DEAR1 is a dominant regulator of acinar morphogenesis and an independent predictor of local recurrence-free survival in early-onset breast cancer.

BACKGROUND: Breast cancer in young women tends to have a natural history of aggressive disease for which rates of recurrence are higher than in breast cancers detected later in life. Little is known about the genetic pathways that underlie early-onset breast cancer. Here we report the discovery of DEAR1 (ductal epithelium-associated RING Chromosome 1), a novel gene encoding a member of the TRIM (tripartite motif) subfamily of RING finger proteins, and provide evidence for its role as a dominant regulator of acinar morphogenesis in the mammary gland and as an independent predictor of local recurrence-free survival in early-onset breast cancer. METHODS AND FINDINGS: Suppression subtractive hybridization identified DEAR1 as a novel gene mapping to a region of high-frequency loss of heterozygosity (LOH) in a number of histologically diverse human cancers within Chromosome 1p35.1. In the breast epithelium, DEAR1 expression is limited to the ductal and glandular epithelium and is down-regulated in transition to ductal carcinoma in situ (DCIS), an early histologic stage in breast tumorigenesis. DEAR1 missense mutations and homozygous deletion (HD) were discovered in breast cancer cell lines and tumor samples. Introduction of the DEAR1 wild type and not the missense mutant alleles to complement a mutation in a breast cancer cell line, derived from a 36-year-old female with invasive breast cancer, initiated acinar morphogenesis in three-dimensional (3D) basement membrane culture and restored tissue architecture reminiscent of normal acinar structures in the mammary gland in vivo. Stable knockdown of DEAR1 in immortalized human mammary epithelial cells (HMECs) recapitulated the growth in 3D culture of breast cancer cell lines containing mutated DEAR1, in that shDEAR1 clones demonstrated disruption of tissue architecture, loss of apical basal polarity, diffuse apoptosis, and failure of lumen formation. Furthermore, immunohistochemical staining of a tissue microarray from a cohort of 123 young female breast cancer patients with a 20-year follow-up indicated that in early-onset breast cancer, DEAR1 expression serves as an independent predictor of local recurrence-free survival and correlates significantly with strong family history of breast cancer and the triple-negative phenotype (ER(-), PR(-), HER-2(-)) of breast cancers with poor prognosis. CONCLUSIONS: Our data provide compelling evidence for the genetic alteration and loss of expression of DEAR1 in breast cancer, for the functional role of DEAR1 in the dominant regulation of acinar morphogenesis in 3D culture, and for the potential utility of an immunohistochemical assay for DEAR1 expression as an independent prognostic marker for stratification of early-onset disease.

A novel DNMT3B subfamily, DeltaDNMT3B, is the predominant form of DNMT3B in non-small cell lung cancer.

De novo promoter DNA methylation represses gene transcription and is a common mechanism to inactivate tumor suppressor genes in tumorigenesis. DNMT3B plays an important role in de novo DNA methylation. We report here the identification of a novel DNMT3B subfamily, termed DeltaDNMT3B, whose expression is initiated through a promoter located at intron 4 and exon 5 of the DNMT3B gene. At least 7 transcriptional variants of DeltaDNMT3B have been observed as the result of alternative pre-mRNA splicing. Predicted proteins derived from these variants suggest that 4 of the variants share a conservative enzymatic domain but contain a variable PWWP motif, a putative DNA binding structure, whereas 3 of the variants lack the enzymatic domain due to predicted premature translational termination. In non-small cell lung cancer (NSCLC) cell lines, DeltaDNMT3B variants are frequently expressed and are the predominant forms of DNMT3B. Similarly, DeltaDNMT3B variants are frequently expressed in primary NSCLC but are not detectable or are expressed at low levels in corresponding normal lung tissue. Our results indicate that DeltaDNMT3B is the major expression form of DNMT3B in NSCLC and may play an important role in the development of aberrant promoter methylation during lung tumorigenesis.

A novel C/T polymorphism in the core promoter of human de novo cytosine DNA methyltransferase 3B6 is associated with prognosis in head and neck cancer.

New biomarkers for head and neck squamous cell carcinoma (HNSCC) patients are being sought to help predict disease outcome, guide treatment, and develop new treatments. In the present study, the association between a novel functional C/T polymorphism in the core promoter of cytosine DNA methyltransferase (DNMT) 3B6 and overall survival of HNSCC patients was investigated. Genomic DNA was extracted from tumor tissue and leukocytes from each HNSCC patient. We used the PCR-restriction fragment length polymorphism (PCR-RFLP) assay to determine the DNMT3B genotype. Kaplan-Meier product-limit method and univariate/multivariate Cox proportional hazard models were used to correlate DNMT3B genotype with overall survival of HNSCC patients who underwent surgical resection. There was a marked association between DNMT3B C/T polymorphism and overall survival of HNSCC patients (P=0.004). The homozygotes (CC-genotype and TT-genotype) survived significantly longer than the heterozygotes (CT-type). The multivariate Cox proportional hazard modeling showed that HNSCC patients with CT-genotype had a hazard ratio of 4.829 over patients with CC-genotype or TT-genotype. A DNMT3B C/T polymorphism has been correlated with survival differences in HNSCC patients. If validated in larger studies, this polymorphism might be used to identify deleterious patterns of gene silencing by methylation in HNSCC.

Aberrant promoter methylation of multiple genes in bronchial brush samples from former cigarette smokers.

Promoter hypermethylation plays an important role in the inactivation of tumor suppressor genes during tumorigenesis. Recent data suggest that such epigenetic abnormality may occur very early in lung carcinogenesis. To determine the extent of promoter hypermethylation in early lung tumorigenesis, we analyzed promoter methylation status of the p16, death-associated protein kinase (DAPK) and glutathione S-transferase P1 (GSTP1) genes using methylation-specific PCR in bronchial brush samples obtained from 100 former smokers enrolled in a chemoprevention clinical trial. We found that 17% of the samples showed methylation for p16 and 17% the same for DAPK, whereas only 6% of the samples displayed methylation for GSTP1. A total of 32% of the samples had methylation in at least one of the three genes tested, and 8% of the samples had methylation in two genes. The methylation status of p16 was correlated with that of DAPK (P = 0.04, Fisher's exact test). p16 methylation was higher in former smokers with a history of previous cancer than in former smokers without a history of cancer (P = 0.04, Fisher's exact test), and methylation of DAPK was detected more frequently in older patients than it was in younger patients (P = 0.01, Wilcoxon rank-sum test). Surprisingly, no correlation was found between methylation in any of these genes and the smoking characteristics of the individuals analyzed (packs per day, pack-years, smoking years, quitting years). The precise meaning of methylated genes in the bronchial brush samples of former smokers must be sought by means of careful follow-up of these individuals.