De novo expression of aromatase in gastric carcinoma

Path. Res. Pract. 188, 53-60 (1992)

De N ovo Expression of Aromatase in Gastric Carcinoma Light and Electron Microscopic Immunohistochemical and Immunoblot Study y. Saitoh 1,2, H. Sasano2 , H. Naganuma2 , H. Ohtani 2 , N. Sasano2 , A. OhuchP and S. Matsuno 1 1First Department of Surgery, 2Second Department of Pathology, Tohoku University School of Medicine, Sendai, Japan

SUMMARY We have performed immunohistochemical and immunochemical studies of steroidogenic enzymes involved in es trogen biosynthesis in 30 eases of gastric carcinoma in order to investigate possible in situ production of estradiol (E 2) in carcinoma cells. Positive incidence of immunoreactivity for E 2, testosterone (T), eholesterol side-ehain cleavage enzyme (P-450 sec) and aromatase (P-450 arom) were 17/30 (56.7%), 11/30 (36.7%), 3/30 (10.0%) and 23/30 (76.7%), respectively on light mieroscopy. Estrogen reeeptor (ER) immunoreactivity was not observed in any of the 30 eases examined. Normal gastrie mueosa was negative for P-450 arom and P-450 sec. Examination of serial seetions revealed that immunoreactivity of E 2 and P-450 arom were loeated in the same cells of earcinomatous glands. Immunoelectron microseopy demonstrated that E 2 and P450 arom were loeated along the membrane and cisternae of smooth endoplasmie retieulum (sER). Western blot analysis showed one major band of 55 kDa of P-450 arom in the gastrie eareinoma tissues examined. Retrospective analysis of immunohistoehemistry of E 2 in 108 eases of gastrie carcinoma revealed that E 2 positive carcinoma eases were likely to demonstrate better survival rate than negative eases. These results above strongly suggest that E 2 is produeed by de novo expressed aromatase in gastric carcinoma cells and is possibly involved in the biology of gastric carcinoma cells.

Introduction

Estrogen and the estrogen receptor have been well known as important factors for carcinogenesis and growth of breast and endometrial cancer. Estrogen receptor in human breast carcinoma was frist demonstrated by Jensen et al. in the 1960s 7, and many studies have reported estrogen receptors in various kinds of human or animal carcinomas of so-called non-targeting organs such as stomaeh, colon, pancreas and kid ney 1,14,15,27,29, Recently, immunohistochemical studies of estradiol (E 2) and biochemical assay of estrogen receptor (ER) in gastric carcinoma have been reported 21 ,30,32, Immunohis© 1992 by Gustav Fischer Verlag, Stuttgart

tochemical methods of steroid hormones themselves have many technical problems including the fixation and preservation of steroid hormones through the process. In addition, immunoreactivity detected by this method may indicate steroid hormones bound to protein as well as those produced in the cells 13, but despite these problems all previous studies indicated that immunoreactive E 2 in gastric carcinoma cells represents receptor-bound E 22 l,30,32, From these observations, anti-estrogenic therapy was proposed and has actually been performed on gastric carcinomas in some institutes ll ,12, However, epidemiologie study did not show any relationship between immunoreactivity of E 2 and prognosis of gastric carcino0344-0338/92/0188-0053$3.50/0

54 . Y. Saitoh et al.

ma 30 • Although receptor-bound E 2 exists in the nudeus, results of previous reports studying immunohistochemical localization of E 2 demonstrated cytoplasmic stain rather than nudear stain in gastric cancer21 ,30. Therefore, immunoreactivity of E 2 may not represent receptor bound E 2 in gastric carcinoma. Therefore in the present study, we investigated the immunohistochemical localization of E 2, testosterone, which is the precursor of E 2, estrogen receptor, aromatase, which is the keyenzyme for synthesizing E 2 and cholesterol side-chain deavage enzyme, which is the enzyme of the initial step of steroidogenesis both at light and electron microscopic level in order to study what immunoreactive E 2 represents in gastric carcinoma. We have also re-examined the relationship between the presence of immunoreactive E 2 and dinical behaviors in 108 cases of gastric carcinoma. In addition, we performed immunoblotting study of aromatase in fresh frozen tissue of gastric carcinoma to confirm the results of immunohistochemistry.

Material and Methods Material Fresh specimens were obtained from surgically resected thirty gastric carcinomas (one early case and 29 advanced cases) and were used for immunohistochemical analysis of E 2, T, ER, p-450 sec and p-450 ar'Üm. All the patients were Japanese. Sex andhistology of each casewere shown in Table 1. In addition, one hundred and eight cases of gastric cancer, which were fixed in 10% formalin and embedded in paraffin, were retrieved from surgical pathology files of the Department of Pathology, Tohoku University School of Medicine in order to study the relationship between immunoreactive E 2 and survival rate. All these ca ses were also Japanese and operated at the 1st Department of Surgery, Tohoku University School of Medicine, from 1975 to 1982. Sex and histological type of these ca ses were shown in Table 2. All ca ses of gastric cancer were histopathologically classified according to the general rules proposed by the Japanese Research Society for Gastric Cancer8,9 and the Lauren's classification l8 .

Antibodies. Antibodies to estradiol and testosterone were purchased from Cambridge Medical Diagnosis (USA), and antibody to estrogen receptor was from Abott Laboratories (North Chicago, IL). Antibodies to P-450 SCC24-26 were generously provided by Prof. Mitsuhiro Okamoto (Osaka University, Jpn) and antibody to P-450 arom 20 ,24,25 was kindly provided by Prof. Evan R. Simp-

Table 1. Histological feature of 30 ca ses of gastric carcinoma which were used for immunohistochemical analysis Histological feature

Male

Intestinal type Diffuse type

12 6

4 8

16 14

Total

18

12

30

Female

Total

Table 2. Histological feature of 108 gastric carcinomas which were examined for clinical survival rate. One hundred and eight ca ses consisted of 35 early and 73 advanced gastric carcinomas. Mean age of the ca ses were 55.9 years old for males and 50.6 years old for females respectively. These cases were examined for relationship berween immunoreactive E 2 and survival rate Histological feature

Male

Female

Total

Intestinal type Diffuse type

29 43

7 29

72

Total

72

36

108

36

son (University of Texas Southwestern Medical School, Dallas, USA).

Methods Fresh specimens from surgically resected gastric cancer were immediately processed in three ways as folIows: 1) For light microscopy, specimens were cut into 20 X 10 X 15 mm in size, and were fixed in 10% formalin and/or periodate lysine paraformaldehyde solution (PLP) (McLean and Nakane, 1974)19 for 1-3 days. After dehydration they were embedded in paraffin; 2) For immunoelectron microscopy, specimens were cut into 5 X 5 x 2 mm in size, fixed in PLP for 6-8 hrs at 4°C and were then washed in phosphate-buffered saline (PBS) containing 10%, 15% and 20% sucrose. They were embedded in O.C.T. compound (Miles Inco., Elkhart, USA) and frozen in dry ice-ethanol. They were stored at -70°C until use. 3) For immunoblot study, specimens were immediately cut into sm all pieces (1 cm x 1 cm x 1 cm) and stored at -70°C until analysis.

Light Mieroseopic Immunohistochemistry for P-450 sec, P-450 arom, E 2 and T After deparaffinization, sections 2.5 ~tm in thickness were immersed into methanol containing 0.3% hydrogen peroxide for 30 minutes to block the endogenous peroxidase activity. Immunostaining was performed with Biotin Strept-Avidin kit (Bio. Genex Labo., USA). Primary antibodies against P-450 sec, P-450 arom, E 2 and T were diluted at 1 : 500, 1: 100,1: 2000, 1: 1000 respectively, with incubation time for 18 h at 4 oe. Methanol and deparaffinization process employing xylen and methanol are considered to remove free estradiol and testosterone. For negative control of the staining, the primary antibodies were replaced by non-immune serum and/or PBS and specificity of immunostain was confirmed. For positive control of immunostain, human full-term plaeenta and human normal ovary were used in P-450 arom and E 2 and human normal testis were employed in T and P-450 scc.

Immunohistoehemistry for ER Frozen materials fixed in PLP were used as was described previously, and four-micron seetions were cut with a cryostat and mounted on valbumin-coated glass slides. Peroxidase-antiperoxidase methods were carried out using ER-ICA kit (Abort labo., USA). For the negative control, the primary antibody was replaced by non-immune serum and/or PBS solution. For positive controls, breast tissues adjacent to breast carcinoma were used, which were processed in the same way as was described above.

Estrogen synthesis in gastric carcinoma . 55

Immunoelectron Microscopy for P-450 arom and E 2 Six ca ses which showed positive staining for both P-450 arom and E 2 by light immunohistochemistry were examined by pre-embedding method. After immersion in 10% normal go at serum, the cryostat sections (6 11m in thickness) were incubated with the ptimary antibodie~ (P-450 arom 1: 100, E 2 1 : 2000) for 24 h at 4 °C and then with peroxidase conjugated Fab fragment of antirabbit IgG (1: 200) (Cappel, Burlingam) for 18 h at 4 oe. They were washed in PBS in each step. The sections were postfixed with 1 % glutaraldehyde for 5 minutes, washed, and subsequently incubated in 0.03 % 3,3' -diaminobenzidine (DAB) solution without hydrogen peroxide for 20 minutes followed by DAB solution containing 0.005% hydrogen peroxide for 3-6 minutes. After washing, the sections were postfixed with 1 % osmium tetroxide in PBS for 30 minutes, dehydrated in a graded ethanol series and embedded in Epon 812. The ultrathin sections were stained with lead citrate for one minute. For the specificiry control, the primary antibodies were replaced by non-immune serum and/or PBS.

Immunoblot Study for P-450 arom in Gastric Carcinoma Five cases with positive stainings of P-450 arom were chosen among stored tissues. The frozen tissues were homogenized and microsomal fractions were obtained by successive centrifugation. Finally, microsomal fractions were resuspended in buffer and 35 ~lg of protein was applied to one lane of 10% polyacrylamide gel containing sodium dodecylsulphate electrophoresis (SDSPAGE). For contral, microsomal fraction from human placental tissue were applied simultaneously. Protein concentration was measured by the method of Lowry et a1. 17 • The molecular markers used were obtained from Sigma Chemical Co. (USA) and consisted of carbonic anhydrase (29 kDa), egg albumin (45 kDa), bovine albumin (66 kDa), phosphorylase (97.4 kDa), and beta galactosidase (116 kDa). After SDS-Page, sampies were transferred to nitrocellulose membrane and reacted to anti-P-450 arom (diluted 1: 100) for 18 hat 4 oe. Then, immunoperoxidase methods were carried out using avidin strept-avidin kit. Molecular weight was calculated using molecular weight standard curve.

Table 3. Positive incidence for E 2, Testosterone, P-450 scc and P-450 arom Scc

Aro

n

E2

T

Male

18

11 (61.1)

9 (50.0)

1 (5.5)

13 (72.2)

Female

12

6 (50.0)

2 (16.7)

2 (16.7)

10 (83.3)

Total

30

17 (56.7)

11 (36.7)

3 (10.0)

23 (76.7)

E 2 = estradiol, T = testosterone, SCC = P-450 scc, Aro = P-450 arom (%).

of one well, one moderately, four poody differentiated adenocarcinomas and one undifferentiated carcinoma. Examination of consecutive serial sections revealed that immunoreactive E 2, T and P-450 arom were located in the same cells of the carcinomatous glands (Fig.2). No staining of ER was observed in any of the 30 cases of gastric carcinomas. Positive results were obtained in the nuclei of mammary ductules in simultaneously performed immunohistochemical analysis of the breast (Fig. 3). Correlation of immunoreactivities among P-450 arom, T and E 2 were as shown in Table 4. Of 23 positive cases for PA50 arom, 9 ca ses (male 7, female 2) showed positive staining for both E 2 and T. Eight cases (male 4, female 4) were negative for E 2 and T. On the other hand, among seven cases negative for PA50 arom, 4 cases (male 2, female 2) showed negative immunoreactivity for E 2 and T.

Results Light Microscopic Immunohistochemistry

Immunoreactivity for E 2 was observed diffusely in cytoplasm of tumor cells forming glandular structure in 17 of 30 cases (Fig. 1). No obvious immunoreactivity was observed in the nucleus of the cells. Immunoreactivities for T, PA50- scc, and P-450 arom were also observed in similar pattern. As was shown in Table 3, positive incidence for E 2, T, P-450 scc and P-450 arom were 17/30 (56.7%), 11/30 (36.7%), 3/30 (10.0%) and 23/30 (76.7%), respectively. Positive staining for T was found in only two (16.7%) of 12 female ca ses and in 50.0% of the male cases. The histological types of gastric cancers in P-450 scc positive ca ses were all differentiated adenocarcinomas, either weil or moderately differentiated type, which also showed positive immunoreactivity for the other antibodies. In contrast, s-even cases which were negative for P-450 arom immunohistochemically were composed

Fig. 1. Immunoreactiviry for E 2 in gastric carcinoma. Immunoreactiviry for E 2 was observed diffusely in cytoplasm of tumor cells forming glandular structure (x 80).

56 . Y. Saitoh et al.

Fig. 2. Immunoreactivity für E 2, Testosterone and P-450 arom. A: E 2, B: Testosterone, C: P-450 arom. Examination of consecutive serial sections revealed that immunoreactive E 2, T and P-450 arom and were located in the same ceUs of the carcinomatous gland (x 80).

Immunoelectran Microscopic Study for P-4S0 aram and E 2

The positive immunoreactivity for P-450 arom was observed on the membrane and cisternae of smooth

•

• •••

endoplasmic reticulum (sER) (Fig. 4), and that for E 2 was also located along the membrane of sER. Immunoblot Study for P-450 arom in Gastric Carcinoma

Six cases with positive staining of P-450 arom were examined. One major band of 55 kDa was found in each sampie of gastric carcinoma tissue (Fig. 5). Same minor bands were also observed . Correlation Between Staining for E 2 and Pragnosis in Gastric Carcinoma

In both curatively resected cases and advanced cases, E 2 positive group showed better survival rate than E 2 Table 4. Correlation of immunoreactivities among P-450 arom, Testosterone and E 2

Aro (+) (n = 23)

Aro (- ) 01 = 7) Fig. 3 . Immunoreactivity for ER. Right: Mammary gland, left: Gastric carcinoma. No staining of ER was observed in any of the 30 gastric carcinomas, whereas positive staining was obtained in thc nuclei of mammary ductules (x 80).

Aro

=

+

+

P-450 arom, E 2

=

T

Male

Female Total

+

7 1

2 4

9 5

+

1 4

0 4

8

+

1 2

0 0

1 2

+

0 2

0 2

0 4

estradiol, T

=

testosterone.

1

Estrogen synth esis in gastric carcinoma . 57

Fig.4. Immunoelectron microscopic study for P-450 arom and E 2 in gastric carcinoma. Right: E 2 (x 22,500), left: P-450 arom (x 50,000). The positive immunoreactivity for P-450 arom and E 2 were observed on the sER.

negative one (Fig. 6). Statistically significant differences of survival rate were observed only in the patients with curative resection between E 2 positive and negative cases (p < 0.05). Discussion Estradiol is synthesized by aromatase in the ovary and placenta but also in peripheral non-steroid producing

Immune Blot

1

2

3

4

5

PL

&&-kDa-

Fig.5. Immunoblot srudy for P-450 arom in gastric carcinoma. Lane 1 to 5 represents cases oE gastric carcinoma. One major band of 55 kDa was fOllnd in e ach sampIe of gastric carcinoma tisslle.

tissues including adipose tissue 3 . Some studies previously described immunoreactivity of E 2 in gastric carcinoma and that E 2 positivity has been demonstrated to be associated with favorable clinical outcome 21 ,29,30. Similar results were also obtained in OUf stlldy. Therefore, it is important to know what immunoreactive E 2 represents in the gastric carcinoma cells. Previous immunohistochemical studies of E 2 proposed that E 2 immunoreactivity represented receptor-bound E 2 21 , 30 and several reporrs showed occllrrence of es trogen receptors in gastric cancerslI, 12,27,29. However, a11 previous reports showed positive staining for E 2 in the cytoplasm of gastric cancer, which indicates the fact that immunoreactive E 2 in gastric carcinoma is not necessarily receptor-bound form, because estrogen receptor has been reported to exist in the nucleus of many tissues both biochemically and immunohistochemically2, 5, 10, 17, 18,23,3 1. In addition, our present investigation revealed no estrogen receptor immunoreactivity in any of the 30 cases of gastric carcinomas including the cases which showed immunoreactivity of E 2. This demonstrates that gastric carcinomas have no or undetectable es trogen receptor and reported immunoreactivity of E 2 is by no me ans associated with receptor-bound E 2. Therefore we studied immunoreactivity of aromatase, and cholesterol side-chain c1eavage enzyme to study whether observed immunoreactivity of E 2 represents E 2 synthesized locally in the gastric carcinoma cells. Estrogen is generally the final product in the pathway of ovarian steroidogenesis (Fig. 7) and if gastric carcinoma produces estrogen from cholesterol as in the ovary, the carcinoma celk should express cholesterol side-chain cleavage enzyme, which is an initial step of ovarian steroidogenesis. On the other hand, aromatase catalyzes the final step of estrogen biosynthesis but is present in non-steroid producing tissues associated with peripheral conversion of testos-

58 . Y. Saitoh et al. ........ E,(-)

. .t.

10'

..

-~-

!O

6- -

_'.l. - - - - - - - - - _L-L-L..

-1 ____

.J.'W~ U

Ja

51

50

CD

CD

3D

30

20

2.

..

"

c loaD

lUD

E,(+)

(n =44)

!O

11

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().--Q

.. 10

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........ E,(-)

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(n =11) • p
, 11

a

Stage 3,4

SURVIVAL RATE (%)

Ed+)

(>,--0

CURATIVE RESECTION

SURVIVAL RATE (%)

''''

4...

200'

3000

OAYS AFTER GASTRECTQMY

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-4000 OAYS AFTER GASTRECTOMY

........ E,(-) E,( +)

0---0

SURVIVAL RATE (,,)

(n=41)

....

IO'

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b...-' ____ ...JUl1.U

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Fig. 6. Correlation betwecn staining for E 2 and prognosis. Survival rate differences between E 2 positive (0---0) and negative (e-e) group among curatively resected (a), Stagc 1, 2 (b) and Stage 3,4 (c) cases. In curatively resected cases and advanced cases, E 2 positive group showcd better survival rate than E 2 negative one. Methods of obtaining survival curves and rates of the patients and definition of "curative resection " and "advanced cases" were based on the general rules proposed by the ] apanese Society for Gastric Cancer8,9 .

5. 4. 30

2.

"

b

1000

mo

31..

4000 OAYS AFTER GASTRECTOMY

Cholesterol

L -__

~

____

~

________

C 17-

17 er - hydroxylase

~

2ll lyase

steroids 17P - hydroxysteroid dehydrogenase

Testosterone

~

)

Deoxycortisol (5 )

Corticosterone 18 ) C1S - hY1roxYlase

Cortisol (F)

ll-hydroxy ~' - androstenedione

)

18 - hydroxycorticosterone Esterone (E,)

Estradiol (E 2 )

Aldosterone

Fig. 7. Schema of sterowogenesis pathway. Cholesterol side-chain c1eavage enzyme is the enzyme of the initial step of steroidogenesis and aromatase is the final step of estrogen synthesis. Testosterone is th e precursor of E 2.

Estrogen synthesis in gastric carcinoma . 59

terone to estradiol. Our results demonstrated that P450 arom was present in most of the gastric carcinomas, whereas P-450 sec was tarely present. Therefore, E 2 is possibly synthesized in gastric carcinoma cells by conversion of circulating testosterone. In addition, testosterone, E 2 and P-450 arom were localized in the same cells of the carcinomatous gland. Moreover, immunoelectron microscopy confirmed the co-Iocalization of p-450 arom and E 2 in sER of the same tumor cells, although the possibility that process of tissue preparation including fixation dislocates E 2 from its normal intracellular position cannot be completely ruled out. Western blot study of P-450 arom, despite so me minor bands observed, revealed one major band of 55 kDa, which is equal to the reported molecular weight of aromatase. All these results strongly suggest that gastric carcinoma may produce E 2 locally by aromatase which was present in the carcinoma cell possibly with circulating testosterone as its precursor and immunoreactive E 2 observed is considered to represent E 2 synthesized locally. Inutsuka et a1. 6 reported that serum testosterone level of patients with gastric ca rein omas was significantly lower than in the control group and that the postoperative serum testosterone level of patients was higher than that of the preoperative level. This report is consistent with our hypothesis that testosterone is actively converted to estradiol by aromatase locally present in gastric carcinoma cells. What is more, aromatase is not expressed in normal gastric mucosa but only fn gastric carcinoma. Therefore, this phenomenon, expression of aromatase in gastric carcinoma cells may be considered within the same biological spectrum of ectopic production of polypeptide hormone, amine, HCG, and chromogranin A reported in gastric carcinoma 4, 22, 28. If so, this is the first report on ectopic steroidogenic enzyme expression associated with malignant transformation of stornach to tile best of our knowledge. In addition, E 2 positive cases showed better clinical prognosis but further investigations are necessary to analyze the possible biological roles of locally produced estradiol in gastric carcinoma cells. In summary, we have demonstrated the possibility that E 2 is produced in gastric carcinoma cells by armatase; and immunoreactive E 2 reported in gastric carcinoma by no means represents receptor-bound form. Acknowledgement We thank Dr. Evan Simpson (The University of Texas, Southwestern Medical School, DaIIas, Texas) for generously providing the antibody for aromatase and Dr. Mitsuhiro Okamoto (Department of Physiological Chemistry, Osaka University School of Medicine) for kindly providing P-450 scc antibody used in this study.

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Received December 5, 1990 . Accepted February 13, 1991

Key words: Gastric carcinoma - Aromatase - Steroidogenesis estrogen - Estrogen receptor Yoshihiro Saitoh, M.l}., The First Department of Surgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aobaku, Sendai, 980 Japan