Hisashi Hirakawa

Estrogen-related receptor alpha in human breast carcinoma as a potent prognostic factor.

Estrogen-related receptor α (ERRα) was identified as a gene related to estrogen receptor α (ERα) and belongs to a class of nuclear orphan receptors. ERRα binds to estrogen responsive element(s) (ERE) and is considered to be involved in modulation of estrogenic actions. However, biological significance of ERRα remains largely unknown. Therefore, we examined the expression of ERRα in human breast carcinoma tissues using immunohistochemistry (n = 102) and real-time reverse transcription-PCR (n = 30). ERRα immunoreactivity was detected in the nuclei of carcinoma cells in 55% of breast cancers examined, and relative immunoreactivity of ERRα was significantly (P = 0.0041) associated with the mRNA level. Significant associations were detected between ERα and ERE-containing estrogen-responsive genes, such as pS2 (P < 0.0001) and EBAG9/RCAS1 (P = 0.0214), in breast carcinoma tissues. However, no significant association was detected between ERα and pS2 (P = 0.1415) in the ERRα-positive cases (n = 56) or between ERα and EBAG9/RCAS1 (P = 0.8271) in the ERRα-negative group (n = 46). ERRα immunoreactivity was significantly associated with an increased risk of recurrence and adverse clinical outcome by both uni- (P = 0.0097 and P = 0.0053, respectively) and multi- (P = 0.0215 and P = 0.0118, respectively) variate analyses. A similar tendency was also detected in the group of breast cancer patients who received tamoxifen therapy after surgery. Results from our study suggest that ERRα possibly modulates the expression of ERE-containing estrogen-responsive genes, and ERRα immunoreactivity is a potent prognostic factor in human breast carcinoma.

Aromatase Localization in Human Breast Cancer Tissues: Possible Interactions between Intratumoral Stromal and Parenchymal Cells

Aromatase is a key enzyme in intratumoral estrogen production required for the production of estrogens through the conversion of serum androgens in postmenopausal breast cancer patients. There have been, however, controversies regarding the intratumoral localization of aromatase in human breast carcinoma tissues. Therefore, we have first examined the intratumoral localization of aromatase mRNA/protein in 19 breast carcinomas using laser capture microdissection/quantitative reverse transcription-PCR (RT-PCR) and immunohistochemistry. Aromatase mRNA and protein were detected in both intratumoral stromal and parenchymal cells in breast carcinoma tissues. Subsequent microarray expression profiling and clustering analyses, in addition to quantitative RT-PCR studies, showed a significant positive correlation between aromatase and estrogen-related receptor alpha mRNA expression in isolated carcinoma cells. We further examined an interaction between stromal cells isolated from human breast carcinoma tissues and breast carcinoma cell lines using a coculture system to study the biological characteristic of aromatase expression in carcinoma cells. Aromatase mRNA and enzyme activity and 17beta-hydroxysteroid dehydrogenase type 1 mRNA in breast carcinoma cell lines, including MCF-7 and SK-BR-3 cells, were up-regulated in the presence of patient-derived 32N or 74T intratumoral stromal cells. The results from steroid conversion assays were also consistent with the findings above. The results of our study also showed that aromatase inhibitors were more effective in inhibiting aromatization induced by coculture in MCF-7 than that in stromal 32N. The examination of the localization of aromatase and its regulation, including the interactions existing between different cell types in human breast carcinoma tissues, may provide important information as to achieving better clinical response to aromatase inhibitors in breast cancer patients.

5α-Reductase type 1 and aromatase in breast carcinoma as regulators of in situ androgen production

Previous in vitro studies demonstrated that bioactive androgen 5α‐dihydrotestosterone (DHT) exerted antiproliferative effects through an interaction with androgen receptor (AR) in breast carcinoma cells. However, AR status has not been examined in association with DHT concentration in breast carcinoma tissues, and significance of androgenic actions remains unclear in breast carcinomas. Therefore, in our study, we first examined intratumoral DHT concentrations in 38 breast carcinoma tissues using liquid chromatography/electrospray tandem mass spectrometry. Intratumoral DHT concentration was positively associated with 5α‐reductase type 1 (5αRed1), and negatively correlated with aromatase. We then examined clinical significance of AR and 5αRed1 status in 115 breast carcinoma tissues by immunohistochemistry. Breast carcinomas positive for both AR and 5αRed1 were inversely associated with tumor size or Ki‐67. These patients showed significant associations with a decreased risk of recurrence and improved prognosis for overall survival, and the AR / 5αRed1 status was demonstrated an independent prognostic factor. Moreover, we examined possible regulation of DHT production by aromatase in in vitro studies. DHT synthesis from androstenedione in MCF‐7 cells was significantly inhibited by coculture with aromatase‐positive stromal cells, which was significantly reversed by addition of aromatase inhibitor exemestane. These results suggest that intratumoral DHT concentration is mainly determined by 5αRed1 and aromatase in breast carcinoma tissues, and antiproliferative effect of DHT may primarily occur in the cases positive for both AR and 5αRed1. Aromatase inhibitors may be more effective in these patients, possibly due to increasing local DHT concentration with estrogen deprivation.

Intratumoral concentration of sex steroids and expression of sex steroid-producing enzymes in ductal carcinoma in situ of human breast

It is well known that sex steroids play important roles in the development of invasive ductal carcinoma (IDC) of the human breast. However, biological significance of sex steroids remains largely unclear in ductal carcinoma in situ (DCIS), regarded as a precursor lesion of IDC, which is partly due to the fact that the intratumoral concentration of sex steroids has not been examined in DCIS. Therefore, in this study, we first examined the intratumoral concentrations of estradiol and 5alpha-dihydrotestosterone (DHT) using liquid chromatography/electrospray tandem mass spectrometry in DCIS. Intratumoral concentrations of both estradiol and DHT were threefold higher in DCIS than non-neoplastic breast tissues and estrogen-producing enzymes (aromatase, steroid sulfatase, and 17beta-hydroxysteroid dehydrogenase type 1 (17betaHSD1)), and androgen-producing enzymes (17betaHSD5 and 5alpha-reductase type 1 (5alphaRed1)) were abundantly expressed in DCIS by real-time PCR and immunohistochemical analyses. The intratumoral concentration of DHT was significantly lower in IDC than DCIS, while the expression of aromatase mRNA in carcinoma cells and intratumoral stromal cells was significantly higher in IDC than those in DCIS. Immunohistochemistry for sex steroid-producing enzymes in DCIS demonstrated that 5alphaRed1 immunoreactivity was positively correlated with Ki-67 labeling index and histological grade and was also associated with an increased risk of recurrence in patients with DCIS examined. Results of our study suggest that intratumoral concentrations of estradiol and DHT are increased in DCIS, which is possibly due to intratumoral production of these steroids. Therefore, estradiol and DHT may play important roles in the development of DCIS of the human breast.

Invasive micropapillary carcinoma of the breast: clinicopathological and immunohistochemical study.

Invasive micropapillary carcinoma (IMPCa) of the breast refers to a unique variant of invasive ductal carcinoma, but its biological behavior has not been elucidated well. We analyzed 16 IMPCa cases (10 pure type, six mixed type). The incidence of IMPCa was 1.0% of all primary breast carcinoma. High nuclear grade (75.0%), as well as poorly differentiated histological grade (81.3%), was frequently seen. Lymph node metastases were evident in 92.9% of the examined cases, and about half of them showed more than 10 positive nodes. Comparison between serially experienced invasive ductal carcinoma, not otherwise specified (IDC‐NOS), revealed that both high nuclear grade and poor histological grade were significantly more frequent (P < 0001), there was a lower frequency of positive estrogen receptor/progesterone receptor (P < 0.05, P < 0.01), a higher frequency of HER‐2 overexpression (P < 0.025), and more frequent lymph node metastases (P < 0.05) in IMPCa. The comparison between lymph node positive IDC‐NOS did not show any statistically significant differences in frequency for positive p53, matrix metalloproteinase protein‐2 (MMP‐2), vascular endothelial growth factor (VEGF) or E‐cadherin. However, IMPCa showed a significantly increased number of blood vessels counted by CD34 immunostains (P < 0.05). These results suggest that IMPCa is, at least, the same or more aggressive than lymph node positive cases of IDC‐NOS. Hence, not only the high incidence of lymph node metastases but also distant, blood‐borne metastases may be important.

LIN28: a regulator of tumor-suppressing activity of let-7 microRNA in human breast cancer.

A tumor-suppressor gene, let-7 microRNA (miRNA) family, is often inactivated in various human malignancies. LIN28 is a RNA-binding protein that has been well characterized for regulation of let-7 maturation in undifferentiated embryonic stem cells at post-transcriptional level. Oncogenic regulation of let-7 miRNAs has been demonstrated in several human malignancies but their correlation with LIN28 has not been studied in breast cancer. We therefore explored a possible mechanism of tumorigenesis in breast carcinoma tissue via an alternation of let-7 miRNA precursor processing by LIN28 in this study. A total of 26 breast cancer surgical pathology specimens were evaluated for LIN28 and LIN28B expression using immunohistochemistry. We then isolated carcinoma cells in 21 cases using laser capture microdissection, and the miRNAs from these samples were profiled using PCR array analysis. LIN28 status was positively correlated with ERα, PR, and Ki-67 status and inversely correlated with HER2 status. These results suggest the possible involvement of LIN28 in regulation of sex steroid dependent cell proliferation of breast carcinoma cells. We further demonstrated that expression of let-7a, let-7c, let-7d (P=0.026) and let-7f (P=0.016) were inversely correlated with those of LIN28. These results also suggest that LIN28 promotes tumorigenic activity by suppressing let-7 miRNA maturation in breast carcinoma cells.

Prognostic significance of tumor-infiltrating CD8 + and FOXP3 + lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: a retrospective multicenter study

IntroductionThe status of tumor-infiltrating lymphocytes (TILs) has been recently proposed to predict clinical outcome of patients with breast cancer. We therefore studied the prognostic significance of CD8+ TILs and FOXP3+ TILs in residual tumors after neoadjuvant chemotherapy (NAC) and the alterations in these parameters before and after NAC in patients with triple-negative breast cancer (TNBC).MethodsOne hundred thirty-one TNBC patients who received NAC at three institutions were examined. CD8+ TIL and FOXP3+ TIL in residual tumors and biopsy specimens were evaluated by double-staining immunohistochemistry. The CD8+ TIL and FOXP3+ TIL status of the residual tumors was assessed, and the rates of their changes before and after NAC were calculated.ResultsTNBC patients with high CD8+ TIL levels or a high CD8/FOXP3 ratio in residual tumors had significantly better recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) than patients with low values of these parameters. In multivariate analyses, CD8+ TIL exhibited strong prognostic significance for RFS, with a hazard ratio (HR) of 3.09 (95 % confidence interval (CI) 1.537–6.614, P=0.0013). The CD8/FOXP3 ratio was also significantly correlated with RFS (HR=2.07, 95 % CI 1.029–4.436, P=0.0412). TNBC with larger residual tumor size and positive lymph node status, which are known prognostic factors, was independently associated with worse RFS (P=0.0064 and P=0.0015, respectively). High CD8+ TIL levels were a markedly powerful indicator of improved BCSS, with an HR of 3.59 (95 % CI 1.499–9.581, P=0.0036). Nodal status was also associated with BCSS (P=0.0024). TNBC with a high rate of CD8+ TIL changes was associated with significantly better RFS compared with the low group (P=0.011). Higher rates of changes in the CD8/FOXP3 ratio were significantly correlated with both better RFS and BCSS compared with lower rates (P=0.011 and P=0.023, respectively).ConclusionsThis is the first study to demonstrate that high CD8+ TIL and a high CD8/FOXP3 ratio in residual tumors and increment of these parameters following NAC and accurately predict improved prognosis in TNBC patients with non-pathological complete response following NAC. These parameters could serve as a surrogate one for adjuvant treatment in patients with residual disease in the neoadjuvant setting.

Increased intratumoral androgens in human breast carcinoma following aromatase inhibitor exemestane treatment

Sex steroids play important roles in the development of many human breast carcinomas, and aromatase inhibitors are used for the anti-estrogen therapy. Recent studies have demonstrated that aromatase suppressed 5alpha-dihydrotestosterone (DHT) synthesis in breast carcinoma cells, but intratumoral concentration of androgens and its significance have not been reported in the breast carcinoma patients treated with aromatase inhibitors. Therefore, we examined androgen concentrations in breast carcinoma tissues treated with exemestane, and further performed in vitro studies to characterize the significance of androgen actions. Intratumoral DHT concentration was significantly higher in breast carcinoma tissues following exemestane treatment (n=9) than those without the therapy (n=7), and 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2) status was significantly altered to be positive after the treatment. Following in vitro studies showed that 17betaHSD2 expression was dose dependently induced by both DHT and exemestane in T-47D breast carcinoma cells, but these inductions were not additive. DHT-mediated induction of 17betaHSD2 expression was markedly suppressed by estradiol (E(2)) in T-47D cells. E(2)-mediated cell proliferation was significantly inhibited by DHT in T-47D cells, associated with an increment of 17betaHSD2 expression level. These findings suggest that intratumoral androgen actions are increased during exemestane treatment. 17betaHSD2 is a potent DHT-induced gene in human breast carcinoma, and may not only be involved in anti-proliferative effects of DHT on breast carcinoma cells but also serve as a potential marker for response to aromatase inhibitor in the breast carcinoma patients.

Vasohibin-1 as a potential predictor of aggressive behavior of ductal carcinoma in situ of the breast.

Vasohibin‐1 is a recently identified negative feedback regulator of angiogenesis induced by VEGF‐A and bFGF. In this study, we first evaluated mRNA expression of vasohibin‐1 and CD31 in 39 Japanese female breast carcinoma specimens including 22 invasive ductal carcinoma (IDC) and 17 ductal carcinoma in situ (DCIS) using a real‐time quantitative RT‐PCR (QRT‐PCR) with LightCycler system. In addition, we also immunolocalized vasohibin‐1 and CD31 and compared their immunoreactivity to nuclear grades and histological grades of 100 carcinoma cases (50 IDC and 50 DCIS). There were no statistically significant differences of CD31 mRNA expression and the number of CD31 positive vessels between DCIS and IDC (P = 0.250 and P = 0.191, respectively), whereas there was a statistically significant difference in vasohibin‐1 mRNA expression and the number of vasohibin‐1 positive vessels in DCIS and IDC (P = 0.022 and P ≤ 0.001, respectively). There was a significant positive correlation between vasohibin‐1 mRNA level and Ki‐67 labeling index in DCIS (r2 = 0.293, P ≤ 0.001). In addition, vasohibin‐1 mRNA expression was correlated with high nuclear and histological grades in DCIS cases and a significant positive correlation was detected between the number of vasohibin‐1 positive vessels and Ki‐67 labeling index or nuclear grade or Van Nuys classification of carcinoma cells (P ≤ 0.001, respectively). These results all indicate the possible correlation between aggressive biological features in DCIS including increased tumor cell proliferation and the status of neovascularization determined by vasohibin‐1 immunoreactivity.

Androgens in human breast carcinoma

Sex steroids play important roles in the development of human breast carcinoma. Androgen receptor (AR) is expressed in a majority of breast carcinoma tissues. However, the significance of androgen actions remains largely unclear in breast carcinoma, differing from estrogen actions. Therefore, in this review, we summarized recent studies on androgens in breast carcinoma. Concentration of a potent androgen, 5α-dihydrotestosterone (DHT), was significantly higher in breast carcinoma tissue than in plasma, and DHT is considered to be locally produced from circulating androstenedione by 17β-hydroxysteroid dehydrogenase type 5 and 5α-reductase. On the other hand, aromatase was recently reported as a negative regulator for intratumoral DHT production by possibly reducing the precursor testosterone. Androgens predominantly show antiproliferative effects in breast carcinoma cells, but association between AR status and the clinical outcome of the patient remains controversial, perhaps partly because AR status does not necessarily reflect androgenic action in breast carcinoma. Recently, molecular apocrine breast carcinoma was identified by microarray analysis. Molecular apocrine carcinoma was characterized by being estrogen receptor (ER) negative and AR positive and by being associated with increased androgen signaling and apocrine features. Therefore, androgenic actions may also be involved in apocrine features in breast carcinoma.