OBJECTIVES:The phosphatidylinositol 3-kinase/AKT axis is an important cell-signaling pathway that mediates cell proliferation and survival, two biological processes that regulate malignant cell growth. The phosphatidylinositol 3-kinase CA gene encodes the p110α subunit of the phosphatidylinositol 3-kinase protein. There are phosphatidylinositol 3-kinase CA mutations in several types of human tumors, and they are frequently observed in breast cancer. However, these mutations have not been investigated in Brazilian breast cancer patients.METHODS:PCR-SSCP and direct DNA sequencing were performed to identify phosphatidylinositol 3-kinaseCA exon 9 and exon 20 mutations in 86 patients with sporadic breast cancer. The relationships between PIK3CA mutations and patient clinicopathological characteristics and survival were analyzed. The presence of the TP53 mutation was also examined.RESULTS:Twenty-three (27%) of the 86 primary breast tumors contained PIK3CA mutations. In exons 9 and 20, we identified the hotspot mutations E542K, E545K, and H1047R, and we identified two new missense mutations (I1022V and L1028S) and one nonsense (R992X) mutation. Phosphatidylinositol 3-kinase CA exon 20 mutations were associated with poor overall survival and TP53 gene mutations.CONCLUSIONS:Phosphatidylinositol 3-kinase CA mutations are common in tumors in Brazilian breast cancer patients, and phosphatidylinositol 3-kinase CA and TP53 mutations are not mutually exclusive. Phosphatidylinositol 3-kinase CA exon 20 mutations are associated with poor survival, and they may be useful biomarkers for identifying breast cancer patients with aggressive tumors and for predicting the response to treatment with PI3K pathway inhibitors.

Linkage studies have indicated that a gene on chromosome arm 17q, designated BRCA1, confers susceptibility to familial breast and ovarian cancer. To investigate the possible involvement of the BRCA1 gene in sporadic breast cancer we have analysed loss of heterozygosity (LOH) in a panel of 100 sporadic primary breast tumours using 10 PCR-based polymorphic markers from 17q12-21. Allele losses were detected in 40 of 100 tumours informative for at least one of the markers analysed. Of these 40 deleted tumours, 27 showed partial or interstitial loss on 17q. The pattern of LOH in the tumours with partial or interstitial LOH revealed three putative distinct deleted regions on 17q12-21. The first lies on the proximal long arm between D17S250 and THRA1; the second one lies between D17S776 and D17S579, the region containing the BRCA1 gene; and the third is telomeric to D17S733. The most frequently deleted region overlaps with the minimal region containing the BRCA1 gene, suggesting that this gene might also be associated with the development or progression of a proportion of sporadic breast tumours.

The members of the DnaJ/Hsp40 proteins are highly conserved through evolution, expressed in several tissues and act as co-chaperone regulating protein folding, transport, translational initiation and gene expression. Recently, using cDNA microarray we identified differences in the expression of the JDP1 (DNAJC12) gene, a member of the DnaJ/Hsp40 family, between ER-positive and ER-negative breast tumours. In this study, using quantitative real-time PCR (qPCR) we evaluated the expression pattern of the JDP1 gene in a series of 72 primary breast tumours and investigated the effects of 17ß-estradiol on the expression of the JDP1 in MCF-7 breast cancer cells. Three patterns of JDP1 mRNA expression were identified in the primary breast tumours analysed: normal expression was found in 14% of the cases, under-expression in 50%, and over-expression in 36% of the cases. High levels of JDP1 mRNA expression were significantly associated with estrogen receptor-positive status (p=0.02). No relationship was found between JDP1 mRNA expression and any other clinicopathological characteristics of the patients. Sequence analysis of the promoter region of the JDP1 gene revealed the presence of potential estrogen response elements (EREs), suggesting it to be under the control of estrogen action. We also assessed the effects of 17ß-estradiol (10 nM) on JDP1 mRNA expression in MCF-7 breast cancer cells. The JDP1 transcripts were found to be up-regulated in a time-dependent fashion in MCF-7 cells exposed to 17ß-estradiol treatment. Here we show for the first time that JDP1 is a estrogen target gene and that its expression might be used as a marker of the ER transactivation activity and may have a predictive value for response to hormonal therapy.

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Over-expression of ERBB2, a member of the family of transmembrane receptor tyrosine kinases, occurs in 15-30% of primary breast tumors and is associated with poor prognosis and chemoresistance to a variety of anticancer drugs. In this study, aiming to identify differentially-expressed genes involved in erbB2-mediated transformation of the breast, we generated SAGE libraries from two human mammary cell lines, derived from normal luminal cells, expressing different levels of erbB2. The parental cell line HB4a expresses basal levels and the C5.2 expresses high levels of erbB2. A total of 161,632 tags was generated by sequencing, 81,684 from HB4a cells (30,854 unique tags) and 79,948 from C5.2 cells (30,568 unique tags). The comparison between the HB4a and C5.2 libraries revealed 334 distinct transcripts more expressed in HB4a cells and 328 distinct transcripts more expressed in C5.2 cells. The expression pattern of some of these transcripts was further validated by RT-PCR. The C5.2 cell line, which overexpress ERBB2, showed in comparison to HB4a cells a higher percentage of genes involved in transport, RNA processing, apoptosis and protein folding. A higher percentage of the genes more expressed in HB4a cells compared to C5.2 were found to be involved in signal transduction and cytoskeleton organization. The use of SAGE analysis allowed us to identify a significant number of genes implicated in different cellular pathways up-or down-regulated in the presence of ERBB2 over-expression, including genes not previously implicated in breast cancer that could be considered as potential candidate markers for prognosis and therapy.