Purpose:In an effort to additionally determine the global patterns of CpG island hypermethylation in sporadic breast cancer, we searched for aberrant promoter methylation at 10 gene loci in 54 primary breast cancer and 10 breast benign lesions.Experimental Design: Genomic DNA sodium bisulfate converted from benign and malignant tissues was used as template in methyl-specific PCR for BRCA1, p16, ESR1, GSTP1, TR␤1, RAR␤2, HIC1, APC, CCND2, and CDH1 genes.Results: The majority of the breast cancer (85%) showed aberrant methylation in at least 1 of the loci tested with half of them displaying 3 or more methylated genes.The highest frequency of aberrant promoter methylation was found for HIC1 (48%) followed by ESR1 (46%), and CDH1 (39%). Similar methylation frequencies were detected for breast benign lesions with the exception of the CDH1 gene (P ‫؍‬ 0.02). The analysis of methylation distribution indicates a statistically significant association between methylation of the ESR1 promoter, and methylation at CDH1, TR␤1, GSTP1, and CCND2 loci (P < 0.03). Methylated status of the BRCA1 promoter was inversely correlated with methylation at the RAR␤2 locus (P < 0.03).Conclusions: Our results suggest a nonrandom distribution for promoter hypermethylation in sporadic breast cancer, with tumor subsets characterized by aberrant methylation of specific cancer-related genes. These breast cancer subgroups may represent separate biological entities with potential differences in sensitivity to therapy, occurrence of metastasis, and overall prognosis.

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The Ku70/80 heterodimer is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK) and its DNAbinding activity mediates DNA double-strand breaks repair. Although Ku80 was recently proposed as a caretaker gene involved in the control of genome integrity, no data are available on Ku70/80 DNAbinding activity in human tumors. Heterodimer DNAbinding activity and protein expression were assayed by electrophoretic-mobility-shift-assay (EMSA) and Western blot analysis, in nuclear and cytoplasmic extracts from eight breast, seven bladder primary tumors and three metastatic nodes from breast cancers. Corresponding normal tissues of the same patients were used as controls. Ten out of 15 tumors showed nuclear Kubinding activity 3 ± 10 times higher than in the normal tissues, irrespective of bladder or breast origin. Conversely, in 5/15 primary tumors and in all the metastatic nodes analysed, nuclear Ku-activity was 1.5 ± 4.5-fold lower than in the corresponding normal tissues. Cytoplasmic heterodimer activity signi®cantly diered between tumor and normal tissues, displaying a 2 ± 10-fold increase in neoplastic tissues. Three dierent patterns combining both Ku expression and activity with tumor characteristics were identi®ed. In low aggressive breast tumors p70/p80 proteins were expressed in tumor but not in normal tissues. The heterodimer bindingactivity matched the protein levels. In non-invasive bladder carcinomas no signi®cant dierences in protein expression between tumor and the corresponding normal tissues were found, however heterodimer binding-activity was increased in tumor samples. In breast and bladder tumors, at the advanced stage and in node metastases, the binding activity was strongly reduced in tumor biopsies, however no dierences were demonstrated between normal and tumor protein levels. Our results suggest a dierent modulation of Ku70/80 DNA-binding activity in human neoplastic tissues, possibly related to tumor progression. Findings provide further data on tissue-speci®c protein expression and post-translational regulation of heterodimer activity. Oncogene (2001) 20, 739 ± 747.

Our results suggest that COX-2 is highly expressed in DCIS and takes part in the molecular pathway implicated in progression of breast cancer and may provide a rationale for targeting COX-2 in preinvasive breast cancer therapy.

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