Takanori Ishida

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.

Expression of the Steroid and Xenobiotic Receptor and Its Possible Target Gene, Organic Anion Transporting Polypeptide-A, in Human Breast Carcinoma

Steroid and xenobiotic receptor (SXR) or human pregnane X receptor (hPXR) has been shown to play an important role in the regulation of genes related to xenobiotic detoxification, such as cytochrome P450 3A4 and multidrug resistance gene 1. Cytochrome P450 enzymes, conjugation enzymes, and transporters are all considered to be involved in the resistance of breast carcinoma to chemotherapeutic or endocrine agents. However, the expression of SXR/hPXR proteins and that of its target genes and their biological or clinical significance have not been examined in human breast carcinomas. Therefore, we first examined SXR/hPXR expression in 60 breast carcinomas using immunohistochemistry and quantitative reverse transcription-PCR. We then searched for possible SXR/hPXR target genes using microarray analysis of carcinoma cells captured by laser microscissors. SXR/hPXR was detected in carcinoma tissues but not in nonneoplastic and stromal cells of breast tumors. A significant positive correlation was detected between the SXR/hPXR labeling index and both the histologic grade and the lymph node status of the carcinoma cases. Furthermore, in estrogen receptor-positive cases, SXR/hPXR expression was also positively correlated with expression of the cell proliferation marker, Ki-67. Microarray analysis showed that organic anion transporting polypeptide-A (OATP-A) was most closely correlated with SXR/hPXR gene expression, and both OATP-A mRNA and protein were significantly associated with SXR/hPXR in both breast carcinoma tissues and its cell lines. These results suggest that SXR/hPXR and its target gene, such as OATP-A, may play important roles in the biology of human breast cancers.

Sensitivity and specificity of mammography and adjunctive ultrasonography to screen for breast cancer in the Japan Strategic Anti-cancer Randomized Trial (J-START): a randomised controlled trial

BACKGROUND Mammography is the only proven method for breast cancer screening that reduces mortality, although it is inaccurate in young women or women with dense breasts. We investigated the efficacy of adjunctive ultrasonography. METHODS Between July, 2007, and March, 2011, we enrolled asymptomatic women aged 40-49 years at 42 study sites in 23 prefectures into the Japan Strategic Anti-cancer Randomized Trial (J-START). Eligible women had no history of any cancer in the previous 5 years and were expected to live for more than 5 years. Randomisation was done centrally by the Japan Clinical Research Support Unit. Participants were randomly assigned in 1:1 ratio to undergo mammography and ultrasonography (intervention group) or mammography alone (control group) twice in 2 years. The primary outcome was sensitivity, specificity, cancer detection rate, and stage distribution at the first round of screening. Analysis was by intention to treat. This study is registered, number UMIN000000757. FINDINGS Of 72,998 women enrolled, 36,859 were assigned to the intervention group and 36,139 to the control group. Sensitivity was significantly higher in the intervention group than in the control group (91·1%, 95% CI 87·2-95·0 vs 77·0%, 70·3-83·7; p=0·0004), whereas specificity was significantly lower (87·7%, 87·3-88·0 vs 91·4%, 91·1-91·7; p<0·0001). More cancers were detected in the intervention group than in the control group (184 [0·50%] vs 117 [0·32%], p=0·0003) and were more frequently stage 0 and I (144 [71·3%] vs 79 [52·0%], p=0·0194). 18 (0·05%) interval cancers were detected in the intervention group compared with 35 (0·10%) in the control group (p=0·034). INTERPRETATION Adjunctive ultrasonography increases sensitivity and detection rate of early cancers. FUNDING Ministry of Health, Labour and Welfare of Japan.

Nuclear cyclin B1 in human breast carcinoma as a potent prognostic factor

Cyclin B1 is translocated to the nucleus from the cytoplasm, and plays an essential role in cell proliferation through promotion of mitosis. Although overexpression of cyclin B1 was previously reported in breast carcinomas, the biological significance of the intracellular localization of cyclin B1 remains unclear. Therefore, in this study, we examined cyclin B1 immunoreactivity in 109 breast carcinomas, according to the intracellular localization, that is, nucleus, cytoplasm or total (nucleus or cytoplasm). Total cyclin B1 was detected in carcinoma cells in 42% of breast carcinomas examined, whereas nuclear and cytoplasmic cyclin B1 were positive in 17 and 35% of the cases, respectively. Total or cytoplasmic cyclin B1 were positively associated with histological grade, mitosis, Ki‐67, p53, c‐myc or 14‐3‐3σ, and inversely correlated with estrogen or progesterone receptor. Nuclear cyclin B1 was significantly associated with tumor size, lymph node metastasis, histological grade, mitosis, Ki‐67 or polo‐like kinase 1. Only nuclear cyclin B1 was significantly associated with adverse clinical outcome of the patients, and multivariate analyses of disease‐free and overall survival demonstrated nuclear cyclin B1 as the independent marker. A similar tendency was detected in the patients receiving adjuvant therapy after surgery. These results suggest that an onocogenic role of overexpressed cyclin B1 is mainly mediated in nuclei of breast carcinoma cells, and the nuclear translocation is regulated by polo‐like kinase 1 and 14‐3‐3σ. Nuclear cyclin B1‐positive breast carcinoma is resistant to adjuvant therapy, and nuclear cyclin B1 immunoreactivity is a potent prognostic factor in breast carcinoma patients. (Cancer Sci 2007; 98: 644–651)

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.

Vasohibin-1 in human breast carcinoma: a potential negative feedback regulator of angiogenesis.

Vasohibin‐1 is a recently identified negative feedback inhibitor or suppressor of angiogenesis induced by vascular endothelial growth factor (VEGF)‐A. The status of vasohibin‐1 in human breast carcinoma has not been examined. We examined 151 breast specimens including 98 cases of invasive ductal carcinoma (IDC), 12 of ductal carcinoma in situ (DCIS), 16 of fibroadenoma (FA), six of inflammatory lesion, nine of fibrocystic change and seven of non‐pathological breast tissue. We immunolocalized vasohibin‐1 and compared its immunoreactivity to that of VEGF‐A, basic fibroblastic growth factor (bFGF), VEGF receptor 2 (Flk‐1), CD31, CD34 and Ki‐67/MIB‐1. The correlation of vasohibin‐1 immunoreactivity with overall survival (OS), and disease‐free survival (DFS) of the patients with breast carcinoma was also evaluated. In addition, we evaluated Ki‐67 and CD31, and Ki‐67 and vasohibin‐1 double‐immunostaining for further characterization of neovascularization. Vasohibin‐1 was detected in endothelial cells of human breast and its immunodensity was significantly higher in IDC and inflammatory lesions than the other types (P < 0.001). In addition, a significant positive correlation was detected between vasohibin‐1 and VEGF‐A, bFGF or Flk‐1 (P < 0.001). There was also positive associations between vasohibin‐1 and OS (P = 0.004) and between vasohibin‐1 and DFS (P ≤ 0.001) in carcinoma cases. Results of double‐immunostaining demonstrated the ratio of Ki‐67‐positive cells among vasohibin‐1‐positive endothelial cells (46.5%) was significantly higher than those among CD31‐positive cells (23.5%). This is the first study demonstrating the status of vasohibin‐1 in human breast lesions, which indicates that vasohibin‐1 is associated with neovascularization and may especially play important roles in the regulation of intratumoral angiogenesis in human breast cancer. (Cancer Sci 2009; 100: 88–94)

Human liver-specific organic anion transporter-2 is a potent prognostic factor for human breast carcinoma.

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 LST‐2 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 estrone‐3‐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)

Isolation of temperature-sensitive p53 mutations from a comprehensive missense mutation library.

Temperature-sensitive (ts) mutations have been used as a genetic and molecular tool to study the functions of many gene products. Each ts mutant protein may contain a temperature-dependent intramolecular mechanism such as ts conformational change. To identify key ts structural elements controlling the protein function, we screened ts p53 mutants from a comprehensive mutation library consisting of 2,314 p53 missense mutations for their sequence-specific transactivity through p53-binding sequences in Saccharomyces cerevisiae. We isolated 142 ts p53 mutants, including 131 unreported ts mutants. These mutants clustered in β-strands in the DNA-binding domain, particularly in one of the two β-sheets of the protein, and 15 residues (Thr155, Arg158, Met160, Ala161, Val172, His214, Ser215, Pro223, Thr231, Thr253, Ile254, Thr256, Ser269, Glu271, and Glu285) were ts hot spots. Among the 142 mutants, 54 were examined further in human osteosarcoma Saos-2 cells, and it was confirmed that 89% of the mutants were also ts in mammalian cells. The ts mutants represented distinct ts transactivities for the p53 binding sequences and a distinct epitope expression pattern for conformation-specific anti-p53 antibodies. These results indicated that the intramolecular β-sheet in the core DNA-binding domain of p53 was a key structural element controlling the protein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function.

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.

Biophoton detection as a novel technique for cancer imaging.

Biophoton emission is defined as extremely weak light that is radiated from any living system due to its metabolic activities, without excitation or enhancement. We measured biophoton images of tumors transplanted in mice with a highly sensitive and ultra‐low noise CCD camera system. Cell lines employed for this study were AH109A, TE4 and TE9. Biophoton images of each tumor were measured 1 week after carcinoma cell transplantation to estimate the tumor size at week 1 and the biophoton intensity. Some were also measured at 2 and 3 weeks to compare the biophoton distribution with histological findings. We achieved sequential biophoton imaging during tumor growth for the first time. Comparison of microscopic findings and biophoton intensity suggested that the intensity of biophoton emission reflects the viability of the tumor tissue. The size at week 1 differed between cell lines, and the biophoton intensity of the tumor was correlated with the tumor size at week 1 (correlation coefficient 0.73). This non‐invasive and simple technique has the potential to be used as an optical biopsy to detect tumor viability.