Faculty Publications

VILIP-1, a member of the neuronal Ca++ sensor protein family, acts as a tumor suppressor gene in an experimental animal model by inhibiting cell proliferation, adhesion and invasiveness of squamous cell carcinoma cells. Western Blot analysis of human tumor cells showed that VILIP-1 expression was undetectable in several types of human tumor cells, including 11 out of 12 non-small cell lung carcinoma (NSCLC) cell lines. The down-regulation of VILIP-1 was due to loss of VILIP-1 mRNA transcripts. Rearrangements, large gene deletions or mutations were not found. Hypermethylation of the VILIP-1 promoter played an important role in gene silencing. In most VILIP-1-silent cells the VILIP-1 promoter was methylated. In vitro methylation of the VILIP-1 promoter reduced its activity in a promoter-reporter assay. Transcriptional activity of endogenous VILIP-1 promoter was recovered by treatment with 5'-aza-2'-deoxycytidine (5'-Aza-dC). Trichostatin A (TSA), a histone deacetylase inhibitor, potently induced VILIP-1 expression, indicating that histone deacetylation is an additional mechanism of VILIP-1 silencing. TSA increased histone H3 and H4 acetylation in the region of the VILIP-1 promoter. Furthermore, statistical analysis of expression and promoter methylation (n = 150 primary NSCLC samples) showed a significant relationship between promoter methylation and protein expression downregulation as well as between survival and decreased or absent VILIP-1 expression in lung cancer tissues (p<0.0001). VILIP-1 expression is silenced by promoter hypermethylation and histone deacetylation in aggressive NSCLC cell lines and primary tumors and its clinical evaluation could have a role as a predictor of short-term survival in lung cancer patients.

The partner and localizer of BRCA2 (PALB2) gene was recently identified as a BRCA2-interacting protein and subsequently shown to be a Fanconi anemia gene (FANCN). Disease-associated point mutations resulting in protein truncation have been found in BRCA1/2 mutation-negative breast cancer families identifying PALB2 as a susceptibility gene for breast cancer. Aberrant promoter hypermethylation is a mechanism of inactivation of many tumor suppressor genes, including BRCA1 and P16(INK4a), in breast and ovarian cancer. We therefore investigated the methylation status of a 1512 bp typical CpG island located in the promoter and exon 1 region of the PALB2 gene in 130 sporadic and familial breast and ovarian primary tumors, 9 cell lines, and 10 normal cell specimens. We found two primary breast tumors from BRCA2 mutation carriers, four sporadic primary breast tumors, and four sporadic primary ovarian tumors showed hypermethylation of the core promoter region of PALB2. All 10 norma!

The partner and localizer of BRCA2 (PALB2) gene was recently identified as a BRCA2-interacting protein and subsequently shown to be a Fanconi anemia gene (FANCN). Disease-associated point mutations resulting in protein truncation have been found in BRCA1/2 mutation-negative breast cancer families identifying PALB2 as a susceptibility gene for breast cancer. Aberrant promoter hypermethylation is a mechanism of inactivation of many tumor suppressor genes, including BRCA1 and p16(INK4a), in breast and ovarian cancer. We therefore investigated the methylation status of a 1512 bp typical CpG island located in the promoter and exon 1 region of the PALB2 gene in 130 sporadic and familial breast and ovarian primary tumors, 9 cell lines, and 10 normal cell specimens. We found two primary breast tumors from BRCA2 mutation carriers, four sporadic primary breast tumors, and four sporadic primary ovarian tumors showed hypermethylation of the core promoter region of PALB2. All 10 normal tissue DNA had an unmethylated PALB2 promoter region. Quantitative real-time reverse transcription-PCR showed PALB2 expression to be reduced 28-fold in primary breast tumor with PALB2 promoter hypermethylation compared with matched normal breast tissue RNA. Aberrant promoter hypermethylation of PALB2 is more frequent than the reported level of PALB2 point mutations in breast tumors from BRCA1/2-negative families and is similar to the frequency of BRCA1 hypermethylation in inherited and sporadic breast and ovarian cancers.

Molecular studies of many types of cancer have revealed that clinically evident tumours carry multiple genetic and epigenetic abnormalities, including DNA sequence alterations, chromosome copy number changes and aberrant promoter hypermethylation. Together, these aberrant changes result in the activation of oncogenes and inactivation of tumour-suppressor genes (TSG). In many cases these abnormalities can be found in premalignant lesions and even in histological normal adjacent cells. Many tumour types are difficult to detect early and are frequently resistant to available chemotherapy and radiotherapy. Therefore, the early detection, chemoprevention and the design of new therapeutic strategies based on the increased understanding of cancer molecular changes are one of the great challenges nowadays. Insertions of a methyl group at the fifth carbon of cytosines within the dinucleotide 5 ' CpG-3 ' is the best studied epigenetic mechanism. DNA methylation acts together with others mechanisms like histone modification, chromatin remodelling and microRNAs to mould the DNA structure according to the functional state required. The aberrant methylation of the CpG islands located at the promoter region of specific genes is a common and early event involved in cancer development. Thus, hypermethylated DNA sequences from tumours are one of the most promising markers for early detection screenings as well as tumour classification and chemotherapy response in many types of cancer.

Aberrant promoter hypermethylation is a common mechanism for inactivation of tumor suppressor genes in cancer cells. To generate a global profile of genes silenced by hypermethylation in renal cell cancer (RCC), we did an expression microarray-based analysis of genes reactivated in the 786-0, ACHN, HRC51, and HRC59 RCC lines after treatment with the demethylating drug 5-aza-2 deoxycytidine and histone deacetylation inhibiting drug trichostatin A. Between 111 to 170 genes were found to have at least 3-fold up-regulation of expression after treatment in each cell line. To establish the specificity of the screen for identification of genes, epigenetically silenced in cancer cells, we validated a subset of 12 up-regulated genes. Three genes (IGFBP1, IGFBP3, and COL1A1) showed promoter methylation in tumor DNA but were unmethylated in normal cell DNA. One gene (GDF15) was methylated in normal cells but more densely methylated in tumor cells. One gene (PLAU) showed cancer cell-specific methylation that did not correlate well with expression status. The remaining seven genes had unmethylated promoters, although at least one of these genes (TGM2) may be regulated by RASSF1A, which was methylated in the RCC lines. Thus, we were able to show that up-regulation of at least 6 of the 12 genes examined was due to epigenetic reactivation. The IGFBP1, IGFBP3, and COL1A1 gene promoter regions were found to be frequently methylated in primary renal cell tumors, and further study will provide insight into the biology of the disease and facilitate translational studies in renal cancer. (Cancer Res 2006; 66(10): 5021-8).

Methods for the detecting hypermethylated promoters of tumor suppressor genes assocd. with ovarian cancer using methylation-specific PCR are provided. Several cancer genes including BRCA1, RASSF1A, APC, p14ARF, CDKN2A and DAP-kinase have hypermethylated CpG islands within the promoter region which are normally unmethylated. Hypermethylation can be identified by the sensitive methylation-specific PCR method which can identify 1 methylated allele in 1000 unmethylated alleles in body fluids. [on SciFinder (R)]

Purpose: Breast cancer is the most common malignancy in American women and the second leading cause of death from cancer. The genetic and epigenetic alterations that initiate and drive cancer can be used as targets for detection of neoplasia in bodily fluids. Tumor cell-specific aberrant promoter hypermethylation can be detected in nipple aspirate and ductal lavage from breast cancer patients. In this study, we examine serum, a more readily accessible bodily fluid known to contain neoplastic DNA from individuals with cancer, for methylation-based detection of breast neoplasia. Experimental Design: We examined the promoter methylation status of three normally unmethylated biologically significant cancer genes, PAS association domain family protein 1A (RASSF1A), adenomatous polyposis coli (APC), and death-associated protein kinase (DAP-kinase), by sensitive methylation-specific PCR in 34 breast tumor and paired preoperative serum DNA. The 34 patients comprised 7 ductal carcinoma in situ (CIS), 3 lobular CIS, 5 stage I and 15 stage II to IV invasive ductal carcinomas, and 4 invasive lobular carcinomas. Normal and benign tissue and serum control DNA were also examined to determine the specificity of hypermethylation. Results: Hypermethylation of one or more genes was found in 32 of 34 (94%) breast tumor DNA. APC was hypermethylated in 15 of 34 (47%), RASSF1A in 22 of 34 (65%), and DAP-kinase in 17 of 34 (50%) tumors. Twenty-six (76%) of the corresponding serum DNA were positive for promoter hypermethylation, including ductal CIS, lobular CIS, stage I disease, and lobular carcinoma patients. No hypermethylation of APC, RASSF1A, or DAP-kinase was observed in serum DNA from normal healthy women and patients with inflammatory breast disease or nonneoplastic breast tissue specimens. A gene unmethylated in the tumor DNA was always found to be unmethylated in the matched serum DNA (100% specificity). Conclusions: Tumor cell specific promoter hypermethylation of APC, RASSF1A, and DAP-kinase is present in ductal CIS, lobular CIS, and all grades and stages of invasive breast cancer. Hypermethylation can be detected by methylation-specific PCR analysis in serum DNA from patients with preinvasive and early-stage breast cancer amenable to cure. If confirmed in additional studies, hypermethylation-based screening of serum, a readily accessible bodily fluid, may enhance early detection of breast cancer.

JC - cnf, 2984705r SB - IM LM - library has some holdings; check journal list for details CP - United States MH - Adolescent MH - Adult MH - Aged MH - Aged, 80 and over MH - Ascitic Fluid/ge [Genetics] MH - Ascitic Fluid/me [Metabolism] MH - *DNA Methylation MH - DNA, Neoplasm/bl [Blood] MH - DNA, Neoplasm/me [Metabolism] MH - Female MH - *Genes, BRCA1 MH - Human MH - Middle Aged MH - Ovarian Neoplasms/bl [Blood] MH - *Ovarian Neoplasms/ge [Genetics] MH - Support, Non-U.S. Gov't MH - Support, U.S. Gov't, P.H.S. MH - *Tumor Suppressor Proteins/ge [Genetics] AB - Because existing surgical and management methods can consistently cure only early-stage ovarian cancer, novel strategies for early detection are required. Silencing of tumor suppressor genes such as p16INK4a, VHL, and hMLH1 have established promoter hypermethylation as a common mechanism for tumor suppressor inactivation in human cancer and as a promising target for molecular detection in bodily fluids. Using sensitive methylation-specific PCR, we screened matched tumor, preoperative serum or plasma, and peritoneal fluid (washes or ascites) DNA obtained from 50 patients with ovarian or primary peritoneal tumors for hypermethylation status of the normally unmethylated BRCA1 and RAS association domain family protein 1A tumor suppressor genes. Hypermethylation of one or both genes was found in 34 tumor DNA (68%). Additional examination of one or more of the adenomatous polyposis coli, p14ARF, p16INK4a, or death associated protein-kinase tumor suppressor genes revealed hypermethylation in each of the remaining 16 tumor DNA, which extended diagnostic coverage to 100%. Hypermethylation was observed in all histologic cell types, grades, and stages of ovarian tumor examined. An identical pattern of gene hypermethylation was found in the matched serum DNA from 41 of 50 patients (82% sensitivity), including 13 of 17 cases of stage I disease. Hypermethylation was detected in 28 of 30 peritoneal fluid DNA from stage IC-IV patients, including 3 cases with negative or atypical cytology. In contrast, no hypermethylation was observed in nonneoplastic tissue, peritoneal fluid, or serum from 40 control women (100% specificity). We conclude that promoter hypermethylation is a common and relatively early event in ovarian tumorigenesis that can be detected in the serum DNA from patients with ovary-confined (stage IA or B) tumors and in cytologically negative peritoneal fluid. Analysis of tumor-specific hypermethylation in serum DNA may enhance early detection of ovarian cancer

Because existing surgical and management methods can consistently cure only early-stage ovarian cancer, novel strategies for early detection are required. Silencing of tumor suppressor genes such as p16(INK4a), VHL, and hMLH1 have established promoter hypermethylation as a common mechanism for tumor suppressor inactivation in human cancer and as a promising target for molecular detection in bodily fluids. Using sensitive methylation-specific PCR, we screened matched tumor, preoperative serum or plasma, and peritoneal fluid (washes or ascites) DNA obtained from 50 patients with ovarian or primary peritoneal tumors for hypermethylation status of the normally unmethylated BRCA1 and RAS association domain family protein 1A tumor suppressor genes. Hypermethylation of one or both genes was found in 34 tumor DNA (68%). Additional examination of one or more of the adenomatous polyposis coli, p14(ARF), p16(INK4a), or death associated protein-kinase tumor suppressor genes revealed hyp ermethylation in each of the remaining 16 tumor DNA, which extended diagnostic coverage to 100%. Hypermethylation was observed in all histologic cell types, grades, and stages of ovarian tumor examined. An identical pattern of gene hypermethylation was found in the matched serum DNA from 41 of 50 patients (82% sensitivity), including 13 of 17 cases of stage I disease. Hypermethylation was detected in 28 of 30 peritoneal fluid DNA from stage IGIV patients, including 3 cases with negative or atypical cytology. In contrast, no hypermethylation was observed in nonneoplastic tissue, peritonea] fluid, or serum from 40 control women (100% specificity). We conclude that promoter hypermethylation is a common and relatively early event in ovarian tumorigenesis that can be detected in the serum DNA from patients with ovary-confined (stage IA or B) tumors and in cytologically negative peritonea] fluid. Analysis of tumor-specific hypermethylation in serum DNA may enhance early detection of ovarian cancer.

Kidney cancer is curable by surgical resection and therapy, if detected at an early stage. Using sensitive methylation-specific polymerase chain reaction, we screened matched tumor DNA and preoperative urine DNA from 50 kidney cancer patients, for hypermethylation of a panel of six normally unmethylated tumor suppressor genes: VHL, p16/CDKN2a, p14ARF, APC, RASSF1A, and Timp-3. When compared to the tumor DNA, an identical pattern of gene hypermethylation was found in the matched urine DNA from 44 of 50 patients (88% sensitivity) including 27 of 30 cases of stage I disease. By contrast, hypermethylation was not observed in normal and benign disease controls (100% specificity). We conclude that promoter hypermethylation is a common and early event in kidney tumorigenesis and can be detected in the urine DNA from patients with organ-confined renal cancer of all histologic types.

A review on anticancer therapeutic regimens such as chemo-, radiation- and immunotherapy, and cancer-cell resistance to apoptosis. The resistance of tumor cells to anticancer therapies may result from a failure to activate apoptotic pathways in response to drug treatment. A better understanding of the mol. mechanisms of cell death in response to these anticancer therapeutic strategies will help to avoid ineffective treatment regimens and provide a mol. basis for the new therapeutic modalities targeting apoptosis-resistant forms of cancer. [on SciFinder (R)]