P5-01-18: Identification of Pax6 as a Human Breast Cancer-Testis Antigen.
Authors: Yang M , Wang Y 
Published: June 2012

Abstract
Background
Cancer-testis antigens (CTAs) are specifically expressed in the normal testis and various types of cancer cells, but not or expressed with very low level in the other normal tissues. CTAs are considered to be promising targets for immunotherapy. Little is known on CTAs which specifically expressed in breast cancer. Studies show that the transcriptional activation by CpG island hypomethylation is believed to be an important mechanism of their increased expression.
Objective: To find CTAs which specifically expressed in breast cancer; prove the increase of its expression in human breast cancer, and explore the possibility for it to be developed as target for the immunodiagnosis and immunotherapy of breast cancer.
Methods and results Genomic DNAs were isolated from testis tissue, breast cancer and normal breast tissues respectively. To perform CpG island microarray assays, genomic DNAs were prepared from 15 paired breast cancer biopsies and their normal controls. After obtaining sequence information for those clones from the CpG island database, they were then used to search for surrounding genes in the gene banks. We found several testis specific genes, such as, Pax6, NDRG3 and Synaptojanin 2. Since the last two genes had been extended investigated, they were not the subjects of this study. The Pax6 Gene Was Activated in Established and Primary Breast Cancer Cell Lines. First, we examined Pax6 gene expression levels in a breast cancer cell line panel that included Hs578T, MDA-231, MDA-436, MDA-468, T47D, MCF7 and MCF7/Adr, by performing RT-PCR where b-actin served as control. In addition, the RNAs obtained from normal breast and testis tissues were used as negative and positive controls.We found that Pax6 was re-expressed in MDA-436, MDA-468, T47D, and MCF7/Adr. To further test the Pax6 gene's behavior in primary breast cancer cells, we isolated breast cancer epithelial cells from the effusions of 8 patients (E4, E5, K151, K259, K473, K573, K596 and K605). cRNAs were prepared from those cells and the normal human breast cell line (NHBEC) for RT-PCR assays, where cRNAs of paired MCF7 cells and b-actin served as controls. Results showed that Pax6 was expressed in K259, K573, K596 and K605 cells to various degrees. Pax6 Was Expressed in a Majority of Breast Cancer Biopsies. To test if Pax6 was also expressed in breast cancer tissues, IHC assays were carried out using 19 patients’ biopsies where human testes served as positive controls. We found that the Pax6 protein existed mainly in the cytoplasm of breast cancer cells, but was not detectable in normal breast tissue sections.
Conclusion CpG islands of pax6 promoter region were hypomethylated in human breast cancer. The mRNA level of Pax6 in human breast cancer was significantly increased contrast to normal breast tissue, contrast to all other analyzed normal tissues, suggesting Pax6 is a CTA for breast cancer and might be applied as molecular target in immunotherapy against human breast cancer.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-18.