Nana Rokutanda

Expression and Mutations of KCNJ5 mRNA in Japanese Patients with Aldosterone-Producing Adenomas

CONTEXT Mutations of the KCNJ5 gene have recently been identified in patients with aldosterone-producing adenomas (APA). OBJECTIVE Our objective was to investigate the expression and mutations of the KCNJ5 gene in Japanese patients with APA. DESIGN AND PATIENTS We sequenced KCNJ5 cDNA and measured KCNJ5 mRNA levels in 23 patients with APA operated on at Gunma University Hospital. MAIN OUTCOME MEASURES Mutations and mRNA levels of the KCNJ5 gene were examined and compared to those in cortisol-producing adenomas (Cushings syndrome) and pheochromocytomas. RESULTS Of the 23 patients with APA, 15 (65.2%) had two somatic mutations of the KCNJ5 gene: 12 cases of p.G151R (eight with c.451G>A, and four with c.451G>C) and three cases of p.L168R (c.503T>G). Levels of KCNJ5 mRNA were significantly higher in the APA with mutations than those without. Immunohistochemistry also showed a stronger staining of KCNJ5 on the cell membrane in the tumor with a mutation. Furthermore, a PCR-restriction fragment length polymorphism assay with c.503T>G revealed the mutant mRNA to be expressed at a similar level to the wild type. The level of KCNJ5 mRNA in cortisol-producing adenomas was approximately 30% of that in APA, and almost no expression was observed in pheochromocytomas. CONCLUSION We found that: 1) a significant number of patients with APA had somatic mutations of the KCNJ5 gene; 2) KCNJ5 mRNA levels were higher in the APA with KCNJ5 mutations; and 3) the expression of KCNJ5 mRNA was significantly higher in APA than cortisol-producing adenomas and pheochromocytomas.

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.

Tamoxifen activates CYP3A4 and MDR1 genes through steroid and xenobiotic receptor in breast cancer cells

Cytochrome P450 monooxygenase 3A4 (CYP3A4) and P-glycoprotein, encoded by multidrug resistance 1 (MDR1) gene, are responsible for the metabolism of endogenous steroids, prescribed drugs, and xenobiotics. Both genes are regulated by steroid and xenobiotic receptor (SXR), a member of nuclear hormone receptors. Various endogenous steroids and drugs function as ligands of SXR. Although CYP3A4, MDR1, and SXR are expressed mainly in the liver and the small intestine, these gene products are also expressed in breast cancer cells. Because tamoxifen (TAM) is known to be metabolized by CYP3A4 and P-glycoprotein, weinvestigated the effect of TAM on these SXR-targeted genes in breast cancer cells. Transient transfection-based reporter gene assays showed 4-hydroxy TAM activated the SXR-mediated transcription through CYP3A4 and MDR1 promoters in a ligand- and receptor concentration-dependent manner. We confirmed the binding of 4-hydroxy TAM to SXR by ligand binding assay. Moreover, semiquantitative RT-PCR studies revealed that 4-hydroxy TAM activated the expression of CYP3A4 and MDR1 mRNA in MCF-7 cells. These results suggest that TAM induces CYP3A4 and MDR1 gene expression through SXR, which may affect TAM metabolic pathway in breast cancer cells.

Liquid chemiluminescent DNA pull-down assay to measure nuclear receptor-DNA binding in solution

Electrophoretic mobility shift assays (EMSAs) are commonly used to investigate protein-DNA binding in vitro. However, EMSA can generate considerable amounts of undesirable waste, particularly when toxic compounds are examined. We therefore developed a novel in vitro protein-DNA binding assay called liquid chemiluminescent DNA pull-down assay, which is based on solution hybridization between digoxigenin-labeled DNA and glutathione S-transferase (GST)-fused DNA binding protein bound to glutathione-Sepharose beads.

Reversible Cardiomyopathy After Epirubicin Administration

Anthracycline-containing chemotherapy can cause irreversible and progressive left ventricular dysfunction. Epirubicin, which is widely used for breast cancer chemotherapy, is an anthracycline that has less cardiac toxicity than doxorubicin. The present report describes the case of a 70-year-old woman with breast cancer who developed severe congestive heart failure and severe cardiac dysfunction at 6 weeks from epirubicin final administration. Left ventricular function gradually improved after intensive treatment for heart failure and recovered completely within 2 months. To the best of our knowledge, this is the first report to describe epirubicin-induced subacute reversible cardiotoxicity.

Abstract P3-06-29: Change of circulating tumor cells before and after neoadjuvant chemotherapy in patients with primary breast cancer

Background: Circulating tumor cells (CTCs) in peripheral blood may represent the possible presence of early tumor dissemination. However, relatively few studies were designed to investigate the change of CTC status by adjuvant chemotherapy in operable breast cancer patients. Patients and methods: Peripheral blood (7.5ml) was collected from 95 patients with stage II/III breast cancer before neoadjuvant chemotherapy (NAC). NAC consisted of anthracycline and paclitaxel chemotherapy and additional trastuzumab treatment for patients with HER2-positive tumors. Results: The average age of patients was 52.6 year old (median 52.0). One or more CTCs were detected in 18 (18.9%) of 95 patients. CTCs were detected in 6 (12.0%) of 50 patients with clinical stage II disease and 12 (26.7%) of 45 patients with clinical stage III disease. According to tumor subtypes, CTCs were detected in 5 (17.9%) of 28 patients with hormone receptor (HR)-positive and HER2-negative tumors (L subtype), 3 (12.5%) of 24 patients with HR-positive and HER2-positive tumors (L-H subtype), 4 (22.2%) of 18 patients with HR-negative and HER2-positive tumors (H subtype), and 6 (24.0%) of 25 patients with HR-negative and HER2-negative tumors (TN subtype). After NAC, 17 (94.4%) of 18 patients who were CTC-positive before chemotherapy changed into negative status. 30 (31.6%) of 95 patients had a pathologic complete response (pCR) after NAC. There was no correlation between CTC status before NAC and pathological response. At the median follow up of 27 months, distant metastasis was observed in 9 patients (9.5%). Patients with clinical stage III, TN subtype, or non-pCR had a significantly worse disease-free survival (DFS). However, CTC status before NAC was not a significant prognostic factor. Conclusion: NAC has a significant impact on CTC status irrespective of tumor subtypes. CTC status before NAC was not a significant prognostic factor in this study. The reason of which may be that most of patients showing positive for CTCs before NAC have changed into negative after NAC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-29.

Abstract P2-05-07: Comparison of hormone receptor and HER-2 expression in primary breast cancers and sentinel lymph node metastases

Background: Recently, it is a hot topic that the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor type 2 (HER2) may differ between the primary tumors and the paired recurrent metastatic tumors. On the other hand, the concordance of the ER, PR, and HER2 status between the primary tumor and ipsilateral axillary lymph node metastases is reported to be high in previous study. However, in these reports, the majority of lymph nodes metastases examined are macrometastases obtained from axillary lymph node dissection. The aim of the present study is to compare the ER, PR, and HER2 status of the primary breast tumors with that of sentinel lymph node micrometastases and macrometastases. Methods: A total of 770 breast cancer patients underwent sentinel lymph node biopsy between 2006 and 2010 at our hospital. Among them, 87 patients who had only one node positive were eligible for our study. ER, PR, and HER2 status were determined by immunohistochemistry (IHC) and/or FISH. Result: We analyzed 76 patients with 50(66%) patients with macrometastasesn and 26 (34%) micrometastases, except 11 cases those were indeterminate for IHC. ER status of primary tumor and the paired metastatic lymph node were almost full concordance except one case, in which ER was negative in primary tumor but ER was positive in lymph node. Discordance for PR was 15.8% (n = 12). Among these, six patients had PR-positive on primary and PR-negative in lymph node while six patients had PR-negative in primary and PR-positive in lymph node. Discordance for HER2 between primary tumor and metastatic lymph node was 5.3% (n = 4). Among these, three had negative in primaries and positive in lymph nodes while one had positive in primary and negative in lymph node. A discordance between primary and lymph node in HER2, ER, or PR status was observed in 14 of 67 (18.4%) cases. Conclusions: The concordance in HER2, ER, and PR was high between the primary tumor and sentinel lymph nodes. However some cases had discordance of receptor status and these result may cause of discordance for distant metastases or poor prognosis. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-07.

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.