Hiroki Odawara

Activation of Aromatase Expression by Retinoic Acid Receptor-related Orphan Receptor (ROR) α in Breast Cancer Cells IDENTIFICATION OF A NOVEL ROR RESPONSE ELEMENT

Estrogen is a key regulator of the proliferation and differentiation of breast cancer cells. In addition to the estrogen supply from the ovary, estrogen is produced locally from androgen by aromatase. However, the regulation of aromatase gene expression in breast cancer has not yet been fully clarified. Retinoic acid receptor-related orphan receptor (ROR) alpha plays an important role in the differentiation of many organs by regulating the transcription of target genes. Because aromatase and RORalpha are expressed in breast cancer, the effect of RORalpha on aromatase gene expression was studied. RORalpha significantly augmented the expression of aromatase mRNA, particularly those containing exon I.4, in MCF7 cells, and aromatase activities in T47D and MCF7 cells. RORalpha also stimulated the proliferation of these cells. Transient transfection-based reporter gene assays using the promoter at exon I.4 showed that RORalpha augmented the transcription. A series of truncated mutation studies revealed that RORalpha activated the transcription through -147 to +14 bp of the promoter I.4. Furthermore, RORalpha bound to the fragment containing -119 to -107 bp of the promoter in vitro, indicating that this region may contain a novel ROR response element. Chromatin immunoprecipitation assay showed that RORalpha bound to the region containing this site of the promoter I.4 in MCF7 cells. Moreover, we examined clinical samples and found a correlation between RORalpha and aromatase expression. These results suggest that RORalpha directly activates the aromatase expression to accelerate the local production of estrogen, which results in the proliferation of breast cancer cells.Estrogen is a key regulator of the proliferation and differentiation of breast cancer cells. In addition to the estrogen supply from the ovary, estrogen is produced locally from androgen by aromatase. However, the regulation of aromatase gene expression in breast cancer has not yet been fully clarified. Retinoic acid receptor-related orphan receptor (ROR) α plays an important role in the differentiation of many organs by regulating the transcription of target genes. Because aromatase and RORα are expressed in breast cancer, the effect of RORα on aromatase gene expression was studied. RORα significantly augmented the expression of aromatase mRNA, particularly those containing exon I.4, in MCF7 cells, and aromatase activities in T47D and MCF7 cells. RORα also stimulated the proliferation of these cells. Transient transfection-based reporter gene assays using the promoter at exon I.4 showed that RORα augmented the transcription. A series of truncated mutation studies revealed that RORα activated the transcription through −147 to +14 bp of the promoter I.4. Furthermore, RORα bound to the fragment containing −119 to −107 bp of the promoter in vitro, indicating that this region may contain a novel ROR response element. Chromatin immunoprecipitation assay showed that RORα bound to the region containing this site of the promoter I.4 in MCF7 cells. Moreover, we examined clinical samples and found a correlation between RORα and aromatase expression. These results suggest that RORα directly activates the aromatase expression to accelerate the local production of estrogen, which results in the proliferation of breast cancer cells.

APOBEC3B high expression status is associated with aggressive phenotype in Japanese breast cancers

BackgroundThe members of AID/APOBEC protein family possess cytidine deaminase activity that converts cytidine residue to uridine on DNA and RNA. Recent studies have shown the possible influence of APOBEC3B (A3B) as DNA mutators of breast cancer genome. However, the clinical significance of A3B expression in Japanese breast cancer has not been studied in detail.MethodsNinety-three primary breast cancer tissues (74 estrogen-receptor (ER) positive, 3 ER and HER2 positive, 6 HER2 positive, and 10 triple negative) including 37 tumor-normal pairs were assessed for A3B mRNA expression using quantitative real-time RT-PCR. We analyzed the relation between A3B expression, mutation analysis of TP53 and PIK3CA by direct sequencing, polymorphic A3B deletion allele and human papillomavirus (HPV) infection in tumors.ResultsA3B mRNA was overexpressed in tumors compared with normal tissue. Patients with high A3B expression were associated with subtype and progression of lymph node metastasis and pathological nuclear grade. However, the expression was not related to any other clinicopathological factors, including mutation of TP53 and PIK3CA, polymorphic A3B deletion allele, HPV infection and survival time.ConclusionThe expression of A3B in breast cancer was higher than in non-cancerous tissues and was related to the lymph node metastasis and nuclear grade, which are reliable aggressive phenotype markers in breast cancer. Evaluation of A3B expression in tumor may be a marker for breast cancer with malignant potential.

The Effect of Nicorandil on Ischemia-Reperfusion Injury in a Porcine Total Hepatic Vascular Exclusion Model

BACKGROUND We evaluated the effectiveness of nicorandil, which has both K(ATP) channel opener-like and nitrate-like properties, in liver ischemia-reperfusion (IR) injury using a porcine total hepatic vascular exclusion (THVE) model. METHODS Mexican hairless pigs weighing 25-55 kg were used in this study. The animals were divided into three groups. In the nicorandil group (n = 6), a 100 μg/kg bolus of nicorandil was injected intravenously 30 min before the ischemia, and then a continuous infusion (10 μg/kg/min) was administered intravenously for 30 min until just before the ischemia. In the control group (n = 6), a saline solution was injected in the same manner. In the glibenclamide group (n = 6), glibenclamide (0.1 mg/kg), which closes the K(ATP) channel gate, was orally administered 180 min before the hepatic ischemia, and then nicorandil was injected in the same manner as in the nicorandil group. THVE was performed for 120 min, and animals were observed until 360 min after reperfusion. Serum AST and LDH levels, hepatic tissue blood flow (HTBF), and histologic analyses were compared among the three groups. RESULTS Serum AST and LDH levels in the nicorandil group were significantly lower than in the other two groups after reperfusion, while no significant difference was observed between the control and the glibenclamide groups. HTBF in the nicorandil group was also significantly higher than in the other two groups after reperfusion, while no significant difference was observed between the control and glibenclamide groups. Additionally, histopathologic analyses revealed that the hepatic tissue was better maintained in the nicorandil group than in the other two groups. CONCLUSION Our results using a porcine THVE model suggest that nicorandil inhibits hepatic IR injury. The K(ATP) channel-opener aspect of nicorandil might be primarily responsible for the hepatoprotective effect.

Isolated retromammary lymph node metastasis of breast cancer without axillary lymph node involvement: a case report with a false-negative sentinel lymph node biopsy

A 54-year-old woman visited our hospital with a palpable tumor in her left breast, which was diagnosed as invasive ductal carcinoma. Breast-conserving surgery was performed, in association with a sentinel lymph node (SLN) biopsy and back-up dissection of the axillary lymph nodes. One dyed axillary lymph node with high radioactivity was defined as an SLN, and intraoperative frozen-section analysis of the SLN was negative for metastasis. The final pathological diagnosis of the tumor was invasive ductal carcinoma, and one small lymph node, located in the retromammary space, just under the tumor, was positive for metastasis. The backup axillary lymph nodes were not metastatic. This patient was diagnosed false-negative by SLN biopsy, despite being positive for retroMLN metastasis. It should be recognized that retroMLNs are difficult to detect preoperatively, or intra-operatively, using dye or radiocolloid, if they are located in the post-tumoral retro-mammary space. RetroMLNs may be a pitfall in SLN biopsies.