Loss of annexin A1 expression in human breast cancer detected by multiple high-throughput analyses

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Abstract

To test the efficacy of combined high-throughput analyses (HTA) in target gene identification, screening criteria were set using >fivefold difference by microarray and statistically significant changes (p < 0.01) in SAGE and EST. Microarray analysis of two normal and seven breast cancer samples found 129 genes with >fivefold changes. Further SAGE and EST analyses of these genes identified four qualified genes, ERBB2, GATA3, AGR2, and ANXA1. Their expression pattern was validated by RT-PCR in both breast cell lines and tissue samples. Loss of ANXA1 in breast cancer was further confirmed at mRNA level by Human Breast Cancer Tissue Profiling Array and at protein level by immunohistochemical staining. This study demonstrated that combined HTA effectively narrowed the number of genes for further study, while retaining the sensitivity in identifying biologically important genes such as ERBB2 and ANXA1. A distinctive loss of ANXA1 in breast cancer suggests its involvement in maintaining normal breast biology.

Section snippets

Materials and methods

Sample collection and RNA extraction. Human breast tissues were collected according to NIH guidelines, and by protocols approved by the UCLA Institutional Review Board. Fresh surgical samples of breast cancer and normal breast tissues were grossly dissected to remove as much fat tissues as possible and then stored in liquid nitrogen. TriZol (Invitrogen, Carlsbad, CA) was used for total RNA extraction according to the manufacturer’s instructions. RNA quality was examined by

Differential gene expression detected by multiple HTA

CGAP employed several approaches, including EST and SAGE, to catalog all genes expressed during cancer development [3]. Through May 2004, the CGAP database has collected about four million EST and 12 million SAGE tags. To explore the feasibility of using this resource for measuring gene expression levels, Virtual Northern was used to study the expression of a panel of 30 known breast cancer-related genes in cancerous and benign breast tissues (Table 2). Microarray, SAGE, and EST vNorthern each

Multiple HTA

HTA has proven to be a powerful tool in novel gene discovery, classification, and prognosis prediction in cancers. Recent studies focused on identification of disease-related genes [17], [18], expressional profiling to classify cancer into various subtypes [19], [20], [21], and to predict prognosis [22], [23], [24]. Since different HTA have different sensitivities and/or specificities in target gene discovery, we hypothesized that a properly combined use of these technologies might improve the

Conclusion

In summary, four differentially expressed genes in human breast cancer, ERBB2, GATA3, AGR2, and ANXA1, were detected by multiple HTA consisting of microarray and bioinformatic SAGE and EST. The loss of ANXA1 was further confirmed both at the mRNA level by real-time RT-PCR and human breast cancer profiling array, and at the protein level by immunohistochemical staining. Our study suggests that multiple HTA is a useful strategy for discovering biomarkers with clinical relevance in cancer

Acknowledgments

We thank the UCLA DNA microarray core for technical assistance in microarray analysis and Rebecca Radbod for statistical analysis. This study is supported in part by grants from the National Cancer Institute (5R01 CA093736), the Gonda Foundation, the UCLA Human Gene Medicine Program, and the Early Detection Research Network (NCI CA986366).

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    Abbreviations: ANXA1, annexin A1; BCRG, breast cancer-related gene; CGAP, cancer genome anatomy project; DCIS, ductal carcinoma in situ; EST, expressed sequence tag; HTA, high-throughput analysis; IDC, invasive ductal carcinoma; RT-PCR, reverse transcriptase polymerase chain reaction; SAGE, series analysis of gene expression; vNorthern, virtual Northern.

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