Expression of the estrogen receptor in human thyroid neoplasms

CANCER LETTERS

Cancer Letters 84 (1994) 59-66

ELSEVIER

Expression of the estrogen receptor in human thyroid neoplasms Katsunari Yane*a, Yoshiteru Kitahorib, Noboru Konishib, Kunio Okaichi”, Takeo Ohnishi”, Hiroshi Miyahara”, Takashi Matsunagaa, Jung-Chung Lind, Yoshio Hiasab ‘Department of Otorhinolaryngology, bSecond Department of Pathology. ‘Department of Biology, Nara Medical University, 840 Shijyo-cho, Kashihara. Nara 634, Japan ‘Tumor Virology, Centers for Disease Control and Prevention, Building 1. MS-DIO. I600 Clifton Road, Atlanta, GA 30333, USA

Received 21 April 1994; revision received 6 June 1994; accepted 8 June 1994

Abstract The expression and quantitation of the estrogen receptor (ER) in human thyroid tumors were examined by biochemical, immunohistochemical, and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. For this study, neoplasms, adenomatous goiters and adjacent normal thyroid tissues were obtained from 35 patients which included 10 cases of papillary carcinomas, 17 cases of adenomas and 8 cases of adenomatous goiters. Regardless of the histopathological subtype, ER was detected in 19% (5/27) of the neoplastic tissues with the mean value of ER content of 5.0 f 1.3 fmol/mg protein and the mean K,, value of 0.38 f 0.28 nM. ER was also detected, but at a lower concentration (2.8 f 1.6 fmol/mg protein), in the surrounding normal tissues. There was no significant difference between the neoplasms and adenomatous goiters with respect to the incidence of ER positivity and ER content. Furthermore, ER-positive specimens, as determined by both biochemical and immunohistochemical techniques, also showed the expression of ER mRNA detected by RT-PCR method. These results demonstrate that both ER mRNA as well as ER protein are expressed in thyroid neoplasms. This suggests the possibility that estrogen may affect the tumorigenesis or the progression of some thyroid neoplasms. Keywords:

Human thyroid neoplasms; Estrogen receptor; Reverse transcriptase-polymerase

1. Introduction

It is well recognized that the incidence of human thyroid disorders is higher in females than in males, and the prognosis of differentiated thyroid * Corresponding 81 7442 4 6844.

author.

Tel. 81 7442 2 3051 ext 3435. Fax

0304-3835/94/$07.00 0 1994 Elsevier Science Ireland SSDl 0304-3835(94)03463-T

chain reaction; mRNA

carcinomas is better in females during the reproductive age and worse in males over 50 years of age [ 1,7,10,30]. Although thyroid stimulating hormone (TSH) appears to play an important role in regulating the growth and biological functions in thyroid cells [4,5], the effect of age and sex on the incidence and prognosis of thyroid neoplasms can not be explained by the increased level of serum

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K. Yane et al/ Cancer Lett. 84 (1994) 59-66

TSH. Epidemiological data as well as clinical and experimental lines of evidence indicate that sex hormones play a role in the development of thyroid neoplasms [3,9,29]. Thus, if sex hormones can affect the thyroid tissue directly, the cells should contain receptors for the hormone. In fact, several studies have reported the presence of estrogen receptors in both the benign and malignant human thyroid neoplasms [2,6,8,14,17-19,23-26,32,33]. In this study, we tried to determine the expression and quantitation of ER in human thyroid tissues using the dextran coated charcoal (DCC) assay, and the immunohistochemical and reverse transcriptase-polymerase chain reaction (RTPCR) techniques.

Fig. 2. Histologic features of adenomatous goiter (Table 1; case no. 29), showing indistinct encapsulation and poor demarcation from adjacent normal thyroid tissue (HE, x 8).

2. Materials and methods 2.2. DCC assay for estrogen receptor

2.1. Collection of tissue samples Surgical specimens consisting of 10 papillary carcinomas, 17 adenomas, and 8 adenomatous goiters (nodular type) were frozen immediately in liquid nitrogen and stored at -80°C and the remaining parts of the tissues were fixed in 10% buffered-formalin and embedded in paraffin. Histopathologic diagnosis of thyroid lesions was made according to the World Health Organization (WHO) criteria (Figs. 1 and 2) [15].

Fig. I. Histologic features of adenoma (Table 1; case no. 22), showing well-defined fibrous capsule and demarcation from adjacent normal thyroid tissue (HE, x 8).

[2,4,6,7,16,17-3H]Estradiol (141.9 Ci/mmol, New England Nuclear, Boston, MA) was used as radioactive ligand. All specimens were minced and homogenized in cold TEDM buffer (10 mM Tris-HCl, 1.5 mM EDTA, 0.5 mM dithiothreitol, 10 mM sodium molybdate, pH 7.4). The homogenate was centrifuged at 105 000 x g for 1 h. The protein content of the resultant supernatant, the cytosolic fraction, was determined by the method of Lowry et al. [22]. The cytosolic fraction was incubated for 16 h at 4°C with different concentrations (l-40 nM) of radioactive estradiol in the presence or absence of lOO-fold excess of a nonradioactive competitor. The DCC solution was then added to the mixture and incubation was continued for an additional 10 min. Unbound ligands were removed by centrifugation at 3000 rev./min at 4°C for 10 min. The radioactivity in the supernatants was determined by liquid scintillation counting. Uterus was used as a positive control. The content and the dissociation constant (&) of the receptors were determined by Scatchard analysis [31]. Arbitrarily, a receptor content more than 1 fmol/mg protein was regarded as positive. 2.3. Immunohistochemical study Immunohistochemical analysis of ER on paraffinembedded sections was described previously

K. Yane et al/Cancer

[ 17,181. Briefly, sections were incubated with 10% protease (Sigma, St. Louis, MO) solution at 37°C for 10 min, exposed for 30 min to an excess of normal rabbit serum, and then incubated for 2 h at 37°C with the monoclonal antibody H222 (ERICA Monoclonal Kit, Abbott Lab., Chicago, IL). Successively, the sections were incubated with secondary antibody and avidin-biotin peroxidase complex (ABC Kit, Vector Lab., Burlingame, CA). The sections were stained with 3,3’diaminobenzidine and counterstained with Harris hematoxylin. Cultured MCF-7 cells were used as a positive control. The staining results were assessed semiquantitatively based on the number of stained cells within one microscopic field at a magnification of x400; more than three stained cells per field was regarded as a positive reaction. 2.4. RT-PCR analysis of ER mRNA Two pairs of ER-specific oligonucleotide primers were synthesized and designated as follows: Pl (sense), 5 ‘-CCTAACTTGCTCTTGG ACAGGAA-3’ (nucleotide positions 1216-1239); P2 (antisense), 5 ‘-CACCACGTTCTTGCACTTCATGCT-3 ’ (1602- 1579); P3 (sense), 5 ‘-GGAGACATGAGAGCTGCCAAC-3’ (850-870); P4 (antisense), 5 ‘-CCAGCAGCATGTCGAAGATC-3 ’ (1288-1269). The PUP2 primer pair encompasses a 387 bp fragment which is specific for the DNA sequence of ER gene and the hormonebinding domain [ 13,20,21]. In contrast, the P3/P4 pair spanning a 439 bp fragment is specific for both the DNA- and hormone-binding domains [12,13,20,21]. Total cellular RNA was isolated by the RNAzol method (Biotecx Lab., Houston, TX). RT-PCR method was performed as previously described [ 161. Briefly, synthesis of cDNA was carried out in a 10 ~1 reaction volume containing 1 pg of total RNA, 2 PM primer, and 5 units of AMV RT. After heat inactivation of the RT at 92°C 2.5 units of Taq DNA polymerase was added to 50 ~1 of the PCR reaction mixture (100 PM dNTPs, 2 PM primers) for a final reaction volume of 60 ~1. Forty-five cycles of PCR amplification were performed with the parameters 94°C 54°C and 72°C for 1 min each. The amplified products were

Lett. 84 (1994) 59-66

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analyzed by electrophoresis in 4% agarose gel (NuSieve), and detected by ethidium bromide staining. 2.5. Restriction enzyme digestion analysis RT-PCR amplified products (387 bp and 439 bp fragments) were digested with the restriction enzyme PstI or XbaI at 37°C for 2 h, and the reaction products were analyzed by agarose gel electrophoresis as described above. 2.6. Statistical analysis Statistical analyses were performed by X’-test or Student’s t-test. All data were presented as mean values f standard deviation. 3. Results 3.1. DCC assay of ER

The content and dissociation constant (Kd) of ER measured by DCC assay in neoplasms, adenomatous goiters and adjacent normal thyroid tissues are summarized in Table 1. Regardless of the histopathological subtype, ER was detected in 19% (5/27) of the neoplastic tissues with the ER content ranging from 3.8 to 7.1 fmol/mg protein, and K,, values ranging from 0.1 to 0.7 nM. The mean values of ER content and Kd in ER-positive neoplasms were 5.0 f 1.3 fmol/mg protein and 0.38 f 0.28 nM, respectively. The surrounding normal tissues were assayed in parallel with their corresponding neoplastic regions. Regardless of the histopathological subtype, ER was also detected in 19% (5/27) of the normal tissues with a mean ER content of 2.8 + 1.6 fmol/mg protein and a mean Kd value of 0.44 f 0.48 nM. The mean content of ER in neoplastic tissues was higher than that in normal tissues (P < 0.05). ER was detected in 38% (3/8) of the adenomatous goiters with a mean ER content of 3.5 f 2.2 fmol/protein and a mean K,, value of 0.81 + 0.79 nM. There was no significant difference between the neoplasms and adenomatous goiters with respect to the incidence of ER positivity and ER content. Among the different histological types of thyroid lesions, ER positivity was detected in 30%

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Table 1 Detection of ER in thyroid tissues Case

Sex

Age

ER-ICAa

Papillory carciiKlma F I

2 3 4 5 6 7 8 9 IO Adenoma 11

12 13 14 I5 16 17 18 19 20 21 22 23 24 25 26 27

Normal tissue

Neoplastic tissue ER cont. (fmol/mg protein)

Kd (nW

ER cont. (fmol/mg protein)

4.8 _

21

-

F F F F F F M F F

49 62 43 34

4.5 -

0.15

3.8

0.31

F F F F F F F F F M M M M F F M F

39

Adenomatous goiter F 28 F 29 F 30 F 31 F 32 F 33 M 34 F 35

0.72

41 46 51 29 72

70 62 29 40 45 40 40 54

-

0.11

-

1.9 -

0.38

+

67 64 76 64

0.26

4.5 5.2

0.63 0.10

43 57 51 64

73 49 76 38 43 68 35 70

-, not detectable. Coefficient of variation, 11.2%. “ER-ICA, ER-immunocytochemical assay.

4.2 _

1.29

-

2.9 -

0.54

1.7 -

0.19

5.9 -

1.7

1.7

0.2

2.5

0.11

K. Yam et al/Cancer

(3/10) of papillary carcinoma, 12% (2/17) of follicular adenoma, and 38% (3/8) of adenomatous goiter (Table 1). No significant difference in age was observed between the ER-positive and ERnegative patients (49 f 14 versus 52 f 15 years). Despite the small number of samples from male patients assayed, the incidence of ER positivity was higher in male (3/7) than in female (5/28), but the difference was not significant. 3.2. Immunohistochemical localization of ER Immunohistochemical staining with a monoclonal antibody (H222) against ER demonstrated that the specific staining for ER was localized exclusively in the nuclei, but not in the cytoplasm or in the plasma membrane (Fig. 3). ER positivity in the thyroid lesions was found in 20% (2110) of papillary carcinoma, 29% (5/17) of adenoma and 50% (418) of adenomatous goiter (Table 1). Eightyeight percent (7/8) of ER-positive cases detected by DCC assay were positive by immunoreactive staining as well, and 85% (23/27) of ER-negative cases determined by DCC assay were also negative by the staining method. The concordance between DCC assay and immunohistochemical study was 86% (30135) for all cases. A few ER-positive cells in the normal thyroid tissues were found in case nos. 2, 22 and 29 (data not shown).

Lett. 84 (1994) 59-66

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neoplastic lesions detected by DCC and immunohistochemical methods correlated with the expression of ER mRNA, the RT-PCR technique was used. As shown in Fig. 4(A), PCR products of 387 bp obtained with the primer pair PUP2 were found in uterus used as positive control (lane l), and in ER-positive specimens (lanes 2 and 3; Table

Ml23456

3.3. Detection of ER mRNA To determine whether the presence of ER in the Fig. 4. Expression of ER mRNA in human thyroid neoplasms. M, molecular weight marker (6X 174IHaeIII digested fragments). (A) Detection of ER mRNA was performed by RTPCR amplification method. PCR product obtained with primer pair PI/P2 was a 387 bp fragment. Lane I. human uterus; lane 2 (case no. 6) and lane 3 (case no. 4). ER-positive papillary carcinomas; lane 4 (case no. IO), ER-negative papillary carcinoma ;lanes 5-7 (case nos. 14, IS, and 18). ER-negative adenomas;

Fig. 3. Immunohistochemical staining of ER in papillary carcinoma of the thyroid. Specific staining for ER is localized exclusively in the nuclei of cancer cells (arrows) (x 100).

lane 8, normal thyroid tissue. (B) PCR products from the ERpositive papillary carcinoma (case no. 6) were digested with PHI and XbaI. Lane 1, 387 bp fragment amplified from uterus with primer pair Pl/P2; lane 2, &I-digested products (171, 109 and 107 bp) of 387 bp fragment from uterus; lane 3, 439 bp fragment amplified from papillary carcinoma with P3/P4; lane 4, XbaI-digested products (285 and I54 bp) of 439 bp fragment from papillary carcinoma; lane 5, 387 bp fragment amplified from papillary carcinoma with PlIP2; lane 6. &I-digested products (171, 109 and 107 bp) of 387 bp fragment from papillary carcinoma.

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K. Yane et al/ Cancer Leti. 84 (1994) 59-66

1, case nos. 6 and 4) detected by both DCC and immunohistochemical methods, but not in ERnegative specimens (lanes 4-8; cases nos. 10, 14, 15, 18 and normal thyroid tissue). Representative results of analysis by restriction enzyme digestion are shown in Fig. 4(B). With primer pair Pl/P2, a 387 bp fragment was amplified from a positive control of uterus (lane 1) and from an ER-positive papillary carcinoma of the thyroid (lane 5; case no. 6). Similarly, the primer pair P3/P4 amplified a 439 bp fragment from an ER-positive papillary carcinoma (lane 3; case no. 6). Based on the nucleotide sequence of ER gene, Two PstI restriction sites were present in the 387 bp fragment and one X&z1restriction site was present in the 439 bp fragment. Each of the 387 bp fragments from uterus (lane 2) and papillary carcinoma (lane 6) generated three fragments of expected sizes (171 bp and a doublet of 109 and 107 bp) after digestion with the restriction enzyme PstI. Digestion of the 439 bp fragment from papillary carcinoma with XbaI yielded two fragments of 285 bp and 154 bp (lane 4). In case no. 4, a similar result was obtained by analysis with restriction enzyme digestion (data not shown). 4. Discussion Previous studies utilizing ligand binding assays to assess the presence of ER have generated conflicting results. Clark et al. [6] reported that neoplastic thyroid tissue had approximately fourfold higher ER level than non-neoplastic thyroid tissue removed from the same patients, and that the K,, for ER did not differ in the neoplastic and nonneoplastic cases. In contrast, Miki et al. [23] reported that there was no significant difference between the neoplastic and non-neoplastic thyroid lesions with respect to the concentration and Kd of ER. In this study, normal thyroid tissues also contained measurable ER, however, the mean content of ER in normal tissues was lower than that in neoplastic tissues. Interpretation of results obtained from ligand binding assays are difficult in cases where receptor concentration is low or only a specific cell within a tissue contains ER. To circumvent the limitation of this technique, we adopted an immunohisto-

chemical method, using a monoclonal antibody (H222) with specificity for the ER protein, to determine the cellular localization of ER. Our results clearly indicate that ER is localized exclusively in the nuclei, but not in the cytoplasm or in the plasma membrane which is in agreement with previous reports [8,18,26]. Improvements in the level of detection of ER in thyroid neoplasms by the immunohistochemical method has been recently documented [ 181. In this study, we detected four additional ER-positive cases using this method compared with DCC assay (Table 1). Furthermore, we employed RT-PCR analysis to detect the expression of ER mRNA and confirmed that ER mRNA was practically present in ERpositive specimens in which ER molecules were also detected by DCC assay and immunohistochemical staining. A large amount of study material was needed for the DCC assay or immunohistochemical staining (0.5-l g), but not for the RT-PCR technique (about 50 mg). Therefore, the detection of ER mRNA using RT-PCR in some cases may be a more useful method for determining ER status when a limited amount of tumor tissue is available. Our findings as well as previous studies demonstrated the presence of ER in human thyroid tissues. Although the amount of ER in these tissues was low, compared to that in breast cancer, these results indicate that estrogen may exert its effect directly on thyroid tissues. If the progression of thyroid tumors is found to be dependent on estrogen, it is conceivable that endocrine therapy using anti-estrogens may be an effective approach for the management of thyroid tumors. Recent studies have demonstrated that ovariectomy reduced the incidence of thyroid carcinoma, and the progression of malignancy of thyroid tumors correlated positively with the ER content in experimental animals [ 11,271. Furthermore, it is documented that estradiol treatment induces overexpression of various protooncogenes in the target tissues [28,34]. The biological evolution of thyroid tumors in relation to ER content needs to be established. These data, taken together, indicate that the development of some thyroid neoplasms may be affected by estrogen. Especially in females, the change in the level of circulating estrogen by

K. Yane et al/Cancer Len. 84 (1994)

the menstrual cycle or pregnancy may influence the tumorigenesis and biological interaction of thyroid neoplasms with ER, and may account for the differences in both the incidence and prognosis between the two sexes. Acknowledgments

The authors thank Drs. Shinzaburo Noguchi and Yasuko Nishizawa of the Center of Adult Disease, Osaka, for technical support. This work was supported by a Grant-in-Aid from the Ministry of Education of Japan. References

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