found that whites had a 67% incidence of cytoplasmic estrogen receptor positive tumors compared with only 49% in blacks and 41% in asians. The proportion of tumors which contained a full complement of receptors (cytoplasmic and nuclear ER and cytoplasmic PR), however, were similar in blacks, whites, and asians in each menopausal group [255]. Crowe et al. [256] found a trend favoring higher ER positivity in whites (77%) vs. blacks (68%), although this was not statistically significant. Black patients also had

105

a significantly worse disease-free survival and overall survival rates compared with white patients (64% vs. 74% and 75% vs. 86%, respectively). Levine et al. [257] found ER levels in Tunisians were generally lower than those in Americans as reflected by both mean levels and percentages with high and low levels (p 5 cm, 66% and 4496;p < 0.0001 and p < 0.0003 for 2977 patients. Thorpe and Rose [225] found a comparable distribution using the same categories, also highly significant (p < 0.0001 and p lOO= 83%). Whether the ER assay was performed on the original primary tumor or on a metastatic lesion did not significantly alter the results, indicating that ER assays on the primary tumor predicted response to subsequent therapy for metastatic disease. Vollenweider et al. [282] found the correct prediction percentages of the response of patients to endocrine therapy were 77% if ER + ,69% if PR + , and 79% if both ER+ and PR+. They felt the correct prediction for percent of response to endocrine therapy appeared less good when measured on the metastatic lesion instead of the primary tumor. Both of these reports, however, point out that the information about receptor status from the primary tumor can be used to treat metastatic

disease. This concept has been reinforced with results from many adjuvant trials. Williams et al. [288] found the objective response among stage IV patients to endocrine therapy was 32% among ER+ vs. 10% in ER-, p 201= 61%. Also, there was a clear relationship between patients with ER+ tumors and prolonged survival after onset of distant metastases. Among 122 patients with ER + tumors receiving first line endocrine therapy, 50% were alive at 18 months vs. 18% of 82 patients with ER- tumors. Vollenweider et al. [282], however, while noting a 74% response rate among ER+ patients (vs. 13% for ER - patients) found that variation in ER concentration (in ER+ patients) did not influence the response rate. The ability of progesterone receptor status to predict response rate has also been examined in many trials. Horwitz [87] recently summarized the findings of 15 series totaling 2430 patients in which response to endocrine therapy was correlated with ERjPR status. This is reproduced in Table 5. As noted, PR appears to make a significant contribution to predicting response to therapy. ER+ /PR + or PR + alone had response rates (69% and 66%, respectively) which were greater than ER+ (53.0%) or ER+/PR(32.0%) alone. Response to therapy among receptor negative tumors was low. Osborne et al. [297] found that PR could also predict response rate (PR+ = 80% vs. 31% for PR-), and that the presence of PR appeared to be a better discriminator for objective response than quantity of ER alone. Vollenweider et al. [282] found response to endocrine therapy to be greater for PR + ( > 15 fm/mg) tumors (70%) than for PR- tumors (< 5 fmol/mg= 30$:, p ~0.01). For ER+/PR+ =784;; vs. 18% for ER-/PR(p 10 fmol/mg. Corle et al. [300] evaluated response to adriamycin/cytoxan among 324 patients and found a direct correlation between ER positivity and response: 0 fm/mg= 50%; l50=62%; >50=79%, ~~0.017). If four drugs were used, the response in the > 50 fm/mg group was 88%. They concluded that there appeared to be a benefit from increased numbers of drugs and from quantitatively higher ER levels. Rosner et al. [301] reviewed the value of ER in 182 patients with metastatic breast cancer. They found there was no significant difference in overall response between ER+ (57.7%) and ER- (63%) patients. However, there was a significant trend toward a higher degree of response in ER - patients (more complete response: 18% vs. 7%) and fewer failures (12% vs. 34% p< 0.006) than in ER+ patients. This better response for ER - patients was offset by an earlier relapse which was reflected in a longer duration of remission, time to treatment failure, and survival in favor of ER + patients. ER - patients, therefore, appeared to respond better but relapse sooner than ER + patients. Blarney et al. [291], Vollenweider et al. [282], and Hilf et al. [302] found no correlation between ER status and response to chemotherapy. Nomura et al. [303] examined reponse of patients to medroxyprogesterone acetate (MPA) according to steroid receptor status and also reviewed nine other series in the literature. They found that although there was no significant difference in the response rate in connection with the presence or absence of PR in the primary tumor, the combination of ER and PR, as well as ER, correlated well with response and was effective in differentiating the response to MPA in second-line therapy. They found no predictive value of ER or PR

for response to chemotherapy. Davidson and Lippman [304] reviewed 17 series totaling 1492 patients. They concluded that overall there appeared to be no significant correlation between estrogen receptor status and response to combination chemotherapy. Use of receptor status to predict response to therapy in adjuvant trials

The steroid receptor status of breast cancer also been used to predict response to adjuvant therapy, both chemotherapy and endocrine therapy. Among trials evaluating chemotherapy, Fisher et al. [305] reported among node positive patients treated with L-PAM, 5FUf tamoxifen, that patients with tumors containing 2 IO fmol/mg of either ER or PR had a better outcome through 5 years for DFS and OS than those with t&9 fmol/mg. ER, PR, and nuclear grade had an independent influence on outcome, and a more accurate assessment of outcome was obtained when more than one marker was used (Fig. 10). In a recent randomized trial evaluating a single perioperative course of adjuvant CMF. an improvement in disease-free survival was noted, with the magnitude of this effect largest among patients with no or low ER content in the primary tumor [306]. Jakesz et al. [307] found a benefit with chemotherapy in stage I,11 ER- patients. An ECOG trial evaluating CMFP/CMFPT/observation also reported an improvement in DFS with chemotherapy among ERnegative patients [309]. Several trials have evaluated the predictive importance of receptor status for adjuvant endocrine therapy

c

I

I

30YEARS0

I

I

2

3

4

5

0

I

2

3

Fig. 10. Relationship

of ER and PR status to overall survival

cancer with adjuvant

chemo/endocrine

expressed

as O-9 or 2 10 fmol/mg,

therapy. and nuclear

5

in breast

ER and PR status are grade is good or poor.

Patients received adjuvant phenylalanine mustard (F).+tamoxifen (T). Reproduced with permission. [305].

4

(P), S-fluoruracil Fisher B., et al.

112 ER++

A

PGR+

+

:. 80 8 .k

70-

x cl

60-

:_ :.

.~ :. i.

b

10 o0

z

60-

s 2 -

3020

........ Control ’ 24

:

c?

TM

’ 12

f..

2 io% 2 40-

.

30 20

70-

& :.: :.

50-

2 40 aJ y 8

i......i

.. .,

E L

‘;

% 0

_

’ 36

’ 60

’ 72

’ 84

’ 96

’ 108

-

TM

I0t .‘.‘.‘...

p = 0.005 ’ 48

-

01 0

1 120

’

12

’

24

’

36

Months

Fig. 11. Disease-free

survival

according

with early breast cancer were randomized or no therapy

(Control).

Disease-free

to ER and PR status.

Patients

to receive tamoxifen

(TAM)

survival

expressed

according

to

trials. In a randomized trial from Naples, Italy, ER-, PR-, or ER/PR-positive patients had a significantly greater disease-free survival at 10 years than ER-negative or PR-negative patients when 100 fmol/mg was used as the cutoff (p200 fm/mg), little difference in survival was found between the latter two groups, suggesting that simply the presence of PR signified a better prognosis, and that absolute levels above 10 fmol/mg were not significant from this point of view. Chambers et al. [372] studied 188 patients with stage I-II carcinoma according to ER and PR status. The overall survival at 2 years and 5 years according to ER and PR status is given in Table 12. It can be seen that survival was significantly dependent upon ER and PR status. If ER status was divided at tS19,2&100, > 100 fmol/mg, survival was significantly different between the low range groups and the other two groups. If PR status was divided at &6, 7-50, > 50 fm/mg, survival was significantly different between the first two groups and the high range. They

felt survival in endometrial carcinoma was better predicted by ER status than grade. Creasman et al. [359] found that, among patients with stage I-II carcinoma a significant 2 year disease-free survival benefit was present for ER positivity (ER+ =9096, ER- =67$, p < 0.01) and for PR positivity (PR+ =92X, PR = 56%, p 70 fmol/mg combined with a PR vaue of more than 30 were independently associated with improved survival. They concluded that for maximum prognostic information, both ER and PR content should be measured. The above series as a group indicate that receptor information (ER and PR) are useful prognostic indicators for endometrial carcinoma. In general, receptor-rich tumors have a better prognosis than receptor poor tumors. This applies for all stages, although exceptions have been noted for stage I and II. These findings further support the recommendations that ER and PR determinations be performed routinely on endometrial carcinoma specimens. Epidermal growth factor receptor has also been identified in endometrial carcinoma. This has been found to correlate inversely with ER content [379]. One series found EGFR to vary inversely with grade [380], however, another review did not [381]. The latter series also found no correlation with depth of myometrial invasion, ER - PR status, the presence of extrauterine metastases, or the development of recurrent disease [381].

120

Because endometrial carcinoma is a hormonally responsive tumor, further studies are definately indicated and should clarify the relationship of EGFR to the behavior of these tumors. Response to therapy

The experience with the use of ER or PR to predict the response to hormonal therapy in endometrial carcinoma is not nearly as extensive as it is for breast cancer. Nevertheless it does appear to indicate that the receptorpositive tumors have higher response rates to systemic therapy than receptor-poor tumors. Whether ER and/or PR is the best predictor is not clear. In 1980 Vihko et al. [382], in a preliminary study indicated that patients with progestin rich tumors responded more frequently to progestin administration than patients with receptor poor tumors. Conversely, patients with low tumor ER or PR concentration responded more often to combination cytotoxic chemotherapy than patients with higher tumor receptor levels. Ehrlich et al. [352] reviewed their experience as well as that of six other series in the literature totaling 152 patients. This indicated that, among PR positive patients, the likelihood of response to progestin was 72% vs. 1291;for PR-negative tumors. Response to progestin, however, appeared to be independent of ER content (Table 13). Benraad et al. [366], analyzing a small series of 13 patients, felt ER positivity, as well as PR positivity predicted response to progestin therapy. Kauppilla et al. [386] evaluated response to cytotoxic chemotherapy (ACV + 5FU) and found patients with low ERjPR (< 30 fmol/mg) had a significantly greater response (70%) than did patients with higher re-

ceptor levels (20%; p < 0.05). In a recent review of all series in the literature, Kauppila [387] concluded that in identifying responders to progestin therapy, a positive PR result appeared to give more precise information than a positive ER, the accuracy being about 75% for PR. The use of the combination of ER and PR determination as a prognostic indicator or predictor of sensitivity to progestin therapy does not significantly increase the information available by PR measurement alone. These findings, therefore, appear to indicate that endometrial carcinoma is a hormonally responsive tumor, and that the receptors are functional and are useful for predicting response to therapy. Receptor-rich tumors have increased response to endocrine therapy, whereas receptor-poor tumors appear to have increased response to chemotherapy. III-C. Ovarian carcinoma Ovarian carcinoma frequently contains estrogen and/ or progesterone receptors, The findings from 24 series totaling 1429 patients are summarized in Table 14. It

TABLE

14

Overall incidence

of steroid receptors

Series

n

Progesterone trial carcinoma:

and response

published

Author

Responders

et al. [376]

Benraad Creasman Kauppila

therapy

in endome-

series

PR+ Martin

to progestin

et al. [366] et al. [383] et al. [347]

Nonresponders PR-

PRf

PR-

49.0%

[388]

37

68.0%

[389]

82

56.1

57.3

45.0

64.4

Schwartz

[390]

101

Kuhnel[39

l]

94

55.0

54

46.0

Lantta

[392]

carcinoma ER+/PR+

52.0

4.0

90.0

41.0

26.0

55.4

53.3

27.0

31

52.0

48.0

32.0

Anderl[395]

51

63.0

38.0

44

68.8

35.0

100

52.0

40.0

Galli [398]

18

67.0

50.0

Quinn [399]

43

51.5

34.4

23.0

Spona [400]

68

58.8

39.7

32.4

Willcocks

49

57.1

29.0

20.4

Pollow [402]

28

65.0

39.0

39.0

Hamilton

19

32.0

Janne [404]

21

71.4

38.1

38.1 81.4

[396] [397]

[401] [403]

27.0 72.0

1

1

4

Richman

[405]

36

31.0

2

5

Kauppila

[406]

59

89.8

86.4

1

0 2

7

Jones [407]

81.0

43.0

36.0

1

2

16

Bizzi [408]

42 97

55.0

65.0

44.0

91.0

Pollow et al. [384]

0

0

13

Vierrko

(4091

51

89.0

0

7

13

Harding

[410]

89

52.8

39.3

32.5

Ehrlich et al. [352]

6

4

Rose [4 1 I]

123

61.0

28.0

16.0

McCarty

0

1

26 8

Total Total

45

11

20

92

80%

20%

18%

82%

Mean

1429 59.3+2.947.7*3.8

27.9

88.0

Quinn et al. [385] [362]

AR+

91.5

92

[393]

_

3.7

Iversen [394]

Toppila receptors

PR+

Slotman

Sutton

13

ERf

Masood

Friedlander

TABLE

in ovarian

34.4k4.2

121

can be seen that approx. 59.3% of tumors are ER positive (range:31.&89.8%) and 47.7% are PR positive (range 28.0%-91.0%). Approx. 31.9% of tumors contained both receptors. This is to be compared with findings for normal tissue or benign tumors which are summarized for seven series in Table 15. While there is a substantial range for both receptors in ovarian carcinoma, the value of ER is generally lower, whereas those for PR are more comparable, to those for other malignant tumors such as breast or endometrial carcinoma. Androgen receptors were measured in five series and found to be present, on the average, in 73.9% of cases. EGF receptor has also been identified in ovarian carcinoma. Kohler et al. [413] recently reported EGFR-binding sites were present in 36.0% of ovarian carcinomas. Increased expression of EGFR specific mRNA was detectable in 33.3% of ovarian carcinomas. A positive correlation between the amounts of EGFR mRNA and EGFR binding was also found. Histology

Several series have examined the relationship of histologic type of ovarian carcinoma to the presence of steroid receptors. Among 13 series, six found no correlation between ER and PR status and type of tumor [388,393,402,404,405,414]. Among the remaining seven series [392,396,399,406,411,412,415] it is difficult to make generalizations, however, in several cases endometrioid tumors often had greater amounts of ER, whereas mutinous tumors tended to have lower levels than other histologies. The results of the latter series are as follows. Ford et al. [415] reported a low incidence of any detectible ER- and PR-binding activity in mutinous and serous carcinoma of the ovary, whereas in eight patients with endometrioid carcinoma all had detectible ER and 4/S had PR activity. Rose et al. [41 l] found PR more frequently positive (53%) in tumors of endometroid his-

TABLE

I5

Distribution

of ER and PR in benign/normal

Series

n

ovarian

ER+

PR+

11

45.0%

36.0%

9

0.0

0.0

Pollow [402] Galli 13981

IO 5

30.0 40.0

60.0 20.0

Lantta

30

24.0

63.0

Willcocks [4011

32

22.0

Janne [404]

83.0

86.0

MeankS.E.

34.9q6f9.748.6%%111.7

Anderl[395] Agarwal

[412]

[392]

tissue

AR+

GR+

100.0

100.0

tology than with other histologic types @ = 0.01). Kaupilla et al. [406] found primary serous and endometrioid carcinoma had higher concentrations of cytolsolic ER than did mutinous tumors. Quinn et al. [399] found serous tumors were significantly more likely to be ER+ than mutinous tumors, but the incidence of PR and AR positivity was similar in serous and mutinous and endometrioid tumors. The mean ER content of serous tumors was significantly higher than that of endometroid tumors. Agarwal et al. [412] reported that serous tumors were more often ER (55.5%) and PR (88.9%) positive than mutinous tumors (33.3% and 33.3% respectively). Sutton et al. [396] found endometrial adenocarcinoma to be PR positive significantly more often (80%) then serous, mutinous, or undifferentiated histologic variants (20%), and had a significantly higher PR level @ < 0.02). Lantta et al. [392] found the following distribution for ER positivity according to histology: endometrioid, 63.6%; serous, 3 1.8%; mutinous, 50%; mesonephroid, 50%; and undifferentiated, 80%; for PR, the respective values were 45.5%, 54.5%, 33.3%, 33.3% and 60%. ln summary, among the series examining this question, at least half found no correlation, and among the remainder there does not appear to be any consistent pattern of distribution of ER or PR according to histology. Whether sampling or methodological problems contribute to this is not known. Histology is, therefore, not considered to be an important determinant of ER content in ovarian carcinoma. Stage

Among 14 series evaluating receptor content according to stage [363,388,393-396,400,405-409,410,416], eleven found no correlation between ER and PR content and stage, and one found no correlation for ER (only receptor examined). One series (Spona et al. [400]) found that patients with stage I,11 had a higher frequency of ER and PR (ER + PR + = 42.6%) than subjects who were stage III-IV (29.5%). In stage I and II the percentage of patients with detectible ER but undetectible PR was greater than in patients with higher stages. A second series (Harding et al. [410]) reported an inverse association between the proportion of PR-positive tumors and advancing stage, but no relationship between ER positivity and stage. Whether the findings in these two series are statistically significant was not stated. The overall findings from all series, however, would indicate that ER and PR content of ovarian carcinoma does not correlate with stage of disease.

15.0

Grade

The relationship of histologic grade to receptor content in ovarian carcinoma has been examined. Among

122

17 series [363,388,393,394,396,398,405,407-410,4144191 eleven of these found the ER and PR content to be independent of grade. The findings of the remaining six series are as follows: Schwartz et al. [416] in 1982 reported that, among 30 patients, ER-rich tumors were independent of grade, however, in a subsequent report in 1985 [414], for a larger series of 113 tumors, indicated that grade IV cancer had a statistically greater likelihood of containing ER (p=O.O3) than did lower grade cancers. Grade III tumor samples containing abundant (3 + or 4 + ) mitoses had a significantly greater number of ER - cancers (p =O.Ol) than did cancer containing none to moderate (O--2+) mitoses. Vierikko et al. [409] reported that the concentration of cytosol progesterone receptors were lower in the most anaplastic group compared with the more differentiated categories @ < 0.05). This was evident in the group of all epithelial carcinomas (including serous, mutinous and endometrial carcinoma analyzed separately). Vihko et al. [363] found a parallelism between the histopathologic differentiation of certain ovarian tumors and ER and PR. In anaplastic serous carcinoma PR was signifcantly lower (p < 0.01) than in differentiated tumors, and in anaplastic endometriod carcinoma ER (p~O.01) and PR (PC 0.05) were lower than in corresponding differentiated tumors. Friedman et al. [419] reported that grading correlated directly with cytosol PR and indirectly with ER. Ford et al. [415] found that one half of well differentiated serous tumors had ER, while none of poorly differentiated tumors had measurable binding. In serous carcinoma, PR was only detected in well differentiated lesions. Iverson et al. [394] noted that ER was found more often in highly differentiated malignant tumors (86%) than in poorly differentiated lesions. In summary, while the majority of series found no correlation between receptor content and histologic grade, the remainder indicate that the receptor content is higher in the well differentiated lesions, lower in the more anaplastic tumors. The latter findings are comparable to those for breast cancer. Menopausal status, age, parity, and race

A number of series have examined the relationship of menopausal status and age to receptor content in ovarian carcinoma. Among eight series evaluating age [388,395,396,400,405,407,410,416] seven found no correlation between ER and PR content and age. Spona et al. [400] reported a higher incidence of ER and PR positivity in patients >60 years of age (63%) than in younger patients (36@, however, this was not analyzed statistically. With regard to menopausal status, among eight series [391,392,395,396,402,408,412,410] six found no correlation. Agarwal et al. [412] analyzed 15 cases and noted that all eight postmenopausal women were

ER and/or PR positive vs. 317 premenopausal women women who were ER + or PR+. Pollow et al. [402] found among ER+ or PR+ cases (> 5 fm/mg prot) there existed a significant difference in receptor content between pre- and postmenopausal women. Jones et al. [407] and Masood et al. [388] found no correlation between receptor status and parity, and Masood et al. [388] found no correlation between race and receptor status. Menopausal status, age, and parity therefore do not appear to be important determinants of ER or PR content in ovarian cancer. Recurrent or metastatic ovarian carcinoma

In general, the receptor content of recurrent or metastatic ovarian carcinoma is lower than that of the primary tumor. Kauppila et al. [406] found the primary epithelial ovarian carcinomas were more often ER, PR positive (81% vs. 44%) and had higher receptor concentrations then recurrent epithelial carcinomas. Vihko et al. [363] found the concentration of ER, (60+ 11 fm/mg prot) and PR, (107 f 30 fm/mg) among primary epitheha1 tumors significantly higher than in recurrent tumors (22-L-7 and 27+30 fmol/mg protein, respectively, p < 0.05). Holt et al. [418] found 8/16 primary ovarian tumors were ER and/or PR positive vs. 33.3% of metastatic lesions. Richman et al. [405] reported that, if the primary tumor contained ER > 3 fmol/mg, so did the metastastic lesions. The primary tumor, however, contained more ER than did the metastases in lo/14 (71%) of cases. The ER of the primary tumor was lower than that of the metastases in 29% of patients. Rose et al. [41 l] noted synchronous and metachronous assays for ER and PR were in agreement in 60-79s of cases. Finally, Quinn et al. [420] reviewed eight cases and found that among two that were ER-, the metastases were also ER - , whereas in six cases which were ER + , the concentration of ER in the metastases was less in five and greater in one. For PR, among three cases which were PR - , the metastases were also PR - , whereas in four cases which were PR + , three metastases had lower levels of PR and one had greater levels. These findings indicate a reasonable degree of concordance for ER and PR content between primary and metastatic lesions, although perhaps lower than that for breast cancer. Discordance is usually represented by lower receptor content in the metastatic/recurrent lesion. These findings also indicate the importance of determining the receptor content, when possible, of recurrent lesions when treatment decisions are being made, rather than relying exclusively on information from the primary tumor. Prognosis

The ER and PR status of ovarian carcinoma has been

123

analyzed for its prognostic value in a manner analagous to that for breast carcinoma. Among 13 series in the literature [388-390,394,395,400,405,406,408,411,416,417, 4211 eight showed a positive correlation. That is, ER or PR positivity was found to correlate with better prognosis. Slotman et al. [421] found, among 100 patients with primary ovarian carcinoma followed for a mean period of 5.4 years, a better survival rate in PR (p 2 cm. Patients with ER+ and PR+ tumors survived longer than the group which was ER - PR - (n = 97 patients). Spona et al. [400] found, among 68 patients with stage I-IV ovarian carcinoma, ER/PR receptor positivity was greater among survivors (44.4%) than among those who died (21.791;). Schwartz et al. [390] reported that patients with stage I,11 disease whose tumor contained elevated levels of PR had improved survival (3 years = 83%) over tumors containing < 7 fm/mg (47%; ~~0.04). For patients with advanced ovarian carcinoma (stage III, IV), those with low cytosol PR (< 7 fm/ mg) had significantly longer survival (4 years= 82%) than those with high PR (> 7 fmol, 10%;p < 0.018). Slotman et al. [389], in an initial report, found no significant correlation between the presence or absence of ER, PR or AR and survival. Among PR+ patients, high levels of PR (> 50 fm/mg) were associated with greater survival (5 years= 85%) than < 50 fm/mg (50%, p 10 fmol/mg. Most series, therefore, indicate a prognostic benefit to the presence of ER and PR in ovarian carcinoma. Response to therapy

There is little published information on the relationship of receptor status to response to systemic therapy in ovarian carcinoma. There are no major clinical studies available in which patients with epithelial ovarian carcinoma have been treated with hormone manipulation and the results of endocrine therapy compared with

124

pretreatment ER and PR content of the cancer [388]. Rose et al. [41 l] recently reported on 123 patients with epithelial ovarian cancer. Positive ER, PR, or both did not predict response to chemotherapy, negative secondlook findings, or survival. Positive receptors did not predict hormonal response or disease stabilization. Two studies have examined the effect of therapy on receptor content in ovarian carcinoma. Richman et al. [405] and Sutton et al. [396] examined the effect of chemotherapy on receptor content. In the latter study, excluding endometrioid carcinoma, tissue from patients treated with chemotherapy (L-PAM, cytoxan or &platinum) contained less ER (8 fm/mg) and less PR (0) than tissue from untreated patients (32.9 and 85.1, respectively, p < 0.05). ER + tumors from three patients assayed before cytoxan or combination chemotherapy became ER negative after therapy. Richman et al. [405], however, found that in 314 patients who had specimens analyzed before and after chemotherapy, there was little difference between the levels of ER in the initial sample and in samples taken after l&32 months of chemotherapy. There was no discernible effect of treatment on the prevalence or amount of ER in metastases. In conclusion, the usefulness of receptor content to predict response to systemic therapy, and the effect of therapy on receptor content in ovarian carcinoma remain inmportant but unanswered questions. III-D. Cervical carcinoma

Several studies have reported the presence of steroid receptors in carcinoma of the cervix. Six series totaling 582 patients are summarized in Table 16. The average incidence of ER was 48.5% and PR, 43.5%. Potish et al. [426] found that squamous cell carcinoma had a greater proportion of ER positivity than did adenocarcinoma, carcinoma adenosquamous, or undifferentiated @=O.OOl). Martin et al. [422], however, found that adenocarcinoma of the cervix was more often ER+

TABLE 16 Steroid receptor Series

content

in cervical carcinoma n

ER+

PR

34.6%

4.1%

Martin

[422]

246

Yajima

[423]

30

43.0

Twiggs [424]

44

45.5

61.4

Hunter [425] Potish [426]

70 61

41.0 50.0

30.0 55.4

131

76.9

66.6

48.5%*6&I

43.5sk11.6

Dame

[427]

Mean k SE

ER, 0

L

I

:

c 50 Q ;1

! I .-------~

t

.i:j

ER,O

, , , , , , , , , ( , , , , , , , ,

0

6

12

18

24

30

36

42

48

54

Months

Fig. 15. Survival positivity survival

(>6

in cervical

fmol/mg

(p~O.05).

prot)

Reproduced

carcinoma

according

was associated

to ER status.

with improved

with permission,

Twiggs

ER

overall

L.L., et al.

[424].

than squamous cell carcinoma. Other groups [424,425] found no correlation between histologic type and receptor content. The receptor content in cervical carcinoma was found not to correlate with age [426], menopausal status [423,425,426], histologic grade [424426] or lymph node metastases [426]. Efforts have been made to correlate steroid receptor status with prognosis in carcinoma of the cervix. Twiggs et al. [424] found, among stage Ib cervical carcinoma patients, those that were ER+ had improved overall survival compared with those that were ER- (p < 0.05; Fig. 15). No statistically significant differences in survival were noted according to PR status. Potish et al. [426] found ER to be of prognostic value but only in premenopausal patients. Hunter et al. [425] found a weak @=0.063) correlation between the presence of PR and length of survival, but no correlation between ER, and survival. Martin et al. [422], however, reviewing 246 women with primary carcinoma of the cervix, found the survival curves for patients with ER + /ER - and PR + or PR- tumors virtually superimposable. Darne et al. [427] similarly found no evidence that PR status affected prognosis. In summary, the number of series examining the prognostic role of ERjPR in cervical carcinoma are limited and do not consistently indicate a useful correlation. Additional studies may clarify this relationship. EGF Receptor in cervical carcinoma

Battaglia et al. [428] reviewed 14 cases of carcinoma of the cervix and reported that 78.6% expressed EGF receptor. Pfeiffer et al. [429] studied 52 cases of squamous cell carcinoma of the cervix and found EGF receptor was significantly increased compared with normal tissues. EGF was also increased in patients with lymph node metastases, in whom survival was reduced. Ir-

125

respective of tumor stage, patients with a very high level of EGF receptor (> 100 fmol/mg protein) were more likely to have recurrences later or to die from disease: recurrence or death occurred in five of seven patients with high capacity and in two of 45 patients with low capacity. They felt level of EGF receptor was indicative of the level of biological aggressiveness. III-E. Leukemia Glucocorticoids have been used in the treatment of leukemia for over 40 years. Accordingly, there has been much interest in the nature and role of glucocorticoid receptors (GR) in this disease. GR has been identified in all major forms of leukemia, including ALL, CLL, ANLL, and CML and in both adult and pediatric populations. Stevens and Stevens [ 1061have presented an excellent review of series analyzing GR levels in leukemias according to histology. This indicates that the number of receptors per cell covers a wide range in any given series of patients, although the median number is usually 200&20,000 sites per cell. The dissociation constant (&) is usually in the range of l-20 nM. Whether these characteristics are similar to those found in the nonneoplastic state is not clear because the corresponding nonneoplastic cell, in which malignant transformation occurs, is not known and. therefore, comparisons can not be made [430]. There is evidence that glucocorticoid receptor (GCR) number in leukemia may vary with cell type as well as some biologic/clinical characteristics. Sherman et al. [43 1] found that the mean and median cytosolic receptor concentration in 12 patients with acute lymphoblastic leukemia lacking the standard B-cell or T-cell markers (‘null cells’) were approx. 4-fold higher than 23 other leukemic cell specimens. Yarbro et al. [432] reported GCR sites in null lymphoblasts ranged from 4096 to 21,869 sites/cell (median = 757 1), whereas 18 patients with T lymphoblasts had binding sites ranging from & 5887 (median =2173, p 1920 sites/cell) and those with low GR levels (< 1920 sites/cell). They concluded that determination of GR provides no reliable indicator for clinical response to regimens with glucocorticoid as a component in patients with CLL and immunocytoma [437]. Bloomfield [430] considered the glucocorticoid receptors in hematologic malignancy to be biologically normal, as determined by their molecular weights and ability to form cytoplasmic glucocorticoid receptor complexes. This is supported by the findings of Sherman et al. [431], who studied GCR from six normal subjects and 35 high risk leukemic patients. They found glucocorticoid receptor complexes of similar size and shape (e.g., analyzed with regard to stokes radius, sedimentation coefficient, molecular weight and axial ratio) in all clinical specimens tested. They concluded that intrinsic structural defects in the receptors are an unlikely explanation for the unresponsiveness of some types of leukemia to steroid therapy. Others, however, have suggested alterations in the GR may be present. McCaffrey et al. [438] analyzed DNA binding of GCR fractions separated by DEAE chromatography from 62 cases of acute leukemia. They found the patterns of 35 of 62cases were abnormal, and hypothesized that these individuals may not respond to glucocorticoid therapy. Distelhurst et al. [439] demonstrated that malignant cells from lo/25 patients with leukemia contained electrophoretically abnormal glucocorticoid receptor having a molecular weight of 55,000 in addition to the normal size receptor (M, = 97,000). This abnormality was not restricted to a particular type of leukemia. This 52,000 receptor fragment was derived from intact glucocorticoid receptor (97,000) by the action of serine proteases [440]. The presence of GR in leukemic cells has been used to predict the response of this malignancy to hormonal therapy or chemotherapy. Lippman et al. [441], in 1978, demonstrated that patients with high receptor levels tend to have ‘null’ cell ALL and a long remission duration. Patients with low receptor levels had T-lymphoblasts and a short remission duration. Patients with in-

126

termediate receptor levels had an intermediate and identifiable remission duration, regardles of cell type. They concluded that GR levels appeared to have clinical significance independently of age, WBC or cell type, and may represent a biological marker associated with other factors, which are related to chemotherapy response, such as rate of growth or biochemical differentiation. Iacobelli et al. 14421found a direct correlation between receptor level and response to combination chemotherapy. Forty-seven of 50 patients with leukemic cells containing more than 6000 receptor sites achieved remission, and 22 of 36 patients with cells containing less than 6000 receptor sites achieved remission. Quddus et al. [433] reported that among subtypes of ALL, within the early pre-b group 291 patients with a median receptor number of 9.9 x lo3 achieved remission, while 13 with a median receptor number of 4.8 x 1O3(p = 0.034) did not. Within pre-B and T-cell groups the distributions of receptor numbers for responders and nonresponders were not significantly different. They concluded that each immunological subtype has a characteristic receptor distribution. High receptor numbers within the null group is associated with the ability of the patient to achieve remission. Mastrangelo et al. [443] studied 19 children with ALL and found that the presence of even a substantial number of GR does not necessarily indicate a clinical response to corticosteroids. On the other hand, patients with a low number of glucocorticoid receptors, in agreement with the results of Lippman’s, appeared invariably resistent to a short term course of glucocorticoids. Bloomfield et al. [444] found that response to glucocorticoid therapy in adult ALL appeared to correlate with lymphoblast levels. Pui et al. [445], however, in a study of 193 evaluable children with ALL separated into ‘standard’ vs. ‘high’ risk, found receptor levels were not related to treatment outcome. They considered the GR level in childhood leukemia to have prognostic value, but was not an independent factor and its importance was related to the efficacy of treatment. Simmonsson et al. [436] found no correlation between GR content and clincial response. Leukemic cells have also been examined for the presence of other steroid receptors. ER, PR, and AR have been detected in these cells, however, their clinical significance is unclear. Rosen et al. [446] found ER in 73% of specimens from patients with CLL, with binding ranging from 4.3 to 43 1 fmol/mg. One patient was treated with tamoxifen and achieved a minor response. Dane1 et al. [447] found the following incidence of steroid receptors in ALL and ANLL, respectively: ER, 100% and 69%; PR, 38% and 32%; AR, 50% and 38%. Zaniboni et al. [448] found ER activity in 52% and PR activity in 26% of patients with CLL. No correlations

between ERjPR status and other parameters such as age, sex, stage, AR and GCR, lymphocyte count or plasma estradiol and progesterone levels were noted. Seventeen consecutive patients out of 23 with a known receptor status and one with an unknown receptor status had been treated with tamoxifen 30 mg/day for 3 months. No objective response was achieved. Several groups have evaluated the sensitivity of leukemic cells to glucocorticoids in vitro, however, the results have been conflicting. Crabtree et al. [449] studied cells from 36 patients with untreated ANLL and found in vitro responses to glucocorticoid in leukemic blasts in 26 of 28 cases. These responses varied from near complete cell killing to stimulation of proliferation. They felt these studies might be useful in identifying those patients with ANLL likely to derive benefit from steroid therapy. Homo et al. [450] found the degree of steroid action as well as the extent of spontaneous and dexamethasone-induced cell death may be related to the number of cells in the S phase of the cell cycle. But in a subsequent report they stated that there was no clear correlation between the level of GCR and the in vitro action of steroids in normal and neoplastic lymphoid tissue. Finally, Nanni et al. [451], examining cells from patients with either CLL, ALL, lymphosarcoma cell leukemia, ANLL, or CML found no clear relationship among GR pattern, in vitro sensitivity to glucocorticoids, and clinical hematologic parameters in myeloid leukemic bearing patients. III-F, Lymphoma

Our understanding of the role of glucocorticoid receptors in lymphoma is limited despite the fact that glucocorticoids are an important part of the treatment regimen for many of these tumors. The largest experience is that of Bloomfield et al. [452]. They have summarized their findings as follows: (i) all lymphomas appear to have measurable numbers of glucocorticoid receptors pretreatment. (ii) Lymphoma cells have significantly more receptors than lymphocytes from nonmaiignant lymph nodes. (iii) Among lymphomas there is a wide range of receptor number which is not predicted by histology. (iv) Tumor receptor levels obtained simultaneously from different tissues in the same patient often vary. (v) In vivo administration of glucocorticoid rapidly results in a fall in receptor levels in lymphoid tissue which persists for as long as 17 days, thus patients should not have received glucocorticoid therapy for at least 3 weeks before receptors are assayed. (vi) Tumor receptor levels at relapse are usually lower than at diagnosis in patients who have previously received glucocorticoid therapy and responded. (vii) Tumor glucocorti-

127

coid levels are significantly higher in patients who respond to a short course of glucocorticoid than they are in patients who fail to respond. Homo-Delarche [453] studied lymphocytes from normal lymph nodes and from patients with nonHodgkins lymphoma and found, on the average, higher numbers of binding sites from patients with lymphoma than in cells isolated from control lymph nodes (median values were 4110 and 2082 sites per cell, respectively). In addition, ‘null cell’ lymphomas, as in ALL patients, usually contained more receptors than did T-cell lymphomas. They also found that sequential investigations (at diagnosis and relapse) showed a decrease in the number of GR after treatment. Finally, Dane1 et al. [454] found NHL cells from several patients contained 19-327 fm/mg protein of androgen receptor with an average & of 0.29 nM. The ligandbound complex migrated as a 7S species on low salt, and as a 4s species on high salt, sucrose density gradients. The clincial significance of of these androgen receptors is unknown. III-G. Prostate

Prostatic carcinoma is an important hormone-dependent tumor. While several steroid hormone receptors have been identified in prostate carcinoma, the androgen receptor (AR) appears to be the most significant. Many groups have examined the AR content of prostatic tumors, and the methodological problems encountered in analysis of receptors in prostatic tissue have been reviewed [455]. These problems notwithstanding, a large body of information on AR content is available which has been important in the management of these patients. Among seven series totaling 202 patients, AR was detectible in 70.8 + 4.5% of cases (range 45.5-81.8s; Table 17). The values are rather consistent among the

TABLE

17

Incidence

of steroid receptor

Series

,1

Pertschuk Ekman

[456] [457]

positivity

in prostate

AR

77

81.8%

25

80.0

Castellanos

[458]

36

69.4

Gustaffson

[459]

16

75.0

carcinoma

ER

Wolf [460]

13

100.0

Harper

10

10.0

Habib Ermtage Kumar Total

[461] [462] [463] [464]

13

74.0

10.0

24

70.0 45.5

55.0 82.0

11 225

70.8 k 4.6

PR

92.3

series despite the fact that the source of tissue may have varied between prostatectomy vs. cold punch resectascope vs. hot loop resectascope. Whether the incidence of AR in prostatic carcinoma is increased over that in benign tissue is not clear. Jasper et al. [465] reported that the mean levels of AR of prostatic carcinoma (35.5 fm/ mg tissue) were greater than that of benign tissue (16.0 fm/mg, p< 0.01). Benson et al. [466] found the mean binding of AR in cytoplasmic and nuclear samples was significantly higher in patients with cancer than in those with BPH. Ermitage et al. [463] found no difference in distribution for AR between cytoplasmic and nuclear receptors in either BPH or in cancer. Kirdani et al. [467] reported no difference in ER or AR between BPH and prostatic carcinoma. Radman et al. [469] found no significant difference in the association or dissociation constants between BPH and carcinoma for cytoplasmic samples, however, nuclear samples from benign tissue had a greater dissociation constant and lower receptor content than that for prostatic cancer. The cytoplasmic receptor complexes prepared from normal tissue sedimented in the 8-9s range, whereas those from malignant tissue sedimented 50% in the 8-9s area and 50% in 4S area. Estrogen receptors have been identified in prostatic carcinoma tissue by some but not all laboratories. Wolf et al. [460] and Habib et al. [462] identified ER in 100% and 7496 of samples respectively. Benson et al. [466] found binding of estradiol was low or absent and did not appear to be a significant phenomena. Jasper et al. [465] found the mean ER content of malignant tissue (11 .O fm/mg) greater than that for benign tissue. Ermitage et al. [463] and Kirdan et al. [467], however, noted no difference in the levels or the prevalence of ER between benign or malignant tissue. Wolf et al. [460] exmained 13 patients for the presence of progesterone receptor and found a 92.3% incidence, not significantly different from that of benign tissue. Epidermal growth factor has also been detected in prostate carcinoma cells. In a study of 19 patients the expression of EGFR was significantly higher in benign prostatic hypertrophy than in carcinoma of the prostate [470]. In the latter, expression of EGFR varied according to histologic grade of the cancer: well differentiated tumors demonstrated more receptors (84 + 13 fmol/mg prot) than poorly differentiated tumors (22 f 5 fmol/mg protein, p < 0.01). Relationship of AR to stage and histology of prostate carcinoma

100.0

Several studies have examined the relationship of stage of prostatic carcinoma to androgen receptor content. Among five series, three found a positive correla-

128

tion and two did not. Benson et al. [466,358] found cytosolic AR content increased progressively from stage A through stage D (8.5 fmol/mg protein, 9.8, 15.0, 17.5, respectively, p