Novel molecular markers of malignancy in histologically normal and benign breast.

To detect the molecular changes of malignancy in histologically normal breast (HNB) tissues, we recently developed a novel 117-gene-malignancy-signature. Here we report validation of our leading malignancy-risk-genes, topoisomerase-2-alpha (TOP2A), minichromosome-maintenance-protein-2 (MCM2) and "budding-uninhibited-by-benzimidazoles-1-homolog-beta" (BUB1B) at the protein level. Using our 117-gene malignancy-signature, we classified 18 fresh-frozen HNB tissues from 18 adult female breast cancer patients into HNB-tissues with low-grade (HNB-LGMA; N = 9) and high-grade molecular abnormality (HNB-HGMA; N = 9). Archival sections of additional HNB tissues from these patients, and invasive ductal carcinoma (IDC) tissues from six other patients were immunostained for these biomarkers. TOP2A/MCM2 expression was assessed as staining index (%) and BUB1B expression as H-scores (0-300). Increasing TOP2A, MCM2, and BUB1B protein expression from HNB-LGMA to HNB-HGMA tissues to IDCs validated our microarray-based molecular classification of HNB tissues by immunohistochemistry. We also demonstrated an increasing expression of TOP2A protein on an independent test set of HNB/benign/reductionmammoplasties, atypical-ductal-hyperplasia with and without synchronous breast cancer, DCIS and IDC tissues using a custom tissue microarray (TMA). In conclusion, TOP2A, MCM2, and BUB1B proteins are potential molecular biomarkers of malignancy in histologically normal and benign breast tissues. Larger-scale clinical validation studies are needed to further evaluate the clinical utility of these molecular biomarkers.

Distinguishing between cancer driver and passenger gene alteration candidates via cross-species comparison: a pilot study.

BACKGROUND: We are developing a cross-species comparison strategy to distinguish between cancer driver- and passenger gene alteration candidates, by utilizing the difference in genomic location of orthologous genes between the human and other mammals. As an initial test of this strategy, we conducted a pilot study with human colorectal cancer (CRC) and its mouse model C57BL/6J ApcMin/+, focusing on human 5q22.2 and 18q21.1-q21.2. METHODS: We first performed bioinformatics analysis on the evolution of 5q22.2 and 18q21.1-q21.2 regions. Then, we performed exon-targeted sequencing, real time quantitative polymerase chain reaction (qPCR), and real time quantitative reverse transcriptase PCR (qRT-PCR) analyses on a number of genes of both regions with both human and mouse colon tumors. RESULTS: These two regions (5q22.2 and 18q21.1-q21.2) are frequently deleted in human CRCs and encode genuine colorectal tumor suppressors APC and SMAD4. They also encode genes such as MCC (mutated in colorectal cancer) with their role in CRC etiology unknown. We have discovered that both regions are evolutionarily unstable, resulting in genes that are clustered in each human region being found scattered at several distinct loci in the genome of many other species. For instance, APC and MCC are within 200 kb apart in human 5q22.2 but are 10 Mb apart in the mouse genome. Importantly, our analyses revealed that, while known CRC driver genes APC and SMAD4 were disrupted in both human colorectal tumors and tumors from ApcMin/+ mice, the questionable MCC gene was disrupted in human tumors but appeared to be intact in mouse tumors. CONCLUSIONS: These results indicate that MCC may not actually play any causative role in early colorectal tumorigenesis. We also hypothesize that its disruption in human CRCs is likely a mere result of its close proximity to APC in the human genome. Expanding this pilot study to the entire genome may identify more questionable genes like MCC, facilitating the discovery of new CRC driver gene candidates.

Evaluation of malignancy-risk gene signature in breast cancer patients.

We recently developed a malignancy-risk gene signature that was shown to identify histologically-normal tissues with a cancer-like profile. Because the signature was rich with proliferative genes, we postulated it might also be prognostic for existing breast cancers. We evaluated the malignancy risk gene signature to see its clinical association with cancer relapse/progression, and cancer prognosis using six independent external datasets. Six independent external breast cancer datasets were collected and analyzed using the malignancy risk gene signature designed to assess normal breast tissues. Evaluation of the signature in external datasets suggested a strong clinical association with cancer relapse/progression, and prognosis with minimal overlap of signature gene sets. These results suggest a prognostic role for the malignancy risk gene signature in the assessment of existing cancer. Proliferative biology dominates not only the earliest stages of tumor development but also later stages of tumor progression and metastasis.

Proliferative genes dominate malignancy-risk gene signature in histologically-normal breast tissue.

Historical data have indicated the potential for the histologically-normal breast to harbor pre-malignant changes at the molecular level. We postulated that a histologically-normal tissue with "tumor-like" gene expression pattern might harbor substantial risk for future cancer development. Genes associated with these high-risk tissues were considered to be "malignancy-risk genes". From a total of 90 breast cancer patients, we collected a set of 143 histologically-normal breast tissues derived from patients harboring breast cancer who underwent curative mastectomy, as well as a set of 42 invasive ductal carcinomas (IDC) of various histologic grades. All samples were assessed for global gene expression differences using microarray analysis. For the purpose of this study we defined normal breast tissue to include histologically normal and benign lesions. Here we report the discovery of a "malignancy-risk" gene signature that may portend risk of breast cancer development in benign, but molecularly-abnormal, breast tissue. Pathway analysis showed that the malignancy-risk signature had a dramatic enrichment for genes with proliferative function, but appears to be independent of ER, PR, and HER2 status. The signature was validated by RT-PCR, with a high correlation (Pearson correlation = 0.95 with P < 0.0001) with microarray data. These results suggest a predictive role for the malignancy-risk signature in normal breast tissue. Proliferative biology dominates the earliest stages of tumor development.

Epithelial progeny of estrogen-exposed breast progenitor cells display a cancer-like methylome.

Estrogen imprinting is used to describe a phenomenon in which early developmental exposure to endocrine disruptors increases breast cancer risk later in adult life. We propose that long-lived, self-regenerating stem and progenitor cells are more susceptible to the exposure injury than terminally differentiated epithelial cells in the breast duct. Mammospheres, containing enriched breast progenitors, were used as an exposure system to simulate this imprinting phenomenon in vitro. Using MeDIP-chip, a methylation microarray screening method, we found that 0.5% (120 loci) of human CpG islands were hypermethylated in epithelial cells derived from estrogen-exposed progenitors compared with the non-estrogen-exposed control cells. This epigenetic event may lead to progressive silencing of tumor suppressor genes, including RUNX3, in these epithelial cells, which also occurred in primary breast tumors. Furthermore, normal tissue in close proximity to the tumor site also displayed RUNX3 hypermethylation, suggesting that this aberrant event occurs in early breast carcinogenesis. The high prevalence of estrogen-induced epigenetic changes in primary tumors and the surrounding histologically normal tissues provides the first empirical link between estrogen injury of breast stem/progenitor cells and carcinogenesis. This finding also offers a mechanistic explanation as to why a tumor suppressor gene, such as RUNX3, can be heritably silenced by epigenetic mechanisms in breast cancer.

Mapping geographic zones of cancer risk with epigenetic biomarkers in normal breast tissue.

PURPOSE: Genetic alterations were previously identified in normal epithelia adjacent to invasive cancers. The aim of this study was to determine DNA methylation in histologically normal tissues from multiple geographic zones adjacent to primary breast tumors. EXPERIMENTAL DESIGN: First, methylation status of a 4-kb region of RASSF1A promoter was interrogated using oligonucleotide-based microarray in 144 samples (primary tumors, 47; adjacent normals, 69; reduction mammoplasty tissues, 28). Second, allelic imbalance (AI)/loss of heterozygosity (LOH) surrounding RASSF1A promoter were analyzed in 30 samples (tumors, 8; adjacent normals, 22). Third, global methylation screening of 49 samples (tumors, 12; adjacent normals, 25; reduction mammoplasty, 12) was done by differential methylation hybridization. Real-time quantitative methylation-specific PCR was used to validate the microarray findings. RESULTS: DNA methylation in the core RASSF1A promoter was low in reduction mammoplasty tissues (P=0.0001) when compared with primary tumors. The adjacent normals had an intermediate level of methylation. The regions surrounding the core were highly methylated in all sample types. Microsatellite markers showed AI/LOH in tumors and some of the adjacent normals. Concurrent AI/LOH and DNA methylation in RASSF1A promoter occurred in two of six tumors. Global methylation screening uncovered genes more methylated in adjacent normals than in reduction mammoplasty tissues. The methylation status of four genes was confirmed by quantitative methylation-specific PCR. CONCLUSIONS: Our findings suggest a field of methylation changes extending as far as 4 cm from primary tumors. These frequent alterations may explain why normal tissues are at risk for local recurrence and are useful in disease prognostication.

Protein-protein interactions of a Kvbeta subunit in the cochlea.

Accessory subunits associated with voltage-gated potassium (Kv) channels can influence the biophysical properties and promote the surface expression of channel-forming alpha-subunits. Previously, we cloned several alpha-subunits and a beta-subunit from a cDNA library of the chicken cochlea. In the present study, we raised an antibody against the N-terminus of chicken Kvbeta1.1 (cKvbeta1.1) and characterized the Kvbeta-related polypeptide in cochlear tissues and heterologous cells. The anti-cKvbeta1.1 antibody recognizes a 45-kDa polypeptide in chick cochlear extracts as well as in Chinese hamster ovary (CHO) cells transfected with cKvbeta1.1. The accessory subunit was localized to the ganglion cells of the chick cochlea using immunohistochemistry and in situ hybridization. Coimmunoprecipitation studies show that Kvbeta1.1 interacts with Shaker channel members Kvalpha1.2 and 1.3, both of which colocalize with beta to the cochlear ganglion cells. Additionally, coimmunoprecipitation studies show that Kvalpha1.2 and 1.3 interact with each other, suggesting that these ion channels are formed by heteromultimers. In comparison, Kvbeta did not coprecipitate with a member of the Shal subfamily. The presence of Kvbeta in the cochlea suggests that this subunit contributes to the modulation of auditory signals in the ganglion cells, presumably by regulating properties of inactivation as well as surface expression of Kvalpha channels.