aromatase activity in receptor negative breast and endometrial

228
Exp Oncol 2003
25, 3, 228-230
Experimental Oncology 25, 228-230, 2003 (September)
AROMATASE ACTIVITY IN RECEPTOR NEGATIVE BREAST
AND ENDOMETRIAL CANCER
L.M. Berstein1, *, A. Kovalevskij1, A. Larionov1 ,
E. Tsyrlina1, D. Vasilyev1, T. Zimarina1, J.H.H. Thijssen2
1
Laboratory of Oncoendocrinology,
N.N. Petrov Research Institute of Oncology, St.Petersburg 197758, Russia
2 ASL Endocrinology, University Medical Center, Utrecht 3508 AB, The Netherlands
ÀÊÒÈÂÍÎÑÒÜ ÀÐÎÌÀÒÀÇÛ Â ÊËÅÒÊÀÕ ÐÅÖÅÏÒÎÐÍÅÃÀÒÈÂÍÛÕ
ÎÏÓÕÎËÅÉ ÌÎËÎ×ÍÎÉ ÆÅËÅÇÛ È ÝÍÄÎÌÅÒÐÈß
Ë.M. Áåðøòåéí1, *, A. Êîâàëåâñêèé1, A. Ëàðèîíîâ1,
E. Öûðëèíà1, Ä. Âàñèëüåâ1, T. Çèìàðèíà 1, Äæ. Ã.Ã. Òèññåí2
1
Ëàáîðàòîðèÿ îíêîýíäîêðèíîëîãèè, Íàó÷íî-èññëåäîâàòåëüñêèé èíñòèòóò îíêîëîãèè
èì. ïðîô. Í.Í. Ïåòðîâà, Ñàíêò-Ïåòåðáóðã, Ðîññèÿ
2
Ëàáîðàòîðèÿ ýíäîêðèíîëîãèè, Ìåäèöèíñêèé öåíòð óíèâåðñèòåòà, Óòðåõò, Íèäåðëàíäû
Among the factors for estrogen and progesterone receptors (ER and PR) negativity of tumors of reproductive
tissue special attention is attracted by ability of the tumor produce estrogens (as intratumoral regulators of ER and
PR) through reaction of aromatization. 101 samples of tumor tissue (64 cases of breast cancer and 37 cases of
endometrial carcinomas) mostly from postmenopausal women were studied. When combined group of the tumors
was divided on the basis of receptor-negativity or positivity (with cut-point correspondingly ≤ and > 10 fM/mg
protein) among samples with higher aromatase activity (> 7 fM/mg protein/h), a tendency to higher number of
ER-negative tumors was found (p = 0.07). Such association was significant for breast (p = 0.04) but not for
endometrial cancer (p > 0.5) samples. No evidence of PR contents dependence of tumor aromatase activity was
revealed which may be connected with disturbance of estrogen signal transfer. Thus, inverse relation between
steroid receptor levels and aromatase activity may be tissue- and receptor type-dependent.
Key words: aromatase, estrogen receptors, progesterone receptors, breast cancer, endometrial cancer.
Ñðåäè ôàêòîðîâ, ñïîñîáñòâóþùèõ âîçíèêíîâåíèþ ðåöåïòîðíåãàòèâíûõ íîâîîáðàçîâàíèé ðåïðîäóêòèâíîé ñèñòåìû,
îñîáîå âíèìàíèå ïðèâëåêàåò ñïîñîáíîñòü îïóõîëåâîé òêàíè ïðîäóöèðîâàòü ýñòðîãåíû (êàê âíóòðèîïóõîëåâûå
ðåãóëÿòîðû ýêñïðåññèè ðåöåïòîðîâ ýñòðîãåíà è ïðîãåñòåðîíà) íà îñíîâå ðåàêöèè àðîìàòèçàöèè àíäðîãåííûõ
ïðåäøåñòâåííèêîâ. Â íàñòîÿùåé ðàáîòå èññëåäîâàëè 101 îáðàçåö îïóõîëåâîé òêàíè ïðåèìóùåñòâåííî îò áîëüíûõ
ðàêîì ìîëî÷íîé æåëåçû (n = 64) è ýíäîìåòðèÿ (n = 37) â ïîñòìåíîïàóçàëüíûé ïåðèîä. Ïîñëå ðàçäåëåíèÿ âñåõ
îïóõîëåé íà îñíîâàíèè îòñóòñòâèÿ èëè ïðèñóòñòâèÿ â íèõ ðåöåïòîðîâ (ñ ãðàíèöåé íà óðîâíå 10 ôÌ/ìã áåëêà) â
íîâîîáðàçîâàíèÿõ ñ áîëåå âûñîêîé àêòèâíîñòüþ àðîìàòàçû (> 7 ôÌ/ìã áåëêà/÷) áûëî îáíàðóæåíî áîëüøåå
÷èñëî ÝÐ-íåãàòèâíûõ îïóõîëåé. Òàêàÿ çàêîíîìåðíîñòü äîñòèãàëà ñòàòèñòè÷åñêîé çíà÷èìîñòè â îïóõîëÿõ ìîëî÷íîé
æåëåçû, íî íå áûëà ñâîéñòâåííà íîâîîáðàçîâàíèÿì ýíäîìåòðèÿ. Çàâèñèìîñòè óðîâíÿ ÏÐ îò àêòèâíîñòè àðîìàòàçû
â îïóõîëåâîé òêàíè îáíàðóæåíî íå áûëî, ÷òî ìîæåò áûòü àññîöèèðîâàíî ñ íàðóøåíèÿìè â ïåðåäà÷å ýñòðîãåííîãî
ñèãíàëà. Èíâåðñíûå âçàèìîîòíîøåíèÿ ìåæäó ýñòðîãåíïðîäóöèðóþùåé ñïîñîáíîñòüþ îïóõîëè è ñîäåðæàíèåì â
íåé ñòåðîèäíûõ ðåöåïòîðîâ ìîãóò, òàêèì îáðàçîì, áûòü òêàíå- è ðåöåïòîðñïåöèôè÷íûìè.
Êëþ÷åâûå ñëîâà: àðîìàòàçà, ðåöåïòîðû ýñòðîãåíîâ è ïðîãåñòåðîíà, ðàê ìîëî÷íîé æåëåçû, ðàê ýíäîìåòðèÿ.
Steroid receptor status remains the most reliable
predictor of response to hormonal therapy both in breast
and endometrial cancer [1, 2]. Receptor-negative cancers of breast and endometrium make-up at least 30–
40% of all these tumors cases [3, 4] and demonstrate
significantly worsier prognosis in comparison with receptor-positive observations [1, 2]. This explains an
importance of the evaluation of possible factors which
ma y influence contents, initial presence or loss of steroid receptors in tumor tissue.
Received: July 9, 2003.
*Correspondence:
Fax 7-812-596-8947;
E-mail: levmb@endocrin.spb.ru
Abbreviations used: ER — estrogen receptors; PR — progesterone receptors.
Generally, two hypotheses have been raised about
the relationship between steroid receptor-positive and
receptor-negative cancers. One hypothesis considers
receptor, and namely estrogen (ER) or progesterone
receptor (PR), status as an indicator of a different stage
of the disease. The other regards R-positive and
R-negative tumors as different entities [5]. Although
the latter possibility suggests that receptor status of a
tumor is determined early in its natural history [6] at the
same time it underlines also the need for search of the
factors of hormonal nature under which action receptor
status of the neoplasm can be changed into positive or
negative one.
Estrogens should be considered among such factors first of all due to their ability influence inversely
Experimental Oncology 25, 228-230, 2003 (September)
et al [16] with minor modifications [17]. The receptor activity was expressed as fM/mg protein. Protein content
was determined by the Lowry method.
Statistical analysis was performed by methods allowing for means and standard errors. The significance
of the differences between the groups was tested using
xi-square approach by computerized program (SigmaPlot). The differences with p ≤ 0.05 were considered
as significant.
Individual values of aromatase activity in studied
breast and endometrial cancer samples varied between
0 and 41.7 fM/mg protein/h. Analysis of the distribution
of individual variants revealed that median of aromatase
activity in collected material was equal 7.2 fM/mg protein/h. According to this, further analysis of the association with steroid receptor content was performed in
groups with aromatase activity levels below and higher
than 7 fM/mg protein/h (Table, Figure).
As shown in Table, among combined group of breast
and endometrial carcinomas with aromatase activity
> 7 fM/mg protein/h incidence of appearance of estrogen receptor-negative tumors (with ER content
< 10 fM/mg protein) demonstrated tendency to decrease
in comparison with cancers characterized with lower level
of estrogen-producing activity (p = 0.07). Incidence of
progesterone receptor-negative and PR-positive tumors
in this material did not differ and actually was practically
the same (p > 0.9). These observations were confirmed
also by χ2-coefficients values which were equal correspondingly 2.96 (for ER) and 0.004 (for PR).
At the same time, when groups of breast and endometrial carcinomas were evaluated separately the following picture emerged. In breast cancer group ER-negative tumors appeared in 12 of 33 cases when aromatase activity was higher that 7 fM/mg protein/h and
in 5 of 31 cases when this activity was < 7 fM/mg protein/h (χ 2 4.47, p = 0.04; see Figure). Progesterone re30
χ 2 4.47
χ 2 2.03
25
Number of cases
expression of ER and PR in breast cancer cell culture
[7]. Suppressive effect of estrogen on ER expression
and content depends though of estrogen concentration and several other conditions and it is of interest
that long-term estrogen replacement therapy in menopause not decreases but increases number of ER-positive tumors [8]. On the other side, although PR is as a
rule induced by estrogens and is regarded as a marker
of estrogen-responsiveness, decrease in tumor PR
content may be related not only to estrogen deficiency
but also to defects in estrogen signal transduction [7].
Intratumoral estrogenic influences on ER and PR
expression and content can be important as well. In regard to this it should be mentioned that estrogen-producing ability of tumor, or in other words its aromatase
activity, was studied so far rather rarely as a determinant of receptor positivity or negativity. Besides, although
conclusions which have been made were not monotonous [9–11], in two papers [12, 13] negative association
between ER content and aromatase activity in breast
cancer tissue was revealed. On the other side, in only
data we are aware of related to endometrial cancer [14]
no connection between steroid receptor content and
activity of aromatase in tumor tissue was discovered.
Taking into account above mentioned information
we evaluated changes in aromatase activity characteristic for receptor-negative breast and endometrial
cancer.
Totally tumor samples from 101 patients (64 with
breast cancer and 37 with endometrial cancer) have
been evaluated. 87 patients (or 86.3%) had been postmenopausal for at least 1 year. The majority of the patients were at clinical stage I-II. 92% of breast tumors
have had size T1–2, 60% did not demonstrate damage of regional lymphatic nodes. Among endometrial
cancers, morphologically only endometrioid adenocarcinomas mostly in grades G1–2 of differentiation were
present in this material. Collected tissue samples were
immediately transferred into laboratory and were placed
into liquid nitrogen for further processing.
Aromatase activity in tumor tissue was estimated by
measuring 3H2O release from 3H-1-β-androstenedione
(NEN, Boston, USA; specific activity, 25.4 Ci/mM) as described [15]. Briefly, the reaction mixture (which contained
tumor homogenate, NADPH regeneration system and
labeled androgenic precursor) was incubated for 2 h at
37 °C. Then reaction was stopped by adding 5 vol of cold
chloroform, and 5% suspension of activated charcoal
(Norit A) was added to the water phase. The fraction containing 3H2O was separated by centrifugation, and counting
was performed with dioxane scintillator. Results were presented in fM/mg protein/h. Estrogen and progesterone
receptor contents in tumor tissue were evaluated by the
dextrane-charcoal radioligand assay according to Saez
229
20
15
10
5
0
ER(–)
ER(+)
Arom <
PR(–)
PR(+)
Arom >
Figure. Distribution of receptor-negative and receptor-positive
cases in regard to aromatase activity in the group of studied breast
cancers. χ2 -coefficient demonstrates significant difference when it is
≥ 3.84. ER (–) and PR (–): ≤ 10 fM/mg protein; ER (+) and PR (+):
> 10 fM/mg protein; Arom <: less than 7 fM/mg protein/h; Arom >:
equal or higher than 7 fM/mg protein/h
Table. Incidence of recept or-negative and receptor-posit ive cases among breast and endometrial cancers wit h different aromat ase activ ity
Parameter
Aromatase activity is lower ( ≤ 7 fm/mg protein/h)
Aromat ase activit y is higher (> 7 fm/mg protein/ h)
ER
PR
ER
PR
> 10 fM
> 10 f M
> 10 fM
> 10 fM
≤ 10 fM
≤ 10 f M
≤ 10 fM
≤ 10 fM
Number of cases
11
37
26
30
16
27
20
25
Incidence of R(–) tumors,%
22.9 ± 6.1*
46.4 ± 6.6
37.2 ± 7.3*
44.4 ± 6.6
*p = 0.07.
230
ceptor-negative breast cancers were revealed correspondingly in 13 of 33 cases (aromatase activity
> 7 fM/mg protein/h) and in 8 of 33 cases (aromatase
activity < 7 fM/mg protein/h), χ 2 2.03, p = 0.09. In endometrial cancer group distribution of ER-negative and
ER-positive cases as well as PR-negative and PR-positive cases in tumors with aromatase activity higher or
lower than median value was practically equal, and this
was confirmed by corresponding χ2 values (for ER
χ 2 0.43, p > 0.4; for PR χ2 1.45, p > 0.1).
In summary, it may be concluded that association
between estrogen-producing activity of studied tumors
and their steroid receptor content is characterized according to received data by receptor- and tumor-speci ficity: namely, reciprocal relationships between these
parameters are revealed only in relation of estrogen (and
not progesterone) receptors and in regard of breast (and
not endometrial) cancer. Thus, higher rates of intratumoral estrogens produced through aromatase mechanism can lead to inhibition of estrogen receptor expression first of all in breast cancer tissue apparently due to
higher importance of ER’s in breast than in endometrial
cancers [2, 4, 6]. Absence of significant relationship bet ween aromatase activity and progesterone content independently of tissue context demonstrates, as it seems,
that estrogen dependence of PR in cancer tissue is lost
more fast (that it appears in regard of ER) although does
not explain, at the same time, why ER(+),PR(–) cancers
are more frequent than ER(–), PR(+) ones [18]. The latter observation may be explained by other reasons, including mechanisms related to so called phenomenon
of switching of estrogen effect [19].
ACKNOWLEDGEMENTS
These studies were partly supported by grants from
RFBR (04-49282), INTAS (01-434) and MS (program 29).
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