Ji-Yeon Bae

DNA copy number alterations and expression of relevant genes in triple-negative breast cancer

Triple‐negative breast cancer (TNBC) is defined by a lack of expression of estrogen, progesterone, and HER2 receptors, and genetically most of them fall into the basal subgroup of breast cancer. The important issue of TNBC is poorer clinical outcome and absence of effective targeted therapy. In this study, we sought to identify DNA copy number alterations and expression of relevant genes characteristic of TNBC to discover potential therapeutic targets. Frozen tissues from 114 breast cancers were analyzed using high‐resolution array comparative genomic hybridization. The classification into subtype was determined by estrogen and progesterone receptor expression, and by the presence or absence of gain on the ERBB2 containing clone. The ACE algorithm was used for calling gain and loss of clones. Twenty‐eight cases (25%) were classified as TNBC. Recurrent gains (≥25%) unique to TNBC were 9p24‐p21, 10p15‐p13, 12p13, 13q31‐q34, 18q12, 18q21‐q23, and 21q22. Two published gene expression array data sets comparing basal subtype versus other subtype breast cancers were used for searching candidate genes. Of the genes upregulated in the basal subtype, 45 of 686 genes in one data set and 59 of 1,428 in the second data set were found to be located in the gained regions. Of these candidate genes, gain of NFIB (9p24.1) was specific for TNBC in a validation set by real‐time PCR. In conclusion, we have identified recurrently gained regions characteristic of TNBC, and found that NFIB copy number and expression is increased in TNBC across the data sets. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

Genomic alterations identified by array comparative genomic hybridization as prognostic markers in tamoxifen-treated estrogen receptor-positive breast cancer

BackgroundA considerable proportion of estrogen receptor (ER)-positive breast cancer recurs despite tamoxifen treatment, which is a serious problem commonly encountered in clinical practice. We tried to find novel prognostic markers in this subtype of breast cancer.MethodsWe performed array comparative genomic hybridization (CGH) with 1,440 human bacterial artificial chromosome (BAC) clones to assess copy number changes in 28 fresh-frozen ER-positive breast cancer tissues. All of the patients included had received at least 1 year of tamoxifen treatment. Nine patients had distant recurrence within 5 years (Recurrence group) of diagnosis and 19 patients were alive without disease at least 5 years after diagnosis (Non-recurrence group).ResultsPotential prognostic variables were comparable between the two groups. In an unsupervised clustering analysis, samples from each group were well separated. The most common regions of gain in all samples were 1q32.1, 17q23.3, 8q24.11, 17q12-q21.1, and 8p11.21, and the most common regions of loss were 6q14.1-q16.3, 11q21-q24.3, and 13q13.2-q14.3, as called by CGH-Explorer software. The average frequency of copy number changes was similar between the two groups. The most significant chromosomal alterations found more often in the Recurrence group using two different statistical methods were loss of 11p15.5-p15.4, 1p36.33, 11q13.1, and 11p11.2 (adjusted p values <0.001). In subgroup analysis according to lymph node status, loss of 11p15 and 1p36 were found more often in Recurrence group with borderline significance within the lymph node positive patients (adjusted p = 0.052).ConclusionOur array CGH analysis with BAC clones could detect various genomic alterations in ER-positive breast cancers, and Recurrence group samples showed a significantly different pattern of DNA copy number changes than did Non-recurrence group samples.

Polymorphisms in the estrogen receptor-alpha gene and breast cancer risk

The estrogen receptor-alpha (ERalpha) has been known to play a role in the development and progression of breast cancer. Several genetic polymorphisms in the ERalpha gene have been related to breast cancer risk and/or different tumor characteristics. In this study, PCR and direct sequencing based methods were used to examine this issue further in a Korean study population consisting of 155 women, 110 with breast cancer and 45 without cancer. We also assessed the potential role of the ERalpha genotype in ER, PR, p53, c-erbB2, and bcl-2 expression. Only one of the allelic variants of ERalpha gene was found in our study subjects; the (C(975)G) change was present in half of the study subjects. Although this allele had no direct effect in individual breast cancer risk, it was positively associated with tumor PR (P for trend=0.04) and ER expression (P for trend=0.06) and negatively associated with p53 expression (P for trend=0.02).

Autoantibody to tumor antigen, alpha 2-HS glycoprotein: a novel biomarker of breast cancer screening and diagnosis.

We sought to identify a new serum biomarker for breast cancer screening and diagnosis using stepwise proteomic analysis of sera from breast cancer patients to detect the presence of autoantibodies that react with urinary protein. Two-dimensional immunoblotting was done for screening autoimmunogenic tumor antigens in the urine of breast cancer patients. Reactive spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Among urinary proteins separated by two-dimensional electrophoresis, 13 spots showed strong reactivity with pooled sera from breast cancer patients or control sera. By mass spectrometry, we identified α 2-HS glycoprotein (AHSG) as a tumor antigen. Peripheral blood was obtained from 81 women diagnosed with breast cancer before surgery and 73 female donors without evidence of any malignancy for the individual analysis. In one-dimensional Western blot analysis, AHSG autoantibody was detected in 64 of 81 breast cancer patients (79.1%) and in 7 of 73 controls (9.6%). The sensitivity of this test in breast cancer patients was 79.0%. Our results suggest that AHSG and anti-AHSG autoantibody may be useful serum biomarkers for breast cancer screening and diagnosis. (Cancer Epidemiol Biomarkers Prev 2009;18(5):1357–64)

Genomic copy number alterations as predictive markers of systemic recurrence in breast cancer

We tried to establish models that predict systemic recurrence in breast cancer by selecting marker clones with DNA copy number alterations (CNAs) using an array comparative genomic hybridization (CGH). Array CGH containing 4,044 human bacterial artificial chromosome clones was used to assess CNAs in 62 primary breast cancer tissues from 31 patients with systemic recurrence within 5 years after surgery and clinicopathologically well matched 31 patients who had no evidence of disease for at least 5years. Fourteen significant clones (11 clones showing gain and 3 showing loss) were identified by systemic recurrence‐free survival (SRFS) analysis and 23 significant clones (17 clones showing gain and 6 showing loss) identified by χ2 test and FDR test were selected as predictive markers of systemic breast cancer recurrence. The significant CNAs were found in the chromosomal regions of 5p15.33, 11q13.3, 15q26.3, 17q25.3, 18q23 and 21q22.3 with gain and 9p12, 11q24.1 and 14q32.33 with loss. We devised 2 prediction models for the systemic recurrence of breast cancer based on the 14 clones and the 23 clones, respectively. The survivals of the patients were significantly separated according to the scores from each model at the optimal cut off values in SRFS and overall survival analysis. We found candidate clones and genes of which CNAs were significantly associated with systemic recurrence of breast cancer. The devised prediction models with these clones were effective at differentiating the recurrence and nonrecurrence.

Peroxiredoxin I and II inhibit H2O2‐induced cell death in MCF‐7 cell lines

Apoptosis is known to be induced by direct oxidative damage due to oxygen‐free radicals or hydrogen peroxide or by their generation in cells by the actions of injurious agents. Together with glutathione peroxidase and catalase, peroxiredoxin (Prx) enzymes play an important role in eliminating peroxides generated during metabolism. We investigated the role of Prx enzymes during cellular response to oxidative stress. Using Prx isoforms‐specific antibodies, we investigated the presence of Prx isoforms by immunoblot analysis in cell lysates of the MCF‐7 breast cancer cell line. Treatment of MCF‐7 with hydrogen peroxide (H2O2) resulted in the dose‐dependent expressions of Prx I and II at the protein and mRNA levels. To investigate the physiologic relevance of the Prx I and II expressions induced by H2O2, we compared the survivals of MCF10A normal breast cell line and MCF‐7 breast cancer cell line following exposure to H2O2. The treatment of MCF10A with H2O2 resulted in rapid cell death, whereas MCF‐7 was resistant to H2O2. In addition, we found that Prx I and II transfection enabled MCF10A cells to resist H2O2‐induced cell death. These findings suggest that Prx I and II have important functions as inhibitors of cell death during cellular response to oxidative stress. J. Cell. Biochem. 101: 1038–1045, 2007.

Berberine inhibits growth of the breast cancer cell lines MCF-7 and MDA-MB-231.

The effects of berberine on the behavior of breast tumors have not yet been established. To determine whether this compound is useful in the treatment of breast cancer, we analyzed the impact of berberine on the human breast cancer cell lines MCF-7 and MDA-MB-231 cells. Berberine was added to proliferating MCF-7 and MDA-MB-231 cells in culture. Following treatment, changes in cell growth characteristics such as proliferation, cell cycle duration, and the degree of apoptosis were assayed. Following berberine treatment, a time-dependent reduction in proliferation was observed in both cell lines at differing concentrations: 20 microM for MCF-7 and 10 microM for MDA-MB-231 cells. Annexin V staining showed an increase in apoptosis in both cell lines of 31 % in MCF-7 and 12 % in MDA-MB-231 cells compared to their respective controls. In addition, 12 % of the MCF-7 cells were arrested at G0/G1, compared to 62 % of control cells. These results demonstrate that treatment with berberine inhibits growth in both MDA-MB-231 and MCF-7 cells. In addition, they show that this partly occurs through the induction of apoptosis in MDA-MB-231 cells, and through both cell cycle arrest and induction of apoptosis in MCF-7 cells. Thus, berberine may be a novel therapeutic drug for breast cancer.

Proteomic analysis of breast cancer tissue reveals upregulation of actin‐remodeling proteins and its relevance to cancer invasiveness

There is an emerging interest in protein expression profiling with the aim of identifying novel diagnostic markers and therapeutic targets in breast cancer. We analyzed breast cancer tissues by 2‐D DIGE using a narrow range IPG strip (pH 5.5–6.7) after the immunodepletion of serum albumin and Ig. Sixty‐three protein spots were detected with more than ±1.8‐fold differences (p <0.05 for three technical replicates) from a set of tissue samples in which three tumor and three nontumor samples were randomly selected from six breast cancer subjects and pooled separately. Of these, 53 proteins were successfully identified by MS. Among the proteins whose levels were increased, we identified three novel WD‐repeat‐motif‐bearing proteins that have been known to be involved in actin remodeling: Arp2/3 complex subunit 2 (p34‐Arc), coronin‐1A and WD‐repeat protein 1 (Wdr1). Significantly increased amounts of p34‐Arc and coronin‐1A in breast cancer were also shown by Western blot analysis of matched tumor and nontumor tissue samples (N = 11, p <0.05), and were consistent with the mRNA levels retrieved from publicly available microarray databases. The siRNA knockdown of p34‐Arc attenuated the invasion of SK‐BR3 breast cancer cells into Matrigel. In contrast, the overexpression of coronin‐1A increased this invasive activity. Taken together, the cellular levels of p34‐Arc and coronin‐1A were linked to cancer development and migration. The data obtained from the present study provides new insight into the management of breast cancer.

Prognosis of Secondary Acute Myeloid Leukemia is Affected by the Type of the Preceding Hematologic Disorders and the Presence of Trisomy 8

BACKGROUND Differences in the clinical course of secondary acute myeloid leukemia according to the type of the preceding disorders are not defined. We compared the outcomes of therapy-related acute myeloid leukemia, acute myeloid leukemia following myelodysplastic syndrome and acute myeloiod leukemia following myeloproliferative neoplasm. We also intended to find prognostic factors in secondary acute myeloid leukemia overall. METHODS Retrospective medical record review at Seoul National University Hospital was performed. We assessed response to induction chemotherapy and overall survival. RESULTS Ninety-five secondary acute myeloid leukemia patients (median age of 56.4 years) were analyzed. Twenty-six, 57 and 12 patients had therapy-related leukemia, leukemia following myelodysplastic syndrome and myeloproliferative neoplasm, respectively. For patients receiving induction chemotherapy, complete remission rate was 47.5% and complete remission rate was different according to the type of the preceding disorders (P = 0.004). Compared to therapy-related leukemia (P = 0.027) and leukemia following myelodysplastic syndrome (P = 0.050), leukemia following myeloproliferative neoplasm had shorter overall survival. In secondary leukemia, presence of trisomy 8 had a prognostic impact (P = 0.003) along with cytogenetic risk group (P = 0.016). In multivariate analysis, the type of the preceding disorders (P = 0.026), 5q deletion (P = 0.015) and trisomy 8 (P = 0.040) were independent prognostic factors. CONCLUSIONS Prognosis of secondary acute myeloid leukemia was different according to the type of the preceding disorders with the worst prognosis in leukemia following myeloprolfierative neoplasm. Along with cytogenetic risk grouping, trisomy 8 had a poor prognostic impact in secondary acute myeloid leukemia.

CD24 cross-linking induces apoptosis in, and inhibits migration of, MCF-7 breast cancer cells

BackgroundThe biological effects of CD24 (FL-80) cross-linking on breast cancer cells have not yet been established. We examined the impact of CD24 cross-linking on human breast cancer cell line MCF-7.MethodsMCF-7 and MDA-MB-231 cells were treated with anti-rabbit polyclonal IgG or anti-human CD24 rabbit polyclonal antibodies to induce cross-linking, and then growth was studied. Changes in cell characteristics such as cell cycle modulation, cell death, survival in three-dimensional cultures, adhesion, and migration ability were assayed after CD24 cross-linking in MCF-7.ResultsExpression of CD24 was analyzed by flow cytometry in MDA-MB-231 and MCF-7 cells where 2% and 66% expression frequencies were observed, respectively. CD24 cross-linking resulted in time-dependent proliferation reduction in MCF-7 cells, but no reduction in MDA-MB-231 cells. MCF-7 cell survival was reduced by 15% in three-dimensional culture after CD24 cross-linking. Increased MCF-7 cell apoptosis was observed after CD24 cross-linking, but no cell cycle arrest was observed in that condition. The migration capacity of MCF-7 cells was diminished by 30% after CD24 cross-linking.ConclusionOur results showed that CD24 cross-linking induced apoptosis and inhibited migration in MCF-7 breast cancer cells. We conclude that CD24 may be considered as a novel therapeutic target for breast cancer.