Human liver-specific organic anion transporter-2 is a potent prognostic factor for human breast carcinoma Blackwell Publishing Asia
Mitsuhisa Muto,1,5 Tohru Onogawa,1,5 Takashi Suzuki,3 Takanori Ishida,2 Toshiki Rikiyama,1 Yu Katayose,1 Noriaki Ohuchi,2 Hironobu Sasano,3 Takaaki Abe4 and Michiaki Unno1,6 Divisions of 1Gastroenterological Surgery and 2Surgical Oncology, and Departments of 3Pathology and 4Nephrology, Endocrinology and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan (Received February 17, 2007/Revised 1 May, 2007/Accepted June 11, 2007/Online publication August 19, 2007)
Human liver-specific organic anion transporter-2 (LST-2/OATP8/ SLCO1B3) has been demonstrated to be expressed in various gastrointestinal carcinomas and also to play pivotal roles in the uptake of a wide variety of both endogenous and exogenous anionic compounds, including bile acids, conjugated steroids and hormones, into hepatocytes in the human liver. However, the biological significance of LST-2 in human carcinomas remains unknown. In the present study, we examined the expression of LST2 in 102 cases of breast carcinoma using immunohistochemistry and correlated the findings with various clinicopathological parameters in order to examine the possible biological and clinical significance of LST-2. LST-2 immunoreactivity was detected in 51 cases (50.0%); of these 51 positive cases, LST-2 immunoreactivity was inversely correlated with tumor size (P = 0.0289). In addition, LST-2 immunoreactivity was significantly associated with a decreased risk of recurrence and improved prognosis by both univariate (P = 0.02 and P = 0.01) and multivariate (P = 0.03 and P = 0.01) analyses. In the estrogen receptor-positive groups, the LST-2-positive patients showed good prognoses. Considering that LST-2 transports estrone3-sulfate, these results suggest that LST-2 overexpression is associated with a hormone-dependent growth mechanism of the breast cancer. The results of our present study demonstrate that LST-2 immunoreactivity is a potent prognostic factor in human breast cancer. (Cancer Sci 2007; 98: 1570–1576)
specifically on the sinusoidal membrane of hepatocytes.(14) In addition, we have also isolated an LST-1 subtype, human LST2, and examined its expression and functions.(15) LST-2 is only expressed in the sinusoidal membrane of the hepatocytes around the central vein in human liver. In addition, the expression of LST-2 (but not LST-1) has been demonstrated in gastric carcinoma, colonic carcinoma, pancreatic carcinoma and biliary carcinoma. LST-1 and LST-2 transport not only organic anions such as taurocholate, conjugated steroids and eicosanoids, but also an anticancer agent, methotrexate. Recently, Nozawa et al. suggested that the uptake of E1-S across the plasma membrane in estrogen-dependent T47D breast cancer cells was mediated by a specific transport mechanism. This transporter-mediated uptake of E1-S in estrogen-dependent cells may also regulate the proliferation of breast cancer cells. (16) It has been reported that LST-2 transport E1-S.(17) However, there has been no report of a hormonal function of LST-2 in carcinoma cells. Therefore, in the present study, we examined the immunolocalization of LST-2 in 102 cases of human breast cancer and correlated the status of LST-2 immunoreactivity in carcinoma cells with the various clinicopathological parameters of the cases, including clinical outcome, in order to clarify the clinical and biological significance of LST-2 in human breast carcinoma. Materials and Methods
B
reast cancer is a major type of malignancy in women, and two-thirds of breast cancer cases are estrogen dependent. (1) Estrogens play an important role in the development of hormonedependent breast carcinomas.(2,3) The biologically active form of estrogen is E2. In premenopausal women, the ovaries are the main source of circulating estrogens,(4) and estrogens produced in the ovaries are transported to breast cancers and stimulate their proliferation. However, in postmenopausal women, the great majority of estrogens in circulation are present in the sulfated form, or as E1-S.(5) Previous studies have demonstrated that intratumoral tissue concentrations of E2 in breast cancers are 10 times higher than the levels detected in plasma,(6,7) suggesting that the estrogens in human breast cancer tissues may be produced in situ. Intratumoral estrogen is already known to be produced through two main pathways: one is the aromatase pathway,(6 –8) in which aromatase converts androgens to estrogens, and the other is the sulfatase pathway,(9,10) in which STS converts E1-S to estrone. Because sulfated steroid conjugates, such as E1-S, carry a net negative charge at physiological pH, their transfer across cell membranes is considered to be carrier mediated. Sulfated steroids have been identified as substrates for characteristic members of two organic anion carrier gene families: the organic anion transporting polypeptide superfamily, classified within the solute carrier 21 A gene family,(11,12) and the organic anion transporter genes.(13) Recently, we isolated a novel type of human organic anion transporting polypeptide, termed LST-1. LST-1 is markedly expressed in the liver, Cancer Sci | October 2007 | vol. 98 | no. 10 | 1570–1576
Patients and tissue preparation. One hundred and two specimens of ‘not other specified’-type(18) invasive ductal carcinoma of the breast were obtained from female patients who underwent mastectomy from 1984 to 1989 at Tohoku University Hospital (Sendai, Japan). The mean age of the patients was 53.1 ± 1.7 years (mean ± SD; range 27–82 years). The mean follow-up time was 106 ± 18.9 months (range 5–154 months). Clinical data were retrieved from the charts of the patients. None of the patients examined in the present study took oral contraceptives. The patients did not receive irradiation or chemotherapy before surgery. Review of the charts revealed that 85 patients received adjuvant chemotherapy (mitomycin C, methotrexate and fluorouracil: n = 80; cyclophosphamide, doxorubicin and fluorouracil: n = 3; and cyclophosphamide, mitomycin C and fluorouracil: n = 2), 13 patients received radiation therapy and 11 patients received tamoxifen therapy after the surgery. Disease-free survival data were available for all patients. The histological grade of each specimen was evaluated based on the method of Elston and Ellis.(19) The research protocols for the present study were approved by the Ethics Committee at Tohoku
5
Mitsuhisa Muto and Tohru Onogawa contributed equally to this paper. To whom correspondence should be addressed. E-mail:
[email protected] Abbreviations: E1-S, estron-3-sulfate; E2, 17β-estradiol; ER, estrogen receptor; EST, estrogen sulfotransferase; HSD1, hydroxsteroid dehydogenase type 1; LI, labeling index; LST, liver-specific organic anion transporter; PR, progesterone receptor; STS, steroid sulfatase.
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doi: 10.1111/j.1349-7006.2007.00570.x © 2007 Japanese Cancer Association
University School of Medicine. All specimens were fixed with 10% formalin and embedded in paraffin wax. Antibodies. Rabbit polyclonal antibody for LST-2 was raised against the synthetic carboxyl-terminal peptide of human LST-2, as described previously.(15) Rabbit polyclonal antibody for EST was raised against the synthetic NH2-terminal peptide of human EST, as described previously.(20) The monoclonal antibody for STS (KM1049 antibody) was purchased from Kyowa Hakko Kogyo (Tokyo, Japan).(21) 17β-HSD1 antibody against a rabbit polyclonal antibody was kindly provided by Dr Poutanen at the University of Oulu (Oulu, Finland).(22) Monoclonal antibodies for ERα (ER1D5), PR (MAB429) and Ki-67 (MIB1) were purchased from Immunotech (Marseille, France), Chemicon (Temecula, CA, USA) and DAKO (Carpinteria, CA, USA), respectively. Immunohistochemistry for HER2 was carried out using the HercepTestII kit (K5204; DAKO). Immunohistochemistry. A Histofine Kit (Nichirei, Tokyo, Japan), with the streptavidin–biotin amplification method, was used for the identification of EST, STS, ERα, PR, 17β-HSD1 and Ki-67 immunoreactive staining. EnVision plus (DAKO) was used for LST-2 immunohistochmical staining. ERα, PR and Ki-67 immunostaining was carried out by heating the slides in an autoclave at 120°C for 5 min in citric acid buffer (10 mM citric acid, pH 6.0). Similarly, antigen retrieval for EST immunostaining was carried out by heating the slides in a microwave oven for 15 min in a citric acid buffer. No antigen retrieval was carried out for LST-2, STS and 17β-HSD1 immunostaining. The dilutions of the primary antibodies used in the present study were as follows: LST-2, 1:500; EST, 1:1500; STS, 1:9000; ERα, 1:50; PR, 1:130; Ki-67, 1:50; and 17β-HSD1, 1:800. 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. A human liver specimen was used to confirm the specificity of the antibodies (data not shown). Normal rabbit or mouse IgG was used as a negative control for immunostaining and no specific immunoreactivity was detected. Scoring of immunoreactivity. After completely reviewing the entire slides of the immunostained sections for each carcinoma, three of the authors (T. O., M. M. and S. T.) independently and blindly classified the carcinoma cases into the following two groups: those in which the percentage of cells positive for LST2, EST, STS and 17β-HSD1 was more than 10% were defined as the positive group, and the negative group comprised those with fewer than 10% positive cells. HER2 immunoreactivity was evaluated according to a grading system proposed in the kit, and moderately or strongly circumscribed membrane staining of HER2 in more than 10% carcinoma cells was considered positive. Discordant results were mainly attributable to differences in the evaluation of weak immunopositive or -negative staining (background). Cases with discordant results among the observers were reevaluated simultaneously using a multiheaded microscope. ERα, PR and Ki-67 immunoreactivity was scored in 500 carcinoma cells for each case and counted independently by the same three authors. The percentage of immunoreactivity, that is, the LI, was determined. The specimens with 10% or more LI were considered positive cases in the evaluation of ERα or PR, in accordance with a report by Allred et al.(23) Statistical analysis. Values for patient age, tumor size and LI for Ki-67 were summarized as mean ± SEM. Statistical differences between the immunoreactivity for LST-2 and menopausal status, stage, lymph node status, histological grade, ERα status, PR status, EST, STS or 17β-HSD1, or HER2 were evaluated in a cross table using the χ2-test. Overall curves were generated according to the Kaplan–Meier method using Statview 5.0 (SAS Institute, Cary, NC, USA). Univariate and multivariate analyses were evaluated by Cox’s proportional hazard model using Statview 5.0. Differences with P < 0.05 were considered to be significant. Muto et al.
Fig. 1. Immunohistochemistry for liver-specific organic anion transporter (LST)-2 in breast cancer in invasive ductal carcinoma. (A) LST-2 immunoreactivity was detected in the cytoplasm and membrane of the breast cancer tissue. (B) LST-2 immunoreactivity was also detected in intraductal components of invasive ductal carcinoma tissues. (C) In morphologically normal glands (arrow), LST-2 immunoreactivity was not detected. Scale bar = 200 µm, original magnification, ×100.
Results Immunohistochemistry for LST-2 in 102 breast carcinoma cases.
As shown in Fig. 1A, immunoreactivity for LST-2 was detected in the cytoplasm and on the membrane of invasive ductal carcinoma cases. According to the criteria, the number of Cancer Sci | October 2007 | vol. 98 | no. 10 | 1571 © 2007 Japanese Cancer Association
Fig. 2. (A) Disease-free and (B) overall survival of 102 patients with breast cancer according to liver-specific organic anion transporter (LST)-2 immunoreactivity (Kaplan–Meier method). LST-2 immunoreactivity was associated with decreased recurrence and improved prognosis. (C) Disease-free and (D) overall survival of estrogen receptor (ER)-positive patients (n = 79) with breast cancer according to LST-2 immunoreactivity (Kaplan–Meier method). (E) Disease-free and (F) overall survival of postmenopausal patients (n = 53) with breast cancer according to LST-2 immunoreactivity (Kaplan–Meier method).
LST-2-positive cases was 51 (50.0%), and 51 cases were negative (50.0%). As shown in Fig. 1B, LST-2 immunoreactivity was also detected in intraductal components of invasive ductal carcinoma tissues. In morphologically normal glands, LST-2 immunoreactivity was not detected in the epithelial cells (Fig. 1C). LST-2 immunoreactivity was not detected in stroma or adipose tissues adjacent to the carcinoma. Correlation between LST-2 status of carcinoma cells and survival of the patients. We then examined the correlation between the
status of LST-2 and the survival of patients. The disease-free survival curves indicated that there was a significant difference between LST-2-positive and -negative cases in 102 breast cancers (P = 0.02, log-rank test) as demonstrated in Fig. 2A. LST-2 immunoreactivity was associated with a decreased risk of recurrence. As demonstrated in Fig. 2B, the overall survival curves indicated that there was also a significant positive difference between the LST-2-positive and -negative cases in this study (P = 0.02, log-rank test). LST-2 immunoreactivity was associated with an improved prognosis. 1572
Correlation between LST-2 immunoreactivity and clinical outcome according to ER status. The disease-free survival curves indicated
that there was a significant difference between LST-2-positive and -negative cases in the ER-positive group as demonstrated in Fig. 2C. LST-2 immunoreactivity was associated with a decreased risk of recurrence in ER-positive patients (P = 0.01, log-rank test). We then examined the correlation between LST-2 immunoreactivity and the clinicopathological parameters of the cases in the ER-positive group (Table 1). A significant inverse correlation was detected between LST-2 immunoreactivity and tumor size (P = 0.03). In addition, an inverse correlation was detected between the expression of LST-2 and Ki-67 LI (P = 0.01). However, in the ER-negative group, no correlation was detected. In overall survival, a similar tendency was detected (Fig. 3D and Table 1). Correlation between LST-2 immunoreactivity and clinical outcome according to menopausal status. In Fig. 2E, the disease-free sur-
vival curves showed that LST-2 immunoreactivity was associated with a decreased risk of recurrence in postmenopausal patients doi: 10.1111/j.1349-7006.2007.00570.x © 2007 Japanese Cancer Association
Table 1. Statistical association between liver-specific organic anion transporter 2 immunoreactivity and clinicopathological parameters according to menopausal and estrogen receptor (ER) status in 102 breast carcinomas Parameter Age (years) Menopausal status Tumor size (mm) Lymph node status Histological grade ER status PR status 17β-HSD type 1 Steroid sulfatase Estrogen sulfotransferase Ki-67 labeling index HER2
ER-positive group (n = 73)
ER-negative group (n = 29)
Premenopausal women (n = 49)
Postmenopausal women (n = 53)
0.94 0.33 0.03 0.06 0.20 – 0.87 0.10 0.25 0.31 0.01 0.88
0.54 0.97 0.44 0.72 0.56 – 0.72 0.50 >0.99 0.41 0.43 0.81
0.37 – 0.81 0.19 0.59 0.67 0.70 0.16 0.28 >0.99 0.84 >0.99
0.26 – 0.01 0.49 0.09 0.62 0.88 0.59 0.59 0.07 0.004 0.78
Data are presented as P-values. P-values less than 0.05 were considered significant and are shown in bold. HSD, hydroxysteroid dehydrogenase; PR, progesterone receptor.
Fig. 3. (A,C) Disease-free and (B,D) overall survival curves according to liver-specific organic anion transporter (LST)-2 immunoreactivity (Kaplan– Meier method). (A,B) LST-2 immunoreactivity was associated with decreased recurrence and improved prognosis in the breast cancer patients with tamoxifen therapy. (C,D) Disease-free and overall survival of estrogen receptor (ER)-negative patients who received adjuvant chemotherapy. LST-2 immunoreactivity was not significantly correlated with recurrence and prognosis.
(P = 0.01, log-rank test). In Fig. 2F, the overall survival curves indicated that LST-2 immunoreactivity was associated with good prognosis in postmenopausal patients (P = 0.004, log-rank test). However, in premenopausal patients, no correlation was detected (disease-free, P = 0.45; overall, P = 0.18; data not shown). Associations between LST-2 immunoreactivity and clinicopathological parameters in postmenopausal patients are summarized in Table 1. LST-2 immunoreactivity was inversely correlated with tumor size (P = 0.01) and Ki-67 LI only in the postmenopausal patients. Muto et al.
Relationship between LST-2 immunoreactivity and clinical outcome according to adjuvant therapies. Eleven patients received tamo-
xifen therapy after surgery, and these cases were ER-positive breast cancers. The disease-free and overall survival curves of these patients are summarized in Fig. 3. LST-2 immunoreactivity was also markedly associated with a decreased risk of recurrence and good prognosis in the group of breast cancer patients who received tamoxifen therapy, although P-values were not available because no patient had recurrence or died in the group with LST-2-positive breast cancers. ER-negative Cancer Sci | October 2007 | vol. 98 | no. 10 | 1573 © 2007 Japanese Cancer Association
Table 2. Association between liver-specific organic anion transporter 2 immunoreactivity and clinicopathological parameters in 102 breast carcinomas Parameter Age† (years) Menopausal status Premenopausal Postmenopausal Tumor size† (mm) Stage 1 2 3 Lymph node status Positive Negative Histological grade 1 2 3 ERα status Positive Negative PR status Positive Negative 17β-HSD type 1 Positive Negative Steroid sulfatase Positive Negative Estrogen sulfotransferase Positive Negative Ki-67 labeling index† HER2 Positive Negative
Positive (n = 51)
Negative (n = 51)
53.6 ± 1.5
53.6 ± 1.8
24 (23.6%) 27 (26.5%) 22.8 ± 1.4
25 (24.5%) 26 (25.5%) 28.2 ± 2.0
15 (14.7%) 30 (29.4%) 6 (5.9%)
11 (10.8%) 33 (32.4%) 7 (6.9%)
19 (18.6%) 32 (31.4%)
27 (26.5%) 24 (23.5%)
15 (14.7%) 17 (16.7%) 19 (18.6%)
10 (9.8%) 19 (18.6%) 22 (21.6%)
34 (33.3%) 17 (16.7%)
39 (38.2%) 12 (11.8%)
36 (35.3%) 15 (14.7%)
36 (35.3%) 15 (14.7%)
36 (35.3%) 15 (14.7%)
26 (25.5%) 25 (24.5%)
8 (7.8%) 43 (42.2%)
8 (7.8%) 43 (42.2%)
26 (25.5%) 25 (17.7%) 23.8 ± 2.4
18 (24.5%) 33 (32.4%) 30.3 ± 2.4
17 (16.7%) 34 (33.3%)
19 (18.6%) 32 (31.4%)
P-value 0.9868 0.8429
0.0289 0.6578
0.1106
0.5123
0.2715
>0.99
0.0426
>0.99
0.1089
0.0585 0.678
†
Data are presented as mean ± SEM. All other values represent the number of cases and percentage. ER, estrogen receptor; HSD, hydroxysteroid dehydrogenase; PR, progesterone receptor. Significant values are shown in bold.
patients received adjuvant chemotherapy after surgery in all cases. However, the association between LST-2 immunoreactivity and clinical outcome of the patients was not significantly changed regardless of the status of adjuvant chemotherapy after surgery in this study (Fig. 3C,D). LST-2 immunoreactivity and clinicopathological parameters in 102 breast carcinoma cases. We then examined the correlation
between LST-2 immunoreactivity and the clinicopathological parameters of the cases (Table 2). A significant inverse correlation was detected between LST-2 immunoreactivity and tumor size (22.8 ± 1.4 vs 28.2 ± 2.0, P = 0.0289). Tumor size in the LST-2-positive population was statistically smaller than in the LST-2-negative group. The LST-2 and 17β-HSD1 immunoreactivities were positively correlated (P = 0.0426). In addition, a weak inverse correlation was detected between the expression of LST-2 and Ki-67 LI, but it did not reach statistical significance (23.8 ± 2.4 vs 30.3 ± 2.4, for LST-2-positive, LST-2-negative, P = 0.0585). After univariate analysis using Cox’s proportional hazard model (Table 3), lymph node status (P < 0.0001), EST immunoreactivity (P = 0.001), STS immunoreactivity (P = 0.01), LST-2 1574
immunoreactivity (P = 0.02) and tumor size (P = 0.001) were all demonstrated to be significant prognostic factors for diseasefree survival in this group of patients. Multivariate analysis further demonstrated that lymph node status (P = 0.001), LST-2 immunoreactivity (P = 0.03) and EST immunoreactivity (P = 0.03) were the only independent prognostic factors for disease-free survival with relative risks >1. Other factors were not significant. Univariate analysis (Table 4) using Cox’s proportional hazard model demonstrated that lymph node status (P = 0.001), EST immunoreactivity (P = 0.003), LST-2 immunoreactivity (P = 0.01), STS immunoreactivity (P = 0.03), HER-2 status (P = 0.03) and tumor size (P = 0.04) were all significant prognostic parameters for the overall survival of the patients after curative operation. Multivariate analysis subsequently demonstrated that lymph nodes status (P = 0.01), EST immunoreactivity (P = 0.01) and LST-2-immunoreactivity (P = 0.01) were independent prognostic factors for overall survival with relative risks >1. Discussion
Previously, it was reported that LST-2 plays a key role in the uptake a variety of endogenous and exogenous compounds, including bile acids, prostaglandins, conjugated steroids, some kinds of drugs, and hormones into cells. (15,24–27) LST-2 is expressed weakly in normal human liver but strongly in gastrointestinal cancers.(15) However, the role of LST-2 in cancer cells has not been clarified. LST-2 immunoreactivity was detected in breast carcinoma cells of 50% of the 102 invasive ductal carcinoma cases examined in the present study, but LST-2 immunoreactivity was not detected in epithelial cells of morphologically normal glands. LST-2 was distributed specifically in breast carcinoma cells. As shown in Table 2, LST-2 immunoreactivity was inversely correlated with tumor size (P = 0.03) and was associated with a decreased risk of recurrence or improved prognosis (Fig. 2A,B). However, LST-2 was positively correlated with 17β-HSD1 (P = 0.04). 17β-HSD1 immunoreactivity was reportedly inversely correlated with both Ki-67 LI and histological grade, and the 17β-HSD1-positive cases were relatively well differentiated.(28) These were well-established diagnostic modalities, associated with good prognosis. Multivariate analyses demonstrated that LST-2 immunoreactivity was a strong independent prognostic factor for both recurrence and overall survival, as was lymph node status, and all were strongly related to patient mortality.(29) We found that histological grade, Ki-67 and HER2 were not significant, possibly because the present study was carried out with a relatively small number of subjects. LST-2 immunoreactivity was inversely correlated with tumor size. LST-2 immunoreactivity was also weakly inversely correlated with Ki-67 LI. It has been reported that Ki-67 is a nuclear antigen associated with cell proliferation.(30) Ki-67 LI is already known to be a useful prognostic factor in human breast cancer.(31) Consequently, it is suggested that LST-2 may be associated with cell proliferation in breast carcinoma. Next, we examined the relationship between LST-2 immunoreactivity and ER status. As shown in Fig. 2, in the ER-positive patients LST-2 immunoreactivity correlated with decreased recurrence and improved prognosis. However, in the ER-negative patients, LST-2 immunoreactivity did not correlate with decreased recurrence and clinical outcome (Fig. 3C,D). LST-2 status affected the prognosis only in the ER-positive patients. Table 1 shows that LST-2 immunoreactivity was inversely correlated with tumor size (P = 0.03) and Ki-67 (P = 0.01) only in the ER-positive group. In addition, a similar tendency was detected in the postmenopausal group. Therefore, these results suggest that the estrogenic actions of breast cancer cells are affected by LST-2. Because the adjuvant therapies for breast cancer were heterogeneous among these cases, we tested for a doi: 10.1111/j.1349-7006.2007.00570.x © 2007 Japanese Cancer Association
Table 3. Univariate and multivariate analyzes of disease-free and overall survival in 102 breast carcinoma patients examined Variable
Univariate P-value
Multivariate P-value
Relative risk (95% CI)
Lymph node status (positive/negative) Tumor size (>20 mm/10/
