Increased chemosensitivity via BRCA2-independent DNA damage in DSS1- and PCID2-depleted breast carcinomas.

Breast cancer, the most common malignancy among women, is closely associated with mutations in the tumor suppressor gene BRCA. DSS1, a component of the TRanscription-EXport-2 (TREX-2) complex involved in transcription and mRNA nuclear export, stabilizes BRCA2 expression. DSS1 is also related to poor prognosis in patients with breast cancer owing to the induction of chemoresistance. Recently, BRCA2 was shown to be associated with the TREX-2 component PCID2, which prevents DNA:RNA hybrid R-loop formation and transcription-coupled DNA damage. This study aimed to elucidate the involvement of these TREX-2 components and BRCA2 in the chemosensitivity of breast carcinomas. Our results showed that compared with that in normal breast tissues, DSS1 expression was upregulated in human breast carcinoma, whereas PCID2 expression was comparable between normal and malignant tissues. We then compared patient survival time among groups divided by high or low expressions of DSS1, BRCA2, and PCID2. Increased DSS1 expression was significantly correlated with poor prognosis in recurrence-free survival time, whereas no differences were detected in the high and low BRCA2 and PCID2 expression groups. We performed in vitro analyses, including propidium iodide nuclear staining, single-cell gel electrophoresis, and clonogenic survival assays, using breast carcinoma cell lines. The results confirmed that DSS1 depletion significantly increased chemosensitivity, whereas overexpression conferred chemoresistance to breast cancer cell lines; however, BRCA2 expression did not affect chemosensitivity. Similar to DSS1, PCID2 expression was also inversely correlated with chemosensitivity. These results strongly suggest that DSS1 and PCID2 depletion is closely associated with increased chemosensitivity via BRCA2-independent DNA damage. Together with the finding that DSS1 is not highly expressed in normal breast tissues, these results demonstrate that DSS1 depletion confers a druggable trait and may contribute to the development of novel chemotherapeutic strategies to treat DSS1-depleted breast carcinomas independent of BRCA2 mutations.

Identification of targets of tumor suppressor microRNA-34a using a reporter library system.

miRNAs play critical roles in various biological processes by targeting specific mRNAs. Current approaches to identifying miRNA targets are insufficient for elucidation of a miRNA regulatory network. Here, we created a cell-based screening system using a luciferase reporter library composed of 4,891 full-length cDNAs, each of which was integrated into the 3' UTR of a luciferase gene. Using this reporter library system, we conducted a screening for targets of miR-34a, a tumor-suppressor miRNA. We identified both previously characterized and previously uncharacterized targets. miR-34a overexpression in MDA-MB-231 breast cancer cells repressed the expression of these previously unrecognized targets. Among these targets, GFRA3 is crucial for MDA-MB-231 cell growth, and its expression correlated with the overall survival of patients with breast cancer. Furthermore, GFRA3 was found to be directly regulated by miR-34a via its coding region. These data show that this system is useful for elucidating miRNA functions and networks.

Effects of serum estradiol and progesterone on estrogen-regulated gene expression in breast cancers of premenopausal patients.

BACKGROUND: Expression of estrogen receptor α in breast cancer is essential for estrogen-dependent growth and partially determines the breast cancer subtype. In premenopausal women, expression of estrogen-regulated genes in estrogen receptor-positive breast cancer tissues are reportedly influenced by the menstrual cycle. METHODS: We investigated correlations between serum estradiol (E2; tested on the day of surgery) and expression of estrogen-regulated genes and proliferation genes in strongly estrogen receptor α-positive breast cancer tissues from 91 premenopausal women by quantitative reverse transcription-polymerase chain reaction. We also investigated correlations between serum progesterone levels on the day of surgery and mRNA expression of progesterone-regulated genes and proliferation genes. RESULTS: The serum E2 level affected expression of estrogen-regulated genes, including progesterone receptor (P = 0.016, Rs = 0.07) but showed no correlation with expression of genes associated with proliferation. We also observed strong positive correlations between mRNA expression of ESR1 and that of estrogen-regulated genes (P < 0.0001, Rs = 0.329-0.756) and proliferation genes (P < 0.0001, Rs = 0.753-0.843). The serum progesterone level affected expression of RANKL mRNA. However, we observed no correlations between serum progesterone and expression of Wnt-4 or proliferation genes. CONCLUSIONS: The serum E2 level on the day of surgery influences estrogen-regulated gene expression moderately in patients found to be strongly positive for estrogen receptor α by immunohistochemistry. Changes in serum E2 levels might influence the results of molecular profiling tests in premenopausal women with breast cancer.

Prognostic impact of TP53INP1 gene expression in estrogen receptor α-positive breast cancer patients.

BACKGROUND: Tumor protein 53-induced nuclear protein 1 (TP53INP1) is a key stress protein with tumor suppressor function. Several studies have demonstrated TP53INP1 downregulation in many cancers. In this study, we investigated the correlations of TP53INP1 mRNA expression in breast cancer tissues with prognosis and the correlations of microRNAs that regulate TP53INP1 expression in breast cancer patients with long follow-up. METHODS: A total of 453 invasive breast cancer tissues were analyzed for TP53INP1 mRNA expression. We examined correlations of clinicopathological factors and expression levels of TP53INP1 mRNA in these samples. The expressions of miR-155, miR-569 and markers associated with tumor-initiating capacity were also analyzed. The median follow-up period was 9.0 years. RESULTS: We found positive correlations between low expression of TP53INP1 mRNA and shorter disease-free survival and overall survival in breast cancer patients (P = 0.0002 and P < 0.0001, respectively), as well as in estrogen receptor α (ERα)-positive patients receiving adjuvant endocrine therapy (P = 0.01 and P = 0.0008, respectively). No correlations were found in ERα-negative patients. Low TP53INP1 mRNA levels positively correlated with higher grade and ERα-negativity. Multivariate analysis indicated that TP53INP1 mRNA level was an independent risk factor for overall survival both in overall breast cancer patients (hazard ratio, 2.13; 95% confidence interval, 1.17-3.92) and ERα-positive patients (hazard ratio, 2.34; 95% confidence interval, 1.18-4.64). CONCLUSIONS: We show that low expression of TP53INP1 is an independent factor of poor prognosis in breast cancer patients, especially ERα-positive patients. TP53INP1 might be a promising candidate biomarker and therapeutic target in ERα-positive breast cancer patients.

[Three Poor Performance Status(PS)Cases of Metastatic Breast Cancer Controlled with Adjustment of Dosing Interval and Dosage of Bevacizumab and Paclitaxel].

A standard symptomatic therapy regimen of bevacizumab(BV)plus paclitaxel(PTX)was planned for use in 3 cases of metastatic breast cancer. Due to poor patient performance status(PS)because of malignant pleural effusion and ascites, the initial standard regimen was determined to be unsuitable. However, adjustment and fine-tuning of the BV plus PTX interval and dosage were found to be effective in improving symptoms, and consequently obtained good efficacy. Adverse effects were managed with drug withdrawal and symptomatic therapy. The 3 clinical cases all included females aged 62-76 years old, with a median age of 67.6. One case was classified as PS 3, and 2 were classified as PS 4. The main deciding factors for initiating the regimen of BV plus PTX were 2 cases of malignant pleural effusion and 1 case of malignant ascites, which contributed to worsening of the overall PS. With adjustment and fine-tuning of the BV plus PTX interval and dosage, we were able to safely achieve symptomatic improvement in 3 metastatic breast cancer cases, in which the overall PS grade was unsuitable for standard chemotherapy.

Prognostic impact of a single-nucleotide polymorphism near the CTSO gene in hormone receptor-positive breast cancer patients.

BACKGROUND: Tamoxifen can reduce the occurrence of breast cancer by a half in high-risk women. Recently, a genome-wide association study identified two single-nucleotide polymorphisms (SNPs) near or in the CTSO and ZNF423 genes that were associated with breast cancer risk during tamoxifen therapy. We hypothesized that these two SNPs could be associated with increased recurrence in breast cancer patients who received adjuvant tamoxifen therapy. METHODS: A total of 586 breast carcinomas were available for SNP genotyping assays. TaqMan pre-designed SNP genotyping assays were used to identify the presence of CTSO rs10030044 and ZNF423 rs8060157. We then investigated the relationship between CTSO rs10030044 genotypes and mRNA expression levels of CTSO and BRCA1 in 290 breast cancer patients. RESULTS: We found a positive correlation between the variant GG genotype of CTSO rs10030044 and shorter disease-free survival, or overall survival in hormone receptor-positive breast cancer patients receiving adjuvant tamoxifen therapy. In contrast, this genotype was not associated with prognosis in hormone receptor-negative breast cancer patients. Multivariate Cox regression analysis revealed that this genotype was an independent factor indicating a poor prognosis in hormone receptor-positive breast cancer patients receiving adjuvant tamoxifen therapy. No association was found between CTSO genotype and mRNA expression of CTSO and BRCA1. ZNF423 rs8060157 genotype was not associated with prognosis in this study. CONCLUSION: We show that a SNP near the CTSO gene is a poor prognostic factor in breast cancer although further research might help to reveal the factors linking this genotype and prognosis.

Immunohistochemical determination of the miR-1290 target arylamine N-acetyltransferase 1 (NAT1) as a prognostic biomarker in breast cancer.

BACKGROUND: There are many molecular differences between estrogen receptor α (ERα)-positive and ER-negative breast cancers. Recent analyses have shown that the former can be divided into two subtypes, luminal A and luminal B. These differ in response to endocrine therapy and chemotherapy, and in prognosis. In a previous study, we found that microRNA (miR)-1290 that was significantly down-regulated in luminal A tumors and its potential target arylamine N-acetyltransferase 1 (NAT1). The aim of the present study was to determine whether NAT1 is a bona fide target of miR-1290, and to investigate the impact of NAT1 on breast cancer prognosis. METHODS: Luciferase reporter assays were employed to validate NAT1 as a putative miR-1290 target gene. Expression of NAT1, ERα, progesterone receptor (PgR) and HER2 was analyzed in 394 breast cancer samples by immunohistochemistry. RESULTS: NAT1 was confirmed to be a direct target of miR-1290. Levels of expression of NAT1 were positively correlated with those of ERα (P < 0.0001) and PgR (P < 0.0001), but negatively correlated with both tumor grade and size (P < 0.0001). Kaplan-Meier analysis showed that the presence of NAT1 was significantly associated with increased overall survival (OS) (P = 0.0416) in these patients. Similarly, significant associations of NAT1 with disease-free survival (DFS) (P = 0.0048) and OS (P = 0.0055) in those patients who received adjuvant endocrine therapy with tamoxifen (n = 176) were found. Moreover, NAT1 was also significantly associated with increased DFS (P = 0.0025) and OS (P = 0.0007) in the subset of lymph node-positive patients (n = 147). Univariate and multivariate analyses showed significant associations between levels of NAT1 and DFS (P = 0.0005 and 0.019, respectively). CONCLUSIONS: We report that miR-1290 directly targets the NAT1 3'-UTR and that NAT1 protein expression is correlated with improved OS of breast cancer patients. NAT1 is a possible prognostic biomarker for lymph node-positive breast cancer. Thus, miR-1290 and its target NAT1 are associated with important characteristics of breast cancer.

High expression of LMTK3 is an independent factor indicating a poor prognosis in estrogen receptor α-positive breast cancer patients.

OBJECTIVE: Over 70% of breast cancers are estrogen receptor alpha-positive, and endocrine therapy targeting estrogen action decreases mortality from breast cancer. Recently, a novel protein kinase that regulates estrogen receptor alpha activity, lemur tyrosine kinase-3, has been identified. In this study, we investigated whether messenger RNA expression and polymorphisms of the gene encoding the kinase, LMTK3, are associated with prognosis in breast cancer patients during long-term follow-up. METHODS: First, we investigated the relationship between messenger RNA expression of LMTK3 and patient outcome in 219 breast cancers. The effects of several variables on survival were tested by Cox proportional hazards regression analysis. Next, we performed LMTK3 genotyping in 471 breast cancers to clarify the prognostic role of these polymorphisms. RESULTS: Our data showed that LMTK3 expression level was not associated with prognosis in all patients. We then analyzed the impact of LMTK3 mRNA expression on the prognosis of breast cancer according to estrogen receptor alpha status. Both disease-free survival and overall survival were significantly shorter in estrogen receptor alpha-positive patients with high LMTK3 expression receiving adjuvant endocrine therapy than in those patients with low LMTK3 expression. Multivariate Cox regression analysis revealed that high LMTK3 expression was an independent poor prognostic factor in estrogen receptor alpha-positive breast cancer patients. We did not find any correlation between LMTK3 genotypes and prognosis of breast cancer patients in our series. CONCLUSIONS: Our results show that high expression of LMTK3 is an independent prognostic factor in estrogen receptor alpha-positive breast cancer patients receiving adjuvant endocrine therapy.

HER2 mutation status in Japanese HER2-negative breast cancer patients.

OBJECTIVE: Human epidermal growth factor receptor 2 (HER2) gene amplification is a major therapeutic target in breast cancer, and has been introduced as a predictive biomarker to identify patients who may benefit from therapy with anti-human epidermal growth factor receptor 2 agents. Human epidermal growth factor receptor 2 somatic mutations have been reported in patients without human epidermal growth factor receptor 2 gene amplification. Since these are activating mutations, these patients may also benefit from human epidermal growth factor receptor 2-targeted drugs. METHODS: In this study, we searched for human epidermal growth factor receptor 2 mutations in a group of 286 Japanese breast cancer patients with human epidermal growth factor receptor 2-negative tumors. The activating mutations of human epidermal growth factor receptor 2 identified were analyzed by direct Sanger sequencing of two major areas: the extracellular domain at 309-310 and the kinase domain between 755 and 781. RESULTS: Two tumors were found to have a human epidermal growth factor receptor 2 somatic mutation; one with I767M mutation and another with D769Y. No mutation was observed in the extracellular domain. One of these patients with human epidermal growth factor receptor 2 mutation recurred early with liver metastasis. CONCLUSIONS: Better knowledge of human epidermal growth factor receptor 2 mutation status will help us to choose personalized molecular targeted therapy for use in human epidermal growth factor receptor 2-negative Japanese breast cancer patients.

HER2 mutation status in Japanese HER2-positive breast cancer patients.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) gene amplification/overexpression is a major therapeutic target in breast cancer, and has been introduced as a predictive biomarker to identify patients who may benefit from therapy with anti-HER2 agents. HER2 somatic mutations have been reported, and these may influence the effect of HER2-targeted drugs. METHODS: Here, we sought HER2 mutations in a group of 135 Japanese breast cancer patients with HER2-positive tumors. We analyzed HER2 mutations by direct Sanger sequencing of two major areas, the extracellular domain at position 309-310 and the kinase domain between 755 and 781. RESULTS: Two patients with the HER2 somatic mutation S310F in the extracellular domain were found in this series. One patient with the S310F mutation had a node-negative invasive ductal carcinoma classified as HER2 2+ by the HercepTest and fluorescence in situ hybridization (FISH) positive, and which was estrogen receptor (ER)-negative and progesterone receptor (PgR)-negative. Another patient with the S310F mutation had an apocrine carcinoma with seven lymph nodes positive for metastasis, classified as HER2 3+ by the HercepTest, but which was FISH-negative, as well as ER-negative and PgR-negative. Both patients had received adjuvant single-agent trastuzumab therapy, and had no local recurrence or distant metastasis for five and three years after surgery, respectively. CONCLUSIONS: Our data show that HER2 mutations are rare in HER2-positive Japanese breast cancer patients. The two mutations found in this study were identical, S310F. We suggest that in vitro experiments to determine whether the S310F mutation could be involved in resistance to anti-HER2 drugs are worthwhile in future.

Detection of parasternal metastatic lymph nodes by sentinel lymph node methods in a patient with recurrence in the conserved breast.

We herein report a case of second sentinel lymph node biopsy (SLNB). A 57-year-old woman underwent breast-conserving surgery including axillary clearance at Aichi Cancer Center on October 20, 2003. Recurrent tumor in the conserved breast was diagnosed in March 2006. She received SLNB using radioactive tracer. Preoperative lymphoscintigraphy detected 2 parasternal lymph nodes as hot spots. No abnormal lymph nodes were revealed on preoperative computed tomography. Salvage mastectomy was performed along with dissection of the Rotter and infraclavicular lymph nodes and biopsy of the detected parasternal lymph nodes. Micrometastases were discovered in both parasternal lymph nodes detected as sentinel lymph nodes. No more metastases were seen in the other lymph nodes. Reoperative SLNB offers the possibility of detecting metastasis in residual lymph nodes and determining whether chemotherapy should be used.