Jeong Eon Lee

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.

Downregulation of the RUNX3 Gene by Promoter Hypermethylation and Hemizygous Deletion in Breast Cancer

The RUNX3 gene is regarded as a tumor suppressor gene in many human solid tumors, and its inactivation is believed to be related with solid tumor carcinogenesis. As little information is available about the role of the RUNX3 gene in breast cancer, we investigated the relationship between the RUNX3 gene and breast cancer. We performed reverse transcriptase-polymerases chain reaction (RT-PCR), methylation specific PCR, and bicolor fluorescent in situ hybridization analysis in an effort to reveal related mechanisms. Forty breast tissue samples and 13 cell lines were used in this study. Eighty-five percent of breast cancer tissues showed downregulated RUNX3 gene expression, whereas it was downregulated in only 25% of normal breast tissues by RT-PCR assay. Sixty-seven percent of breast cancer cell lines showed downregulated RUNX3 expression, but the RUNX3 gene was not expressed in two normal breast cell lines. Hypermethylation was observed in 53% of breast cancer tissues and 57% of breast cancer cell lines. Hemizygous deletion was observed in 43% of breast cancer cell lines. Hypermethylation and/or hemizygous deletion was observed in 5 of 7 breast cancer cell lines, and the four of these five examined showed no RUNX3 gene expression. We suggest that various mechanisms, including methylation and hemizygous deletion, could contribute to RUNX3 gene inactivation.

Neogenin expression may be inversely correlated to the tumorigenicity of human breast cancer

BackgroundNeogenin is expressed in cap cells that have been suggested to be mammary stem or precursor cells. Neogenin is known to play an important role in mammary morphogenesis; however its relationship to tumorigenesis remains to be elucidated.MethodsTo compare the expression levels of neogenin in cells with different tumorigenicity, the expression levels in M13SV1, M13SV1R2 and M13SV1R2N1 cells, which are immortalized derivatives of type I human breast epithelial cells, were evaluated. Then we measured the expression level of neogenin in paired normal and cancer tissues from eight breast cancer patients. Tissue array analysis was performed for 54 human breast tissue samples with different histology, and the results were divided into four categories (none, weak, moderate, strong) by a single well-trained blinded pathologist and statistically analyzed.ResultsThe nontumorigenic M13SV1 cells and normal tissues showed stronger expression of neogenin than the M13SV1R2N1 cells and the paired cancer tissues. In the tissue array, all (8/8) of the normal breast tissues showed strong neogenin expression, while 93.5% (43/46) of breast cancer tissues had either no expression or only moderate levels of neogenin expression. There was a significant difference, in the expression level of neogenin, in comparisons between normal and infiltrating ductal carcinoma (p < 0.001).ConclusionNeogenin may play a role in mammary carcinogenesis as well as morphogenesis, and the expression may be inversely correlated with mammary carcinogenicity. The value of neogenin as a potential prognostic factor needs further evaluation.

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.

βPix‐a enhances the activity of phospholipase Cγ1 by binding SH3 domain in breast cancer

Phospholipase C‐γ1 (PLCγ1) plays a critical role in cell growth and proliferation by generating the second messengers, diacylglycerol and 1, 4, 5‐inositol triphosphate. To investigate the roles of Src homology domain 2 and domain 3 of PLCγ1 in PLCγ1‐mediated cell signaling, we characterized some proteins binding to these domains in the MCF7 and MDA‐MB‐231 breast cancer cell lines. Of the several proteins that bind to glutathione‐S‐transferase‐SH2/SH2/SH3, we identified an 85 kDa protein that binds to the SH3 domain of PLCγ1 as the guanine nucleotide exchange factor, p21‐activated protein kinase‐interacting exchange factor‐a (βPix‐a). βPix‐a co‐immunoprecipitated with PLCγ1 in breast cancer tissues extracts and in MCF7 and MDA‐MB‐231 cell extracts. In addition, PDGF‐stimulated PLCγ1 activity was elevated in βPix‐a‐overexpressing NIH3T3 cells. Our results suggest that βPix‐a binds to the Src homology domain 3 of PLCγ1 and promotes tumor growth in breast cancer by enhancing the activity PLCγ1. J. Cell. Biochem. 94: 1010–1016, 2005.