Faculty Bibliography

INTRODUCTION: Cyclin E, a G1 cyclin essential for G1-S phase transition, is known to have a profound effect on tumorigenesis. Elevated levels of cyclin E have been associated with breast cancer, and chromosomal instability observed in breast cancer is suggested to be associated with constitutive expression of cyclin E. It was previously demonstrated that SUM149PT human breast cancer cells show very high levels of cyclin E expression by western analysis and that they express a nonfunctional cyclin E ubiquitin ligase due to a mutation in the F-box protein hCdc4. METHODS: We examined cyclin E expression in both MCF10A and SUM149PT cells using western blot analysis and flow cytometry. Immunofluorescence was utilized for the localization of cyclin E in both normal and breast cancer cells. In addition, array comparative genomic hybridization analysis was performed to compare chromosome copy number alterations with levels of cyclin E expression among a panel of breast cancer cell lines. RESULTS: SUM149PT cells overexpress cyclin E on a cell per cell basis for the duration of the cell cycle. High cyclin E levels are maintained throughout the S phase, and SUM149PT cells exhibit an S phase delay or arrest probably due to cyclin E overexpression. In addition, comparative genomic hybridization indicated that SUM149PT cells exhibit many chromosome copy number alterations, which may reflect prior or ongoing genomic instability. However, no direct correlation was observed between cyclin E levels and genomic copy number alteration in a panel of human breast cancer cell lines. CONCLUSIONS: Cyclin E is overexpressed at high levels throughout the cell cycle in SUM149PT cells, which is in stark contrast to cyclin E degradation observed in the mid to late S phase of normal cells. SUM149PT cells are unable to regulate cyclin E and also exhibit many copy number alterations. However, there was a lack of direct correlation between cyclin E overexpression and chromosomal instability across a panel of other breast cancer cell lines examined.

Using the human bacterial artificial chromosome (BAC) fingerprint-based physical map, genome sequence assembly and BAC end sequences, we have generated a fingerprint-validated set of 32 855 BAC clones spanning the human genome. The clone set provides coverage for at least 98% of the human fingerprint map, 99% of the current assembled sequence and has an effective resolving power of 79 kb. We have made the clone set publicly available, anticipating that it will generally facilitate FISH or array-CGH-based identification and characterization of chromosomal alterations relevant to disease.

Prostate cancer is the most commonly diagnosed non-cutaneous neoplasm among American males and is the second leading cause of cancer-related death. Prostate specific antigen screening has resulted in earlier disease detection, yet approximately 30% of men will die of metastatic disease. Slow disease progression, an aging population and associated morbidity and mortality underscore the need for improved disease classification and therapies. To address these issues, we analyzed a cohort of patients using array comparative genomic hybridization (aCGH). The cohort comprises 64 patients, half of whom recurred postoperatively. Analysis of the aCGH profiles revealed numerous recurrent genomic copy number aberrations. Specific loss at 8p23.2 was associated with advanced stage disease, and gain at 11q13.1 was found to be predictive of postoperative recurrence independent of stage and grade. Moreover, comparison with an independent set of metastases revealed approximately 40 candidate markers associated with metastatic potential. Copy number aberrations at these loci may define metastatic genotypes.

We have constructed a high-resolution genomic microarray of human chromosome 16q, and used it for comparative genomic hybridization analysis of 16 prostate tumors. We demarcated 10 regions of genomic loss between 16q23.1 and 16qter that occurred in five or more samples. Mining expression array data from four independent studies allowed us to identify 11 genes that were frequently underexpressed in prostate cancer and that co-localized with a region of genomic loss. Quantitative expression analyses of these genes in matched tumor and benign tissue from 13 patients showed that six of these 11 (WWOX, WFDC1, MAF, FOXF1, MVD and the predicted novel transcript Q9H0B8 (NM_031476)) had significant and consistent downregulation in the tumors relative to normal prostate tissue expression making them candidate tumor suppressor genes.

We constructed a tiling resolution array consisting of 32,433 overlapping BAC clones covering the entire human genome. This increases our ability to identify genetic alterations and their boundaries throughout the genome in a single comparative genomic hybridization (CGH) experiment. At this tiling resolution, we identified minute DNA alterations not previously reported. These alterations include microamplifications and deletions containing oncogenes, tumor-suppressor genes and new genes that may be associated with multiple tumor types. Our findings show the need to move beyond conventional marker-based genome comparison approaches, that rely on inference of continuity between interval markers. Our submegabase resolution tiling set for array CGH (SMRT array) allows comprehensive assessment of genomic integrity and thereby the identification of new genes associated with disease.

Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130) (pRb(f), collectively), play a pivotal role in regulating eukaryotic cell cycle progression, apoptosis, and terminal differentiation. While aberrations in the pRb-signaling pathway are common in human cancers, the consequence of pRb(f) loss in the mammary gland has not been directly assayed in vivo. We reported previously that inactivating these critical cell cycle regulators in divergent cell types, either brain epithelium or astrocytes, abrogates the cell cycle restriction point, leading to increased cell proliferation and apoptosis, and predisposing to cancer. Here we report that mouse mammary epithelium is similar in its requirements for pRb(f) function; Rb(f) inactivation by T(121), a fragment of SV40 T antigen that binds to and inactivates pRb(f) proteins, increases proliferation and apoptosis. Mammary adenocarcinomas form within 16 mo. Most apoptosis is regulated by p53, which has no impact on proliferation, and heterozygosity for a p53 null allele significantly shortens tumor latency. Most tumors in p53 heterozygous mice undergo loss of the wild-type p53 allele. We show that the mechanism of p53 loss of heterozygosity is not simply the consequence of Chromosome 11 aneuploidy and further that chromosomal instability subsequent to p53 loss is minimal. The mechanisms for pRb and p53 tumor suppression in the epithelia of two distinct tissues, mammary gland and brain, are indistinguishable. Further, this study has produced a highly penetrant breast cancer model based on aberrations commonly observed in the human disease.

Prostate cancer is known for its highly heterogeneous histological appearance. Data concerning the cytogenetic content of areas with different histology are sparse. We have genetically evaluated 10 prostatic adenocarcinomas with intermediate histopathological grades (Gleason score 7) that showed two distinctive growth patterns with different pathologies, that is, Gleason grades 3 and 4 (G3 and G4). The G3 and G4 tumor specimens were taken from spatially separated regions within the cancer mass. Array-based comparative genomic hybridization (aCGH) was performed to obtain genotypes from the 10 pairs of G3 and G4 cancer areas. The cancer DNAs were retrieved from formalin-fixed and paraffin-embedded tissues allowing optimal recognition and selection of target cells. A genome-wide 2,400-element BAC array that provided high-resolution detection of both deletions and amplifications was used. In the 20 G3 and G4 areas, 252 genomic aberrations (88 gains, 164 deletions) were noted, of which 86 were concurrent in G3 and G4 areas (34% overlap). Ninety-five of the 252 alterations were defined by a single BAC clone (54 gains, 41 deletions). Overlapping changes were more frequent for deletions (46%) than for gains (13%). Frequent coinciding deletions (> or = 20% of tumors) were seen on 8p (60%), 6q (30%), 1p (20%), 2q (20%), proximal 8q (20%), 10q (20%), 13q (20%), 16q (20%), and 18q (20%). A frequent overlapping gain (> or = 20% of tumors) was detected on distal 13q (20%). The patterns of imbalance could be found to coincide in the G3 and G4 areas of the majority of cancers. Array-based CGH can be used as a tool for the evaluation of genetic patterns in prostate cancer.

Gene amplification is an important mechanism of oncogene activation in breast and other cancers. Characterization of amplified regions of the genome in breast cancer has led to the identification of important oncogenes including erbB-2/HER-2, C-MYC, and fibroblast growth factor receptor (FGFR) 2. Chromosome 8p11-p12 is amplified in 10-15% of human breast cancers. The putative oncogene FGFR1 localizes to this region; however, we show evidence that FGFR inhibition fails to slow growth of three breast cancer cell lines with 8p11-p12 amplification. We present a detailed analysis of this amplicon in three human breast cancer cell lines using comparative genomic hybridization, traditional Southern and Northern analysis, and chromosome 8 cDNA microarray expression profiling. This study has identified new candidate oncogenes within the 8p11-p12 region, supporting the hypothesis that genes other than FGFR1 may contribute to oncogenesis in breast cancers with proximal 8p amplification.