Paola Parrella

Nonrandom Distribution of Aberrant Promoter Methylation of Cancer-Related Genes in Sporadic Breast Tumors

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, TRβ1, RARβ2, 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, TRβ1, GSTP1, and CCND2 loci (P < 0.03). Methylated status of the BRCA1 promoter was inversely correlated with methylation at the RARβ2 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.

Frequent epigenetics inactivation of KEAP1 gene in non-small cell lung cancer

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.

Changes in CpG Islands Promoter Methylation Patterns during Ductal Breast Carcinoma Progression

Aberrant promoter methylation of several known or putative tumor suppressor genes occurs frequently during carcinogenesis, and this epigenetic change has been considered as a potential molecular marker for cancer. We examined the methylation status of nine genes (APC, CDH1, CTNNB1, TIMP3, ESR1, GSTP1, MGMT, THBS1, and TMS1), by quantitative methylation specific PCR. Synchronous preinvasive lesions (atypical ductal hyperplasia and/or ductal carcinoma in situ) and invasive ductal breast carcinoma from 52 patients, together with pure lesions from 24 patients and 12 normal tissues paired to tumor and 20 normal breast distant from tumor were analyzed. Aberrant promoter methylation was detected in both preinvasive and invasive lesions for genes APC, CDH1, CTNNB1, TIMP3, ESR1, and GSTP1. However, hierarchical mixed model and Generalized Estimating Equations model analyses showed that only APC, CDH1, and CTNNB1 promoter regions showed a higher frequency and methylation levels in pathologic samples when compared with normal breast. Whereas APC and CTNNB1 did not show differences in methylation levels or frequencies, CDH1 showed higher methylation levels in invasive tumors as compared with preinvasive lesions (P < 0.04, Mann-Whitney test with permutation correction). The analysis of APC, CDH1, and CTNNB1 methylation status was able to distinguish between normal and pathologic samples with a sensitivity of 67% (95% confidence interval, 60-71%) and a specificity of 75% (95% confidence interval, 69-81%). Our data point to the direct involvement of APC, CDH1, and CTNNB1 promoter methylation in the early stages of breast cancer progression and suggest that they may represent a useful tool for the detection of tumor cells in clinical specimens. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2694–700)

Mutations of the D310 mitochondrial mononucleotide repeat in primary tumors and cytological specimens

A mononucleotide repeat (D310) in mitochondrial DNA has been recently identified as a mutational hot spot in primary tumors. We analyzed 56 tumors for insertion/deletion mutations in the D310 repeat. A total of 13 mutations were detected. The highest frequency of mutations was found for cervical cancer, followed by bladder tumors, breast cancer and endometrial neoplasia. No alterations were observed in four patients suspected of malignancy but without evidence of malignant tumor. We detected identical changes in four of four urine sediments from patients with bladder cancer and in three of three fine needle aspirates of patients with breast cancer. Our results indicate that D310 abnormalities are detectable in cytology specimens from patients with cancer and support the notion that D310 analysis may represent a new molecular tool for cancer detection.

Regulation of KEAP1 expression by promoter methylation in malignant gliomas and association with patient’s outcome

In light with the view that KEAP1 loss of function may impact tumour behavior and modify response to chemotherapeutical agents, we sought to determine whether KEAP1 gene is epigenetically regulated in malignant gliomas. We developed a Quantitative Methylation Specific PCR (QMSP) assay to analyze 86 malignant gliomas and 20 normal brain tissues. The discriminatory power of the assay was assessed by Receiving Operating Characteristics (ROC) curve analysis. The AUC value of the curve was 0.823 (95%CI: 0.764-0.883) with an optimal cut off value of 0.133 yielding a 74% sensitivity (95%CI: 63%-82%) and an 85% specificity (95%CI: 64%-95%). Bisulfite sequencing analysis confirmed QMSP results and demonstrated a direct correlation between percentage of methylated CpGs and methylation levels (Spearman’s Rho 0.929, P=0.003). Remarkably, a strong inverse correlation was observed between methylation levels and KEAP1 mRNA transcript in tumour tissue (Spearman’s Rho -0.656 P=0.0001) and in a cell line before and after treatment with 5-azacytidine (P=0.003). RECPAM multivariate statistical analysis studying the interaction between MGMT and KEAP1 methylation in subjects treated with radiotherapy and temozolomide (n=70), identified three prognostic classes of glioma patients at different risk to progress. While simultaneous methylation of MGMT and KEAP1 promoters was associated with the lowest risk to progress, patients showing only MGMT methylation were the subgroup at the higher risk (HR 5.54, 95% CI 1.35-22.74). Our results further suggest that KEAP1 expression is epigenetically regulated. In addition we demonstrated that KEAP1 is frequently methylated in malignant gliomas and a predictor of patient’s outcome.

Electroporation of skeletal muscle induces danger signal release and antigen-presenting cell recruitment independently of DNA vaccine administration.

Background: Plasmid DNA vaccination combined with electroporation (EP) provides a promising approach for the prevention of infectious diseases and for cancer immunotherapy. This technology has been described as being effective in activating humoural and cellular immune response in the host as well as in enhancing expression of the encoded antigen. Several reports showed EP has adjuvant-like properties when combined with plasmid DNA injection although the effect in the absence of DNA has not been investigated. Objective: The aim of this study is to clarify whether the application of EP alone to the skeletal muscle is able to recruit and trigger cells involved in antigen presentation and immune response. Methods: Mouse skeletal muscle treated by EP were observed and processed for clinical, histological and immunohistochemistry analysis at different time points. Results: We demonstrate that EP induces transient morphological changes in the muscle with early production of endogenous cytokines responsible for signalling danger at the local level. Moreover, it causes the recruitment of inflammatory cells independently of the DNA injection and the activation of a danger pro-inflammatory pathway, resulting in T-lymphocyte migration. Conclusions: Our data indicate EP by itself is able to recruit and trigger cells involved in antigen presentation and immune response; hence, the idea that EP has adjuvant-like properties owing to a moderate tissue injury and generation of a pro-inflammatory context with cytokine release that enhances the immune response. We suggest EP may be of practical use in clinical protocols, contributing to the development of DNA vaccination strategies.

The Fragile X Protein binds mRNAs involved in cancer progression and modulates metastasis formation

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.

Aberrant Keap1 methylation in breast cancer and association with clinicopathological features

Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein that mediates the ubiquitination/degradation of genes regulating cell survival and apoptosis under oxidative stress conditions. We determined methylation status of the KEAP1 promoter in 102 primary breast cancers, 14 pre-invasive lesions, 38 paired normal breast tissues and 6 normal breast from reductive mammoplasty by quantitative methylation specific PCR (QMSP). Aberrant promoter methylation was detected in 52 out of the 102 primary breast cancer cases (51%) and 10 out of 14 pre-invasive lesions (71%). No mutations of the KEAP1 gene were identified in the 20 breast cancer cases analyzed by fluorescence based direct sequencing. Methylation was more frequent in the subgroup of patients identified as ER positive-HER2 negative tumors (66.7%) as compared with triple-negative breast cancers (35%) (p = 0.05, Chi-square test). The impact of the interactions between Er, PgR, Her2 expression and KEAP1 methylation on mortality was investigated by RECPAM multivariable statistical analysis, identifying four prognostic classes at different mortality risks. Triple-negative breast cancer patients with KEAP1 methylation had higher mortality risk than patients without triple-negative breast cancer (HR = 14.73, 95%CI: 3.65–59.37). Both univariable and multivariable COX regressions analyses showed that KEAP1 methylation was associated with a better progression free survival in patients treated with epirubicin/cyclophosfamide and docetaxel as sequential chemotherapy (HR = 0.082; 95%CI: 0.007–0.934). These results indicate that aberrant promoter methylation of the KEAP1 gene is involved in breast cancerogenesis. In addition, identifying patients with KEAP1 epigenetic abnormalities may contribute to disease progression prediction in breast cancer patients.

High RAD51 mRNA expression characterize estrogen receptor‐positive/progesteron receptor‐negative breast cancer and is associated with patient's outcome

Mutations in DNA double‐strand breaks (DSB) repair genes are involved in the pathogenesis of hereditary mammary tumors, it is, however, still unclear whether defects in this pathway may play a role in sporadic breast cancer. In this study, we initially determined mRNA expression of 15 DSB related genes by reverse transcription quantitative polymerase chain reaction in paired normal tissue and cancer specimen from 20 breast cancer cases to classify them into homogeneous clusters. G22P1/ku70, ATR and RAD51 genes were differentially expressed in the three branches recognized by clustering analysis. In particular, a breast cancer subgroup characterized by high RAD51 mRNA levels and estrogen receptor (ER)‐positive/progesteron receptor (PR)‐negative phenotype was identified. This result was confirmed by the analysis of G22P1/ku70, ATR and RAD51 mRNA levels on paired normal and tumor specimens from an extended breast cancer cohort (n = 75). RAD51 mRNA levels were inversely associated with PR status (p = 0.02) and the highest levels were, indeed, detected in ER‐positive/PR‐negative tumors (p = 0.03). RAD51 immunostaining of a tissue microarray confirmed the inverse relationship between high RAD51 expression and negative PR status (p = 0.002), as well as, the association with ER‐positive/PR‐negative phenotype (p = 0.003). Interestingly, the analysis of microarray expression data from 295 breast cancers indicate that RAD51 increased mRNA expression is associated with higher risk of tumor relapse, distant metastases and worst overall survival (p = 0.015, p = 0.009 and p = 0.013 respectively). Our results suggest that RAD51 expression determination could contribute to a better molecular classification of mammary tumors and may represent a novel tool for evaluating postoperative adjuvant therapy for breast cancer patients.

Genome-wide methylation profiling and the PI3K-AKT pathway analysis associated with smoking in urothelial cell carcinoma.

Urothelial cell carcinoma (UCC) is the second most common genitourinary malignant disease in the USA, and tobacco smoking is the major known risk factor for UCC development. Exposure to carcinogens, such as those contained in tobacco smoke, is known to directly or indirectly damage DNA, causing mutations, chromosomal deletion events and epigenetic alterations in UCC. Molecular studies have shown that chromosome 9 alterations and P53, RAS, RB and PTEN mutations are among the most frequent events in UCC. Recent studies suggested that continuous tobacco carcinogen exposure drives and enhances the selection of epigenetically altered cells in UCC, predominantly in the invasive form of the disease. However, the sequence of molecular events that leads to UCC after exposure to tobacco smoke is not well understood. To elucidate molecular events that lead to UCC oncogenesis and progression after tobacco exposure, we developed an in vitro cellular model for smoking-induced UCC. SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. Morphological alterations and increased cell proliferation of non-malignant urothelial cells were observed after 4 months (mo) of treatment with CSE. Anchorage-independent growth assessed by soft agar assay and increase in the migratory and invasive potential was observed in urothelial cells after 6 mo of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found that 26 genes were upregulated and 22 genes were downregulated after 6 mo of CSE exposure of HUC1 cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were downregulated in the transformed cells, while AKT1, AKT2, HRAS, RAC1 were upregulated. Validation by RT-PCR and western blot analysis was then performed. Furthermore, genome-wide methylation analysis revealed MCAM, DCC and HIC1 are hypermethylated in CSE-treated urothelial cells when compared with non-CSE exposed cells. The methylation status of these genes was validated using quantitative methylation-specific PCR (QMSP), confirming an increase in methylation of CSE-treated urothelial cells compared to untreated controls. Therefore, our findings suggest that a tobacco signature could emerge from distinctive patterns of genetic and epigenetic alterations and can be identified using an in vitro cellular model for the development of smoking-induced cancer.