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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.

The KEAP1/Nrf2 pathway is a master regulator of several redox-sensitive genes implicated in resistance of tumor cells against chemotherapeutic drugs. Recent data suggest that epigenetic mechanisms may play a pivotal role in the regulation of KEAP1 expression. We performed a comprehensive genetic and epigenetic analysis of the KEAP1 gene in 47 non-small cell lung cancer tissues and normal specimens. Promoter methylation analysis was performed using a quantitative methylation specific PCR assay in real time. Methylation at the KEAP1 promoter region was detected in 22 out of the 47 NSCLCs (47%) and in none of the normal tissues analyzed. Somatic mutations were detected in 7 out of the 47 tumors (15%) and loss of heterozygosity (LOH) in 10 out of the 47 (21%) of the cases. Overall, we found at least one molecular alteration in 57% of the cases. Approximately one third of the tumors had two alterations and this feature was associated with higher risk of disease progression in univariate COX regression analysis (HR = 3.62; 95% CI 1.24-10.65, p = 0.02). This result was confirmed by Kaplan-Meier analysis, which demonstrated an association between worst outcome and KEAP1 double alterations (p = 0.01, Log rank test). Our results further suggest that deregulation of the NRF2/KEAP1 system could play a pivotal role in the cancerogenesis of NSCLC. In addition identifying patients with KEAP1 genetic and epigenetic abnormalities may contribute to disease progression prediction and response to therapy in lung cancer patients.

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CpG island hypermethylation is emerging as one of the main mechanisms for inactivation of cancer related genes in breast tumorigenesis. We examined the changes in methylation patterns during ductal breast cancer progression from atypical ductal hyperplasia to in situ and invasive carcinoma. Paired samples of synchronous pre invasive lesions (Atypical Ductal Hyperplasia and/or Ductal Carcinoma in situ) and invasive ductal breast carcinoma from 31 patients, together with isolated lesions from additional 24 patients were studied. Overall, 95 pathological samples and 20 normal breast tissues were analyzed by Quantitative Methylation Specific PCR (QMSP) on a panel of 9 gene promoters (ESR1, APC, CDH1, CTNNB1, GSTPI, THBS1, MGMT, TMS1 and TIMP3). APC, CDH1, and CTNNB1 promoter regions showed an increase in frequency of methylation and increased methylation levels in pathological samples when compared with normal breast tissues. The analysis of the syncronous paired breast lesions demonstrated also an increase in methylation frequency and level for APC, CDH1, and CTNNB1 genes during progression. By establishing a cutoff value, we were able to distinguish among -invasive and invasive lesions. Synchronous methylation of APC, CDH1, and CTNNB1 was associated only with invasive lesions, whereas simultaneous methylation of APC and CDH1 or APC and CTNNB1 were more frequent in ductal carcinoma in situ and invasive carcinoma. Our data point to direct involvement of APC, CDH1, and CTNNB1 CpG island promoter methylation in the early stages of breast cancer progression, and suggest that these molecular alterations might be involved in the transition to an invasive phenotype.

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression.

These data suggest that BRAF T1796A activating mutation is not common in primary uveal melanoma. These findings are in accord with known differences in tumorigenesis between uveal and cutaneous melanomas.