Abstract
Aromatase is one of the key estrogen-producing enzymes and is regarded as one of the therapeutic targets in estrogen receptor-positive breast cancer patients. Human colon carcinoma has also been recently proposed as being an estrogen-responsive malignancy, but the detailed status of aromatase has not yet been reported. Therefore, in this study, we evaluated the aromatase expression in colon carcinoma using immunohistochemistry and real-time polymerase chain reaction. Aromatase mRNA was significantly higher (p=0.03) in colon carcinoma than in the corresponding non-neoplastic mucosa (n=31). Aromatase immunoreactivity tended to be positively associated with the intratumoral concentration of estrogens (n=53), and in particular, the concentration of estradiol was significantly higher (p=0.02) in aromatase-positive cases in men. Aromatase immunoreactivity was detected in the cytoplasm of the carcinoma cells in 217/328 (65%) examined colon carcinoma cases. Aromatase immunoreactivity was significantly positively correlated with tubular differentiation, and inversely correlated with Ki-67 labeling index, although not necessarily correlated with the clinical outcome of the patients. All these results demonstrate that colon carcinoma expresses functional aromatase, and that estrogens are locally synthesized in the tumor tissues. The findings reported here could contribute to a better understanding of the actions of estrogen in colon carcinoma.
Aromatase is one of the key estrogen-producing enzymes. In breast cancer, estrogens are locally produced from circulating inactive steroids through two major pathways, the aromatase and the sulfatase pathways (1). In the aromatase pathway, aromatase converts androstenedione and testosterone into estrone and estradiol, the most potent estrogen, respectively. In the sulfatase pathway, steroid sulfatase (STS) synthesizes estrone from circulating estrone sulfate, or estrogen sulfotransferase (EST) inversely inactivates estrone into estrone sulfate. Estrone is subsequently converted to estradiol by reductive 17β-hydroxysteroid dehydrogenases (17βHSDs) (1). Results of previous studies in post-menopausal breast cancer patients (2) demonstrated that the intratumoral concentration of estradiol in breast carcinoma tissue was 2.3-fold higher than that in the corresponding non-neoplastic breast tissue. Therefore, in situ production of estrogens is considered to play pivotal roles in providing estrogens for carcinoma tissue, and these enzymes are generally regarded as important therapeutic targets in estrogen-dependent neoplasms (3). The STS activity was reported to be 50 to 200 times greater than the aromatase activity (4), but the aromatase inhibitor has been widely used for patients with breast cancer, and has significantly improved their survival (3).
An increasing body of evidence suggests the importance of estrogen actions in human colon carcinoma. Results of a number of observational studies (5-7) and a randomized trial (8-9) demonstrated that hormone replacement therapy (HRT) affected the overall incidence of colon carcinoma and the recurrence of colorectal adenoma (10) in post-menopausal women. In addition, a great majority of colon carcinoma specimens and cell lines expressed estrogen receptor β (ERβ) (11), and some of these cell lines were actually responsive to estrogens (12-14). Moreover, colon carcinoma has been shown to express aromatase (15, 16), STS (16-18), EST (16), and 17βHSDs (15, 19). We previously demonstrated (16) that concentrations of total estrogen (estrone and estradiol), and estrone in colon carcinoma tissue were significantly (2.0- and 2.4-fold, respectively) higher than those in normal colonic mucosa, and higher intratumoral concentrations of total estrogen and estrone were significantly associated with poorer survival. We also reported that the STS/EST status determined the intratumoral concentration of total estrogen in colon carcinoma, and was significantly associated with clinical outcome of the patients. All these results indicated that estrogens are locally produced in colon carcinoma, mainly through the sulfatase pathway and play important roles in the progression of the disease. Therefore in this study, we examined the clinical significance of another estrogen-producing pathway, the aromatase pathway, in order to obtain a better understanding of intratumoral estrogens in colon carcinoma.
Materials and Methods
Patients and tissues. Detailed characteristics of the patients and the tissues used in this study have been published previously (16). Briefly, colon carcinoma specimens were obtained from 328 consecutive patients (184 men and 144 women including 138 post-menopausal women) who underwent surgery between 1994 and 2000. Two hundred and six patients were operated on at Miyagi Cancer Center (Natori, Japan) and 122 patients at Tohoku University Hospital (Sendai, Japan). The mean age of the patients was 66.0 years (range, 22-91 years). The mean follow-up time was 78.8 months (range, 0-161 months), and survival data of all the patients were available. The surgical specimens were fixed in 10% formalin and embedded in paraffin-wax. Snap-frozen tissues and serum were not obtainable in these cases.
Another set of colon carcinoma tissues was obtained from 53 patients (28 men and 25 post-menopausal women) who underwent surgery from 2000 to 2008 at Tohoku University Hospital (mean age=69.1 years; range, 45-83 years). Among these 53 cases, specimens of non-neoplastic colonic mucosa were available in 31 (16 men and 15 women). The specimens used for measurement of estrogen concentrations and real-time polymerase chain reaction (PCR) were snap-frozen and stored at −80°C until use. For immunohistochemistry, 10% formalin fixed and paraffin-wax embedded tissues were used. Serum samples of these patients were not available in this study.
Patients clinically suspected of having hereditary non-polyposis colorectal cancer, carcinoma associated with inflammatory bowel disease, or rectal carcinoma were excluded from this study. Review of the patients' charts revealed that no patients used oral contraceptives, received HRT, nor underwent irradiation or chemotherapy prior to the surgery. Informed consent was obtained from all the patients examined in this study, and the research protocol for this study was approved by the Ethics Committees at both Tohoku University School of Medicine (2007-369) and the Miyagi Cancer Center (2007-6).
Immunohistochemistry. The characteristics of the antibodies to aromatase, STS and EST used for immunohistochemistry, have been described previously (16, 20, 21). Briefly, the aromatase monoclonal antibody (#677) was raised against recombinant baculovirus-expressed human aromatase protein (20). The affinity-purified monoclonal STS (KM1049) antibody was raised against the STS enzyme purified from human placenta and recognizes the peptides corresponding to amino acids 414–434. A rabbit polyclonal antibody for EST (PV-P2237) was purchased from the Medical Biological Laboratory (Nagoya, Japan) (21). Mouse monoclonal antibodies for ERβ (MS-ERB13-PX1) and Ki-67 (MIB1) were purchased from GeneTex (San Antonio, TX, USA) and DAKO (Carpinteria, CA, USA), respectively.
A Histofine Kit (Nichirei, Tokyo, Japan), which employs the streptavidin-biotin amplification method was used for immunohistochemistry in this study. Antigen retrieval for ERβ and Ki-67 immunostaining was performed by heating the slides in citric acid buffer (2 mM citric acid and 9 mM trisodium citrate dehydrate, pH 6.0), in an autoclave at 120°C for 5 min. Antigen retrieval for EST was carried out by heating the slides in a microwave oven for 15 min in citric acid buffer. The dilutions of the primary antibodies used in this study were as follows: aromatase: 1/6,000; STS: 1/9,000; EST: 1/1,500; ERβ: 1/1,000; and Ki-67: 1/50. The antigen-antibody complex was visualized with 3,3’-diaminobenzidine solution [1 mM 3,3’-diaminobenzidine, 50 mM Tris-HCl buffer (pH 7.6), and 0.006% H2O2] and counterstained with hematoxylin. As a negative control for immunostaining, normal rabbit or mouse IgG was used instead of the primary antibodies.
Immunoreactivities for aromatase, STS and EST were detected in the cytoplasm, and the cases that had more than 10% of positive carcinoma cells were considered as being positive (16, 22). Immunoreactivities for ERβ and Ki-67 were detected in the nucleus, which were evaluated in more than 1,000 carcinoma cells for each case, and subsequently the percentage of the positive cells, i.e. labeling index (LI), was determined (22). Cases with ERβ LI of more than 10% were considered ERβ-positive colon carcinoma in this study (11, 16).
Total RNA extraction and real-time PCR. RNA was extracted from the 31 paired colon carcinoma specimens and from corresponding non-pathological colonic mucosa using the RNeasy Mini Kit (Quiagen, Duesseldorf, Germany). A reverse transcription kit (Superscript II Preamplification system; Invitrogen, San Diego, CA, USA) was used in the synthesis of cDNA. Real-time PCR was carried out using the LightCycler System (Roche Diagnostics GmbH, Mannheim, Germany). The primer sequences of aromatase and ribosomal protein L13a (RPL13A) were reported previously (23). The cDNAs of known concentrations for target genes were used to generate standard curves for real-time quantitative PCR, and the quantity of the target cDNA transcripts were determined. The mRNA level in each case was determined as previously described (23). Negative controls, in which the reaction mixture lacked cDNA template, were included to rule out the possibility of exogenous DNA contamination.
Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS) analysis. Concentrations of estrone and estradiol were measured using LC-ESI-MS at Teikoku Hormone Medical (Kawasaki, Japan) as described previously (16, 22, 24, 25). Results were also reported in the previous studies (16).
Statistical analysis. Values for patients' age, estrogen concentrations, mRNA level, and Ki-67 LI are presented as the mean±SEM. Associations between these parameters and aromatase immunoreactivitiy were evaluated using Welch's t-test. Paired t-test was used for analysis of the paired samples. Associations between aromatase immunoreactivity and other clinicopathological parameters were evaluated in a cross-table using the χ2 test. Overall survival curves were generated according to the Kaplan-Meier method and the log-rank test was used for analysis. The StatView 5.0 software (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses and differences with p-values<0.05 were considered significant.
Results
Immunoreactivity for aromatase and correlation with clinicopathological factors and clinical outcome in 328 colon carcinomas. We performed immunohistochemistry for aromatase in 328 colon carcinomas in order to examine the clinical and prognostic significance of the aromatase status in colon carcinoma. Aromatase immunoreactivity was detected in the cytoplasm of carcinoma cells in 217 (66%) out of 328 cases (Figure 1a). Non-neoplastic colonic epithelium was weakly positive for aromatase in approximately one third of the cases examined, as described previously (Figure 1b) (16). Aromatase immunoreactivity was negative in stromal cells both in carcinoma and non-neoplastic tissues. As summarized in Table I, aromatase immunoreactivity in the carcinoma cells was inversely correlated with Ki-67 LI (p=0.02). Aromatase-positive cases were frequently detected in tubular adenocarcinoma (p=0.01) and in cases other than STS−/EST+ carcinomas (p=0.001). Other clinicopathological factors, including gender, Dukes' stage, and ERβ immunoreactivity, were not associated with the aromatase status in this group of patients.
The aromatase status was not significantly associated with the clinical outcome of the 328 examined colon carcinoma patients (Figure 2).
mRNA expression of aromatase in 31 pairs of colon carcinomas. We evaluated the aromatase expression using real-time PCR in the 31 paired samples. As demonstrated in Table II, aromatase mRNA level was significantly (p=0.03 and 5.2-fold) higher in carcinoma tissue than in non-neoplastic mucosa. When the cases were divided according to gender, aromatase mRNA level was significantly higher in male (p=0.03) but not in female patients.
Correlation between aromatase status and intratumoral estrogen concentrations in the 53 colon carcinoma cases. Since aromatase converts androgens into estrogens, we examined the correlation between aromatase immunoreactivity of the carcinoma tissue and intratumoral concentrations of estrogens in the 53 cases. Aromatase immunoreactivity was detected in 36 (68%) examined cases. Intratumoral concentration of estrogens tended to be higher in aromatase-positive cases, as described previously (16). When the cases were stratified by gender, the intratumoral estradiol concentration was significantly (p=0.02) higher in aromatase-positive cases in men (Table III).
We previously reported that the intratumoral concentrations of estrogens were strongly regulated by the sulfatase pathway, represented by STS/EST status (16). Therefore, we divided the cases according to the STS/EST status, and further examined associations between intratumoral estrogen levels and aromatase status. As demonstrated in Table IV, the intratumoral estradiol level was 1.8 to 2.0 times higher in aromatase-positive cases, and the difference was significant (p=0.02) in the cases other than the STS−/EST+ group.
Discussion
This is the first study to evaluate the clinical and prognostic significance of aromatase in human colon carcinoma. In the present study, aromatase immunoreactivity was detected in 217 out of 328 (66%) colon carcinoma cases, while it was weakly and focally detected in normal colonic epithelium, and the aromatase mRNA level was significantly (5.2-fold) higher in colon carcinoma than in the corresponding non-neoplastic mucosa. Intratumoral aromatase has been reported in several malignancies, including breast (2, 4, 22), lung (26, 27), gastric (28), ovarian carcinomas (29), and others (30). As for colon carcinoma, English and colleagues (15) reported that human colon epithelial and carcinoma tissue had aromatase activity and converted androstenedione into estrone. They also demonstrated aromatase activity in some colon cancer cell lines, such as Caco-2, HT-29 and SW620. Fiorelli and colleagues (31) showed that human colon carcinoma cell lines HCT8 and HCT116 had aromatase mRNA and enzymatic activity, although the maximum capacity was 10- and 4-fold lower than that of adipose tissue. Our present results were consistent with these previous findings, and also suggested that aromatase was frequently overexpressed in human colon adenocarcinoma.
We revealed that the intratumoral concentration of estorogens tended to be higher in aromatase-positive colon carcinomas, and the estradiol level was significantly associated with the aromatase status in men. In human lung carcinoma, Niikawa (27) and colleagues demonstrated that intratumoral estradiol concentration was closely associated with the aromatase mRNA level, and was significantly higher in men than in post-menopausal women. The serum concentrations of androstenedione and testosterone, the substrates for aromatase, are 1.7 and 20 times higher in men than in postmenopausal women, respectively (32, 33). Therefore, circulating testosterone is reasonably postulated to be a major precursor substrate of local estradiol production by aromatase in colon carcinoma. Our results also demonstrated that aromatase immunoreactivity was frequently detected in the colon carcinomas other than the STS−/EST+ group, and that the aromatase status was positively associated with the intratumoral estradiol level in these cases. EST is the only sulfotransferase that displays affinity for estradiol in a physiological concentration range, and the sulfating activity for estradiol was stronger than that for estrone (34-36). Thus, EST might offset the estradiol production through the aromatase pathway, but it awaits further studies for clarification. The results of the present study suggested that aromatase was also involved in the local estradiol production in cooperation with the sulfatase pathway in colon carcinoma.
It is well known that aromatase is expressed not only in the carcinoma cells but also in the intratumoral stromal cells in breast carinoma, and their interaction regulates the local production of estrogens (37). However, aromatase immunoreactivity was detected only in the colon carcinoma cells in our study, and similar localization has been also reported in lung (38), gastric (28), and oral squamous cell carcinomas (30). Therefore, the characteristics of stromal cells in breast carcinoma may be different from those in non-classical estrogen-dependent malignancies including colon carcinoma.
In our current study, aromatase immunoreactivity was not correlated with survival of the patients. Aromatase inhibitors are frequently used in patients with breast carcinoma, and improves their clinical outcome (3), but the actual correlation between intratumoral aromatase expression and prognosis of the patients remains unclear. Salhab and colleagues (39) demonstrated that higher aromatase mRNA expression in breast carcinoma tissues was correlated with poorer survival. However, Miyoshi and colleagues (40) failed to show that intratumoral aromatase mRNA expression had prognostic value, and Marigilka and colleagues (41) reported the absence of correlation between aromatase enzymatic activity and survival of the patients. Hence, our present result does not necessarily deny the significance of aromatase in colon carcinoma. Considering that aromatase expression was associated with the intratumoral estrogen levels, aromatase is considered to play an important role in the pathogenesis of colon carcinoma. Further investigations are required to clarify the clinical importance of estrogen actions and aromatase in colon carcinoma.
Acknowledgements
This work was partly supported by a grant for Research on Intractable Diseases from Japanese Ministry of Health, Labour and Welfare. We appreciate the skillful technical assistance of Mr. Katsuhiko Ono and Ms. Miki Mori at the Department of Pathology, Tohoku University School of Medicine and Ms. Keiko Inabe, Ms. Emiko Shibuya and Ms. Midori Chiba at the Department of Surgery, Tohoku University School of Medicine.
Footnotes
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Disclosure Statement
The Authors have no conflicts of interest.
- Received April 9, 2012.
- Revision received May 27, 2012.
- Accepted May 28, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved